AlphaScreen™ technology

Packard Website, AlphaScreen Technology - Application Note AC-012, Analysis of Potential Compound Interference of AlphaScreen Signal, http //www.perkinelmer.co.jp/tech/tech ls/protocol collection/asc-012.pdf, 2001. 

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]. 

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... 

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. 

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. 

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. 

Format/signal generation technologies Heterogenous— wash, e.g., ELISA, SEAP reporters colorimetric, absorbance homogeneous— luminescence, fluorescence, FRET Homogeneous—Imninescence, fluorescence, TR-FRET, FPA, AlphaScreen ... 

---