Development of a time-resolved fluorescent assay for measuring tyrosine-phosphorylated proteins in cells

We have developed a time-resolved fluorescent assay using Wallac's DELFIA system (DELFIA assay) to monitor changes in the phosphorylation level of insulin receptor from rat hepatoma (KRC-7) cells in response to ligand and the nonspecific, protein-tyrosine phosphatase inhibitor pervanadate. In this system, a biotinylated antiinsulin receptor antibody was used to capture the insulin receptor and an europium-labeled antiphosphotyrosine antibody was used to assess tyrosine phosphorylation. This assay provides a highly sensitive, nonradioactive readout of receptor phosphorylation. We have validated the DELFIA assay by directly comparing receptor phosphorylation using the well-established technique of immunoblotting. The utility of the DELFIA assay in measuring the phosphorylation status of other receptors has also been demonstrated using epidermal growth factor receptor from A431 cells.