AlphaScreen as a Pharmaceutical Screening Tool

An AlphaScreen assay is generally conducted in a 384-well plate in a total volume of 25 ]xL, but can be scaled down to the 1536-well format with assay volumes of 5 pL. Since the donor beads are light sensitive, the assay has to be performed under dimmed light After all components have been added, the luminescence signal is measured, usually after an incubation of 1 h. 

The first consideration when setting up an AlphaScreen assay is the choice of an assay format In most cases, the decision will depend on the interaction partners under investigation and on the biological tools available for their detection. The interaction partners can be coupled to the beads directly via reductive amination of reactive aldehyde groups, similar to the immobilization on a Biacore sensor chip (see above). The usefulness of this approach is limited by the reaction conditions, which may not be appropriate for maintaining the biologically active conformation of the biomolecule. Therefore the biomolecule of interest is usually not coupled to the beads directly, but instead captured via an antibody, also preventing steric hindrance. While not strictly necessary, it is often convenient to use a biotinylated molecule which can be captured by streptavidin-coated donor beads. 

B Upon addition of cAMP (or a cAMP agonist), C and R subunits dissociate, resulting in a decrease in luminescence as the singlet oxygen decays without an acceptor bead in its vicinity. 

C Luminescence signal obtained (counts per second, cps) is plotted against the logarithm of agonist concentration and fitted to a sigmoidal dose-response model. 

The components involved are streptavidin-coated donor beads and anti-GST acceptor beads (Perkin Elmer LAS, Boston, USA) and - as the biological system -the PKA subunits as cAMP sensors. Upon binding of cAMP to each regulatory (R) subunit, the PKA holoenzyme dissociates releasing the catalytic (C) subunits. 

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