AlphaScreen

The Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen ) developed by BioSignal, is a bead-based, non-radioactive assay technology. It uses the principle of luminescent oxygen channeling, which was first described in 1994 by Ullman [173], who two years later demonstrated a broad spectrum of applications for such immunoassays [174]. This principle senses the proximity of two beads, a donor and an acceptor bead, which is mediated by the interaction of molecules on the surfaces of the two beads. 

The AlphaScreen beads are reagent-coated polystyrene microbeads of 250 nm in diameter. When the biological interactions of the surface molecules hold the 

AlphaScreen applications include assays for enzymes such as protein kinases and proteases, immunoassays such as cAMP detection, and protein-protein and protein-DNA interactions. A recent literature example is a comparison of AlphaS-creen, TR-FRET, and TRF as assay methods for FXR nuclear receptors. In this comparison, the AlphaScreen system showed the highest sensitivity and the broadest dynamic range [149]. Another recent publication concerns a high-throughput binding assay for a TNF receptor [175]. 

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A more recently introduced format is the AlphaScreen assay. The assay principal behind this technology has previously been described above. In the kinase format a biotinylated peptide is bound to a streptavidin donor bead, and a phopshospecific antibody is bound to the acceptor bead. When the substrate is phosphorylated, the beads come in close proximity and a signal is generated. An example using the assay for the detection of inhibitors of serine kinases is presented by Von Leo-prechting [26]. 

There are cAMP assays that can be measured using a variety of techniques [35], including HTRF (CisBio) [36], bioluminescence (cAMP-Glo, Promega) [37], TR-FRET (CisBio) [38], LANCE [39], Alphascreen (Perkin Elmer) [40] and Enzyme Fragment Complementation (EFC) [41] (Hithunter, DiscoverX). This review will focus on the LANCE, the TR-FRET and ECF assays as they are the most used in our laboratories. 

The truth most likely lies somewhere in between. Bender [67] published the most quantitative study to date on the success of HTS at Novartis. Several conclusions could be drawn. Particular target types and assay technologies have a great impact on screening success, and this was not always correlated to the number of identifying hits in the HTS runs. For assay formats used a minimum of five times, LC/MS readouts succeed 83% of the time, followed by FP assays, which succeed in 72% of the cases. TR-FRET showed a success rate of 70%, with FLIPR assays (61%), fluorescence intensity readouts (59%), and AlphaScreen (60%) performing... 

Parameters Radiometric proximity assays (SPA, Flashplate) Fluorescence polarization (FP) Time- resolved fluorescence (HTRF) Amplified luminescence (ALPHAScreen) Enzyme (p-galactosidase) complementation Electrochemilumines cence... 

Flashplate and AlphaScreen assays (Perkin Elmer Life and Analytical Sciences Walthan, MA). 

AlphaScreen (amplified luminescent proximity homogeneous assay) PerkinElmer Life Sciences Donor beads produce singlet oxygen that induces fluorescence in proximal acceptor beads bead-bead proximity results from engagement of binding partners immobilized on the two bead types... 

Rouleau, N., Turcotte, S., Mondou, M.H., Roby, P., and Bosse, R., Development of a versatile platform for nuclear receptor screening using AlphaScreen, /. Biomol. Screen., 8,191, 2003. 

In order to avoid the described problems, antibody-based assays have been developed. As mentioned above, usually a primary antibody recognizes the modification, which in turn is then quantitated. One of these approaches is based on the technology of AlphaScreen (Amplified luminescent Proximity Homogeneous Assay) [51, 52], also known as luminescent oxygen channeling immunoassay (LOCI) [53], a method that can be used to study protein-protein interactions in general. In this case the enzymatic transfer of acetyl groups to a histone peptide is determined. 

Warner, G., Illy, C., Pedro, L., Roby, P. and Bosse, R. (2004) AlphaScreen kinase HTS platforms. Current Medicinal Chemistry, 11, 721-730. 

Spannhoff A., Valkov, V., Trojer, P., Bauer, I., Brosch, G. and Jung, M. (2009) In-vitro screening assays for histone acetyl- and methyltransferases using Alphascreen. 

Kinase or phosphatase assays based on the AlphaScreen principle are similar to TR-FRET assays in that they usually require a biotinylated substrate peptide and an anti-phosphoserine or tyrosine antibody. These two reagents are sandwiched between biotin and protein A-functionalized acceptor and donor beads. A kinase assay would show an enzyme-dependent increase in antibody binding (and thus signal) over time and a phosphatase assay would show an enzyme-dependent decrease in antibody binding over time. In some cases, the phosphorylation of an epitope will block the antibody binding and thus a phosphatase assay in principle can be constructed as a signal increase assay (Von Leoprichting and Kumpf, 2004 Warner et al., 2004). 

Cell lysis is required when using the ELISA, TR-FRET, or AlphaScreen readouts to detect the relative quantity of a specific intracellular phosphorylated protein sequence. A typical 2X to 4X RIPA buffer (commonly used for immunoprecipitation) containing a non-ionic detergent such as NP-40 or Tween20 is used. The lysis solution should contain protease and phosphatase inhibitors so that all further metabolism of the analyte is stopped. Often, an MTP shaking step is required to efficiently lyse the cells. 

Trends in biochemical screening assays seem to favor the use of multi-function PMT-based readers that allow for various MTP well densities (96, 384, and 1536 well plates), can handle a number of readout formats such as prompt fluorescence, luminescence, fluorescence polarization, time-gated fluorescence, and luminescent oxygen channeling or AlphaScreen. Examples of this type including the Perkin Elmer EnVision, TECAN-Ultra, BMG FluoStar, and LJL Analyst GT can be employed for a variety of the assay technologies described above. 

Von Leoprechting, A. et al. 2004. Miniaturization and validation of a high-throughput serine kinase assay using the AlphaScreen platform. J. Biomol. Screen. 9, 719-725. 

False positives may be observed with the AlphaScreen histidine detection kit certain inhibitors of Ni chelation to histidine are known. Compounds such as imidazoles can displace histidine from nickel chelates, resulting in a loss of signal. Compounds of interest should be tested for AlphaScreen interference by testing against a biotinylated (His)6 peptide provided in the kit. 

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