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Homogeneous Cell-Based Fluorescence Polarization Assay for the Direct Detection of cAMP

PerkinElmer Life Sciences, Montreal, Quebec, Canada

Annie Gagne

PerkinElmer Life Sciences, Montreal, Quebec, Canada

Patricia Kasila

PerkinElmer Life Sciences, Boston, MA

Li-An Yeh

PerkinElmer Life Sciences, Boston, MA

Peter Banks

PerkinElmer Life Sciences, Montreal, Quebec, Canada

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.

Journal of Biomolecular Screening, Vol. 6, No. 2, 75-82 (2001)
DOI: 10.1177/108705710100600203


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