This report describes the integration of la- ser-scanning fluorometric cytometry and nonseparation li- gand-binding techniques to provide new assay methods adapt- able to miniaturization and high-throughput screening. Re- ceptor-bound, cyanine dye-labeled ligands, [Cy] ligands, were discriminated from those free in solution by measuring the accumulated fluorescence associated with a receptor- containing Particle. To illustrate the various binding formats accommodated by this technique, saturation- and competi- tion-binding analyses were performed with [Cy]ligands and their cognate receptors expressed in CHO cells or as fusion proteins coated on polystyrene microspheres. We have suc- cessfully applied this technique to the analysis of G protein- coupled receptors, cytokine receptors, and SH2 domains. Multiparameter readouts from ligands labeled separately with Cy5 and Cy5.5 demonstrate the simultaneous analysis of two target receptors in a single well. In addition, laser- scanning cytometry has been used to assay enzymes such as phosphatases and in the development of single-step fluores- cent immunoassays.
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