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 tput of the two instruments matched.
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 strument were observed as shown in Figure 4.
Discussion and Conclusions
The AlphaQuest-HTS is a high throughput-screening instrument designed to measure exclusively AlphaScreen reagents in miniaturized formats. Although it is capable of measuring AlphaScreen samples in 96-, 384-well format for assay development purposes, it is not capable of measuring other assay methods. For this reason, the Fusion system was developed to offer versatility and AlphaScreen capability in one system. The versatility of the Fusion system provides the ability to create and optimize assays based on methods currently being used such as prompt fluorescence, absorbance, FP and luminescence. In addition, the Fusion can now be equipped with the AlphaScreen option, which creates the opportunity for assay developers to delve into this unique chemistry, while continuing to use current assay methods.
The significant difference between the Fusion α and the AlphaQuest-HTS for the detection of AlphaScreen assays is in the throughput capabilities as shown in Table 2. When the first AlphaScreen assay is ready to screen, the AlphaQuest-HTS incorporates two lasers and four PMTs to measure four samples simultaneously for screening purposes, and can measure almost 1000 samples/hour.
Graphical representations of the percent bound curves, similar IC50 values, signal to noise, signal to background and ability to detect similar concentrations for an unknown sample confirm that the AlphaQuest-HTS and the Fusion α obtain the same results for a given assay. In addition, the regression analysis of raw data and percent bound values showed an extremely high correlation between the two instruments. This means that any assay that is developed on a Fusion α can be screened directly in the AlphaQuest-HTS without further development and optimization.
1. PerkinElmer Life Sciences, Inc. (BioSignal Packard). Whole Cell cAMP Functional Assay, AlphaScreen Technical Note, AN001-ASC. Montreal, CN, (2000).
2. Bosse, R., et al. AlphaScreen to Measure cAMP Induction with SignalScreen Dopamine D1 Receptor Membranes. Poster. The Society for Biomolecular Screening Annual Conference, Vancouver, CN. 2000.
3. Rawlins, P., et al. A Comparison of Cyclic AMP Accumulation Assays for High Throughput Screening. Poster. The Society for Biomolecular Screening Annual Conference, Vancouver, CN. 2000.
4. Rawlins, P., et al. AlphaScreen: A Novel Non-radioactive Technology for High Throughput Screening. Poster. The Society for Biomolecular Screening Annual Conference, Vancouver, CN. 2000.