Monoclonal antibody binding assays using advanced flow cytometry 

Monoclonal antibodies (mAbs) are used as therapeutics in many areas, including immuno-oncology, autoimmune disorders, cardiovascular disease, inflammation and infectious diseases. There is potential to raise an antibody against virtually any extracellular target, meaning development of mAbs against novel antigens is significant in the field of drug discovery. High-throughput techniques for screening and ranking mAbs based on their binding to the target are essential during these drug development processes. 

Conventional techniques for measuring antibody binding often:

  • Have low-throughput acquisition and long sample times due to the requirement of large sample volumes (e.g. traditional flow cytometry)
  • Measure binding to purified, truncated or tagged recombinant forms of the target protein
  • Are laborious and time-consuming, requiring steps such as protocol optimization, fixation and repetitive washes
  • Necessitate the use of large volumes of precious sample and antibody

The iQue® antibody binding assays and workflows can help identify and characterize antibody binding to targets. Here we present two simple assays that utilize the iQue® Advanced Flow Cytometry Platform and validated reagents to measure binding of unlabeled therapeutic mAbs to your target on live cells.  The first is a direct antibody binding assay which enables ranking of mAbs based on binding to target cells, with the ability to analyze binding to multiple cell types in a single well. The second is a competitive binding assay which can reveal mAbs that target different epitopes. We utilized the  iQue® Cell Encoding and Proliferation Dye (optional for direct antibody binding) to distinguish multiple cell types  and the iQue® Cell Membrane Integrity Dye  to determine live and dead cells. Combining the high-throughput of the iQue® with rapid data analysis using the integrated iQue Forecyt® software will streamline your antibody discovery processes.

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A. Direct antibody binding assay workflow

Figure 1a. Simple workflow to assess binding of test mAbs to live cells using the iQue® Advanced Flow Cytometry Platform

B. Competition antibody binding assay workflow

Figure 1b. iQue® Competitive binding assay workflow

A competing antibody is pre-labeled in a tube with a fluorophore-conjugated secondary antibody, prior to combination with the unlabeled test antibody of interest in the assay plate. The target cells are then added to this antibody mixture and the labeled competing mAb and the test mAb will compete for binding to the target antigen, provided that they both target the same epitope. Therefore, the greater the concentration of the test antigen, the less of the labeled competing antibody will bind, resulting in a lower MFI value for the secondary antibody fluorophore. If the two antibodies don’t compete for the same epitope, the MFI should be unaffected by test mAb concentration. 

Key Advantages

Key Advantages

Streamline data acquisition and analysis

Collect and analyze complex data more quickly to make faster decisions.

Figure 2. Comparing binding of anti-CD20 biosimilars using iQue® Forecyt software with novel data visualization features

A direct mAb binding assay was used to measure the binding of anti-CD20 mAb Rituximab and one of its biosimilars, Truxima, to high CD20 expressing Raji cells. (A) Heat map of the percent of live cells that are positive for binding of the antibody over a gated threshold. (B) Histograms show that the MFI for the secondary antibody increases with both Rituximab and Truxima concentration, but not with the IgG control. (C) Concentration-response curves show the % of cells positive for mAb binding over the gated threshold. (D) Table of values exported directly from iQue® Forecyt details fit properties of curves in (C). 

Enhance your productivity

Screen libraries of antibodies for cell surface binding in minimal time as part of high throughput antibody discovery workflows.

Figure 3. Screen antibodies using the competition binding assay format to reveal differences in epitope binding

Three anti-HER2 therapeutic mAbs (Trastuzumab, Kadcyla and Pertuzumab) were labeled with RPE-conjugated secondary Ab (1:1 labelling ratio, 1.5 µg/mL) and added to a 384 well plate alongside iQue® Cell Membrane Integrity (R/Red) Dye and a range of concentrations of unlabeled mAbs. High HER2 expressing AU565 cells were added and the plate was incubated on ice for 45 minutes before data was collected using the iQue® platform. 

The heat map shows median fluorescence intensity (MFI) for the secondary antibody on live cells (dark grey = high MFI; light grey = low MFI). The data shows that there is competition between Trastuzumab and Kadycla (a Trastzumab based ADC) for binding to the same HER2 epitope. Pertuzumab binds to a different HER2 epitope and did not compete with the other mAbs. Heating Trastuzumab caused a minor reduction in the amount of competition with other mAbs compared to the unheated mAb.

Flexible assay format 

Characterize multiple therapeutic antibodies in either suspension or adherent cell models.

Figure 4. Assess specificity of antibody binding to target antigens on both suspension and adherent cells

Binding and specificity of two therapeutic antibodies: Rituximab and Trastuzumab, was assessed using suspension and adherent cancer cell line models. CD20 positive Ramos cells and HER2 positive AU565 cells were labeled with iQue® Cell Proliferation and Encoder Dye (V/Blue) to distinguish them from antigen negative Jurkat and MDA-MB-468 cells. 

MFI due to RPE-conjugated secondary Ab binding showed a concentration dependent increase in mAb binding on antigen positive cells, whilst binding on antigen negative cells was comparable to the IgG control. 

Maximize usage of precious samples 

Make the most of your samples by simultaneously assessing antibody binding to distinct cell types co-cultured in a single well.

Figure 5. Analyze antibody binding to a range of antigen expressing cell types in a single well

(A) HER2 expression on three adherent cell lines (AU565, MCF7 and BT474) was analyzed using a conjugated anti-HER2 antibody and compared to expression on HER2 negative Ramos cells. Relative HER2 expression: AU565 > BT474 > MCF7. (B) To distinguish cells in the antibody binding assay, AU565 cells were labeled with a high concentration of iQue® Cell Proliferation and Encoder Dye (V/Blue), MCF7 cells were labeled with a lower concentration and BT474 cells were left unlabeled. (C) Binding of Trastuzumab to the three cell lines reflected their relative levels of HER2 expression (AU565 > BT474 > MCF7). (D) EC50 values for Trastuzumab binding were comparable across the three cell types (±95% confidence interval).

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Literature and Documentation


First Edition Advanced Flow Cytometry Handbook

A Guide to Advanced Flow Cytometry Assays and Workflows

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iQue® Reagent Kits

Complete product listing of iQue® Reagents

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iQue® Brochure

Drive your research forward with advanced flow cytometry – miniaturize sampling, multiplex bead and cell based assays,

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Beyond Monoclonal Antibodies - Exploring the Next Generation of Antibody Therapeutics

Beyond Monoclonal Antibodies

Exploring the next generation of antibody therapeutics

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Application Note

Advanced Flow Cytometry and BLI Label-Free Detection for Binding and F...

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Characterization of Anti-HER2 Antibodies Using iQue® & Octet® Plat...

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Application Note

Live Cell Antibody Binding

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

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