A novel high throughput chemiluminescent assay for the measurement of cellular cyclic adenosine monophosphate levels.
The second messenger 3', 5'-cyclic AMP (cAMP) is a highly regulated molecule that is governed by G protein-coupled receptor activation and other cellular processes. Measurement of cAMP levels in cells is widely used as an indicator of receptor function in drug discovery applications. We have developed a nonradioactive ELISA for the accurate quantitation of cAMP levels produced in cell-based assays. This novel competitive assay utilizes chemiluminescent detection that affords both a sensitivity and a dynamic assay range that have not been previously reported with any other assay methodologies. The assay has been automated in 96- and 384-well formats, providing assay data that are equivalent to, if not better than, data generated by hand. This report demonstrates the application of this novel assay technology to the functional analysis of a specific G protein-coupled receptor, neuropeptide receptor Y1, on SK-N-MC cells. Our data indicate the feasibility of utilizing this assay methodology for monitoring cAMP levels in a wide range of functional cell-based assays for high throughput screening.
Chiulli
A C
AC
Advanced Discovery Sciences, Applied Biosystems Tropix Division, Bedford, MA 01730, USA. AChiulli@appliedbiosystems.com
Trompeter
K
K
Palmer
M
M
D002460:Cell Line; D000242:Cyclic AMP / Q000032:analysis*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D008163:Luminescent Measurements; D017476:Receptors, Neuropeptide Y / Q000032:analysis; D012680:Sensitivity and Specificity
D017476:Receptors, Neuropeptide Y; C097212:neuropeptide Y-Y1 receptor; D000242:Cyclic AMP