Homogeneous proximity tyrosine kinase assays: Scintillation proximity assay versus homogeneous time-resolved fluorescence

Two homogeneous proximity assays for tyrosine kinases, scintillation proxim ity assay (SPA) and homogeneous time-resolved fluorescence (HTRF), have bee n developed and compared. In both formats, the kinase assay was performed u sing biotinylated peptide substrate, ATP ([P-33]ATP in the case of SPA), an d tyrosine kinase in a 96-well assay format. After the kinase reaction was stopped, streptavidin-coated SPA beads or europium cryptate-labeled anti-ph osphotyrosine antibody and streptavidin-labeled allophycocyanin were added as detection reagents for SPA or HTRF assays, respectively. Since the assay signal was detected only when the energy donor (radioactivity for SPA, Eu for HTRF) and the energy acceptor molecules (SPA beads for SPA, allophycocy anin for HTRF) were in close proximity, both assays required no wash or liq uid transfer steps. This homogeneous ("mix-and-measure") nature allows thes e assays to be much simpler, more robust, and easier to automate than tradi tional protein kinase assays, such as a filter binding assay or ELISA. Both assays have been miniaturized to a 384-well format to reduce the assay vol ume, thereby saving the valuable screening samples as well as assay reagent s, and automated using automated pipeting stations to increase the assay th roughput. Several advantages and disadvantages for each assay are described . (C) 1999 Academic Press.