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 binding to either intact cells or receptor-containing membrane fragments by analysis of particle brightness. As an example, the affinities and inhibition constants 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. Although the results were generally comparable between the two approaches, the cell-based measurements revealed a more complex pattern of both ligand and inhibitor titration curves, pointing to the influence of intracellular regulatory events. Both the vesicle- and cell-based membrane receptor assays were successfully miniaturized to a total volume of 1 microl without compromising 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 reagent could potentially enable the use of primary cells for future HTS/ultra-HTS efforts.