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A. Gagne, E. Robitaille, M. Harvey, J.-A. St. Pierre, D. Wenham and P. Banks
Introduction
High throughput screening is an indispensable tool for modern drug discovery1-3. The process is typically highly automated and can provide screening rates over 100,000 samples in a 24 hour period. To achieve this, assays must be simple, preferably mix and read type assays, where all reagents necessary to perform the assay are added sequentially to the microtitre plate without the need for transfer, aspiration, centrifugation or wash steps. In the same vein, the fewer the addition steps, the higher the screening rate achievable. Furthermore, assays must be miniaturizable in order to be adopted by most screening laboratories, since lower reagent consumption is an ever increasingly important attribute. Moreover, the use of radiometric detection tends to increase screening costs due to the need for significant safety and waste disposal costs, thus assays should be non-radiometric.
Arguably, fluorescence polarization (FP) detection is the best performer in that most of the ideal attributes for G-protein coupled receptor (GPCR)-ligand binding assay platforms (homogeneous, few addition steps required, miniaturizable, non-radiometric and ultra high throughput) are realized. There is no question that this assay is a simple non-radiometric mix and read type assay with few addition steps4-6. Ultra high throughput operation has been demonstrated for real screening applications7 and satisfactory performance with competitive economy has been demonstrated for the demanding application of GPCR ligand binding. Using red-shifted fluorescent labels, such as BODIPY-TMR, colored compound interference and autofluorescence from cell membrane fragments can be minimized7-9. Furthermore, nM binding affinities can be assayed with satisfactory accuracy and precision10'"/>
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