Thrombosis and haemostasis vol:84 issue:6 pages:1082-1086
Two monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) for the quantitation of porcine plasminogen activator inhibitor-1 (PAI-I) antigen and activity in plasma were constructed and validated. The intra-assay, interassay, and interdilution coefficients of variation were 4.3, 13, and 8%, respectively, for the antigen ELISA and 5, 16, and 11% for the activity assay. Assay recoveries, in the antigen ELISA, of either latent or active recombinant porcine PAI-I (10 and 50 ng/ml) added to plasma were 86 +/- 9% and 92 +/- 22%, respectively, for the latent form and 89 +/- 9% and 87 + 7% for the active form (mean + SD, n = 3 to 4). In the immunofunctional assay, recoveries for the same concentrations of active PAI-1 were 108 +/- 16% and 92 +/- 21%, respectively. In male porcine plasma the level of PAI-I antigen was 31 +/- 11 ng/ml and the activity, 34 +/- 16 ng/ml (mean a SD, n = 10). In female plasma PAI-1 antigen levels were 20 +/- 5.2 ng/ml and the PAI-I activity 42 +/- 17 ng/ml (n = 13). A linear correlation was found between PAI-1 antigen and activity levels in male (r = 0.60) and female (r = 0.70) plasma. Immunodepletion resulted in a decrease of >95% of the original PAI-1 antigen or activity levels. Incubation of plasma samples at 37 degrees C for 16 h resulted in a significant decrease (70 to 85%) of PAI-I activity. Under these conditions (37 degrees C, 16 h) PAI-I antigen levels remained unchanged in males whereas the response of the female samples in the PAI-I antigen assay increased two-fold.