Sensitive and specific enzyme-linked immunosorbent assay for urokinase-type plasminogen activator and its application to plasma from patients with breast cancer.
An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of human urokinase-type plasminogen activator (u-PA) in plasma and serum. Microtiter plates were coated with a monoclonal antibody and incubated with standard or sample. Bound u-PA was quantitated with polyclonal antibodies conjugated with biotin, followed by avidin-peroxidase. The assay was 10 times as sensitive as previously reported immunoassays, the detection limit being approximately 1 pg u-PA in a volume of 100 microliter, with a linear dose-response up to 15 pg u-PA. The assay detected active u-PA and its inactive proenzyme form equally well, and the recovery of both forms was higher than 90% in plasma. It also detected u-PA complexed with plasminogen activator inhibitor type 1. Various structurally related proteins, including t-PA, were tested, but no reaction was observed with proteins other than u-PA and its amino-terminal fragment. The intra-assay and interassay coefficients of variation for determination of u-PA in plasma were 7.6% and 8.4%, respectively. The ELISA was used to measure the concentration of u-PA in plasma from 34 healthy donors and 92 patients with breast cancer with a varying extent of disease. The mean value for the healthy donors was 1.1 +/- 0.3 ng/ml (SD) of u-PA in plasma. This value is substantially lower than those previously reported. The mean value for the patients with breast cancer was 1.3 +/- 0.4 ng/ml. This moderate increase was statistically significant at the 1% level. Approximately one quarter of the patients had plasma u-PA concentrations above the range observed for the healthy controls. There was a positive correlation between the mean u-PA plasma concentration and the extent of disease in different groups of patients.
D000293:Adolescent; D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal; D001782:Blood Donors; D001943:Breast Neoplasms / Q000097:blood / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D005260:Female; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D008875:Middle Aged; D010960:Plasminogen Activators / Q000097:blood* / Q000276:immunology; D012967:Sodium Dodecyl Sulfate; D014568:Urokinase-Type Plasminogen Activator / Q000097:blood* / Q000276:immunology
D000911:Antibodies, Monoclonal; D007202:Indicators and Reagents; D012967:Sodium Dodecyl Sulfate; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator