Ligand binding to transmembrane receptors on intact cells or membrane vesicles measured in a homogeneous 1-microliter assay format

We have developed homogeneous miniaturized assays: to measure ligand bindin g to either intact cells or receptor-containing membrane fragments by analy sis of particle brightness. As an example, the affinities and inhibition co nstants of fluorescently labeled interleukin-8 (IL-8;) and a low-molecular- weight antagonist toward the receptors CXCR1 and CXCR2, which belong to the superfamily of G protein-coupled receptors (GPCRs), were determined. Altho ugh the results were generally comparable between the two approaches, the c ell-based measurements revealed a more complex pattern of both ligand and i nhibitor titration curves, pointing to the influence of intracellular regul atory events. Both the vesicle- and cell-based membrane receptor assays wer e successfully miniaturized to a total volume of 1 mu cl without compromisi ng their sensitivity, indicating that screening of transmembrane receptors in these formats is feasible. This is the first report of a cellular ligand -binding assay performed in such low volumes. The resulting savings in reag ent could potentially enable the use of primary cells for future HTS/ultra- HTS efforts.