Open AccessHomogeneous Cell-Based Fluorescence Polarization Assay for the Direct Detection of cAMP
Author(s) -
Linda Prystay,
Annie Gagne,
Patricia Kasila,
Li-An Yeh,
Peter Banks
Publication year - 2001
Publication title -
slas discovery
Language(s) - English
Resource type - Journals
eISSN - 2472-5560
pISSN - 2472-5552
DOI - 10.1177/108705710100600203
Subject(s) - agonist , high throughput screening , fluorescence , recombinant dna , chemistry , bioassay , hek 293 cells , homogeneous , receptor , fluorescence anisotropy , cell culture , ligand binding assay , chromatography , assay sensitivity , microbiology and biotechnology , biochemistry , biology , gene , thermodynamics , medicine , alternative medicine , pathology , physics , genetics , quantum mechanics , membrane
A fluorescence polarization-based functional assay for cyclic AMP (cAMP) production in cells has been proven effective for the detection of agonist-stimulated cAMP production in a HEK 293 recombinant cell line expressing the corticotropin-releasing factor subtype 2cr (CRF2a) receptor. Assays were completed in a single well of a 384-well microplate with no transfer, separation, or wash steps incurred. The assay performance is excellent for adaptation to the high throughput screening environment in terms of speed of analysis, magnitude of displaced signal, precision, and detection limits for cAMP quantitation. Relative potencies of agonists and antagonists are maintained with respect to radiometric assays. The assay withstands up to 5% DMSO and up to 10 μM concentrations of highly colored compound. These attributes suggest that accurate assessment of drug binding can be measured using this assay.