By Laura Gaidar, Kristin Prasauckas,
Kelly Carter-Allen and Loraine Upham
PerkinElmer Life Sciences, Inc.
AlphaScreen technology is a highly sensitive, non-isotopic homogenous assay method developed for assay development and high throughput screening applications. PerkinElmer Life Sciences has developed two instruments for the detection of AlphaScreen reagents. The AlphaQuest-HTS system measures 96-, 384- and 1536-well plates with four detectors simultaneously to maximize throughput, and is dedicated to the detection of AlphaScreen reagents. The Fusion α Multilabel Reader is a multi-detection system, capable of measuring absorbance (Vis and UV), prompt fluorescence, fluorescence polarization, time-resolved fluorescence, luminescence and AlphaScreen reagents in 96-, 384- and, in most modes, 1536-well plates, with a single detector. The following study was conducted to compare the performance of each system for the detection of cAMP using the AlphaScreen cAMP detection kit.
Materials and Methods
The cAMP standard curves were prepared as described in the AlphaScreen cAMP detection kit (PerkinElmer #6760600M) technical data sheet instructions. Unlabeled cAMP (Sigma, St. Louis, MO #A9501) was used to compete against the biotinylated cAMP provided in the AlphaScreen cAMP kit. Total reagent volume was 25 μl in 384-well white OptiPlate (PerkinElmer #6005214). Competition of unlabeled cAMP at various concentrations against a fixed concentration of biotinylated cAMP was measured using two Fusion α systems and an AlphaQuest-HTS system. Figure 1 illustrates the AlphaScreen cAMP assay(1). One unknown cAMP sample was also run with each curve to test the accuracy of the interpolation. A regression analysis of the raw and transformed values was performed to determine how closely the ou