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Thromb Haemost 2000; 84(06): 1082-1086
DOI: 10.1055/s-0037-1614174
Review Article
Schattauer GmbH

Immunoassays for the Quantitation of Porcine PAI-1 Antigen and Activity in Biological Fluid Samples

Henry M. J. Leng
1   From the Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium
,
Els Brouwers
1   From the Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium
,
Isabelle Knockaert
1   From the Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium
,
Paul J. Declerck
1   From the Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium
› InstitutsangabenHenry M. J. Leng was a recipient of a post-doctoral grant from the Flemish Community Government and a travel grant from the South African Foundation for Research and Development. The authors gratefully acknowledge Mrs. T. Stassen and Dr. I. De Scheerder (Dept. of Cardiology) for collection of the samples.
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Publikationsverlauf

Received 18. April 1999

Accepted after revision 29. Juni 2000

Publikationsdatum:
13. Dezember 2017 (online)

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Summary

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 ± 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-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° C for 16 h resulted in a significant decrease (70 to 85%) of PAI-1 activity. Under these conditions (37° 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.

Key words

Fibrinolysis - pig - PAI-1 - atherosclerosis - ELISA
 

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