A generic particle-based nonradioactive homogeneous multiplex method for high-throughput screening using microvolume fluorimetry

We have developed a novel fluorescence-based homogeneous binding assay for high-throughput screening of chemical compounds. In this assay, a Cy5- or C y5.5-labeled ligand binds to receptor immobilized on a particle, either a b ead or a cell. The resulting localized signal can be detected by a modified microvolume fluorimeter (MVF). When a molecule which competes with the lab eled ligand is present, the localized fluorescence on cells or beads is red uced. Image processing software enumerates events and analyzes fluorescence intensity. We describe MVF assays for the IL-1 and IL-5 receptors. Using s ynthetic peptides with a range of affinities for the IL-1 receptor, we obta ined IC50 data consistent with those determined by radioligand binding assa ys. Because the image processing software can discriminate among events wit h different diameters, we were able to develop a multiplex assay, in which the IL-1R and IL-5R assays were carried out in the same well with each rece ptor immobilized on a different size of bead. IC50 values generated in the multiplex assay for ligands specific to each receptor were comparable to th ose determined independently. Finally, similar IC50 values were obtained in a 16-mu l volume in an 864-well plate. This homogeneous, nonradioactive, m iniaturizable, and multiplex-capable assay holds much promise for screening of combinatorial libraries and compound collections. (C) 1999 Academic Pre ss.