Simultaneous determination of free tissue-type and free urokinase-type plasminogen activators in biological fluids by a solid-phase immunoassay.
Tissue-type and urokinase-type plasminogen activators (t-PA and u-PA) coexist in numerous biological fluids, where their fibrinolytic activities are determined by the concentration of the free, uncomplexed species. A simple, sensitive method has been developed for the simultaneous determination of free t-PA and u-PA concentrations in biological fluids using a solid-phase immunoassay. Microtiter plates were coated with polyclonal goat antibodies and incubated with PA standards or unknown samples. The absorbed PAs were then assayed by incubation with a mixture of plasminogen, poly-L-lysine, and the chromogenic substrate H-D-norleucylhexahydrotyrosyllysine-p-nitroanilide. Free t-PA and free u-PA were detectable in human plasma and urine, and in conditioned media from different endothelial cell cultures. The method is sensitive, with lower limits of quantitation being 0.76 mU/ml (1.25 pg/ml) for free t-PA and 0.16 mU/ml (2.0 pg/ml) for free u-PA. There was no cross-reaction between the two PA species and the recovery in plasma was greater than 95% for both. The intra- and interassay coefficients of variation for t-PA and u-PA were 3.5-12.2 and 3.2-11.3%, and 2.4-11.8 and 1.6-10.4%, respectively. The presence of PA-inhibitor complexes and PA inhibitors in biological fluids did not interfere with the assay. Application of the assay has demonstrated that free u-PA levels are several fold higher than free t-PA levels in human plasma and in conditioned media of human vascular endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Kuo
B S
BS
Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Bjornsson
T D
TD
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000201:enzymology; D004792:Enzyme Precursors / Q000032:analysis; D006801:Humans; D007118:Immunoassay / Q000379:methods; D015151:Immunoblotting; D008297:Male; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D010959:Tissue Plasminogen Activator / Q000032:analysis* / Q000097:blood / Q000652:urine; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis* / Q000097:blood / Q000652:urine
D004792:Enzyme Precursors; D012967:Sodium Dodecyl Sulfate; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator