Immunoassays for the quantitation of porcine PAI-1 antigen and activity inbiological fluid samples

Two monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) f or the quantitation of porcine plasminogen activator inhibitor-1 (PAI-I) an tigen and activity in plasma were constructed and validated. The intra-assa y, interassay, and interdilution coefficients of variation were 4.3, 13, an d 8%, respectively, for the antigen ELISA and 5, 16, and 11% for the activi ty assay. Assay recoveries, in the antigen ELISA, of either latent or activ e 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 ass ay, 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 an tigen 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 P AI-I activity 42 +/- 17 ng/ml (n = 13). A linear correlation was found betw een 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 degree s C for 16 h resulted in a significant decrease (70 to 85%) of PAI-I activi ty. Under these conditions (37 degrees C, 16 h) PAI-I antigen levels remain ed unchanged in males whereas the response of the female samples in the PAI -I antigen assay increased two-fold. In lysed platelet-rich plasma males had 990 +/- 470 ng/ml antigen and 160 a 80 ng/ml activity and females, 920 +/- 500 ng/ml antigen and 150 +/- 98 ng /ml activity corresponding to 2.1 +/- 0.77 fg PAI-I antigen per platelet. O nly 16% of PAI-1 released from platelets was found to be active. Linear cor relations between PAI-1 antigen and activity were found for both males (r = 0.61) and females (r = 0.67). The assays are both sensitive and specific a nd may, therefore, aid the elucidation of the pathophysiological role of PA I-1 in swine experimental models of atherosclerosis and other thrombotic di sorders.