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Cell multiplexing in homogeneous antibody binding assays

mirrorball

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Monoclonal antibody therapy represents one of the most rapidly expanding and exciting segments of the
pharmaceutical industry today. This has placed unprecedented demands on the antibody discovery process that has traditionally relied on assay formats such as ELISA and flow cytometry. When screening against antigens expressed in cells, these formats have many disadvantages notably tedious wash steps, detachments of cells from plate surfaces and a requirement for large numbers of cells. To address these challenges we have employed a mirrorball® high sensitivity microplate cytometer (TTP Labtech) to carry out homogenous cell-based FMAT assays originally developed for the now obsolete ABI 8200 instrument. The combination allows researchers to screen antibody supernatants with minimal reagent additions, no wash steps and achieve excellent sensitivity with only 5 –10 microlitres of sample.


Using the mirrorball’s unique ability to scan simultaneously with up to three lasers, it is also possible to multiplex cell
lines, for example to screen against parental and transfected cells in the same well. Cell encoding is achieved by labelling with different fluorophores or a range of fluorophore concentrations. This multiplexing capability results in higher throughput, reduced reagent requirements and more robust data by eliminating interplate variability.
To demonstrate the technique, we have developed a homogeneous assay to quantify binding of human antiEGFR
antibody to receptors expressed in A549 or A431 cells. Jurkat cells served as a negative control for EGFR expression.