A CHEMILUMINESCENT MICROTITER PLATE ASSAY FOR SENSITIVE DETECTION OF PROTEIN-KINASE ACTIVITY

A chemiluminescent protein kinase assay using biotinylated substrate p eptides captured on a streptavidin-coated microtiter plate and monoclo nal antibodies to detect their phosphorylation is described. Assay con ditions were optimized and validated for sensitive measurement of prot ein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kina se II (CAMKII), receptor interacting protein, and src activities. The newly developed chemiluminescent assay has several advantages over cur rently used radioactive or colorimetric methods. It is highly sensitiv e at low enzyme and substrate concentrations and high, close to physio logical ATP levels. It is fast, simple to perform and amenable to auto mation and high-throughput drug screening. The assay is also robust, e xhibiting minimum interference from solvents and test substances from various sources. Overall, among the presently available methods for th e detection of protein kinase activity, chemiluminescence was found to provide the highest sensitivity under conditions most closely mimicki ng the intracellular environment. This assay is expected to be useful in both academic and industrial laboratories, especially in identifyin g novel classes of protein kinase inhibitors. (C) 1997 Academic Press