MONOCLONAL ANTIBODY-BASED IMMUNOASSAYS FOR THE SPECIFIC QUANTITATION OF RAT PAI-1 ANTIGEN AND ACTIVITY IN BIOLOGICAL SAMPLES

Two enzyme-linked immunosorbent assays (ELISAs) for the quantitation o f rat plasminogen activator inhibitor-1 (PAI-1) antigen and activity, respectively, in biological fluids were developed using monoclonal ant ibodies raised against recombinant rat PAI-1. These assays had a lower limit of sensitivity in plasma of 0.3 and 0.15 ng/ml, respectively. T he intra-assay, inter-assay and inter-dilution coefficients of variati on were 9, 14 and 9%, respectively, for the antigen assay and 8, 17 an d 13%, respectively for the activity assay. Assay recoveries of recomb inant rat PAI-1 (5 to 20 ng/ml) added to plasma were 73 to 88% and 89 to 93% for the antigen and the activity assay, respectively. The level of PAI-1 antigen in rat plasma was 1.8 +/- 0.9 ng/ml (mean +/- SD, n = 18), with a corresponding value of 1.0 +/- 0.5 ng/ml for PAI-1 activ ity. In lysed platelet-rich rat plasma PAI-1 antigen was 6.0 +/- 1.0 n g/ml (n = 8) and PAI-1 activity was 2.3 +/- 0.4 ng/ml (n = 8). Endotox in injection (0.5 mg/kg) induced a time-dependent increase of both PAI -1 antigen and PAI-I activity levels in rat plasma, eventually resulti ng in a 100- to 200-fold increase (p <0.0001 vs, baseline). A linear c orrelation was found between PAI-1 antigen and activity levels in norm al plasma (r = 0.63, n = 18, p <0.01) and in plasma from endotoxin-tre ated rats (r = 0.90, n = 35, p <0.001). Application of these assays fo r the analysis of gel filtration experiments of plasma from endotoxin- treated rats demonstrated that PAI-1 antigen eluted as two peaks (with corresponding Mr of similar to 430 kDa and 61 kDa) whereas PAI-1 acti vity eluted as a single peak corresponding with the high molecular wei ght antigen form. Thus, these unique assays allowing the specific dete rmination of rat PAI-1 antigen and rat PAI-1 activity may constitute i mportant tools for further investigations on the pathophysiological ro le of PAI-1 in a variety of experimental rat models.