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 Cy5.5-labeled ligand binds to receptor immobilized on a particle, either a bead or a cell. The resulting localized signal can be detected by a modified microvolume fluorimeter (MVF). When a molecule which competes with the labeled ligand is present, the localized fluorescence on cells or beads is reduced. Image processing software enumerates events and analyzes fluorescence intensity. We describe MVF assays for the IL-1 and IL-5 receptors. Using synthetic peptides with a range of affinities for the IL-1 receptor, we obtained IC(50) data consistent with those determined by radioligand binding assays. Because the image processing software can discriminate among events with 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 receptor immobilized on a different size of bead. IC(50) values generated in the multiplex assay for ligands specific to each receptor were comparable to those determined independently. Finally, similar IC(50) values were obtained in a 16-microl volume in an 864-well plate. This homogeneous, nonradioactive, miniaturizable, and multiplex-capable assay holds much promise for screening of combinatorial libraries and compound collections.