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 determination of protein tyrosine kinase activity originating from the presence of src-like tyrosine kinases in biological samples. In this assay a peptide derived from p34cdc2, 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 substrate with sample and ATP, the amount of phosphorylated tyrosyl residues is determined with phosphotyrosine specific antibodies and a secondary peroxidase-labeled antibody. The assay appears to be very sensitive 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 which the broad-specificity tyrosine kinase substrate poly(GluNa,Tyr)4:1 was coated. The results of both assays in 27 cytosolic breast cancer samples correlated very well (r = 0.94), in accordance with the predominant expression of src kinase activity in breast cancers (Ottenhoff-Kalff et al., Cancer Res. 52 (1992), 4773-4778). The present assay provides an easy, reproducible, and quick alternative for the usual radioactive methods used for the determination of src-kinase activities including immunecomplex kinase assay and TCA-precipitation assays. It allows the determination of src-like activities in human tumors for routine diagnostic purposes.
Rijksen
G
G
Dept. Hematology, University Hospital Utrecht, The Netherlands.
Adriaansen-Slot
S S
SS
Staal
G E
GE
D000818:Animals; D001681:Biological Assay; D001943:Breast Neoplasms / Q000175:diagnosis / Q000201:enzymology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D011099:Polyglutamic Acid / Q000032:analysis; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013154:Spleen / Q000201:enzymology; D014443:Tyrosine / Q000032:analysis; D019061:src-Family Kinases / Q000378:metabolism*
D011099:Polyglutamic Acid; D014443:Tyrosine; D019061:src-Family Kinases