Flow Cytometry
screens. Using the HyperCyt HTFC screening for therapeutic antibodies is now possible and at the same time offers all of the full armoury associated with flow cytometry providing a more robust and sensitive screening platform.
During hybridoma screening it is usual to test specific binding of secreted antibodies from each hybridoma supernatant to a positive human target cell (eg, a transient cell line expressing the target protein of interest), a negative expressing cell (expressing a similar related protein) and one or two orthologue species expression the protein of interest. This would normally result in screening hybridoma supernatants several times in separate assays to test for binding. Using flow cytometry all the different cell types can be mixed together and run as one, thus increasing the throughput of the assay, making significant time savings and saving costly reagents. This is accomplished by using a technique known as fluorescent cell bar coding15, where different populations of cells can be identi- fied using a different fluorescent signature. For example, each cell population can be labelled with a different concentration of the fluorogenic sub-
strate calcein AM. The cells are then mixed and aliquoted into microtitre plate wells. Test antibody supernatants plus controls are added to the mix- ture and then analysed using HTFC. Each popula- tion can be differentiated based on their fluores- cent intensity, and the corresponding antibody binding to each cell type can be quantified simulta- neously (Figure 6).
Discussion and future perspectives Traditional flow cytometry has an important role throughout the drug development cycle and is rep- resented at each phase in some capacity. Often flow cytometry is the preferred technology to use for drug screening when running phenotypic assays, but throughput limitations have led to the development of less desirable recombinant assays. The introduction of plate-based sampling on flow cytometers began to realise the potential of flow cytometry as a drug screening platform. The intro- duction of faster sampling technologies, such as the HyperCyt and the use of peripheral automation and liquid handling has transformed flow cytome- try as a drug screening tool. HTFC is still at ‘the
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Drug Discovery World Spring 2013
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