Stable, Sensitive, Fluorescence-Based Method for Detecting cAMP
cAMP is a universal secondary messenger that connects changes in the extracellular environment, as detected by cell surface receptors, to transcriptional changes in the nucleus. Since cAMP-mediated signal transduction plays a role in critical cell functions and human diseases, monitoring its activity can aid in understanding these responses and the process of drug discovery. This report examines the performance of a fluorescence-based competitive immunoassay in 384-well microplate format. Using
... plate format. Using purified cAMP as a competitor, the estimated detection limit was determined to be 0.1 nM and Z′-factor was greater than 0.83, which indicates that the assay is of high quality and one of the most sensitive assays currently on the market. Of note, the results obtained were similar whether the reaction was allowed to proceed for 10 min or up to 60 min. Next, HEK 293 cells were treated with the promiscuous adenylate cyclase activator, forskolin, and the β-adrenoceptor agonist, isoproterenol. The resultant average EC 50 values were 11 µM and 123 nM, respectively, which correspond to those found in the literature. Together, these results demonstrate that this assay is a fast, accurate, nonradioactive method that is ideal for highthroughput screening. Figure 3. Dose response curves for isoproterenol-treated HEK 293 cells. Each point on the dose response curves represents the average cAMP of eight replicate samples for one concentration of the agonist. The R 2 value for both experiments was 0.98 for a 4-parameter curve fit. The dose response curves shown were obtained after 60 min incubation of samples with Stoplight Red substrate. The curve shown in panel A was obtained using Gemini XS, and the curve shown in panel B was obtained using Analyst AD. The cAMP values shown on the Y-axis were obtained by interpolation of the original data. The EC 50 values were (A) 123 nM and (B) 123 nM for Gemini XS and Analyst AD, respectively.