AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR THE DETERMINATION OF SRC-FAMILY TYROSINE KINASE-ACTIVITY IN BREAST-CANCER

An enzyme-linked immunosorbent assay is described for the determinatio n of protein tyrosine kinase activity originating from the presence of src-like tyrosine kinases in biological samples. In this assay a pept ide derived from p34(cdc2), cdc2(6-20)NH2, is coupled to the wells of a maleic anhydride-activated microtiter plate. This particular peptide has been described as an efficient and specific substrate for protein tyrosine kinases belonging to the src family kinases (Cheng et al.,J. Biol. Chem. 267 (1992) 9248-9256). After incubation of the coated sub strate with sample and ATP. the amount of phosphorylated tyrosyl resid ues is determined with phosphotyrosine specific antibodies and a secon dary peroxidase-labeled antibody. The assay appears to be very sensiti ve and is linear with sample protein concentration and phosphorylation time. Intra-assay variation is <5%, whereas day-to-day variation is < 10%. The results of the assay have been compared with an ELISA in whic h the broad-specificity tyrosine kinase substrate poly(GluNa,Tyr)4:1 w as coated. The results of both assays in 27 cytosolic breast cancer sa mples correlated very well (r = 0.94). in accordance with the predomin ant expression of src kinase activity in breast cancers (Ottenhoff-Kal ff ct at, Cancer Res. 52 (1992), 4773-4778). The present assay provide s an easy, reproducible, and quick alternative for the usual radioacti ve methods used for the determination of src-kinase activities includi ng immunecomplex kinase assay and TCA-precipitation assays. It allows the determination of src-like activities in human tumors for routine d iagnostic purposes.