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W550:Bowen:A Mix and Read Cell-Based Assay for Antibody Screening Against Epidermal Growth Factor Receptor

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Wayne Bowen, David Onley, Tristan Cope

Antibodies against a wide range of protein targets have been either approved or are currently under development for therapeutics. The conventional antibody screening assay based on antibody-antigen binding has been enzyme-linked immunosorbent assay (ELISA). While tedious and consuming, ELISA has proved sufficient for the identification of antibodies directed against secreted antigens. However, cell surface antigens (e.g. GPCRs) provide challenges for ELISA primarily due to the shortage of soluble antigens and high variability resulting from loss of cells during wash procedures. Mix-and-read assays – popularized as FMAT® assays – overcome such problems, while at the same time offering simplified workflows for automation.

We have developed a mix-and-read method for the screening of antibodies against cell surface proteins expressed on live cells. The method uses a high sensitivity microplate cytometer to quantify cellular fluorescence in cultures seeded in microplates. Here, we describe its use for the determination of human epidermal growth factor receptor (EGFR) antibody binding in A549 cells which are known to express high levels of EGFR. A549 cells were incubated with mouse anti-EGFR antibody and fluorescently-labelled anti-mouse IgG antibody. Without washing away unbound antibodies, plates were scanned and fluorescence of each cell quantified. Clear concentration-dependent antibody binding was observed with low assay variability. With its simple operation and high sensitivity, this new method is well-suited for high throughput antibody screening.

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