Developing and Running Cross-competition/Epitope Binning Assays:
New Application Note

Rashi Takkar, Marketing Application Scientist, rashi_takkar@pall.com

Scientists at Igenica, Inc. and Pall ForteBio LLC recently co-authored Application Note 16, Cross-competition or Epitope Binning Assays on the Octet HTX System. This new note provides all the information you need to develop and run cross-competition (epitope binning) assays, analyze the data generated, and interpret your results.

Running epitope binning assays on the Octet HTX System provides several compelling advantages over ELISA and SPR as summarized in Table 1. The Octet HTX system is significantly faster than competing methods, compatible with crude matrices, allows discrete, independent, parallel interactions to be monitored simultaneously and is easy to operate.

Three types of binning assay formats are presented, allowing you to choose the format that suits your experimental needs for a particular antigen and panel of antibodies. The flow chart in Figure 1 outlines various factors in deciding the appropriate assay format and also highlights considerations and recommendations for each. Classical sandwich and in-tandem formats are recommended for crude monoclonal antibody (mAb) supernatants and purified mAbs, and premix assays are best used with purified mAbs. Specific details are provided on choosing the right biosensor (i.e. capture-based, streptavidin-based or amine coupling-based) to immobilize the antigen or mAb, as well as how to optimize antigen loading or mAb immobilization. Best practices are also provided to ensure your mAb and antigen remain active, setting the appropriate concentration and duration of association steps, ensuring complete self-blocking, minimizing cross-reac-

  Octet HTX System ELISA SPR
Labeled detection reagents No Yes No
Monitoring of all steps Yes No Yes
Real-time monitoring Yes No Yes
Run time (32x32) 8 hours 24 hours 14 hours*
Crude lysate compatibility (hybridoma supernatants, lysates) Yes No No; clogging issues when using crude, unpurified samples
Detection of low-affinity mAbs Yes No Yes
Parallel interactions Discrete, independent, parallel interactions monitored simultaneously Discrete, independent interaction MAY be monitored simultaneously but throughput is compromised; for higher throughput, restricted number of multiple, independent interactions analyzed Combined flow paths restrict ability to monitor multiple, independent interactions simultaneously
Samples are recoverable and can be re-used Yes No No
Biosensor choices Many biosensor chemistries available N/A Limited choice of chip biosensor chemistries
Regeneration
  • Biosensors are inexpensive and may be regenerated
  • Can tailor specific regeneration buffers for each mAb
  • Cannot regenerate and re-use samples
  • Chips are expensive and have to be regenerated
  • Cannot tailor regeneration for each mAb (universal regeneration solution needed)
Versatility Compatible with all binning formats More amenable to sandwich and premix binning as capturing antigen on plate surface for in-tandem binning may distort its conformation Compatible with all binning formats; rarely used for in-tandem assay because throughput is significantly compromised
Other considerations
  • Easy experiment set-up
  • Minimal user-training required
  • Extensive analyst time needed
  • Minimal user training required
  • Automation required for high throughput runs
  • Need experienced users
  • Requires optimization of surface regeneration

Table 1: Octet HTX system advantages over ELISA and SPR.

*20-channel SPR system.

tivity between mAbs, biosensor regeneration and assay set up based on the assay format and biosensor you select.

Binning assay data is easily visualized and analyzed using the new epitope binning features available in Octet Data Analysis software version 8.0 and higher. The application note walks you through analysis step by step, including the calculating of nm shifts at user-specified report points, flagging problematic data, normalizing raw binding data and creating two-dimensional matrices for large binning datasets. Information on analyzing exported data using the Pearson function and the statistical computing program R for generating a clustergram with the pvclust package are also provided.

The application note was developed in collaboration with Sindy Liao-Chan and Jan-Willem Theunissen of Igenica, Inc. and is available on the Pall ForteBio website.

EP Binning Workflow

FIGURE 1: Assay format flow chart.