Application Note: Utilizing Advanced High-Throughput Flow Cytometry to Quantify Direct and Competitive Live Cell Antibody Binding


Monoclonal antibody (mAb) therapeutics are one of the most rapidly growing drug classes. Amongst FDA approved mAb products, are treatments for an enormously wide range of diseases, such as immune disorders and some cancers. 

Within these categories, mAbs are raised against a wide variety of target antigens, such as anti-HER2 (human epidermal growth factor receptor 2), mAbs to target breast cancers, and anti-TNF (tumor necrosis factor) antibodies to treat symptoms of inflammatory disease.

The success of mAbs as a class of therapeutics is largely due to their high specificity and affinity for the target antigen. Because of this, evaluation of mAb target binding is a critical part of the antibody development process, requiring robust, high-throughput techniques to facilitate rapid screening of antibody libraries.

Utilizing such techniques as early as possible in the drug discovery pathway can enhance the quality of ‘hits’ generated for mAbs with desirable characteristics, providing increased likelihood of candidates being progressed through the clinical pipeline.

Here we present two simple assays that utilize the iQue® Advanced Flow Cytometry Platform and validated reagents, to enable high-throughput measurement of the binding of unlabeled therapeutic mAbs to your target on live cells. The direct binding workflow provides robust quantification of binding to native protein on live cells, with simple generation of pharmacological readouts, such as EC₅₀ values using the iQue Forecyt® software.

This streamlined process enhances the speed and quality of hits generated during antibody discovery.

Page count: 10
Read time: 10 minutes
Authors: Kirsty McBain, Daryl Cole and Nicola Bevan

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Key Takeaways

This application note highlights the following topics:

  • Assessment of mAb binding and specificity in both adherent and suspension cell models
  • Measuring antibody binding to a range of target cells in a single well
  • Competition binding assay reveals antibodies that bind to the same epitope

Target audience:

Immunologists, Immuno-oncology, virology researchers, neuroscientists and cancer immunotherapy drug discovery researchers, in biotechnology, pharmaceuticals, biopharma, research institutes, core facilities and academia. 

Applications covered:

Flow cytometry

Direct Antibody Binding iQue® Assay

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