Crystal Structure of cGMP-dependent Protein Kinase Iβ Cyclic Nucleotide-binding-B Domain: Rp-cGMPS Complex Reveals an Apo-like, Inactive Conformation
Campbell JC, et al., FEBS Lett., 591(1):221-230, 2017
The R-diastereomer of phosphorothioate analog of cGMP (Rp-cGMPS) is a well known inhibitor of cGMP-dependent protein kinase I (PKG I). However, no structural information is currently available to fully understand its mechanism of inhibition. Described herein is the determination of the crystal structure of the PKG Iβ cyclic nucleotide-binding domain (PKG Iβ CNB-B): Rp-cGMPS complex. The binding affinity of Rp-cGMPS and cGMP towards PKG Iβ CNB-B was studied by Surface Plasmon Resonance (SPR) using a Pall ForteBio Pioneer FE instrument equipped with HisCap sensor chips. His-tagged PKG Iβ CNB-B was immobilized onto activated HisCap sensor chips via standard amine coupling chemistry. Subsequently, cGMP and RpcGMPS samples at different concentrations were injected in triplicates over the sensor chip using OneStepTM injections. SPR data obtained were fitted to a mass transport limited model using QDAT software. The equilibrium dissociation constants (KD) for the binding of Rp-cGMPS and cGMP to PKG Iβ CNB-B were determined. Collectively, the crystal structure and NMR data suggest that Rp-cGMPS inhibits PKG I by stabilizing an apo-like, inactive conformation of CNB-B.
PubMed
Fructose-1, 6-bisphosphate Couples Glycolytic Flux to Activation of Ras
Peeters K, et al. , Nat Commun., 8(1):922, 2017
Both yeast and mammalian cells share Ras, a key regulator of cell proliferation and a prime proto-oncogene product. It is known that yeast and mammalian cancer cells favor fermentation of sugar over respiration. It remains divisive whether high fermentative activity is a cause or a symptom of a cancerous state since no direct relationship between glycolysis and proteins controlling cell proliferation has been reported. Described herein is the use of the yeast tps1Δ mutant to identify the molecular link between glucose fermentation and activation of Ras. Results demonstrate that fructose-1,6-bisphosphate (Fru1,6bisP) triggers activation of Ras proteins via its Cdc25/Sos1 guanine nucleotide exchange factor. The disruption of the H-Ras/Sos1 complex by Fru1,6bisP was studied by Bio-Layer Interferometry (BLI) using a Pall ForteBio Octet RED96 instrument equipped with Ni-NTA (NTA)Biosensor probes. Biosensor tips were first loaded with HIS-tagged H-Ras followed by Sos1. Subsequently, biosensors were immersed in wells containing increasing concentrations of Fru1,6bisP and dissociation of the H-Ras/Sos1 complex was studied. The equilibrium dissociation constant (KD) was determined by plotting a titration curve and fitting it to a Langmuir equation. Similar protocols were followed to investigate the effect of Fru6P, DHAP, GAP and glucose on the H-Ras/Sos1 complex. Overall findings of this study suggest that Fru1,6bisP activation of Ras lead to enhanced fermentation rate that stimulate oncogenic potency.
PubMed
P15 Peptide Stimulates Chondrogenic Commitment and Endochondral Ossification
Zhang J, et al., Int Orthop., 41(7):1413-1422, 2017
PP15, a synthetic 15 amino acid biomimetic peptide sequence derived from the alpha (α)-1 chain of collagen I has been known to enhance osteogenic differentiation and bone formation via an assumed interaction with α2β1 integrin. Reported herein is a study of the putative direct binding mechanism of P15 peptide to α2β1 integrin and the effect of P15 on bone formation. Surface Plasmon Resonance (SPR) was used to study the binding interaction between P15 and α2β1 integrin. All SPR experiments were performed using a Pall ForteBio Pioneer instrument equipped with COOH1 sensor chip. The sensor chip surface was activated using a 1:1 mixture of EDC/NHS. Subsequently, neutrAvidin was allowed to bind to the activated surface until a response plateau was reached. The residual active sites were blocked by using Tris-HCl (pH 8.5). The biotinylated P15 was injected over the neutrAvidin-coated sensor chip until all available biotin-binding sites were saturated. Bovine serum albumin (BSA) was used to block any non-specific binding sites. The kinetics of P15-α2β1 integrin binding was analyzed by injecting an active recombinant variant of α2β1 integrin in increasing concentrations over the P15 immobilized sensor chip surface. After each assay, the sensor chip surface was regenerated by injecting HCl. SPR data obtained were globally fitted. The association rate constants (kon), dissociation rate constants (koff) , and the equilibrium dissociation constant (KD) values were determined. Overall results of this study demonstrate that though there is no direct binding interaction between P15 and α2β1 integrin, P15 enhance integrin signaling, thereby increasing the differentiation of both osteoblasts and chondrocytes.
PubMed
Surface Plasmon Resonance: A Useful Strategy for the Identification of Small Molecule Argonaute 2 Protein Binders
Poser E, et al., Methods Mol Biol., 1517:223-237, 2017
The Argonaute proteins play a key role in RNA silencing processes. Small non-coding RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs) guide Argonaute proteins such as Argonaute 2 protein (AGO2) to their specific targets through base pairing resulting in silencing due to mRNA cleavage. Described herein is the use of Surface Plasmon Resonance (SPR) methods to characterize the binding interaction between Argonaute 2 and the small molecule, BCI-137. A Pall ForteBio Pioneer system equipped with COOH5 sensor chip was used to perform all SPR experiments. AGO2 was immobilized onto sensor chip surface using standard amine coupling chemistry (NHS/EDC). Any residual active sites were blocked using ethanolamine. FastStep injections of BCI-137 were performed using seven serial doubling dilutions. Sucrose was used as a bulk reference standard and internal control for the experiment. Data obtained were globally fitted using a simple 1:1 Langmuir model. The equilibrium dissociation constant was determined using a scatchard analysis of the dependence of Req on the concentration of analyte concentration. Overall results of this study demonstrate how SPR technology can be used to monitor biomolecular interactions in real time.
PubMed
Thermal Shift as an Entropy-Driven Effect
Redhead M, et al., Biochemistry, 56(47):6187-6199, 2017
The goal of this study was to provide more clarity and quantitative interpretation to the data obtained from Thermal Shift Assays (TSAs). The models and methodologies were designed to determine the ligand affinity, enthalpy, and molecular mechanism of ligand pairs. Duplex DNA as well as two protein models, carbonic anhydrase II (CAII) and glutathione S-transferase (GST) were chosen as model systems. Surface Plasmon Resonance (SPR) and Isothermal Calorimetry (ITC) were chosen as orthogonal biophysical techniques to verify TSA data. The binding of furosemide to CAII was investigated by SPR using a Pall ForteBio Pioneer FE instrument. CAII was immobilized onto the sensor chip surface using standard amine coupling chemistry. Ethanolamine was injected to cap any residual active sites. Subsequently, furosemide was injected using a OneStep injection protocol in the presence or absence of guanidine. SPR data obtained were analyzed using the QDat software. The kinetic rate constants (ka and kd) and and the KD values were determined. Overall results obtained from assay systems ranging from protein?small molecule interactions to duplex DNA stability, demonstrate the utility of TSAs in all stages of drug discovery.
PubMed
Unravelling the Molecular Basis of High Affinity Nanobodies against HIV p24: In Vitro Functional, Structural, and in Silico Insights
Gray ER, et al., ACS Infect Dis., 3(7):479-491, 2017
The p24 capsid protein is one of the earliest biomarkers of HIV-1 infection and widely used in HIV-1 diagnostic tests. Described herein is the development of high affinity nanobodies to detect HIV-1 p24. An initial semiquantitative screen was performed to identify lead nanobody candidates using ELISA. Bio-Layer Interferometry (BLI) was used to better understand the biophysical characteristics, thermodynamics and kinetics of the identified lead nanobody candidates. The binding interactions of nanobodies and mAbs with p24 subtype B was compared by BLI using a Pall ForteBio Octet RED96 system equipped with Amine Reactive 2G (AR2G) Biosensor probes. AR2G Biosensor tips were activated by incubating in a freshly mixed sulfo-NHS-EDC solution. Subsequently, p24 subtype B was immobilized onto sensor tips. Ethanolamine-HCl (pH 8.5) was used to cap any residual active sites. Biosensor tips immobilized with p24 subtype B were then dipped in various concentrations of analyte (nanobodies and mAbs) samples. BLI data obtained were fitted to a 1:1 binding model. Kinetic rate constants (kon and koff) and the Equilibrium Dissociation Constant Values (KD values) were determined. Overall results of this study indicate that the high affinity HIV-1 anti-p24 nanobodies identified have broad implications for use as capture tools in HIV-1 diagnostics.
PubMed
Zika Virus Activates De Novo and Cross-Reactive Memory B Cell Responses in Dengue-Experienced Donors
Rogers TF, et al., Sci Immunol., 2(14). pii: eaan6809, 2017
Zika virus (ZIKV) is closely related to dengue virus (DENV), suggesting an understanding of how previous DENV exposure affects the B cell response to ZIKV will be important for the development of therapeutic interventions for DENV-immune populations. Described herein is a longitudinal study of the ZIKV-induced B cell responses in three DENV-experienced donors using single B cell cloning and large-scale antibody isolation. Bio-Layer Interferometry (BLI) was used to study the binding affinities of IgGs. A Pall ForteBio Octet HTX instrument equipped with Anti-Human IgG Fc (AHQ) Biosensor probes was used to perform all BLI experiments. High-throughput binding affinity screening was performed by immobilizing IgGs on AHQ sensor tips and dipping them in ZIKV E, DENV1-4 E, or NS1 protein samples for an association step, followed by a dissociation step in PBSF buffer. BLI data obtained were fitted to a 1:1 binding model and the kinetic rate constants (association and dissociation rate constants) and the equilibrium dissociation constants (KD values) were determined. Collectively,the results of this investigation have significant implications for the development of ZIKV vaccines for DENV-immune individuals.
PubMed
Strategies Using Bio-Layer Interferometry Biosensor Technology for Vaccine Research and Development
Petersen RL, Biosensors (Basel), 7(4). pii: E49, 2017
Over the years, Bio-Layer Interferometry (BLI) biosensor technology has contributed immensely to vaccine research and development in numerous ways. Reviewed herein are some key BLI based strategies that are commonly used in human vaccine research and development such as epitope-design/epitope-capture approaches, antibody-design /antibody-capture strategies, virus capture strategies, and nucleic acid capture approaches. Dip and Read BLI assay format can handle crude samples and offers high-throughput high-precision real-time biomolecular interaction analysis, making it a very powerful platform in the field of vaccine research and development.
PubMed
AraC-like Transcriptional Activator CuxR Binds c-di-GMP by a PilZ-like Mechanism to Regulate Extracellular Polysaccharide Production
Schaper S, et al., Proc Natl Acad Sci U S A, doi: 10.1073/pnas.1702435114, 2017
This study aims to unravel the structure and mechanism of action of a c-di-GMp responsive AraC-like protein, the CuxR, that plays a role in bacterial biofilm generation. The interaction of c-di-GMP, a bacterial second messenger, with CuxR promotes CuxR dimerization and DNA binding. The binding mode of c-di-GMP to CuxR and PilZ-domain, both with similar topologies, is suggesting convergent evolution. In this work biotinylated c-di-GMP was immobilized on SA biosensors and a dilution series of CuxR was used to measure affinity of c-di-GMP to CuxR (KD=6.7 μM) on a Blitz system. Thereby demonstrating unambiguously the binding of c-di-GMP to CuxR.
PubMed
All Three Human Scavenger Receptor Class B Proteins Can Bind and Transport all Three Macular Xanthophyll Carotenoids
Rajalekshmy S, et al. , Arch Biochem Biophys., https://doi.org/10.1016/j.abb.2017.09.013 , 2017
Plant carotenoids are important antioxidant molecules required in the human diet for multiple physiological functions and health benefits. The goal of this study was to identify the role of Scavenger Receptor Class B proteins (SRBs) in the uptake into circulation, and the transport and accumulation of xanthophyll carotenoids into retinal tissue. Using purified human proteins, a Pall Fortebio Pioneer was used to determine binding kinetics of three xanthophyll carotenoids (lutein, zeaxanthin, and mesozeaxanthin) to three SRBs (SR-B1, SR-B2, and CD36). All three SRBs were immobilized onto biosensors and were capable of binding the carotenoids at low micromolar affinities characteristic of transport proteins. Cell-based assays were then performed to examine the uptake of carotenoids by SRBs using a cell line overexpression system in SRB null cells (HEK-293T) compared to retinal cells endogenously expressing SRBs (ARPE19). Additionally, immunohistochemistry of human donor retinal tissue identified areas of carotenoid accumulation in different zones of retinal tissue.
ScienceDirect
NOX4 Functions as a Mitochondrial Energetic Sensor Coupling Cancer Metabolic Reprogramming to Drug Resistance
Shanmugasundaram K, et al., Nat Commun., 8(1):997, 2017
Reported herein is an analysis of the functional interconnections between the ATP-binding motif within the NADPH oxidase isoform (NOX4), metabolic reprogramming, and cancer drug-resistance using VHL-deficient human-renal carcinoma cells as a model system. Renal cell carcinoma is caused by a mutation or absence of the von Hippel-Lindau (VHL) tumor suppressor gene. The binding affinities of WTNOX4 and mutNOX4 to ATP were studied by surface plasmon resonance (SPR) using a Pall ForteBio Pioneer system equipped with a HisCap sensor chip. The sensor chip surface was activated using nickel ions and NHS/EDC. Subsequently, the protein (wild-type or mutant) was injected over activated surface until the maximal loading is achieved. Any residual active sites on the sensor chip surface was deactivated by injecting ethanolamine. A dilution series of ATP samples were injected using the FastStep injection mode (discontinued). Overall results of this investigation demonstrate that NOX4 functions as a mitochondrial energetic sensor that couple the metabolic switch to cancer cell survival and provide useful insights to understand drug-resistance in glycolytic cancers.
PubMed
Alteration of RNA Splicing by Small-Molecule Inhibitors of the Interaction between NHP2L1 and U4
Diouf B, et al., SLAS Discov., doi: 10.1177/2472555217735035, 2017
Described herein is the development of time-resolved FRET (TR-FRET) assay to assess the interaction between proteins NHP2L1 and U4 (RNA), the two key components of spliceosome. Furthermore, a high-throughput TR-FRET screen was used to identify small molecules that disrupt the interaction between NHP2L1 and U4(RNA) and verified the results by using orthogonal methods. Topotecan and related camptothecin derivatives were identified as the most effective small molecules that interfere with NHP2L1 and U4 interaction. Topotecan binding to NHP2L1 protein or to U4 5' SL was studied by surface plasmon resonance (SPR) using a Pall ForteBio Pioneer instrument equipped with a COOH5 sensor chip. The sensor chip surface was activated using NHS and EDC. Neutravidin was immobilized onto COOH5 sensor chip by using a routine amine coupling chemistry. Any remaining active sites on the sensor chip surface were blocked with ethanolamine. Biotin-NHP2L1 and U4 5?SL-biotin were injected on separate flow channels. One flow channel on the chip was used as a reference. Topotecan was prepared in running buffer and injected using the OneStep injection feature, which exploits Taylor dispersion to generate a continuous analyte titration in a single injection. SPR data obtained were globally fitted to a 1:1 binding model and equilibrium dissociation constants (KD) were determined. Overall results of this study provide new insights into how small molecules can interfere with the interaction between NHP2L1 and U4. The findings may provide important implications for the development of therapeutics that alter RNA splicing and thereby gene function.
PubMed
Synthetic Single Domain Antibodies for the Conformational Trapping of Membrane Proteins
Zimmermann I, et al., bioRxiv, doi: https://doi.org/10.1101/168559, 2017
The group developed a novel in vitro selection platform, which builds on synthetic nanobodies called sybodies. Inspired by the shape diversity of natural nanobodies, three sybody libraries exhibiting different randomized surface shapes were engineered for high thermal stability. The conformation-selective, high affinity sybodies were engineered against the human glycine transporter GlyT1, the human equilibrative nucleotide transporter ENT1 and a bacterial ABC transporter. This platform is built exclusively on commercially available reagents and enables nonspecialized labs to generate conformation-specific binders against previously intractable protein targets. The Octet RED96 System was applied for competition measurements. Streptavidin sensors were loaded with biotinylated MBP and then dipped in wells containing Sb_MBP#1 which leads to the formation of the Sybody-MBP complex. Sensors containing the complex were sequentially dipped in a row of wells containing Sb_MBP#1 and increasing concentrations of maltose (0.1, 1, 10, 100, 1000 μM). The reversibility of the competition was shown by decreasing maltose concentrations (1000, 100, 10, 1, 0.1, 0 μM) again in the presence of Sybody Sb_MBP#1. BLI measurements were conducted for biophysical analysis of sybody-MBP interactions.
bioRxiv
Novel In Vitro Booster Vaccination to Rapidly Generate Antigen-specific Human Monoclonal Antibodies
Nandin IS, et al., J Exp Med. , 214(9):2811, 2017
Identification of novel pathogen antigens and production of human therapeutic antibodies are important in the continued development of vaccines to prevent infections disease. In this study a novel in vitro approach using human B-Cells induced to produce antigen specific antibodies is described. Antigens were linked to streptavidin coated beads along with a TLR ligand, CpG, known to induce B-Cell proliferation and differentiation into plasma cells. It is hypothesized that B-cell receptor specific internalization of CpG will lead to activation and proliferation of antigen specific B-Cells within a population. Initially a common B-Cell receptor kappa chain was targeted with a particle coated with anti-ƙ light chain antibody and CpG to allow optimization of B-Cell activation. B-Cell activation was determined by expression of CDs by flow cytometry, phosphorylation of key intracellular signalling proteins and RNA-seq analysis of stimulated B-Cells. Subsequently, the ability of the system to induce proliferation and differentiation of B-Cell subsets for tetanus toxoid, influenza haemaglutinin subtypes H1N1, H5N1, H7N9 and gp120 was tested. Plasma cells were generated and produced antibodies shown to be antigen specific by ELISA. The affinity of the generated antibodies for the antigen was determined using SPR or BLI (Octet) technologies. The Octet data was generated using streptavidin biosensors hydrated in 1X kinetics buffer for at least 10min and loaded with 12.3 μM antigen. Association of antibody from 0.094 to 60nM in 2-fold dilutions and subsequent dissociation in 1X kinetics buffer was used to generate sensorgrams for analysis of kinetics parameter ka, kd and KD.
PubMed
Semi-synthetic vNAR Libraries Screened Against Therapeutic Antibodies Primarily Deliver Anti-Idiotypic Binders
Konning D, et al., Sci Rep., 7(1):9676, 2017
Development of anti-idotypic binders which specifically recognize the variable region of monoclonal antibodies derived from VNAR libraries. BLI used to determine the binding affinity constants.
PubMed
A Covalently Bound Inhibitor Triggers EZH2 Degradation Through CHIP-mediated Ubiquitination
Wang X, et al., EMBO J, 36(9):1243-1260, 2017
EZH2 is a histone methyltransferase that plays a role in regulating chromatin structure during gene expression in normal tissue. Elevated and aberrant EZH2 expression has also been found in malignant tumors, therefore making it a promising drug target. This study identified gambogenic acid (GNA) and a derivative, GNA002, as chemical inhibitors that specifically and covalently bind EZH2 in its methyltransferase domain resulting in protein degradation through CHIP-mediated ubiqitination. Several binding analyses were performed including a competition assay using a Pall Fortebio Octet. Binding curves were generated by capturing biotinylated-GNA on SSA biosensors and titrated against the EZH2 methyltransferase domain in the presence of GNA or GNA002. The results demonstrate that both GNA and GNA002 compete with biotinylated-GNA to bind EZH2 in its methyltransferase domain with GNA002 having a stronger inhibition effect than GNA. The Octet platform allowed for all binding assays to be performed in parallel and carried out with controls for non-specific binding and signal drift over time.
PubMed
Potent Single-domain Antibodies that Arrest Respiratory Syncytial Virus Rusion Protein in its Prefusion State
Rossey I, et al., Nat Commun, 8:14158, 2017
Human respiratory syncytial virus (RSV) is a major cause of respiratory infection in children. The prefusion form of RSV F protein is a known target for development of antiviral therapies. Rossey et. al. used an Octet HTX instrument to perform a cross-competition assay between two camelid antibodies that they created and several monoclonal antibodies that bind F protein, allowing them to distinguish which antibodies bind to the same epitopes as their camelid antibodies.
PubMed
A Dock and Coalesce Mechanism Driven by Hydrophobic Interactions Governs Cdc42 Binding with its Effector Protein ACK
Tetley GJN, et al., J Biol Chem, doi: 10.1074/jbc.M117.789883, 2017
Cdc42 is a s small G protein belonging to the Rho-family which has been widely studied for its role in controlling the actin cytoskeleton and playing an taking part in several potentially oncogenic signaling networks. The aim of this study was to complete description of Cdc42-binding site on ACK. Data suggested that the binding affinity of ACK relies on several conserved residues that are critical for stabilizing the quaternary structure. ForteBio’s Octet Red system was applied to study interactions between Cdc42 and GST fusion constructs of ACK, WASP, and a basic region mutant of WASP. GST sensors were used for immobilization of GST-tagged proteins. The analysis was performed at 25 °C in the following buffer: 50mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.02% Tween 20, 0.1% BSA, and 0.05% sodium azide. Data were collected for sensors loaded with GST alone and this was subtracted from the experimental traces (by using double referencing) to subtract the effects of nonspecific binding. Sensors were regenerated in 10 mM glycine, pH 2. Interaction analysis was conducted using BLI and SPA (direct scintillation proximity assay) method and results on both platforms were fairly comparable.
PubMed
A Multidimensional Analytical Comparison of Remicade and the Biosimilar Remsima
Pisupati K, et al., Anal Chem, 89(9):4838-4846, 2017
Biosimilars are biological drugs that are “equivalent” with regard to quality, efficacy, and safety to an original innovator product. Due to their low market price, biosimilars are much more affordable to patients who want these life-changing treatments. In recent years, biosimilar monoclonal antibodies (mAbs) increasingly earn regulatory approval and prompted a need for the development of rapid approaches to characterize the innovator and biosimilar products, and to identify their clinically relevant differences. Reported herein is a multidimensional analytical comparison of biosimilar mAb, Remsima (RS) and the original innovator product, Remicade (RC). The methods used to evaluate biosimilarity include, mass spectrometry (MS), ion mobility, quantitative peptide mapping, collision-induced unfolding (CIU) analyses, as well as biophysical techniques such as Bio-Layer Interferometry (BLI). The binding affinities of different lots of RC and RS towards FcγIIIa receptor (FcγRIIIa) were assessed by the BLI. A Pall ForteBio BLItz instrument equipped with Protein G (ProG) Biosensor probes was used to perform all BLI assays. ProG sensor tips were immobilized with the RC or RS samples. Subsequently, association and dissociation kinetics of FcγRIIIa was analyzed by immersing the biosensor tips in various concentrations of FcγRIIIa (FcγRIIIa-V158 variant). The experimental results were fitted to a 1:1 binding model. The ka, kd, and KD values were determined for these binding interactions. Overall results of this investigation provide a template for future analytical comparisons of biosimilar mAbs.
PubMed
Cell Attachment Domains of the Porcine Epidemic Diarrhea Virus Spike Protein Are Key Targets of Neutralizing Antibodies
Li C, et al., J Virol, doi: 10.1128/JVI.00273-17, 2017
Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes enteric disease in pigs, resulting in high mortality rates, especially in suckling piglets. PEDV infections have resulted in significant economic losses to the swine industry. The spike (S) glycoprotein on the virion surface plays a key role in the virus cell entry, making it an excellent target for neutralizing antibodies. Described herein is the development and characterization of a panel of neutralizing and non-neutralizing mouse monoclonal antibodies raised against the spike protein’s S1 receptor binding region. The competitive binding of monoclonal antibodies to PEDV S1 antigen was evaluated by the Bio-Layer Interferometry (BLI). A Pall ForteBio Octet QK system equipped with Protein A (ProA) Biosensor probes was used to perform BLI assays. ProA sensor tips were immobilized with PEDV GDU S1-Fc protein. Subsequently, unoccupied sites were blocked using polyclonal feline antibodies. The S1-Fc-loaded sensor tips were exposed to a first antibody until a saturation was reached, followed by exposure to a second antibody. This process was repeated using different pairs of antibodies. The investigation reveals antibody epitope landscape of the PEDV S1 subunit. It also highlights the importance of cell-attachment sites on PEDV spike protein as key targets of potent neutralizing antibodies.
PubMed
Effects from Metal Ion in Tumor Endothelial Marker 8 and Anthrax Protective Antigen: BioLayer Interferometry Experiment and Molecular Dynamics Simulation Study
Jia Z, et al., J Comput Chem, 38(15):1183-1190, 2017
Anthrax is a bacterial infectious disease caused by Bacillus anthracis. Anthrax intoxication initiates when anthrax protective antigen (PA), a component of anthrax toxin, binds to anthrax toxin receptors such as the tumor endothelial marker 8 (TEM8) located on the cell surface. TEM8 is also known to play a key role in the angiogenic processes that can enhance tumor growth, hence making it an excellent target for tumor-specific therapies. TEM8 is a von Willebrand factor type A protein that contains a divalent cation in a metal-ion dependent adhesion site (MIDAS domain). By using a molecular dynamics simulation study, reported herein is the effects of different divalent cations on the interaction between TEM8 and PA. Also the binding affinity between TEM8 and PA in the presence of different metal ions (Mg2+ and Ca2+) was studied experimentally by using Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED instrument equipped with Anti-GST (GST) Biosensor probes was used to perform the BLI experiments. GST sensor tips were immobilized with GST-tagged TEM8 (TEM8-GST). Association and dissociation kinetics of PA were analyzed by immersing the TEM8-GST loaded sensor tips into various concentrations of PA solutions in the presence of MgCl2, CaCl2, or both. The BLI data were fitted globally. The kon, kdiss, and KD values were determined. Collectively, the results of this study suggest that the metal ions play a significant role in PA-TEM8 binding affinity, and the PA-TEM8 interaction prefers Mg2+ over Ca2+ as the divalent cation.
PubMed
Phage Display Analysis of Monoclonal Antibody Binding to Anthrax Toxin Lethal Factor
Goldstein J, et al., Toxins, 9(7), 221, 2017
Anthrax is caused by a gram-positive bacterium called Bacillus anthracis. AVR1674 and AVR1675 are murine monoclonal antibodies (mAbs) that are highly specific to anthrax toxin lethal factor (LF) and lethal toxin (LTx). These mAbs have been used for the development of mass spectrometry-based anthrax toxin detection tests. Reported herein is the ability of AVR1674 and AVR1675 mAbs to improve LF cleavage of specific peptide substrates in vitro, their remarkable similarity in sequence and functional activity. A panel of anti-LF mAbs binding to rLF, competitive binding of mAbs (AVR1674 and AVR1675) to rLF, and binding kinetics of AVR1675 mAb-peptide interactions were assessed using Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED system equipped with Streptavidin (SA) Biosensor probes was used to perform all BLI assays. The binding of anti-LF mAbs to rLF was studied by immobilizing biotinylated rLF onto SA sensor tips. Association kinetics were studied by dipping the biotinylated rLF loaded sensor tips in a concentration series of purified mAb IgG solutions. Inorder to determine the competition for available LF binding sites, sensor tips with preformed LF-IgG complex was immersed in AVR1675. To study mAb-peptide interactions, SA sensor tips were loaded with biotinylated synthetic oligopeptides. Subsequently, oligopeptide loaded sensor tips were immersed in various concentrations of purified mAb (AVR1675) solutions. The Octet data were fitted globally to a 1:1 Langmuir binding model. The kinetic rate constants (kon and koff) and the binding constant, KD (KD = kon/koff) were determined for all interactions. Overall findings of this study is expected to contribute significantly to future development of function-based LF detection assays with enhanced analytical sensitivity.
MDPI
Characterization of A Stable HIV-1 B/C Recombinant, Soluble, and Trimeric Envelope Glycoprotein (Env) Highly Resistant to CD4-Induced Conformational Changes
Kumar R, et al., J Biol Chem., 292(38):15849-15858, 2017
The group reports on an HIV-1 B/C recombinant, native-like trimeric Env protein that is highly resistant to CD4-induced conformational changes but displays epitopes recognized by a diverse array of bnAbs. Such features make this B/C recombinant trimeric Env a useful addition to the pool of other recently identified native-like HIV-1 Env trimers suitable for use as antigenic bait for bnAb isolation, structural studies, and use as potential immunogens. The engagement of the HIV-1 gp120 glycoprotein to the host CD4 protein triggers conformational changes in gp120 that allow its binding to co-receptors and is necessary for virus entry to establish infection. In the present study, his6-tagged protein sCD4 (D1-D2) was immobilized on Ni-NTA sensors and its binding to the varying concentrations (500, 250, 125, 62.5, 32.25, 16, and 8 nM) of either BG505 or LT5.J4b12C SOSIP.664 was measured using Octet Red96 instrument. To achieve double referencing, blank Ni-NTA sensors were also dipped into the same SOSIP samples to subtract background (nonspecific) binding of SOSIP Envs to the Ni-NTA biosensor. Kinetic analysis was conducted with samples diluted in phosphate-buffered saline, pH 7.2, containing 0.01% (w/v) bovine serum albumin and 0.002% (v/v) Tween 20. For data analysis a 1:1 binding model was applied to fit the association and dissociation curves. Kinetics measurements on BLI showed that LT5.J4b12C SOSIP.664 binds to sCD4 with slower on-rate and faster off-rate (resulting in app. 3-fold lower affinity) compared with a binding of BG505 SOSIP.664 under the same experimental conditions. These data indicate that increased resistance of LT5.J4b12C SOSIP.664 to CD4-induced conformational changes is associated with less favorable binding kinetics to CD4.
PubMed
A High-Throughput Platform for Population Reformatting and Mammalian Expression of Phage Display Libraries to Enable Functional Screening as Full-Length IgG
Xiao X, et al., MAbs, 9(6):996-1006, 2017
Phage display antibody libraries have become a powerful tool that can be used to rapidly select and evolve therapeutic and reagent antibodies. Authors have developed a novel high-throughput platform that would enable population reformatting of diverse scFv populations from phage selections into full-length IgG. This strategy preserves pairing of the variable heavy (VH) and variable light (VL) chains from phage libraries while maintaining the diversity of different sequences in a phage library output. Quantitation of IgG expression levels in cell culture supernatants were performed by using Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED384 instrument equipped with Protein A (ProA) Biosensor probes was used to conduct the quantitation experiments. The binding of IgG to the ProA sensors was measured. The sample IgG concentrations were determined by using a standard curve. Overall findings of this study demonstrate that this new high-throughput platform lead to the discovery of diverse and functional antibodies that are comparable or better than those obtained by traditional methods.
PubMed
Biosimilarity Under Stress: A Forced Degradation Study of Remicade® and Remsima™
Pisupati K, et al., MAbs, 9(7):1197-1209, 2017
RemsimaTM (infliximab) is a biosimilar monoclonal antibody and Remicade® is its reference product. Described herein is the use of humidity and temperature induced forced-degradation studies to evaluate biosimilarity of RemsimaTM and Remicade® under stress. Structural and chemical changes that occurred due to stress were analyzed in order to understand infliximab’s degradation pathways, and to identify possible modifications in the sequence. Furthermore, the effect of these modifications on biologic activity was assessed by gauging how stress affects infliximab’s potential to bind tumor necrosis factor (TNF) and FcγRIIIa. The binding affinity of infliximab to FcγRIIIa was studied by using Bio-Layer Interferometry (BLI). A Pall ForteBio BLItz system equipped with Protein G (ProG) Biosensor tips was used to perform all BLI assays. ProG sensor tips were immobilized with infliximab. The association and dissociation kinetics of FcgRIIIa was measured by immersing the infliximab loaded biosensor probes into various concentrations of FcγRIIIa solutions. The biosensor tips were regenerated after each cycle. Binding data obtained were globally fitted to a 1:1 binding model. The dissociation constant (KD) was also determined. Overall results suggest that the Remicade® and RemsimaTM behaved similarly in the forced degradation studies and provide a comprehensive biosimilarity comparison of the two mAbs.
PubMed
Computational Design of Trimeric Influenza-Neutralizing Proteins Targeting the Hemagglutinin Receptor Binding Site
Strauch EM, et al., Nat Biotechnol., 35(7):667-671, 2017
Hemagglutinin (HA) is a glycoprotein found on the surface of the influenza viruses that enable viral entry into host cells. Binding sites on HA are highly conserved and they present excellent targets for lead design. Reported herein is a computational design of high-avidity trimeric influenza-neutralizing proteins targeting the HA receptor binding site of influenza A. The binding affinity of H3 HK68 HA towards monomeric HSB.2 and trimeric HSB.2 was studied by using Bio-layer Interferometry (BLI). A Pall ForteBio Octet RED96 instrument equipped with Streptavidin (SA) Biosensor probes were used to perform binding assays. Biotinylated HA was immobilized onto SA sensor tips. BLI data were fitted using a 1:1 binding model. KD values were determined. Authors suggest that the designed trimeric proteins have important implications toward diagnostic and therapeutic applications.
PubMed
Maximizing In Vivo Target Clearance by Design of pH-dependent Target Binding Antibodies with Altered Affinity to FcR
Yang D, et al., MAbs, 9(7):1105-1117, 2017
Reported herein is the identification of a series of mouse-derived high affinity antibodies with pH-dependent target binding characteristics against two soluble antigens. Studies carried out in cynomolgus (cyno) monkeys demonstrate that the accumulation of total antigen in plasma can be reduced by increasing the affinity of antibodies to FcRn at neutral or acidic pH. Binding affinities of mAbs to FcRn (human and cyno) at pH 7.4 and pH 6.0 were analyzed by using Bio-Layer Interferometry (BLI). A Pall ForetBio Octet RED384 system equipped with Anti-Human Fab-CH1 Biosensor probes was used to perform all BLI experiments. Sensor tips were immobilized with mAbs. Subsequently, mAb loaded sensor tips were dipped into microplate wells containing human or cyno FcRn at pH 7.4 and pH 6.0. The sensor tips were regenerated with glycine (pH 1.5) after each binding cycle. Tighter binding interactions were fitted using a 1:1 Langmuir kinetic model, while Equilibrium binding analysis (steady-state analysis) was performed on the weaker interactions. The KD values were generated for each antibody Fc variant that represent binding affinities of the mAb and FcRn at pH 7.4 and pH 6.0. Overall results of this investigation provide guidance for optimal selection, design, and evaluation of sweeping antibodies.
PubMed
Non-neutralizing Antibodies Alter the Course of HIV-1 Infection In Vivo
Horwitz JA, et al., Cell, 170(4):637-648.e10, 2017
The glycoprotein spikes located on the surface of HIV-1 virus are composed of two non-covalently associated subunits, gp41 and gp120. HIV virion uses gp120 to attach itself to CD4 receptors on the host cells. Most of the individuals infected by HIV-1 develop non-neutralizing antibodies (nnAbs) during initial stages of the infection. Described herein are the effects of nnAbs on the course of HIV-1 infection in vivo. Authors have engineered a recombinant HIV-1 reporter virus that expresses a heterologous hemagglutinin-tag (HA-tag) on the surface of infected cells and virions. Binding kinetics of soluble 2-domain CD4 (2dCD4) binding to the YU2.SOSIP.664 constructs were studied by using Bio-Layer Interferometry (BLI). A Pall ForteBio Octet QKe system equipped with Ni-NTA (NTA) biosensors was used to perform all BLI assays. NTA sensor tips were loaded with HIS-tagged 2dCD4 until saturation. Association and dissociation kinetics were assessed by dipping the loaded NTA sensor tips in a concentration series of SOSIP.664 trimer samples. BLI data analyses were executed using nonlinear regression curve fitting using the Graphpad Prism software. Equilibrium dissociation constants (KD values) for the binding interactions were determined. Overall results of this study suggest that nnAbs can protect against and alter the course of HIV-1 infection by applying immune pressure on the infecting virus.
PubMed
Human Leukocyte Antigen F Presents Peptides and Regulates Immunity through Interactions with NK Cell Receptors
Dulberger CL, et al. , Immunity, 46(6):1018-1029.e7, 2017
A major histocompatibility complex (MHC) molecule, HLA-F (human leukocyte antigen F) is involved in wide range of immunoregulatory functions by signaling through natural killer cell receptors (NKRs). Current study reports the molecular description of HLA-F and provides a model to demonstrate how peptide binding modulates HLA-F recognition by NKRs. Structural, biochemical, and evolutionary studies reveal that HLA-F can exist in an open conformer (OC) and peptide-bound state. Binding interactions between HLA-F and the inhibitory NKR, LIR1 were measured using Bio-Layer Interferometry (BLI). A Pall ForteBio BLItz instrument equipped with Streptavidin (SA) Biosensor probes was used to perform all BLI experiments. SA sensor tips were immobilized with biotinylated LIR1 protein until saturation. Subsequently, loaded sensor tips were dipped in different dilutions of HLA-F, HLA-F OC and HLA-F W62R mutant solutions. Equilibrium dissociation constants (KD values) were determined by nonlinear regression using Prism software. Overall findings of this investigation provide molecular details of HLA-F to understand how HLA-F could regulate immunity via interactions with NKRs.
PubMed
Multimechanistic Monoclonal Antibodies (MAbs) Targeting Staphylococcus aureus Alpha-Toxin and Clumping Factor A: Activity and Efficacy Comparisons of a MAb Combination and an Engineered Bispecific Antibody Approach
Tkaczyk C, et al., Antimicrob Agents Chemother., 61(8). pii: e00629-17, 2017
Staphylococcus aureus is a bacterial pathogen that causes a number of infectious diseases including skin infections, bloodstream infections, and respiratory infections. The virulence factors, secreted alpha-toxin, and surface expressed clumping factor A (ClfA) play key roles in S. aureus bloodstream infections. Described herein is the engineering of bispecific antibodies (BiSAbs) with anti-alpha-toxin and anti-ClfA activities by combining multimechanistic MAbs targeting alpha-toxin and ClfA. Furthermore, the activities and efficacies of the engineered BiSAbs were compared with different antibody pairs targeting the same antigens. Anti-ClfA MAbs 11H10 and SAR114 were analyzed in a competition assay for binding to ClfA001 by using Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED96 instrument equipped with Amino-Propyl-Silane (APS) Biosensor probes were used to perform all BLI assays. APS sensor tips were immobilized with the first MAb (SAR114 or 11H10). Blocking agent, bovine serum albumin (BSA) was used to block any unoccupied Biosensor binding sites. Subsequently, sensor tips were dipped in ClfA001. Biosensor probes were then moved into wells containing the second MAb (SAR114 or 11H10). According to the competition assay results, both MAbs (11H10 and SAR114) bind to an overlapping epitope on ClfA001. Overall results of this investigation suggest that a BiSAb approach is not always superior to a MAb combination approach, and appropriate method selection likely depend on many factors, including antigen location, antigen concentration, and the binding affinities of MAb components.
PubMed
Inhibition of Pax2 Transcription Activation with a Small Molecule that Targets the DNA Binding Domain
Grimley E, et al., ACS Chem Biol, 12(3):724-734, 2017
The Pax gene family represents crucial embryonic developmental control genes that encode DNA binding transcription factors. In vertebrates, Pax2 plays a key role in the development of the kidney and the reproductive systems. Authors hypothesize that Pax2 could be an excellent target in the development of therapeutics for renal diseases. Described herein is the use of a homology model of the Pax2 paired domain and a structure based virtual screening to identify small molecules that can inhibit Pax2 transcription activation by targeting the Pax2 DNA binding domain. The EG1 was identified as a compound that can effectively block Pax2 activity and the DNA binding. Binding affinity and kinetics of EG1 interacting with the Pax2 paired domain were studied using Bio-Layer Interferometry (BLI) with a Pall ForteBio Octet RED system. Biotinylated Pax2 protein was immobilized on to Streptavidin Biosensor probes. Subsequently, sensor tips were incubated in various concentrations of EGI. The binding buffer contained PBS with 0.1% DMSO. kon, koff, and KD values were determined by global fitting of the binding curves. A steady-state analysis of the binding curves (Req. vs. Concentration) provided an affinity constant consistent with that obtained from on- and off-rates. Overall results of this study suggest that the targeting of tissue-specific developmental control genes has a significant potential to minimize off-target activity and to be more effective in treatments.
PubMed
Vesicular Stomatitis Virus N Protein-specific Single-domain Antibody Fragments Inhibit Replication
Hanke L, et al., EMBO Rep, 18(6):1027-1037, 2017
Vesicular stomatitis virus (VSV) single-stranded RNA genome is tightly encapsidated by nucleoprotein N to form a nucleocapsid (N-RNA). N-RNA serves as the template for RNA synthesis. In the absence of nucleoprotein N, full-length RNAs will not be produced and therefore nucleoprotein N is considered as a target of intervention of the virus life cycle. Described herein is the use of cytosolically expressed variable region of the heavy chain of camelid heavy-chain-only antibodies (VHHs) to target the viral nucleoprotein N. Four of the VHHs identified are specific for VSV. Affinity and kinetic parameters were determined by using Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED96 instrument equipped with Streptavidin Biosensor probes was used to perform all BLI experiments. Biosensor tips were loaded with biotinylated WT and escape mutant versions of N-RNA variants. Association and dissociation of VHHs were studied over a series of concentrations. Experimental data obtained were fitted using the 2:1 heterogeneous ligand binding global fit model. kon, koff, and KD values were determined. The binding sites on nucleoprotein N for some of the identified VHHs were characterized and the information was used to present a molecular explanation for the inhibitory effects of the N-specific VHHs. Overall, these findings reveal new features on the N protein surface that may have the potential to be used as an antiviral intervention options.
PubMed
Tumor-secreted Anterior Gradient-2 Binds to VEGF and FGF2 and Enhances Their Activities by Promoting Their Homodimerization
Guo H, et al., Oncogene, doi: 10.1038/onc.2017.132, 2017
Human anterior gradient-2 (AGR2) is a protein that overexpresses in human adenocarcinomas. The exact function of secreted AGR2, including its mechanism of action is not well understood. Reported herein is the mechanism for the tumor-promoting function of extracellular AGR2. Furthermore, authors demonstrate that extracellular AGR2 directly binds to vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) and amplify their activities. Binding affinity of AGR2 to various growth factors were studied by using the Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED96 instrument equipped with Ni-NTA biosensors was used to perform all BLI experiments. Biosensor tips were immobilized with His-AGR2. Subsequently, the AGR2-loaded biosensor tips were dipped in solutions of VEGF121, VEGF165, VEGF189, FGF2, EGF, IGF-1 and TGFβ1, respectively. KD values were determined for all the binding interactions. Collectively, findings of this investigation suggest that the secreted AGR2 is a promising antitumor target, which affects the function of extracellular signaling networks.
PubMed
Structural Basis for Dimerization and RNA Binding of Avian Infectious Bronchitis Virus nsp9
Hu T, et al., Protein Sci, 26(5):1037-1048, 2017
Coronaviruses (CoVs) are positive-strand RNA viruses with an ability to infect animals as well as humans. CoVs produce nonstructural proteins (nsps) that play a key role in assembling the RNA replicase complex, which is essential for viral replication and transcription. Nsp9 forms a homodimer, and its dimeric form is critical for RNA binding. An investigation directed towards the exploration of the structure and oligomerization state of a gamma-CoV, reported herein is an avian infectious bronchitis virus (IBV) nsp9. The crystal structure is solved for IBV nsp9. According to the crystal structure, a hydrophobic region and two parallel α-helices are vital for nsp9 dimerization. The dimeric state was further studied using various biochemical techniques. Binding interactions of wild-type and mutant nsp9 with RNA were investigated by using Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED96 system equipped with Streptavidin (SA) Biosensor probes were used to perform BLI assays. Biotinylated 20-mer RNA was loaded onto SA Biosensor tips. Sensor tips were immersed in various concentrations of nsp9 to determine association and dissociation rates. KD values were calculated for the binding interactions. Data obtained by BLI was confirmed also by Surface Plasmon Resonance (SPR) method. Overall results of this study suggest that the dimeric form of nsp9 is essential for its binding to RNA. Furthermore, three conserved residues within the dimeric interface of IBV nsp9 were revealed pivotal for both dimerization and RNA interactions.
PubMed
Enhancement of Antibody Functions Through Fc Multiplications
Wang Q, et al., Mabs, 9(3):393-403, 2017
The crystallizable fragment (Fc) domain of an IgG antibody molecule presents an excellent target for protein engineering to develop antibodies with better therapeutic effects. Described herein is the engineering of tandem IgG1 Fc molecules by adding one or two extra copies of Fcs and assessing the effects of Fc multiplication (or tandem Fc) on antibody functions. Furthermore, authors have investigated the functional activities of these newly developed antibodies both in vitro and in vivo. All expressed IgG variants were quantified by using Bio-Layer Interferometry (BLI). A Pall ForteBio Octet QK384 system equipped with Protein A (Pro A) Biosensor probes was used to perform BLI experiments. Authors have observed a significant enhancement of protection provided by the antibodies with 3 Fc tandem repeats in a Klebsiella pneumoniae mouse therapeutic model. The observations of this study reveal a new Fc engineering technology with an unique ability to expand the development of improved antibody therapeutics.
PubMed
Immunodominant Protein MIP_05962 from Mycobacterium Indicus Pranii Displays Chaperone Activity
Sharma A, et al., FEBS J, 284(9):1338-1354, 2017
Mycobacterium indicus pranii (MIP) is a nonpathogenic, saprophytic, cultivable organism and it has attracted attention as a potential intervention against many diseases including tuberculosis. Reported herein is the biophysical and biochemical characterization of an immunodominant MIP protein, MIP_05962 as a molecular chaperone. The interaction of MIP_05962 protein with one of its substrate proteins, citrate synthase (CS) was studied by using the Bio-Layer Interferometry (BLI) platform. A Pall ForteBio BLItz instrument equipped with his-tagged MIP_05962 protein immobilized Ni-NTA Biosensor probes was used to perform all BLI experiments. CS (native or non-native) was allowed to interact with MIP_05962 captured biosensor tips in an equimolar ratio. BLI data revealed that MIP_05962 binds only to non-native proteins. Overall results of this investigation confirms MIP_05962 as a molecular chaperone.
PubMed
Structural Basis of the Human Endoglin-BMP9 Interaction: Insights into BMP Signaling and HHT1
Saito T, et al., Cell Rep, 19(9):1917-1928, 2017
Endoglin (ENG)/CD105 is an essential type I transmembrane glycoprotein of the vascular endothelium, which plays a critical role in angiogenesis and vascular homeostasis. A mutated ENG exists in the genetic disorder HHT1 and involved in tumor angiogenesis and preeclampsia. It has a high potential to be used as a target in anti-angiogenic cancer therapy. It is important to expand the structural biology knowledge of ENG, and how it interacts with the key ligand bone morphogenetic protein 9 (BMP9). Reported herein are the crystal structures of the ectodomain of human ENG and its complex with human BMP9. Binding affinity of BMP9 to purified ENG constructs and mammalianized maltose binding protein (mMBP) control expressed in HEK293T cells were studied by using Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED96 instrument equipped with High Precision Streptavidin (SAX) Biosensor probes was used to perform BLI assays. BMP9 was biotinylated and loaded onto SAX biosensors. Subsequently, sensor tips were dipped in biocytin to block any unoccupied binding sites in order to reduce nonspecific binding events. Association and dissociation of each ENG construct were studied over a series of ENG concentrations. kon, koff, and KD values were determined. Collectively, the findings of this investigation provide new insights into the molecular basis of the BMP signaling pathway, thus contributing to the future development of therapeutic interventions.
PubMed
Binding of the Methyl Donor S-Adenosyl-l-Methionine to Middle East Respiratory Syndrome Coronavirus 2'-O-Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10
Aouadi W, et al., J Virol, 91(5). pii: e02217-16, 2017
Nonstructural protein 16 (nsp16) of Middle East respiratory syndrome coronavirus (MERS-CoV) is an S-adenosyl-L-methionine (SAM)-dependent 2'-O-methyltransferase (2'-O-MTase). Nsp 16 transforms virus cap-0 into cap-1 structure by methylating the ribose 2'-OH of the first transcribed nucleotide (N1) of viral RNA cap-0 structures, which blocks virus detection by cell innate immunity mechanisms. Reported herein are the biochemical characterization of nsp16 and the demonstration of nsp10 as a cofactor required for nsp16 2'-O-MTase enzymatic activity. Kinetic analysis of nsp10-nsp16 interaction was carried out using the Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED96 system equipped with Streptavidin-coated Biosensor probes was used in all BLI experiments. Biotinylated nsp10 was immobilized onto Streptavidin Biosensor tips. Association and dissociation of nsp16 was studied over various concentrations of nsp16. kon, koff and KD values were determined for the binding interactions. All binding curves were fitted using a 1:1 binding model. The steady-state curve of nsp10-nsp16 interaction was fitted using a site-specific binding equation. The effects of SAM and SAH on the off-rates of nsp16 were also evaluated. Overall results of this study might lead to the development of molecular inhibitors with an ability to target cap 2'-O-methylations and to restore the host antiviral response.
PubMed
Single Amino Acid Substitution in LC-CDR1 Induces Russell Body Phenotype that Attenuates Cellular Protein Synthesis Through eIF2α Phosphorylation and Thereby Downregulates IgG Secretion Despite Operational Secretory Pathway Traffic
Hasegawa H, et al., Mabs, doi: 10.1080/19420862.2017.1314875, 2017
Differences in Amino acid sequence in the variable region of immunoglobulin (Ig) can have dramatic effects on stability, efficiency of biosynthesis (due to differences in secretion outputs), and on the antigen-binding characteristics. Described herein is a study directed towards the investigation of underlying mechanisms of secretion-level variance between two distinct human IgG2k monoclonal antibodies (mAbs). The two mAbs differ only by one amino acid residue in the light chain complementarity-determining region 1 (LC-CDR1). Human IgG in the harvested cell culture media were quantified by Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED96 system equipped with Protein A (ProA) biosensor probes was used to perform BLI experiments. Although the primary sequences of the parental mAb and the variant mAb are nearly identical, variant mAb induced the aggregation of enlarged globular structures called Russell bodies (RBs), leading to 20-fold less IgG secretion. Overall findings of this study demonstrate that a single amino acid substitution in the LC-CDR1 is sufficient to change the efficiency of IgG secretion.
PubMed
Virus-like Particles Identify an HIV V1V2 Apex-Binding Neutralizing Antibody that Lacks a Protruding Loop
Cale EM, et al., Immunity, 46(5):777-791.e10, 2017
Neutralizing antibodies (NAbs) targeting V1V2 apex of the HIV-1 envelope (Env) trimer typically use long protruding loops to penetrate the glycan shield of HIV-1 and to contact underlying protein. Reported herein is the identification of an HIV V1V2 apex-directed NAb, N90-VRC38.01-11, by using virus-like particles and stabilized Env trimers. N90-VRC38 lineage targets the trimer apex without protruding binding loops. A crystal structure of N90-VRC38.01 with scaffolded V1V2-Env apex reveals a previously unknown binding mechanism involving side-chain-to-side-chain interactions. Binding stoichiometry of VRC38.01 to BG505 SOSIP trimers was investigated by using three techniques, including the Bio-Layer Interferometry (BLI). A Pall Foretbio Octet instrument equipped with HIS1K Biosensor probes was used to perform BLI assays. BG505.T332N.His8x SOSIP.664 was immobilized onto Biosensor probes. Biosensor tips were immersed in saturating concentrations of various mAbs (IgG or Fab fragments). Mean binding response were plotted against the known number of binding sites occupied by each mAb or mAb cocktail in order to develop a standard curve for trimer occupancy. By interpolation from a linear regression equation, it was determined that the two copies of VRC38.01 (Fab or IgG) occupy the trimer. Overall results of this study reveal a new immunological solution to target the HIV-1 V1V2 apex that may contribute significantly to the development of new vaccine strategies.
PubMed
Lung Cancer-Targeting Peptides with Multi-subtype Indication for Combinational Drug Delivery and Molecular Imaging
Chi YH, et al., Theranostics, 7(6):1612-1632, 2017
Histopathologically, lung cancer can be divided into two main types; small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC). NSCLC is further subdivided into large cell carcinoma (LCC), adenocarcinoma, and squamous cell carcinoma (SCC). Currently, there are no targeted therapies available to treat LCC or SCLC. Described herein is the identification of three new lung cancer-targeting peptides , HSP1, HSP2, and HSP4, which possess diagnostic and therapeutic potential in both SCLC and NSCLC. The binding of HSP peptides (HSP1, HSP2, and HSP4) to H460 LCC cell membrane extract were studied using Bio-Layer Interferometry (BLI). A Pall ForteBio Octet HTX system was used to perform all BLI experiments. Sensorgrams of serial dilutions of HSP peptides binding to membrane components extracted from H460 cells were reported. The association rates (ka), dissociation rates (kd), and dissociation constants (KD) were determined for each binding interaction. Overall findings of this investigation suggest that these newly discovered lung cancer-targeting peptides have the potential to play a key role in the design of targeting therapeutics, molecular imaging, and clinical detection of SCLC and NSCLC.
PubMed
Lin28a Uses Distinct Mechanisms of Binding to RNA and Affects miRNA Levels Positively and Negatively
Nowak JS, et al., RNA, 23(3):317-332, 2017
Cell lineage abnormal 28a (Lin28a) is an essential RNA-binding protein, which plays a key role in the regulation of biogenesis of miRNAs from the let-7 family. Reported herein are the molecular and biophysical data to demonstrate that Lin28a uses two distinct mechanisms of binding to pre-let-7a and pre-miRNA-9. Binding affinity of recombinant Lin28 to pre-miR-9 (wild-type and mutant) and pre-let-7-a (wild-type and mutant) were studied by Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED96 system equipped with Streptavidin Biosensor probes was used to perform all BLI experiments. Biosensor tips were immobilized with biotinylated pre-miRNA-9 or pre-let-7a-1 RNAs and immersed in a concentration series of Lin28a. Equilibrium binding isotherms were obtained by plotting the signal intensity (nm shift) against analyte concentration (μM). The KD values were determined by fitting the curve using non-linear regression. Results obtained by the BLI were validated using electrophoretic mobility shift assays (EMSA). Overall findings provide a better understanding of the roles played by Lin28a in terms of differentiating the cells and the regulation of RNA processing in cells.
PubMed
Protection of Calves by a Prefusion-stabilized Bovine RSV F Vaccine
Zhang B, et al., NPJ Vaccines, doi:10.1038/s41541-017-0005-9, 2017
The Bovine respiratory syncytial virus (bRSV) is the leading cause of respiratory disease in calves. bRSV is closely related to human RSV (hRSV), which cause severe respiratory illness in young children. Authors have recently reported a structure-based design to engineer thermostable versions of the pre-fusion (pre-F) hRSV fusion (F) glycoprotein. Reported in this article is the use of structure-based design, antigenic characterization, and X-ray crystallography to develop a prefusion-stabilized bRSV F vaccine. Pre-F-stabilizing mutations previously designed for hRSV F were transplanted into bRSV F. The binding kinetics of each purified bRSV F glycoprotein immunogen against D25, AM14, MPE8, and Mz antibodies were studied by using Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED384 instrument equipped with Ni-NTA Biosensor probes was used to perform all BLI assays. Ni-NTA sensor tips were immobilized with bRSV F variants and incubated in various Fabs in solution. KD values were determined using a 1:1 binding model. Overall results of this investigation demonstrate that pre-F-stabilized DS2 bRSV F induces high levels of neutralizing antibodies in calves. Furthermore, authors suggest that protection against hRSV may be boosted significantly in humans by vaccination through DS2-like hRSV F immunogens.
Nature
In Vitro Evolution of an Influenza Broadly Neutralizing Antibody is Modulated by Hemagglutinin Receptor Specificity
Wu NC, et al., Nat Commun, 8:15371, 2017
The discovery of human broadly neutralizing antibodies (bnAbs) against influenza virus has provided significant contributions toward the development of a universal influenza vaccine. C05, is a bnAb that targets the influenza haemagglutinin (HA) receptor-binding site (RBS). Reported in here is an investigation of the evolution of bnAb C05 by analyzing its functional sequence space. Binding affinities of C05 variants against hemagglutinins from different influenza strains were studied by using Bio-Layer Interferometry (BLI). All BLI assays were performed using a Pall ForteBio Octet RED system. Biotinylated HA0 was immobilized onto Streptavidin biosensor probes and dipped in samples (supernatants from transfected cells or purified Fab). KD value was determined using a 1:1 binding model. When the binding affinities were relatively weak or the non-specific binding (NSB) was observed, a 2:1 heterogeneous ligand model was used to obtain a better fitting. Overall data of this study suggest that the highly conservative substitutions in HA RBS among different influenza strains may modulate the evolution of bnAbs differently.
PubMed
Autophosphorylation-based Calcium (Ca2+) Sensitivity Priming and Ca2+/Calmodulin Inhibition of Arabidopsis thaliana Ca2+-dependent Protein Kinase 28 (CPK28)
Bender KW, et al., J Biol Chem, 292(10):3988-4002, 2017
Calcium (Ca2+)-dependent protein kinases (CPKs) are proteins that play a key role in Ca2+ signaling and protein phosphorylation in plants. Described herein is a screening of a panel of plant CPKs for calmodulin (CaM) binding in order to explore a possible role of CaM in regulation of CPKs. Furthermore, the contribution of autophosphorylation to CPK function has been investigated. Recombinant Arabidopsis thaliana CPK28 was identified as a high affinity Ca2+/CaM binding protein. Bio-Layer Interferometry (BLI) was used to perform the binding analysis of the CaM6-CPK28 binding interaction. A Pall ForteBio Octet instrument equipped with biotinylated CaM6 immobilized Streptavidin Biosensor probes were used. After immobilization of CaM6, the Biosensor tips were immersed in a dilution series of purified recombinant His6-CPK28. The KD value of CPK28-CaM interaction was determined using a steady-state binding model equation. The study has revealed new scenarios pertaining to the regulation of CPK28 by Ca2+, autophosphorylation, and CaM binding.
PubMed
AZ17: a new bispecific drug targeting IL-6 and IL-23 with potential clinical use—improves psoriasis in a human xenograft transplantation model
Karin Stenderup et. al., Protein Engineering, Design and Selection, 28(10):467-480, 2015
Psoriasis is a multifactorial disease in which interleukin (IL)-6 and IL-23 are important disease mediators because they facilitate development of Th17 cells. The group aimed to develop clinically relevant bispecific antibodies targeting IL-6 and IL-23 exhibiting high affinity, high stability and the ability to be produced in high yield. Thus, the bispecific drug molecule AZ17 has the potential to alleviate psoriasis. Stability of the drug and its long residence time in vivo was achieved by introducing flexible poly-ethylene glycol molecule into the structure. ForteBio Pioneer technology (SensiQ Pioneer system) was applied to measure affinity of AZ17 towards IL-6 andIL-23. Kinetic parameters were measured using a carboxylated COOH1 sensor. The sample running buffer was Hepes-buffered saline (HBS), containing 10 mM Hepes, 150 mM NaCl (pH 7.4), 0.001% Tween 20 and 3 mM EDTA. The assay temperature was maintained at 25°C with an analyte flow rate of 50 μl/min, including a 2 min association phase and a 10–30 min dissociation phase. Binding affinity studies resulted in determination of KD of 215 pM for IL-6 and KD of 92 pM for IL-23. Additionally, the kinetic parameters of anti-IL-6-scFv were measured and obtained KD = 98 pM, demonstrated a reduction in the IL-6 affinity in AZ17.
peds
Measurement of Free Versus Total Therapeutic Monoclonal Antibody in Pharmacokinetic Assessment is Modulated by Affinity, Incubation Time, and Bioanalytical Platform
Talbot JJ, et al., AAPS J, 17(6):1446-54, 2015
Ligand binding assays (LBAs) play a critical role in quantifying serum therapeutic protein (TP) levels in pharmacokinetic and pharmacodynamic assessments. Described herein is the impact of the bioanalytical platform, reagent-antibody affinity, and incubation time on obtaining free or total TP levels. An ELISA-based LBA that quantifies humanized anti-sclerostin monoclonal antibody (TPx) was used as the model system. All kinetic experiments were performed by Bio-Layer Interferometry (BLI) on a Pall ForteBio Octet RED384 system equipped with Streptavidin (SA) Biosensor probes. The sensor tips were loaded with biotinylated-Anti-Fc mAb, which were subsequently captured with TPx. Sensor probes were then dipped in wells containing Sclerostin (Scl) protein. Kinetic parameters (kon, koff, and KD values) were determined. The importance of having a complete picture of the binding interaction from a BLI kinetic assay as compared to the use of end-point data from ELISA is higlighted. Overall results of this investigation provide a better understanding of how the choice of reagents and the platform for LBAs can affect accurate quantitation of either the free or total TP concentrations.
PubMed
Dengue Virus prM-Specific Human Monoclonal Antibodies with Virus Replication-Enhancing Properties Recognize a Single Immunodominant Antigenic Site
Smith S, et al., J Virol, 90(2):780-9, 2015
Dengue is a mosquito-borne disease caused by four dengue virus (DENV) serotypes (DENV1 to DENV4). Currently there are no specific dengue therapeutics. It is crucial to have a better understanding of the pathogenesis of severe dengue in order to develop DENV vaccines. It is believed that cross-reactive antibodies produced during a primary infection play an important role in the pathogenesis of severe dengue. Described herein is the isolation of a large panel of dengue premembrane (prM) protein-specific human monoclonal antibodies (MAbs) from post-infected individuals in order to explore antigenic sites on the prM protein that is recognized by human antibodies. All competition (binding) assays were performed by Bio-Layer Interferometry(BLI) using a Pall ForteBio Octet RED instrument equipped with Streptavidin Biosensor probes. Biotinylated mouse anti-dengue virus prM MAb 2H2 was immobilized onto Biosensor tips. The sensor tips were then immersed in a solution of crude DENV2 S-16803 viron particles. After a washing step, the first human anti-DENV MAb was introduced. Then exposed to a second human MAb, in order to study binding interference. Results obtained from competition binding assays demonstrate that only a single antibody molecule can be bound to each prM protein molecule at any given time. Overall, the findings of this study indicate a distinct overlapping epitope that lies within a single major antigenic site. This antigenic domain was also suggested to represent an immunodominant site of the prM protein.
PubMed
Structural Basis Underlying the Binding Preference of Human Galectins-1, -3 and -7 for Galβ1-3/4GlcNAc
Hsieh TJ, et al., PLoS One, 10(5):e0125946, 2015
Galectins are a large family of β-galactoside-binding proteins, known to participate in a broad variety of biological functions including cell adhesion, cell growth regulation, and apoptosis. Previous studies suggest that human galectins play a critical role in processes linked to many types of cancers. It is vital to identify how each galectin member interacts with various glycan structures in connection with cancer progression for the development of new cancer therapeutics. All galectins are known to recognize Galβ1-3/4GlcNAc disaccharides, namely type 1 and 2 Lac-NAc (abbreviated as LN1 and LN2, respectively). Reported herein is the crystal structures of full-length human galectin-1 (hGal1), galectin-7 (hGal7), and the C-terminal carbohydrate recognition domain (CRD ) domain of galectin-3 (hGal3-CRD) in complex with LN1. Binding affinity of LN1 and LN2 to hGal1, hGal3 and hGal7 were studied by Bio-Layer Interferometry (BLI) and Fluorescence polarization (FP)-based competition assays. A Pall ForteBio Octet RED system equipped with biotinylated galectins captured Super Streptavidin Biosensor probes was used in the BLI assays. The free sites on the biosensor tips were blocked with biocytin in order to avoid non-specific binding. Galectin bound biosensor tips were then immersed in a concentration series of 3-fold diluted LN1/LN2 solutions. Dissociation constants were determined for the binding interactions. The dissociation constants obtained by BLI and FP-based competition assays demonstrated a highly consistent trend. Overall results from this structural study reveals why galectins display a binding preference for Galβ1-3/4GlcNAc disaccharides.
PubMed
Bispecific Antibodies Targeting Different Epitopes on the HIV-1 Envelope Exhibit Broad and Potent Neutralization
Asokan M, et al., J Virol., 89(24):12501-12, 2015
The high antigenic diversity of human immunodeficiency virus type 1 (HIV-1) makes the development of a single-antibody that potently neutralizes nearly all strains of HIV-1 a major challenge. Therefore, the physical combination of two broadly neutralizing antibodies (bNAbs) could potentially broaden neutralization coverage against majority of viral strains. Described herein is the construction of four different bispecific immunoglobulins (IgGs) using the CrossMab format. Newly constructed bispecific IgGs were based on four parental HIV-1 bNAbs, one each to the CD4 binding site (CD4bs) , glycan-dependent site near the variable loop 3 (V3-glycan), a variable-region (V1V2) glycan-dependent site on the trimer apex (V1V2-glycan), and membrane-proximal external region (MPER) on the HIV-1 envelope glycoprotein (Env). Each bispecific IgG was designed with a different combination of two antigen binding fragments. Bio-Layer Interferometry (BLI) was used to confirm the bispecificity of the bispecific antibodies and to determine the binding affinities of such antibodies to various ligands. BLI experiments were performed on a Pall ForteBio Octet RED384 instrument. For bispecificity confirmation studies, Streptavidin (SA) Biosensor probes were immobilized with biotinylated RSC3, then immersed in the antibody, followed by probing with a ligand against the second arm of the antibody. For 10E8 x PG916RSH, Amine Reactive 2G (AR2G) Biosensor probes were captured with 1VH8 x CAP256SU and dipped in antibody followed by 3AGJ-GFP. For the determination of binding affinities, Anti-Human Fc (AHC) biosensors were immobilized with either the parental or bispecific antibodies and then immersed in a serial dilutions of corresponding ligand (RSC3 for VRC07, 3AGJ-MPER-GFP for 10E8, and BG505.SOSIP for PGT121). For assays involving PG9-16-containing antibodies, 1VH8_CAP256SU was captured onto AR2G Biosensor tips and then coupled with a serial dilution of PG9-16-Fab or MAbs. The study demonstrates that newly developed bispecific antibodies possess binding specificities of the two parental antibodies. A single bispecific antibody having the optimal combination of VRC07 x PG9-16 was found to neutralize more viruses (97%) than either parental bNAb with a high potency similar to those of the parental bNAbs. These findings suggest that bispecific antibodies are promising candidates for future development of HIV-1 therapeutics.
PubMed
A Search for Synergy in the Binding Kinetics of Trastuzumab and Pertuzumab Whole and F(ab) to Her2
Lua WH, et al., npj Breast Cancer, doi:10.1038/npjbcancer.2015.12, 2015
It has been reported that the survival rate of Her2 overexpressing breast cancer patients can be improved significantly by combining the antibodies Trastuzumab and Pertuzumab in their treatments. However, the exact molecular mechanism underlying this clinical observation is not understood well. A recent molecular modeling study hypothesized colocalization of the two antibodies onto the extracellular domain of Her2 may lead to enhanced affinities. Reported in the current study are the efforts towards experimental characterization of the interaction between antibodies, Trastuzumab and Pertuzumab, their binding kinetics, and also the binding of their F(ab)s to the extracellular domain of Her2. Bio-Layer Interferometry (BLI) was used to assess the binding affinities. A Pall ForteBio BLItz instrument equipped with HIS-tagged Her2 captured Ni-NTA biosensor probes was used to perform BLI assays. Antibodies were successively loaded onto Biosensor tips in order to measure synergistic binding of whole antibodies and their F(ab)s. Kinetic parameters (ka, kd, and KD) were determined based on the binding curves. Overall results demonstrate that both Trastuzumab and Pertuzumab, whether it is the whole antibody or the fragments can bind simultaneously to Her2. However they do not enhance the binding of each other.
npj Breast Cancer
The Structural Basis of DNA Binding by the Single-stranded DNA-binding Protein from Sulfolobus Solfataricus
Gamsjaeger R, et al., Biochem J, 465(2):337-46, 2015
Single-stranded DNA-binding proteins (SSBs) are a class of proteins that are present in all organisms. SSBs are known to have one or more characteristic conserved oligonucleotide/oligosaccharide-binding (OB) folds. Single stranded DNA (ssDNA) binds to these conserved OB domains with high affinity. The SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus (SsoSSB) has a single OB fold coupled to a flexible C-terminal tail. Recent studies have identified two new human SSBs with protein sequences that are similar to SsoSSB. Described herein is the determination of the structure of the OB domain of SsoSSB bound to ssDNA using NMR spectroscopy. SsoSSB mutational analysis was performed by Bio-Layer Interferometry (BLI) using a Pall ForteBio BLItz instrument. Streptavidin Biosensor tips were immobilized with 5' biotinylated ssDNA oligonucleotide (5'-AAATTTTTT-3'). The sensor tips were then exposed to serial dilutions of the protein. The KD value was calculated for the binding interaction. This study reveals that the ssDNA binding to SsoSSB is regulated by base-stacking of three key aromatic residues. In contrast, the human SSBs (RPA and hSSB1) involve only two aromatic residues. The results also demonstrate SsoSSB recognition of ssDNA with a footprint of five bases.
PubMed
Restoring Anticancer Immune Response by Targeting Tumor-Derived Exosomes With a HSP70 Peptide Aptamer
Gobbo J, et al., J Natl Cancer Inst, 108(3). pii: djv330, 2015
According to recent studies, myeloid-derived suppressor cells (MDSCs) suppress tumor immunity of cancer patients. Exosomes are cell-derived nanovesicles that are discharged into the extracellular environment. Exosomes, through heat shock protein 70 (HSP70) on their membrane, binds to the toll-like receptor 2 (TLR2) on MDSCs, in order to modulate antitumor immune responses. Described herein is the analysis of exosomes isolated from mouse (C57Bl/6), breast/lung/ovarian cancer patient samples, and cultured cancer cells, using various methods such as the Nanoparticle tracking analysis, Bio-Layer Interferometry, FACS, and electron microscopy. Bio-Layer Interferometry (BLI) was used to study protein-protein binding interactions. A Pall ForteBio Octet RED instrument equipped with Streptavidin Biosensor probes were used to perform BLI assays. Biosensor tips were allowed to capture biotinylated peptide aptamer ligand (A8) or TLR2. The sensor tips were then incubated with exosomes (from cancer cell lines, normal cells, mice blood, or human urine) or HSP70 or cmHSP70. The KD values were determined for each binding interaction. Overall results of this investigation demonstrate that the peptide aptamer A8 tightly binds to HSP70 present in Tumor-derived exosomes (TDEs) and in turn blocks the MDSCs stimulation. By using an in vivo study, authors report that, associating A8 with a chemotherapeutic drug such as cisplatin or 5-fluorouracil, anticancer immune response can be restored. Finally, the potential of using A8 to quantify TDEs in human samples was also demonstrated.
PubMed
Peptides of Presenilin-1 Bind the Amyloid Precursor Protein Ectodomain and Offer a Novel and Specific Therapeutic Approach to Reduce β-Amyloid in Alzheimer's Disease
Dewji NN, et al., PLoS One, 10(4):e0122451, 2015
Alzheimer's disease (AD) pathogenesis is widely accepted to be triggered by the accumulation of the amyloid-β (Aβ) in the brain. Aβ is a set of oligopeptides each of which is proteolytically cleaved from β-amyloid precursor protein (APP) by β-secretase and γ-secretase. Presenilin (PS)-1 or -2 act as the catalyst in this process. Most of the current approaches used to treat AD aim to inhibit the effects of Aβ by altering the activities of β- or γ-secretase. This article reveals a novel and completely different approach to reduce the production of Aβ without affecting the catalytic activities of β- or γ-secretase. The binding affinities of synthetic peptides P1, scrambled P1, P4, P5, P7 and P8 for the soluble ectodomain of APP or control IgG were studied by Bio-Layer Interferometry (BLI) on a Pall ForteBio Octet RED96 instrument. Streptavidin (SA) Biosensor probes were immobilized with biotinylated peptides. Biosensor tips were then immersed in various concentrations of APPs. Kinetic parameters (KD, kon, and koff) were determined for each binding interaction. Overall findings of this study demonstrate that the two small peptides from PS-1 NH2-terminal domain, P4 and P8 effectively reduce the production of Aβ in AD model systems. These peptides and their derivatives offer promising new drug leads for the development of future AD therapeutics.
PubMed
Conserved Role of an N-Linked Glycan on the Surface Antigen of Human Immunodeficiency Virus Type 1 Modulating Virus Sensitivity to Broadly Neutralizing Antibodies against the Receptor and Coreceptor Binding Sites
Townsley S, et al., J Virol., 28;90(2):829-41, 2015
Earlier studies have reported that the removal of a potential N-linked glycan (PNLG) site at amino acid residue 197 (N7) in the conserved region of gp120 of HIV-1 envelope glycoprotein (Env) increases neutralization sensitivity of the antibodies directed at CD4 binding site (CD4bs) relative to its wild-type (WT). Described herein is an investigation of the role of N-linked glycan at amino acid position 197 (N7) in modulating the antigenicity of HIV-1 Env pertaining to a panel of HIV-1 strains. Broadly neutralizing antibodies (bNAbs) with defined epitope specificities were used. Binding affinity of N7-glycosylated and deglycosylated gp120 to CD4-IgG2 was tested by Bio-Layer Interferometry. A Pall ForteBio Octet instrument equipped with Anti-Human IgG Fc Biosensor probes were used for the binding assays. CD4-IgG2 immobilized Biosensor tips were dipped in a 2-fold dilution series of gp120. The association rate constant (ka) and the dissociation rate constant (kd) were measured. The KD values were calculated for the binding interactions. The findings of this study revealed a conserved role played by the N7 glycan in masking epitopes present in both CD4bs and variable loops.
PubMed
Immunogenicity of a Prefusion HIV-1 Envelope Trimer in Complex with a Quaternary-Structure-Specific Antibody
Cheng C, et al., J Virol., 90(6):2740-55, 2015
The HIV-1 envelope trimer (Env) is the main target of virus-directed neutralizing antibodies. One of the commonly used glycoprotein-mimics of HIV-1 Env is the SOSIP.664-stabilized soluble trimer from HIV-1 strain BG505. Reported in here is the enhancement of antigenic specificity of BG505 SOSIP.664 by minimizing the exposure and reactivity of the variable loop 3 (V3) region on Env. To stabilize BG505 SOSIP.664 trimer, it was coupled with the antigen-binding fragment (Fab) of broadly neutralizing antibody, PGT145. The stabilities, antigenicities, and immunogenicities of BG505 SOSIP.664 (BG505 trimer) and the PGT145-trimer complex were compared. Antigenic analysis of BG505 trimer and PGT145-trimer complex was performed by Bio-Layer Interferometry (BLI) using a Pall ForteBio Octet HTX instrument equipped with Anti-Human Fc-Capture biosensors (AHC). The binding of the ligand-free BG505 trimer and the PGT145-trimer complex to a panel of purified monoclonal antibodies (VRC01, PGT145, PGT151, F105, and 17b) were studied. Antibodies were immobilized onto AHC Biosensor probes. The sensor tips were subsequently dipped in six different concentrations of trimer. Overall results of this study reflects an improved antigenic specificity of the PGT145-trimer complex for neutralizing antibodies as compared to wild type BG505 trimer. Data also suggest that the immune response on crucial neutralization epitopes can be improved by introducing appropriate modifications to the trimer immunogens.
PubMed
A Neglected Modulator of Insulin-degrading Enzyme Activity and Conformation: The pH
Grasso G, Satriano C, and Milardi D, Biophys Chem, 203-204:33-40, 2015
Concentration Analysis of Rapid-Equilibrium Binding between Warfarin and Human Serum Albumin Using a Gradient Surface Plasmon Resonance Technique
Vachali P and Martin A, Single Cell Biology, doi 10.4172/2168-9431.1000i103, 2015
Structure of Human N-acylphosphatidylethanolamine-hydrolyzing Phospholipase D: Regulation of Fatty Acid Ethanolamide Biosynthesis by Bile Acids
Magotti P, et al., Structure, 23(3):598-604, 2015
Cytokine Refacing Effect Reduces Granulocyte Macrophage Colony-stimulating Factor Susceptibility to Antibody Neutralization
Heinzelman P, Carlson S, and Cox G, Protein Eng Des Sel, 28(10):461-6, 2015
Engineering Superactive Granulocyte Macrophage Colony-Stimulating Factor Transferrin Fusion Proteins as Orally-Delivered Candidate Agents for Treating Neurodegenerative Disease
Heinzelman P and Priebe M, Biotechnol Prog, 31(3):668-77, 2015
A Rapid and Sensitive Assay for the Detection of Benzylpenicillin (PenG) in Milk
Pennacchio A, et al., PLoS One, 10(7):e0132396, 2015
Detection Of Biological Molecules Via Molecularly Imprinted Polymers Coupled With Surface Plasmon Resonance
Cenci L, et al., J Nanobiotechnology, 13:51, 2015
H5N1 Vaccine-Elicited Memory B Cells Are Genetically Constrained by the IGHV Locus in the Recognition of a Neutralizing Epitope in the Hemagglutinin Stem
Wheatley A, et al., J Immunol, 195(2):602-10, 2015
Although it is becoming evident by the recent research that viral hemagglutinin (HA) could be the target for broadly neutralizing antibody responses, the frequency and phenotype of HA stem-specific B cells in vivo are yet to be revealed. This article focuses on HA stem-specific B cell responses following H5N1 vaccination and explains the re-expansion of memory B cells specific for stem epitopes. The kinetic characterization of binding between mAbs and HA were investigated using a Pall ForteBio Octet HTX system equipped with Streptavidin Biosensor probes. Biotinylated HA-trimers captured on to Streptavidin probes were used to screen against Fab of mAbs.
PubMed
Heat Shock Protein 90 Mediates the Apoptosis and Autophage in Nicotinic-Mycoepoxydiene-Treated HeLa Cells
Sun Y, et al., Acta Biochim Biophys Sin (Shanghai), 47(6):451-8, 2015
This article characterizes the interaction between Heat shock protein 90 (Hsp90) and its inhibitor nicotinicmycoepoxydiene (NMD). A Pall ForteBio Octet RED system equipped with Super-Streptavidin (SSA) biosensors was used to capture biotinylated Hsp90 binding to NMD. The KD value obtained from the Octet RED system is comparable to that obtained from a Fluorescent assay. Overall results suggest a novel mechanism pertaining to Hsp90 inhibition by NMD.
PubMed
Helicobacter Pylori FlhA Binds the Sensor Kinase and Flagellar Gene Regulatory Protein FlgS with High Affinity
Tsang J, et al., J Bacteriol, 197(11):1886-92, 2015
This study provides additional insights into flagellar assembly in Helicobacter pylori. The high affinity interaction between Flagellar Gene Regulatory Protein (Flgs) and FlhA suggest an additional role for FlhA in flagellar assembly. A Pall ForteBio Octet QK system equipped with Streptavidin Biosensor probes was used to capture biotinylated peptides representing residues 1 to 25 of FlhA. The analytes used in this investigation had FlgS or a shorter version called FlgSc.
PubMed
Heterogeneous IgG Interacts with FcRn and Its Transport Across Gastrointestinal Barrier
Pang G, et al., Food and Agricultural Immunology, DOI:10.1080/09540105.2014.918588, 2015
This investigation focuses on the pathway for transporting of heterogeneous IgG across gastrointestinal barrier and hence to their destination in vivo upon consumption of IgG products from various mammalian origins. The human FcRn interactions with various IgG from different animal species were evaluated using a Pall ForteBio Octet RED system equipped with Streptavidin Biosensor probes. Biotinylated IgG captured on to the Streptavidin biosensors were used to bind human FcRn in solution. The results suggest that heterogeneous IgG could be transported into the blood stream by neonatal Fc receptor (FcRn).
Food and Agricultural Immunology
High Throughput Cross-Interaction Measures for Human IgG1 Antibodies Correlate with Clearance Rates in Mice
Kelly R, et al., MAbs, 7(4):770-7, 2015
The development of monoclonal antibodies (mAbs) into drugs can often be difficult due to developability issues. Therefore implementing early stage developability screening assays is important to minimize downstream risks. In this study the authors have selected a panel of 16 monoclonal antibodies (mAbs) exhibiting different developability profiles, and investigated their later stage behavior from expression titer to accelerated stability and pharmacokinetics in mice. A clone self-interaction by Bio-Layer Interferometry (CSI-BLI) assay was perfomed using a Pall ForteBio Octet HTX system equipped with AHQ biosensors. The results indicate that the assays tested could be used to screen panels of lead antibodies at a reasonably early stage in the drug discovery process.
PubMed
High-Throughput Kinetic Screening of Hybridomas to Identify High-Affinity Antibodies Using Bio-Layer Interferometry
Lad L, et al., J Biomol Screen, 20(4):498-507, 2015
In this study authors have compared a traditional solid-phase enzyme-linked immunosorbent assay (ELISA) against Bio-Layer Interferometry (BLI) to screen high-affinity antibodies from hybridoma clones. A Pall ForteBio Octet RED384 instrument was used as their kinetic screening platform. This instrument was equipped with anti-mouse immunoglobulin G (IgG) Fc (AMC)-coated biosensor tips which was used to capture atibodies from hybridoma supernatants. This article also discusses about the advantages and limitations of different methods and platforms available for screening antobodies such as the flow cytometry, ELISA, electrochemiluminescence using Meso Scale Discovery (MSD) platform, and homogeneous time-resolved fluorescence (HTRF). Overall results suggest that the kinetic screening is an efficient method for identifying high-affinity antibodies.
PubMed
High-Throughput mAb Expression and Purification Platform Based on Transient CHO
Barnard GC, Hougland M, Rajendra Y, Biotechnol Prog, 31(1):239-47, 2015
This article describes a coupled approach based on high-throughput transient transfection process involving CHO cells and a high-throughput protein A purification process, which could be used as a mAb drug discovery tool. Antibody titers were measured using a Pall ForteBio Octet QK system equipped with Protein A biosensor probes in the Quantitation mode. Calibration curves generated involve appropriate mAb standards.
PubMed
Identification and Structure Solution of Fragment Hits Against Kinetoplastid N-Myristoyltransferase
Robinson D, Wyatt P, Acta Crystallogr F Struct Biol Commun , 71(Pt 5):586-93, 2015
The article describes identification of new chemical hits that could occupy DDD85646 binding site of Trypanosoma brucei N-myristoyltransferase (TbNMT). This is a therapeutic target for the treatment of human African trypanosomiasis. Fragment screening was performed by ligand-observed NMR spectroscopy. Crystal structures of the identified hits were reported in complex with closely related NMT from Leishmania major. A Pall ForteBio Octet RED384 instrument equipped with Super-Streptavidin (SSA) Biosensor probes was used to obtain the binding measurements. Biotinylated TbNMT and Leishmania major N-myristoyltransferase (LmNMT) were captured onto the SSA biosensor tip surface. The fragment hits obtained from this study are smaller and possess limited complexity. Results suggest both binding-site configurations could be induced upon ligand binding, hence demonstrating conformational plasticity of the peptide-binding site.
PubMed
Identification of a Common Epitope Between Enterovirus 71 and Human MED25 Proteins Which May Explain Virus-Associated Neurological Disease
Fan P, et al., Viruses, 27;7(4):1558-77, 2015
This article describes for the first time, the identification of a common epitope (PPGAPKP) of human mediator complex subunit 25 (MED25) and Enterovirus 71 VP1. A Pall ForteBio Octet RED96 system was used to determine the affinity of the monoclonal antibody (2H2) to both MED25 and VP1. Biotinylated 2H2 mAb immobilized onto Streptavidin Biosensor probes was screened against a dilution series of the proteins.
PubMed
Identification of Malaria Parasite-Infected Red Blood Cell Surface Aptamers by Inertial Microfluidic SELEX (I-SELEX)
Birch C, et al., Sci Rep, 1;5:11347, 2015
Plasmodium falciparum (malaria parasite) enters the human red blood cells (RBCs) and traffic parasite-synthesized proteins to the RBC surface. These parasite-generated proteins present on the surface of infected RBCs tend to interact with various receptors on host cells and contribute to disease pathogenesis. This study introduces a new strategy called inertial microfluidic SELEX (I-SELEX) to identify high affinity aptamers that selectively recognize distinct surface protein targets present on parasite infected RBCs. Aptamer binding studies were carried out using a Pall ForteBio BLItz instrument equipped with thrombin-loaded Streptavidin (SA) biosensors. Those high affinity aptamer reagents will be useful for mapping malaria parasite-infected RBC surface proteome in detail.
PubMed
Identification of the Base-Pairing Requirements for Repression of hctA Translation by the Small RNA IhtA Leads to the Discovery of a New mRNA Target in Chlamydia trachomatis
Grieshaber N, et al., PLoS One , 10(3):e0116593, 2015
This study demonstrates IhtA mediated inhibition of HctA translation by base pairing with a seven nucleotide CAUGGCG sequence centered around AUG start codon. A deeper understanding of the translation repression in Chlamydia is crucial to unravel chlamydial development cycle. The RNA-RNA interactions monitored by a Pall ForteBio Octet QKe system was equipped with Streptavidin (SA) biosensor tips. RNAs were annealed to a biotinylated oligo sequence in order to capture onto SA biosensors. Association was monitored by incubating with 1500 nM sRNA IhtA.
PubMed
Identifying Low-Level Sequence Variants via Next Generation Sequencing to Aid Stable CHO Cell Line Screening
Zhang S, et al., Biotechnol Prog, doi: 10.1002/btpr.2119, 2015
This article describes for the first time, the application of transcriptome sequencing (RNAseq) in an IgG1 monoclonal antibody (mAb) cell line development (CLD) process to assist stable Chinese hamster ovary (CHO) cell line screening, by identifying low-level sequence variants. Batch titers were measured using a Pall ForteBio Octet instrument. Results suggest that RNAseq is a rapid and highly sensitive technique to detect genetic mutation de novo, and it is useful for cell line screening at various stages of CLD.
PubMed
Immunotherapy Based on Bispecific T-Cell Engager with hIgG1 Fc Sequence as a New Therapeutic Strategy in Multiple Myeloma
Zou J, et al., Cancer Sci, 106(5):512-21, 2015
This is an immunotherapy investigation describing the development of a single-chain variable fragment (ScFv) combination of anti-CD3 ScFv and anti-CD138 ScFv, using hIgG1 Fc (hIgFc) sequence. The bispecific T-cell engager (BiTE) with the addition of single-chain fragment hIgFc (BiTE-hIgFc, STL001) can target T cells, natural killer cells, and multiple myeloma cells. Bio-Layer Interferometry was used to determine dissociation constant (KD) of aCD138-ScFv-hIgFc and BiTE-hIgFc (STL001) antibody. A Pall ForteBio Octet RED96 instrument equipped with Streptavidin Biosensor probes was used in the kinetic characterization. The biotinylated rCD138 antigen was immobilized on to Streptavidin probes.
PubMed
Improving Target Cell Specificity Using a Novel Monovalent Bispecific IgG Design
Mazor Y, et al., MAbs, 7(2):377-89, 2015
This article introduces a novel platform called DuetMab for efficient production of monovalent bispecific IgGs. It uses knobs-into-holes (KIH) methodology to heterodimerize two distinct heavy chains while increasing the efficiency of heavy and light chain pairing with an engineered disulfide bond. A Pall ForteBio Octet RED384 instrument was used in the Quantitation and Kinetic Characterization of antibodies. The concentration of DuetMab antibodies in cell-culture supernatants were measured by Protein A biosensor probes in the Quantitation mode. Whereas, the kinetic measurements of DuetMab binding to EGFR and HER2 were performed using the Octet RED384 equipped with anti-human IgG Fc capture (AHC) biosensor probes. Concurrent binding of antibodies to EGFR and HER2 were also tested on the Octet RED384 equipped with AHC and SA biosensor probes.
PubMed
In Silico Discovery and Validation of Potent Small-Molecule Inhibitors Targeting the Activation Function 2 Site of Human Oestrogen Receptor α
Singh K, et al., Breast Cancer Res, 17:27, 2015
Described herein is the identification of more potent and selective small-molecule inhibitors for a coactivator-binding pocket on oestrogen receptor-alpha (Erα). Commonly known as the activation function 2 (AF2), the interaction between AF2-binders, and the ERα protein were analyzed by Bio-Layer Interferometry (BLI) using a Pall ForteBio Octet RED instrument equipped with Super Streptavidin biosensors. Fifteen inhibitors of ERα AF2 were identified from this study out of which, the inhibitor VPC-16230 was identified as the lead ERα AF2 inhibitor demonstrating a substantial down regulation of ERα transcriptional activity.
PubMed
Inhibition of Type VI Secretion by an Anti-TssM Llama Nanobody
Nguyen V, et al., PLoS One , 10(3):e0122187, 2015
Reported in this article is the selection and structural analyses of two specific nanobodies. Llama immunizations carried out with a purified TssM periplasmic domain allowed further understanding of an inner membrane protein called TssM from enteroaggregative Escherichia coli (EAEC). The binding characterization involved a Pall ForteBio Octet RED96 system equipped with Streptavidin (SA) Biosensor tips. Biotinylated TssJ captured onto SA tips was screened against TssM. In addition, the TssM/nanobody competition assays were also involved the Octet RED96.
PubMed
Insights into the Molecular Basis of a Bispecific Antibody's Target Selectivity
Mazor Y, et al., MAbs, 7(3):461-9, 2015
A series of affinity-reduced variants of the anti-CD4 ibalizumab mAb were generated in order to understand target selectivity seen with bispecific antibodies. Particularly, the target selectivity of anti-CD4/CD70 DuetMab variants were evaluated for their binding affinities, selectivity, and depletion via antibody-dependent cell-mediated cytotoxicity (ADCC). A Pall ForteBio Octet RED384 system was used for antibody quantitation in cell-culture supernatants and affinity ranking of the variants. The binding of IgG and DuetMab to monomeric CD4 and CD70 were characterized by capturing IgGs on to anti-human IgG Fc biosensor probes and by titrating against serial dilutions of CD4 or CD70. Whereas for the DuetMab characterization, biotinylated CD4 and CD70 were captured onto Streptavidin Biosensor probes and analyzed against serial dilutions of DuetMab.
PubMed
Interaction of AIM with Insulin-Like Growth Factor-Binding Protein-4
You Q, et al., Int J Mol Med, Epub ahead of print, 2015
This investigation has uncovered first ever Insulin-like growth factor-binding protein (IGFBP) binding partners to apoptosis inhibitor of macrophages (AIM) by co-immunoprecipitation (co-IP) and Bio-Layer Interferometry (BLI). The interaction between AIM and IGFBP-4 was evaluated by a Pall ForteBio Octet RED system equipped with Super-Streptavidin (SSA) Biosensor probes. Biotinylated IGFBP-4 captured on to SSA biosensors allowed screening against a dilution series of AIM. Authors believe that these findings will provide clues on the mechanism of action for AIM-mediated cell survival.
PubMed
Isolation and Characterization of Broad and Ultrapotent Human Monoclonal Antibodies with Therapeutic Activity Against Chikungunya Virus
Smith S, et al., Cell Host Microbe, 18(1):86-95, 2015
A panel of human monoclonal antibodies isolated from a human infected with Chikungunya Virus (CHIKV) was characterized for their ability to treat immunodeficient mice inoculated with a lethal dose of CHIKV. The antigenic site for recognition by human antibodies was identified as the A domain of E2. The group of antibodies bound to the same antigenic site as revealed by a competitive binding assay performed on a Pall ForteBio Octet RED system. The CHIKV-LR2006 E2 ectodomain protein containing a polyhistidine-tag captured on to Anti-Penta-His Biosensor probes was used in the competitive binding assay.
PubMed
Key Mutations Stabilize Antigen-Binding Conformation During Affinity Maturation of a Broadly Neutralizing Influenza Antibody Lineage
Xu H, et al., Proteins, 83(4):771-80, 2015
The mechanism of affinity gain in a human B-cell lineage was investigated in which, three mature antibodies with the ability to neutralize a broad range of H1 influenza viruses have been used. Kinetic measurements obtained using a Pall ForteBio BLItz system involves Fab immobilization on to Ni-NTA Biosensor probes. The association and dissociation of varying concentrations of H1 influenza hemagglutinin (HASI) head molecules against different Fab variants yielded the kinetic parameters.
PubMed
Kinetics Characterization of Ligand-Receptor Interactions Using Oblique-Incidence Reflectivity Difference Method
Liu S, Yang G, Methods in Pharmacology and Toxicology, pp 111-118, 2015
This book chapter introduces Oblique-incidence reflectivity difference (OIRD) platform as an alternate label-free and high-throughput technology to SPR or BLI.
Springer Link
Label-Free Kinetic Analysis of an Antibody-Antigen Interaction Using Bio-Layer Interferometry
Kumaraswamy S, Tobias R, Methods Mol Biol, 1278:165-82, 2015
This is a book chapter describing the fundamentals of BLI technology, kinetic characterization, assay design considerations, assay development, data analysis, and key factors to keep in mind for a successful antibody-antigen assay development effort. It provides a quick-start guide for life science researchers on kinetic characterizations performed using the BLI platform.
PubMed
Label-Free Technologies: Which Technique to Use and What to Watch Out for!
Halai R, Cooper M, Methods in Pharmacology and Toxicology, pp 3-15, 2015
This is a book chapter on label-free technologies. It describes solution phase and biosensor surface based methodologies and their utility in studying ligand-receptor interactions. The chapter highlights BLI capabilities of affinity measurements in crude samples.
Springer Link
L-Citrulline Increases Hepatic Sensitivity to Insulin by Reducing the Phosphorylation of Serine 1101 in Insulin Receptor Substrate-1
Yoshitomi H, et al., BMC Complement Altern Med, 18;15:188, 2015
An investigation on the beneficial effects of L-citrulline (L-Cit) on improved insulin resistance is described. A Pall ForteBio Octet RED system equipped with Streptavidin Biosensor (SA) probes was used to determine the effect of L-Cit on insulin and its receptor (INSR) binding. The interaction between insulin and INSR was investigated in the presence and absence of L-Cit by capturing INSR on to SA biosensors.
PubMed
Mapping and Quantitation of the Interaction Between the Recombination Activating Gene Proteins RAG1 and RAG2
Zhang Y, et al., J Biol Chem, 290(19):11802-17, 2015
Described herein is an investigation of interaction between the Recombination Activating Gene (RAG) proteins, RAG1 and RAG2 using Bio-Layer Interferometry (BLI) and pulldown assays. A Pall ForteBio BLItz instrument was used to measure the RAG1-RAG2 binding affinity. In these experiments, GST-R2c and MBP-R1c-His6 were immobilized on to Anti-GST or Anti-His Biosensor probes, respectively. Overall results suggests that the RAG1-RAG2 interaction requires only a small portion of RAG1 flanking the RAG1 catalytic region. The binding affinity (KD) for the RAG1-RAG2 interaction is around 0.4 μM.
PubMed
Maturation and Diversity of the VRC01-Antibody Lineage Over 15 Years of Chronic HIV-1 Infection
Wu X, et al., Cell , 161(3):470-85, 2015
An investigation of the VRC01-antibody lineage that targets the site of CD4 engagement on HIV-1 is reported. Although germline-reverted variants of VRC01-class antibodies were reported to have no binding affinity to gp120, the current investigation provides evidence of binding to the early autologous gp120 molecule, d45-01dG5. A Pall ForteBio Octet RED384 system equipped with Anti-Human IgG probes was used to measure binding constants of HIV-1 gp120 extended core and the germline-reverted antibodies. The affinities of 1995 and 2008 antibodies showed improvements in the nanomolar range. Structure data suggest mutations in 20 amino acids are responsible for the affinity maturation.
PubMed
Maturation of the Proteasome Core Particle Induces an Affinity Switch That Controls Regulatory Particle Association
Wani P, et al., Nat Commun , doi: 10.1038/ncomms7384, 2015
Described herein is a conceptual model of the role of Pba1-Pba2 dimer during proteasome assembly. Although same binding surface is used, the dimer binds immature core particles (CPs) while the regulatory particle (RP) can bind only to the mature core particles during the assembly process. A Pall ForteBio BLItz system equipped with Ni-NTA Biosensor probe was used to capture His-tagged Pba1-Pba2. The kinetic characterization involved a serial dilution of mature CP.
PubMed
Measuring Protein-Protein and Protein-Nucleic Acid Interactions by Bio-Layer Interferometry
Sultana A, Lee J, Curr Protoc Protein Sci, 79:19.25.1-19.25.26, 2015
This is a detailed write-up explaining the stepwise procedure of BLI-based advanced kinetic analyses of DNA-protein and protein-protein interactions using Pall ForteBio BLItz and the Octet systems. The preparation of the biotinylated ligands, choices for controls, assay optimization approaches, strategies applicable to regeneration of Streptavidin-based biosensors, and assay troubleshooting approaches are described. Authors recommend BLI as a versatile platform providing accurate measurements of binding affinities (KD), rates of association, and rates of dissociation (kon and koff) of protein-protein and protein-nucleic acid interactions in minutes by utilizing relatively small quantities of sample.
PubMed
Mechanism of Human Antibody-Mediated Neutralization of Marburg Virus
Flyak A, et al., Cell, 160(5):893-903, 2015
This article describes the molecular basis of Marburg virus (MARV) neutralization by human antibodies. A large panel of human neutralizing antibodies (nAbs) from B cells of a human survivor of MARV infection was isolated. A Bio-Layer Interferometry based binding competition assay was performed using a Pall ForteBio instrument equipped with biotinylated glycoprotein (GP) or GPΔmuc immobilized onto Streptavidin Biosensor tips. Some of the isolated human survivor antibodies bind to Ebola virus GP. Overall results suggest that MARV-neutralizing antibodies inhibit virus by binding to MARV GP at the predicted region of the receptor-binding site and hence demonstrate the mechanism of filovirus inhibition.
PubMed
Mechanisms Mediating Enhanced Neutralization Efficacy of Staphylococcal Enterotoxin B by Combinations of Monoclonal Antibodies
Dutta K, et al., J Biol Chem , 13;290(11):6715-30, 2015
Reported in this article are two different mechanisms of how a combination of different mAbs can enhance efficacy of mAb-mediated protection against Staphylococcal enterotoxin B (SEB). Combining two SEB-specific mAbs enhanced the overall efficacy, even though one of the two mAbs alone has no effect. A Pall ForteBio BLItz instrument equipped with a Streptavidin Biosensor probe was used to perform affinity determination experiments and competitive binding assays involving TCR- β chain, MHC-II, and the mAbs toward SEB. Biotinylated SEB was captured onto the biosensor tip. Understanding the mechanisms of how different combinations of mAbs enhance toxin neutralization is important to the development of new high efficacy antibody cocktails.
PubMed
Milk Matrix Effects on Antibody Binding Analyzed by Enzyme-Linked Immunosorbent Assay and Bio-Layer Interferometry
Brandon D, Adams L, J Agric Food Chem , 63(13):3593-8, 2015
This article is reporting complementary results obtained from BLI and ELISA on ricin binding to an antibody or a receptor in the presence of milk in the matrix. A Pall ForteBio Octet QK system equipped with either, Amine-Reactive (AR2G) or Streptavidin (SA) sensor tips was used to analyze ricin binding to asialofetuin (ASF), mimic a sandwich ELISA assay, and for the investigation of milk as a ""sink"".
PubMed
Molecular Determinants of Polyubiquitin Recognition by Continuous Ubiquitin-Binding Domains of Rad18
Thach T, et al., Biochemistry, 54(12):2136-48, 2015
Described herein is the mechanism by which Rad18 recognizes polyubiquitin at a molecular level and how it functions in the DNA damage response (DDR) pathway. A Pall ForteBio BLItz instrument was used to perform Bio-Layer Interferometry (BLI) studies. For linkage-specific diubiquitin binding to Rad18 experiments, amine reactive sensor tips were immobilized with linear, K48-linked, or K63-linked Ub2. Ni-NTA biosensors immobilized with His-Ub, His-Ub2, His-Ub3, or His-Ub4 were used to investigate length-dependent binding of linear polyubiquitin chains to Rad18. The KD values were determined by the steady state analysis of Req values. The results demonstrate continuous ubiquitin binding domains (comprising UBZ and ELRM of Rad18) outcompete RAP80 in polyubiquitin recognition, suggesting a potential regulation of the DDR pathway.
PubMed
Moraxella catarrhalis Binds Plasminogen to Evade Host Innate Immunity
Singh B, et al., Infect Immun, Epub ahead of print, 2015
The article describes on a series of clinical isolates of the respiratory pathogen (Moraxella catarrhali) with the ability to attract plasminogen from human serum and causing an inactivation of complement components. Plasminogen interacts with surface protein A2 (UspA2) and a hybrid of UspA2 (UspA2H) that are present on the outer membrane of Moraxella. Bio-Layer Interferometry (BLI) was used to study the interaction between plasminogen and the bacterial proteins (UspA2 and UspA2H). A Pall ForteBio Octet RED96 instrument equipped with plasminogen immobilized Amine-Reactive (AR2G) biosensor probes was utilized in these experiments. Overall results suggest that the recruitment of plasminogen by M. catarrhalis may be contributing to increased virulence.
PubMed
mRNA Maturation in Giant Viruses: Variation on a Theme
Priet S, et al., Nucleic Acids Res, 20;43(7):3776-88, 2015
This investigation was aimed at identifying the Mimivirus and M. chilensis predicted viral polyA polymerases (vPAPs) with an ability to perform polyadenylation. Based on a series of biochemical and structure biology investigations, the vPAPs are found to be homodimeric and self-processive. A Pall ForteBio BLItz system equipped with Ni-NTA Biosensor probes was used in the binding experiments. His tagged versions of proteins Mg18 or R341 were captured onto Ni-NTA biosensors followed by screening against dilution series of Mg561 or Mg18, respectively.
PubMed
Mycobacterium tuberculosis RpfE Promotes Simultaneous Th1- and Th17-Type T-cell Immunity via TLR4-Dependent Maturation of Dendritic Cells
Choi H, et al., Eur J Immunol, 45(7):1957-71, 2015
Described herein is the role of resuscitation-promoting factor (Rpf) E in the host immune response against Mycobacterium tuberculosis (Mtb) infection. RpfE stimulates the Th1 and Th17 immune responses via maturation of dendritic cells, in a TLR4-dependent manner. A Pall ForteBio BLItz instrument equipped with a HIS Biosensor probe was used to study the binding of recombinant human TLR4/MD2 or TLR4 itself to RpfE. Both TLR4/MD2 complex and TLR4 alone were HIS-tagged for these experiments. Overall findings of this investigation indicate that the RpfE protein could be used to develop more effective multi-antigenic Mtb subunit vaccines.
PubMed
Nanocell Targeting Using Engineered Bispecific Antibodies
Taylor K, et al., MAbs, 7(1):53-65, 2015
Reported in here is the development of several bispecific antibodies (BsAbs) to target the EnGeneIC delivery vehicle (EDVTMnanocell) via epidermal growth factor receptor (EGFR). Since the EDVTMnanocell is coated with lipopolysaccharide (LPS), BsAb design formats included scFvs derived from an anti-LPS antibody (1H10) and an anti-EGFR antibody ABX-EGF. Bio-Layer Interferometry was used to study the binding of mAbs and BsAbs to LPS molecules using a Pall ForteBio Octet instrument equipped with Aminopropylsilane (APS) biosensors. LPS molecules were captured on to APS Biosensor probes. Due to natural stickiness of LPS to glass and plastic, the investigators were unable to use Biacore microfluidics systems to perform this assay. Overall results provide clues for the development of new BsAbs capable of efficiently targeting nanoparticles that would play an important role in future nanomedicine.
PubMed
Nonbinding Site-Directed Mutants of Transferrin Binding Protein B Exhibit Enhanced Immunogenicity and Protective Capabilities
Frandoloso R, et al., Infect Immun, 83(3):1030-8, 2015
Described herein is the effect of binding host transferrin (Tf) on the immunogenicity of Tf binding proteins. A mutant Haemophilus parasuis Tf binding protein B (TbpB) demonstrated an improved B-cell and T-cell response in pigs compared to native TbpB. The dissociation constant (KD) of wild-type and mutant TbpBs was determined by Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED instrument equipped with Streptavidin Biosensor probes and immobilized with pTf was used in these experiments. A dilution series of TbpB was used for the determination of binding between TbpB-pTf . Overall findings are expected to influence the engineering of new vaccines against pathogens in the future.
PubMed
Novel Mechanism of Regulation of Tomato Bushy Stunt Virus Replication by Cellular WW-Domain Proteins
Barajas D, et al., J Virol, 89(4):2064-79, 2015
Described herein is the role of cellular WW-domain proteins in regulating tomato bushy stunt virus (TBSV) replication process. The WW is a highly conserved protein domain involved in protein-protein interactions. The WW domain could also inhibit TBSV replication in yeast or in a cell-free replication assay. Kinetic experiments involve a Pall ForteBio BLItz instrument equipped with GST-tagged yeast proteins immobilized onto Anti-GST Biosensor probes. Kinetic parameters such as the association rate constant (ka), dissociation rate constant (kd), and interaction affinity constant (KD) values were determined. Overall assessment suggests a new mechanism to describe the effect of proviral factors and WW-domain proteins in the regulation of tombusviruses viral replicase complex (VRC) assembly.
PubMed
Oligomerization of FtsZ Converts the FtsZ Tail Motif (CCTP) into a Multivalent Ligand with High Avidity for Partners ZipA and SlmA
Du S, Park K, Lutkenhaus J, Mol Microbiol, 95(2):173-88, 2015
Described herein is an investigation to differentiate affinities between ZipA binding to full-length FtsZ versus a short peptide located at the carboxy terminus of FtsZ, referred to as the CCTP (conserved carboxy-terminal peptide). The interaction of SlmA and FtsZ was also evaluated. A series of Bio-Layer Interferometry assays performed using a Pall ForteBio BLItz instrument equipped with Ni-NTA biosensors. Thereby binding of FtsZ, FtsZ mutants, MalE-CCTP, or MinDΔ10-linker-CCTP to His tagged ZipA were studied. In a reciprocal binding evaluation, His-FtsZ was captured on to Ni-NTA biosensors and binding to untagged ZipA was analyzed. In the assays with SlmA, the His-SlmA/SBS complexes were loaded on to Ni-NTA Biosensor tips and binding of FtsZ, FtsZ mutants, or MinDΔ10-linker-CCTP was tested. Overall results suggest that the oligomerization of FtsZ is important for the enhanced affinity of FtsZ towards ligands that bind the CCTP.
PubMed
Oncogenic and RASopathy-Associated K-RAS Mutations Relieve Membrane-Dependent Occlusion of the Effector-Binding Site
Mazhab-Jafari M, et al., Proc Natl Acad Sci U S A, 112(21):6625-30, 2015
Described herein are the NMR-derived models pertaining to plausible interactions between Kirsten rat sarcoma viral oncogene homolog 4B (K-RAS4B) and the lipid membrane biolayer. The report also evaluates how theose interactions could affect oncogenic and RASopathy-associated K-RAS mutations. Binding of K-RAS4B to Anti-GST antibody-coated biosensor immobilized ARAF-RBD was studied by Bio-Layer Interferometry (BLI) using a Pall ForteBio Octet RED96 instrument. Overall results suggest that the tendency of K-RAS4B to interact with the membrane in a way that occludes its effector binding site may reveal a new therapeutic target.
PubMed
Overexpression of Cryoglobulin-Like Single-Chain Antibody Induces Morular Cell Phenotype via Liquid-Liquid Phase Separation in the Secretory Pathway Organelles,2015,Upstream Processing,Quantitation of other proteins,,,FEBS J,"Hasegawa H
Hasegawa H, Patel N, Lim A, FEBS J, doi: 10.1111/febs.13332, 2015
The identification and characterization of a single-chain antibody (scFv-Fc) is reported. Upon multimerization, scFv-Fc promotes unexpected cryoglobulin-like properties. Modified scFv-Fc-stp, with a secretory tailpiece (stp) stimulates a morular cell phenotype during protein biosynthesis and also causes cryo-precipitation of secreted proteins in cell culture media causing a liquid-liquid phase separation (LLPS) event. Bio-Layer Interferometry was used to evaluate the concentration of Fc-fusion proteins in cell culture media by using a Pall ForteBio Octet RED96 instrument equipped with Protein A biosensors. Overall findings of this investigation suggest a probable reason as to why morular Russell body phenotype is seen in patients with B-lymphoproliferative disorders and plasma cell dyscrasias.
PubMed
Passive Immunization with Phospho-Tau Antibodies Reduces Tau Pathology and Functional Deficits in Two Distinct Mouse Tauopathy Models
Sankaranarayanan S, et al., PLoS One, 10(5):e0125614, 2015
This is a study on phospho-tau (p-tau) antibodies (PHF6 and PHF13) with ability to target pT231 and pS396 sites, respectively. These two p-tau antibodies are capable of preventing the induction of tau pathology in primary neuron cultures and in two distinct transgenic mouse tauopathy models. Antibody affinity studies were carried out with a Pall ForteBio Octet Red instrument equipped with Streptavidin Biosensor probes. Biotinylated phospho-peptides were immobilized on to Streptavidin probes. Association & dissociation kinetics and the binding affinity constant (KD) of respective antibodies were determined. Overall results contribute significantly towards the probable use of tau immunotherapy for the treatment of Alzheimer's disease (AD) and related tauopathies.
PubMed
Patient-Specific Neutralizing Antibody Responses to Herpes Simplex Virus are Attributed to Epitopes on Either gD, gB, or Both and Can Be Type-Specific
Cairns T, et al., J Virol, pii: JVI.01213-15, 2015
Described herein is a detailed study involving human sera obtained from HSV (Herpes simplex virus) infected patients and tests for the ability to bind to glycoproteins and compete with anti-gD and anti-gB neutralizing mAbs. Some of the examined infected individuals had no recurrent disease. A Pall ForteBio Octet RED96 instrument equipped with biotinylated glycoproteins captured onto Streptavidin biosensors was used to investigate the serum samples. Authors describe several advantages of using the Octet system as compared to the BIAcore system that was used in their previous studies. Authors recognize Octet system as a powerful method to carry out their assays. Overall results demonstrate the significance of gD and gB serotypes as immunogens, bringing new insights for future HSV vaccine development.
PubMed
Peptides of Presenilin-1 Bind the Amyloid Precursor Protein Ectodomain and Offer a Novel and Specific Therapeutic Approach to Reduce β-Amyloid in Alzheimer's Disease
Dewji N, et al., PLoS One, 10(4):e0122451, 2015
Accumulation of β-Amyloid (Aβ) in the brain is believed to be the initiator of Alzheimer's disease (AD). This study demonstrates that two independent peptides, P4 and P8 from within the amino-terminal domain of Presenilin-1 (PS-1) exhibit reduced β-Amyloid (Aβ) production in vitro and also in vivo in the brains of β-amyloid precursor protein (APP) transgenic mice. Bio-Layer Interferometry determined the binding affinities of peptides (P1, scrambled P1, P4, P5, P7, and P8) and APP. A Pall ForteBio Octet RED96 instrument equipped with Streptavidin (SA) biosensors allows capture of biotinylated peptides. The kinetic parameters such as the association rate constant (kon), dissociation rate constant (koff), and binding affinity, KD, were determined. Overall results indicate peptides P4 and P8 have the potential to become drug candidates for the development of successful Alzheimer's disease-modifying therapies.
PubMed
Physicochemical and Biological Characterization of a Biosimilar Trastuzumab
Lopez-Morales C, et al., Biomed Res Int, 2015:427235, 2015
This is a comparability study of a biosimilar trastuzumab with respect to its reference product to evaluate the physicochemical and biological aspects. Trastuzumab is a monoclonal antibody that interferes with the human epidermal growth factor receptor (HER2), which overexpresses in certain invasive forms of breast cancers. A Pall ForteBio Octet QK384 instrument equipped with Streptavidin Biosensor probes captured with Biotinylated FcRn investigated the binding of trastuzumab to FcRn. Overall results of this investigation suggest that there is a high degree of similarity in the physicochemical properties of the biosimilar trastuzumab with respect to the reference product as evident from comparable biological activity.
PubMed
Plasmodium falciparum Adhesion Domains Linked to Severe Malaria Differ in Blockade of Endothelial Protein C Receptor
Sampath S, et al., Cell Microbiol, DOI: 10.1111/cmi.12478, 2015
Domain cassette 8 (DC8) and DC13 of Plasmodium falciparum are responsible for severe malaria and demonstrate different endothelial protein C receptor (EPCR) blockade activity. Cysteine-rich interdomain regions (CIDRα1) of DC8 and DC13 were expressed and their interactions with EPCR were investigated. Results indicate each domain interact with EPCR in a distinct manner resulting in different levels of inhibition of the activated protein C (APC)-EPCR interaction. Bio-Layer Interferometry was used to study the binding kinetics of the CIDR-EPCR interaction. A Pall ForteBio Octet QKe instrument equipped with biotinylated EPCR captured Streptavidin biosensor probes were used in these assays to determine relevant kinetic parameters (kon, koff, and KD). Overall results of this investigation reveal a functional heterogeneity in the interaction between P. falciparum and EPCR, which would help in the development of novel treatment methods for cerebral malaria.
PubMed
Predicting the Uncertain Future of Aptamer-Based Diagnostics and Therapeutics
Bruno J, Molecules, 20(4):6866-87, 2015
This is a review article elaborating the prospects of aptamers as diagnostics and therapeutic agents. The so-called conformational changes of a ligand upon analyte binding could cause a blue shift (instead of a red shift) and yield inverted binding curves on the Octet. The kinetic characterization and data analysis of a binding event involving an aptamer and a peptide is described. The author describes this observation as an induced fit binding mechanism pertaining to longer aptamers.
PubMed
Preexisting Immunity, More Than Aging, Influences Influenza Vaccine Responses
Reber A, et al., Oxford Journals, doi: 10.1093/ofid/ofv052, 2015
An assessment of physiological factors that affect immune responses of older adults to the 2008-2009 seasonal trivalent inactivated influenza vaccine (TIV) is reported. The hemagglutination inhibition (HI) assay revealed vaccine's capability to produce an immune response. A Pall ForteBio Octet RED instrument was used in the analysis of binding between serum antibodies and hemagglutinin (HA). Overall results demonstrate a major impact coming from pre-vaccination responses on the post-vaccination antibody levels regardless of the age group.
Oxford Journals
Antigenicity and Immunogenicity of a Trimeric Envelope Protein from an Indian Clade C HIV-1 Isolate
Sneha Priya R, et al., J Biol Chem, 290(14):9195-208, 2015
This article characterizes gp145 from a CCR5 Indian clade C HIV-1 (93IN101) isolated from 293 cells. Data suggest the ability of gp145 to induce broadly neutralizing antibodies by exposing conserved regions of the protein. The findings also suggest the potential of trimeric gp145 to be used as a multiclade candidate HIV-1 vaccine. The characterization of binding affinities between 93IN101 glycoproteins with CD4 or various monoclonal antibodies was achieved by a Pall ForteBio Octet RED system. The glycoproteins (gp120 and gp145) were biotinylated and captured onto Streptavidin Biosensor probes. These binding assays involved screening against varying concentrations of IgG.
PubMed
Aptamers as a Replacement for Antibodies in Enzyme-Linked Immunosorbent Assay
Toh s, et al., Biosens Bioelectron, 64:392-403, 2015
This is a review article describing the utility of aptamers in place of antibodies in Enzyme-Linked Apta-Sorbant Assay (ELASA). Authors are drawing parallels between ELISA and ELASA in terms of their capability to identify target molecules such as antigens on cellular surfaces, allergens in environmental samples, and contaminants in food. A special emphasis is given to Bio-Layer Interferometry (BLI) based ELASA due to its applicability to aptamer based diagnostic assay development.
PubMed
Assembly of Cellulases with Synthetic Protein Scaffolds In Vitro
Yu T, et al., Bioresources and Bioprocessing, 2:16, 2015
The construction of an artificial protein scaffold and its applicability to assembly of a complex comprised of three-enzyme cellulases is described. The functionality of such protein scaffolds was established with an apparent increase in enzymatic activity. Binding interactions between protein candidates were estimated by a Pall ForteBio Octet QKe system equipped with Streptavidin Biosensor probes.
Springer Link
Bacterial Cytoplasmic Display Platform Retained Display (ReD) Identifies Stable Human Germline Antibody Frameworks
Beasley M, et al., Biotechnol J, 10(5):783-9, 2015
Discussed in this article is the development of a novel cytoplasmic display platform namely, the Retained Display (ReD). The methodology eliminates certain difficulties inherent to conventional antibody display approaches. As a proof of concept, the authors applied this methodology to screen for human scFv frameworks. The kinetic characterization of ?-mAG1 involved a Pall ForteBio BLItz system. The Streptavidin Biosensor probes used in these assays captured biotinylated ?-mAG1. The lysate containing soluble ?-mAG1 scFv were subjected to binding with immobilized ?-mAG1 target protein. Authors highlight the ability of this method to rapidly develop antibodies against infectious diseases and agents of bioterrorism concerns.
PubMed
Bactericidal Monoclonal Antibodies Specific to the Lipopolysaccharide O Antigen from Multidrug-Resistant Escherichia coli Clone ST131-O25b: H4 Elicit Protection in Mice
Szijarto V, et al, Antimicrob Agents Chemother, 59:3109-3116, 2015
Described herein is the generation and selection of a humanized Monoclonal Antibody (MAb) capable of binding to a conserved antigen called lipopolysaccharide O25b. In particular, the development of an antibody therapeutic against multidrug-resistant bacteria (gram-negative) species is discussed. Bio-Layer Interferometry (BLI) measured the binding of candidate antibodies. The polysaccharide antigen O25b was biotinylated and captured onto Streptavidin biosensors. The monitoring of association and dissociation of MAbs at varying concentrations allowed determination of the KD. Additionally, the BLI evaluation of polyreactivity (against commercially available unrelated antigens) was achieved by immobilizing the MAbs via Anti-human Capture Biosensors.
Antimicrob. Agents Chemother
Bio-Layer Interferometry of a Multivalent Sulfated Virus Nanoparticle with Heparin-Like Anticoagulant Activity
Groner M, etal., Anal Bioanal Chem, 407(19):5843-7, 2015
With the intention of finding an alternative to heparin, the authors investigated the feasibility of sulfated virus-like nanoparticles (sulf-VLP) using Pall ForteBio BLItz platform in the advanced kinetic mode. Although protamine is the ideal ligand, the authors decided to use CDK5 as an alternative cationic peptide due to its availability in biotinylated form. Biotinylated CDK5 captured onto Streptavidin biosensors was used in the affinity measurements against varying concentrations of VLP. The competitive binding was evaluated using protamine and antithrombin.
PubMed
Biophysical Measurement of the Balance of Influenza A Hemagglutinin and Neuraminidase Activities
Benton D, et al., J Biol Chem, 290(10):6516-21, 2015
The application of Bio-Layer Interferometry to study the interdependence of hemagglutinin (HA) and neuraminidase (NA) activities pertaining to Influenza A virus (IAV) is reported. In order to mitigate pandemic strains, it is important to have a better understanding of characteristics such as the HA/NA balance. Particularly the binding and release of virus from receptor analogs immobilized onto a biosensor surface were investigated. An Octet RED system equipped with Streptavidin biosensors was used to capture biotinylated receptor analogs (sugars). The binding of virus monitored in real time in the presence and absence of NA inhibitors allowed understanding of the mechanistic aspects of virus transmissibility.
PubMed
Biophysical Methods for Identifying Fragment-Based Inhibitors of Protein-Protein Interactions
Pfaff S, et al., Methods Mol Biol, 1278:587-613, 2015
This book chapter describes most common fragment screening methods that are widely used in the Fragment-Based Lead Discovery (FBLD). It highlights the use of Pall ForteBio Octet RED like platforms as alternatives to Biacore 4000 SPR with additional capabilities such as the throughput, automation, sensitivity, and innovativeness in execution.
PubMed
Bioreactor Process Parameter Screening Utilizing a Plackett-Burman Design for a Model Monoclonal Antibody Measurement
Agarabi C, et al., J Pharm Sci, 104(6):1919-28, 2015
The authors report the use of Plackett-Burman design to screen cell culture parameters and highlight the importance of eleven different variables toward achieving this goal. In particular, the authors found temperature variations could affect glycan-modification and distribution profiles. This approach is expected to be useful in monoclonal antibody manufacturing processes. IgG3 antibody quantitation was achieved by a Pall ForteBio Octet RED96 system equipped with Protein A biosensor probes.
PubMed
BMI1-RING1B is an Autoinhibited RING E3 Ubiquitin Ligase
Taherbhoy A, Huang O, Cochran A, Nat Commun, 5.542361111, 2015
The authors investigated enzymatic properties of all six RING-RING heterodimers (RING1B-PCGF1 through RING1B-PCGF6) involved in the ubiquitanation process. Affinity measurements carried out using a Pall ForteBio Octet RED384 system equipped with anti-GST biosensors helped reveal subtle differences. Binding curves were generated by capturing GST-RING1B-PCGF constructs and titrating against UbcH5c(S22R/C85K) or UbcH5c(S22R/C85K)~Ub. A steady state analysis of the binding curves was used to obtain KD values.
PubMed
BolA Is a Transcriptional Switch That Turns Off Motility and Turns on Biofilm Development
Dressaire C, et al, MBio, 6(1):e02352-14, 2015
This investigation has focused on target interaction and global effects of BolA, widespread gene product of bolA, initially identified in E. coli that promotes round morphology upon overexpresssion. With the help of ChIP-seq and transcriptomic analysis, the authors revealed BolA involvement in the regulation of flagellar and curli biosynthesis pathways. The affinity measurements of BolA with biotinylated DNA fragments were achieved using a Pall ForteBio BLItz system equipped with Streptavidin biosensors.
PubMed
Boosting Antibody Developability Through Rational Sequence Optimization
Seeliger D, et al., MAbs, 7(3):505-15, 2015
This study reports computational approaches based on sequence analyses that could be used to modify an antibody sequence to prevent it from precipitation and to improve its stability in vitro. A Pall ForteBio Octet system equipped with Protein A biosensors was used to estimate productivity of cell cultures by titer measurements.
PubMed
Broadly Neutralizing Human Immunodeficiency Virus Type 1 Antibody Gene Transfer Protects Non-Human Primates from Mucosal Simian-Human Immunodeficiency Virus Infection
Saunders K, et al., J Virol, doi: 10.1128/JVI.00908-15, 2015
This is a proof-of-concept study to show anti-HIV antibody gene transfer as a viable option to protect primates against infection by HIV. A Pall ForteBio Octet RED384 system was used to investigate IgG binding to plasma samples. Amine coupling was used to capture antibodies to AR2G biosensors. The macaques plasma samples were diluted into a 1% BSA-PBS to minimize nonspecific interactions.
PubMed
Cellular Uptake and Ultrastructural Localization Underlie the Pro-Apoptotic Activity of a Hydrocarbon-Stapled BIM BH3 Peptide
Edwards A, et al., ACS Chem Biol, DOI: 10.1021/acschembio.5b00214, 2015
This article demonstrates a stepwise approach to the development of clinically relevant stapled peptides by comparing biochemical, biophysical, cellular, and mechanistic investigations. Binding measurements were made using a Pall ForteBio Octet RED384 system equipped with Super Streptavidin (SSA) biosensors. The biotinylated peptides captured onto SSA biosensors were dipped into a serial dilution of BCL-XL ΔC protein.
PubMed
Characterization of Protective Immune Response Elicited by a Trimeric Envelope Protein from an Indian Clade C HIV-1 Isolate in Rhesus Macaques
Menon V, et al., J Med Primatol, doi: 10.1111/jmp.12178, 2015
This is an extension to a previous investigation carried out by the authors to demonstrate the immunogenic nature of gp145 in rhesus macaques. Trimeric gp145 binding to sCD4 and several HIV-1 monoclonal antibodies were evaluated by Bio-Layer Interferometry(BLI). A Pall ForteBio Octet RED system equipped with Streptavidin biosensors was used to capture biotinylated 93IN101 gp145. The sensors were dipped into serial dilutions of sCD4 or IgG. The results suggest a strong antibody response triggered by Env proteins in macaques.
PubMed
Circulating Levels of sFlt1 Splice Variants as Predictive Markers for the Development of Preeclampsia
Souders C, et al., Int J Mol Sci, 16(6):12436-53, 2015
This article describes the development and application of diagnostic antibodies that are specific to the splice variants of soluble fms-like tyrosine kinase 1 (sFlt1), namely sFlt1-1 and sFlt1-14. A Pall ForteBio Octet QK system equipped with anti-murine or anti-human biosensors was used to determine binding affinities. The authors assume that these isoform specific biomarkers could be used to predict preeclampsia in pregnant women.
PubMed
Comparable Antigenicity and Immunogenicity of Oligomeric Forms of a Novel, Acute HIV-1 Subtype C gp145 Envelope for Use in Preclinical and Clinical Vaccine Research
Wieczorek L, et al., J Virol, 89(15):7478-93, 2015
The production of a subtype C HIV-1 envelope immunogen from mammalian cells is described. It also demonstrates assembly of gp145 protein into trimeric forms without any engineered trimerization motifs. The gp145 binding to several broadly neutralizing MAbs were measured using a Pall ForteBio Octet RED system equipped with Streptavidin biosensors. Biotinylated gp145 (unfractionated, dimer, trimer) and gp120 were captured onto Streptavidin biosensors and dipped into dilution series of MAbs and sCD4.
PubMed
Conditional Trimerization and Lytic Activity of HIV-1 gp41 Variants Containing the Membrane-Associated Segments
Dai Z, et al., Biochemistry, 54(8):1589-99, 2015
This report has revealed the fact that HIV-1 gp41 subunit can disintegrate into a monomeric form as a result of changes in pH in membrane environments. It also suggests that the monomeric form still remains active and represents a folded form. A Pall ForteBio BLItz system was employed to measure affinities between gp41 constructs and MAbs. The antibodies were captured using Anti-human Fc capture biosensors (at pH 7 or pH 4) and dipped into varying concentrations of gp41.
PubMed
Construction of an Antimyoglobin Single-Chain Variable Fragment with Rapid Reaction Kinetics
Jang J, et al., Biotechnol Appl Biochem, DOI: 10.1002/bab.1349, 2015
This article describes the development of an IgG-type monoclonal anti-human myoglobin antibody using the hybridoma technology. The cloned gene for a single-chain variable fragment (scFv) was originated from IgG-Myo2-7Ds. The gene product is fused to Protein-A (ProA) tag at the C-terminus and expressed in E. coli. The purified scFv was characterized for binding with immobilized Myo using a Pall ForteBio Octet RED system equipped with aminopropyl silane (APS) biosensor probes.
PubMed
Crystal Structure of the Bloom's Syndrome Helicase Indicates a Role for the HRDC Domain in Conformational Changes
Newman J, et al., Nucleic Acids Res, 43(10):5221-35, 2015
The crystal structure of Bloom's Syndrome Helicase (BLM) in complex with adenosine diphosphate (ADP) and a llama antibody single domain fragment (nanobody) as well as two other structures showing BLM in complex with DNA are reported. The data suggest tight packing between helicase and RNase D C-terminal (HRDC) and the two RecA like domains D1 and D2. A Pall ForteBio Octet RED384 system equipped with Streptavidin biosensors was used to capture biotinylated HRDC and screened against a dilution series of RecA.
PubMed
Crystal Structure, Conformational Fixation and Entry-Related Interactions of Mature Ligand-Free HIV-1 Env
Do Kwon Y, et al., Nat Struct Mol Biol, 22(7):522-31, 2015
The crystal structure of conformationally fixed ligand free HIV-1 Env trimer and its receptor interactions are reported. In order to investigate the physical stability of the prefusion, the proteins were subjected to a variety of pharmaceutically relevant stress conditions such as high temperature, repeated freeze thaw cycles, and extreme pH. The fraction of binding to V1V2-antibody (CAP256-VRC26.09) retained after stress treatment was investigated with a Pall ForteBio Octet HTX system.
PubMed
C-Terminal Amino Acids Are Essential for Human Heat Shock Protein 70 Dimerization
Marcion G, et al., Cell Stress and Chaperones, pp 61-72, 2015
This article describes the heterologous expression and purification of soluble, full length, non-tagged human inducible heat shock protein 70 (hHsp70). Homodimerizing behavior of hHsp70 and important C-terminal residues of interest were demonstrated using multi angle light scattering and Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED system equipped with Streptavidin Biosensor probes was used to capture biotinylated Hsp70 or DnaK and then dipped into wells containing a serial dilution of Hsp70 and DnaK. In another experiment, the dimerized species (Hsp70 or DnaK) were tested for binding with a biotinylated peptide-aptamer ligand A17.
Springer
Design of a Potent Antibiotic Peptide Based on the Active Region of Human Defensin 5
Wang C, et al., J Med Chem, 58(7):3083-93, 2015
The design of a novel antibiotic peptide based on the active region of human defensin 5 (HD5) is described in this article. It provides a useful guide as to how the increase in electropositive charge can be compensated for an impaired ability to assemble and hence for future defensin-based antibiotic design. A Pall ForteBio Octet RED96 system equipped with AR2G biosensors was used to determine binding kinetics between the peptides and bacterial lipid A and lipoteichoic acid (LTA).
PubMed
Design, Synthesis, and Structure-Activity Relationship of Novel LSD1 Inhibitors Based on Pyrimidine-Thiourea Hybrids as Potent, Orally Active Antitumor Agents
Ma L, et al., J Med Chem, 58(4):1705-16., 2015
The design and synthesis of small molecule inhibitors for histone lysine specific demethylase 1 (LSD1) is reported. One of the compounds from the novel inhibitor series based on pyrimidine-thiourea hybrids showed highest potency, selective inhibition of LSD1, and cytotoxicity against LSD1 overexpressed gastric cancer cells. A Pall ForteBio Octet RED96 equipped with SSA Biosensor probes was used to measure the interaction between small molecules and LSD1. Biotinylated LSD1 was captured onto SSA biosensors. These sensors subsequently blocked with biocytin. An unloaded set of sensors blocked with biocytin used for double referencing.
PubMed
Detection of Mycobacterium tuberculosis Based on H37Rv Binding Peptides Using Surface Functionalized Magnetic Microspheres Coupled with Quantum Dots - A Nano Detection Method for Mycobacterium Tuberculosis
Yang H, et al., Int J Nanomedicinev, 10:77-88, 2015
This article describes the generation of phage display derived peptide ligands for Mycobacterium tuberculosis (MTB) cells with improved limit of detection (LOD) and specificity. A Pall ForteBio Octet QK system equipped with Streptavidin biosensors was used to capture biotinylated peptides. The bacteria binding events were monitored in real time to determine affinity and specificity for H37Rv, BCG , P. aeruginosa, and E. coli.
nih.gov
Development of a VHH-Based Erythropoietin Quantification Assay
Kol S, et al., Mol Biotechnol, 57(8):692-700, 2015
The development and implementation of a robust assay for erythropoietin (EPO) quantification using a Pall ForteBio Octet RED96 system is reported. Traditionally, this type of quantification is performed with either ELISA or HPLC. However, the current method is capable of detecting EPO in Chinese hamster ovary cell supernatants with better sensitivity in a pH-dependent manner. The capture antibody used in this assay development is a camelid single-domain antibody fragment that is specific for EPO. A Pall ForteBio Octet RED96 system equipped with Streptavidin biosensors was used to capture biotinylated anti-EPO VHH or anti-EPO IgG ligands. The assays carried out under various conditions suggest a much broader applicability of the BLI platform.
PubMed
Directed Evolution of a Yeast-Displayed HIV-1 SOSIP gp140 Spike Protein Toward Improved Expression and Affinity for Conformational Antibodies.
Grimm S, Battles M, Ackerman M, PLoS One, 10(2), 2015
The use of yeast display technology to demonstrate the ability to display HIV spike protein variants and the display of a JR-FL gp140 variant with SOSIP mutations in particular is reported. A Pall ForteBio Octet RED96 system equipped with Protein A biosensor probes was used to capture a panel of HIV gp120 antibodies (IgG) in order to perform binding studies with S. cerevisiae or P. pastoris-secreted gp120. This study is expected to facilitate solving of other spike protein structures and rational vaccine design approaches.
PubMed
Directed Evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process
Houlihan G, et al., Protein Eng Des Sel, pii: gzv029 Epub ahead of print, 2015
The application of SNAP display technology for in vitro evolution of Designed Ankyrin Repeat Proteins (DARPins) targeting HER2 is reported. The KD values of the mutant DARPins were investigated against biotinylated HER2 using a Pall ForteBio Octet RED system equipped with Streptavidin Biosensor probes.
PubMed
Discovery of MINC1, a GTPase-Activating Protein Small Molecule Inhibitor, Targeting MgcRacGAP
van Adrichem A, et al., Comb Chem High Throughput Screen, 18(1):3-17, 2015
The identification of a Male germ cell Rac GTPase-activating protein (MgcRacGAP) inhibitor with the hope of dissecting its biological role is reported. The interaction between MgcRacGAP and Rac1 (Q61L) was analyzed using a Pall ForteBio Octet RED384 system equipped with Anti-GST biosensor probes. MgcRacGAP, together with Rac1 and two other protein candidates, regulates mitosis. The MgcRacGAP labeled with a GST is loaded onto the Anti-GST biosensors and analyzed against Rac1Q61L. The discovery of this inhibitor (MINC1) and its ability to interfere with biological functions of the protein in cells suggest the possibility of developing inhibitors for other small GTPase GAPs.
PubMed
Dose-Dependent Biphasic Leptin-Induced Proliferation Is Caused by Non-Specific IL-6/NF-κB Pathway Activation in Human Myometrial Cells
Barrichon M, et al., Br J Pharmacol, 172(12):2974-90, 2015
The article emphasizes the importance of leptin in the pharmacological management of preterm labour (PTL) due to its ability to resist mechanisms pertaining to labour, remodeling, apoptosis, and contractions. A Pall ForteBio Octet RED system equipped with Streptavidin biosensors was used to evaluate the binding affinities of leptin or SHLA (leptin receptor antagonist) to the leptin receptor or IL-6 receptor alpha. Leptin and SHLA were biotinylated to enable capture using Streptavidin biosensors and titrated against a serial dilution of each receptor.
PubMed
During Cytochrome c Maturation CcmI Chaperones Class I Apocytochromes Until the Formation of Their b-Type Cytochrome Intermediates
Verissimo A, Shroff N, Daldal F, J Biol Chem, 290(27):16989-7003., 2015
The authors investigated the heme effects on interactions involving CcmI, apoCcmE, and different c-type apocytochromes in order to understand how R. capsulatus Ccm System I matures structurally dissimilar c-type cytochromes. The protein-protein interactions were monitored by a Pall ForteBio Octet RED96 system equipped with Streptavidin coated biosensors. Biotinylated c-type apocytochromes captured on to these SA biosensors were incubated with a dilution series of CcmI or apoCcmE.
PubMed
Exploiting Light Chains for the Scalable Generation and Platform Purification of Native Human Bispecific IgG
Fischer N, et al., Nat Commun, doi: 10.1038/ncomms7113, 2015
This article describes a novel platform to generate fully human bispecific IgG antibodies (BiAbs) that are unmodified and compatible with large scale manufacturing/purification processes. It uses in vitro display libraries to isolate candidates with the same heavy chain and carrying either κ or λ light chains in order to possess different target specificities. A Pall ForteBio Octet RED96 system was used to obtain the affinities and other kinetic parameters. Depending on the nature of ligand under consideration, two types of biosensor chemistries (anti-human Fc capture or Streptavidin) were employed. Biosensor tips were preconditioned/regenerated using a solution of 10mM glycin at pH 1.7.
PubMed
Exploration of the HIF-1α/p300 Interface Using Peptide and Adhiron Phage Display Technologies
Kyle H, et al., Mol Biosyst, DOI: 10.1039/C5MB00284B, 2015
This study has explored the use of peptide and adhiron phage-display capabilities to deepen the understanding of HIF-1α/p300 protein-protein interaction. This interaction is crucial for tumor metabolism and plays as a drug target for anticancer drug development. A Pall ForteBio Blitz system equipped with Ni-NTA Biosensor probes was used to capture adhirons and to estimate binding with p300.
PubMed
Five Birds, One Stone: Neutralization of α-Hemolysin and 4 bi-Component Leukocidins of Staphylococcus aureus with a Single Human Monoclonal Antibody
Rouha H, et al., MAbs, 7(1):243-54, 2015
This article describes the discovery of human mAbs with an ability to cross neutralize Alpha-hemolysin (Hla) and multiple leukocidins. Given the high level of pathogenesis of Staphylococcus aureus, the authors used murine models of S. aureus pneumonia and sepsis to make their case. A Pall ForteBio Octet RED96 system was used to determine binding affinities, cross reactivity, specificity (epitope binning), and the binding inhibition by phosphocholine. In these experiments, the biotinylated antigen or the antibody was captured using either Streptavidin or Anti-Human Capture (AHC) biosensor probes and screened against the antibody, the antigen, or the toxin using the Octet system.
PubMed
Following Natures Lead: On the Construction of Membrane-Inserted Toxins in Lipid Bilayer Nanodiscs
Akkaladevi N, et al., J Membr Biol, 248(3):595-607, 2015
The article highlights the use of BLI (or the BLItz system in particular) to demonstrate important prerequisites such as the immobilization, orientation, the state of folding (unfolding/refolding), and solution kinetics that make cellular entry of toxins feasible. A clear understanding of these steps is crucial for the generation of pure membrane-inserted toxins in nanodiscs for electron microscopy and for the rational design of inhibitors that target toxin membrane-insertion transitions.
PubMed
Fragment-Based Drug Discovery and Molecular Docking in Drug Design
Wang T, et al., Curr Pharm Biotechnol, 16(1):11-25, 2015
This is a review highlighting recent developments in the Fragment-Based Drug Discovery (FBDD) methods and distinct advantages of the Bio-Layer Interferometry (BLI) in achieving success with high-throughput screening.
PubMed
Functional Label-Free Assays for Characterizing the In Vitro Mechanism of Action of Small Molecule Modulators of Capsid Assembly
Lad L, et al., Biochemistry, 54(13):2240-8, 2015
A series of label-free assays were reported that would help dissecting the mechanism of action of small-molecule and peptide antiviral agents developed against the HIV capsid protein (CA). In addition to distinct assay development advantages of the Octet platform, this study has provided yet another testimony to the comparable sensitivity of the Octet platform towards small-molecule and peptide detection and binding characterization. A Pall ForteBio Octet RED384 system equipped with Super Streptavidin Biosensor probes, was used to capture biotinylated wild type CA and hexameric CA.
PubMed
Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine
Chi X, et al., Clin Vaccine Immunol, 22(5):553-60, 2015
This is an investigation of a large panel of monoclonal antibodies, generated from an individual immunized with recombinant anthrax protective antigen (rPA), to evaluate their neutralizing potential. The characterization involved identifying domain specificities, affinities, neutralization mechanism, and possible synergistic effects of the protective human monoclonal antibodies (hMAbs). A Pall ForteBio Octet system equipped with anti-hIgG Fc capture biosensors was used to evaluate binding between hMAbs and PA.
PubMed
Glutathione Activates Virulence Gene Expression of an Intracellular Pathogen
Reniere M, et al., Nature, 517(7533):170-3, 2015
The study aimed at the biochemical mechanism by which the transcription factor PrfA detects the intracellular environment. A genetic approach involving a selection in macrophages helped to understand how Listeria monocytogenes recognizes and responds to the mammalian cell cytosol. The requirement of glutathione for virulence was suggested. The binding of glutathione to the wild-type or PrfA(C/A)4 proteins were investigated using a Pall ForteBio Octet RED384 system equipped with Ni-NTA biosensor probes. His-tagged proteins immobilized onto the Ni-NTA biosensors were used to evaluate the binding with glutathione or reduced glutathione.
PubMed
Group Selection and Contribution of Minority Variants During Virus Adaptation Determines Virus Fitness and Phenotype
Borderia A, et al., PLoS Pathog, 11(5):e1004838, 2015
By using the deep sequencing technology, the authors investigated the Coxsackievirus B3 (CVB3) adaptation to host environment in A549 cells. Based on the findings, the adaptation occurs in response to a differential expression of viral receptors. Also found is the fact that a group of genotypes instead of a single dominant genome causes the increased fitness. Interaction between the co-receptor, Decay Accelerating Factor (DAF) and the CVB3 (parental strain) or CVB3-E76G (mutant strain) were evaluated by a Pall ForteBio BLItz system equipped with Ni-NTA biosensors. Purified His-tagged DAF captured onto Ni-NTA biosensors were dipped into virus containing solutions.
PubMed
3, 4-dihydroxyl-phenyl Lactic Acid Restores NADH Dehydrogenase 1 α Subunit 10 to Ameliorate Cardiac Reperfusion Injury
Yang X, et al., Sci Rep, 5:10739, 2015
The role of 3, 4-dihydroxyl-phenyl lactic acid (DLA) in cardioprotection against ischemia/reperfusion (I/R) injury and its ability to interact and activate sirtuin1 (SIRT1) were demonstrated in this study. The activation of SIRT1 causes enhanced expression of NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10 (NDUFA10) and mitochondrial function after I/R. An Octet RED instrument equipped with super Streptavidin Biosensor probes was used to capture biotinylated SIRT1 antibody followed by human recombinant SIRT1. The DLA analyte binding to SIRT1 was monitored in real time for K determination. In addition to identifying DLA mediated signaling pathway, the findings provide clues for designing new medicines for I/R induced cardiac injury.
PubMed
A Broadly Neutralizing Human Monoclonal Antibody Exhibits In Vivo Efficacy Against Both Human Metapneumovirus and Respiratory Syncytial Virus
Schuster J, et al., J Infect Dis, 211(2):216-25, 2015
The article describes neutralizing activity and affinity kinetics of a human mAb known as 54G10 against human metapneumovirus (HMPV). In vivo efficacy studies were carried out using a new mouse model called DBA/2. An Octet RED96 instrument equipped with Streptavidin biosensors was used to capture biotinylated B2F?TM. The titrations involve varying concentration of the mAb. The 54G10 showed subnanomolar affinity for the recombinant fusion protein HMPV B2F?TM.
PubMed
A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence-Quencher Probe as a Tool for Functional Antibody Screening
Li Y, et al., J Biomol Screen, 20(7):869-75, 2015
This article describes the development of a novel fluorescence pair to evaluate the extent of antibody internalization and degradation in target cells. An Octet system was used for antibody quantitation in supernatants. Human IgG was used as the standard.
PubMed
A Combination of Three Fully-Human Toxin A- and Toxin B-Specific Monoclonal Antibodies Protects against Challenge with Highly Virulent Epidemic Strains of C. difficile in the Hamster Model
Anosova N, et al., Clin Vaccine Immunol, 22(7):711-25, 2015
The development of an 3-antibody cocktail (3-MAbs) against highly virulent strains of Clostridium difficile is reported. The cocktail targets C. difficile toxins A and B. Authors used a high-throughput B-cell cloning strategy to isolate monoclonal antibodies (MAbs) from healthy human donors. Affinity measurements involved a Pall ForteBio Octet RED96 system equipped with Protein A biosensors to capture MAbs. These MAb captured sensors were subsequently immersed in solutions containing toxin A or toxin B for kinetic measurements.
PubMed
A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus
Stewart-Jones G, et al., PLoS One, 10(6):e0128779, 2015
The authors designed a cycteine zipper motif by carefully analyzing engineered coiled-coils for Respiratory Syncytial virus fusion (RSV F) glycoprotein and by studying the in vivo immunogenicity. They claim successful implementation of structure-based rational design to improve this RSV vaccine candidate. An Octet RED384 instrument equipped with anti-human Fc biosensor probes was used for the antigenic characterization. The probes initially loaded with Motavizumab IgG were used to capture RSV F variants. A dilution series of RSV F glycoproteins were titrated with D25 neutralizing antibody Fab using the Octet.
PubMed
A Fully Human Monoclonal Antibody with Novel Binding Epitope and Excellent Neutralizing Activity to Multiple Human IFN-α Subtypes: a Candidate Therapy for Systemic Lupus Erythematosus
Du P, et al., MAbs, 7(5):969-80, 2015
This article introduces a neutralizing antibody named AIA22 which consist of an alternate epitope and a different neutralizing profile to those observed in anti interferon alpha (IFN-α) antibodies currently in clinical trials. IFN-α protein plays a central role in the pathogenesis of systemic lupus erythematosus (SLE). A series of competitive binding assays were carried out using an Octet QKe system in order to identify AIA22 binding region on the IFN-α. The Octet QKe system equipped with anti-human-IgG (AHC) biosensors was used to capture purified AIA22 followed by monitoring of its binding with IFN-α2b.
PubMed
A Generic Approach to Engineer Antibody pH-Switches Using Combinatorial Histidine Scanning Libraries and Yeast Display
Schroter C, et al., MAbs, 7(1):138-51, 2015
This is a strategy to develop antibodies with reversible pH-sensitive antigen binding capabilities. Combinatorial histidine substitution libraries made out of VH and VL portions of adalimumab was used in this proof of concept experiment. Adalimumab is a human IgG antibody that targets tumor necrosis factor (TNF). The authors isolated VH and VL variants with high affinity at pH 7.4 and a considerable increase in antigen release at pH 6 after 3-rounds of yeast-display screening. The pH sensitive antigen binding was confirmed using an Octet RED system equipped with anti-human Fc (AHC) biosensors. These biosensors captured the antibody allowing concentration dependent titrations with rhTNF. Furthermore, the authors used the Octet platform to provide evidence of reversible binding at varying pH conditions.
PubMed
A High Cell Density Transient Transfection System for Therapeutic Protein Expression Based on a CHO GS-Knockout Cell Line: Process Development and Product Quality Assessment
Rajendra Y, et al., Biotechnol Bioeng, 112(5):977-86, 2015
This article describes the optimization of transfection and protein expression in CHO K1SV GS KO cells by using a variety of strategies such as the optimization of DNA, polyethyleneimine (PEI), cell density as well as several other process-development strategies. The protein concentration estimations in culture media were carried out using an Octet QK system.
PubMed
A High-Affinity Native Human Antibody Neutralizes Human Cytomegalovirus Infection of Diverse Cell Types
Kauvar L, et al., Antimicrob Agents Chemother, 59(3):1558-68, 2015
By using a previously reported platform known as the single-B-cell screening, the authors cloned 30 different native human MAbs that bind to site I of AD-2 epitope on viral glycoprotein B (gB). Despite a reasonably high affinity, one of the antibodies (TRL345) also exhibited the best in vitro potency allowing the authors to carry out this detailed biological and biochemical evaluation. The KD measurement were achieved by an Octet QK system equipped with Streptavidin biosensors. This has allowed them to immobilize C-terminal biotinylated AD-2 peptide (HRANETIYNTTLKYGD-biotin) and monitor binding with TRL345 in a concentration dependent manner.
PubMed
A Human Monoclonal IgG That Binds Aβ Assemblies and Diverse Amyloids Exhibits Anti-Amyloid Activities In Vitro and In Vivo
Levites Y, et al., J Neurosci, 22;35(16):6265-76, 2015
This article reports isolation of an affinity matured human monoclonal antibody with the ability to recognize pan-amyloid epitope, and with a demonstrated specificity for aggregated amyloid forms. The authors used an affinity matured IgG known as 3H3 in this study. Several techniques including the ELISA, immunohistochemistry, and competitive binding assays employed in this investigation. An Octet RED 384 system equipped with Streptavidin biosensors was used to carry out affinity determinations and off-rate screening assays. The monomeric forms of Aβ40 or Aβ42 were biotinylated and captured via Streptavidin biosensors. Binding experiments involved various forms of 3H3 or pan-Aβ antibody dilutions prepared in a 384-well plate.
PubMed
A Human-Infecting H10N8 Influenza Virus Retains a Strong Preference for Avian-Type Receptors
Zhang H, et al., Cell Host Microbe, 11;17(3):377-84, 2015
This work is an attempt to understand the mechanism of human infection and transmission capabilities of the H10N8 virus. These authors determined hemagglutinin (HA) crystal structures with human or avian receptor analogs and investigated receptor-binding properties as well. An Octet RED system equipped with Streptavidin Biosensor probes was used to capture biotinylated avian and human linear glycan receptor analogs, α2-3-sialylated di-N-acetyllactosamine (SLNLN) and α2-6 SLNLN, respectively and to monitor glycan binding specificity of recombinant H10 HA.
PubMed
A Multi-Pronged Investigation into the Effect of Glucose Starvation and Culture Duration on Fed-Batch CHO Cell Culture
Fan Y, et al., Biotechnol Bioeng, 112(10):2172-84, 2015
In this study, the effects of glucose starvation and culture duration on monoclonal antibody production in CHO cell cultures were investigated. The effects of these process parameters on the titer, N-glycosylation, proteomic signature, and the intrinsic properties of the cells were evaluated. Monoclonal antibody production in the cell culture was evaluated by an Octet QK384 system equipped with Protein A biosensors.
PubMed
A Mutant Tat Protein Inhibits HIV-1 Reverse Transcription by Targeting the Reverse Transcription Complex
Lin M, et al., J Virol, 89(9):4827-36, 2015
This study has focused on the mechanism of reverse transcription inhibition by Nullbasic in HIV-1. Direct binding of Nullbasic to HIV-1 Reverse Transcriptase (RT) was demonstrated by a variety of techniques including the BLI. An Octet RED system equipped with SA biosensors was used to capture the biotinylated RT. Titrations carried out in the presence of Nullbasic as analyte confirmed direct binding between the two proteins. This interaction appears to be uncoating the viral core and destabilizes the viral reverse transcription complex (RTC).
PubMed
A Novel Antibody Engineering Strategy for Making Monovalent Bispecific Heterodimeric IgG Antibodies by Electrostatic Steering Mechanism
Liu Z, et al., J Biol Chem, 290(12):7535-62, 2015
The article describes a novel approach for the production of bispecific heterodimeric IgG antibody in mammalian cells. The antibody consists of two different heavy chains and two different light chains that were paired by an antibody engineering approach. The protein expression levels in harvested media were estimated using an Octet RED96 equipped with Protein A biosensors.
PubMed
A Novel In Vitro Assay to Predict Neonatal Fc Receptor-Mediated Human IgG Half-Life
Souders C, et al., MAbs, 7(5):912-21, 2015
The neonatal Fc receptor (FcRn) binding to therapeutic IgG and the pH-dependence of this process had been investigated in depth. Using the BLI platform, the authors performed a high throughput assay to determine the association rate at an acidic pH and the dissociation rate at physiological pH and correlated those rates to half-lives obtained from a phase I clinical study. This method development used an Octet QK system equipped with nickel-nitrilotriacetic acid (Ni-NTA) biosensors. FcRn was immobilized on to the biosensor probes and the mAb solutions were loaded onto a 96-well plate for binding assessments.
PubMed
A Novel Synthetic Small Molecule YH-306 Suppresses Colorectal Tumour Growth and Metastasis Via FAK Pathway
Dai F, et al., J Cell Mol Med, 19(2):383-95, 2015
With the intention of finding a small molecule drug candidate against colorectal cancer (CRC) metastasis, the authors performed a comprehensive study on an aryl tetrahydro-β-carboline called YH-306 using a variety of assays. The estimation of binding between YH-306 and Arp2/3 protein complex was achieved by using an Octet QK system equipped with Streptavidin Biosensor probes. Biotinylated Arp2/3 protein complex was captured on to Streptavidin probes while YH-306 or CK-636 (positive control) analytes were prepared in binding buffer (25 mM Na HEPES (pH 8.0), 50 mM arginine-glutamate and 150 mM NaCl).
PubMed
A PfRH5-Based Vaccine is Efficacious Against Heterologous Strain Blood-Stage Plasmodium Falciparum Infection in Aotus Monkeys
Douglas A, et al., Cell Host Microbe, 17(1):130-9, 2015
This is a proof-of-concept vaccine study performed in Aotus monkeys, a nonhuman primate (NSP) to obtain evidence that it is possible to develop a strain-transcending blood-stage vaccine against Plasmodium falciparum. The in vivo efficacy studies suggest the ability of P. falciparum reticulocyte binding protein homolog 5 (PfRH5) to protect monkeys against malaria. During growth inhibitory activity (GIA50) assays, the IgG concentration in plasma samples were estimated by a Pall ForteBio BLItz instrument equipped with Protein A biosensors.
PubMed
Acetylproteomic Analysis Reveals Functional Implications of Lysine Acetylation in Human Spermatozoa (sperm)
Yu H, et al., Mol Cell Proteomics, 14(4):1009-23, 2015
This investigation has focused on identifying the global lysine acetylation profile (acetyl proteome) of human sperm with the help of a pan-anti-acetyllysine monoclonal antibody. The authors discuss several factors correlating lysine acetylation and sperm functions. An Octet RED96 system equipped with Streptavidin Biosensor probes was utilized to characterize the binding between mAb and BSA conjugated acetyllysine peptide. After synthesis, BSA-P1 and BSA-P1(Ac) biotinylated and captured onto Streptavidin Biosensor probes. A serial dilution of antibody 1G5 was used as the analyte.
PubMed
Aerobic Growth of Escherichia coli Is Reduced and ATP Synthesis Is Selectively Inhibited When Five C-Terminal Residues Are Deleted from the ε Subunit of ATP Synthase
Shah N, Duncan T, J Biol Chem , 290(34):21032-41, 2015
This study suggests that in different bacterial species the C-terminal domain of ε-subunit (εCTD) of ATP synthase could be fine-tuned to regulate ATP synthesis as well as ATP hydrolysis. The study also found that such regulation of functions is based on the unique metabolic and environmental demands of each bacterial species. A variety of molecular biology approaches including mutagenesis, phenotyping, expression, and purification of enzymes were used in this investigation. The interaction between F1(-σε) and biotinylated ε variants were estimated by an Octet RED system equipped with Streptavidin Biosensor probes. The dissociation phase in each interaction was analyzed to obtain the off-rates using nonlinear regression fits for two phases of exponential decay.
PubMed
Amino Acid and Glucose Metabolism in Fed-Batch CHO Cell Culture Affects Antibody Production and Glycosylation
Fan Y, et al., Biotechnol Bioeng, 112(3):521-35, 2015
By using two different IgG-producing cell lines and two different culture media, the authors investigated the differences in cell growth, IgG expression, nutrient media consumption, sugar availability, and IgG glycosylation. This article provides the information that may help growth media development and IgG expression optimization. An Octet QK384 system equipped with Protein A biosensors was used to estimate IgG titer levels.
PubMed
Amplification Systems of Weak Interaction Biosensors: Applications and Prospects
Wang X, et al., Emerald Insight, DOI: http://dx.doi.org/10.1108/SR-03-2014-629, 2015
This is a review article on the prospects of biosensor methods that are being used for the investigation of weak interactions. It describes BLI platform (Pall ForteBio) as a viable alternative to Biacore SPR with distinct advantages such as the multichannel detection, high quality reagent availability, absence of the microfluidics, low cost, etc. The authors also appreciate the fact that there is an increased market share for BLI biosensors. This article highlights a signal amplification strategy known as the G protein signaling cascade amplification system for weaker interactions.
Emerald Insight
Antibody Mimetic Receptor Proteins for Label-Free Biosensors
Raina M, et al., Analyst, 140(3):803-10, 2015
The authors describe a detailed characterization of a model receptor protein based on a non-antibody scaffold protein (which binds to anti-myc tag antibody) as the target specific receptor molecule in a label-free electrochemical biosensor. A Pall ForteBio BLItz system was used to characterize binding interactions between the receptor proteins and the anti-myc tag antibody. HIS-tagged receptor proteins were captured using the Ni-NTA biosensor probes and different concentrations of anti-myc tag antibody solutions were used as analyte.
PubMed
Antibody-Mediated Targeting of Iron Oxide Nanoparticles to the Folate Receptor Alpha Increases Tumor Cell Association In Vitro and In Vivo
Ndong C, et al., Int J Nanomedicine , 10:2595-617, 2015
This article demonstrates molecular targeting of Iron oxide nanoparticles (IONPs) to the folate receptor alpha (FOLRα) biomarker for cancer by using an engineered antibody fragment (Ffab). The IONPs used in this study were functionalized with the fab fragment of a humanized antibody called Farletuzumab. A series of in vitro as well as in vivo assays were used for the characterization. In addition to ELISA, an Octet RED system equipped with Streptavidin Biosensor probes was used to obtain affinity measurements for Ffab. Varying concentrations of rFOLR1-his were used in a 96-well plate as analyte solutions.
PubMed
Anticancer Activity of a Novel Selective CYP17A1 Inhibitor in Preclinical Models of Castrate-Resistant Prostate Cancer
Toren P, et al., Mol Cancer Ther, 14(1):59-69, 2015
This article reports the preclinical results of a novel small molecule CYP17A1 enzyme inhibitor called VT-464. Particularly, the anticancer activity of VT-464 was compared with that of ABI in prostate cancer cell lines and in xenograft models. The reversible interaction between small molecules and the androgen receptor was evaluated using an Octet RED system. The authors used a biotinylated androgen receptor (produced using the AviTag technology), which was captured onto super Streptavidin biosensors as the ligand. The results suggest a better performance by VT-464 as compared to ABI.
PubMed
Anti-Idiotypic Nanobody as Citrinin Mimotope from a Naive Alpaca Heavy Chain Single Domain Antibody Library
Xu Y, et al., Anal Bioanal Chem, 407(18):5333-41, 2015
The article describes development of a strategy to obtain β-AI VHH as a CIT mimotope using a phage display VHH library. The authors claim several advantages of this strategy as compared to conventional AId development methods. A Pall ForteBio BLItz system was used to estimate binding kinetics of VHH or CIT-OVA to McAb. The Ni-NTA and AR2G biosensors were used to capture HIS-tagged VHH and CIT-OVA, respectively.
PubMed
Immunogenicity Assessment of Biotherapeutic Products: An Overview of Assays and Their Utility
Wadhwa M, et al., Biologicals, 43(5):298-306, 2015
This review article elaborates on the evaluation of immunogenicity in biotherapeutic products (BTPs). In pharmaceutical industry, BTPs represent a fastest growing market. However, the immunogenicity of BTPs continues to present a challenge. In this article, the authors describe the platforms used for the evaluation of immunogenicity. The article also describes their advantages and limitations. The authors highlight high tolerance shown by the Bio-Layer Interferometry (BLI) platform (Pall ForteBio Octet) against residual therapeutics in samples.
PubMed
Reverse Engineering of Vaccine Antigens Using High Throughput Sequencing-Enhanced mRNA Display
Nini Gu, et al., Science Direct, 2(8):857-65, 2015
This article reports the use of mRNA display and high throughput sequencing to identify peptides for a hepatitis C virus neutralizing monoclonal antibody (mAb41). This antibody prevents hepatitis C virus infection in cell cultures. Immunization of mice with the peptides produced mouse antibodies with neutralizing characteristics similar to the original antibody. The affinity characterizations performed on an Octet RED96 system equipped with Streptavidin biosensors allowed immobilization of biotinylated peptides. The analyte used in the binding assays consist of mAb41 antibody Fab segments prepared using a mouse IgG1 kit.
PubMed
Structural Analysis of the Unmutated Ancestor of the HIV-1 Envelope V2 Region Antibody CH58 Isolated from an RV144 Vaccine Efficacy Trial Vaccinee
Nicely N, et al., EBioMedicine, 20;2(7):713-22, 2015
In this article, the authors are reporting the structural and biophysical characterization of an ancestral HIV-1 envelope V2 region human monoclonal antibody known as CH58 (CH58-UA). This antibody was isolated from an RV144-vaccine efficacy trial vaccinee. The crystal structures of CH58-UA antibody with or without V2 peptide allowed understanding of how V2 responses are developed. The epitope mapping experiments carried out on a BIAcore 4000 SPR instrument served as the basis for Ala scanning experiments. A Pall ForteBio Octet RED96 instrument equipped with Streptavidin biosensors allowed identification of binding partners via affinity maturation. Biotinylated gp120165-182 peptides captured by the Streptavidin biosensors were screened against CH58-UA and mature CH58 Fabs.
PubMed
Therapeutic Intradermal Delivery of Tumor Necrosis Factor-Alpha Antibodies Using Tip-Loaded Dissolvable Microneedle Arrays
Korkmaz E, et al., Acta Biomater, pii: S1742-7061(15)00284-6, 2015
This investigation is aimed at finding a biologically effective means of delivering anti-TNF- α antibodies to the intradermal microenvironment of the skin in humans. These antibodies are potent tumor necrosis factor (TNF) inhibitors and efficient therapeutics for many inflammatory diseases. Due to inefficiencies pertaining to existing methods, the authors evaluated the applicability of tip-loaded dissolvable microneedle arrays (MNAs) for localized intradermal delivery of the antibodies. MNAs with microneedles that incorporate antibodies in the needle tip were generated from carboxymethylcellulose (CMC). An Octet QK system (Pall ForteBio) was used to verify the activity of anti-TNF- α Ab in tip-loaded CMC-MNAs. A biotinylated version of the recombinant mouse TNF-α protein immobilized onto Streptavidin biosensors was used against a dilution series of dissolved MNAs.
PubMed
pH Recycling Aqueous Two-Phase Systems Applied in Extraction of Maitake β-Glucan and Mechanism Analysis Using Low-Field Nuclear Magnetic Resonance
Hou 1, Cao X, J Chromatogr A, 1405:40-8, 2015
This article describes the utility of recyclable, pH-responsive, two-phase aqueous polymer systems (PADB and PMDM) to extract β-Glucan from G. frondosa. Low-field nuclear magnetic resonance (LF-NMR), Fourier transform infrared (FT-IR), and Pall ForteBio Octet systems were used to elucidate the partition mechanism. The Octet was used to verify the interactions between β-Glucan and different polymer materials. Due to presence of carboxyl groups, the polymers under investigation were captured via amino groups on APS sensors.
PubMed
Clinical Immunosensing of Tuberculosis CFP-10 Antigen in Urine Using Interferometric Optical Fiber Array
Kim J, et al., Science Direct, 10.1016/j.snb.2015.04.046, 2015
This article demonstrates development and utility of an immunosensing diagnostic platform which is readily applicable to a clinical setting for early detection of tuberculosis (TB). The TB-biomarker under investigation, also known as the CFP-10 antigen, is found in the tissues/fluids (e.g. urine) of TB patients. A detailed investigation of the three interferometric immunoassays involves an Octet RED96 system (Pall ForteBio) equipped with amine-reactive (AR), aminopropylsilane (APS), and anti-human IgG Fc capture (AHC) biosensors. The newly uncovered clinical diagnostic method involving clinical urine samples suggest a much better sensitivity with indirect immobilization, presumably due to right orientation and high density attachment of the 2nd recognition layer. The linear correlation between the interferometry signal and acid-fast bacilli (AFB) staining stage suggest a robust TB diagnostic tool.
Science Direct
Modulation of the Binding of Basic Fibroblast Growth Factor and Heparanase Activity by Purified λ-Carrageenan Oligosaccharides
Niu T, eta;., Carbohydr Polym, 125:76-84, 2015
A group of seven λ-Carrageenan oligosaccharides (λ-COs) with different molecular weights were investigated in the present study for their ability to bind to basic fibroblast growth factor (bFGF) and to affect the activity of bFGF and heparanase, and their cell invasion characteristics. λ-COs are heavily-sulfated oligosaccharides with demonstrated ability to act as bFGF antagonists and as heparanase inhibitors. The interaction between λ-COs and bFGF was determined by a Pall ForteBio Octet system equipped with super Streptavidin biosensors. Recombinant bFGF was biotinylated and captured onto super Streptavidin biosensors and different analyte concentrations of λ-COs were screened for their ability to interact with bFGF.
PubMed
Recognization of Receptors on Bone Marrow-derived Dendritic Cells Bound with Pholiota Nameko Polysaccharides
Li H, et al., Int J Biol Macromol, 72:649-57, 2015
Three major polysaccharides, namely PNPS-1, PNPS-2, and PNPS-3 isolated from a mushroom called Pholiota nameko were investigated for their ability to recognize receptors on bone marrow derived dendritic cells (BMDCs). The binding affinities derived from a Pall ForteBio Octet RED system suggest PNPSs ability to interact with TLR2, Dectin-1, and Mannose receptors. Biosensors with the ability to recognize Fc-tag and HIS-tag fusion proteins were used throughout the study. Some insights onto mechanism of action were also revealed based on the recognition patterns.
PubMed
Enhanced Humanization and Affinity Maturation of Neutralizing Anti-hepatitis B Virus PreS1 Antibody Based on Antigen-antibody Complex Structure
Kim J, et al., FEBS Lett, 589(2):193-200, 2015
This article describes a biochemical investigation that lead up to an enhancement of the humanized nature and binding affinity of a broadly neutralizing hepatitis B virus (HBV)-specific humanized antibody called HzKR127. Further humanization was achieved by a methodology known as specificity-determining residue (SDR) drafting. The affinity maturation was based on the crystal structure of antigen-antibody complex and that was achieved by mutating two residues in heavy-chain complementarity-determining regions (CDR). An Octet RED system equipped with anti-human Fc-coated biosensor tips was used to capture the antibody. A serial dilution of preS1 fused onto GST was screed against the captured antibody ligand. This study provides insights onto the methods for engineering antibody therapeutics for HBV immunoprophylaxis.
PubMed
Identification of Outer Membrane Porin D as a Vitronectin-binding Factor in Cystic Fibrosis Clinical Isolates of Pseudomonas Aeruginosa
Paulsson M, et al., J Cyst Fibros, pii: S1569-1993(15)00120-4, 2015
This investigation has focused on the identification of Pseudomonas aeruginosa surface receptors and their ability to bind to vitronectin. Although vitronectin is a naturally occurring plasma protein primarily involve in cell adhesion and wound healing, increased release of it by the respiratory epithelial cells suggest inflammation caused by lung diseases. A proteomics approach in conjunction with 2D SDS-PAGE allowed identification of the receptors. An outer membrane protein (OMP) called Porin D (OprD) was identified as a biding partner for vitronectin. Affinity measurements carried out by a Pall ForteBio Octet RED96 platform equipped with AR2G biosensors allowed vitronectin immobilization and KD determination.
PubMed
A Saxitoxin-Binding Aptamer with Higher Affinity and Inhibitory Activity Optimized by Rational Site-Directed Mutagenesis and Truncation
Zheng X, et al., Toxicon, 101:41-7, 2015
This study focuses on a previously selected DNA aptamer (APTSTX1) against a toxin know as saxitoxin (STX). Using site-directed mutation and truncation, the authors revealed an aptamer (M-30f) with a 30-fold higher affinity and with better inhibitory properties. Subsequent to optimization, the interaction was monitored through Bio-Layer Interferometry (BLI), ELISA, cell, and mouse bioassays. An Octet RED96 system equipped with super Streptavidin biosensors was used to capture biotinylated aptamers. Varying concentrations of STX subjected to binding with immobilized aptamers revealed precise kinetic parameters.
PubMed
Rapid and Sensitive Detection of Norovirus Antibodies in Human Serum with a Biolayer Interferometry Biosensor
Auer S, et al., Science Direct, doi:10.1016/j.snb.2015.06.088, 2015
This article discusses the use of Bio-Layer Interferometry (BLI) for rapid detection of antibodies in human serum that are specific to norovirus. The authors have taken measures to enhance the detection of antibodies from serum. Rapid detection using the BLI as compared to ELISA suggests robustness of the Octet platform in complex matrices such as the serum. An Octet RED384 equipped with Ni-NTA biosensors allowed capture of HIS-tagged NoV-VLPs or NoV P-particles. This article also highlights the use of diaminobenzidine (DAB) metal chelator as a method to enhance BLI signal from complex samples.
Science Direct
Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex
Kolumam G, et al., EBioMedicine, 2(7):730-43, 2015
This article describes generation of a humanized antibody named bFKB1 with the ability to activate fibroblast growth factor receptor 1 (FGFR1)/βKlotho (KLB) complex both in vitro as well as in vivo. Activation of this molecule in mice induced energy expenditure in brown adipose tissue (BAT), weight loss, and improvements in obesity related metabolic ailments. Phage derived antibodies were isolated and characterized for their interaction with the complexes FGFR1-KLB-bFKB1 and bFKB1-KLB-FGF using SPR and BLI technologies, respectively. An Octet RED system equipped with amine reactive biosensor tips allowed bFKB1 immobilization. Alternatively, KLB-ECD captured using the same biosensor tips were analyzed against human FGF21. This approach is expected to be a useful tool for the treatment of type2 diabetes and obesity related disorders.
PubMed
Optimization of Cell Line Development in the GS-CHO Expression System Using a High-Throughput, Single Cell-Based Clone Selection System
Nakamura T, Omasa T, J Biosci Bioeng , 120(3):323-9, 2015
This report describes the application of high-throughput ClonePix FL system for mammalian cell line development to yield high-producing clones with high monoclonality by using CHO-K1SV cells. The CHO-K1SV is a CHO-K1 derived cell line that utilizes a glutamine synthase gene expression system (GS-CHO). An OctetQK system equipped with Protein A biosensors was used to measure antibody concentrations during this process development.
PubMed
Overexpression of Serpinb1 in Chinese Hamster Ovary Cells Increases Recombinant IgG Productivity
Lin N, et al., J Biotechnol, 193:91-9, 2015
This is a proof of concept study to generate a stable pool of overexpressing SERPINB1 using a lentiviral vector. Serpinb1 (serpin peptidase inhibitor, clade B, member 1) is an intracellular serine protease inhibitor with an ability to inhibit elastase in neutrophils while also involved in protecting bone marrow neutrophil reserves. The authors used a SERPINB1 host cell line for IgG expression for verification purposes. An increased IgG productivity and extended culture longevity were notable improvements in this study. A Pall ForteBio Octet system was used to evaluate volumetric IgG productivity.
PubMed
Optimal Fusion of Antibody Binding Domains Resulted in Higher Affinity and Wider Specificity
Dong J, et al., J Biosci Bioeng, pii: S1389-1723(15)00141-3, 2015
With the aim of improving the affinity and specificity of binding domains, the authors generated novel fusion proteins (PAxPG) with a flexible linker between the two immunoglobulin binding domains derived from Protein A and Protein G. Once constructed, these fusion proteins were evaluated for their binding affinity to human Fab and certain mouse IgG subclasses (IgG1). A Pall ForteBio BLItz system equipped with Streptavidin biosensors was used in the analyses of binding interactions. Biotinylated PAxPG were captured onto Streptavidin biosensors and screened against the analytes such as the Human anti-vimentin Fab, human IgG1 Fc, and mouse IgG1. The data offers crucial insights into the linker lengths that can be used in the fusion proteins.
PubMed
Translational Development of an ADAMTS-5 Antibody for Osteoarthritis Disease Modification
Larkin J, et al., Osteoarthritis Cartilage, 23(8):1254-66, 2015
This article describes generation of selective inhibitors that may help to understand the roles of aggrecanase (ADAMTS-4 and ADAMTS-5) in human osteoarthritis (OA). Accordingly, the inhibition of both recombinant and native aggrecanase activity by high affinity monoclonal antibodies (mAb) were determined. Additionally, features such as the target engagement and modulation of disease endpoints in human and other preclinical systems were also determined. Kinetic characterization of antibody-antigen interactions was achieved by an OctetQK system equipped with Streptavidin biosensors. The results suggest ADAMTS-5 as a protease involved in cartilage degradation. Also the mAb GSK2394002 selected against ADAMTS-5 shows the potential to be used as a disease modifying osteoarthritis drug.
PubMed
Selection and Characterization of Human PCSK9 Antibody from Phage Displayed Antibody Library
Cao Y, et al., Biochem Biophys Res Commun, 7;463(4):712-8, 2015
The discovery of a novel antibody for Proprotein convertase subtilisin/kexin type 9 (PCSK9) is reported in this article. The authors used a phage-display antibody library against recombinant PCSK9 to obtain a fully human IgG1-PA4 antibody that is specific for PCSK9. A BLItz system equipped with Protein A biosensors allowed IgG1-PA4 capture and kinetic characterization of its binding to PCSK9.
PubMed
Highly Stable Aptamers Selected from a 2'-Fully Modified fGmH RNA Library for Targeting Biomaterials
Friedman A, Kim D, Liu R, Biomaterials, 36:110-23, 2015
This article describes an in vitro selection of fGmH RNA aptamers with enhanced binding affinity and higher specificity to Staphylococcus aureus Protein A (SpA) protein. The study used a 2'-fully modified RNA library (with N40 randomized) transcribed from a dsDNA library by His-tagged LAR T7 RNA polymerase mutant. The data suggest greater stability of RNA aptamers against alkaline hydrolysis and serum nucleases. Additionally these aptamers can perform as SpA affinity-based delivery reagents of antimicrobial agents. Both SPR (Biacore 3000) and BLI (Octet QK) platforms were used in the affinity characterizations. Authors underscore features such as the high throughput nature, enhanced automation, and relatively low cost as reasons for using the Octet platform. An Octet QK equipped with Protein A biosensors were used for affinity measurements. Specificity measurements involved ProA, ProG, and ProL biosensors.
PubMed
Structure and Receptor Binding Preferences of Recombinant Human A (H3N2) Virus Hemagglutinins
Yang H, et al., Virology, 477:18-31, 2015
The article describes glycan microarray analysis of recombinant A(H3N2) hemagglutinins (HAs) for their structural differences and changes in receptor binding. The authors cloned, expressed, and analyzed the glycan preference of 19 HAs chosen from WHO vaccine library that were observed from 1968 to 2012. An Octet RED instrument equipped with Streptavidin biosensors was used to perform glycan binding analyses. Biotinylated glycans captured on to Streptavidin biosensors were screened against recombinant HAs.
PubMed
A Newly Designed Molecule J2326 for Alzheimer's Disease Disaggregates Amyloid Fibrils and Induces Neurite Outgrowth
Chang P, et al., Neuropharmacology, 92:146-57, 2015
This study has focused on developing a multifunctional ligand with an ability to induce β-amyloid fibril (fAβ) disaggregation and to stimulate neurite outgrowth in fAβ-driven neurodegeneration. Such features do have the potential to be a promising therapeutic strategy for fAβ-driven AD patients. The authors synthesized a hydroxyalkylquinoline J2326; subsequently characterized its neurodegenerative potential in vitro and in vivo. The effects on aggregation kinetics, Aβ interaction analyses, and precise K determination involved SPR and BLI platforms. Equipped with super Streptavidin biosensors, the Pall ForteBio Octet system used in this investigation captured biotinylated Aβ1-42. The binding assays involved transferring of Aβ1-42 captured sensors into well containing varying concentrations of J2326.
PubMed
Aptamers as a Replacement for Antibodies in Enzyme-Linked Immunosorbent Assay
Toh S, et al., Biosens Bioelectron, 64:392-403, 2015
This is a comprehensive review on the application of aptamers in assays where antibodies are originally used. Aptamers are nucleic acid based affinity agents with several advantages as compared to antibodies such as smaller size, susceptibility to modifications, very low production costs, ability to generate against a variety of target molecules. The review elaborates on several different ELISA-inspired approaches and platforms collectively known as ELASA (enzyme-linked apta-sorbent assay). Authors also highlighted the advantages of using a BLI platform to carry out ELASA assays by using a BLI-ELISA study.
PubMed
Label-Free Detection of Small-Molecule Binding to a GPCR in the Membrane Environment
Heym R, et al., Biochim Biophys Acta, 1854(8):979-86, 2015
This article reports a comparative analysis of G-protein-coupled receptor (CXCR4) binding to a small molecule ligand AMD3100 using SAW, SPR, and BLI technologies. The data obtained from SAW and SPR compared directly with previously published data from BLI and radioligand-binding experiments. Since the current data is in agreement with prior data, the authors concluded that SAW can be as reliable as more established techniques such as SPR, BLI, and radioligand binding experiments.
PubMed
Robust Antibody-Antigen Complexes Prediction Generated by Combining Sequence Analyses, Mutagenesis, in Vitro Evolution, X-ray Crystallography and in Silico Docking
Loyau J, et al., J Mol Biol , 427(16):2647-62, 2015
This is a detailed investigation on structural and mechanistic elements contributing to the interaction between Hu15C1 neutralizing antibody and Toll-like receptor 4 (TLR4). The authors used a combined approach consist of site-directed and combinatorial mutagenesis along with X-ray crystallography and affinity measurements to dissect the molecular basis of this interaction. The information obtained from these experiments along with computational molecular docking lead to a mechanistic model. The model predicts that the antibody binding interferes with dimerization of the receptor, which is a crucial step for its activation. Both SPR and BLI systems were used for the kinetic characterization of the interaction. Epitope binning experiments were carried out using a Pall ForteBio Octet RED96 system equipped with Protein A biosensor tips.
PubMed
Precise and Efficient Antibody Epitope Determination Through Library Design, Yeast Display and Next-Generation Sequencing
Van Blarcom T, et al., J Mol Biol, 427(6 Pt B):1513-34, 2015
This article describes a methodology for epitope mapping of a panel of antibodies over a course of several weeks. It relies on rational library design, yeast display, and the next-gen DNA sequencing. The model system used in this article includes several neutralizing antibodies that bind to alpha toxin from Staphylococcus aureus. The affinity estimate is expected to serve as a predictor of antibody potency and hence the best clinical candidates. The epitope binning experiments performed using an Octet RED384 instrument equipped with Streptavidin biosensors allowed capture of biotinylated antibodies. Then the alpha toxin was bound to these captured antibodies. The pair wise binding was assessed in a classical sandwich orientation using the unbiotinylated antibodies.
PubMed
Fasxiator, a novel factor XIa inhibitor from snake venom, and its site-specific mutagenesis to improve potency and selectivity
Chen W, et al., J Thromb Haemost, 13(2):248-261, 2014
Standard anticoagulant drugs are commonly associated with a higher risk of excessive bleeding. Alternatives that can target intrinsic coagulation factors are constantly being pursued in order to slowdown coagulation cascade in the event of an intravascular thrombosis while lowering the bleeding risks during an injury. Described herein is the identification of a novel factor XIa (FXIa) specific inhibitor, Fasxiator, which was isolated from the venom of the banded krait snake, Bungarus fasciatus. Furthermore, the Fasxiator was subjected to a systematic site-specific mutagenesis in order to design a series of variants with an enhanced potency and selectivity. FasxiatorN17R,L19E mutant demonstrated the best balance in-between the potency and selectivity. Binding interactions of rFasxiator with FXIa and chymotrypsin was studied by Surface Plasmon Resonance (SPR) spectroscopy. A Pall ForteBio Pioneer system equipped with COOH5 sensor chip was used for the SPR assays. By using standard amine coupling chemistry, rFasxiator was immobilized onto the COOH5 sensor chip surface. Subsequently, different concentrations of FXIa or chymotrypsin diluted in running buffer were injected. The sensor chip surface was regenerated with glycine/HCl buffer. KD values were determined for the binding interactions assuming a simple 1:1 stoichiometry and one-step binding. Overall results of this investigation predict Fasxiator as a promising anticoagulant drug candidate.
PubMed
The Identification, Analysis and Structure-Based Development of Novel Inhibitors of 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase
Yun MK, et al., Bioorg & Med Chem, 22: 2157-2165, 2014
The microbial folate biosynthetic pathway is commonly known as an ideal target for the development of antimicrobial agents. Both dihydropteroate synthase (DHPS) and 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) are essential enzymes involved in the folate biosynthetic pathway.Authors have previously discovered that a DHPS pterin-pocket inhibitor can also engage the pterin pocket of Francisella tularensis HPPK (FtHPPK). Reported in this current study is the screening of a library of DHPS pterin-pocket binding molecules in order to identify new HPPK inhibitors. Two related compounds were identified as potential inhibitors of HPPK. Using a structure-based approach, a number of variants were synthesized. Binding affinity of those compounds to HPPK were analyzed by surface plasmon resonance (SPR) using a Pall ForteBio Pioneer system. Neutravidin was covalently immobilized onto the surface of the COOH5 sensor chip using standard amine coupling chemistry. Any residual active sites were capped by reaction with ethanolamine. Biotinylated HPPK was injected. Unoccupied biotin-binding sites of neutravidin were capped with amine-PEG2-biotin to minimize non-specific binding of the compounds to be tested. KD values were determined. The structure activity relationships derived from these derivative molecules will contribute greatly for the future design of new HPPK inhibitors.
PubMed
Human β-defensin HBD3 Binds to Immobilized Bla g2 from the German Cockroach (Blattella germanica)
Dietrich D, Martin A, and Brogden K, Peptides, 53:265-269, 2014
The Identification, Analysis and Structure-Based Development of Novel Inhibitors of 6-Hydroxymethyl-7,8-Dihydropterin Pyrophosphokinase
Yun M, et al., Bioorg Med Chem., 22(7):2157-65, 2014
Histatin 5 Binds to Porphyromonas Gingivalis Hemagglutinin B (Hagb) and Alters Hagb-inducedchemokine Responses
Borgwardt D, et al., Sci Rep, 4:3904, 2014
Predicting the Origins of Anti-blood Group Antibody Specificity: A Case Study of the ABO A-and B-antigens Supplementary Material
Makeneni S, et al., Front Immunol, 22;5:397, 2014
This article describes unique insights into the affinity and specificity of a mAb raised against blood group A antigen using computational modeling and the BLI. Despite versatility of this carbohydrate-antibody interaction in human blood transfusion medicine, very little information is known about the factors contributing to their high specificity. The 3D models generated for blood group A and B trisaccharides in complex with the antibody variable region, scFv AC1001 were able to identify key residues and interactions such as hydrogen bonding that are contributing to higher affinity and specificity. The affinity measurements involving the scFv (immobilized on to amine reactive AR2G sensors) and the blood group A trisaccharide (conjugated to BSA) were obtained using a BLI (Octet RED96) system. The kinetic binding constants were determined assuming a 1:1 interaction. The BSA-LeX trisaccharide conjugate is used as the negative control.
PubMed
Recognition of the Small Regulatory RNA RydC by the Bacterial Hfq Protein
Dimastrogiovanni D, et al., Elife, 3 doi: 10.7554/eLife.05375, 2014
The authors describe structural and biochemical characterization of RydC (a small RNA from Salmonella sp. and E. coli) binding to Hfq chaperone protein and hence to the cognate mRNA called cfa. The small RNA (sRNA) molecules often use Hfq to facilitate their interaction with mRNA and therefore a precise understanding of the interaction has been warranted. Several biophysical experiments along with X-ray crystallography have eluded into the proposed model in this report. The interaction between cfa mRNA (in vitro transcribed, biotinylated, and immobilized onto streptavidin biosensors) and Hfq analyte (WT & mutant) was carried out in the presence or absence of RydC on the Octet RED96 system using Streptavidin biosensors.
PubMed
Rapid Detection of [Beta]-lactam Antibiotic Residue in Milk Using Biolayer Interferometry
Liu, X, et al., Agricultural Biotechnology, 3(1):9-11,15, 2014
The article describes a complementary method that could be used for qualitative detection of β-lactam antibiotics in cow milk. The β-lactam antibiotics such as penicillins, cephalosporins, cephamycins, and carbapenems are widely used to treat bovine mastitis. Traces of these antibiotics could remain in milk if the antibiotics are administered improperly or without adhering to proper guidelines. Therefore techniques that have higher sensitivity, higher specificity, and a high-throughput are required to screen milk samples on a regular basis. The BLI platform used in this method development has an ampicillin-BSA conjugate tethered via hydrophobic interactions on to aminopropylsilane (APS) biosensors. The conjugate was then bound to colloidal gold labeled β-lactam antibiotic receptor. The receptor bound biosensors were subsequently used to qualitatively detect β-lactam antibiotic residues in milk. The method, although a qualitative approach, has a two-fold higher sensitivity and better throughput compared to existing immuno-chromatographic approaches. An Octet RED system was used in the assay development.
ProQuest
Identification of a Misfolded Region in Superoxide Dismutase 1 that is Exposed in Amyotrophic Lateral Sclerosis
Rotunno MS, J Biol Chem, 289(41):28527-38, 2014
A fatal neurodegenerative disorder called ALS (amyotrophic lateral sclerosis) caused by mutations and post-translational modifications within Cu,Zn-superoxide dismutase (SOD1), is an incurable disease and there are challenges in developing effective therapies due to its complex nature. The C4F6 antibody selectively binds to an epitope on misfolded SOD1 from human and mouse ALS cases. A variety of approaches including chemical cross-linking, mass spectrometry, site-directed mutagenesis, and BLI helped in the current study to obtain a detailed understanding of the structural elements and the residues that comprises this epitope. The information obtained will shine light on the mechanism of action of the disease. Such understanding could lead to therapeutic strategies for treating SOD1-mediated ALS. The BLI experiments carried out using the Octet QK system used C4F6 immobilized onto anti-mouse IgG Fc biosensors. The wild-type SOD1 and its variants showed changes in their affinity towards C4F6 as a result of the structure perturbations caused by mutagenesis and by chemical means. The data suggest that C4F6 recognizes and binds to an unusual conformation shared among misfolded variants of SOD1.
PubMed
Kinetic Titration Series with Biolayer Interferometry
Frenzel D, Willbold D., PLoS One, 9(9):e106882, 2014
The article describes execution of a strategy often used in surface plasmon resonance (SPR) known as "Kinetic Titration Series" in a BLI platform, and compares it to the "Parallel Sensor Kinetics" method. In SPR, the Kinetic Titration Series involve sequential injections of analyte on a ligand coated sensor chip without observing a complete dissociation between injections. Application of this methodology using the BLI platform described in this report utilize two pairs of interactions, namely IgG binding to protein G B1 and scFv IC16 binding to amyloid β(1-42). Authors compared the data obtained from Kinetic Titration Series and Parallel Sensor Kinetics for the above two systems using the BLI. The protein G B1 immobilized via amine coupling onto AR2G sensors and the biotinylated Aβ(1-42) immobilized onto SSA sensors were used as ligands. The Binding curves were obtained on an Octet RED96 system. According to the report, the execution of Kinetic Titration Series is relatively straightforward and avoids data evaluation discrepancies associated with sensor heterogeneity. This approach could also minimize the number of sensors and material required for kinetic titration assays. However, the caveat of this approach is that the users have to export the data obtained from this assay format into a BiaEvaluation software package that usually comes with the Biacore SPR system.
PubMed
A Phospha-oseltamivir-biotin Conjugate as a Strong and Selective Adhesive for the Influenza Virus
Streicher H, et al., Bioorg Med Chem Lett, 24(7):1805-7, 2014
The article describes an approach for selective immobilization of influenza virus onto BLI biosensors. In particular, it describes the synthesis of a biotin conjugated influenza neuraminidase (NA) inhibitor known as oseltamivir. The biotinylated version of the molecule inhibits influenza virus neuraminidase in the same range as the parent compound. Due to slow off-rate, the NA bound conjugate (biotinylated oseltamivir) can form a stable NA-coated surface on a streptavidin (SA) biosensor. The article also reports picomolar range binding of purified H3N2 X31 virus on to these pre-loaded biosensors. This strategy could be helpful for immobilization of viruses on to BLI-biosensors and for studying virus-antibody interactions. The compound may be useful in developing preliminary assays for influenza diagnosis and vaccine development. They used an Octet RED system to perform these assays.
PubMed
Separation of Lysozyme from Salted Duck Egg White by Affinity Precipitation Using pH-responsive Polymer with an L-thyroxin Ligand
Ding Z, Li X, Cao X, Science Direct, 138:153-60, 2014
The article describes a lysozyme enrichment method from salted duck egg white. Salted duck egg has a cultural and traditional value in China. The salted duck egg white once separated out from the salted duck egg yolk has become just a by-product and discarded in massive scale. Lysozyme on the other hand, is an egg white protein with applications in industries such as pharmaceutical, food, and biotechnology due to its ability to breakdown the bacterial cell wall. The method uses a pH sensitive affinity purification matrix containing L-thyroxine ligand. The BLI, SEM, FT-IR, CD, and XPS used during the verification and optimization of the process. The BLI assay setup involves a biotinylated resin conjugated to thyroxine (PMMDN-T). The titration involves a serial dilution of Lysozyme (analyte) from the affinity purification. The adsorption and desorption steps were monitored using a ForteBio Octet system using SSA sensors.
Science Direct
Structure and Protein-protein Interactions of Methanol Dehydrogenase from Methylococcus Capsulatus (Bath)
Culpepper M, Rosenzweig A, Biochemistry, 53(39):6211-9, 2014
Based on several biochemical and X-ray crystallography studies on two major protein families involved in the first step of the metabolic pathway in methanotrophs (gram-negative bacteria), the authors are trying evaluate the possible existence of a metabolic super-complex between methane monooxygenases (MMOs) and methanol dehydrogenases (MDHs). BLI experiments carried out using a BLItz system were able to estimate the protein-protein interactions between MDH and pMMO as well as between MDH and spmoB (truncated recombinant periplasmic domains of pMMO). Each BLI assay involves immobilization of recombinant MDH onto amine reactive second-generation (AR2G) biosensors followed by the association with a particular concentration of pMMO.
PubMed
The Spleen Tyrosine Kinase (Syk) Regulates Alzheimer Amyloid-β Production and Tau Hyperphosphorylation
Paris D, et al., J Biol Chem, 289(49):33927-44, 2014
The authors have previously reported that the L-type calcium channel (LCC) antagonist nilvadipine reduces brain amyloid-β (Aβ) accumulation. Nilvadipine affects both Aβ production and Aβ clearance across the blood-brain barrier (BBB). Further studies pertaining to the mechanism of action revealed inhibitory effects on spleen tyrosine kinase (Syk) by the Nilvadipine. The Octet RED platform has confirmed the binding and inhibitory effects of Nilvadipine on Syk. A biotinylated version of Syk is loaded onto SSA biosensors to estimate binding with Nilvadipine and BAY61-3606 (positive control). The data identifies Syk as a potential therapeutic target for major pathological lesions that define Alzheimer disease.
PubMed
Strand-specific Recognition of DNA Damages by XPD Provides Insights into Nucleotide Excision Repair Substrate Versatility
Buechner C, et al., J Biol Chem , 289(6):3613-24, 2014
The article looked into the mechanisms by which the helicase XPD (xeroderma pigmentosum group D) protein accomplish broad substrate adaptability during nucleotide excision repair (NER). Timely recognition and repair of DNA damages caused by UV radiation or chemical substances is crucial for cell viability. The authors used biophysical methods such as single molecule analysis by atomic force microscopy and BLI to investigate different recognition strategies of XPD for different types of damage. The BLI estimates on an Octet RED system allowed understanding of the affinities between immobilized DNA (single stranded or double stranded oligonucleotides containing different motifs or chemical alterations) and serial dilutions of XPD. The DNA ligands (labeled with biotin) immobilized onto SA biosensors.
PubMed
Seed Sequence-matched Controls Reveal Limitations of Small Interfering RNA Knockdown in Functional and Structural Studies of Hepatitis C Virus NS5A-MOBKL1B Interaction
Chung H, et al., J Virol, 88(19):11022-33, 2014
The authors describe a very important concept pertaining to siRNA-mediated gene silencing experiments. It shows an alternate dimension called "siRNA off-target effect" that could lead to false interpretations of the biological data. In a previous investigation, authors identified a cellular protein called MOBKL1B as a binding partner for NS5A protein of hepatitis C virus. They used MOBKL1B siRNA (in the absence of sufficient control experiments) to demonstrate its ability to reduce viral replication. Although they believed MOBKL1B-NS5A interaction is crucial for the siRNA effects observed, a variant lacking this interaction was not been able to replicate after treatment with MOBKL1B siRNA. By repeating the experiments with appropriate controls the authors were able to demonstrate that the MOBKL1B siRNAs previously reported had off-target inhibitory effects on viral replication. The BLI assays helped in the identification of a highly conserved binding site in NS5A comprising residues implicated in CypA-dependent HCV RNA replication. The BIACORE 2000 and ForteBio Octet RED 384 instruments were helpful in determining the binding interactions.
PubMed
Chemical Biology. A bump-and-hole Approach to Engineer Controlled Selectivity of BET Bromodomain Chemical Probes
Baud M, et al., Science, 346(6209):638-41, 2014
The article describes an approach to obtain selective inhibitors for Bromo and Extra-terminal (BET) bromodomain proteins. The BET proteins are important targets due to their roles in transcriptional regulation, epigenetics, and cancer. The authors used a chemical genetic approach to achieve shape complementarity between the bromodomain and a small-molecule inhibitor based on a "bump and hole" strategy. In other words, the bromodomain is mutated to a smaller residue to generate a "hole" on the protein. The mutated protein is targeted with analogs of a known ligand having a sterically bulky group ("bump") that can be accommodated by the pocket engineered on the mutated protein. The BLI experiments helped them to generate a heat map and hence identify BET mutants with similar binding profiles to their respective wild-type protein. An Octet RED 384 system equipped with SA sensors were used to immobilize biotinylated histone H4 peptides.
PubMed
Structural and Biochemical Insights into the Activation Mechanisms of Germinal Center Kinase OSR1
Li C, et al., J Biol Chem , 289(52):35969-78, 2014
This article describes the details of a regulatory mechanism involving the OSR1 based on structural and biochemical studies. The oxidative stress-responsive 1 (OSR1) is a mammalian kinase protein. It modulates ion homeostasis and the cell volume in mammal. The study has found that the OSR1 activity is regulated by autoinhibition, with no lysine (WNKs), and mouse protein 25 (MO25). As part of the mechanistic dissection, the interactions between purified proteins of MO25 and OSR1 were analyzed using an Octet RED96 instrument. In one mode of analysis, they used biotinylated wild-type OSR1 protein immobilized onto SA biosensors and screened against varying concentration of wild type and mutant MO25. In the second mode of analysis, they swapped the order and used a biotinylated wild type MO25 with wild type and mutant OSR1 proteins.
PubMed
Probing the Sites of Interactions of Rotaviral Proteins Involved in Replication
Viskovska M, et al., J Virol, 88(21):12866-81, 2014
This investigation reveals additional protein partners that are involved and directly interact with the non-structural protein 2 (NSP2) during the replication and packaging of rotavirus genome in cytoplasmic compartments called viroplasms. These viroplasms generally formed during virus infection. A complex interactome coordinates the replication and packaging processes by making use of the non-structural proteins (NSPs) and structural proteins (VPs). With the help of isothermal calorimetry (ITC), biolayer interferometry (BLI), and peptide array screening, they examined the interactions between NSP2, NSP5, NSP6, VP1, and VP2. The BLI experiments carried out using an Octet RED96 instrument utilized biotinylated proteins immobilized on to SA biosensors. The binding assays carried out between NSP2-VP1, NSP2-VP2, and NSP2-NSP6 involved immobilization of VP1, NSP2, and NSP6, respectively on to SA sensors.
PubMed
Development of a Protein Nanoparticle Platform for Targeting EGFR Expressing Cancer Cells
Jakob W, et al., Chemical Technology and Biotechnology, 90(7):1230-36, 2014
This investigation provides evidence that the antibody fragments targeting the epidermal growth factor receptor (EGFR), once tethered to E2 nanoparticles are still capable of targeting/recognizing EGFR overexpressing cancer cells. There are previous reports where protein-based nanoparticles that are being developed for cancer drug delivery and diagnostics. Particularly, the E2 protein derived from pyruvate dehydrogenase complex in B. subtilis assembles into a 60-subunit protein cage-structure that is capable of encapsulating cancer therapeutics. In this particular study, they conjugated variants of the anti-EGFR antibody fragment with E2 protein (containing specific cysteine residues) via a maleimide-specific crosslinker. The binding between conjugated E2 particle and recombinant EGFR was monitored using a BLItz instrument. The recombinant EGFR-Fc and the recombinant VEGFR-Fc (negative control) proteins immobilized on to Protein A biosensors helped in determining the binding interactions. The wild-type EGFR (from cancer cells) binding to E2 particles was confirmed by a Flow cytometry assay
Wiley Online Library
Glypican-3-targeting F(ab')2 for 89Zr PET of Hepatocellular Carcinoma
Sham J, et al., J Nucl Med , 55(12):2032-7, 2014
This article reports the development of a 89Zr PET imaging probe that only uses the F(ab')2 region of anti GPC3 mAb (αGPC3) to address several limitations shown by the whole-αGPC3 targeting moiety during hepatocellular carcinoma (HCC) imaging. The use of whole monoclonal antibody that target GPC3 in PET imaging is a promising approach. These mAbs have lengthy circulation times and that is considered as a drawback in this whole-antibody approach. Affinity characterizations of αGPC3 whole-antibody and F(ab')2 region against purified human carrier-free glypican-3 protein performed on an Octet RED96 instrument suggest compatibility of the antibody fragment (F(ab')2 region) in PET imaging.
PubMed
Plasmodium Falciparum Aldolase and the C-terminal Cytoplasmic Domain of Certain Apical Organellar Proteins Promote Actin Polymerization
Diaz S, et al., Mol Biochem Parasitol, 197(1-2):9-14, 2014
This investigation tried to deepen the understanding of the interactome involved in apicomplexan motility and host cell invasion pertaining to Plasmodium falciparum (Pf). In particular, they investigated the interactions between actin, several cytoplasmic tail domains (CTD), and aldolase. Using an Octet RED96 system, they showed that Pf aldolase binds with high affinity to both rabbit and Pf actin. The investigation also determined that Pf aldolase has a similar affinity for filamentous (F-) actin and globular (G-) actin. Both G and F actin were biotinylated and immobilized on to SA sensors during this investigation.
PubMed
Hyperglycosylated Stable Core Immunogens Designed to Present the CD4 Binding Site are Preferentially Recognized by Broadly Neutralizing Antibodies
Ingale J, et al., J Virol, 88(24):14002-16, 2014
In this study, the investigators were trying to obtain antibodies that are capable of focusing mainly the gp120 CD4 binding site (CD4bs), while eliciting a broader neutralization of HIV. They used structure-based engineering of gp120 cores to present cysteine-stabilized CD4bs and glycan masking of the non-neutralizing determinants. By using an Octet RED system, they evaluated the binding interactions between hyperglycosylated core analytes and various CD4bs antibodies. The CD4bs antibodies were captured onto anti-human Fc capture sensors. This approach is successfully focusing antibodies to the CD4bs. However, those antibodies did not neutralize the HIV nor they bound to unmodified gp120.
PubMed
A von Willebrand Factor Fragment Containing the D'D3 Domains is Sufficient to Stabilize Coagulation Factor VIII in Mice
Yee A, et al., Blood, 124(3):445-52, 2014
This article reports the effect of a series of von Willebrand factor (VWF) fragments on plasma factor VIII (FVIII) binding in vitro as well as their effect in vivo using mouse models. Typically, FVIII and VWF circulate together as a complex preventing FVIII from degradation and rapid clearance from plasma. The results indicate that a fragment containing only about 20% of the VWF sequence is sufficient to support FVIII stability in vivo. Binding assessments performed using an Octet RED system suggested that VWF propeptide processing of VWF D'D3 fragment is still a requirement for optimal FVIII affinity. Immobilized onto anti-FLAG biosensors, the VWF fragments (purified from transiently transfected HEK293T cells) allowed analysis of the association and dissociation of various concentrations of recombinant FVIII during these assays.
PubMed
Exposure of Epitope Residues on the Outer Face of the Chikungunya Virus Envelope Trimer Determines Antibody Neutralizing Efficacy
Fong R, et al., J Virol, 88(24):14364-79, 2014
The investigation was able to produce seven human monoclonal antibodies (MAbs) against envelope glycoproteins E2 and E1 found in chikungunya virus (CHIKV), by using the phage display technology. CHICKV is a mosquito-borne pathogen and the most important alphavirus-affecting the humans. It results in a chronic arthritic condition that can persist for months or even years. The study went on to locate the binding region of one of the antibodies (IM-CKV063), that showed most neutralizing features, highest affinity binding. IM-CKV063 antibody is also capable of providing therapeutic and prophylactic protection in multiple animal models. As part of the characterization, the authors used an Octet RED system to assess the kinetics of MAb binding to E2/E1 displayed in its native state on the CHIKV virion surface. The noninfectious CHIKV VLP, immobilized onto amine reactive (AR2G) biosensors using a human MAb against CHIKV was the ligand in these assays. In addition to the dissociation constants, the specific binding of IM-CKV063 (analyte) onto CHIKV VLP was also confirmed during this investigation.
PubMed
MM-141, an IGF-IR- and ErbB3-directed Bispecific Antibody, Overcomes Network Adaptations that Limit Activity of IGF-IR Inhibitors
Fitzgerald J, et al., Mol Cancer Ther, 13(2):410-25, 2014
The article describes the discovery and development of the first tetravalent bispecific antibody (named MM-141) which is capable of co-inhibiting two growth factor receptor families, namely IGF-IR and ErbB3. The MM-141 prevents PI3K/AKT/mTOR network adaptation by blocking the survival pathways and by degrading the receptor tyrosine kinase (RTK) complexes. Heregulin (HRG) is the ligand partner for ErbB3. HRG-ErbB3 autocrine loops are observed in ovarian cancer cells. Using an Octet system, authors were able to show that the antibody (MM-141) has the ability to block HRG binding to ErbB3. The HRG used for ligand blocking experiment was biotinylated and immobilized onto SA biosensors and the assays performed in the presence or in the absence of MM-141. Based on their detailed investigation, the authors suggest the potential of MM-141 to become an effective therapeutic agent for patients with solid tumors.
PubMed
Towards Engineering Hormone-binding Globulins as Drug Delivery Agents
Chan W, Zhou A and Read R., PLoS One, 9(11):e113402, 2014
This is a proof-of-concept study to exploit the mechanism of targeted release of cortisol by corticosteroid binding globulin (CBG) in order to carry therapeutic agents toward predetermined locations. They altered the reactive centre loop of CBG by site-directed mutagenesis to facilitate the cleavage by other proteases such as the prostate specific antigen (PSA, a prostate specific serine protease) and thrombin (a major protease in the blood coagulation cascade). They used fluorescence spectroscopy and biolayer interferometry (BLI) to investigate CBG-ligand interactions. The BLI approach was particularly useful when the fluorescence quenching experiments did not work due to mutation of Trp 371. The Trp 371 is the main source of intrinsic fluorescence in the CBG binding pocket. An Octet RED system (equipped with SA biosensors) was used to immobilize biotinylated cortisol. A serial dilution of CBG variants used for the KD determination revealed that the protein carrier binds to its ligand with a high affinity in the S-state. It is subjected to a larger decrease in binding affinity upon reactive centre-loop-cleavage in order to release much of the compound bound to the target site in the R-state.
PubMed
REAL-Select: Full-Length Antibody Display and Library Screening by Surface Capture on Yeast Cells
Rhiel L, et al., PLoS One, 9(12):e114887, 2014
This report describes a novel technology called REAL-Select (Reversible Expression of Antibody Libraries for Selection) for displaying full-length IgG-molecules or libraries for screening and clone characterization. This methodology does not require genetically encoded anchor proteins or intracellular antibody modifications. The cell wall anchoring of secreted antibodies is achieved by chemical coupling of Fc-binding ZZ domains (an artificial variant of protein A-derived B-domain with a high affinity to the Fc-region of IgG) that allow capturing of secreted antibodies onto the yeast cell surface. The affinity-matured antibodies obtained from REAL-Select retain their native form while allowing the detection of subtle changes in affinity by flow cytometry. An Octet RED system allowed the determination of binding kinetics of IgG analogs (subcloned and purified) with a high affinity and specificity to cMet protein. IgG analogs were captured onto anti-human-FC (AHC) biosensors. This methodology does not rely on the genetic background of the antibody-expressing host. Therefore, it is expected to be compatible with other eukaryotic expression hosts such as the mammalian cells and P. pastoris.
PubMed
Envelope Variants Circulating as Initial Neutralization Breadth Developed in Two HIV-infected Subjects Stimulate Multiclade Neutralizing Antibodies in Rabbits
Malherbe D, et al., J Virol, 88(22):12949-67, 2014
Based on the hypothesis that the B cells are developing broad antibodies by exposure to ever evolving viral envelope population, the authors tested the ability to use multiple envelops from two different subjects who developed neutralization breadth within a few years of HIV infection. They compared several different combinations of envelops from each subject to determine the most effective regimens. This approach revealed more-potent antibodies and they are modestly cross neutralizing. Surface plasmon resonance (SPR) and biolayer interferometry (BLI) techniques were used in the antigenic characterization of gp140 trimers as well as in the binding analyses of polyclonal antibodies. Anti-human IgG Fc capture (AHC) biosensors were used to capture different antibodies during the antigenic characterization. On the other hand, SA sensors were used to capture biotinylated SF162 gp140 trimer during the binding analyses.
PubMed
Mechanism of Inhibition for BMS-791325, a Novel Non-nucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase
Rigat K, et al., J Biol Chem, 289(48):33456-68, 2014
By using several biochemical and biophysical techniques, the authors were able to dissect the mechanism of action of BMS-791325 (a non-nucleoside inhibitor) on HCV (hepatitis C virus) NS5B, an RNA-dependent RNA polymerase. Particularly, this study revealed a non-competitive time-dependent inhibition mechanism for BMS-791325. In addition, it uncovered all parameters pertaining to inhibitor binding kinetics. The BLI kinetic measurements obtained from an Octet RED system eliminated the discrepancies shown by the kinetic parameters generated from the activity assay and stopped flow studies. Biotinylated NS5B enzymes were immobilized onto SSA biosensors. Although complementary, the stopped-flow and BLI techniques uncovered different steps of the mechanism giving rise to a full characterization of the interaction between BMS-791325 and NS5B polymerase.
PubMed
TACI Deficiency Enhances Antibody Avidity and Clearance of an Intestinal Pathogen
Tsuji S, et al., J Clin Invest, 124(11):4857-66, 2014
The authors performed an investigation to understand the phenotype of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) deficiency in mice. The mice lacking TACI exhibit hypogammaglobulinemia and generate defective long-lived antibody responses. These features are consistent with the human phenotype of common variable immunodeficiency (CVID). Those individuals with CVID express either the C104R or A181E mutants of TACI. Since TACI deficient mice can produce bursts of IgG, the authors tested whether those antibodies produced by TACI deficient mice attain the same avidity and functions of those produced by normal mice. The study revealed such antibodies produced in bursts attain higher avidity and the mice were able to rapidly clear Citrobacter rodentium, a pathogen known for severe human enteritis. An Octet RED system was used in the determination of KD for 4-hydroxy-3-nitrophenylacetyl (NP) antigen specific IgGs. A biotinylated version of NP4-BSA captured onto SSA biosensors used in the analyses.
PubMed
Mining the Human Autoantibody Repertoire: Isolation of Potent IL17A-neutralizing Monoclonal Antibodies from a Patient with Thymoma
Beerli R, et al., Mabs, 6(6):1608-20, 2014
This article describes the therapeutic potential of neutralizing anticytokine autoantibodies based on the observations pertaining to interleukin-17A (IL17A)-neutralizing autoantibodies isolated from a thymoma patient. IL17A is a proinflammatory cytokine associated with several chronic inflammatory diseases such as Rheumatoid arthritis, psoriasis, and multiple sclerosis. A mammalian cell display allowed isolation of three specific clonotypes that are capable of neutralizing the IL17A. Several methods including biolayer interferometry (BLI) were employed to characterize several potent IL17A-neutralizing antibodies. Selected IgG1 antibodies were captured using an Octet system equipped with anti-hIgG-Fc-capture (AHC) biosensors and they were screened against recombinant human IL17A analyte. This study demonstrates the potential of human autoantibodies in therapeutic and clinical applications.
PubMed
Resolving Self-association of a Therapeutic Antibody by Formulation Optimization and Molecular Approaches
Casaz P, et al., Mabs, 6(6):1533-9, 2014
This article describes plausible approaches to resolve self-association of therapeutic antibodies. In particular, it demonstrates how formulation optimization and molecular approaches such as framework change, targeted mutagenesis, and isotype switch can benefit during the development of high concentration monoclonal-antibody formulations. The C4 human-antibody used in this investigation has the ability to neutralize diphtheria toxin and shows a strong potential to become a therapeutic antibody. When formulated at >30 mg/mL, C4 forms a white semi-solid gel at low temperatures. The affinities between C4 or C4-derivatives (generated by aforementioned approaches) and the antigen were measured using the BLI technology. An Octet system equipped with anti-human IgG biosensors was used in the capturing of C4 and its derivatives.
PubMed
HIV-1 Receptor Binding Site-directed Antibodies Using a VH1-2 Gene Segment Orthologue are Activated by Env Trimer Immunization
Navis M, et al., PLoS Pathog, 10(8):e1004337, 2014
This investigation aims at finding possible explanations for the lack of broadly neutralizing activity in broadly neutralizing antibodies (bNAbs) following vaccination. They isolated a series of monoclonal antibodies encoded by a particular gene segment and demonstrated that one of the Ab (GE356) binds with a different mode of interaction from other CD4-binding-site-directed bNAbs. These findings will lead to the development of an HIV-1 vaccine that stimulates the production of Abs that are capable of neutralizing various HIV-1 strains. The binding kinetics between MAbs (from NHP and human) and Env variants were assessed by an Octet RED96 system. Anti-human IgG Fc capture biosensors were used to immobilize MAbs.
PubMed
Assembly and Architecture of the EBV B Cell Entry Triggering Complex
Sathiyamoorthy K, et al., PLoS Pathog, 10(8):e1004309, 2014
The entry of Epstein-Barr Virus (EBV) into B cells of the immune system was investigated using several biochemical, structural, and functional experiments. These lipid-enveloped viruses in general require coordination of the receptor recognition, viral protein activation, and significant protein-conformational changes that can drive fusion of the membrane bilayer. Three viral glycoproteins (gH, gL, and gp42) contribute to the complex formation with the host receptor know as the human leukocyte antigen (HLA). The biolayer interferometry (along with electron microscopy analyses) was used to dissect the assembly mechanism. An Octet RED96 system equipped with SA biosensors was used to capture biotinylated HLA-DQ2 or EBV gHgL depending on the interaction under consideration. The binding interaction analyses involved gp42 or its mutants (C114S or I159C) and HLA-DQ2 in the presence or absence of gHgL. Based on the current data, the authors proposed a model for EBV entry into B cells.
PubMed
Structural and Functional Insight into TAF1-TAF7, a Subcomplex of Transcription Factor II D
Bhattacharya S, et al., Proc Natl Acad Sci U S A, 111(25):9103-8, 2014
This article is reporting the crystal structure of the yeast TAF1/7 heterodimer and provides also a detailed biochemical analysis to probe its function. During eukaryotic transcriptional initiation, TATA binding protein-associated factors 1 and 7 (TAF1 and TAF7) together with two subunits of the transcription factor IID (TFIID) form a preinitiation complex (PIC). Current structure suggests that TAF1 is not a histone acetyltransferase (HAT). Instead, the new findings suggest new roles for the TAF1/7 complex in the regulation of PIC assembly. An Octet RED384 system equipped with SA biosensors was used to demonstrate the peptide binding to native or mutant yTAF1/7 protein complexes.
PubMed
Susceptibility to HLA-DM Protein is Determined by a Dynamic Conformation of Major Histocompatibility Complex Class II Molecule Bound with Peptide
Yin L, et al., J Biol Chem, 289(34):23449-64, 2014
These authors investigated the sequence and structural determinants of human leukocyte antigen DM (HLA-DM) interaction. HLA-DM arbitrates the exchange of peptides loaded onto major histocompatibility complex class II (MHCII) molecules during antigen presentation. These molecules typically found on antigen-presenting cells such as the B cells, dendritic cells, endothelial cells, and phagocytes. Non-optimal peptide interactions often lead to low MHCII binding affinities and hence kinetic instability. These peptides are inclined to HLA-DM-mediated peptide exchange. The biochemical and biophysical approaches used in this investigation helped to identify changes that are often accompanying a conformational alteration. The binding studies involved assays from both Biacore and Pall ForteBio Octet systems. Binding of the wild type or mutant HLA-DM to immobilized HLA-DO (DM negative regulator) were performed on an Octet system.
PubMed
High-content Analysis of Antibody Phage-display Library Selection Outputs Identifies Tumor Selective Macropinocytosis-dependent Rapidly Internalizing Antibodies
Ha K, et al., Mol Cell Proteomics, 13(12):3320-31, 2014
This article describes a novel screening strategy for the identification of phage antibodies with the ability to bind and rapidly enter tumor cells via macropinocytosis. The antibodies used throughout the investigation were quantified using a Blitz system.
PubMed
HSP70 Sequestration by Free α-globin Promotes Ineffective Erythropoiesis in β-thalassaemia
Arlet J, et al., Nature, 514(7521):242-6, 2014
Based on their in vitro studies, the authors demonstrate that the heat shock protein70 (HSP70) interacts with free α-globin chains during the maturation of human β-TM erythroblasts. As a result, HSP70 is secluded in the cytoplasm and GATA-1 transcription factor is no longer protected, causing an end-stage maturation arrest and apoptosis. Mutants of HSP70 and GATA-1 restore the maturation of β-TM erythroblasts and hence provide a platform for novel β-TM targeted therapeutics. An Octet RED instrument (equipped with SA sensors) was used to conduct protein interaction assays. HSP70 or AHSP proteins were biotinylated and captured onto SA sensors.
PubMed
Manipulation of Receptor Oligomerization as a Strategy to Inhibit Signaling by TNF Superfamily Members
Warren J, et al., Sci Signal, 7(339):ra80, 2014
This article focuses on the interaction between the receptor-ligand pair known as receptor activator of nuclear factor- κB (RANK) and its ligand RANKL. RANKL is a member of the TNF superfamily of cytokines and it stimulates osteoclast formation and bone resorption. The authors engineered a single protein by encoding all three monomers of RANKL as a single polypeptide chain. By using an unbiased forward genetic approach, they identified mutants of RANKL with a 500-fold increase in affinity for RANK. The affinity parameters of RANKL variants with RANK were determined by the surface plasmon resonance (SPR) and biolayer interferometry (BLI) techniques. An Octet RED system equipped with SSA biosensors was used to capture biotinylated GST-RANKL fusion proteins. The BLI experiments helped in the determination of apparent K
D values for the binding of RANK-Fc or OPG-Fc with RANKL variants.
PubMed
Monoclonal Antibody Targeting of Fibroblast Growth Factor Receptor 1c Ameliorates Obesity and Glucose Intolerance via Central Mechanisms
Lelliott C, et al., PLoS One, 9(11):e112109, 2014
This article describes the phage display identification of a novel fully human fibroblast growth factor receptor 1 (FGFR1c) targeting monoclonal antibody called R1c mAb. It causes significant body fat and weight loss in diet-induced obese mice due to reduced food intake. The receptor binding assays performed using an Octet RED system involved label-free analysis between R1c mAb and FGFR1c. The R1c mAb or a control mAb captured on to SA biosensors via a biotinylated Protein G was instrumental in monitoring the association and dissociation of receptor.
PubMed
Characterization and Epitope Mapping of the Polyclonal Antibody Repertoire Elicited by Ricin Holotoxin-based Vaccination
Cohen O, et al., Clin Vaccine Immunol, 21(11):1534-40, 2014
This is an investigation to obtain information and a plausible strategy for the development of an improved holotoxin-based vaccine against ricin. Ricin is one of the most lethal toxins known to date and yet, there is no cure against ricin exposure despite less effective therapies based on neutralizing antibodies elicited by active vaccination or those given passively are available. An epitope mapping analysis (using phage display random peptide libraries) performed on anti-ricin antibodies revealed immunodominant epitopes in both subunits A and B of ricin. The study also helped to determine the roles of these epitopes in ricin neutralization. An Octet RED instrument equipped with SA biosensors was used to capture biotinylated ricin. These titrations were carried out using serial dilutions of ricin-purified fractions of antibodies.
PubMed
Tools for High-Throughput Process and Medium Optimization
Jordan M and Stettler M, Methods Mol Biol, 1104, 77-88, 2014
The article described two different conditions for shaking cultured cells under scaled down conditions in order to increase sample throughput. Media conditions, shaking speeds, aeration of the cultures and the vessels used to house the cells were all compared. Titers were determined using the Octet QK system.
PubMed
Application of the Log-normal Model for Long Term High Affinity Antibody/Antigen Interactions using Bio-Layer Interferometry
Wallner J, et al., J Math Chem, 52(2):575-587, 2014
Optical biosensors are commonly used to characterize biomolecular interactions and are an important tool for quality control and process development groups. In this report, the authors compared two different theoretical binding models to determine which method most accurately interprets the results of the interaction. All binding studies were performed on the Octet QK system using Streptavidin biosensors. The results suggested that for reactions of high affinity and slow kinetics, the log-normal model provided better curve fitting tools over the more commonly used simple 1:1 interaction model.
Springer
Screening and Optimization of Chemically Defined Media and Feeds with Integrated and Statistical Approaches
Xiao Z, et al., Methods Mol Biol, 1104, 117-35, 2014
The authors described a case study detailing the design of experiment and revised workflow to improve titer and protein yields by varying culture media selection and feeding conditions performed by bioprocessing and production groups for batch culturing of cells. Protein titers were determined using the Octet QK system and custom biosensors.
PubMed
Production and Characterization of Recombinant Human Beta-defensin DEFB120
Liu H, et al., J Pept Sci, doi: 10.1002/psc.2611, 2014
The authors performed structural and biological characterization on recombinantly generated DEFB120, a novel human beta-defensin protein. Beta defensins are involved in innate immunity and represent a new class of possible therapeutics to be developed that can overcome the evolving multi-antibiotic resistances of many strains. Detection and interaction between DEFB120 and LPS was carried out on the Octet RED96 system using Streptavidin biosensors.
PubMed
An Unusual Repressor Controls the Expression of a Crucial Nicotine-degrading Gene Cluster in Pseudomonas Putida?S16
Wang L, et al., Mol Microbiol, doi: 10.1111/mmi.12533, 2014
The authors performed structural and functional analysis on NicR2, a transcriptional regulator in Pseudomonas putida. The binding characteristics and regulation of nicotine degradation by NicR2 provided more insight on protein-DNA binding interactions and gene expression in prokaryotes. Binding studies were performed on the Octet RED96 system. Streptavidin biosensors were used.
PubMed
Rapid Homogenous Time-Resolved Fluorescence (HTRF) Immunoassay for Anthrax Detection
Cohen N, et al., J Fluoresc, doi: 10.1007/s10895-014-1354-7, 2014
This report described the development and testing of a new rapid and sensitive homogenous time-resolved fluorescence (HTRF) assay for the detection of bacterial toxin-PA and bacterial spores from Bacillus anthracsis, which can induce acute anthrax disease. Rapid, accurate and sensitive diagnosis of anthrax at early stages of the disease is critical to allow for an effective treatment. Binding characterization of the antibodies used in the assay development was performed on the Octet RED system and Streptavidin biosensors were used.
PubMed
Global Identification of O-GlcNAc Transferase (OGT) Interactors by a Human Proteome Microarray and the Construction of an OGT Interactome
Deng RP, et al., Proteomics, doi: 10.1002/pmic.201300144, 2014
In this report, the authors screened a human proteome microarray to identify proteins that interact with OGT (O-GlcNac transferase) the key enzyme responsible for O-GlcNacylation and uncovered 25 novel targets of OGT that are known to play important roles in protein transportation/localization and transcriptional regulation providing greater insight in the role of OGT. Kinetic binding interactions were measured on the Octet system and Streptavidin biosensors were used.
PubMed
The Development and Application of High Throughput Cultivation Technology in Bioprocess Development
Long Q, et al., J Biotechnol, pii: S0168-1656(14)00158-8, 2014
This review discusses the recent advances and technologies for scaling up and developing higher throughput cultivation and bioprocessing procedures for protein production without compromising data quality. The key factors behind improving process development, sample scaling, screening strategies and maximizing timelines during the processing steps are discussed in detail.
PubMed
Directed Evolution of Multivalent Glycopeptides Tightly Recognized by HIV Antibody 2G12
Horiya S, et al., J Am Chem Soc, 136(14):5407-15, 2014
The authors presented a screening strategy to identify and select multivalent glycopeptides that showed strong picomolar to low nanomolar affinity for the HIV broadly neutralizing antibody 2G12 as a method to generate new HIV vaccine candidates. Binding studies were performed on the Blitz system, using Streptavidin biosensors.
PubMed
Talin-bound NPLY Motif Recruits Integrin-signaling Adapters to Regulate Cell Spreading and Mechanosensing
Pinon P, et al., J Cell Biol, 205(2):265-81, 2014
The authors examined the functional activities of integrin protein as it relates to cell adhesion and cell spreading activity by monitoring binding and complex formation after performing mutational analysis on a targeted cytoplasmic sequence motif of the protein. Binding analysis was performed on the Octet RED384 system using Ni-nitrilotriacetic acid (NTA) biosensors.
PubMed
Ca2+-Calmodulin Regulates SNARE Assembly and Spontaneous Neurotransmitter Release via v-ATPase Subunit V0a1
Wang D, et al., J Cell Biol, 205(1):21-31, 2014
In this report, the authors performed structural and functional analysis to examine the signaling cascade of Ca2+dependent release and the involvement of soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) complexes on neutrotransmission release. Binding studies were performed on the Octet QK system using Ni-nitrilotriacetic acid (NTA) biosensors.
PubMed
GlycoDelete Engineering of Mammalian Cells Simplifies N-glycosylation of Recombinant Proteins
Meuris L, et al., Nat Biotechnol, doi: 10.1038/nbt.2885, 2014
This report described a new methodology called GlycoDelete, a glycoengineering strategy used as an approach to solve the issues of N-glycosylation heterogeneity in mammalian cell-based glycoprotein production. The goal of the protocol is to produce more consistent therapeutic protein preparations in process development settings. Real-time binding was performed on an Octet RED96 system. Anti-penta his biosensors were used.
PubMed
Optical Biosensors for Label-free Detecion of Biomolecular Interactions
Sun, YS, Instrumentation Science & Technology, doi:10.1080/10739149.2013.843060, 2014
In this review, the author presented an overview on optical biosensor methods for monitoring biomolecules and the regulatory events that take place in the cell. While commonly used fluorescence based technologies offer sensitivity, optical biosensors provide a label-free platform and real time and kinetic detection of molecules without affecting the intrinsic properties of the target.
Taylor Francis Online
Receptor mimicry by antibody F045-092 Facilitates Universal Binding to the H3 Subtype of Influenza Virus
Lee PS, et al., Nat Commun, 5:3614, 2014
The authors performed structural and functional characterization of F045-092, a monoclonal antibody isolated from a human B lymphocyte derived phage-display library which showed strong broadly neutralizing activity against H3 influenza virus and providing greater insight into the development of a pan-H3 vaccine. Binding kinetics were determined on the Octet RED system. Streptavidin biosensors were used.
PubMed
Structure of Vaccinia Virus A46, an Inhibitor of TLR4 Signaling Pathway, Shows the Conformation of VIPER Motif
Kim Y, et al., Protein Sci, doi: 10.1002/pro.2472, 2014
In this report, the authors performed a detailed study on the specific interactions and binding characteristics of the vaccinia virus protein A46 to better understand the role of this protein in interfering with Toll receptor binding and the host immune response system. Binding analysis was performed on the Blitz system using Ni-NTA biosensors.
PubMed
Therapeutic Targeting of BET Bromodomain Proteins in Castration-resistant Prostate Cancer
Asangani IA, et al., Nature, doi: 10.1038/nature13229, 2014
In this report, the authors described the treatment and screening of prostate cancer and control cell lines and the identification of Bromodomain and extraterminal (BET) inhibitors that act on cell proliferation and knock down expression of specific cancer biomarkers suggesting new possibilities in alternative treatments for castration-resistant prostate cancer (CRPC). Binding studies were performed on the Octet RED system. Super Streptavidin biosensors were used.
PubMed
The Crystal Structure of S. cerevisiae Sad1, a Catalytically Inactive Deubiquitinase that is Broadly Required for pre-mRNA Splicing
Hadjivassiliou H, Rosenberg OS, and Guthrie C, RNA, 20(5):656-69, 2014
The authors performed structural and functional analysis on the zinc finger ubiquitin binding domain (Znf-UBP) of Sad1, an essential splicing factor from Saccrharomyces cerevisiae to better understand the mechanisms of spliceosome assembly and the regulation of pre-mRNA splicing. Binding studies were done on the Octet384 system using Streptavidin biosensors.
PubMed
ZMYND11 Links Histone H3.3K36me3 to Transcription Elongation and Tumour Suppression
Wen H, et al., Nature, 508(7495):263-8, 2014
The authors identified and performed structural and functional characterization of ZMYND11, an adenovirus early region 1A (E1A) interacting protein acts as a histone H3.3-specific reader of H3.3K36me3 (trimethylation modification of the histone). Regulation of RNA polymerase II elongation control by ZMYND11 was linked to tumor suppression. Binding kinetic studies were performed on the Octet RED96 system using Anti-GST antibody-coated biosensors.
PubMed
Alternative Recognition of the Conserved Stem-Epitope in Influenza A Hemagglutinin by a VH3-30-Encoded Heterosubtypic Antibody
Wyrzucki A, et al., J Virol, doi: 10.1128/JVI.00178-14, 2014
The authors isolated a monoclonal antibody designated MAB3.1 from phage display with immobilized hemagglutinin from H2N2 influenza virus used as the target. They also utilized de novo in siico-design and affinity-matured synthetic proteins to determine the antibody binding characteristics in order to gain more insight into the development of pan-influenza virus vaccines. Binding studies were performed on the Octet RED instrument and Streptavidin biosensors were used.
PubMed
Physical Stability Comparisons of IgG1?Fc Variants: Effects of N?Glycosylation Site Occupancy and Asp/Gln Residues at Site Asn 297
Alsenaidy MA, et al., J Pharm Sci, doi: 10.1002/jps.23975, 2014
The authors performed structural integrity and conformational stability studies on various IgG1-Fc proteins and mutant forms produced from the yeast Pichia pastoris to better understand the different glycosylation site occupancies and how these modifications regulate the biological activity of mAbs. Binding interactions were studied on the Blitz instrument. Protein G biosensors were used.
PubMed
Isolation and Optimization for Affinity and Biophysical Characteristics of Anti-CCL17 Antibodies from the VH1-69 Germline Gene
Kehoe JW, et al., Protein Eng Des Sel, doi: 10.1093/protein/gzu012, 2014
The authors described the screening of a targeted designed antibody library and the isolation and characterization of monoclonal variants with a high affinity to CCL17, a homeostatic chemokine associated with many human inflammatory diseases. Antibodies were characterized on the Octet system using Streptavidin biosensors.
PubMed
Sulfolobus replication factor C stimulates the activity of DNA polymerase B1
Xing X, et al., J Bacteriol, doi: 10.1128/JB.01552-14, 2014
The authors performed functional analysis on the replication factor C (RFC) of Sulfolobus solfataricus , a thermoacidophilic crenarchaeon to further elucidate the mechanism and role the RFC plays in the activation of DNA polymerase B and DNA replication. Binding studies were carried out on the Octet RED system and Streptavidin biosensors were used.
PubMed
Modulation of mAb Quality Attributes Using Microliter Scale Fed?batch Cultures
Rouiller Y, et al., Biotechnol Prog, doi: 10.1002/btpr.1921, 2014
The authors optimized a high-throughput process development method to test the effects of different experimental inputs (cell lines and media formulation) on both process performance and product quality of monoclonal antibody production to determine if earlier identification of cell lines and product can be made. Titer quantification of mAbs was performed on the Octet QKe using Protein A biosensors.
PubMed
Modulation of In Vivo IgG Crystallization in the Secretory Pathway by Heavy Chain Isotype Class Switching and N-linked Glycosylation
Hasegawa H, et al., Biochim Biophys Acta, 1843(7):1325-1338, 2014
The authors performed screening and functional analysis to isolate and better characterize the regulatory mechanism of a second human IgG2 that has been linked to hematological disorder and disease conditions when it accumulates intracellularly and forms crystalline bodies. Human IgG concentration was determined on an Octet RED96 system. Protein A biosensors were used.
PubMed
A Non-Active-Site SET Domain Surface Crucial for the Interaction of MLL1 and the RbBP5/Ash2L Heterodimer within MLL Family Core Complexes
Shinsky SA, et al., J Mol Biol, pii: S0022-2836(14)00156-9, 2014
The authors performed mutagenic analysis on mixed lineage leukemia-1 (MLL1) enzyme, a histone H3 lysine 4 monomethyltransferase which has been implicated in many human developmental disorders and cancers in order to better understand its mechanism of action and involvement with the SET1 family core complexes. Binding studies were performed on the Octet RED system and Streptavidin biosensors were used.
PubMed
Structural determinants for binding of Sorting Nexin 17 (SNX17) to the cytoplasmic adaptor protein Krev Interaction Trapped 1 (KRIT1)*
Stiegler AL, et al., J Biol Chem, pii: jbc.M114.584011, 2014
The authors performed biochemical, crystallographic and biophysical analysis to better characterize the interaction between the cytoplasmic adaptor proteins nexin 17 and KRIT1 and understand their roles in the endosomal sorting process. Mutagenesis and biochemical analysis were performed to pinpoint specific regions and motifs that are key sites for promoting interaction. Binding studies were performed on the Blitz system and Anti-GST biosensors were used.
PubMed
Structural and functional insight into TAF1-TAF7, a subcomplex of transcription factor II D
Bhattacharya S, et al., Proc Natl Acad Sci U S A, 111(25):9103-8, 2014
In this report, the authors performed detailed crystallography and biochemical functional analysis on TATA binding protein-associated factors 1 and 7 (TAF1 and TAF7) to better define how these two key subunit proteins of the transcription factor II D complex bind chromatin to control gene expression. Peptide binding studies were carried out on the Octet RED384 system. Streptavidin biosensors were used.
PubMed
Susceptibility to HLA-DM is determined by a dynamic conformation of major histocompatibility complex class II molecule bound with peptide
Yin L, et al., J Biol Chem, pii: jbc.M114.585539, 2014
The researchers performed mutational and functional analysis to demonstrate how the non-classical MHCII protein DM (HLA-DM) acts as a conformational editor to mediate the exchange of peptides loaded onto MHCII molecules during antigen presentation, a key step in the control of adaptive immune responses. Binding studies were performed on the Octet QK system and Streptavidin biosensors were used.
PubMed
Structural Insights into Regulatory Mechanisms of MO25-mediated Kinase Activation
Hao Q, et al., J Struct Biol, 186(2):224-33, 2014
The authors perform structural and functional analysis on MO25, an adapter molecule and key component in the LKB1-STRAD-MO25 complex that acts as an upstream kinase for AMPK family of kinases. The work described the evolution of different regulator mechanisms of kinase activation that MO25 has a role in. Kinetic binding studies were performed on the Octet RED96 system and Streptavidin biosensors were used.
PubMed
Blocking Interaction of Viral gp120 and CD4-expressing T Cells by Single-stranded DNA Aptamers
Zhao N, et al., Int J Biochem Cell Biol, 51C:10-18, 2014
The authors generated and screened single stranded DNA (SSDNA) aptamers using a systematic evolution of ligands by exponential enrichment (SELEX) approach and next generation sequencing. They further demonstrated that the SSDNA aptamers selectively bound CD4 on human cells and disrupted the interaction of viral gp120 to CD4 receptors, which is one of the prerequisite steps in HIV-1 infection and transmission. Binding characterization was performed on the Blitz system using Anti-His biosensors.
PubMed
Design, Synthesis, and Biological Evaluation of Novel FAK Scaffold Inhibitors Targeting the FAK-VEGFR3 Protein-protein Interaction
Gogate PN, et al., Eur J Med Chem., 80C:154-166, 2014
The authors designed, synthesized and screened for potent and selective tyrosine kinase inhibitors of focal adhesion kinase (FAK) and vascular endothelial growth factor receptor 3 (VEGFR3), two key modulators of survival and metastasis signaling in cancer cells. Binding studies were performed using Super Streptavidin biosensors and run on the Octet RED96 system.
PubMed
Identification of Human Neutralizing Antibodies Against MERS-CoV and their role in Virus Adaptive Evolution
Tang XC, et al., Proc Natl Acad Sci U S A, doi: 10.1073/pnas.1402074111, 2014
In this report, the authors screened a human Ab-phage library using a novel panning strategy to identify seven neutralizing antibodies against Middle East Respiratory Syndrome coronavirus (MERS-CoV) which causes severe respiratory disease with ~43% mortality. They further defined key residues that impacted receptor binding, viral fitness and had neutralizing effects leading to new possibilities for human mAb-based immunotherapies. Binding studies were performed on the Oct RED96 using Anti-FLAG biosensors.
PubMed
YlxM is a Newly Identified Accessory Protein that Influences Function of Signal Recognition Particle (SRP) Pathway Components in Streptococcus Mutans
Williams ML, et al., J Bacteriol, 196(11):2043-52, 2014
In this report, the authors focused on better understanding the key protein that regulates the signal recognition particle (SRP) pathway in Streptococcus mutans, a cariogenic oral pathogen that causes tooth decay and whose virulence is determined largely by its membrane composition. The protein YlxM was shown to be one of the important regulatory components of the SRP complex and has an role in modulating GTPase activity. Binding experiments were carried out on the Octet system. Streptavidin biosensors were used.
PubMed
Striatins Contain a Noncanonical Coiled Coil That Binds Protein Phosphatase 2A A Subunit to Form a 2:2 Heterotetrameric Core of Striatin-interacting Phosphatase and Kinase (STRIPAK) Complex
Chen C, et al., J Biol Chem, 289(14):9651-61, 2014
Striatins are novel regulatory subunits of protein phosphatase 2A (PP2A) that form supramolecular complexes called striatin-interacting phosphatase and kinase (STRIPAK) and have been shown to have a role in signal transduction, cytoskeleton dynamics, endocytosis and apoptosis. The authors performed the structural and biochemical studies on key residues of striatins to better understand the STRIPAK complex function. Binding studies were performed on the Octet RED96 system and Streptavidin biosensors were used.
PubMed
Development and Characterization of Monoclonal Antibodies Against Pancreatic Cancer Marker Hippocalcin-like 1 Protein
Chen Y, et al., Monoclon Antib Immunodiagn Immunother, 33(1):20-7, 2014
The authors performed structural and functional analysis and characterized two recombinantly generated monoclonal antibodies to hippocalcin-like 1 protein (HPCAL1), which can be used for early diagnosis screening of pancreatic cancer. Monoclonal antibody pairing studies were carried out on the Octet RED system using Protein A biosensors.
PubMed
Sensor Domain of Histidine Kinase KinB of Pseudomonas A HELIX-SWAPPED DIMER
Tan K, et al., J Biol Chem, 289(18):12232-44, 2014
The authors generated structural, mutagenesis, computational and biophysical data to examine the signaling molecules that interact with KinB, a histidine kinase that acts as a negative regulator of alginate biosynthesis in Psduodomas aeruginosa and to better understand the signal transduction mechanism. Binding studies were performed on the Octet RED system and Super Streptavidin biosensors were used.
PubMed
Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects
Shah NB and Duncan TM, J Vis Exp, (84):e51383, 2014
This video highlights the use of BLI to study inhibitory interactions of the subunit e with the catalytic complex of E. coli ATP synthase as a means to better generate new antibacterial drugs. Binding studies were performed on the Octet RED96 system and Streptavidin biosensors were used.
PubMed
Structure of MST2 SARAH Domain Provides Insights into Its Interaction with RAPL
Liu G, et al., J Struct Biol, 185(3):366-74, 2014
The authors performed structural and biochemical studies on the hetero-interaction and signaling of MST2, a STE20 kinase involved in the HIPPO pathway. Interaction as well as dimerization of MST2 is a catalyst to this kinases' apoptotic function in T cells. Kinetic analysis studies were performed using the Octet RED96 system with Streptavidin biosensors.
PubMed
Validation of Aspergillus Fumigatus Glucosamine-6-phosphate N-acetyltransferase as a Potential Antifungal Target
Lockhart D, et al., The Lancet, 383:S69, 2014
The authors described the screening of small-moleculae ligands that bind to the antifungal target glucosamine-6-phosphate N-acetyltransferase (GNA1) from Aspergillus fumigatus. They showed that GNA is essential for cell viability in A. fumigatus and could also be a possible novel antifungal target. Binding studies were performed on the Octet RED system.
The Lancet
Molecular Determinants of the Interaction Between Doa1 and Hse1 Involved in Endosomal Sorting
Han S, et al., Biochem Biophys Res Commun , 446(1):352-7, 2014
The authors performed structural and functional analysis on the yeast protein Doa1, which has a role in endosomal sorting via an association with Hse1, a component of the endosomal sorting complex required for transport (ESCRT) system. Studies demonstrated that hydrogen binding is a major determinant in the interaction of these two proteins. Binding association was performed using a Blitz system along with Ni-NTA biosensors.
PubMed
Tetraspecific Ligand for Tumor-targeted Delivery of Nanomaterials
Kim D, Friedman AD, and Liu R, Biomaterials, pii: S0142-9612(14)00389-5, 2014
The authors generated a recombinant targeting ligand with tetraspecificity for four different cancer biomarkers to demonstrate synergistic binding effects of the ligand to act on the multiple signaling pathways for different cancer types and also exhibit the potential of nanomaterials with a wider tumor targeting range, potency and efficiency. Binding affinities of the ligands tested were performed on the Octet QK system and Anti-hIgG Fc capture (AHC) biosensors were used.
PubMed
Receptor Binding by H10 Influenza Viruses
Vachieri SG, et al., Nature, 511(7510):475-7, 2014
The authors performed structural and functional analysis to better understand the binding and receptor interaction characteristics of the H10 influenza virus from avians and humans as the avian H10N8 virus has sufficient avidity for human receptors. However, it is the presence of mucins in the human airway and a lack of mutations to the H10 haemagglutinin that need to be constantly monitored to prevent transmission between humans. Binding studies were performed on the Octet RED system and Streptavidin biosensors were used.
PubMed
Polymeric Penetration Enhancers Promote Humoral Immune Responses to Mucosal Vaccines
Klein K, et al., J Control Release, 183:43-50, 2014
This report described the used of three commonly used penetration enhancers to promote trans-muscosal bioavailability of insulin and two vaccine candidates as biomarkers for mucosal absorption to test the theory that the mucosal immune responses is more efficiently activated after direct mucosal application compared to parenteral routes of vaccination. Binding interactions were carried out on the Blitz system and Amine reactive (ARG2) biosensors were used.
PubMed
Probing Structurally Altered and Aggregated States of Therapeutically Relevant Proteins using GroEL Coupled to Bio-Layer Interferometry
Naik S, et al., Protein Sci, doi: 10.1002/pro.2515, 2014
The authors described the functional analysis and characterization of several pharmaceutically relevant proteins used as models to test a novel methodology utilizing the protein chaperonin GroEL coupled with Bio-layer interferometery to detect structurally altered and preaggregate/aggregate formation in protein solutions during slightly elevated to moderate thermal stress conditions. Understanding and possibly controlling protein aggregation events during the development processes can be key to successful therapeutic generation and accurate characterization. Binding studies were performed on the Blitz system using Streptavidin biosensors.
PubMed
Selectively Targeting the DNA Binding Domain of the Androgen Receptor as a Prospective Therapy for Prostate Cancer
Dalal K, et al., J Biol Chem, pii: jbc.M114.553818, 2014
This study detailed the screening, identification and characterization of small molecules that are targeted to bind specifically to the androgen receptor DNA-binding domain (DBD) and abolish androgen receptor transcriptional activity without involving the ligand binding domain (LBD). This work suggests a new approach for the treatment of recurrent and metastatic prostate cancer. Binding studies using Streptavidin biosensors were performed on the Octet RED system.
PubMed
Production of Anti-idiotype Antibodies for Deoxynivalenol and their Evaluation with Three Immunoassay Platforms
Maragos CM, Mycotoxin Res, 30(2):103-11, 2014
The authors focused on developing anti-idiotypes for deoxynivalenol (DON) as a method to monitor mycotoxin and related tichothecenes in commodities and foods. The researchers compared three independent methods (ELISA-the gold standard method, FP and BLI) to determine if anti-idiotypes can replace DON in immunoassays for this toxin. Binding studies were performed on the Octet Red instrument
PubMed
DC-SIGN Plays a Stronger Role than DCIR in Mediating HIV-1 Capture and Transfer
Jin W, et al., Virology, 458-459:83-92, 2014
The authors performed a systematic study comparing the capability between two C-type lectin receptors, DC-SIGN and DCIR expressed in dendritic cells in HIV-1 capture and transfer. Both receptors bind to gp-120 and contribute to HIV-1 capture and transfer but their mechanisms of actions differ slightly providing insight into targets for intervention. Binding kinetics were determined using the Octet RED system along with Streptavidin biosensors.
PubMed
Peptide Microarrays Enable Rapid Mimotope Optimization for Pharmacokinetic Analysis of the Novel Therapeutic Antibody IMAB362
Schnatbaum K, et al., Biotechnol J, 9(4):545-54, 2014
This article described the screening of a peptide library for mimotopes that are detected by the novel cancer therapeutic antibody IMAB362. Mimotopes are candidate substitutions for target proteins that can be difficult to produce or handle and can be used for pre-clinical and clinical assay or for the purification of biological products. Binding studies were performed on the Octet RED system using Streptavidin biosensors.
PubMed
Emergence of Broadly Neutralizing Antibodies and Viral Co-evolution in Two Subjects During the Early Stages of Infection with the Human Immunodeficiency Virus Type 1
Sather DN, et al., J Virol, pii: JVI.01816-14, 2014
The study reported on the epitope response, characteristics and specificity of two HIV-1 positive patients over a several year period and how broadly neutralizing antibody activity evolved, developed and matured over time in these patients. Binding affinities were performed on the Octet QK system using Streptavidin biosensors.
PubMed
Seed-sequence Matched Controls Reveal Limitations of siRNA Knockdown in Functional and Structural Studies of HCV NS5A-MOBKL1B Interaction
Chung HY, et al., J Virol, pii: JVI.01582-14, 2014
The authors performed functional characterization of proteins that interact with the Hepatitis C virus (HCV) nonstructural protein N55A, a key therapeutic target to the HCV complex. Studies utilizing RNA interference mediated gene knockdown revealed off-target inhibitory effects on virus replication suggesting stricter controls for RNAi studies are needed to ensure more reliable results. Binding studies were performed on the Octet RED384 system.
PubMed
Isolation of Dengue Virus-specific Memory B Cells with Live Virus Antigen from Human Subjects Following Natural Infection Reveals the Presence of Diverse Novel Functional Groups of Antibody Clones
Smith SA, et al., J Virol, pii: JVI.00247-14, 2014
The authors performed a focused screening and characterization of inhibitory antibodies to dengue virus infection in humans. The results revealed that humans have the potential to generate very potent antibodies which are antigenic to diverse regions of the dengue virus surface protein. Binding studies were performed on the Octet RED system using Anti-mouse IgG FC (AMQ) biosensors.
PubMed
Heterogeneous IgG Interacts with FcRn and its Transport Across Gastrointestinal Barrier
Qiao D, et al., Food Agric Immunol, doi:10.1080/09540105.2014.918588, 2014
In this report, the authors further examined the application of bovine colostrus and milk immunoglobulin as a passive immunity against diseases in other species, especially humans. The work demonstrated that heterogenous IgG could be transported into the blood by Fc receptors (FcRn) and that the IgGs posse various affinities for human FcRn. Binding assays were performed on the Octet RED system and Streptavidin biosensors were used.
Taylor & Francis
The P2/P2' sites Affect the Substrate Cleavage of TNF- α Converting Enzyme (TACE)
Liu S, et al., Mol Immunol, 62(1):122-128, 2014
The authors performed computation and functional analysis of the P2 and P2' sites of the proteinase tumor necrosis factor-alpha converting enzyme (TACE) to better understand the enzymatic mechanisms, catalytic domains and substrate cleavage and provide insight in to the design of novel TACE inhibitors. Real-time binding assays were performed on the Blitz system and Streptavidin biosensors were used.
PubMed
Reducing Protein Adsorption with Polymer-grafted Hyaluronic Acid Coatings
Ramadan MH, et al., Langmuir, 30(25):7485-95, 2014
The authors described the modification of hyaluronic acid (HA) used for coating a broad range of biomaterial surfaces as a means to reducing protein adsorption and thereby generate more accurate test results. The preparation and characterization of HA was first monitored by chromatography and atomic force microscopy and confirmed by BLI analysis. Real-time binding experiments were performed on the Octet RED system. Aminopropylsilane (APS) biosensors were used.
PubMed
Global identification of CobB Interactors by an Escherichia Coli Proteome Microarray
Liu CX, et al., Acta Biochim Biophys Sin (Shanghai), 46(7):548-55, 2014
Described in this article was the screening of an E. coli proteome microarray to identify proteins that interact with the protein deacetylase CobB and to better understand its role in translational regulation. BLI was used to characterize the six lead candidates identified from the screen. Kinetic binding studies were performed on the Octet system and Streptavidin biosensors were used.
PubMed
Antibody Interactions with Ricinus Communis Agglutinins Studied by Biolayer Interferometry
Brandon D, et al., Anal Lett, doi:10.1080/00032719.2014.886693, 2014
The authors described the screening of several food matrices for mAbs to Ricin, a protein synthesized from castor plants that has been shown to have toxic effects. BLI was used to examine and confirm previously reported ELISA data on the binding characteristics of two ricin-specific mAbs suggesting this method is a potential immunodiagonostic for food matrices. Binding studies were performed on the Octet QK system. Amine-reactive (AR), Streptavidin and Anti-mouse Fc capture (AMC) biosensors were used in the experiments.
Taylor & Francis
Strategic Approaches for Assessment and Minimization of Matrix Effect in Ligand-binding Assays
Shih JY, Patel V and Ma M, Bioanalysis, 6(8):1103-12, 2014
The article examined the potential causes of matrix effects that potentially impact and influence the results of ligand binding assays when quantitating large molecule analytes in biological matrices. Understanding better the root causes of the matrix effect will allow for improved experimental design and data analysis. Binding studies were performed on the Octet RED384 system and Streptavidin biosensors were used.
PubMed
Vaccine-Induced HIV-1 Envelope gp120 Constant Region 1-Specific Antibodies Expose a CD4-Inducible Epitope and Block the Interaction of HIV-1 gp140 with Galactosylceramide
Dennison SM, et al., J Virol, 88(16):9406-17, 2014
The researchers presented data characterizing vaccine-induced antibodies that potentially block HIV-1 Env- galactosylceramide (Galacer) binding by perturbing HIV-1 Env conformation. Antibody identification was done using artificial membranes containing synthetic Galacer and recombinant HIV-1 Env proteins. Binding studies were performed on the Octet RED96 with Aminopropylsilane (APS) biosensors.
PubMed
Identification and Characterization of Human Rad51 Inhibitors by Screening of an Existing Drug Library
Normand A, Riviere E and Renodon-Corniere A, Biochem Pharmacol, pii: S0006-2952(14)00454-7, 2014
The authors screened the Prestwick library to identify small molecule candidates that will inhibit Rad51, a key protein involved in homologous recombination in eukaryotes and thought to be involved in chemo- and radio-resistance of human cancers by perturbing DNA repair processes. Binding of Rad51 to SS DNA was monitored using the Blitz system and ssDNA biosensors.
PubMed
Increasing Stability of Antibody via Antibody Engineering: Stability Engineering on an Anti-hVEGF
Wang S, et al., Proteins, doi: 10.1002/prot.24626, 2014
The authors designed and integrated computer-aided engineering protocols to optimize and improve antibody structure, expression, activity, specificity, thermo-stability and storage throughout the antibody development process. Affinities of the different antibody variants were tested on the Octet QKe system and Streptavidin biosensors were used.
PubMed
Diversity of the Murine Antibody Response Targeting Influenza A (H1N1pdm09) Hemagglutinin
Wilson JR, et al., Virology, 458-459:114-24, 2014
The authors used single-cell cloning of influenza hemagglutinin (HA)-specific B cells to assess the diversity and nature of the antibody response to influenza HA in mice to better understand the cross-reactivity, affinity and molecular nature of the influenza virus-antibody response. Binding studies were performed on the Octet RED system. Anti-Penta-his biosensors were used.
PubMed
Semi-continuous, Label-free Immunosensing Approach for Ca2+-based Conformation Change of a Calcium Binding Protein
Paek SH, et al., Analyst, 139(15):3781-9, 2014
The study detailed the use of BLI to directly measure the interaction of small molecules and calcium binding to a calcium-binding protein (CBP) designated GGBP for analysis of food samples. This novel and reproducible approach of label-free sensing of calcium levels specifically in milk samples suggests that food components can be analyzed with minimal handling and could possibly lead to further advances in automated analysis. Detection of calcium levels was performed on the Octet RED system using Anti-mouse Fc capture (AMC) biosensors.
PubMed
An EGFR Targeting Nanoparticle Self Assembled from a Thermoresponsive Polymer
Goodall S, et al., J Chem Technol Biotechnol, doi: 10.1002/jctb.4509, 2014
The authors described a novel method of creating a self-assembled immunoparticle using a high affinity antibody scFv that is capable of targeting multiple receptors and delivering therapeutic antibody fragments and other therapeutic agents. The study demonstrates an assembly of a thermoresponsive polymer and a portion of the anti-EGFR into a nanoparticle with target specificity. Binding affinities were analyzed on the Octet system using HIS2 biosensors.
Wiley
Gel Filtration Chromatography as a Method for Removing Bacterial Endotoxin from Antibody Preparations
London AS, et al., Biotechnol Prog, doi: 10.1002/btpr.1961, 2014
The authors presented a method for removing endotoxins from antibody preparations both in the presences and absence of aggregates using gel filtration chromatography. The removal of endotoxins from biological samples is important due to their potent biologic activities causing pyrogenic and shock reactions in animals. Secondary screening was performed on the Octet RED384 system.
PubMed
Ribosomal Protein S19 Binding Domain Provides Insights into Hantavirus Nucleocapsid Protein-mediated Translation Initiation Mechanism
Ganaie SS, et al., Biochem J, doi:10.1042/BJ20140449, 2014
The authors performed structural and functional analysis to identify and characterize the ribosomal protein S19 (RPS-19) binding domain in the nucleocapsid (N) protein of hantavirus, the RNA virus responsible for causing haemorrhagic fever and hantavirus cardiopulmonary syndrome. The results demonstrated the interaction of N-RPS19 as a novel target for therapeutic intervention of hantavirus diseases. Binding affinities were determined using the Blitz system along with Streptavidin biosensors.
PubMed
Potent Neutralization of Vaccinia Virus by Divergent Murine Antibodies Targeting a Common Site of Vulnerability in L1 Protein
Kaever T, et al., J Virol, pii: JVI.01491-14, 2014
In this report, the researchers developed and characterized potent neutralizing antibodies against vaccinia virus L1 protein, a key target in small pox vaccine. Multiple methodologies were used to provide a better understanding of the neutralizing epitopes on L1 and the neutralizing mechanizing of anti-L1 antibodies. Real time binding assays were performed using the Octet RED96 instrument and Ni-NTA (NTA) biosensors.
PubMed
Shark Attack: High affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation
Zielonka S, et al., J Biotechnol, pii: S0168-1656(14)00204-1, 2014
The authors described the use of yeast surface display library screening to generate and identify high-affinity binders to antigen-specific shark vNAR (new antigen receptor) domains in vitro. They demonstrated specific binding of the antibodies without compromising the desirable high thermostability of the vNAR scaffold. Binding kinetics were determined on the Octet RED96 system using Streptavidin biosensors.
PubMed
Pharmacologic Profile of the Adnectin BMS-962476, a Small Protein Biologic Alternative to PCSK9 Antibodies for Low-Density Lipoprotein Lowering
Mitchell T, et al., J Pharmacol Exp Ther, 350(2):412-24, 2014
The authors performed structural and functional characterization on a new class of high affinity binding agents known as adnectins which interact with the proprotein convertase subtilisin/kexin type 9 (PCSK9) protein to regulate LDL levels. Adnectins exhibited many of the functional properties of inhibitory mAb to PCSK9 but their molecular size is in between that of peptides and immunoglobulins. The binding studies were performed on the Octet RED 96 system using Super Streptavidin biosensors.
PubMed
Antibody-based Prevention of von Willebrand Factor Degradation Mediated by Circulatory Assist Devices
Rauch A, et al., Thromb Haemost, 112(5), 2014
This report described the screening of protein fragments and monoclonal murine antibodies that interact with von Willebrand factor (VWF), a glycoprotein that has a key role in the formation of platelet-rich thrombin, particularly under conditions of high shear stress. The goal was to develop a strategy to treat bleeding episodes due to shear stress-dependent VWF degradation. Equilibrium binding assays were performed using the Octet QK system. Protein A-coated biosensors were used.
PubMed
A Common Solution to Group 2 Influenza Virus Neutralization
Friesen RH, et al., Proc Natl Acad Sci U S A, 111(1):445-50, 2014
In this study, the authors performed structural and functional analysis including electronic microscopy, mutational analysis, ELISA and FACS binding on a newly identified (only the second one) human group 2 type broadly neutralizing antibody (bnAb) to Influenza A viruses. This new bnAb specifically targets residues at the base of the stem region in the major surface glycoprotein hemagglutinin (HA). Binding studies were performed on the Octet RED system to determine
KD values using Streptavidin biosensors. This study provides insight into group 2 bnAbs and the potential rationale in designing broader spectrum influenza vaccines.
PubMed
Human Antibody Responses to Avian Influenza A (H7N9) Virus, 2013
Guo L, et al., Emerg Infect Dis, 20(2):192-200 , 2014
The authors performed biochemical analysis on a series of human patient samples to evaluate antibody responses against the influenza A (H7N9) virus to better understand the vaccine responses to influenza virus infection and viral pathogensis. The binding activity of three recombinantly generated hemagglutinins were confirmed using the Octet system prior to performing ELISA analysis. The ability to appropriately screen the antibody response to influenza A virus becomes critical in developing vaccines to provide immunity.
PubMed
Motif-directed Redesign of Enzyme Specificity
Borgo B and Havranek JJ, Protein Sci, 23(3):312-20, 2014
A detailed computational sequence analysis examining specific residues and motifs of a methoionine aminopeptidase (eMAP) from E. coli was performed in attempts to better understand the specificity and binding activity of this enzyme. The goal was to switch the specificity from N-terminal methionine to N-terminal leucine using predicted motif-based redesign. Binding kinetics on the native and engineered proteins were performed on the BLItz system using Amine-Reactive biosensors. This motif-based computational design combined with a focused functional study provides protein engineers with new tools to more rapidly develop and test enzyme model systems.
PubMed
The Sphingolipid Receptor S1PR2 is a Receptor for Nogo-A Repressing Synaptic Plasticity
Kempf A, et al., PLoS Biol, 12(1):e1001763, 2014
This study describes how the authors identified and characterized a new G-protein Coupled Receptor (GPCR), S1PR2 which binds to Nogo-A, a membrane protein of the central nervous system (CNS) that possibly has a role in inhibiting nerve fiber regeneration of the CNS after injury. Kinetic binding studies were performed on the Octet RED system using Amine-Reactive biosensors. The results of the study suggest a more dynamic model of GPCR signaling that involves shared membrane components interacting and intramolecular cross talk to control regulation.
PubMed
The Carboxy-terminus of BAK1 Regulates Kinase Activity and is Required for Normal Growth of Arabidopsis
Oh MH, et al., Front Plant Sci, 5:16, 2014
The authors performed structural and functional analysis on the interaction of the brassinosteroid-insensitive 1 (BRI1) receptor kinase and its co-receptor BRI1-associated receptor kinase (BAK1) in order to better understand the plant receptor kinase signaling process as well as the control mechanisms of Arabidopsis growth. Binding studies were performed using the Octet QK system with Streptavidin biosensors to further elucidate the binding kinetics and characteristics of these two targets and the key role the carboxy terminal domain plays in the binding and phosphorylation events.
PubMed
High Temperature SELMA: Evolution of DNA-Supported Oligomannose Clusters Which Are Tightly Recognized by HIV bnAb 2G12
Temme JS, et al., J Am Chem Soc, 136(5):1726-9, 2014
SELMA (SELection with Modified Aptamers) was performed using a modified protocol at 37°C to screen and identify glycocluster clones that exhibit strong target recognition to the neutralizing HIV antibody 2G12. Interestingly, the binding affinity of the synthetic glycocluster constructs are as strong as the HIV envelope protein gp120 yet contain just 3-4 glycans. The binding kinetics of the screened constructs was performed on the BLItz system, using Streptavidin biosensors.
PubMed
Lycium Barbarum Polysaccharide LBPF4-OL may be a New Toll-like Receptor 4/MD2-MAPK Signaling Pathway Activator and Inducer
Zhang XR, et al., Int Immunopharmacol, 19(1):132-141, 2014
The authors report on the first demonstration that Lycium barbarum polysaccharide (LBP) stimulates tumor necrosis factor-alpha (TNF-α) and IL-1β generation and promotes lymphocyte proliferation in the Toll-like Receptor 4 (TLR 4)/MD2-MAPK signaling pathway. LBP is the main active ingredient in L. barbaum has been reported to have immunoregulation and antitumor effects. Kinetic binding experiments were performed on the Octet RED system using Anti-Human IgG FC Capture (AHC) biosensors.
PubMed
Peptide Inhibitor of Japanese Encephalitis Virus Infection Targeting Envelope Protein Domain III
Zu X, et al., Antiviral Res, 104C:7-14, 2014
In this study, the authors screened a phage display library and identified a unique peptide designed "P3" that specifically binds and interacts with the Japanese encephalitis virus (JEV) Domain III envelope glycoprotein (E DIII) specifically at the N terminus to block virus attachment to the cell and inhibit viral infection. This is the first report of a peptide which binds to sites on JEV E DIII but not cellular receptors to interfere with JEV infection. Binding analysis was performed on the Octet QK system.
PubMed
Multiplexed Screening of Natural Humoral Immunity Identifies Antibodies at Fine Specificity for Complex and Dynamic Viral Targets
McCutcheon KM, et al., Mabs, 6(2):460-73, 2014
The authors performed a high throughput multiplexed antigen screen of memory B cell from immune individuals to identify potential antibodies that have a neutralizing effect on viral epitopes. The report describes optimization of the screening method coupled with in vivo experiments as a direction to better design of optimal vaccine antigens. Kinetic binding and affinity studies on recombinant antibodies were performed on the Octet QK system using Human IgG FC capture (AHC) biosensors.
PubMed
The Yeast Ess1 Prolyl Isomerase Controls Swi6 and Whi5 Nuclear Localization
Atencio D, et al., G3 (Bethesda), pii: g3.113.008763v1, 2014
This study outlines the genetic screening and functional binding analysis of the Ess1 prolyl isomerase in Saccharomeyces cerevisiae to better understand the mechanism of how this protein influences RNA polymerase II activity, transcriptional regulation and chromatin modification. Kinetic binding studies were performed on the Octet RED system using Streptavidin biosensors to analyze Ess1 binding and isomerization of phosphor-Ser-Pro motifs resulting in conformational changes that impact nuclear import and export of cell-cycle control genes.
PubMed
PA Tag: A Versatile Protein Tagging System Using a Super High Affinity Antibody Against a Dodecapeptide Derived from Human Podoplanin
Fujii Y, et al., Protein Expr Purif, 95:240-7, 2014
The authors describe the development of a novel affinity tagging system utilizing a unique mAb NZ-1 that is directed against human podoplanin and has an unusually high affinity toward linear dodecapeptide antigen designated "PA". The system has the abililty to purify low abundant proteins at high yield in one step along with its ability for extended reusability, therefore providing new opportunities for protein purification in many biomedical research areas. Binding kinetics were determined on the Octet RED system using Amine Reactive (AR2G) biosensors.
PubMed
A Dynamin Homolog Promotes the Transition from Hemifusion to Content Mixing in Intracellular Membrane Fusion
Kulkarni A, et al., Traffic, 15(5):558-71, 2014
Biochemical analysis of the dynamin homolog-Vps 1 (Vacuolar protein sorting 1) in yeast was performed to characterize the role of this protein on the lipid mixing and content mixing properties of yeast vacuoles and the incorporation of Soluble N-ethylmaleimide sensitive factor Attachment Protein Receptor (SNAREs) into fusogenic complexes. Binding kinetics studies for Vps 1 were performed on the Octet RED96 system. These studies provide further insight into the role of dynamin function in SNARE mediated fusion and fission equilibrium and regulation in yeast.
PubMed
Super Spy Variants Implicate Flexibility in Chaperone Action
Quan S, et al., Elife, 3:e01584, 2014
Genetic screening was performed to isolate variants of the Spy chaperone that better stabilize poorly folded protein clients and prevent degradation in vivo while also having an improved ability to prevent aggregation of proteins in vitro. Termed "super-Spy", the isolated variants examined in binding studies using the Octet RED system clearly demonstrated a stronger affinity to client proteins than the WT form and provided insight into better understanding chaperone function and protein degradation. Streptavidin biosensors were used.
PubMed
Dynamic Evolution and Immunoreactivity of Aptamers Binding to Polyclonal Antibodies Against MPT64 Antigen of Mycobacterium Tuberculosis
Qin LH, et al., Eur J Clin Microbiol Infect Dis, 33(7):1199-209, 2014
In this study, the authors systematically monitored the selection process of aptamers, short single-stranded nucleic acids with high affinity to target molecules, in this case specifically against anti-MPT64 antibodies of Mycobacterium tuberculosis as potential substitutes in the serological diagnosis of TB. The binding affinity of candidate aptamers and the dissociation constant of preponderant aptamers were tested on the Octet platform using Streptavidin biosensors.
PubMed
In Vitro Functional Characterization of Feline IgGs
Strietzel C, et al., Vet Immunol Immunopathol, 158(3-4):214-23, 2014
The authors performed structural and functional analysis to characterize the feline IgG heavy chain constant regions (IGHC) to better understand the pharmacokinetics (PK) and pharmacodynamics (PD) of the IgG subclasses with a vision to develop monoclonal antibody therapeutics to address unmet medical needs for cats. Binding studies were performed on the Octet QK system using Protein A and Protein G biosensors.
ScienceDirect
Discovery of Potent Keap1-Nrf2 Protein-Protein Interaction Inhibitor Based on Molecular Binding Determinants Analysis
Jiang ZY, et al., J Med Chem, 57(6):2736-45, 2014
Structural and functional analysis were performed on a newly identified a potent small molecule E3 ligase inhibitor designated compound 2, which directly targets protein-protein interactions and provides a strategy to develop a novel class of antioxidant, anti-inflammatory and anti-cancer agents. Binding interactions of ligands and the Keap1 DC domain protein were performed on the Octet RED96 system using Super Streptavidin biosensors.
PubMed
Crystal Structure and Biochemical Analysis of the Heptameric Lsm1-7 Complex
Zhou L, et al., Cell Res, 24(4):497-500, 2014
The authors present binding affinity results of the Lsm1-7, a heptameric protein complex that functions in mRNA degradation pathway regulation. A crystallized structure of the S. cerevisiae Lsm1-7 complex components were determined and using the Octet system the differential binding affinity and specificity to oligo repeat sequences were analyzed. This work summarizes the LSM1-7 RNA-binding properties as demonstrated by mutational analysis and provides a better understanding of the mechanistic regulation in RNA metabolism.
PubMed
A Phospha-oseltamivir-biotin Conjugate as a Strong and Selective Adhesive for the Influenza Virus
Streicher H, et al., Bioorg Med Chem Lett, doi: 10.1016/j.bmcl.2014.02.021, 2014
A new compound was identified that is a derivative of a surface neuraminidase that can directly and effectively immobilize the influenza virus with an apparent equilibrium dissociation constant in the low picomolar range. Immobilization of the virus is a key step in diagnosis and influenza vaccine production and has been hindered by the current processes using antibody and sialic acid binding tests. Binding of neuraminidase and virus to the compound were performed on the Octet RED using Streptavidin biosensors. The detected high affinity and slow off-rate of the new compound suggest it is highly suitable for virus particle immobilization studies.
ScienceDirect
Phosphorylation of Sli15 by Ipl1 Is Important for Proper CPC Localization and Chromosome Stability in Saccharomyces cerevisiae
Makrantoni V, et al., PLoS One, 9(2):e89399, 2014
In this study, the authors performed extensive point mutational analysis of the yeast Sli 15 protein, a key component of the chromosomal passenger complex (CPC) to better understand the mechanisms regulating eukaryotic cell division. Studies utilizing the Octet RED384 and Streptavidin biosensors to specifically examine Sli15 (wild-type compared to a series of 15 different mutant forms) binding affinity to microtubules. This work suggests that Sli 15 may play an important role with microtubules in tension sensing as opposed to chromosome biorientation.
PubMed
Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking
Rice EA, et al., PLoS One, 9(4):e94238, 2014
DNA and protein interaction and binding studies were performed to characterize ATHB17, a homeodomain leucine zipper class II (HD-ZIP II) transcriptional repressor in Arabidopsis which contains a newly identified EAR-like motif that requires dimerization for high affinity DNA binding and repression activity and ultimately plant growth and development. DNA binding experiments were performed on the Octet QK system using Streptavidin biosensors.
PubMed
A Neutralizing Anti-gH/gL Monoclonal Antibody is Protective in the Guinea Pig Model of Congenital CMV Infection
Auerbach MR, et al., PLoS Pathog, 10(4):e1004060, 2014
The authors performed structural and functional analysis on a neutralizing monoclonal antibody targeting an guinea pig envelope cytomegalovirus (CMV) glycoprotein H/glycoprotein L that can protect the fetus from infection. Binding of antibodies to guinea pig FcRn complexes was performed on the Octet QK system, using Amine-Reactive biosensors. The guinea pig is a model system for maternal-fetal transmission and the studies may provide insight into therapeutic intervention of neutralizing mAbs for CMV congenital infections in humans.
PubMed
Imperfect Centered miRNA Binding Sites are Common and Can Mediate Repression of Target mRNAs
Martin HC, et al., Genome Biol, 15(3):R51, 2014
Structural and functional studies were carried out to identify the targets of 10 human microRNAs (miRNAs), which are small non-coding RNAs that regulate protein-coding genes in association with the RNA-induced silencing complex (RISC) and have a key role in transcriptional regulation. Biotin pull-down experiments showed that the highly conserved central regions of the different miRNAs have a role in regulating the binding/interaction with mRNAs and could explain the evolutionary conservation for this region and examining the mechanism of transcriptional regulation and evaluating disease-causing mRNA variants. The Octet RED system was used to examine the association and disassociation profiles of the miRNAs and Streptavidin biosensors were used.
PubMed
Pharmacological Characteristics and Efficacy of a Novel Anti-angiogenic Antibody FD006 in Corneal Neovascularization
Wang Q, et al., BMC Biotechnol, 14:17, 2014
The authors examined the pharmacological characteristics and efficacy of FD006, a novel anti-vascular endothelial growth factor (VEGF) that can regulate physiologic and pathologic angiogenesis specifically acting to inhibit corneal neovascularization. The studies demonstrate that FD006 has a higher affinity and better anti-neovascularization function than the well-known VEGF inhibitor becvacizumab/Avastin suggesting that it may be a promising agent in the treatment of corneal neovascularization in the near future. The binding kinetics of FD006 and bevacizumab to VEGF were performed on the Octet RED system, using Anti-Human IgG biosensors.
PubMed
Peptide Microarrays Enable Rapid Mimotope Optimization for Pharmacokinetic Analysis of the Novel Therapeutic Antibody IMAB362
Schnatbaum K, et al., Biotechnol J, 9(4):545-54, 2014
IMAB362 is a therapeutic antibody used to treat gastro-esophageal and stomach cancer. The authors used a combination approach of phage display library and microarray screening to identify peptide candidates to IMAB362. Detailed kinetic binding studies of selected peptide variants were performed on the Octet RED system using Streptavidin biosensors and showed consistent and supportive results to the ELISA data to pinpoint key interactive sequences. This approach using a combination of kinetic and binding assays resulted in improved and more efficient SAR optimization of mimotopes and informative pharmacokinetic studies on IMAB362 in a mouse test system.
PubMed
Potent Dengue Virus Neutralization by a Therapeutic Antibody with Low Monovalent Affinity Requires Bivalent Engagement
Edeling MA, et al., PLoS Pathog, 10(4):e1004072, 2014
In this study, the authors performed analysis to better understand the structural and mechanistic basis for the neutralization activity of E106, a monoclonal antibody which protected against the lethal Dengue virus type 1 infection in mice by bivalent engagement of the adjacent domain III subunits on a single virion. The binding kinetics of the mAB and Fabs were performed on the Octet RED system using Streptavidin biosensors. These studies shed new insight into the humoral response against flaviviruses.
PubMed
Adhiron: A Stable and Versatile Peptide Display Scaffold for Molecular Recognition Applications
Tiede C, et al., Protein Eng Des Sel, 27(5):145-55, 2014
The authors performed structural and functional analysis of a new non-antibody artificial binding protein scaffold designated Adhiron, that was derived from modifications on the consensus sequence of the plant-derived phytocystatin, an protein inhibitor of cysteine proteases. To determine the use of adhirons as research reagents, binding to SUMO protein and as well as various peptide substrates were tested and binding affinities were determine on the BLItz system as a confirmation of the affinities measured by isothermal titration calorimetry. Streptavidin biosensors were used. This work validated the use of the BLItz system for rapid estimation of binding affinity ranges of Adhiron and suggests and efficient method to better drug design.
PubMed
Therapeutic Effect of IVIG on Inflammatory Arthritis in Mice is Dependent on the Fc Portion and Independent of Sialylation or Basophils
Campbell IK, et al, J Immunol, 192(11):5031-8, 2014
The authors performed intravenous IG (IVIG), a common treatment for different chronic and acute autoimmune and inflammatory diseases and demonstrated in two different mouse inflammatory arthritis models that IVIG was protective in a dose-dependent manner and that the protective activity was contained within the Fc fragment and not the F(ab′)2 fragments. The integrity of the lectin-purified Fc molecules and their binding ability was confirmed on the Octet QK platform, along with ELISA experiments.
PubMed
Enhanced Human Receptor Binding by H5 Haemagglutinins
Xiong X, et al., Virology, 456-7:179-187, 2014
Structural and biochemical functional studies were performed on the virus membrane glycoprotein, Haemagglutinin (HA) as well as a number of H5 virus mutants isolated from humans in two geographically distinct regions to demonstrate the differences in receptor binding properties. Detailed binding studies were performed on the Octet RED system using Streptavidin biosensors to examine 12 different wildtype and mutant/double H5 mutant variants and further characterized using X-ray crystallography. Three specific substituted residues in the characterized mutants were shown to dramatically change the avidity for the human vs. avian receptor by affecting the receptor binding pocket as oppose to direct interaction with the receptor. These studies provide additional insight into the evolution of HA receptor binding properties during H5 virus infection of humans.
Virology
Microbial Strain Development Using BioLayer Interferometry
Cantin G, BioProcess International, 12(4):54-57, 2014
The author described a case study for rapid screening of high-producing production strains for expression of granulocyte colony stimulating factor (G-CSF) clones in E. Coli. The Octet platform was used to efficiently screen and quantitate the binding characteristics of the expression clones. Streptavidin biosensors were used. BLI can be used for rapid protein measurement, titer determination, as well as functional analysis applications for a diverse variety of protein types.
BioProcess International
High-Throughput Epitope Binning Assays on Label-Free Array-Based Biosensors can Yield Exquisite Epitope Discrimination that Facilitates the Selection of Monoclonal Antibodies with Functional Activity
Abdiche YN, et al., PLoS One, 9(3):e92451, 2014
Epitope binning assays were performed on large panels of monoclonal antibodies (mAbs) to demonstrate similar epitope profiles correlate to functional activity. Two separate label-free technologies were tested and showed very similar binding characterizations with each technology exhibits its own benefits. The Octet HTX, RED384 and QK384 platforms were used to characterize the binding affinities of mAbs. Amine-Reactive, Streptavidin and anti-species specific biosensors were used in the studies. The experiments demonstrate that label-free epitope binning assays can be performed at both high throughput and high-resolution to further support the discovery and development of therapeutic mAbs.
PubMed
The Histone Chaperones Vps75 and Nap1 Form Ring-like, Tetrameric Structures in Solution
Bowman A, et al., Nucleic Acids Res, 42(9):6038-51, 2014
Biochemical and biophysical techniques were used to examine the structure and function of Vps75 and related Nap 1, a yeast histone chaperone that forms homodimeric tetramers as part of its mechanism in regulating nucleosome assembly and disassembly as well as transcription. The Octet RED384 system was used to calculate the
KD for tetramerization formation. Streptavidin biosensors were used. A molecular model for Vps75/NAP1 was determined using pulse electron-electron double resonance (PELDOR), small angle X-ray scattering (SAXS), multi-angle light scattering (MALS) and structure-guided mutagenesis showing the protein forms discrete tetrameric conformation under physiological ionic conditions.
PubMed
Clearance Kinetics and Matrix Binding Partners of the Receptor for Advanced Glycation End Products
Milutinovic PS, et al., PLoS One, 9(3):e88259, 2014
The authors performed studies examining protein clearance kinetics and matrix binding protein interactions of the receptor for advanced glycation end products (RAGE), which is known to be a key player in numerous disease processes such as cardiomyopathy, inflammation, neurodegeneration and tumor invasion. Binding kinetics of RAGE to various matrix proteins were performed on the Octet system using Amine-Reactive biosensors.
PubMed
FRET-based System for Probing Protein-protein Interactions between σR and RsrA from Streptomyces Coelicolor in Response to the Redox Environment
Wei ZH, et al., PLoS One, 9(3):e92330, 2014
The authors examined the protein-protein interaction between sR and RsrA, two key analytes that trigger the cellular redox response effected by zinc binding and response to peroxide street (ROS) in the Gram Positive bacterium S. coelicolor. The Octet QK system was used to examine and confirm the binding kinetics of two proteins which were further characterized by fluorescence resonance energy transfer (FRET) studies. Streptavidin biosensors were used.
PubMed
The Human Orphan Nuclear Receptor Tailless (TLX, NR2E1) is Druggable
Benod C, et al., PLoS One, 9(6):e99440, 2014
In this study, the authors performed structural and functional analysis identifying three novel small molecules (ccrp 1, ccrp 2 and ccrp 3) that bind Tailless (TLX), a Human orphan nuclear receptor previously thought to have no ligands and therefore was difficult to characterize. The binding studies were carried out on the Octet RED384 system using Super-Streptavidin (SSA) biosensors. The results demonstrated that the ligands bind to TLX, and they enhance TLX repressive actions thereby providing insight into characterizing the receptor and dissecting TLX dependent regulatory pathways.
PubMed
Enhancement of Tumor-specific T Cell-mediated Immunity in Dendritic Cell-based Vaccines by Mycobacterium Tuberculosis Heat Shock Protein X
Jung ID, et al., J Immunol, 193(3):1233-45, 2014
This report details the use of Heat Shock Protein (Hsp) X from Mycobacterium tuberculosis as a potent and strongly selective agonist for CTLs and is highly effective in the induction of T cell immunity as demonstrated by binding to pattern recognition receptor 4 (TLR4) and activating the TLR4 signaling pathway in dendritic cells (DC) in both in vitro and in vivo studies. These results suggest that Hsp X could be a potential adjuvant for dendritic cell-based antitumor immunotherapies. Binding studies were performed on the BLItz system using HIS biosensors.
PubMed
Construction of pH-sensitive Her2-binding IgG1-Fc by Directed Evolution
Traxlmayr MW, et al., Biotechnol J, 9(8):1013-22, 2014
The authors utilized an evolutionary design strategy and a yeast display library to screen for crystallizable fragments of immunoglobulin G1 (Fcab) that were engineered for pH-dependent antigen binding as a means of generating more effective therapeutic proteins that possess longer half-lives. Binding studies were performed on the Octet QK system using Streptavidin and Protein A biosensors.
PubMed
Improving the Anti-Toxin Abilities of the CMG2-Fc Fusion Protein with the Aid of Computational Design
Xi Y, et al., PLoS One, 9(8):e104674, 2014
Computational design and functional analysis was employed in a rat model system to generate a capillary morphogenesis protein 2(CMG2) fusion protein with improved protein-binding affinity and therapeutic efficacy to anthrax infection. Binding kinetics by the mutant fusion proteins for the protective antigen (PA) were performed on the Octet QK system using Anti-Human IgG Fc (AHC) biosensors.
PubMed
A Novel VHH Antibody Targeting the B Cell-Activating Factor for B-Cell Lymphoma
Wu W, et al., Int J Mol Sci, 15(6):9481-96, 2014
The study describes the generation of single-domain anti-B cell activating factor (BAFF) camel antibodies from heavy chain variable domain phage display libraries as well as the panning, expression and functional characterization of the antibodies to understand their specificity to BAFF. The antibodies identified provide new options for targeting tumors for B cell lymphoma diagnosis or therapy. The relative binding affinities of the different sdAb against BAFF was performed on the Octet RED system using Streptavidin biosensors.
PubMed
Predicting the Origins of Anti-blood Group Antibody Specificity: A Case Study of the ABO A- and B-antigens
Makeneni S, et al., Front. Immunol., doi: 10.3389/fimmu.2014.00397, 2014
The authors performed detailed structural and functional analysis on a monoclonal antibody raised against blood group A (BGA) antigen which complexes with scFv AC1001 in order to better understand the origin of antigenicity (the specificity and affinity) in ABO blood typing. Computer and theoretical modeling, crystallization studies, array screening and function binding analysis were employed to better understand the origin of antibody-carbohydrate specificity. Binding studies were performed on the Octet RED96 system using Amine Reactive Second-Generation (AR2G) biosensors.
Frontiers
Application of Docking Methods for Metal Chelate Affinity Precipitation of Endo-glucanase Using pH-response Polymer
Ding Z, Kang L, and Cao X, Colloids Surf B Biointerfaces, 113C, 412-420, 2014
The authors report on molecular simulation docking methods for the identification of an optimal chelator/metal ion system for metal chelate affinity purification on endo-glycanase. Experiments on the Octet system with Super Streptavidin biosensors were performed to confirm the predictions of the docking calculations.
PubMed
Kinetics of Bioconjugate Nanoparticle Label Binding in a Sandwich-type Immunoassay
Näreoja T, et al., Anal Bioanal Chem, 406(2),493-503, 2014
This study investigates the kinetics of nonspecific interactions of typical antibody-functionalized nanoparticles in a well-plate assay, in a BLI assay, and in a dynamic force microscopy setup. The Octet RED384 system and Streptavidin biosensors were used to characterize TSH-nanoparticle binding kinetics.
PubMed
Structural and Functional Analysis of G Protein-coupled Receptor Kinase Inhibition by Paroxetine and a Rationally Designed Analog
Homan KT, et al., Mol Pharmacol, 85, 237-248, 2014
With the long-term goal of developing more selective inhibitors of G protein-coupled receptor kinase 2 (GRK2), the authors investigated the molecular basis for the selectivity of paroxetine for GRK2 by directly determining the affinity of paroxetine for various GRKs, its inhibition constants and mechanisms of inhibition for GRK1 and GRK2, and its atomic structure in complex with GRK1, the GRK most weakly inhibited by paroxetine. The Octet RED system and Streptavidin biosensors were used with biotinylated GRKs to characterize the binding of small molecule GRK inhibitors.
PubMed
Identification of a Small Peptide that Inhibits PCSK9 Binding to the Low Density Lipoprotein Receptor
Zhang Y, et al., J Biol Chem, 289, 942-955, 2014
To identify a peptidic PCSK9 inhibitor, the authors screened phage-displayed peptide libraries and identified the 13-amino acid peptide, Pep2-8. Structural and biochemical characterization studies provided molecular details of its inhibitory mechanism. Affinities for Pep2-8_Fc and Pepctrl_Fc binding to PCSK9 were determined using the Octet RED384 system equipped with Anti-hIgG-Fc Capture biosensors loaded with Pep2-8_Fc or Pep-ctrl_Fc.
PubMed
Soluble Production of a Biologically Active Single-chain Antibody Against Murine PD-L1 in Escherichia Coli
Drees JJ, et al., Protein Expr Purif, 94, 60-66, 2014
Programmed death ligand 1 (PD-L1) is an important regulator of T-cell activation and has emerged as an important target for cancer immunotherapy. In this study, the authors constructed an immunized chicken scFv library and used phage display to isolate a biologically active, high affinity antagonistic scFv against mouse PD-L1. Binding studies with the scFv were performed using the Octet QK
e system and Anti-Human Fc Capture biosensors loaded with PD-L1-Fc.
PubMed
Bisubstrate UDP-peptide Conjugates as Human O-GlcNAc Transferase Inhibitors
Borodkin VS, et al., Biochem J, 457, 497-502, 2014
This study reports on the rational design of new bisubstrate inhibitors of O-GlcNAc transferase (OGT) that combine elements of both substrates. The Octet RED384 system was used to measure binding affinities of inhibitors for hOGT, with biotinylated hOGT loaded onto Super Streptavidin biosensors.
PubMed
Conformation-dependent High-affinity Potent Ricin-neutralizing Monoclonal Antibodies
Hu W, et al., Biomed Res Int, 2013:471346, 2013
Altering Drug Tolerance of Surface Plasmon Resonance Assays for the Detection of Anti-drugantibodies
Barbosa M, et al., Anal Biochem, 441(2):174-9, 2013
Shark Variable New Antigen Receptor (VNAR) Single Domain Antibody Fragments: Stability and Diagnostic Applications
Griffiths K, et al., Antibodies, 2(1), 66-81, 2013
A Protein Engineering of Bacillus Thuringiensis δ-Endotoxin by Conjugating with 4"-O-succinoyl Abamectin
Pan Z, et al., Int J Biol Macromol, 62, 211-6, 2013
In this study, Bt toxin was conjugated with 4-O-succinoyl abamectin to construct a new biocide (BtA). The Octet RED system was used to investigate binding of BtA to its receptors in the brush border membrane vesicles. Biotinylated Bt toxin and BtA were loaded onto Streptavidin biosensors for binding studies.
PubMed
Robust Neutralizing Antibodies Elicited by HIV-1 JRFL Envelope Glycoprotein Trimers in Nonhuman Primates
Chakrabarti BK, et al., J Virol, 87(24), 13239-51, 2013
This study explores DNA priming followed by a rest interval and protein boosting to present JRFL Env spikes to the immune system to better generate HIV-1 cross-clade neutralizing antibodies. Kinetic binding measurements of JRFL gp140-F trimers with selected monomeric Fabs were performed using the Octet RED system. JRFL gp140-F trimers were immobilized on Amine Reactive (AR2G) biosensors for these experiments.
PubMed
Glycan-dependent and Independent Interactions Contribute to Cellular Substrate Recruitment by Calreticulin
Wijeyesakere S, Rizvi S and Raghavan M, J Biol Chem, 288(49), 35104-35116, 2013
The authors investigated different modes of calreticulin-substrate binding, and report that calreticulin binds glycosylated and non-glycosylated proteins with similar affinities but distinct kinetics and P-domain conformations. The Octet RED system and Streptavidin biosensors were used to characterize calreticulin interactions with biotin-labeled glycosylated and non-glycosylated substrates.
PubMed
A Neutralizing Monoclonal Antibody Targeting the Acid-sensitive Region in Chikungunya Virus E2 Protects from Disease
Selvarajah S, et al., PLoS Negl Trop Dis, 7(9):e2423, 2013
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has produced up to 6.5 million cases of acute and chronic rheumatic disease. The authors describe the isolation and characterization of two human monoclonal antibodies, C9 and E8, from CHIKV infected and recovered individuals. C9 was determined to be a potent virus neutralizing antibody. The Octet RED system and AR2G biosensors were used to characterize binding of C9 to intact CHIKV VLPs.
PubMed
Recognition of Sulphated and Fucosylated Receptor Sialosides by A/Vietnam/1194/2004 (H5N1) Influenza Virus
Xiong X, et al., Virus Res, 178(1), 12-14, 2013
This study examines the nature of the interaction of human H5 virus HA with four related Gal2 beta1-4GlcNAc3-linked sialoside receptor analogues. Affinities of the H5 virus for different receptor analogues were estimated using the Octet RED system.
PubMed
Targeting Nuclear Import Shuttles, Importins/Karyopherins Alpha by a Peptide Mimicking the NFκB1/p50 Nuclear Localization Sequence
Zienkiewicz J, Armitage A and Hawiger J, J Am Heart Assoc, 2(5):e000386, 2013
The authors analyzed interaction of a hydrophobic peptide, derived from the bifunctional nuclear transport modifier (NTM) peptide, with endogenous human importins/karyopherins to determine the mechanism of NTM modulation of importin alpha-mediated nuclear transport. Various peptide-protein binding affinities were determined using the Octet RED96 system. Biotinylated peptides were immobilized on Streptavidin biosensors and then incubated target protein or GST control protein solutions for binding studies.
PubMed
Structural Analysis of the Hemagglutinin from the Recent 2013 H7N9 Influenza Virus
Yang H, et al., J Virol, 87(22), 12433-46, 2013
This study is focused on hemagglutinins (HA) from the 2013 H7N9 influenza virus, assessing their receptor binding phenotype in relation to previously studied HAs. Two recombinant A(H7N9) HAs were structurally characterized, and a mutational study of the receptor binding site was performed to analyze important residues that can affect receptor preference and affinity. HA glycan binding selectivities were examined using Streptavidin biosensors loaded with biotinylated glycans on the Octet RED system.
PubMed
New LIC Vectors for Production of Proteins from Genes Containing Rare Codons
Eschenfeldt WH, et al., J Struct Funct Genomics, 14, 135-144, 2013
The authors describe modification of a series of LIC pMCSG vectors currently used in the high-throughput production of proteins to include tRNA genes covering rare codons for Arg and Ile. Ionosine 5'-monophosphate dehydrogenase from B. anthracis was used as a test protein for the biotinylated vector system. Analyses of the binding of inhibitors to IMPDH proteins were performed using the Octet RED system with biotinylated IMPDH loaded onto Super Streptavidin biosensors.
PubMed
Inhibition of Hepatitis C Virus by the Cyanobacterial Protein MVL: Mechanistic Differences Between the High-mannose Specific Lectins MVL, CV-N, and GNA
Kachko A, et al., Mol Pharm, 10, 4590_4602, 2013
The authors investigate the antiviral properties of the newly discovered Microcystis viridis lectin (MVL) along with cyanovirin-N (CV-N) and Galanthus nivalis agglutinin (GNA) against cell culture Hepatitis C Virus (HCV), as well as their binding properties toward viral particles, target cells, and recombinant HCV glycoproteins. The Octet RED96 system was used for various binding and kinetics experiments with biotinylated HCV rE1E2 protein loaded onto Streptavidin biosensors.
PubMed
Structural and Antigenic Variation among Diverse Clade 2 H5N1 Viruses
Shore DA, et al., PLoS One, 8(9):e75209, 2013
The authors assessed the structural, antigenic and receptor-binding properties of three H5N1 HPAI virus hemagglutinins, which were recently selected by the WHO as vaccine candidates. The Octet RED system was used to measure recombinant HA binding to a panel of biotinylated glycans loaded onto Streptavidin biosensors.
PubMed
A Monoclonal Antibody Based Capture ELISA for Botulinum Neurotoxin Serotype B: Toxin Detection in Food
Stanker LH, et al., Toxins (Basel), 5(11), 2212-26, 2013
The authors report the development botulinum neurotoxin B mAbs capable of binding toxin in solution. Binding specificities and affinity constants were determined using the Octet QK system with the mAbs loaded onto Anti-Mouse Fc Capture biosensors.
PubMed
A Polyclonal Antibody Based Immunoassay Detects Seven Subtypes of Shiga Toxin 2 Produced by Escherichia coli in Human and Environmental Samples
He X, et al., PLoS One, 8(10):e76368, 2013
This study describes the development of a sandwich ELISA assay for detecting both O157 and non-O157 strains of Shiga toxin-producing Escherichia coli (STEC) by incorporating a novel polyclonal antibody against Shiga toxin 2 (Stx2). Real-time binding assays between purified antibodies and purified Stx2a protein were performed on the Octet QK system with biotinylated pAb coupled to Streptavidin biosensors.
PubMed
MAIT Recognition of a Stimulatory Bacterial Antigen Bound to MR1
López-Sagaseta J, et al., J Immunol, 191(10), 5268-77, 2013
This study reports on structural and biochemical characterization of MAIT (mucosal-associated invariant T) TCR engagement of MR1 presenting an Escherichia coli-derived stimulatory ligand, rRL-6-CH2OH. For binding analysis between humanized bovine MR1 (hbMR1) and various human MAIT TCR clones, hbMR1 expressed in the presence or absence of the E. coli supernatant was loaded onto a Ni-NTA biosensor for BLItz system studies.
PubMed
The Potent and Broadly Neutralizing Human Dengue Virus-specific Monoclonal Antibody 1C19 Reveals a Unique Cross-reactive Epitope on the bc Loop of Domain II of the Envelope Protein
Smith SA, et al., MBio, 4(6):e00873-13, 2013
This study describes the isolation and characterization of a large panel of naturally occurring human monoclonal antibodies (MAbs) directed to the dengue virus domain II fusion loop envelope protein region. The majority of cross-reactive MAbs exhibited weak neutralizing potency and showed strong antibody dependent enhancement activity. One unusual clone (1C19), however, exhibited ultrahigh neutralization potency against DENV strains corresponding to all four serotypes. The Octet RED system and Anti-Mouse Fc Capture biosensors were used to classify the panel of DII-FL MAbs for their binding properties. Octet experiments included competitive binding studies, using representative full-length MAbs, on either intact DENV particles or recombinant E80 protein.
PubMed
Antibody Recognition of the Pandemic H1N1 Influenza Virus Hemagglutinin Receptor Binding Site
Hong M, et al., J Virol, 87(22), 12471-80, 2013
This study presents the crystal structure of human monoclonal antibody 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the H1N1 pandemic virus, and reports that 5J8 recognizes a conserved epitope in and around the receptor binding site. Octet RED system binding studies of 5J8 Fab and IgG were performed against a panel of 12 H1 strains that spanned from 1918 to 2009. Biotinylated HAs were loaded onto Streptavidin biosensors and incubated with various concentrations of Fab or IgG of 5J8 or CH65 for these experiments.
PubMed
Yeast Mnn9 is Both a Priming Glycosyltransferase and an Allosteric Activator of Mannan Biosynthesis
Striebeck A, et al., Open Biol, 3(9), 130022, 2013
This study reports the structure of the mannosyltransferase domain of yeast Mnn9 in complex with manganese and GDP, and shows that both the presence and the priming activity of ScMnn9 are required for the formation of the alpha-1,6-mannose backbone of mannan proteins. The interactions of ScMnn9 and ScVan1 were studied using the Octet RED384 system. Biotinylated ScMnn9 was loaded onto Streptavidin biosensors for these binding studies.
PubMed
Crystal Structure of the Hendra Virus Attachment G Glycoprotein Bound to a Potent Cross-reactive Neutralizing Human Monoclonal Antibody
Xu K, et al., PLoS Pathog, 9(10):e1003684, 2013
This study reports the crystal structure of the complex of Hendra Virus G (HeV-G) glycoprotein with human mAb m102.3, an m102.4 derivative, and describes NiV and HeV escape mutants. Binding kinetics for wild type or mutant G proteins to m102.3 and m102.4 antibodies or to ephrin-B2 were measured using the BLItz system. Ni-NTA biosensors were used to immobilize the hexa-Histidine fused antibodies and ephrin-B2 proteins.
PubMed
Structural Determinants of HIV-1 Vif Susceptibility and DNA Binding in APOBEC3F
Siu KK, et al., Nat Commun, 4, 2593, 2013
This important study reports the crystal structure of the HIV-1 viral infectivity factor (Vif) binding domain in A3F-CD2, a critical obstructer to retroviral replication. The author performed site-directed mutagenesis to map out the ssDNA-binding site, and developed a novel BLI-based HIV-1 Vif-APOBEC3 binding assay. The BLItz system first was used to determine the equilibrium dissociation constant for A3Fc-CD2/ssDNA binding using biotinylated ssDNA loaded onto Streptavidin biosensors. Second, ssDNA alanine-mutants were generated for each position and characterized similarly. Next, the authors used their BLI-based Vif-binding assay to map the HIV-1 Vif-binding interface at single-residue resolution.
PubMed
Structure and Function of Norrin in Assembly and Activation of a Frizzled 4-Lrp5/6 Complex
Ke J, et al., Genes Dev, 27(21), 2305-19, 2013
This study reports on the expression, purification, crystallization, and structure determination of the growth factor Norrin, and demonstrates that Norrin activates Fz4 through assembly of a complex of Norrin, Fz4, Lrp5/6, and Tspan12. Binding of MBP-Norrin to Fz4-FcH6, Fz8-FcH6, or human IgG was measured using the Octet RED system with Protein A biosensors loaded with Fz4-FcH6, Fz8-FcH6, or human IgG.
PubMed
Structural and Pharmacological Characterisation of Novel Potent and Selective Monoclonal Antibody Antagonists of Glucose-Dependent Insulinotropic Polypeptide Receptor
Ravin P, et al., J Biol Chem, 288, 19760-19772, 2013
The authors describe the generation and characterization of an antagonizing antibody (Gipg013) to the glucose-dependent insulinotropic polypeptide receptor (GIPr). Binding studies by BLI and SPR are included. The Octet RED system was used for ranking of clones during phage and ribosome display selection, and for the determination of Gipg013/Gipr ECD binding constants. Super Streptavidin biosensors were loaded with biotinylated GIPr ECD for the BLI studies.
PubMed
Quantum Dot-induced Viral Capsid Assembling in Dissociation Buffer
Gao D, et al., International Journal of Nanomedicine, 8, 2119-2128, 2013
This study reports that the capsid protein VP1 can assemble into capsids in the presence of quantum dots (QDs), and that the QDs are encapsulated in the SV40 capsids. BLI studies showed a strong interaction between the QDs and SV40 VP1 proteins. Binding kinetics of VP1 to mercaptoproprionic acid-QDs and VP1 pentamers were measured using the Octet RED96 system with biotinylated VP1 loaded onto Streptavidin biosensors.
Dovepress
Signaling of High Mobility Group Box 1 (HMGB1) Through Toll-like Receptor 4 in Macrophages Requires CD14
Kim S, et al. , Molecular Medicine, 19, 88-98, 2013
The authors report that CD14 is required for activation of TLR4-dependent signaling by HMGB1, and that this occurs in association with CD14, TLR4 and MD2 accumulation within lipid rafts. The Octet QK system was used to characterize the binding of various recombinant proteins and antibodies to HMGB1, which was covalently immobilized on Amine Reactive biosensors.
PubMed
Intravenous Immunglobulin Binds Beta Amyloid and Modifies Its Aggregation, Neurotoxicity and Microglial Phagocytosis In Vitro
Cattepoel S, et al., PLoS One, 8(5):e63162, 2013
This study investigates the effects of intravenous immunoglobulins (IVIG) and purified polyclonal amyloid β-specific antibodies on aggregation, toxicity and phagocytosis of Aβ in vitro, thus elucidating some of the potential mechanisms of action of IVIG in Alzheimer's disease patients. The Octet QK
e was used for binding studies of amyloid β-specific antibodies, IVIG, and Aβ. Biotinylated Aβ oligomers were loaded onto Streptavidin biosensors for these experiments.
PubMed
Nck-mediated Recruitment of BCAP to the BCR Regulates the PI(3)K-Akt Pathway in B Cells
Castello A, et al., Nat Immunol, 14(9), 966-75, 2013
This report describes a previously unknown adaptor function for Nck in recruiting BCAP to sites of BCR signaling and thereby modulating the PI(3)K-Akt pathway in B cells. The Octet RED96 system was used to characterize interactions of various recombinant proteins with an immunoglobulin-α 22-residue peptide centered on phosphorylated Tyr204. The biotinylated peptide was loaded onto Streptavidin biosensors for these experiments.
PubMed
Paramagnetic Solid Lipid Nanoparticles as a Novel Platform for the Development of Molecular MRI Probes
Rolla G, et al., Chemistry, 19(34), 11189-93, 2013
In this study, solid lipid nanoparticles (SLNs), based on crystalline triglyceride, are investigated as an alternative delivery system for MRI probes based on Gd(III) complexes. The Octet QK system was used to characterize the targeting properties of folic acid-pSLNs to an anti-folic acid monoclonal antibody. Protein A biosensors were loaded with the anti-folic acid mAb for binding studies.
PubMed
Monovalent Antibody Design and Mechanism of Action of Onartuzumab, a MET Antagonist with Anti-tumor Activity as a Therapeutic Agent
Merchant M, et al., Proc Natl Acad Sci U S A, 110(32), E2987-96, 2013
This paper describes the development and characterization of onartuzumab, an E. coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET. The Octet RED system was used for binding measurements of wild-type and mutants of MET Sema-PSI to onartuzumab. The onartuzumab Fab was biotinylated and loaded onto Streptavidin biosensors for binding studies. Rates of association, dissociation, and binding affinities were reported.
PubMed
Intrinsic Selectivity of Notch 1 for Delta-like 4 over Delta-like 1
Andrawes M, et al., J Biol Chem, 288(35), 25477-25489, 2013
This report utilizes signaling assays and biochemical studies of purified recombinant ligand and receptor molecules to investigate the differences in signaling behavior and intrinsic affinity between Notch1-Dll1 and Notch1-Dll4 complexes. Notch-ligand binding affinities were determined using the BLItz system. Biotinylated EGF6-15 region of Notch1 was immobilized on Streptavidin biosensors and bound to varying concentrations of Delta-like ligands Dll1(1-5) or Dll4(1-5).
PubMed
Autoantibodies to Variable Heavy (VH) Chain Ig Sequences in Humans Impact the Safety and Clinical Pharmacology of a VH Domain Antibody Antagonist of TNF-α Receptor 1
Holland M, et al., J Clin Immunol, 33, 1192-1203, 2013
The authors report on a series of clinical and in vitro studies that investigate the impact of HAVH (human anti-VH) autoantibodies on the pharmacology and safety of GSK1995057, an anti-TNFR1 VH domain antibody. HAVR autoantibody binding kinetics to GSK1995057 were evaluated on an Octet QK384 system using biotinylated GSK1995057-loaded Streptavidin biosensors and various concentrations of HAVR autoantibody.
PubMed
Disassembly of All SNARE Complexes by N-Ethylmaleimide-sensitive Factor (NSF) is Initiated by a Conserved 1:1 Interaction Between α-Soluble NSF Attachment Protein (SNAP) and SNARE Complex
Vivona S, et al., J Biol Chem, 288(34), 24984-91, 2013
This work presents comparative studies of binding constants and disassembly kinetics for four ternary SNARE complexes and one binary (t-SNARE) complex with NSF and α-SNAP. Interaction studies were performed using the Octet RED system. Biotinylated SNARE complex was loaded onto Streptavidin biosensors for binding kinetics experiments.
PubMed
Molecular Mechanism for p202-Mediated Specific Inhibition of AIM2 Inflammasome Activation
Yin Q, et al., Cell Rep, 4(2), 327-39, 2013
To elucidate the role of p202 in the regulation of cytosolic DNA sensing, the authors performed extensive structural, biochemical, and cellular studies. The BLItz system was used for binding studies of HIN (hemopoietic expression, interferon inducibility, nuclear localization) proteins. Human AIM2 HIN, IFI16 HIN1 or HIN2 were biotinylated and immobilized on Streptavidin biosensors for binding studies.
PubMed
Small-molecule Modulation of Wnt Signaling via Modulating the Axin-LRP5/6 Interaction
Wang S, et al., Nat Chem Biol, 9(9), 579-85, 2013
This study investigated the use of small molecules to modulate the differential assemblies of Axin and, therefore, modulate Wnt signaling. Binding kinetics and affinities of small molecule-Axin interactions were determined using the Octet Red system. Biotinylated Axin domains were loaded onto Super Streptavidin biosensors for the interaction studies.
PubMed
Ferredoxin Competes with Bacterial Frataxin in Binding to the Desulfurase IscS
Yan R, et al., J Biol Chem, 288(34), 24777-87, 2013
The authors have characterized the interactions of the bacterial desulferase IscS and the specialized ferredoxin Fdx using a combination of biophysical tools and mutagenesis. The Octet RED system was used for binding experiments. Biotinylated holo-Fdx was loaded onto Streptavidin biosensors to quantify the affinity of the IscS-Fdx interaction and the effect of Fdx on Fe-S cluster assembly.
PubMed
Receptor Binding by an H7N9 Influenza Virus from Humans
Xiong X, et al., Nature, 499(7459), 496-9, 2013
This study examines the receptor-binding properties of the H7N9 influenza virus and compares them with those of an avian H7N3 virus. The authors conclude that the human H7 virus has acquired some of the receptor-binding characteristics that are typical of pandemic viruses, but its retained preference for avian receptor may restrict its further evolution towards a virus that could transmit efficiently between humans. Virus binding to receptor analogues was characterized on an Octet RED system using biotinylated α2,3- and α2,6-linked sialyl lactosamine sugars loaded onto Streptavidin biosensors.
PubMed
Mutational Analysis of 48G7 Reveals that Somatic Hypermutation Affects Both Antibody Stability and Binding Affinity
Sun S, et al., J Am Chem Soc, 135(27), 9980-9983, 2013
The authors analyzed the effects of individual somatic mutations and combinations thereof on the antibody binding affinity and thermal stability of the well-characterized monoclonal antibody 48G7. Hapten binding affinities for 48G7 and mutants were studies using the Octet RED96 system. Biotinylated JWJ1 hapten was immobilization on Streptavidin biosensors for the binding studies.
PubMed
pH-induced Quaternary Assembly of Vitreoscilla Hemoglobin: the Monomer Exhibits Better Peroxidase Activity
Li W, et al., Biochim Biophys Acta, 1834(10), 2124-2132, 2013
In this work, the pH-dependent (pH 6.0-8.0) quaternary assembly of ferric Vitreoscilla hemoglobin (VHb) was investigated. The Octet RED96 system was used to characterize the self-association properties of VHb at various pH levels. VHb was biotinylated using NHS-PeG4-Biotin and loaded onto Streptavidin biosensors for the Octet experiments.
PubMed
Chemical Cross-linking of HIV-1 Env for Direct TLR7/8 Ligand Conjugation Compromises Recognition of Conserved Antigenic Determinants
Feng Y, et al., Virology, 446, 56-65, 2013
This study examines the effects of chemically cross-linking a synthetic TLR7/8 ligand to surface-exposed lysine residues on the external subunit of the HIV-1 envelope glycoprotein, gp120. Unmodified and cross-linker conjugated gp120 were used for kinetics analysis on the Octet RED system. Anti-Human IgG Fc biosensors were used to capture the mAbs VRC01, F105 or 447-52D, which then were incubated with the gp120 proteins.
ScienceDirect
The Depolymerized Fucosylated Chondroitin Sulfate from Sea Cucumber Potently Inhibits HIV Replication via Interfering with Virus Entry
Huang N, et al., Carbohydr Res, 380, 64-69, 2013
This work focuses on the anti-HIV activity of FuCS1, a low molecular weight fragment of a fucosylated chondroitin sulfate derived from the sea cucumber Thelenota ananas. The binding of FuCS1 to HIV-1 gp120 was investigated using the Octet RED96 system. The gp120 protein was loaded onto AR2G biosensors for these measurements.
PubMed
Identify the Key Amino Acid of BAFF Binding with TACI
Wang R, et al., Cell Immunol, 284(1-2), 84-90, 2013
In this study, the key amino acids involved in the binding of B-cell activating factor (BAFF) to its receptor TACI were identified. TACI binding kinetics for various BAFF mutants were studied using the Octet RED system. BAFF receptor TACI-Ig was loaded onto Anti-Human IgG Capture biosensors for the determination of k
on, k
offand K
D.
PubMed
Identification of a Human Monoclonal Antibody to Replace Equine Diphtheria Anti-toxin for the Treatment of Diphtheria Intoxication
Sevigny L, et al., Infect Immun, 81(11), 3992-4000, 2013
The authors describe identification of an anti-diphtheria hMAb (315C4) isolated from antibody-secreting cells obtained from a vaccine-immunized human volunteer. The 315C4 antibody neutralized diphtheria toxin in a cell-based cytotoxicity assay and prevented toxin from binding to the known diphtheria receptor, heparin binding-epidermal growth factor-like growth factor. Octet assays were used to determine the affinity of 315C4 for diphtheria toxin, fragment B, and fragment R. Anti-Human IgG biosensors were used for 315C4 immobilization. Toxin binding competition analysis with a panel of neutralizing antibodies was also performed on the Octet system.
PubMed
Kinetic Analysis of Antibodies from Different Cultured Media
Ho D, Fletcher, T, and Ni, J, BioPharm International , July: 2-5, 2013
In this study, an Octet QK
e system method was established for assessing the effects of media condition on the quality and activity of cultured antibodies. Affinities and binding kinetics of monoclonal antibodies produced from a hybridoma cell line cultured in two different types of medium were measured. With this method, the authors were able to measure the intermolecular binding interactions between the antibodies and targeted protein. Anti-Mouse IgG Fc Capture (AMC) biosensors were used for antibody immobilization. Binding studies with fibronectin used Streptavidin biosensors loaded with biotinylated fibronectin.
BioPharm International
The E3 Ubiquitin Ligase CHIP and the Molecular Chaperone Hsc70 Form a Dynamic, Tethered Complex
Smith M, et al., Biochemistry, 52(32), 5354-64, 2013
The E3 ubiquitin ligase CHIP (C-terminus of Hsc70 Interacting Protein) binds to the molecular chaperone Hsc70, and this complex is important in both the ubiquitination of Hsc70 and the turnover of Hsc70-bound clients. In this study, the authors characterized the binding of Hsc70 to CHIP using the Octet RED96 system, fluorescence polarization, and NMR spectroscopy. For the Octet experiments, Hsc70 proteins and the C-terminal peptide were biotinylated and loaded onto Streptavidin biosensors.
PubMed
Effects of Hyaluronic Acid Conjugation on Anti-TNF-α Inhibition of Inflammation in Burns
Friedrich E, et al., J Biomed Mater Res A, doi: 10.1002/jbm.a.34829, 2013
To elucidate the benefits of a topical antibody treatment, the authors compare the effectiveness of (anti-TNF-α)-hyaluronic acid conjugates to nonconjugated anti-TNF-α antibody and a non-conjugated mixture of hyaluronic acid and anti-TNF-α. Binding affinities were determined using the Octet QK system and biotinylated antibodies loaded onto Streptavidin biosensors.
PubMed
A Novel Rabbit Monoclonal Antibody Platform To Dissect the Diverse Repertoire of Antibody Epitopes for HIV-1 Env Immunogen Design
Chen Y, et al., J Virol, 87(18), 10232-43, 2013
In this study, 12 HIV-1 Env-specific rabbit hybridoma cell lines were produced that recognized a wide range of Env epitopes. Heavy-chain and light-chain genes were cloned and their genetic features were analyzed. Binding kinetics of the rabbit mAb to gp120s from HIV-1 clades A to E were studied using the Octet QK
e system. Protein A biosensors were loaded with the rabbit mAbs and incubated with concentration series of gp120 proteins.
PubMed
Two ScFv Antibody Libraries Derived from Identical VL-VH Framework with Different Binding Site Designs Display Distinct Binding Profiles
Huovinen T, et al., Protein Eng Des Sel, 20(10), 683-693, 2013
In this study of diverse antibody libraries, the authors demonstrate that by targeting different residues of the same antibody gene, it is possible to create sublibraries with distinct profiles in terms of the number and affinities of the library clones against different-sized antigens. Apparent affinities for anti-streptavidin clones were measured on the BLItz system using Streptavidin biosensors.
PubMed
Anti-HIV-1 Activity and Structure-activity-relationship Study of a Fucosylated Glycosaminoglycan from an Echinoderm by Targeting the Conserved CD4 Induced Epitope
Lian W, et al., Biochim Biophys Acta, 1830(10), 4681-91, 2013
Fucosylated glycosaminoglycan (FG) is a novel glycosaminoglycan with a chondroitin sulfate-like backbone and fucose sulfate branches. The aim of this study was to investigate the mechanism and structure-activity relationships of FG for combating HIV-1 infection. The Octet RED96 system was used to study the binding of FGs and gp120, and the effects of CD4 on that binding. For these studies, FGs were biotinylated and loaded onto Streptavidin biosensors.
PubMed
Disruption of Focal Adhesion Kinase and p53 Interaction with Small Molecule Compound R2 Reactivated p53 and Blocked Tumor Growth
Golubovskaya V, et al., BMC Cancer, 13(1), 342, 2013
The authors identified a small molecule (R2) that bound to Focal Adhesion Kinase (FAK), disrupted the binding of FAK and p53, and decreased cancer cell viability and clonogenicity in a p53-dependent manner. The Octet RED384 system was used for binding studies. Human FAK-N-terminal domain protein was biotinylated and loaded onto Super Streptavidin biosensors for the characterization of R2-FAK binding. For detection of FAK-p53 dissociation by R2, p53 protein was biotinylated and bound to Streptavidin biosensors.
PubMed
Monitoring the Kinetics of the pH Driven Transition of the Anthrax Toxin Prepore to the Pore by Biolayer Interferometry and Surface Plasmon Resonance
Naik S, et al., Biochemistry, 52, 6335-6347, 2013
In this comprehensive study, the authors construct immobilized complexes of anthrax toxin prepore and the anthrax protective antigen (PA) binding domain of anthrax lethal factor to monitor the prepore-to-pore kinetic transition in real time, using both BLI and SPR. For BLI experiments, Octet RED96 and BLItz systems were used. Immobilization of PA was achieved via noncovalent binding to sulfhydryl-linked LF
nE126C.
PubMed
Effects on Peptide Binding Affinity for TNFα by PEGylation and Conjugation to Hyaluronic Acid
Elder A, et al., European Polymer Journal, 49, 2968-2975, 2013
This study uses a model peptide inhibitor of TNF-α to investigate the effects of site-specific conjugation to hyaluronic acid and polyethylene glycol. Binding affinities and kinetic rate constants for the conjugated and unconjugated biotinylated peptides were determined using the Octet QK system and Streptavidin biosensors.
ScienceDirect
Off-rate Screening for Selection of High-affinity Anti-drug Antibodies
Ylera F, et al., Anal Biochem, 441(2), 208-213, 2013
To aid in the development of high-affinity anti-drug antibodies, the authors developed a method for off-rate screening of crude E. coli lysate containing monovalent Fab fragments obtained after phage display of an antibody library. The antibody drugs trastuzumab and cetuximab were used as antigen examples. Using the Octet RED384 system, they demonstrate that antibody off-rates can be reliably determined in crude bacterial lysates with high throughput. AR2G biosensors were used for the cetuximab studies; trastuzumab experiments were performed with biotinylated trastuzumab loaded onto Streptavidin biosensors.
PubMed
The Novel Human Beta-defensin 114 Regulates Lipopolysaccharide(LPS)-mediated Inflammation and Protects Sperm from Motility Loss
Yu, H, et al., J Biol Chem, 288(17), 12270-82, 2013
The authors characterize the recombinant human beta-defensin DEFB114 peptide, demonstrating its anti-microbial potential, lipopolysaccharide (LPS) binding and neutralization, and anti-inflammatory effects in vitro and in vivo. Data suggest that DEFB114 could also protect human sperm from motility loss when challenged with LPS. Affinity measurements of interaction between LPS and DEFB114 were performed using the Octet system. Purified DEFB114 was biotinylated and loaded onto Streptavidin biosensors, followed by association/dissociation with purified LPS.
PubMed
Coagulation Factor X Shields Adenovirus Type 5 from Attack by Natural Antibodies and Complement
Xu, Z, et al., Nat Med, 19(4), 452-7, 2013
Adenovirus type 5 (Ad5) can survive attack by natural antibodies and complement, making it a valuable vector for systemic gene therapy. Results presented in this paper demonstrate that resistance of Ad5 to complement is dependent on recruitment of and binding to coagulation factor X (FX). The Octet RED system and Streptavidin biosensors were used to analyze interactions of FX with non-FX-binding mutant Ad vector and corresponding control.
PubMed
In Vitro Selection of Multiple Libraries Created by Genetic Code Reprogramming to Discover Macrocyclic Peptides that Antagonize VEGFR2 Activity in Living Cells
Kawakami, T, et al., ACS Chem Biol, 8(6), 1205-1214, 2013
Multiple highly diverse peptide libraries were constructed using genetic code reprogramming to incorporate non-proteinogenic amino acids. The authors used a TRAP selection scheme to select macrocyclic peptides in vitrothat bind to vascular endothelial growth factor receptor 2 (VEGFR2). Affinity of selected peptides was determined using the Octet system by immobilizing Fc-fused VEGFR2 on Anti-Human IgG Fc biosensors.
PubMed
Removal of a C-terminal Serine Residue Proximal to the Inter-chain Disulfide Bond of a Human IgG1 Lambda Light Chain Mediates Enhanced Antibody Stability and Antibody Dependent Cell-mediated Cytotoxicity
Shen, Y, et al., MAbs, Apr 8, 5(3), 2013
Sequence and structural analysis of human IgG1 λ constant domains suggest three possible mutations in residues proximal to the lambda Lc cysteine may contribute to disulfide bond stability. Here, deletion of the C-terminal serine of lambda constant region is shown to improve stability of the disulfide bond between the light chain and heavy chain at elevated temperature and pH. This mutation also improves transient expression levels and purification yields of IgG1 λ as well as improved Fc function. Protein A biosensors and the Octet platform were used to determine IgG1 λ levels in culture supernatant.
PubMed
ATP-competitive Inhibitors Block Protein Kinase Recruitment to the Hsp90-Cdc37 System
Polier, S, et al., Nat Chem Biol, 9(5), 307-12, 2013
Authors demonstrate that Cdc37 directly antagonizes ATP binding to client protein kinases and inhibits phosphorylation of kinase substrates. ATP-competitive inhibitors are also shown to antagonize Cdc37 interaction with Hsp90-dependent kinases in vitro.The binding of Cdc37 to B-Raf protein kinase domain in the presence of ATP-competitive kinase inhibitors was assessed on the Octet RED96 system using Anti-Penta-HIS biosensors.
PubMed
Synthesis and Initial Evaluation of YM-08, a Blood-Brain Barrier Permeable Derivative of the Heat Shock Protein 70 (Hsp70) Inhibitor MKT-077, Which Reduces Tau Levels
Miyata, Y, et al., ACS Chem Neurosci, 4(6), 930-939, 2013
This study reports on the synthesis and initial characterization of YM-08, a neutral small-molecule Hsp70 inhibitor designed for enhanced blood-brain barrier permeability. The Octet RED system was used to determine YM-08 binding affinities for Hsp70 nucleotide binding domain and for full-length Hsp70. Biotinylated Hsp70 proteins were loaded onto Super Streptavidin biosensors for these measurements.
PubMed
Structural Mechanism of CCM3 Heterodimerization with GCKIII Kinases
Zhang, M, et al., Structure, 21(4), 680-688, 2013
The protein Cerebral Cavernous Malformation 3 (CCM3) can heterodimerize with GCKIII kinases, including MST4. This study presents structural and functional studies of the CCM3-MST4 heterodimeric complex. Interactions between wild-type or mutant MST4 and wild-type or mutant CCM3 were quantified using the Octet RED96 system. Biotinylated CCM3 variants were loaded onto Streptavidin biosensors for these experiments.
PubMed
Identification of Human Monoclonal Antibodies Specific for Human SOD1 Recognizing Distinct Epitopes and Forms of SOD1
Broering, TJ, et al., PLoS One, 8(4):e61210, 2013
Misfolded SOD1 may be implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). The authors generated and characterized a panel of fully human mAbs directed against human SOD1. Relative avidity and cross-competition experiments were performed on the Octet system equipped with Anti-Human Fc Capture (AHC) biosensors. The mAbs were loaded onto the AHC biosensors for avidity measurements. For cross-competition assays, a mAb first was loaded onto the AHC Biosensor, followed by sequential incubation with recombinant SOD1 and the competing mAb.
PubMed
Feasibility Study of Semi-selective Protein Precipitation with Salt-tolerant Copolymers for Industrial Purification of Therapeutic Antibodies
Capito, F, et al., Biotechnol Bioeng, doi: 10.1002/bit.24950, 2013
This publication presents a feasibility study for the use of salt-tolerant copolymers for secondary clarification of mAbs in clarified cell culture fluid. The Octet RED system was used to compare binding of mAbs before and after precipitation and redissolution. Anti-Human Fc Capture biosensors were loaded with mAbs for these measurements.
PubMed
Receptor Binding by a Ferret-transmissible H5 Avian Influenza Virus
Xiong, X, et al., Nature, 497(7449), 392-6, 2013
This publication presents quantitative biophysical measurements of the receptor-binding properties of hemagglutinin from an H5 avian influenza virus transmissible mutant, and reports a dramatic increase in preference for binding of human over avian receptors. Virus binding to defined receptor analogues was measured using the Octet RED96 system. Biotinylated alpha2,3- and alpha2,6-sialic acid structures were loaded onto Streptavidin biosensors to generate equilibrium binding data.
PubMed
Label-free Methods of Reporting Biomolecular Interactions by Optical Biosensors
Citartan, M, et al., Analyst, 138(13), 3576-3592, 2013
The authors present a mini-review of optical biosensor technologies for biomolecular interaction analysis.
PubMed
Dynamin_SNARE Interactions Control Trans-SNARE Formation in Intracellular Membrane Fusion
Alpadi, K, et al., Nat Commun, 4, 1704, 2013
This study explores the molecular mechanism of how the dynamin-like protein Vsp1 controls membrane fusion and coordinates fusion with antagonistic fission processes. The Octet RED96 system was used to characterize the specificity and kinetics of Vsp1 binding to the SNARE protein Vam3. GST-Vam3 was loaded onto Anti-GST biosensors and incubated with various concentrations of His-Vsp1 to determine kinetic rate constants and the equilibrium dissociation constant.
PubMed
14-3-3 Proteins Interact with a Hybrid Prenyl-Phosphorylation Motif to Inhibit G Proteins
Riou, P, et al., Cell, 153(3), 640-53, 2013
This study identifies 14-3-3 proteins as Rnd interaction partners, and further shows that 14-3-3 proteins can act as solubilizing factors for phosphorylated and prenylated proteins from the Ras superfamily. The authors use the Octet RED96 system to characterize binding between 14-3-3 proteins and various phosphorylated, isoprenylated, geranylated, farnesylated and geranylgeranylated peptides. Biotinylated peptides were loaded onto Streptavidin biosensors and interacted with 14-3-3 proteins to generate kinetic and affinity constants.
PubMed
The Molecular Basis for Mucosal-associated Invariant T cell Recognition of MR1 Proteins
Lopez-Sagaseta, J, et al., Proc Natl Acad Sci U S A, 110(19), E1771-8, 2013
This study elucidates the structure of MAIT TRC (mucosal-associated invariant T-cell receptor) bound to bovine MR1 (MHC related-1 protein). The affinity of the F7 MAIT TCR for single bovine MR1 mutants expressing human residues was determined using the BLItz system and compared to the wild-type affinity. Recombinant 12xHIS tagged MR1 proteins were loaded onto Ni-NTA biosensors for these experiments.
PubMed
Structural Characterization of the Self-association of the Death Domain of p75NTR
Qu, Q, et al., PLoS One, 8(3):e57839, 2013
To elucidate the mechanism of regulation for the neurotrophin receptor p75NTR, the authors perform structural and functional analysis of truncations of the intracellular region. Crystal structures of intracellular death domain were reported showing two possible dimer arrangements. The Octet system and Streptavidin biosensors were used to measure kinetics of p75NTR binding to downstream adaptor protein RIP2 (CARD) domain in order to elucidate binding features of various oligomeric conformations of p75NTR intracellular region.
PubMed
F1-ATPase of Escherichia coli: The ε-inhibited State Forms After ATP Hydrolysis, is Distinct from the ADP-inhibited State, and Responds Dynamically to Catalytic-site Ligands
Shah, NB, et al., J Biol Chem, 288(13), 9383-95 , 2013
The authors use BLI experiments on the Octet RED system to study inhibition of E. coli F
1-ATPase by its subunit ε. The binding and dissociation kinetics for F
1-ε were determined and correlated with inhibitory effects for wild type and mutant forms of ε. Biotinylated ε species were loaded onto Streptavidin biosensors for these studies.
PubMed
Peptide Ligands for Pro-survival Protein Bfl-1 from Computationally Guided Library Screening
Dutta, S, Chen, T S, Keating, A E, ACS Chem Biol, 8(4), 778-88 , 2013
The focus of this study was the protein Bfl-1, a pro-survival member of the Bcl-2 family implicated in a variety of cancers. The authors isolated a high affinity peptide that bound to Bfl-1 in preference to other pro-survival proteins, and performed mutational and structural analysis to identify positions important to preferential binding. The BLItz system was used to generate equilibrium and kinetic binding data for peptide-protein interactions. Biotinylated peptide was loaded onto Streptavidin biosensors for these measurements.
PubMed
Optimization and Modification of Anti-rhTNF-α Single Chain Variable Fragment Antibody: Effective In Vitro Affinity Maturation and Functional Expression of Chimeric Fab
Xiaoniu, M, et al., Biomed Pharmacother, 67(5), 437-444, 2013
The CDR-H3 domain of a well-characterized anti-rhTNF-α scFv fragment was mutated by degenerate PCR and the mutant library screened for higher affinity binders by phage display. An improved clone, scFv-G10, was identified and used along with constant regions CH1 and CL of human IgG1 to construct a novel vector for expression of a functional chimeric Fab. Biotinylated rhTNF-α was immobilized onto Streptavidin biosensors and binding affinity of scFv-G10 as compared to the original parent scFv clone was determined using the Octet RED system.
ScienceDirect
Mitoxantrone Targets the ATP-binding Site of FAK, Binds the FAK Kinase Domain and Decreases FAK, Pyk-2, c-Src, and IGF-1R In Vitro Kinase Activities
Golubovskaya V, et al., Anticancer Agents Med Chem, 13(4), 546-554, 2013
Computer modeling and cell viability assays were used to screen NCI small molecule compound database for compounds specific for the ATP binding site of FAK(Focal Adhesion Kinase), which is involved in multiple cellular functions and is a proposed therapeutic cancer target. A mitoxantrone derivative, compound A18, was identified and shown to inhibit cancer cell viability in vitro. The authors show compound A18 decreases FAK kinase function and kinase activity of other enzymes. The authors demonstrated using the Octet RED384 system and Super-Streptavidin biosensors that compound A18 binds to the FAK kinase domain.
PubMed
Targeting the Binding Function 3 (BF3) Site of the Androgen Receptor Through Virtual Screening. 2. Development of 2-((2-phenoxyethyl) thio)-1H-benzimidazole Derivatives
Munuganti, R S, et al., J Med Chem, 56(3), 1136-48, 2013
The authors develop structure-activity relationships that allowed the design of small molecule inhibitors of the human androgen receptor (AR) binding function 3 (BF3). The Octet RED system was used to quantify interactions between the small molecules and the AR. Purified biotinylated AR LBD was loaded onto Super Streptavidin biosensors and BLI dose-response curves were generated.
PubMed
A High-throughput Media Design Approach for High Performance Mammalian Fed-batch Cultures
Rouiller, Y, et al., mAbs, 5(3), 501-511, 2013
The authors describe a new high-throughput blending method for batch-fed cell culture process media development. The method enabled optimization of all components of a CHO fed-batch medium in a single experiment. Titer determinations were performed using an Octet QK
e system with Protein A biosensors.
mAbs
The Regulation of Apoptosis by the Downstream Regulatory Element Antagonist Modulator/Potassium Channel Interacting Protein 3 (DREAM/KChIP3) Through Interactions with Hexokinase I
Craig, T A, et al., Biochem Biophys Res Commun, 433(4), 508-12, 2013
This study explores the mechanism by which the DREAM protein alters apoptosis in neuronal cells. DREAM was found to interact with brain hexokinase I in a calcium-dependent manner. The BLItz system was used to characterize binding of DREAM to hexokinase I. Biotinylated DREAM was loaded onto Streptavidin biosensors and incubated with hexokinase I in the presence of either calcium chloride or EGTA. Association and dissociation constants were determined.
PubMed
Structure of a Classical Broadly Neutralizing Stem Antibody in Complex with a Pandemic H2 Influenza Virus Hemagglutinin
Dreyfus C, Ekiert D C, Wilson I A, J Virol, 87(12), 7149-54 , 2013
The authors present characterization studies on C179, the first antibody identified to cross-neutralize multiple subtypes of influenza A viruses. For kinetic analysis, the Octet RED system was used. Biotinylated hemagglutinins were loaded onto Streptavidin biosensors and incubated with varying concentrations of C179 Fab. To explore the breadth of C179, the authors evaluated C179 Fab binding to a large panel of hemagglutinins, including subtypes H1-H17 of influenza A.
PubMed
Engineering HIV Envelope Protein to Activate Germline B Cell Receptors of Broadly Neutralizing Anti-CD4 Binding Site Antibodies
McGuire, A T, et al. , J Exp Med, 210(4), 655-663, 2013
The authors show that elimination of specific N-linked glycosylation sites on HIV envelope glycoprotein (Env) allows Env binding to, and activation of, B cells expressing the germline-reverted BCRs of two broadly neutralizing antibodies. Env binding studies were performed on the Octet QK
e system with IgG's immobilized on Anti-Human IgG Fc Capture biosensors and wild type/mutant recombinant trimeric Env gp140.
PubMed
Affinity Capture of Biotinylated Proteins at Acidic Conditions to Facilitate Hydrogen/Deuterium Exchange Mass Spectrometry Analysis of Multimeric Protein Complexes
Jensen, P F, et al., Anal Chem, doi: 10.1021/ac303442y, 2013
The authors present a strategy for reducing interferences during hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis of protein complexes. The method is based on streptavidin capture of biotinylated proteins under HDX-MS quench conditions. Interactions of two anti-EGFR antibodies with EGFR were studied as a model system. The Octet QK384 system was used to determine the impact of antibody biotinylation on antibody-EGFR binding. The assay showed that binding of the two biotinylated antibodies to an EGFR-Fc fusion protein loaded onto Protein G biosensors was identical to the binding of unmodified antibodies.
PubMed
Stimulation of the Retinoid X Receptor Facilitates Beta-Amyloid Clearance Across the Blood-Brain Barrier
Bachmeier, C, et al., J Mol Neurosci, 49(2), 270-6, 2013
The impact of retinoid X receptor stimulation on beta-amyloid protein (Aβ) clearance across the blood brain barrier was investigated. The Octet RED96 system was used to determine whether stimulation of Aβ(1-42) in the presence of bexarotene and fluorobexarotene was due to direct binding of the drugs to Aβ(1-42). No binding was apparent, although binding the Congo Red positive control was clearly detected. Super Streptavidin biosensors were loaded with Aβ(1-42) for these experiments.
PubMed
Structure of the MST4 in Complex with MO25 Provides Insights into its Activation Mechanism
Shi, Z, et al., Structure, 21(3), 449-61, 2013
This study focusses on the activation of STE20-like kinase MST4 by the scaffold protein MO25. The Octet RED96 system was used to detect direct interactions between recombinant MO25 and various MST4 fragments. The MST4 fragments were biotinylated and loaded onto Streptavidin biosensors. Association and dissociation rate constants were determined. The Octet RED96 system next was used for mutational analysis of the MST4-MO25 interface using a similar experimental approach.
PubMed
Switchable Elastin-like Polypeptides that Respond to Chemical Inducers of Dimerization
Dhandhukia, J, et. al., Biomacromolecules, 14(4), 976-85 , 2013
A strategy for designing elastin-like polypeptides (ELPs) that respond to specific chemical substrates is presented. The authors engineered a construct (FKBP-ELP) consisting of an engineered protein polymer linked to a fusion protein that homodimerized upon binding of a bifunctional chemical inducer of dimerization (CID). Association/dissociation kinetics and the affinity constant for CID binding to FKBP-ELP were characterized on the BLItz system using biotinylated FKBP-ELP loaded onto Streptavidin biosensors.
PubMed
Selection of a High-affinity WW Domain Against the Extracellular Region of VEGF Receptor Isoform-2 from a Combinatorial Library using CIS Display
Patel, S, et al., Protein Eng Des Sel, 26(4), 307-15 , 2013
The authors demonstrate that the Pin1 WW domain motif can be used as a framework for engineering and selection of high-affinity binders. The Octet RED system was used for real-time binding assays between chemically synthesized peptides and human VEGFR-2-Fc fusion, VEGFR-1-Fc fusion or VEGFR-3-Fc fusion. The biotinylated peptides were loaded onto Streptavidin biosensors for these studies.
PubMed
An Evaluation of the Capability of a Biolayer Interferometry Biosensor to Detect Low-molecular-weight Food Contaminants
McGrath, T F, et al., Anal Bioanal Chem, 405(8), 2535-44, 2013
The authors use the shellfish toxin domoic acid as a model compound to evaluate BLI for low-molecular weight food contaminant detection. Domoic acid was loaded onto Amine Reactive biosensors for analysis on the Octet RED96 system. A competition assay was developed in which the biosensor was dipped into solutions containing a fixed concentration of anti-domoic acid antibodies mixed with different concentrations of free domoic acid.
PubMed
β-Arrestin-1 Directly Interacts with Gαs and Regulates its Function
Li, B, et al., FEBS Lett, 587(5), 410-6, 2013
This study explores how β-arrestin and G protein signaling pathways coordinate with one another. The Octet system was used to conduct kinetic analysis of the interaction between β-arrestin and Gα
s. Biotinylated Gα
s was loaded onto Streptavidin biosensors for these measurements. Association/dissociation rate constants and equilibrium dissociation constants for these interactions were reported.
PubMed
Dip-and-Read Method for Label-Free Renewable Sensing Enhanced Using Complex DNA Structures
Zhang, M, et al., ACS Appl Mater Interfaces, 5(3), 473-8, 2013
The authors demonstrate that DNA tetrahedron structures can be used for signal enhancement in BLI quantitation of sequence-specific DNA oligonucleotides or ATP. The Octet QK
e system was used for these studies. Biotinylated oligonucleotides were loaded onto Streptavidin biosensors for all BLI measurements.
PubMed
Conformational Biosensors Reveal GPCR Signalling from Endosomes
Irannejad, R, et al., Nature, 495(7442), 534-8, 2013
This publication presents results that support the hypothesis that canonical GPCR signaling occurs from endosomes as well as the plasma membrane. A conformation-specific single-domain camelid antibody (Nb80) that selectively binds agonist-occupied β2-adrenoceptor was central to the key experiments. Nb80 binding to unliganded and agonist-occupied β2-adrenoceptor was studied using the Octet RED system. Streptavidin biosensors were loaded with biotinylated β2-AR-rHDL particles and then incubated with or without isoprenaline for 30 minutes before Nb80 association and dissociation steps.
PubMed
Structural Insights into the Regulation of Cohesion Establishment by Wpl1
Chatterjee, A, et al., EMBO J , 32(5), 677-87, 2013
This paper presents structural and biochemical studies of the Wpl1 protein, a member of the cohesin complex. Binding affinities for Wpl1259-647 (corresponding to residues 271-635 of budding yeast Wpl1) and ScSm3-AH (variant of the cohesin protein Smc3) were determined using the Octet RED system. The Wpl1259-647 was amine-coupled to the biosensor surface for affinity measurements.
PubMed
N332-Directed Broadly Neutralizing Antibodies Use Diverse Modes of HIV-1 Recognition: Inferences from Heavy-Light Chain Complementation of Function
Pancera, M, et al., PLoS One, 8(2):e55701, 2013
In this study of broadly neutralizing antibodies that target the N332 site of HIV-1 gp120, complementation between 12 heavy chains and 12 light chains was characterized. Expression levels for a panel of transiently transfected antibodies were quantified using the Octet RED96 system with Anti-human IgG Fc biosensors.
PubMed
Conformational Stabilization of Ubiquitin Yields Potent and Selective Inhibitors of USP7
Zhang, Y, et al., Nat Chem Biol, 9(1), 51-8, 2013
Computational design along with phage display and affinity maturation were utilized to modify ubiquitin to select for variants with altered conformations resulting in high affinity binding to the USP7 tumor-associated deubiquitinase (DUB). High affinity clones were selected, providing tools for elucidating the role of USP7 in the Mdm2-p53 tumorigenic pathway. The Octet RED384 system and Streptavidin biosensors were used to determine binding affinities of ubiquitin variants to USP7 constructs.
PubMed
Structural Plasticity of Histones H3-H4 Facilitates Their Allosteric Exchange Between RbAp48 and ASF1
Zhang, W, et al., Nat Struct Mol Biol, 20(1), 29-35, 2013
Interactions of the Histone H3-H4 complex with the histone chaperone proteins RbAp48 and ASF-1 were studied. RbAp48 was shown to bind the histone H3-H4 complex as a heterodimer, not a heterotetramer. Major conformational changes were shown to take place in the core fold of H3-H4 complex upon binding to RpAp48, indicating an allosteric mechanism facilitating the exchange of H3-H4 complex between the two chaperones. The Octet RED384 system and Streptavidin biosensors were used to characterize RbAp48 and ASF1 binding to acetylated histone H4.
PubMed
Outer Domain of HIV-1 gp120: Antigenic Optimization, Structural Malleability, and Crystal Structure with Antibody VRC-PG04
Joyce, M G , J Virol, 87(4), 2294-306, 2013
The authors describe design and antigenic optimization of an HIV-1 gp120 soluble outer domain as a minimal immunogen that can be recognized by broadly neutralizing antibodies that target the CD4 binding site. The outer domain variant OD4.2.2 was shown to bind known broadly neutralizing antibodies with nanomolar affinity. The Octet RED384 system was used with Anti-Human Fc Capture biosensors to measure binding kinetics of outer domain molecules to broadly neutralizing antibodies VRC01, VRC-PG04 and b12 and also to a group of poorly neutralizing antibodies.
PubMed
Application of Bio-Layer Interferometry for the Analysis of Protein/Liposome Interactions
Wallner, J, et al., J Pharm Biomed Anal, 72, 150-4, 2013
A method to characterize protein/liposome binding interactions is described. Binding of three liposome formulations (DDPC, egg-PC and POPC) to recombinant human erythropoietin (rh-Epo) on the Octet QK platform was the model system. Binding experiments used Streptavidin biosensors loaded with biotinylated rh-Epo, incubated with various liposomes at different concentrations to measure the corresponding association and dissociation profiles.
PubMed
HLA-DO Acts as a Substrate Mimic to Inhibit HLA-DM by a Competitive Mechanism
Guce, A I, et al., Nat Struct Mol Biol, 20(1), 90-8, 2013
This study investigates how the nonclassical MHCII protein HLA-DO modulates the function of another nonclassical MHCII protein, HLA-DM. Binding studies on the Octet QK system showed that HLA-DO acts as a competitive inhibitor of HLA-DM. The effects of several mutations of HLA-DM at the putative MHCII interaction surface also were evaluated using the Octet system. Biotinylated proteins and Streptavidin biosensors were used for interaction experiments.
PubMed
An Inhibitory Antibody against Dipeptidyl Peptidase IV Improves Glucose Tolerance In Vivo
Tang, J, et al., J Biol Chem, 288(2), 1307-16, 2013
The authors generated and characterized tight-binding inhibitory mAbs against rat dipeptidyl peptidase IV (DPP-IV), an enzyme whose suppression is an established strategy to treat type 2 diabetes mellitus. The Octet system was used to study mAb binding to rat DPP-IV, and to identify the key recognition epitope. Protein G biosensors were used for the binding experiments.
PubMed
Postnatally-transmitted HIV-1 Envelope Variants have Similar Neutralization-sensitivity and Function to that of Nontransmitted Breast Milk Variants
Fouda, G G, et al., Retrovirology, 10(1), 3, 2013
In this study, the authors compare the genotype and function of HIV-1 Env variants isolated for postnatally-transmitting mothers, clinically-matched nontransmitting mothers, and postnatally-infected infants. Binding of breast milk HIV-1 gp120 proteins expressed by milk ENV variants to Galcer liposomes was studied using the Octet RED system with Aminopropylsilane (APS) biosensors.
PubMed
A High-Yielding, CHO-K1-Based Transient Transfection System
Agrawal, V, et al., BioProcess International, 11(1), 28-35, 2013
The Aragen team report on a high-yielding transient transfection platform based on suspension-adapted CHO K1 cells. Expressed antibody production levels were determined by analysis of culture supernatants using an Octet RED system with Protein A biosensors.
BioProcess
Mechanistic Study of Broadly Neutralizing Human Monoclonal Antibodies Against Dengue Virus that Target the Fusion Loop
Costina, J M, et al., J Virol, 87(1), 52-66, 2013
The authors identified and characterized three broadly neutralizing, high affinity human monoclonal antibodies to Dengue Virus. The Octet QK system was used to study the binding of these hMAbs to Dengue Virus -1, -2, -3, and -4 soluble E proteins, and to perform antibody binding competition assays. For all Octet experiments, hMAbs were loaded onto Anti-Human IgG Fc Capture (AHC) biosensors.
PubMed
Recombinant HIV Envelope Proteins Fail to Engage Germline Versions of Anti-CD4bs bNAbs
Hoot, S, et al., PLoS Pathog, 9(1): e1003106, 2013
Recombinant HIV envelope glycoproteins (Env) have, so far, not been able to elicit broadly neutralizing antibodies (bNAbs). This publication explores whether and how Env immunogens interact with the predicted germline versions of known bNAbs. The Octet QK
e system was used to characterize binding of mature and germline IgG b12 with a panel of Envs using two methods, one with IgG b12 loaded onto Anti-Human IgG Fc capture (AHC) biosensors, and the other with His-tagged antigens loaded onto Ni-NTA biosensors.
PubMed
A Novel Hemoglobin-binding Peptide Reduces Cell-free Hemoglobin in Murine Hemolytic Anemia
Hanson, M S, et al., Am J Physiol Heart Circ Physiol, 304, H328-36, 2013
In this study, a novel hemoglobin-binding peptide (Hb-B10) was synthesized and used to study the contribution of cell-free hemoglobin to vascular complications associated with hemolysis. The binding of the Hb-B10 peptide to hemoglobin was characterized using the Octet RED96 system. Biotinylated Hb-B10 peptide was loaded onto Streptavidin biosensors and incubated with various concentrations of hemoglobin to determine kinetic binding constants.
PubMed
Evaluation of Taylor Dispersion Injections: Determining Kinetic/Affinity Interaction Constants and Diffusion Coefficients in Label-Free Biosensing
Quinn JG, Anal biochem, 421: 401-410, 2012
Reported herein is the use of Taylor dispersion injection (TDi) to generate a continuous analyte titration that could be used in both steady-state and kinetic analyses of label-free biomolecular interactions. The biophysical characterizations that can be performed by this new TDi-based assay format and standard fixed concentration injection (FCI) format were compared using the following three model interactions: Antibody fragment-receptor interaction, furosemide-carbonic anhydrase II interactions, and warfarin-human serum albumin (HSA) interaction. All binding assays described in this article were performed by surface plasmon resonance (SPR) using a Pall ForteBio Pioneer instrument. In TDi analysis, ethylene tetrafluoroethylene (ETFE) and polyether ether ketone (PEEK) tubing were cut into appropriate lengths and were used to connect the injector valve and the reaction flow cell in the Pioneer system. For the antibody fragment-receptor interaction study, a COOHV sensor chip was immobilized with Protein G using standard amine coupling chemistry. Subsequently, the fusion protein, rhErbB2/Fc was captured onto Protein G immobilized sensor chip, followed by the injection of a serial dilutions of scFv. For the analysis of furosemide-carbonic anhydrase II interactions, carbonic anhydrase II was immobilized onto the sensor chip by amine coupling. Binding data were obtained upon injection of serial dilutions of Furosemide. For the affinity analyses of warfarin-HSA interaction, COOHV chip surface was immobilized with HSA by using standard amine coupling. Subsequently, the serial dilutions of warfarin were injected. The association rate constant (ka), the dissociation rate constant (kd), the maximum biosensor response expected if all ligand sites are occupied (Rmax), the analyte diffusion coefficient (D), and the mass transport constant (km) were determined. Overall results suggest that the TDi-based assay format is capable of providing high-quality biophysical characterizations while reducing assay complexity and run time as compared to the standard FCI approach.
PubMed
Modeling Taylor Dispersion Injections: Determination of Kinetic/Affinity Interaction Constants and Diffusion Coefficients in Label-Free Biosensing
Quinn JG, Anal biochem, 421: 391-401, 2012
Standard fixed concentration injections (FCIs) are commonly used when studying biomolecular interactions by label-free detection methods such as surface plasmon resonance (SPR). With the FCIs, a serial dilution of the analyte is prepared and sequentially injected in order to obtain an analyte concentration range to determine kinetic parameters. Where as in Taylor dispersion injections (TDis), the analyte concentration gradually evolves into a sigmoidal gradient profile enabling kinetic analysis of biomolecular interactions from a minimal number of injections. Pall ForteBio Pioneer instruments incorporate a capillary tube that enables TDi assay formats. Reported herein is the use of a numerical model, Monte Carlo simulations, and statistical profiling in a comparison of Taylor dispersion injection approach with the standard fixed concentration injections approach. Overall results of this study demonstrates the effectiveness of the TDi approach.
PubMed
Interaction of Clusterin and Matrix Metalloproteinase-9 and Its Implication for Epithelialhomeostasis and Inflammation
Jeong S, et al., Am J Pathol, 180(5):2028-39, 2012
Novel High-affinity Binders of Human Interferon Gamma Derived from Albumin-Binding Domain of Protein G
Ahmad J, et al., Proteins, 80(3):774-89, 2012
Noncovalent Assembly of Anti-dendritic Cell Antibodies and Antigens for Evoking Immuneresponses In Vitro and In Vivo
Flamar A, et al., J Immunol, 189(5):2645-55, 2012
Leishmania (Viannia) Braziliensis: Insights on Subcellular Distribution and Biochemical Properties of Heparin-Binding Proteins
de Castro Cortes L, et al., Parasitology, 139(2):200-7, 2012
Simultaneous Screening for T-2/HT-2 and Deoxynivalenol In Cereals Using a Surface Plasmon Resonance Immunoassay
Meneely J, et al., World Mycotoxin Journal, 5(2):117-126, 2012
Surface Plasmon Resonance (SPR) Studies on the Interactions of Carotenoids and Their Binding Proteins
Vachali P, at al., Arch Biochem Biophys, 519(1):32-7, 2012
Label-free Epitope Binning Assays of Monoclonal Antibodies Enable the Identification of Antigen Heterogeneity
Abdiche, Y. N., et al., J Immunol Methods, 382(1-2), 101-16, 2012
Epitope binning assays are a valuable tool for characterizing epitope binding regions of panels of monoclonal antibodies that target a specific antigen. However, some antigen preparations may contain subpopulations of molecules that are either native or denatured, potentially leading to selection of candidates that bind biologically irrelevant epitopes. This article reviews methods to identify antigen heterogeneity through use of epitope binning assays on label-free biosensors. The Octet platform and Streptavidin biosensors were used to screen for binding of antigen to immobilized mAbs in epitope binning experiments.
PubMed
Identification of the Key Functional Domain of BAFF for Binding TACI by Computer-Guided Molecular Modeling Method
Wang, R., et al., J Genet Syndr Gene Ther , 3 (114). doi:10.4172/2157-7412.1000114, 2012
In this study, the key interaction domains of BAFF (B cell activating factor) with its receptor TACI were predicted using computer-guided molecular modeling. Binding kinetics of BAFF and its mutants to TACI then were measured using the Octet RED system. Anti-Human IgG capture biosensors were loaded with TACI-Ig and incubated with various concentrations of BAFF and mutant BAFF proteins to determine kinetic rate constants and affinities.
OmicsOnline
Endoglin Requirement for BMP9 Signaling in Endothelial Cells Reveals New Mechanism of Action for Selective Anti-Endoglin Antibodies
Nolan-Stevaux, O., et al., PloS One, 7(12):e50920, 2012
This study utilized various techniques to analyze the requirement of Endoglin for endothelial SMAD activation. The Octet RED system was used for a series of Endoglin-binding competition assays with the anti-Endoglin antibodies M999, TRC105, SN6 and SN6h. For the binding competition assays, biotinylated recombinant human Endoglin was loaded onto Streptavidin biosensors. The authors found that M999 and TRC105 competed with each other for binding to their epitope on Endoglin, and that both antibodies significantly inhibited BMP9 binding to Endoglin.
PubMed
LUZ-Y, a Novel Platform for the Mammalian Cell Production of Full-length IgG-bispecific Antibodies
Wranik, B. J., et al., J Biol Chem, 287(52), 43331-9, 2012
The authors present a novel bispecific antibody platform that can be expressed in mammalian cells. These antibodies are referred to as LUX-Ys because a leucine zipper is used to induce heterodimerization of different heavy chains. The Octet system was used to confirm that LUZ-Y antibodies maintained their ability to bind to their cognate antigens. Binding specificity and kinetics were characterized using Anti-Human IgG Fc capture biosensors loaded with one armed anti-HER2 and one armed anti-EGFR LUZ-Ys.
PubMed
Protein Microarrays, Biosensors, and Cell-based Methods for Secretome-wide Extracellular Protein-Protein Interaction Mapping
Gonzales, L.C. , Methods, 57(4), 448-58, 2012
This review highlights traditional and emerging technologies that are compatible with secretome-wide screens for extracellular protein-protein interaction discovery. The author notes co-receptor interactions discovered using the Octet system (TIGIT-PVR and Robo4-UNC5B) and discusses the screening capabilities of BLI instrumentation.
PubMed
4F Decreases IRF5 Expression and Activation in Hearts of Tight Skin Mice
Xu, H., et al., PLoS One, 7(12):e52046, 2012
This study investigates the effects of 4F (an apoA1 mimetic) on the transcription factor IRF5 in the hearts of Tsk-/+ mice (a murine model of autoimmune disease). IRF5 and 4F interactions were studied using the Octet RED96 system. Biotin-labeled IRF5 was loaded onto Streptavidin biosensors and incubated with 4F protein in PBS to determine rates of association and dissociation.
PubMed
Influenza Human Monoclonal Antibody 1F1 Interacts with Three Major Antigenic Sites and Residues Mediating Human Receptor Specificity in H1N1 Viruses
Tsibane, T., et al., PLoS Pathog, 8(12):e1003067, 2012
This study focusses on the influenza human monoclonal antibody 1F1, which was isolated from a 1918 influenza pandemic survivor and shows an unusual breadth of inhibitory activity. The Octet QK system was used to characterize binding of recombinant 1918 Fabs to recombinant trimerized HIS-tagged hemagglutinin protein containing sequence of 1918 wild-type or avianized variant strains. HIS-tagged proteins were loaded onto Anti-Penta HIS biosensors for the binding studies.
PubMed
Attaining Next-Level Titers in CHO Fed-Batch Cultures
Barrett, S. L., et al., BioProcess International, 10(10), 56-62, 2012
There are inherent difficulties in providing desired concentrations of critical nutritional components in fed-batch cultures. The authors describe a new functional additive that is highly concentrated and pH neutral, and demonstrate improved titers. The Octet QK system was used to measure antibody titers in multiple media experiments using the additive.
BioProcess
IL-13-induced Airway Mucus Production is Attenuated by MAPK13 Inhibition
Alevy, Y. G., et al., J Clin Invest, 122(12), 4555-68, 2012
Genetic and chemical tools are used to define a signaling pathway controlling mucous production, beginning with IL-13 induction of CLCA1-gene expression to CLCA1-mediated activation of MAPK13, followed by induction of mucin gene expression. Using structure-based drug design, a series of nM affinity MAPK13 inhibitors were developed. Kinetics of MAPK13 binding to these inhibitors was assessed with the Octet system and Super Streptavidin biosensors.
PubMed
Origin of the Conformational Heterogeneity of Cardiolipin-bound Cytochrome C
Hong, Y., et al., J Am Chem Soc, 134(45), 18713-23, 2012
The mitochondrial phospholipid cardiolipin promotes unfolding of cytochrome C, with implications in the cellular apoptosis pathway. BLI combined with TR-FRET and FCS was utilized to understand functional partitioning between compact and extended cardiolipin-bound cytochome C conformations. Specifically, the Octet RED96 system and Streptavidin biosensors were used to determine kinetic constants of cytochrome C binding to liposomes with varied cardiolipin content.
PubMed
Strategic Selection and Development of Immunogenicity Binding Methods
Menendez, A.T. , Bioanalysis, 4(12), 1491-508, 2012
This is a comprehensive review on development of a robust program for immunogenicity testing. New detection technologies with applications in immunogenicity are discussed, including the Octet platform, its capabilities and advantages of label-free technology. The author also describes different assay formats for immunogenicity testing, preparation and storage of critical reagents, feasibility studies, and optimization and validation methods.
PubMed
Translation Inhibition of the Developmental Cycle Protein HctA by the Small RNA IhtA is Conserved Across Chlamydia
Tattersall, J., et al., PLoS One, 7(10):e47439, 2012
HctA, a histone-like protein, is central to differentiation between cellular forms in the developmental cycle of Chlamydia trachomatis. The small RNA lhtA represses translation of HctA, allowing cell differentiation to proceed. This mechanism is shown here to be conserved across pathogenic chlamydial species. lhtA from each species tested was shown to interact with its related hctA target mRNA in vitro using the Octet QK
e system and Streptavidin biosensors.
PubMed
Noncanonical G Recognition Mediates KSRP Regulation of Let-7 Biogenesis
Nicastro, G., et al., Nat Struct Mol Biol, 19(12), 1282-6, 2012
The authors determine the contribution of individual KH domains of the protein KSRP to its interaction with let-7, a tumor-suppressive micro RNA. The Octet platform and Streptavidin biosensors were utilized to assess binding of KSRP mutants to let-7, followed by NMR structural studies to dissect the binding mechanism of KSRP KH domains to the G-rich target sequence on the terminal loop of let-7.
PubMed
Structural Mechanism of ER Retrieval of MHC Class I by Cowpox
McCoy, W. H., et al., PLoS Biol, 10(11):e1001432, 2012
The authors performed structural and functional studies to elucidate the mechanism by which the cowpox virus-encoded protein CPXV203 blocks normal trafficking of MHCI from the endoplasmic reticulum to the plasma membrane in order to evade the immune response. Binding studies with the Octet and Biacore systems were run to determine binding affinities of CPXV203 to different MHCI molecules in a variety of buffer conditions to mimic the ER/golgi environments. Streptavidin biosensors were used.
PubMed
Evolution of the Receptor Binding Properties of the Influenza A(H3N2) Hemagglutinin
Lin, Y.P., et al., Proc Natl Acad Sci USA, 109(52), 21474-9, 2012
The influenza A (H3N2) virus was responsible for the 1968 influenza pandemic. In this fascinating study, the authors analyze the receptor binding characteristics of H3N2 hemagglutinin (HA) and correlate virus avidity changes over time with structural changes caused by mutations in HA. Their data show that the progressive decrease in binding of these viruses to human receptors between 2001 to 2010 correlates with changes in infectivity efficiency and specific structural changes. The Octet RED system was used to characterize virus binding to human and avian receptor analogs. Streptavidin biosensors were loaded with biotinylated α2,3- and α2,6-linked sialyl lactosamine sugars linked to polyacrylamide polymers. Virus binding to the sialyl lactosamine sugars was monitored to generate equilibrium binding curves.
PubMed
Design of a Novel Cyclotide-Based CXCR4 Antagonist with Anti-Human Immunodeficiency Virus (HIV)-1 Activity
Aboye, T. L., et al., J Med Chem, 55(23), 10729-34, 2012
The authors describe the design and synthesis of a novel cyclotide able to inhibit the G-protein-coupled receptor CXCR4, a potential target for inhibition of HIV replication. To assess stability in human serum, the BLItz system was used to determine association and dissociation rate constants of the cyclotide to human serum proteins.
PubMed
A Novel Process for Developing Fully Human Monoclonal Antibodies
Schram, B., et al., Pharmaceutical Technology, 11(36), s28-s31, 2012
The authors describe Neoclone's human-mAb platform for generation of fully human therapeutic antibodies. In the NeoAb approach, antigen supernatants from cultured B cells are tested for affinity and specificity using the Octet QK system. Streptavidin biosensors loaded with biotinylated antigens are employed for binding constant determinations of purifies antibodies and for KD rank-ordering of crude tissue culture supernatants.
PharmTech
Furin-cleaved Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Is Active and Modulates Low Density Lipoprotein Receptor and Serum Cholesterol Levels
Lipari, M.T., et al., J Biol Chem, 287(52), 43482-91, 2012
PCKS9 regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. This publication investigates how cleavage of PCSK9 by furin affects PCSK9 function. LDL receptor binding to PCSK9 was characterized using the Octet RED384 system. Biotinylated LDL receptor (ectodomain) was loaded onto Streptavidin biosensors for these experiments. Kinetic binding constants for LDL receptor interaction with intact and cleaved PCSK9 are reported.
PubMed
Variable Lymphocyte Receptor Recognition of the Immunodominant Glycoprotein of Bacillus Anthracis Spores
RN Kirchdoerfer, et al., Structure, 20(3), 479-486, 2012
In this study, interactions of variable lymphocyte receptor 4 (VLR4) from jawless fish and the Bacillus anthracis spore protein BcIA are characterized using the Octet RED platform. Various biotinylated BcIA C-terminal domain variants were immobilized on Streptavidin biosensors and incubated with a dilution series of truncated monomeric VLR4.
PubMed
XPB Helicase Regulates DNA Incision by the Thermoplasma acidophilum Endonuclease Bax1
HM Roth, et al., DNA Repair, 11(3), 286-293, 2012
In this work, the endonuclease activity of Bax1 is characterized, alone and in combination with XPB helicase. Binding of Bax1 and of the XPB/Bax1 complex to different DNA structures was studied using the Octet RED platform. Biotinylated dsDNA strands with blunt ends or overhangs were immobilized on Streptavidin biosensors and kinetic rate constants for Bax1, XPB, and the XPB/Bax1 complex were determined. The Octet data demonstrated that XPB facilitates binding of Bax1 to DNA.
PubMed
Identification of Galectin-1 and Galectin-3 as Novel Partners for Von Willebrand Factor
N Saint-Lu, et al., Arterioscler Thromb Vasc Biol, 32(4), 894-901, 2012
This publication explores interactions between the heavily glycosylated von Willebrand factor (VWF) and glycan-binding proteins galectin-1 and galectin-3. The Octet QK system was used in parallel with immunosorbent microtiter plate assays for VWF-galectin binding studies. Galectin-1 and galectin-3 were coupled to Amine Reactive biosensors for the Octet platform experiments. VWF bound to immobilized galactin-1 and galactin-3 in a dose-dependent and reversible manner. The binding was significantly reduced after incubation of VWF with N-glycosidases, indicating that galectin-VWF binding is dependent on VWF glycan structures.
PubMed
Calcium-independent Inhibition of PCSK9 by Affinity-improved Variants of the LDL Receptor EGF(A) Domain
Zhang Y, et al., J Molecular Biology, 422(5), 685-96, 2012
To generate more potent EGF(A) domain inhibitors of PCSK9, the authors constructed and screened a phage display library. Several EGF variants with improved binding affinities were identified, with one clone displaying significantly increased inhibition and prevention of LDLR degredation. The Octet system was used to determine binding affinities of EGF-Fc fusion proteins to PCSK9.
PubMed
Determination of Cathepsin S Abundance and Activity in Human Plasma and Implications for Clinical Investigation
J.M. Cox, et al., Analytical Biochemistry, 430(2), 130-137, 2012
To investigate the role of cathepsin S in disease pathogenesis and progression, a sandwich ELISA assay was developed to measure circulating levels in human serum or plasma. An LC-MS method was also developed to help correlate circulating levels with activity of cathepsin S in plasma. Only a small fraction of circulating cathepsin S was found to be active, and attenuated activity correlated with increased association with cystatin C, an endogeneous inhibitor of cathepsin. The Octet QK system was used to determine relative affinity rankings of cathepsin S antibodies to be used for ELISA assay development.
PubMed
Chemical Synthesis and X-ray Structure of a Heterochiral {D-protein Antagonist Plus Vascular Endothelial Growth Factor} Protein Complex by Racemic Crystallography
K. Mandala, et al., Proc Natl Acad Sci USA, 109(37), 14779-14784, 2012
Mirror image phage display was applied as an approach to develop high affinity D-protein antagonists for a biologically active protein target. A D-protein ligand would be an optimal therapeutic - i.e. it can be manufactured, will be resistant to proteolytic degradation and will likely be non-immunogenic. Here, a specific D-protein ligand for VEGF-A was generated that blocks binding to the VEGFR1 receptor. X-ray structure of the antagonist-protein complex was determined. The Octet system was used to show specificity of protein ligand/VEGF-A interactions.
PubMed
Aptamers as a Sensitive Tool to Detect Subtle Modifications in Therapeutic Proteins
R Zichel, et al., PLoS ONE, 7(2), e31948, 2012
This study demonstrate proof-of-principle that DNA aptamers to thromin can be used as surrogate antibodies to characterize thrombin conformational changes. Thrombin binding afinities and kinetic rate constants for six anti-human thrombin aptamers were determined using the Octet RED96 system. Biotinylated aptamers were immobilized on Streptavidin biosensors for these studies. Following heat treatment of recombinant thromin, changes in dissociation rate were reported for 4 of the 6 aptamers.
PubMed
Expression of High-Affinity Human Antibody Fragments in Bacteria
R Rouet, et al., Nature Protocols, 7(2), 364-373, 2012
General protocols are presented with trouble shooting tips for the expression of human antibody fragments in E. coli. The authors discuss the use of BLI and SPR for the determination of antibody fragment-antigen binding affinitites, clone ranking, and off-rate selection, although no data are shown.
PubMed
Practical Quantitative and Kinetic Applications of Bio-Layer Interferometry for Toxicokinetic Analysis of a Monoclonal Antibody Therapeutic
Dysinger, M.; King, L.E., J Immunological Methods, 379(1-2), 30-41, 2012
The authors present rationale and strategies for the development and analytical qualification of a BLI assay for the quantitation of a humanized antibody therapeutic in cynomolgus monkey plasma. They used an Octet QK system, a first-generation instrument based on BLI technology. Reference is made at several points in the publication to the Octet RED-type systems that are capable of higher sensitivity detection in comparison to the Octet QK system. Results of the qualification were compared to those of a validated ELISA used to quantitate the same therapeutic. Selectivity, matrix effect, and precision and accuracy were found to be similar between the two methods. The main difference between the two assays was in the dynamic range (0.1-10 μg/mL for ELISA vs. 0.4-50 μg/mL for BLI). A direct quantitation comparison of sample results for the two methods shows a high degree of agreement (r2=0.979, slope=1.017).
PubMed
Metformin Interacts with AMPK Through Binding to γ Subunit
Zhang, Y., et al., Mol Cell Biochem, 368(1-2), 69-76, 2012
Metformin is a commonly used drug to treat type 2 diabetes. Octet systems and other techniques including CD, fluorescence spectroscopy were employed to study the binding relationship between Metformin and AMPK, a kinase that is a key regulator of energy metabolism and the target of Metformin.
PubMed
Reliable Protein Production in a Pseudomonas Fluorescens Expression System
Retallack, D., Jin, H., Chew, L., Protein Expression and Purification, 81(2), 157-65, 2012
This report discusses Pfenex's Pseudomonas fluorescens expression system. The Octet system with Protein L biosensors was used to select optimal expression strains for an antibody Fab fragment. Binding of Fab fragments to Protein L biosensors was used as an indication of proper folding. Octet system data are used to assess the impact of various host strains and expression vectors on the amount of Fab present in the soluble fraction.
PubMed
Towards a Universal Disulphide Stabilised Single Chain Fv Format: Importance of Interchain Disulphide Bond Location and vL-vH Orientation
Weatherill, E.E., et al., Protein Engineering, Design and Selection, 25(7), 321-329, 2012
Five disulphide bond locations were introduced into one scFv in order to compare their relative effects on expression, thermal stability, % monomer formation and retention of antigen binding. The Octet system was used to determine the expression levels of the scFv with different bond modifications using a Streptavidin Biosensor coupled to biotinylated Protein L to quantitate the scFv forms. The expression levels of scFvs were not significantly affected in various disulphide bond locations with the exception of vH101-vL46 bond location.
PubMed
Evaluation of the Potency of the Anti-idiotypic Antibody Ab2/3H6 Mimicking gp41 as an HIV-1 Vaccine in a Rabbit Prime/Boost Study
Mader, A., Kunert, R., PLoS One , 7(6), e39063, 2012
The authors characterize an anti-ideotypic antibody of interest in HIV-1 vaccine strategies. Affinity analysis and competition assays were performed using the Octet QK system with Streptavidin biosensors. Biotinylated gp140 or a biotinylated epitope of the 2F5 mAb were immobilized on SA biosensors for these studies.
PubMed
Identification of Broadly Protective Human Antibodies to Pseudomonas Aeruginosa Exopolysaccharide Psl by Phenotypic Screening
Digiandomenico, A., et al., J Experimental Medicine, 209(7), 1273-87, 2012
Whole P. aeruginosa cells and single-chain variable fragment phage libraries derived from patients were employed to select for monoclonal antibodies against P. aeruginosa infection. Affinity and epitope specificity for the mAbs against the P. aeruginosa exopolysaccharide PsI were analyzed using the Octet 384 instrument. Crude PsI preparations were immobilized onto APS biosensors for these studies. The authors also present novel carbohydrate capture experiments using the Octet system.
PubMed
A Small Molecule Focal Adhesion Kinase (FAK) Inhibitor, Targeting Y397 site: 1-(2-hydroxyethyl)-3,5,7-triaza-1-azoniatricyclo[3.3.1.13,7]decane; Bromide Effectively Inhibits FAK Autophosphorylation Activity and Decreases Cancer Cell
Golubovskaya, V.M., et al., Carcinogenesis, 33(5), 1004-1013, 2012
Y11 is a small molecule that was identified using computer modeling to target the main autophosphorylation site of FAK (Y397), a protein tyrosine kinase that is overexpressed in most solid tumors. The Octet RED system was used to characterize Y11 binding to the N-terminal domain of FAK containing the Y397 site. Biotinylated FAK N-terminal domain was immobilized on Super Streptavidin biosensors for these measurements.
PubMed
The Sclerostin-bone Protein Interactome
Devarajan-Ketha, H., et al., Biochemical and Biophysical Research Communications, 417 (2), 830-835, 2012
This publication identifies a large number of bone-derived protein binding partners of sclerostin, an important regulator of osteoblastic activity. The authors use the Octet system to determine the dissociation constant for sclerostin-carbonic anhydrase II binding. The assay used Super Streptavidin biosensors with biotinylated carbonic anhydrase II.
PubMed
Exploring the Dynamic Range of the Kinetic Exclusion Assay in Characterizing Antigen-Antibody Interactions
Bee, C., et al., PLoS One , 7(4), e36261, 2012
The binding interactions of Anti-DKK (DS4) antibody to a panel of DKK Ags was characterized. The Octet QK384 syste, was used to determine the activities of various DKK proteins in different preps (carrier-free vs. lyphilized) by conducting stoichiometry-controlled titrations. These values were used to correct apparent ka values obtained by other orthogonal methods.
PubMed
Chemical and Genetic Evidence for the Involvement of Wnt Antagonist Dickkopf2 in Regulation of Glucose Metabolism
Li, X., et al., Proc Natl Acad Sci USA, 109(28), 11402-7, 2012
This study concludes that the naturally-occuring Wnt antagonist Dickkopf2 (DKK2) may be a potential therapeutic target for type 2 diabetes. The authors use the Octet RED system to characterize the binding of DKK2C to repeat domains of the Wnt coreceptor LRP6 , and the inhibition constant for a small molecule inhibitor of that interaction.
PubMed
The N-terminus of the Human RecQL4 Helicase is a Homeodomain-like DNA Interaction Motif
Ohlenschlager, O., et al., Nucleic Acids Research, 40(17), 8309-24, 2012
Mutations in RecQL4 helicase are associated with Rothmund-Thomson and other syndromes. The authors use the Octet RED96 system to study DNA binding characteristics of the N-terminus of RecQL4 (RecQL4_N54). Three forms of biotinylated DNA (Y-shaped, dsDNA and ssDNA) are immobilized on Streptavidin biosensors and then exposed to RecQL4_N54 to determine dissociation constants. The authors note that these experiments could not be performed on their Biocore 2000 system due to nonspecific binding problems.
PubMed
Adenanthin Targets Peroxiredoxin I and II to Induce Differentiation of Leukemic Cells
Liu, C.X., et al., Nature Chemical Biology, 8(5), 486-93, 2012
The authors report that adenanthin, a diterpenoid, binds to Peroxiredoxins I and II and inhibits their peroxidase activities. The Octet RED system was used to characterize initial noncovalent binding of wild-type and mutant Peroxiredoxin I and Peroxiredoxin II to biotin-adenanthin.
PubMed
Bifunctional CD4-DC-SIGN Fusion Proteins Demonstrate Enhanced Avidity to gp120 and Inhibit HIV-1 Infection and Dissemination
Du, T., et al., Antimicrobial Agents and Chemotherapy, 56(9), 4640-9, 2012
Direct binding analysis of gp120 protein to CD4-linker-DC SIGN fusion proteins (CLD) was performed using the Octet RED system. Biotinylated gp120 was immobilized onto a Streptavidin biosensor and interactions with the CLDs were characterized. The results indicated that CLDs interacted with gp120 in a bivalent manner. The strongest binding affinity of CLDs to gp120 was achieved with a 35AA linker; shorter linkers potentially hindered the interactions.
PubMed
Phage-derived Fully Human Antibody scFv Fragment Directed Against Human Vascular Endothelial Growth Factor Receptor 2 Blocked its Interaction with VEGF
Zhang, J., et al., Biotechnology Progress, 28(4), 981-9, 2012
VEGFR-2 is an important target for treatment of angiogenesis-related diseases. The authors discover a single-chain antibody fragment that targets extracellular domain 3 (KDR3) of human VEGFR-2. Binding of the single-chain antibody fragment to KDR3 was studied using the Octet RED system. Biotinylated KDR3 was immobilized on Streptavidin biosensors and exposed to various concentrations of the antibody fragment to determine kinetic rate constants and the dissociation constant.
PubMed
Factor VIII and von Willebrand Factor are Ligands for the Carbohydrate-Receptor Siglec-5
Pegon, J.N., et al., Haematologica, 97(12), 1855-63, 2012
Factor VIII and von Willebrand factor (VWF) are coagulation factors that exhibit sialylated glycan structures. This publication investigates the interactions of Factor VIII and VWF with Siglec-5 (sialic acid binding immunoglobulin-like lectin-5). Using the Octet QK system and dimeric sSiglet-5/Fc immobilized on Protein A biosensors, the authors show that both Factor VIII and VWF are able to interact with Siglec-5 with high affinity.
PubMed
Small-molecule Inhibition of Human Immunodeficiency Virus Type 1 Replication by Targeting of the Interaction between Vif and ElonginC
Zuo, T., et al., Journal of Virology, 86(10), 5497-507, 2012
The objective of this study was to design a small molecule that disrupts binding of HIV viral infectivity factor Vif to the cellular host protein ElonginB/C, and thus inhibit HIV replication. A potent small molecule inhibitor was identified, and its effects on Vif-ElonginB/C binding were characterized using the Octet RED96 system. Inhibition was studied using recombinant ElonginB/C immobilized on Streptavidin biosensors and incubated with Vif protein in the presence or absence of the small molecule inhibitor.
PubMed
Cyclic di-GMP Sensing via the Innate Immune Signaling Protein STING
Yin, Q., et al., Molecular Cell, 46 (6), 735-745, 2012
The structural characteristics of STING (stimulator of interferon genes) cytosolic domain are investigated in this publication. The BLItz system was used to study binding between STING CBD (c-di-GMP binding domain) and IKK-like kinase TBK1. Biotinylated CBD was immobilized on Streptavidin biosensors and incubated with various concentrations of TBK1.
PubMed
Optimization of Affinity, Specificity and Function of Designed Influenza Inhibitors Using Deep Sequencing
Whitehead, T.A., et al., Nature Biotechnology, 30(6), 543-548, 2012
The authors use sequence-function maps generated from deep sequencing studies to optimize the binding affinities of two computationally-designed protein inhibitors of H1N1 hemagglutinin. The Octet RED system was used to study binding specificities for the two inhibitors against a panel of 16 hemagglutinin subtypes. The inhibitors were biotinylated and loaded onto Steptavidin biosensors for these measurements.
PubMed
KH Domains with Impaired Nucleic Acid Binding as a Tool for Functional Analysis
Hollingworth, D., et al., Nucleic Acids Research, 40(14), 6873-86, 2012
In this study, the authors dissect the role of K homology domains in the recognition of RNA targets by RNA-binding proteins. For binding studies, the Octet RED system was used with biotinylated RNA oligonucleotides immobilized onto Streptavidin biosensors. RNA binding constants for wild-type and mutant RNA-binding proteins were determined.
PubMed
Helicobacter Pylori Hydrogenase Accessory Protein HypA and Urease Accessory Protein UreG Compete with Each Other for UreE Recognition
Benoit, S.L., et al., Biochimica et Biophysica Acta (BBA), 1820 (10), 1519-1525, 2012
In this study, BLI and SPR are used to characterize the interactions of accessory proteins implicated in the survival of H. pylori in human stomach mucosa. Proteins studied include the urease accessory proteins UreE, UreF, UreG and UreH, and the hydrogenase accessory proteins HypA and HypB. BLI protein-protein interaction measurements were performed on the Octet QK system using biotinylated accessory proteins immobilized on Streptavidin biosensors. Binding constants for two important binding pairs were determined: HypA-UreE and UreE-UreG.
PubMed
Data Fusion-based Assessment of Raw Materials in Mammalian Cell Culture
Lee, H., Christie, A., Xu, J., Yoon, S., Biotechnology and Bioengineering, 109(11), 2819-28, 2012
This is a process analytical study in which four spectroscopic techniques (NIR, 2D fluorescence, Raman and X-ray fluorescence) were used to predict the raw material quality of soy hydrolysate samples in CHO cell culture. Intergrated viable cell density and IgG production were used as quantitative indices of raw material quality. The Octet QK system was used to determine IgG production levels.
PubMed
Russell Body Inducing Threshold Depends on the Variable Domain Sequences of Individual Human IgG Clones and the Cellular Protein Homeostasis
Stoops, J., Byrd, S., Hasegawa, H., Biochimica et Biophysica Acta (BBA), 1823(10), 1643-57, 2012
Russel bodies are naturally-occuring aggregates of immunoglobulins. In this paper, an IgG panel was used to investigate the propensity of individual clones to induce Russell body formation under various cellular conditions. The Octet RED96 system with Protein A biosensors was used to determine human IgG concentrations in harvested culture media.
PubMed
Cleavage of a Putative Metal Permease in Chlamydia Trachomatis Yields an Iron-dependent Transcriptional Repressor
Thompson, C., et al., Proc Natl Acad Sci USA, 109 (26), 10546-10551, 2012
The authors demonstrate that the C-terminal domain of the CT069 protein in Chlamydia trachomatatis serves as an iron-dependent DNA-binding repressor protein. The Octet QKe system was used to quantitatively characterize binding of the repressor domain of the CT069 protein to a DNA operator sequence. The biotinylated DNA fragment was immobilized using Streptavidin biosensors, and then incubated in solutions containing the repressor domain supplemented with various divalent metals. Only the addition of Fe2+ led to DNA binding. The Octet system then was used to characterize iron-dependent DNA-protein binding kinetics and the dissociation constant.
PubMed
Differential Polyubiquitin Recognition by Tandem Ubiquitin Binding Domains of Rabex-5
Shin, D., et al., Biochem Biophys Res Commun, 423(4), 757-62, 2012
Mutations were introduced in the two ubiquitin binding domains (A20_ZF and MIU) of Rabex-5 protein in an effort to characterize Rabex-5 binding affinities and selectivity for polyubiquitin structures. Tetraubiquitin chains were immobized by coupling to Amine Reactive biosensors on the Octet RED system. Kinetic constants for wild-type and mutant Rabex 5 domain binding to polyubiquitin structures were reported.
PubMed
Gambogic Acid Activates AMP-activated Protein Kinase in Mammalian Cells
Zhao, B., et al., Biochem Biophys Res Commun, 424(1), 100-4, 2012
Gambogic acid (GB), a known antitumor agent, activates AMP-activated protein kinase (AMPK) by increasing the phosphorylation of AMPKa and its downstream substrate in various cell lines. GB does this by physically interacting with the AMPKa subunit as measured by Octet RED system with a Kd of 7.81uM (similar Kd determined by quenching experiments). Kinetics measurements were performed using biotinylated AMPKa subunit immobilized onto Super Streptavidin biosensors. Association and dissociation rate constants were reported.
PubMed
Domain Activities of PapC Usher Reveal the Mechanism of Action of an Escherichia coli Molecular Machine
Volkan, E, et al., Proc Natl Acad Sci USA, 109 (24), 9563-9568, 2012
The Octet RED system was used to investigate the chaperone-usher pathway used by Gram-negative pathogens. Binding interactions of the periplasmic PapC functional domains NTD, CTD2, and Plug were studied to gain insight into the molecular basis of usher-catalyzed pilus biogenesis and how these domains function as a molecular machine in E.coli. All interaction experiments were carried out by capturing the biotinylated ligands (PapC or chaperone-subunit complexes) onto the Super Streptavidin biosensors and then dipping into specific binding partners at various concentrations.
PubMed
p53/HMGB1 Complexes Regulate Autophagy and Apoptosis
Livesey, K.M., et al., Cancer Research, 72(8), 1996-2005, 2012
Direct interaction of HMGB1 and p53 was evaluated in a cell-free system using the Octet QK system. Recombinant HMGB1 was immobilized onto Amine Reactive and Streptavidin biosensors; association and dissociation curves to recombinant p53 were generated to determine the affinity constant. KD values of the reverse orientations were very similar on both the sensors. Oxidation of HMGB1 had minimal effect whereas reduction of p53 abrogated interaction with HMGB1.
PubMed
Structure and Receptor Complexes of the Hemagglutinin from a Highly Pathogenic H7N7 Influenza Virus
Yang, H., et al., Journal of Virology, 86(16), 8645-52, 2012
A H7N7 Influenza virus isolated from a fatal case (NL219) caused a lethal infection in mouse models. A mutation which introduces a potential glycosylation site in the NL219 hemagglutinin was postulated to contribute to its pathogenicity. Kinetic analysis were performed using Octet RED system to compare the receptor binding profiles of the wild-type recombinant NL219 hemagglutinin to a variant with a threonine-to-alanine mutation at position 125, resulting in loss of the glycosylation site. The results suggest that the additional glycosylation sequon increased binding affinity relative to avian-type a2-3-linked sialosides and not to human type a2-6-linked sialosides. The binding affinity was measured by immobilizing biotinylated glycans onto the Streptavidin Biosensor, and then dipping into recombinant hemagglutinin (WT and mutant versions).
PubMed
Recombinant Fab Expression and Secretion in Escherichia Coli Continuous Culture at Medium Cell Densities: Influence of Temperature
Carmona, E. et al., Process Biochemistry, 2012, 47 (3), 446-452, 2012
The aim of this work was to study the production of a model Fab in an E. coli continuous culture at medium cell densities. Temperature was chosen as the working parameter to perform a systematic analysis of the effect of operational factors on the process behavior at cell densities closer to those typically used under bioprocess conditions. An Octet QK system with Streptavidin biosensors was used to characterize Fab fragments produced at different temperatures for functional binding activity.
PubMed
Human B-cell Ontogeny in Humanized NOD/SCID γcnull Mice Generates a Diverse Yet Auto/Poly-and HIV-1-reactive Antibody Repertoire
Chang, H., et al., Genes and Immunity, 13(5), 399-410, 2012
In this study, analysis of V-H and V-kappa gene arrangements in humanized NOD/SCID mice-derived single human B cells sorted at different developmental stages was performed. The Octet RED system was used for affininty measurements of hNSG-derived scFvFcs to HIV-1gp140. Abs were loaded on Protein A biosensors and exposed to recombinant HIV-1gp140 solutions to determine kinetic binding constants.
PubMed
Identifying Bottlenecks in Transient and Stable Production of Recombinant Monoclonal-antibody Sequence Variants in Chinese Hamster Ovary Cells
Mason, M., et al., Biotechnology Progress, 28(3), 846-855, 2012
The cellular mechanisms that control expression and the relationships between antibody sequence and expression level remain poorly understood. In this study, the authors examined an antibody pair in which a single amino acid substitution impacted antibody expression without altering the innate antibody function (e.g., antigen binding). Assays for antibody titer were performed on an Octet QK system with Protein A biosensors over a working range of 1-700 μg/mL.
PubMed
Engineering an Improved IgG4 Molecule with Reduced Disulphide Bond Heterogeneity and Increased Fab Domain Thermal Stability
Peters, S., et al., J Biological Chemistry, 287(29), 24525-24533, 2012
The authors present an IgG4 molecule with increased Fab thermal stability and reduced product heterogeneity that potentially offers advantages for the production of IgG4 molecules. The Octet system with Protein A biosensors was used to determine expression levels for IgG1 wild-type, IgG4 wild-type and IgG4 mutants.
PubMed
Estimation of Raw Material Performance in Mammalian Cell Culture Using Near infrared Spectra Combined with Chemometrics Approaches
Lee, H.W, et al., Biotechnology Progress, 28(3), 824-832, 2012
Soy hydrolysate is a commonly used supplement in mammalian cell cultures, and often exhibits considerable variability in its growth-promoting and production- enhancing activities due to the compositional variability. In this study, both near-infrared spectra and bioassays of multiple soy lots were analyzed using the approaches of multivariate data analysis. Cultures were harvested after 7 days of incubation for IgG titer analysis using an Octet QK system. The Octet system titer values were used as one of the quantifiable culture performance indices throughout the study.
PubMed
Structure of Bradavidin - C-Terminal Residues Act as Intrinsic Ligands
Leppiniemi, J., et al., PLoS ONE, 7(5), e35962, 2012
The biotin-binding pocket occupying peptide (Brad-tag) serves as an intrinsic stabilizing ligand in wild-type bradavidin. The binding of Brad-tag to core-bradavidin was analysed by isothermal titration calorimetry and on the Octet RED 384 system. Anti-Penta HIS biosensors were used to immobilize His-tagged Brad-tag for these studies.
PubMed
Secretion of the Housekeeping Protein Glyceraldehyde-3-phosphate Dehydrogenase by the LEE-encoded Type III Secretion System in Enteropathogenic Escherichia coli
Aguilera, L., et al., Int J Biochem Cell Biol, 44(6), 955-962, 2012
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional housekeeping protein secreted by pathogens and involved in adhesion and/or virulence. The authors demonstrate a novel interaction between GAPDH and the chaperone CesT by means of pull down experiments, overlay immunoblotting and BLI . The Octet RED system with Amine Reactive biosensors was used to study the GAPDH-Cest interaction. Biosensors with immobilized CesT were exposed to various concentrations of purified GAPDH (0-5.5 micromolar).
PubMed
The Protein Zfand5 Binds and Stabilizes mRNAs with AU-rich Elements in Their 3 prime Untranslated Regions
He, G., et al., J Biological Chemistry, 287(30), 24967-24977, 2012
Identification of novel factors that are involved in regulating cytokine mRNA stability and capable of attenuating host inflammatory responses offers potentially important insights into the pathological basis of diverse inflammatory diseases, and could aid in the development of therapeutic strategies. This study reports that Zfand5 stabilizes class II ARE-mRNAs by binding directly to the ARE-mRNA and competing with tristetraprolin (TTP), a zinc finger-containing protein that destabilizes mRNAs with Class II AREs. The affinity constants for binding of Zfand5/ARE and TTP/ARE were determined using the BLItz system. Biotinylated ARE-RNA was immobilized on Streptavidin biosensors for these experiments.
PubMed
Protuboxepin A, a Marine Fungal Metabolite, Inducing Metaphase Arrest and Chromosomal Misalignment in Tumor Cells
Asami, Y., et al., Bioorganic & Medicinal Chemistry, 20(12), 3799-3806, 2012
The authors identified protuboxepin A as a microtubule-stabilizing agent which has a distinctly different chemical structure from previously reported microtubule inhibitors. This publication reports Octet system data for binding of protuboxepin A to tubulin. The reported results indicate that protuboxepin A has potential for being a new and effective anti-cancer drug.
PubMed
Enzyme Kinetics and Interaction Studies for Human JNK1[beta]1 and Substrates ATF2 And cJun
Figuera-Losada, M.; LoGrasso, P.V., J Biological Chemistry, 287(16), 13291-13302, 2012
The authors report on the binding mechanism of c-Jun N-terminal kinase (JNK) with transcription factors ATF2 and c-Jun. Interaction affinity values for these proteins are reported. Both the phosphorylation state of the kinase and the presence of ATP significantly affected the kinetics of association and dissociation of JNK1 1 and its substrates, and therefore the affinity for the interactions, as elucidated by Octet RED system assay data.
PubMed
Heterosubtypic Antibody Recognition of the Influenza Virus Hemagglutinin Receptor Binding Site Enhanced by Avidity
P.S. Lee, et al., Proc Natl Acad Sci USA, 109(42), 17040-5, 2012
Using the Octet RED system, Kd values for S139/1 Fab and IgG binding to various influenza HA strains were determined to assess enhanced virus neutralization effects due to avidity of full IgG vs. Fab. Biotinylated HA and Streptavidin biosensors were used for these measurements. The increased avidity of IgG binding was shown to reduce Kd values and improve ability of S139/1 to recognize different heterosubtypes of the virus. The Kd data correlated with neutralization data.
PubMed
Self-antigen Recognition by Follicular Lymphoma B Cell Receptors
K.L. Sachen, et al. , Blood, 120(20), 4182-90, 2012
To investigate the hypothesis that self-antigen recognition is involved in the pathogenesis of follicular lymphoma, the authors used recombinant tumor IgG's and evaluated reactivity to human tissue antigens. A self antigen recognized by the B-cell receptor from a patient's tumor, myoferlin, was used to perform clonal analysis of antigen recognition and test the ability to transduce signals through the tumor BCR. The Octet QK system was used for equilibrium affinity measurements for binding of a follicular lymphoma tumor IgG to the self-antigen myoferlin. Streptavidin biosensors loaded with biotinylated goat anti-HA were used to capture recombinant myoferlin-HA for the binding studies.
PubMed
Cross-neutralization of Influenza A Viruses Mediated by a Single Antibody Loop
D.C. Ekiert, et al. , Nature, 489(7417), 526-32, 2012
An antibody (C05) that neutralized strains from multiple subtypes of influenza A virus was isolated by phage display . Functional and structural characterization showed that C05 neutralized multiple subtypes by inserting a single extended CDR loop into the receptor binding pocket of the HA1 subunit. The Octet RED system and ITC were used to measure KDs for C05 Fab binding do a panel of influenza HA subtypes. Biotinylated HAs and Streptavidin biosensors were used for the binding studies.
PubMed
Performance Characteristics of Monoclonal Antibodies as Recyclable Binders to Cardiac Troponin I
D.H. Kim, et al., Analytical Biochemistry, 431(1), 11-8, 2012
A method was developed to screen for rapidly reversible antibodies from 22 hybridoma clones against cardiac troponin 1 (cTn1). Antibodies were characterized based on association and dissociation kinetics using the Octet RED system and APS biosensors. A rapidly dissociating antibody was selected and shown to be recyclable. This antibody was used to continuously monitor analyte concentration by recycling the antibody as capture agent on a microfluidic sensor.
PubMed
A Simple and Rapid Detection of Viral Protein Using RNA Oligonucleotide in a Biosensor
C. Roh, S. Kim, S. Jo, J Analytical Chemistry, 67(11), 925-929, 2012
Current HCV detection methods using ELISA are limited by temperature and condition sensitivity, false negatives, and inability to detect at early stages of infection. In this publication, the authors develop an HCV detection method on the Octet RED system using biotinylated RNA oligonucleotides immobilized on Streptavidin biosensors. The method specifically detected HCV viral protein with high sensitivity (to 500 pg/mL).
Springer
Structural Insights into SUN-KASH Complexes Across the Nuclear Envelope
W. Wang, et al., Cell Research, 22(10), 1440-1452, 2012
Structural, biochemical and cell-based studies of the SUN-KASH complex spanning the inner and outer nuclear membranes were performed. After determining the overall structure of the complex, similarity between the complex and the apo-SUN domain was examined as well as the interface between the SUN domain trimer and KASH peptides. Mutational analysis was performed to probe the contributions of individual amino acids to complex formation. The Octet RED96 system and Streptavidin biosensors were utilized for kinetic analysis of wild-type vs mutant SUN domains and KASH domain. The Octet system was also used to determine the affinity of the SUN2 luminal region containing different coiled-coil motifs for the KASH domain.
PubMed
Changes in the Oligomerization Potential of the Division Inhibitor UgtP Co-ordinate Bacillus Subtilis Cell Size with Nutrient Availability
A.C. Chien, et al., Molecular Microbiology, 86(3), 594-610, 2012
The glycotransferase UgtP interacts directly with cell division protein FtsZ to inhibit cell division and increase cell size in Bacillus subtilis. Nutrient-dependent changes to UgtP's potential to oligomerize or bind to FtsZ were found to be regulated by the presence of FtsZ and by levels of UDP-glucose. The Octet RED96 system was used to identify difference in affinity of UgtP for itself in the presence and absence of UDP-glucose using biotinylated Thio-UgtP and Streptavidin biosensors.
PubMed
1-(2,6-Dibenzyloxybenzoyl)-3-(9H-fluoren-9-yl)-urea: A Novel Cyclophilin A Allosteric Activator
M. Lv, et al. , Biochem Biophys Res Commun, 425(4), 938-943, 2012
A small molecule compound 1-(2,6-Dibenzyloxybenzoyl)-3-(9H-fluoren-9-yl)-urea (1a) was found to be an allosteric activator of human CypA, which plays an important role in many physiological processes including protein folding, transportation, immune modulation and cell signaling. Compound 1a was screened by PPIase activity and binding affinity assay using the Octet RED system and Super Streptavidin biosensors. Its effect on ERK phosphorylation was tested in cells. The allosteric binding site and mechanism of compound 1a were also analyzed by mutagenesis in combination with molecular dynamics simulations.
PubMed
Selection and Maturation of Antibodies by Phage Display Through Fusion to pIX
M. Tornetta, R. Reddy, J.C. Wheeler, Methods, 58(1), 34-9, 2012
Functional assessment of phage-derived antibodies can be hindered by low affinities or lack of epitopic diversity. The authors describe an approach to managing primary hits from pIX-mediated phage libraries into epitope bins using the Octet system and Amine Reactive biosensors, followed by high-throughput maturation of clones to isolate epitope- and sequence-diverse panels of high-affinity binders.
PubMed
Structure-Guided Alterations of the gp41-directed HIV-1 Broadly Neutralizing Antibody 2F5 Reveal New Properties Regarding its Neutralizing Function
J. Guenaga, R.T. Wyatt, PLoS Pathogens, 8(7), e1002806, 2012
Mutagenesis and functional analysis of the broadly neutralizing HIV-1 antibody 2F5 was performed. Data were presented describing a new property of the antibody, an ability to first bind downstream of its known core epitope in a two-step process not directly involving the lipid membrane. The Octet RED system was utilized to determine binding affinities to MPER peptides to test variations in length of core binding region and to determine whether 2F5 antibody might interact with residues downstream of its core epitope. For these binding studies, 2F5 wild-type and variant antibodies were immobilized using Anti-Human IgG Fc Capture (AHC) biosensors.
PubMed
Existence of Separate Domains in Lysin PlyG for Recognizing Bacillus anthracis Spores and Vegetative Cells
H. Yang, et al., Antimicrob Agents Chemother, 56(10), 5031-5039, 2012
A 60 amino acid domain of the potent anti-microbial bacteriophage lysin plyG was shown to selectively recognize Bacillus anthracis spores but not vegetative cells. This discovery has implications in identfying new biomarkers for identfiying Bacillus anthracis spores and in developing plyG as a preventive antibiotic to reduce the threat of anthrax in suspected exposures to spores. The Octet system was used to assess binding of various protein domains to biotinylated Bacillus anthracis spores immobilized on Streptavidin biosensors.
PubMed
Bacillus anthracis Inosine 5'-Monophosphate Dehydrogenase in Action: The First Bacterial Series of Structures of Phosphate Ion-, Substrate-, and Product-Bound Complexes
M. Makowska-Grzyska, et al. , Biochemistry, 51(31), 6148-6163, 2012
IMPDH inhibitors have broad clinical applications in cancer treatment, antiviral drug development, and as immuno-supressants. The authors have determined three crystal structures for Bacillus anthracis IMPDH. The newly identified structures include a phosphate ion -bound form (apoenzyme), in complex with its substrate, and in complex with its product. Kinetics of enzymatic reaction of the apoenzyme were also determined, as well as inhibitory effects of two compounds, XMP and MPA. The Octet RED system was utilized to analyze binding of inhibitors to biotinylated IMPDH proteins with Super Streptavidin biosensors.
PubMed
Antibacterial Activity and Mechanism of a Scorpion Venom Peptide Derivative In Vitro and In Vivo
L. Cao, et al., PLoS One, 7(7), e40135, 2012
The authors derived a peptide (Kn2-7) from the scorpion venom antimicrobial peptide BmKn2. The Kn2-7 peptide showed increased antimicrobial activity and decreased hemolytic activity relative to BmKn2. Results from in vitro and in vivo assays indicated that Kn2-7 shows promise as a topical therapeutic agent for treating bacterial infections. The Octet RED system was used to identify the targets for Kn2-7 in S. aureus and E. coli. Streptavidin biosensors and biotinylated Kn2-7 were used for the binding studies.
PubMed
Do GGA Adaptors Bind Internal DXXLL Motifs?
B. Doray, et al., Traffic, 13(10), 1315-1325, 2012
GGA family of clathrin adaptor proteins mediates intracellular trafficking of transmembrane proteins by interaction with DXXLL sorting signals. Recent studies have challenged whether GGA adaptors bind to internal DXXLL signals as an auto-inhibitory mechanism or only to C-terminal DXXLL signals. The authors confirmed in vitro binding between internal DXXLL motif and GGA2 using MBP-peptide fusions on the Octet RED system. Purified mouse Flag-GGA2 was biotinylated and immobilized on Streptavidin biosensors for the Octet binding studies. These data were confirmed using modeling studies and GST pull-down assays. Binding was shown to be modulated by amino acids surrounding the DXXLL motif.
PubMed
Identification of Anti-Alpha Toxin Monoclonal Antibodies that Reduce the Severity of Staphylococcus Aureus Dermonecrosis and Exhibit a Correlation Between Affinity and Potency
C Tkaczyka, et.al., Clin Vaccine Immunol, 19(3), 377-385, 2012
Anti-alpha toxin MAbs were generated using hybridoma technology and characterized as inhibitors of alpha toxin function in Staphylococcus aureus infections. Binding kinetics and epitope competition were evaluated using Biacore and Octet systems. For epitope competition experiments, native alpha toxin was biotinylated and immobilized on Streptavidin biosensors on the Octet system.
PubMed
MAP Kinases Bind Endothelial Nitric Oxide Synthase
Chrestensen, C.A., McMurry, J.L., Salerno, J.C., FEBS Open Bio, 2, 51-55 , 2012
Endothelial nitric oxide synthase (eNOS) is an important signaling molecule involved in control of vascular tone, insulin secretion, and angiogenesis, and contains a motif similar to recognition sequences in known MAPK binding partners. Here the Octet QK system was used to demonstrate MAP kinases ERK1/2 and p38 binding to eNOS, and characterize binding kinetics of these interactions. Also demonstrated was inhibition of p38-eNOS binding by Calmodulin. Biotinylated ERK and p38 kinases were immobilized on Streptavidin biosensors for the binding studies.
ScienceDirect
A Recombinant Clumping Factor A-containing Vaccine Induces Functional Antibodies to Staphylococcus aureus that are not Observed after Natural Exposure
Hawkins J, et al., Clin Vaccine Immunol, 19(10), 1641-50, 2012
ClfA (clumping factor A), a conserved fibrinogen (Fg) binding protein, is being investigated as a possible component of vaccine to Staphylococcus aureus. The authors have developed an assay for ClfA mediated Fg binding, that can be used to measure biologically relevant immune response in mouse and human sera. A panel of anti-ClfA monoclonal antibodies was also characterized. The Octet RED96 system and Anti-Murine IgG biosensors were used to perform epitope mapping of the mAb's against the ClfA antigen and to determine mAb-ClfA kinetic constants.
PubMed
Mapping the HLA-DO/HLA-DM Complex by FRET and Mutagenesis
Yoon T, et al., Proc Natl Acad Sci USA, 109(28), 11276-81, 2012
The authors characterize a putative binding surface and an overall orientation for HLA-DO inhibition of HLA-DM, two key molecules in the class II presentation pathway. HLA-DO/HAL-DM complexes were analyzed by FRET and mutagenesis, resulting in identification of an apparent binding site. Stable mutant HLA-DO proteins were produced by mutation for these studies. The Octet system and Streptavidin biosensors were used to determine KD values of mutant HLA-DO proteins binding to HLA-DM.
PubMed
Structure and Receptor Complexes of the Hemagglutinin from a Highly Pathogenic H7N7 Influenza Virus
Yang, H., et al., Journal of Virology, 86(16), 8645-52, 2012
A H7N7 Influenza virus isolated from a fatal case (NL219) caused a lethal infection in mouse models. A mutation which introduces a potential glycosylation site in the NL219 hemagglutinin was postulated to contribute to its pathogenicity. Kinetic analysis were performed using Octet RED system to compare the receptor binding profiles of the wild-type recombinant NL219 hemagglutinin to a variant with a threonine-to-alanine mutation at position 125, resulting in loss of the glycosylation site. The results suggest that the additional glycosylation sequon increased binding affinity relative to avian-type a2-3-linked sialosides and not to human type a2-6-linked sialosides. The binding affinity was measured by immobilizing biotinylated glycans onto the Streptavidin Biosensor, and then dipping into recombinant hemagglutinin (WT and mutant versions).
PubMed
Development and Characterization of a Novel C-terminal Inhibitor of Hsp90 in Androgen Dependent and Independent Prostate Cancer Cells
Eskew J, et al., BMC Cancer, 11:468, 2011
Development of a Highly Specific Amine-Terminated Aptamer Functionalized Surface Plasmonresonance Biosensor for Blood Protein Detection
Zheng R, et al., Biomed Opt Express, 2(9):2731-40, 2011
Mutagenesis of Surfactant Protein D Informed by Evolution and X-ray Crystallographyenhances Defenses Against Influenza A Virus In Vivo
Crouch E, et al., J Biol Chem, 286(47):40681-92, 2011
Nanocapsules Incorporating Igg Fc-Binding Domain Derived from Staphylococcus Aureus Protein A for Displaying Iggs on Immunosensor Chips
Iijima M, et al., Biomaterials, 32(6):1455-64, 2011
A Broadly Neutralizing Human Monoclonal Antibody that Recognizes a Conserved, Novel Epitope on the Globular Head of the Influenza H1N1 Virus Hemagglutinin
Krause, J.C., et al., Journal of Virology, 85(20), 10905-10908, 2011
The conserved influenza virus hemagglutinin (HA) stem domain elicits cross-reactive antibodies, but epitopes in the globular head typically elicit strain-specific responses because of the hypervariability of this region. The authors isolated human monoclonal antibody 5J8 that neutralized a broad spectrum of 20th century H1N1 viruses and the 2009 pandemic H1N1 virus. Detailed mapping of the interaction unexpectedly revealed a novel epitope on HA. Binding affinities of 5J8 Fab to recombinant trimeric His-tagged HA proteins were determined using Anti-Penta-HIS biosensors on an Octet RED instrument.
PubMed
Identification of Conformational Core Epitope Lys68 in C5a Based on the 3-D Modeling Complex C5a and its Functional Antibody F20
Wei, H., et al., Molecular Immunology, 48(12-13), 1377-1383, 2011
C5a is a naturally occuring peptide with potent inflammatory properties. Inhibition of C5a by antibodies has been demonstrated to dramatically improve survival in various sepsis models in mice and rats. The structural basis of C5a mediated bioactivity and C5a antibody mediated neutralization was investigated in this article. Octet RED platform binding assays were performed between anti-C5a antibodies and C5a mutants to investigate the structural basis of C5a bioactivity. Anti-C5a antibody F20 was loaded onto Anti-Mouse IgG Capture biosensors for these binding experiments.
PubMed
Divergent Activities Of Osteogenic BMP2, And Tenogenic BMP12 And BMP13 Independent Of Receptor Binding Affinities
Berasi, S.P., et al., Growth Factors, 29(4), 128-139, 2011
Bone morphogenic proteins (BMPs) are growth factors that can induce bone and cartilage formation in vitro and in vivo. Some BMP subtypes induce bone formation while other subtypes induce cartilage and tendon formation. Both classes of BMPs initiate intracellular signaling through interactions with the same receptors, but produce such different responses. In this article, the Octet RED platform was used to study the affinity of interaction of various BMPs to different receptors to identify differences in binding behavior that may help explain the differences in behavior of BMP subclasses. Receptor Fc chimeras were immobilized on Anti-Human IgG Fc biosensors and incubated with purified BMPs for the affinity studies.
PubMed
Detection of Deoxynivalenol using Biolayer Interferometry
Maragos, C.M., Mycotoxin Research, 27, 157-165, 2011
Deoxynivalenol (DON) is a toxin produced by Fusarium graminearum and F. culmorum. These fungi can infest wheat, barley, and corn and cause Fusarium Head Blight, a disease of substantial economic significance worldwide. Because of this, and the toxic nature or DON, extensive monitoring is conducted of commodities and foods. As part of efforts to improve detection of the toxin, the author examined the applicability of the Octet RED system. DON spiked in whole wheat flour was successfully measured, suggesting further development of a rapid quantitative Octet assay for DON in wheat may be possible. For these studies, a DON-BSA conjugate was non-covalently immobilized on aminopropylsilane (APS) biosensors.
Springer
Recognition of Ubch5c and the Nucleosome by the Bmi1/Ring1b Ubiquitin Ligase Complex
Bentley, M.L., et al., The EMBO Journal, 30(16), 3285-3297, 2011
This publication studies the transcriptional repressor proteins Bmi1 and Ring1b, and characterizes their interaction with the E2 enzyme UbcH5c and the nucleosome. The Octet RED platform and Streptavidin biosensors were used. Biotinylated Bmi1/Ring1b complexes (wild type and mutants) were immobilized, and association/dissociation kinetic with UbcH5c were determined.
PubMed
Inhibitors of Androgen Receptor Activation Function-2 (AF2) Site Identified through Virtual Screening
Axerio-Cilies, P., et al., Journal of Medicinal Chemistry, 54 (18), 6197-6205, 2011
This study employs computer-aided design to identify small-molecule androgen receptor inhibitors that specifically target the receptor's activation function-2 (AF2) site. Interactions between the inhibitors and the androgen receptor were quantified using the Octet RED platform. Biotinylated androgen receptor LBD was immobilized on Super Streptavidin biosensors and used to characterize direct binding of the small-molecule inhibitors.
PubMed
Characterization of the Human Folate Receptor Alpha via Novel Antibody-Based Probes
O, Oncotarget, 2 (12) 1227-1243, 2011
This publication describes the development of high-affinity mAbs to the human folate receptor alpha (FRA). Epitope binning studies on the Octet QK system were performed to identify mAbs that cross-compete for FRA binding. Biotinylated rFRA was loaded onto Streptavidin biosensors, followed by incubation with a primary saturating anti-FRA mAb. The biosensors then were moved to wells containing secondary competing anti-FRA mAbs.
PubMed
The Structural and Functional Basis of the p97/Valosin-containing Protein (VCP)-interacting Motif (VIM) Mutually Exclusive Binding of Cofactors to the N-Terminal Domain of P97
Hanzelmann, P. and Schindelin, H., Journal of Biological Chemistry, 286(44), 38679-38690, 2011
This study uses BLI and other techniques to study the interaction between p97/VCP and the VCP-interacting motif (VIM), a domain found in several cofactors for p97/VCP. The Octet RED platform with Streptavidin biosensors was used. Biotinylated VIM peptides were loaded onto biosensors to determine kinetic rate constants for the interaction. BLI also was used for detailed mutational analysis of the VIM motif.
PubMed
Functional and Structural Studies of the Nucleotide Excision Repair Helicase XPD Suggest a Polarity for DNA Translocation
Kuper, J., et al., The EMBO Journal, 31(2), 494-502, 2011
The subject of this study is the XPD, a critical protein in transcription and nucleotide excision repair. The authors determine the structure of an XPD protein complexed with a short DNA fragment, and then perform mutational and biochemical analysis of XPD to define details of the XPD-ssDNA interaction. The Octet RED platform was used to determine protein-ssDNA binding constants for wild-type XPD and a panel of variants harboring point mutations. Biotinylated ssDNA was immobilized onto Streptavidin biosensors for these studies.
PubMed
Characterization of Crude Echis Carinatus Venom-induced Cytotoxicity in HEK 293T Cells
Pierce, R.D., et al., Journal of Venom Research, 2, 59-67, 2011
This study explores the cytotoxic responses caused by Echis carinatus (saw-scaled viper) venom. To study cellular attachment effects, polyethyleneimine (PEI) was immobilized onto Amine Reactive biosensors. Specific binding between fetal calf serum and the immobilized PEI was characterized using the Octet QK system.
PubMed
A Stable IgG-like Bispecific Antibody Targeting the Epidermal Growth Factor Receptor and the Type I Insulin-like Growth Factor Receptor Demonstrates Superior Anti-Tumor Activity
Dong J., et al., Mabs, 3(3), 273-88, 2011
The epidermal growth factor receptor (EGFR) and the type I insulin-like growth factor receptor (IGF-1R) are two cell surface receptor tyrosine kinases known to cooperate to promote tumor progression. This publication reports on the development and characterization of a stable IgG-like bispecific antibody dual-targeting EGFR and IGF-1R. IGF-R1 binding assays were performed on the Octet RED platform. The biospecific antibody was captured on Anti-Human IgG Fc biosensors and incubated with different concentrations of the IGF-1R ectodomain.
PubMed
Impact of Gene Vector Design on the Control of Recombinant Monoclonal Antibody Production by Chinese Hamster Ovary Cells
Davies, S.L., et al., Biotechnology Progress, 27 (6), 1689-1699, 2011
In this paper, two vector design strategies for monoclonal antibody synthesis in CHO cells were compared. A dual open reading frame (ORF) expression vector utilizing separate CMV promoters to drive heavy and light chain expression independently produced higher antibody titers than a single ORF vector design employing a single promoter. Stable recombinant antibody titer from the 100 transfectants was analyzed using an Octet QK system equipped with Protein A biosensors.
PubMed
Purification of Tetracysteine-Tagged Proteins by Affinity Chromatography Using a Non-Fluorescent, Photochemically Stable Bisarsenical Affinity Ligand
Ying, L.Q.; Branchaud, B.P., Bioconjugate Chemistry, 22 (5), 987-992, 2011
Thei article describes affinity purification of tetracysteine (CCXXCC) tagged proteins using the nonfluorescent, photochemically stable bisarsenical affinity ligand SplAsH. The Octet instrument was used to measure the binding affinity of SplAsH and tetracysteine-tagged GFP. SplAsH-biotin 5 was immobilized on Streptavidin biosensors and then incubated with purified tetracysteine-tagged GFP. Relative to the interaction between His tag and Ni-NTA, SplAsH showed >1000-fold higher affinity and thus should be much more useful for the capture and purification of low-abundance proteins.
PubMed
A TPO Receptor Agonist, ALXN4100TPO, Mitigates Radiation-Induced Lethality and Stimulates Hematopoiesis in CD2F1 Mice
Satyamitra, M., et al., Radiation Research, 175 (6), 746-758, 2011
A TPO agonist peptide was grafted into the human antibody against anthrax, resulting in a series of 4100TPO agonist variants with utility as radiation countermeasure agents. The binding kinetics of the variants with the human TPO receptor were quantified using the Octet RED platform by immobilizing biotinylated c-Mpl-r on Streptavidin biosensors and then characterizing association and dissociation rates for the different variants.
PubMed
Integrin Binding Human Antibody Constant Domains - Probing the C-terminal Structural Loops for Grafting the RGD Motif
Traxlmayr, M.W., et al., J Biotechnology, 155(2), 193-202, 2011
In this paper, C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc were functionalized to form integrin-binding sites. The impact of this structural change upon IgG1-Fc structural integrity, thermal stability and binding to the ligands Protein A, CD16 and FcRn was probed. Protein A binding for each IgG1-Fc variant was characterized using the Octet QK system with Protein A biosensors.
PubMed
TMEFF2 is a PDGF-AA Binding Protein with Methylation-Associated Gene Silencing in Multiple Cancer Types Including Glioma
Lin, K., et al., PLoS ONE, 6(4), e18608, 2011
TMEFF2 is a protein containing a single EGF-like domain and two follistatin-like modules. The biological function of TMEFF2 remains unclear with conflicting reports suggesting both a positive and a negative association between TMEFF2 expression and human cancers. Here the authors report that the extracellular domain of TMEFF2 interacts with PDGF-AA. Octet platform binding assays were performed to validate the results obtained by ELISA, confirming presence of specific binding between TMEFF2 and PDGF-AA.
PubMed
The Peroxisomal Targeting Signal 1 in Sterol Carrier Protein 2 is Autonomous and Essential for Receptor Recognition
Williams, C.P., et al. , BMC Biochemistry, 12, 12, 2011
The majority of peroxisomal matrix proteins destined for translocation into the peroxisomal lumen are recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. In this study, the contribution of a second receptor-cargo binding motif, SCP2, was investigated. The Octet RED platform was used to determined interaction kinetics between Pex5p(C) wild-type & mutants and four SCP2 variants. Biotinylated Pex5p(C) proteins were immobilized on Streptavidin biosensors for these measurements.
PubMed
Exploiting Nucleotide Composition to Engineer Promoters
Grabherr, M.G., et al., PLoS ONE, 6(5), e20136, 2011
Artificial promoters that provide stable expression across cell lines and can be designed to the desired strength constitute an alternative to the use of viral promoters. This study demonstrates how the nucleotide characteristics of highly active human promoters can be modelled via the genome-wide frequency distribution of short motifs. The Octet QK system was used to characterize binding of the transcription factors TFIIB and TBP to artificial promoter constructs. Streptavidin biosensors were loaded with 5'-biotinylated DNA fragments of various promoter constructs for these experiments.
PubMed
Role of Iron and Sodium Citrate in Animal Protein-Free CHO Cell Culture Medium on Cell Growth and Monoclonal Antibody Production
Bai, Y., et al., Biotechnology Progress, 27(1), 209-219, 2011
The trend for biologics manufacturing is to reduce the use of animal-derived raw material to minimize potential risk of introducing contaminating agents in the final product. Iron plays a critical role in supporting healthy cell growth. In this work, chemically defined iron compounds were investigated for the development of animal protein-free cell culture media to support growth of CHO cells and production of monoclonal antibodies (mAb). MAb production in 96-well plates was analyzed by an Octet system using protein A biosensors.
PubMed
Chaperone Activity of alpha β-Crystallin Is Responsible for Its Incorrect Assignment as an Autoantigen in Multiple Sclerosis
Rothbard, J.B., et al., J Immunology, 186(7), 4263-4268, 2011
The authors study the role of alpha β-crystallin (also called Hsp B5) as an autoantigen in Multiple Sclerosis, an autoimmune disease. Octet platform binding assays were performed to show that binding of anti-HspB5 Abs from 23 MS patients binds not only HspB5, but cross-react with 7 other members of the Hsp family, and, that the binding is temperature dependent. This supports the authors' hypothesis that HspB5 binds antibodies more generally than believed and that this protein is likely not an autoantigen involved in MS.
PubMed
Label Free Inhibitor Screening of Hepatitis C Virus (HCV) NS5B Viral Protein Using RNA Oligonucleotide
Roh, C.; Kim, S.E.; Jo, SK., Sensors (Basel), 11(7), 6685-6696, 2011
The authors demonstrate that the hepititis C viral protein NS5B can be detected and monitored using an immobized RNA aptamer on the Octet QK system. The biotinylated RNA strand was loaded onto Streptavidin biosensors and incubated with a dilution series of NS5B protein. A detection limit of 700 pg/mL was reported.
PubMed
An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody
Krause, J.C., et al., MBio, 2(1), e00345-10, 2011
The authors demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. Comparison of Octet QK system binding data for wild-type 2D1 and del 2D1 mutant binding to hemagglutinin showed that the mutant not harboring the insertion had a 35-fold faster dissociation rate. HIS-tagged hemagglutinin protein was immobilized with anti-Penta-HIS biosensors for these mesurements.
PubMed
Formation of Raloxifene Homo-dimer in CYP3A4, Evidence for Multi-substrate Binding in a Single Catalytically Competent P450 Active Site
Davis, J.A., et al., Arch Biochem Biophys, 513(2), 110-118, 2011
Studies were carried out to understand the interactions the polyaromatic compound raloxifene with CYP3A4, and how those interactions relate to raloxifene homo-dimer formation in vitro. Kinetics of raloxifene/CYP3A4 binding were characterized using the Octet RED platform and biotinylated CYP3A4 immobilized on Super Streptavidin biosensors. Data suggested a 2:1 binding relationship between raloxifene and CYP3A4, supporting the conclusion that raloxifene forms a homo-dimer within the CYP3A4 active site.
PubMed
Production of Rapidly Reversible Antibody and its Performance Characterization as Binder for Continuous Glucose Monitoring
Paek, S.H., et al., Analyst, 136, 4268-4276, 2011
The authors report on the development of a continuous glucose monitoring immunosensor based upon antibodies raised against dextran-protein conjugates. The Octet RED platform was used to determine association and dissociation constants for antibody and concanavalin A binding to immobilized dextran-BSA conjugates using APS biosensors.
PubMed
Lysine Acetylation is a Widespread Protein Modification for Diverse Proteins in Arabidopsis
Wu, X., et al., Plant Physiology, 155(4), 1769-1778, 2011
The authors describe a reversible protein posttranslational modification called Lysine Acetylation (LysAc), that is found to be widespread in small flowering plants called Arabidopsis. Their findings reveal the possibility that reversible LysAc may be an important and previously unknown regulatory mechanism affecting a wide range of cellular pathways and processes in Arabidopsis plants. The paper shows Octet QK system data comparing binding of Calmodulin to eEF peptide before and after acetylation of the lysine. Acetylation was shown to reduce the affinity of the protein-protein interaction.
PubMed
Novartis Evaluation of the ForteBio Octet RED: A Versatile Instrument for Direct Binding Experiments
Cooper, M., et al., Label-Free Technologies for Drug Discovery, Ch 15, Feb 24 Epub, 2011
This publication provides details of the Novartis evaluation of the Octet RED platform for kinetic characterization of small molecule and fragment binding to various proteins, which resulted in adoption of the system at Novartis. The Octet RED platform was evaluated against a number of platforms, including ITC and Biacore for small molecule screening and proper kinetic characterizations. The Octet RED platform gave binding constants and kinetics consistent with other enzymatic and biophysical methods. Primary, medium throughput assay for fragment-based screening was also possible on the Octet platform and produced reliable data.
Wiley
Mutations in the G-H Loop Region of Ephrin-B2 can Enhance Nipah Virus Binding and Infection
Yuan, J., et al., J General Virology, 92(Pt 9), 2142-2152, 2011
Nipah Virus (NiV) first emerged in Asia in 1998-1999 as the causative agent of an outbreak of respiratory disease in pigs and severe encephalitic disease with high case fatality in humans. Ephrin-B2 and ephrin-B3 have been identified as functional receptors for NiV. This study revealed that some alanine-substitution mutations located within the G-H loop of ephrin-B2 were able to enhance virus entry and infection by infectious viruses. This study also potentially provides a new target host-cell platform for either virus isolation or virus entry inhibitor screening and discovery. The authors report binding kinetics data obtained on the Octet RED platform for an envelope glycoprotein called NiV-G binding to wildtype and mutated forms of ephrin-B2. For the Octet platform experiments, biotinylated sNiV-G was immobilized on Streptavidin biosensors. The data show that the mutated form has greater binding affinity and correlates with immunofluorescence staining results that show the mutated protein as having enhanced virus adsorption, entry and infection efficiency.
PubMed
Rate, Affinity and Calcium Dependence of Nitric Oxide Synthase Isoform Binding to the Primary Physiological Regulator Calmodulin
McMurry, J.L, et al. , FEBS Journal, 278(24) 4943-4954, 2011
Calmodulin (CaM) is a calcium-binding messenger protein expressed in all eukaryotic cells. CaM is a multifunctional intermediate messenger protein that transduces calcium signals by binding calcium ions and then modifying its interactions with various target proteins. This publication describes use of an Octet QK system to probe the binding of eNOS and nNOS signal generating enzymes to CaM and their Ca
2+ dependance. Octet platform data are used to understand the binding equilibria driving formation of CaM-NOS complexes. Biotinylated CaM was immobilized on Streptavidin biosensors for these experiments.
PubMed
Robo4 Maintains Vessel Integrity And Inhibits Angiogenesis By Interacting With UNC5B
Koch, A.W., et al., Developmental Cell, 20(1), 33-46, 2011
In this publication, a library of over 1900 purified protein constructs containing Fc or histidine tags was screened for binding to Robo4-Fc with the Octet RED platform - a novel example of a protein-protein interaction screen. Robo4-Fc was loaded to saturation onto anti-human Fc biosensors, then washed in HBS-P buffer for 30 s and placed for 3 min in wells containing library proteins (5 mg/mL).
PubMed
Chronic Intranasal Treatment with an Anti-Aβ30-42 scFv Antibody Ameliorates Amyloid Pathology in a Transgenic Mouse Model of Alzheimer's Disease
Cattepoel, S., et al., PLoS ONE, 6(4), e18296, 2011
The authors characterized a scFv generated by grafting the complementarity determining regions (CDRs) of the VH and VL domains of the 22C4 IgG into a human scFv framework. The resulting 22C4 scFv was expressed in E. coli and characterized in terms of its binding characteristics, and its potential to inhibit Ab aggregation and prevent Ab-induced neurotoxicity. Affinity (
KD) values for 22C4 scFv and 22C4 IgG binding to A-beta(1-42) were determined using the Octet platform. For these studies, biotinylated A-beta(1-42) was immobilized on Streptavidin biosensors.
PubMed
Wnt Antagonists Bind through a Short Peptide to the First β-Propeller Domain of LRP5/6
Bourhis, E., et al., Structure, 19 (10), 1433-1442, 2011
In this study, the Octet system is used to characterize the interactions between the E1 domain of the Wnt coreceptor LRP6 and its consensus binding sequence, a motif found in Wnt inhibitors that bind to LRP5/6. The effects of motif point substitutions on motif-LRP6 E1 binding affinities are characterized using BLI.
PubMed
Soluble Periplasmic Production of Human Granulocyte Colony-stimulating Factor (G-CSF) in Pseudomonas fluorescens
Jin, H., et al., Protein Expression and Purification, 78(1), 69-77, 2011
To identify a strain producing high levels of intact, soluble Met-G-CSF product, more than 150 protease defective host strains from the Pfenex Expression Technology™ toolbox were screened in parallel using the Octet system to quantify active GCSF binding to its receptor. For these measurements, recombinant GCSF receptor was biotinylated and immobilized on Streptavidin biosensors.
PubMed
Biosensor-based Small Molecule Fragment Screening with Biolayer Interferometry
Wartchow, C.A., et al., J Comput Aided Mol Des, 25(7), 669-676, 2011
In this study, BLI was validated for small molecule detection and fragment screening with model systems and well-characterized targets where affinity constants and binding profiles are generally similar to those obtained with SPR. Screens with challenging targets involved in protein-protein interactions including BCL-2, JNK1, and eIF4E were performed with a fragment library of 6,500 compounds, and hit rates were compared for these targets. For eIF4E, a protein containing a PPI site and a nucleotide binding site, results from a BLI fragment screen were compared to results obtained in biochemical HTS screens. Overlapping hits were observed for the PPI site, and hits unique to the BLI screen were identified. Hit assessments with SPR and BLI are described.
PubMed
Design of a Reversible Biotin Analog and Applications in Protein Labeling, Detection, and Isolation
Ying, L.Q.; Branchaud, B. P., Chemical Communications, 47(30), 8593-5, 2011
A reversible biotin analog, N3'-ethyl, was designed so that biotin-streptavidin binding could be reversed under non-denaturing conditions. The Octet system was used to determine the kinetics of this interaction by immobilizing the biotin analog to an Amine Reactive biosensor, and then observing its association to streptavidin and dissociation in PBS or PBS supplemented with biotin. There was a fast on-rate with streptavidin, and a relatively slow off-rate in PBS alone; however, the off-rate was much faster in the presence of 2mM biotin, highlighting the reversibility of the interaction.
PubMed
Structural and Biophysical Analysis of Bst-2/Tetherin Ectodomains Reveals an Evolutionary Conserved Design to Inhibit Virus Release
Swiecki, M., et al., J Biological Chemistry, 286(4), 2987-2997, 2011
BST-2/tetherin is a host antiviral molecule that inhibits the release of enveloped viruses from infected cells. This paper presents detailed sturctural and biophysical analysis of this protein. In one series of experiments, refolding mouse BST-2 ectodomain was assessed for proper folding using anti-mBST-2 mAb 927, a mAb raised against cell surface-expressed mouse BST-2. Biotinylated mAb 927 was immobilized on Streptavidin biosensors for these studies. Binding of various concentrations of mouse BST-2 ectodomain were monitored using the Octet system.
PubMed
Challenges in Developing Bioanalytical Assays for Characterization of Antibody-Drug Conjugates
Stephan, J.P. ; Kozak, K R.; Wong, W.L., Bioanalysis, 3(6), 677-700, 2011
In this article scientists at Genentech review the different bioanalytical strategies that have been implemented to characterize various antibody-drug conjugates (ADCs) and discuss the challenges and issues associated with these approaches. The Octet platform is discussed as a convenient alternative to fluidics-based systems for the analysis of ADCs.
PubMed
Development and Characterization of APRIL Antagonistic Monoclonal Antibodies for Treatment of B-Cell Lymphomas
Guadagnoli, M., et al., Blood, 117(25), 6856-6865, 2011
APRIL (a proliferation-inducing ligand) is a TNF family member that binds TACI and BCMA. APRIL is a growth promoter of solid tumors, and an important survival factor in B-cell malignancies, such as CLL. The authors develop two anti-human APRIL antogonist monoclonal antibodies. Kinetic analysis of mAb association/dissociation rates to hAPRIL was performed on the Octet system by coupling the purified antibodies to Amine Reactive biosensors.
PubMed
In Vitro Analyses of the Dysregulated R206H ALK2 Kinase-FKBP12 Interaction Associated with Heterotopic Ossification in FOP
Groppe, J.C., et al., Cells Tissues Organs, 194 (2-4), 291-295 , 2011
A single arginine-for-histidine substitution in ALK2 kinase is linked to classic fibrodysplasia ossificans progressiva (FOP). Interaction analyses with purified wild-type and R206H ALK2 kinase to FKBP12 inhibitory protein was performed using the Octet QK system. Biotinylated FKBP12 protein was immobilized on Streptavidin biosensors, and then interacted into wild-type or R206H ALK2 kinase. The R206H substitution in ALK2 kinase resulted in diminished binding to FKBP12.
PubMed
High-end pH-controlled Delivery of Glucose Effectively Suppresses Lactate Accumulation in CHO Fed-batch Cultures
Gagnon, M., et al., Biotechnology and Bioengineering, 108(6), 1328-1337, 2011
A method was developed to control lactate accumulation in suspension cultures of CHO cells, based on the culture's pH. When applied to a 12 day fed-batch process with intial high cell densities in a concentrated medium, final titers were almost doubled relative to the previous process. The Octet system was used for titer determinations for various proteins in CHO crude supernatants.
PubMed
Post-Translational Modifications and Lipid Binding Profile of Insect Cell-Expressed Full-Length Mammalian Synaptotagmin 1
Vrljic, M., et al., Biochemistry, 50 (46), 9998-10012, 2011
This publication descibes the expression, purification, and biochemical characterization of Synaptotagmin 1 (Syt1), a protein involved in SNARE-mediated synptic vesicle fusion in neurons. The binding of various liposomes to Syt was assessed using biotinylated Syt1 calcium-binding domains immobilized on Streptavidin biosensors in an Octet system. Figure 5 in the publication presents association curves for liposome-biosensor binding.
PubMed
Profiling Highly Conserved Microrna Expression in Recombinant IgG-Producing and Parental Chinese Hamster Ovary Cells
Lin, N., et al., Biotechnology Progress, 27 (4), 1163-1171, 2011
In this study, microRNA expression was profiled in various CHO cell lines, including IgG-producing and parental lines. Supernatant IgG concentrations from samples under various experimental conditions was determined using the Octet system.
PubMed
Functional Importance of BAK1 Tyrosine Phosphorylation In Vivo
Oh, M.H., et al., Plant Signaling and Behavior, 6 (3), 400-405, 2011
The function of BAK1 as a co-receptor with BRI1 in brassinosteroid signaling in investigated. Binding of BRI1 to BKA1 in vitro was characterized on the Octet system using immobilized GST-BAK1 cytoplamic domain exposed to solutions of Flag-BRI1 cytoplasmic domain.
PubMed
A Strategy for Clone Selection Under Different Production Conditions
Legmann, R., et al., Biotechnology Progress, 27 (3), 757-765, 2011
The authors report on a strategy for dynamic ranking of clones in micro-bioreactors. Multiple clones in multiple culture conditions were monitored for maximum cell density, growth, and titer. The Octet QK system with Protein A biosensors was used for high-throughput determination of Mab concentrations in the crude culture supernatants.
PubMed
Heavy Chain-Only Antibodies and Tetravalent Bispecific Antibody Neutralizing Staphylococcus Aureus Leukotoxins
Laventie, B.J., et al., Proc Natl Acad Sci USA, 108 (39), 16404-16409, 2011
The actions of humanized heavy-chain only antibodies (HCAb) and a tetravalent bispecific HCAb against components of Panton-Valentine leukocidin are studied in the publication. The Octet QK was used to assess the binding of the bispecific HCAb, e.g. to demonstrate that it binds to both antigens simultaneously.
PubMed
Efficient Production of Antibodies Against a Mammalian Integral Membrane Protein by Phage Display
Hotzel, I., et al., Protein Engineering Design and Selection, 24 (9), 679-689, 2011
The authors report on a phage display method for selecting antibody fragments specific for mammalian multispan membrane proteins. The model protein used was claudin-1 (CLDN1), an entry co-receptor used by the hepatitis C virus. For kinetic measurements, biotinylated CLDN1 was loaded onto Streptavidin biosensors on the Octet RED platform and incubated with anti-CLDN1 Fab fragments derived from the screen. Affinity maturation experiments utilized the Octet platform in a similar fashion.
PubMed
Post-translational Modifications and Lipid Binding Profile of Insect Cell-expressed Full-length Mammalian Synaptotagmin 1
Vrljic, M., et al., Biochemistry, 50 (46), 9998-10012, 2011
This publication descibes the expression, purification, and biochemical characterization of Synaptotagmin 1 (Syt1), a protein involved in SNARE-mediated synptic vesicle fusion in neurons. The binding of various liposomes to Syt was assessed using biotinylated Syt1 calcium-binding domains immobilized on Streptavidin biosensors in an Octet system. Figure 5 in the publication presents association curves for liposome-biosensor binding.
PubMed
Surface Plasmon Resonance Biosensor Analysis as a Useful Tool in FBDD
Retra K, et al., Drug Discovery Today, https://doi.org/10.1016/j.ddtec.2010.11.012, 2010
MIRG2010 Study: Molecular Interactions in a Three Component System
Bergqvist S, et al., J Biomol Tech, 21(3 Suppl):S15, 2010
Walkmycin B Targets WalK (YycG), a Histidine Kinase Essential for Bacterial Cell Growth
Okada A, et al., J Antibiot (Tokyo), 63(2):89-94, 2010
Characterization of a Novel Novobiocin Analogue as a Putative C-terminal Inhibitor of Heat Shockprotein 90 In Prostate Cancer Cells
Matthews S, et al., Prostate, 70(1):27-36, 2010
Detection of Low-Affinity Anti-Drug Antibodies and Improved Drug Tolerance in Immunogenicity Testing by Octet Biolayer Interferometry
Li, J., et al., J Pharm Biomed Anal, 54(2), 286-294, 2010
The authors compare performance of the Octet system to ELISA and MSD techniques for detection of anti-drug antibodies (ADA) against an investigational therapeutic human IgG1 mAb called CNTO X. They found that the Octet platform assay was the most sensitive and ELISA the least sensitive for detection of low-affinity ADAs. The assay also tolerated the presence of free drug to 10X greater levels than the MSD assay, and 100X greater levels than the ELISA assay. The MSD and Octet platform assays were applied to the bioanalysis of cynomolgus monkey sera from a pre-clinical multiple dose study of CNTO X. The Octet system indicated 3 positive animals developed ADA as early as day 15 of the dosing phase while drug was present at nearly 1 mg/mL. ECLIA detected only one of these, and only in a day 57 recovery sample after drug had cleared from circulation. The authors conclude that the Octet instrument is a promising platform for detection of lower affinity ADAs and is particularly suitable for ADA detection when drug persists at levels that negatively impact bridging immunoassays.
PubMed
Development and Evaluation of Monoclonal Antibodies against Phosphatidylethanolamine Binding Protein 1 in Pancreatic Cancer Patients
Wang, X., et al., J Immunological Methods, 362, 151-160, 2010
Phosphatidylethanolamine binding protein 1 (PEBP1) is considered to be a prognostic marker in prostate and other cancers. The authors expressed PEBP1 in E. coli and generated a panel of anti-PEBP1 antibodies using hybdridoma technology. The Octet system was used to measure the affinities of antibodies directly from hybridoma supes to PEBP1 to identify antibodies with the highest binding affinities. The authors next identified an optimal antibody binding pair for PEBP1 by performing a sandwich assay on the Octet system. In short, the Octet system enabled screening of antibodies to develop a sensitive immunoassay assay for the cancer biomarker PEBP1.
PubMed
Specificity for Human Hemoglobin Enhances Staphylococcus aureus Infection
Pischany, G., et al., Cell Host Microbe, 8, 544-550, 2010
Iron is required for bacterial proliferation and S. aureus steals this metal from host hemoglobin during invasive infections. The authors demonstrate an enhanced ability of S. aureus to bind hemoglobin derived from humans as compared to other mammals. The Octet QK system was used to characterize staphylococcal hemoglobin receptor IsdB-hemoglobin binding affinity using Streptavidin biosensors loaded with biotinylated hemoglobin.
PubMed
Allosteric Peptide Activators of Pro-Hepatocyte Growth Factor Stimulate Met Signaling
Landgraf, K.E., et al., J Biological Chemistry, 285, 40362-40372, 2010
Hepatocyte growth factor (HGF) activates Met receptor tyrosine kinase in a therapeutically important signaling sequence. The authors performed Met binding assays on the Octet RED system to show that an 8 amino acid peptide allosterically enhances pro-HGF beta binding to Met protein with an apparent affinity constant of 1.6 micromolar. For these experiments, biotinylated Met ECD was immobilized on Streptavidin biosensors.
PubMed
Humanization Strategies for an Anti-idiotypic Antibody Mimicking HIV-1 gp41
Mader, A., Kunert, R., Protein Eng Des Sel, 23(12), 947-954, 2010
Ab2/3H6 is an anti-idiotypic antibody able to mimic the antigen recognition site of the broadly neutralizing HIV-1 antibody 2F5, making it a putative candidate as an HIV-1 vaccine. The mouse variable regions of Ab2/3H6 were subjected to three different humanization approaches in this study. Four different humanized Ab2/3H6 variants were characterized for their binding affinity to 2F5 in comparison to the chimeric Ab2/3H6. Binding constants were determined on the Octet QK system with Streptavidin biosensors and biotinylated mAb 2F5.
PubMed
Molecular Basis of FIR-Mediated c-myc Transciptional Control
Cukier, C.D., et al., Nature Structural and Molecular Biology, 17(9), 1058 - 1064, 2010
This publication investigates components of the far upstream element (FUSE) transcriptional control system. Molecular interaction experiments on the Octet RED platform utilized a biotinylated 40-mer of single-stranded FUSE DNA immobilized on Streptavidin biosensors. Affinity constants and association/dissociation rate constants were determined for various DNA binding domains, alone and in combinations: FBP Nbox-KH1-KH4, FBP KH1-KH4,FBP3 Nbox-KH1-KH4, and FIR RRM1-RRM2.
PubMed
A Diversity of Antibody Epitopes Can Induce Signaling Through the Erythropoietin Receptor
Lim, A.C., et al., Biochemistry, 49(18), 3797-3804, 2010
A panel of single chain Fc fusion constructs were generated to agonize the erythropoietin receptor (EpoR). The Octet RED platform was used to map the epitopes to which the scFcv constructs bound using cross-competition experiments. These studies used immobilize biotinylated EpoR immobilized on Streptavidin biosensors. For competition binding experiments, immobilized EpoR was saturated with an individual scFv-Fc protein, and then incubated with a second scFv-Fc protein.
PubMed
GP369, an FGFR2-IIIb Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling
Bai, A., et al., Cancer Research, 70 19), 7630-7639, 2010
Aberrant activation of FGF receptor 2 (FGFR2) signaling, through overexpression of FGFR2 and/or its ligands, mutations, and receptor amplification, has been found in a variety of human tumors. The authors generated monoclonal antibodies against the extracellular ligand-binding domain of FGFR2 to address the role of FGFR2 in tumorigenesis and to explore the potential of FGFR2 as a novel therapeutic target. Octet QK system analysis revealed specific binding of a highly potent antibody called GP369 to human and mouse FGFR2-IIIb. Further epitope mapping studies were performed on the Octet system with Streptavidin biosensors by binding GP369 to 10 overlapping peptides covering the COOH-terminal half of the third Ig domain of human FGFR2-IIIb. Peptides 4, 5 and 6 showed strong binding, consistent with crystallographic data.
PubMed
High-throughput Screening and Selection of Yeast Cell Lines Expressing Monoclonal Antibodies
Barnard, G.C., et al., J Ind Microbiol Biotechnol, 37(9),961-971, 2010
The goal of the studies reported here was to design an efficient method for identifying clones able to produce therapeutic mAbs at manufacturing scale. To this end, the authors developed and implemented an integrated high- and medium-throughput screening workflow to identify glycoengineered yeast clones that express mAbs using standard microbial fermentation equipment. They used Octet platform assays to perform high throughput titer measurements for primary screening of clones.
PubMed
Wnt Isoform-Specific Interactions with Coreceptor Specify Inhibition or Potentiation of Signaling by LRP6 Antibodies
Gong, Y., et al., PLoS ONE, 5(9), e12682, 2010
Several components of Wnt signaling are implicated in the genesis of human cancer. Wnt signaling includes numerous ligands, receptors and transcriptional effectors. Beta-catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The authors generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. Their study results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. The LRP6 protein binding assays were performed on the Octet RED platform using Streptavidin biosensors loaded with biotinylated, HIS-tagged LRP6 proteins.
PubMed
Flexible Label-free Quantitative Assay for Antibodies to Influenza Virus Hemagglutinins
Carney, P.J., et al., Clinical and Vaccine Immunology, 17(9), 1407-1416, 2010
This article reports the use of a cell-free and label-free flu antibody biosensor assay developed on the Octet RED platform for influenza research and diagnostics that utilizes recombinant hemagglutinin (HA) in conjunction with BLI to measure biomolecular interactions between the HA and specific anti-HA antibodies or sialylated ligands. The authors evaluated the assay to determine anti-HA antibody binding activity in serum or plasma to assess vaccine-induced humoral responses. They note that the Octet platform assay could be used in global surveillance laboratories since preliminary tests on desiccated HA-loaded biosensors showed no loss of activity after >2 months in storage at room temperature, indicating that the same reagent lots could be used in different laboratories to minimize interlaboratory assay fluctuation. They also note that the Octet systems offer a robust platform for influenza surveillance activities.
PubMed
Optical Biosensing: Kinetics of Protein A-IgG Binding Using Biolayer Interferometry
Wilson, J.L.; Scott, I.M.; McMurry, J.L., Biochem Mol Biol Educ, 38(6), 400-407, 2010
Thsi article describes an undergraduate biochemistry laboratory experiment using the Octet QK system, in which students obtain and analyze kinetic data for a protein-protein interaction. This work represents the first published undergraduate teaching experiment utilizing optical biosensing suitable for larger classes.
PubMed
Cytokine Binding by Polysaccharide-Antibody Conjugates
Sun, L.T., et al., Molecular Pharmaceutics, 7(5), 1769-1777, 2010
Monoclonal antibodies against interleukin-1beta and tumor necrosis factor were conjugated to a variety of polysaccharides and tested for cytokine binding and neutralization. Polysaccharide-antibody conjugate/cytokine binding affinites were determined using the Octet QK system. Biotinylated antibody or polysaccharide-antibody conjugates were immobilized on Streptavidin biosensors for these experiments. All conjugates had pM binding affinity constants. Some variation in dissociation constants was noted, suggesting that conjugation to a high MW polysaccharide did not interfere with formation of the complex with cytokine, but could affect the complex's stability.
PubMed
Gambogic Acid Inhibits Hsp90 and Deregulates TNF-alpha/NF-kappaB in HeLa Cells
Zhang, L., et al., Biochem Biophys Res Commun, 403(3-4), 282-287, 2010
Molecular docking, together with the Octet RED platform and spectroscopic methods, were used to study the interaction between a novel small molecule drug called Gambogic Acid (GB) and full-length Hsp90, N-Hsp90, M-Hsp90 and C-Hsp90. Biotinylated protein targets (e.g., Hsp90) were immobilized onto Super Streptavidin biosensors for interaction studies. From the binding energy and dissociation constants, the authors deduce that GB is an inhibitor of Hsp90 and that it binds to the N-terminal ATP-binding domain of Hsp90.
PubMed
Structural Optimization and De Novo Design of Dengue Virus Entry Inhibitory Peptides
Costin, J.M., et al., PLoS Neglected Tropical Diseases, 4(6), e721, 2010
Predictive strategies together with computational optimization of binding "pseudoenergies" were utilized to design two antiviral peptide sequences against the Dengue-2 virus envelope protein. Kinetic binding analysis was performed on the Octet QK system by immobilizing biotinylated DENV-2 S1 E protein onto Streptavidin biosensors, and incubating with the inhibitory peptides. The Octet platform data, along with supporting data, confirmed that these two peptides interact directly with DENV-2 E proteins and are entry inhibitors.
PubMed
Neutralizing and Non-neutralizing Monoclonal Antibodies Against Dengue Virus E Protein Dervied from a Naturally Infected Patient
Schieffelin, J.S., et al., Virology Journal, 7, 28-38, 2010
The authors generate human monoclonal antibodies (mAbs) against dengue virus envelope glycoprotein (E protein), and then characterize mAb binding and netralization. Kinetic binding assays between purified mAbs and purified E proteins were performed on the Octet QK system. E proteins were biotinylated, immobilized on Streptavidin biosensors, and incubated with mAb solutions to determine association and dissociation rate constants.
PubMed
Structural Bases for the Interaction of Frataxin with the Central Components of Iron-Sulphur Cluster Assembly
Prischi, F., et al., Nature Communications, 1, 95, 2010
Frataxin is an essential iron-binding protein that is highly conserved in most organisms, from bacteria to humans. Reduced expression levels of this protein are sufficient to induce Friedreich's ataxia, a relentless and currently incurable neurodegenerative disease. The authors performed experiments using various biophysical techniques such as small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC) and BLI to dissect the intractions of frataxin with important proteins in iron-related signaling pathways. Octet RED platform assays with Streptavidin biosensors helped the authors decipher the binding interactions and clarify the binding mechanism in play. They also found excellent correlation between Octet instrument data and ITC data.
PubMed
Kinetic Characterization of Salmonella FliK-FlhB Interactions Demonstrates Complexity of the Type III Secretion Substrate-Specificity Switch
Morris, D.P., et al., Biochemistry, 49(30), 6386-6393, 2010
In this paper, FliK-FlhB interactions were characterized in Salmonella using wild-type and two variant FlhBs from mutants with severe flagellar structural defects. The Octet system was used for kinetic analysis; biotinylated FliK was immobilized on Streptavidin biosensors and associated with FlhB. The Octet platform data show a rapid association followed by a slow conformational change and dissociation leading to a complex binding mechanism between FliK-FlhBc.
PubMed
Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-Active Site
Maun, H.R., et al., J Biological Chemistry, 285(34), 26570-26580, 2010
A murine/human chimeric 5E1 monoclonal antibody (ch5E1) against the hedhehog pathway was engineered. Binding kinetics and affinities of ch5E1 Fab for various hedgehog ligands were measured using the Octet RED platform. A number of kinetic binding experiments were reproted using biotinylated Sonic hedgehog, biotinylated ch5E1 Fab, or biotinylated hedgehog ligand immobilized on Streptavidin biosensors. Kinetics experiements were confirmed in reverse orientations. In the presence of calcium and zinc ions, ch5E1 binding affinity for Sonic hedgehog increased 10-20-fold, primarily because of a decrease in the dissociation rate.
PubMed
Reconstitution of a Frizzled8-Wnt3a-LRP6 signaling complex reveals multiple Wnt and Dkk1 binding sites on LRP6
Bourhis, E., et al., Journal of Biological Chemistry, 285 (12), 9172-9179, 2010
To dissect Wnt signaling at the cell surface, the authors characterized interactions of the Wnt signaling proteins LRP6, Wnt3a, Wnt9b, Fz8, and DKK1 using the Octet RED platform with Streptavidin and Anti-Human Fc biosensors. Binding constants for several binary complexes were reported. Notably, the authors were able to reconstitue a Fz8-Wnt3a-LRP6 ternary complex on the surface of the Anti-Human FC biosensor and report new detailes about its molecular arrangement and regulation by DKK1.
PubMed
Molecular Mechanism of the Synaptotagmin-SNARE Interaction in Ca2+-Triggered Vesicle Fusion
Vrljic, M., et al., Nature Structural and Molecular Biology, 17(3), 325-331, 2010
This publication characterizes the interactions between SNARE complex and synaptotagmin, proteins involved in the fusion of vesicles in the plasma membrane of neurons. The SNARE-synaptotagmin 3 interaction was studied with the Octet RED platform using biotinylated synaptotagmin 3 immobilied on Streptavidin biosensors.
PubMed
Direct Transactivator-Transcription Factor IID (TFIID) Contacts Drive Yeast Ribosomal Protein Gene Transcription
Layer, J.H., et al., J Biological Chemistry, 285(20), 15489-15499, 2010
The Rap1 binding domains (RBD) of TATA-binding protein associated factors subunits Taf4 and Taf5 were identified and characterized. The Octet system was used to determine the binding affinities Rap1 to wild-type and mutated Taff4 and Taf5. Biotinylated Rap1 was immobilized on Streptavidin biosensors for these measurements. All the mutants of Taf4 and 5 exhibited lower binding affinities to Rap1 compared to WT.
PubMed
Generation of Stable Cell Clones Expressing Mixtures of Human Antibodies
de Kruif, J., et al., Biotechnology and Bioengineering, 106 (5), 741-750, 2010
PerC6 mammalian cells were transfected to generate stable clones expressing mixtures of human antibodies. Total human IgG was quantified using the Octet QK system using Protein A biosensors. Constant ratios of specific antibodies were produced, and total IgG productivity was also similar, averaging about 15 and 20 pg/cell/day in most subclones.
PubMed
Recombinant Respiratory Syncytial Virus F Protein Expression is Hindered by Inefficient Nuclear Export and mRNA Processing
Huang, K., et al., Virus Genes, 40(2), 212-221, 2010
This publication investigates obstacles to expression of recombinant respiratory syncytial virus (RSV) F protein, and proposes a strategy for enhanced expression of the protein. In the study, the Octet system was used to confirm that Eu-labeling of two monoclonal antibodies did not alter their binding properties (data not shown).
PubMed
Autophosphorylation of Tyr-610 in the Receptor Kinase BAK1 Plays a Role in Brassinosteroid Signaling and Basal Defense Gene Expression
Oh, M.h., et al., Proc Nat Acad Sci USA, 107(41), 17827-17832, 2010
The authors study the role of a Tyrosine amino acid on BAK1 kinase protein's function in signaling. Plants contain a large family of receptor-like kinases that are thought to control many aspects of plant growth and development. Octet QK system data were used to show the presence of a binding interaction between wild-type and mutant BAK1 and BRI1 proteins and to measure their binding affinities.
PubMed
Design Principles for Cytokine-Neutralizing Gels: Cross-linking Effects
Sun, L.T., et al., Acta Biomaterialia, 6(12), 4708-4715, 2010
Constructs composed of cytokine-neutralizing antibodies conjugated to high-molecular-weight hyaluronic acid have been shown to be effective at controlling inflammatory responses in vivo. A critical question in the development of this new class of biomaterial is whether crosslinked conjugates have similar anti-inflammatory effects, which would open up a broad range of tissue engineering applications in which the material would have intrinsic inflammation-controlling function. To test this, high-molecular-weight hyaluronic acid was conjugated with monoclonal antibodies to the pro-inflammatory cytokines interleukin-1b and TNF-a in two forms of the material: viscous, non-crosslinked polymer-antibody conjugates and crosslinked, elastomeric polymer-antibody conjugates. IL-1b binding affinities of both constructs were charaterized using the Octet system.
PubMed
Structural Correlates of Antibodies Associated with Acute Reversal of Amyloid beta-related Behavioral Deficits in a Mouse Model of Alzheimer Disease
Basi, G.S., et al., Journal of Biological Chemistry, 285(5), 3417-3427, 2010
The authors study three antibodies that target the same epitope of amyloid-beta peptide, but differ in their ability to reverse amyloid-beta-related behavioral deficits in transgenic mouse models. The Octet system was used for kinetic characterization of antibody binding to soluble myeloid percursor protein. Various antibodies were immobilized using Goat Anti-Mouse biosensors, and then incubated with different concentrations of soluble myeloid percursor protein.
PubMed
Rapid Identification of Production Strains
Kumaraswamy, S.; Allen, J. R., Genetic and Engineering News, June 15, 30 (12), 2010
This article describe the implementation of Octet systems at Pfenex for high-throughput identification of optimal production strains. Details are presented about a Pfenex screen of 240 unique strains for expression of soluble, correctly folded granulocyte colony stimulating factor (GCSF). The authors report that Pfenex has implemented expression screens for diverse classes of proteins, including mAbs, Fab, single-chain antibodies, growth factors, cytokines and vaccine antigens.
GEN
Human Framework Adaptation of a Mouse Anti-Human IL-13 Antibody
Fransson, J., et al., Journal of Molecular Biology, 398(2), 214-231, 2010
This publication describes development of a humanized neutralizing mouse anti-human IL-13 antibody at Centocor. Clarified cell supernatants from transiently transfected HEK293-E and CHO-S cells were analyzed on the Octet system to determine relative amounts of IgG (data not shown).
PubMed
Binding Rate Screen - A High-Throughput Assay in Soluble Lysate for Prioritizing Protein Expression Constructs
Tian-Yu, J., et al., Analytical Biochemistry, 399(2), 276-283, 2010
This publications reports on the Binding Rate Screen, a functional screen for prioritizing expression constructs in crude soluble lysates. In this approach, the association rate of hexahistinine tags provides information on both the tagged protein concentration and the tag accessibility (a probe of protein folding and/or aggregation). A biotinylated penta-His antibody was used with Streptavidin biosensors for binding measurements on the Octet system.
PubMed
