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Use of FlashPlate Technology for In Vitro Measurement of [35S]-GTPγS Binding in CHO Cells Expressing the Human 5-HT1B Receptor

says 5-HT appears as a full agonist, GR127935 acts as a partial agonist and SB-224289 appears as an inverse agonist. The maximum response of 5-HT and GR127935 differs between the two methods (Table 1), but the intrinsic activity of GR127935 is comparable. The maximum response produced by SB-224289 is similar for both assay systems. The comparison between the two methods is summarized in Table 2.

Discussion

This studys aim was to investigate the efficacy of PerkinElmers Basic FlashPlate as a tool for measuring [35S]-GTPγS binding, using CHO cells expressing the human 5-HT1B receptor as the receptor source. After optimal conditions were assessed, significant stimulation of binding was achieved in the presence of an agonist. The FlashPlate system was also sensitive enough to pick up partial and inverse agonists. (Assessment of optimal conditions will be essential when designing other assays on FlashPlate, for different receptors expressed in different cell lines.)

The FlashPlate-based method agreed closely with the conventional filtration method in terms of potencies and intrinsic activities of the compounds tested, but differences in maximum responses were seen. Intra- and inter-assay variation present in the FlashPlate system may play a role in this, and might be attributable to non-uniform coating of membrane protein to wells. The pipetting procedure adopted, or the volume of residual reaction mixture after aspiration may also be factors. This may not be a major concern if the window of response is adequate for the given application. In cases where intra- and inter-assay variation is a problem, it might be addressed by performing assays in duplicate or greater multiples.

Significantly, total assay time was reduced from 12 hours for the filtration method to 4 hours on FlashPlate. Labor time was also reduced significantly. The FlashPlate method also allows greater s
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