In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 96, No. 20 ( 1999-09-28), p. 11122-11127
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.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.96.20.11122
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
1999
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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