Characterization of a specific anti-human urokinase antibody: development of a sensitive competition radioimmunoassay for urokinase antigen.
Specific inhibiting IgG antibodies were raised in a rabbit using purified human high molecular weight urokinase as antigen. The antibodies reacted with both high molecular weight and low molecular weight human urokinase using an Ouchterlony double-immunodiffusion technique in such a way that one line of complete identity was obtained. Neither precipitation nor functional inhibition was observed for the tissue-type plasminogen activator. Kinetic studies with plasminogen as a natural high molecular weight substrate or the synthetic low molecular weight p-nitroanilide substrate pyro-Glu-Gly-Arg-pNA revealed, for both substrates, mainly a competitive type of inhibition for the Fab fragments of the specific anti-urokinase antibodies. This characterized anti-urokinase IgG was employed to develop a competitive radioimmunoassay, for human urokinase with 125I-labeled urokinase as tracer. In this radioimmunoassay, the lower detection limit for urokinase antigen was 10 pg/ml sample; the intrassay variation was 2.8%, and the interassay variation was 5.3%. Applying this radioimmunoassay to plasma samples obtained from healthy young volunteers, urokinase antigen could be measured in a concentration of 7.82 +/- 3.97 ng/ml for mean and 6.66 +/- 2.39 ng/ml for women (mean +/- SD).
D000328:Adult; D000906:Antibodies / Q000302:isolation & purification*; D001667:Binding, Competitive; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000302:isolation & purification; D007457:Iodine Radioisotopes; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D011863:Radioimmunoassay / Q000379:methods*; D013379:Substrate Specificity; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis* / Q000097:blood / Q000652:urine
D000906:Antibodies; D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D014568:Urokinase-Type Plasminogen Activator