Fusion α and AlphaQuest-HTS produced overlaying curves
AlphaScreen cAMP assays have been developed using the PerkinElmer Life Sciences (BioSignal)(2) kit, have been compared with other technologies(3), and have been utilized for high throughput screening applications, such as to show accumulation of cAMP from Gi-coupled chemokine receptor(4).
AlphaScreen cAMP assays were performed. The IC50 values and concentrations of an unknown sample were analyzed using the Fusion and the AlphaQuest-HTS. Percent bound standard curves were created and IC50 values calculated using Fusion Data Analysis software. Both instruments produced comparable percent bound curves, such that the curves overlay (Figure 2).
IC50 values produced from the curves above were both 3.2 nM and the interpolated unknown concentration of cAMP for each instrument is reported in Table 1.
Both % bound and raw values correlated with an R2 of 0.999, showing a high degree of similarity between the AlphaQuest-HTS and Fusion α instruments (Figure 3).
Fusion α and AlphaQuest-HTS achieve similar levels of sensitivity .
Signal to Background ratio (S/B) compares the signal from the lowest (0.01 nM) and the highest (1,000 nM) concentration of unlabeled cAMP. Signal to background ratios were shown to be 23 and 17 for Fusion α and AlphaQuest-HTS, respectively. Signal to noise (S/N) ratio was calculated by taking the net signal of each concentration and dividing by the standard deviation of the 1,000 nM sample, which was in excess of the competitor and hence produced the lowest signal. At various concentrations, insignificant differences between the signal to noise ratios of each in