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, an...
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.
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.
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