Immunoassays for the quantitation of porcine PAI-1 antigen and activity in biological fluid samples.
Two monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) for the quantitation of porcine plasminogen activator inhibitor-1 (PAI-1) 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-1 (10 and 50 ng/ml) added to plasma were 86 +/- 9% and 92 +/- 22%, respectively, for the latent form and 89 +/- 99% 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-1 antigen was 31 +/- 11 ng/ ml and the activity, 34 +/- 16 ng/ml (mean +/- SD, n = 10). In female plasma PAI-1 antigen levels were 20 +/- 5.2 ng/ml and the PAI-1 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-1 activity. Under these conditions (37 degrees C, 16 h) PAI-1 antigen levels remained unchanged in males whereas the response of the female samples in the PAI-1 antigen assay increased two-fold. In lysed platelet-rich plasma males had 990 +/- 470 ng/ml antigen and 160 +/- 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-1 antigen per platelet. Only 16% of PAI-1 released from platelets was found to be active. Linear correlations 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 and may, therefore, aid the elucidation of the pathophysiological role of PAI-1 in swine experimental models of atherosclerosis and other thrombotic disorders.
Leng
H M
HM
Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium.
Brouwers
E
E
Knockaert
I
I
Declerck
P J
PJ
D000818:Animals; D000941:Antigens / Q000032:analysis / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary; D005260:Female; D007118:Immunoassay / Q000379:methods / Q000662:veterinary; D008297:Male; D017395:Plasminogen Activator Inhibitor 1 / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012737:Sex Factors; D013552:Swine / Q000097:blood* / Q000276:immunology*
D000941:Antigens; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins