Determination of ligand binding affinities for endogenous seven-transmembrane receptors using fluorometric microvolume assay technology

We have developed a fluorescence-based mix and read method for the quantita tive determination of receptor-ligand binding interactions. This method was used to determine IC50 values for peptide ligands of two endogenous seven- transmembrane receptors that are expressed in cultured human cancer cells. Substance P, neurokinin A, and galanin were labeled with Cy5 and were shown to retain their native binding affinities. The cell-associated fluorescenc e was quantified using a fluorometric microvolume assay technology (FMAT) s canner that was designed to perform high-throughput screening assays in mul tiwell plates with no wash steps. The binding of fluorescently labeled subs tance P and neurokinin A was tested on the human astrocytoma cell line UC11 that expresses endogenous NK1 receptor. Galanin binding was measured on en dogenous galanin type 1 receptors in the Bowes neuroblastoma cell line. IC5 0 values were determined for substance P, neurokinin A, and galanin and wer e found to correspond well with reported values from radioligand binding de terminations. To demonstrate FMAT as instrumentation for high-throughput sc reening, it was utilized to successfully identify individual wells in a 96- well plate in which Cy5-substance P binding in UC11 cells was competed with unlabeled substance P. In addition, we developed a two-color multiplex ass ay in which cells individually expressing neuropeptide Y and substance P re ceptors were mixed in the same well. In this assay, the fluorescent ligands substance P and neuropeptide Y bound only to their respective cell types a nd binding was specifically competed. Therefore, two different seven-transm embrane receptor targets can be tested in one screen to minimize reagent co nsumption and increase throughput, (C) 1999 Academic Press.