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Ligand-Receptor Binding Measured by Laser-Scanning Imaging

Paul Zuck, Zhege Lao, Stephen Skwish, J. Fraser Glickman, Ke Yang, Jonathan Burbaum and James Inglese
Proceedings of the National Academy of Sciences of the United States of America
Vol. 96, No. 20 (Sep. 28, 1999), pp. 11122-11127
Stable URL: http://www.jstor.org/stable/49000
Page Count: 6
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Ligand-Receptor Binding Measured by Laser-Scanning Imaging
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Abstract

This report describes the integration of laser-scanning fluorometric cytometry and nonseparation ligand-binding techniques to provide new assay methods adaptable to miniaturization and high-throughput screening. Receptor-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 competition-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 successfully 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 fluorescent immunoassays.

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