Phosphorylation of retinoblastoma protein (pRB) by cyclin-dependent kinases (CDKs) at multiple sites leads to activation of transcription of cell-cycle-related genes. Cyclin/CDK complexes thus play a pivotal role in the regulation of progression from G1 to S phase. In the present study, we developed a nonradioactive, sandwich enzyme-linked immunosorbent assay (ELISA) system for measuring activities of cyclin/CDK complexes, in which the immobilized monoclonal antibody works as a trap for phosphorylated pRB containing phosphorylated amino acids at specific sites. For this purpose, we raised monoclonal antibodies that are highly specific to ppRB phosphorylated at Ser780, Thr356, or Ser612 and used them as detectors for the individual reaction products by cyclin/CDK complexes. In particular, this approach proved useful for cyclin D1/CDK4 that specifically recognizes Ser780 in pRB with only very limited phosphorylation of a conventional substrate, histone H1. The study revealed the newly developed sandwich ELISA system to have advantages over the current radioisotope assay in terms of sensitivity, precision, and rapidity. It should find application for inhibitor screening and drug discovery related to CDKs.
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