IMMUNOASSAY OF MURINE T-PA, U-PA AND PAI-1 USING MONOCLONAL-ANTIBODIES RAISED IN GENE-INACTIVATED MICE

Three enzyme-linked immunosorbent assays for the quantitation of murin e tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor 1 (PAT-1), were developed using monoclonal antibodies raised against the autologous pr oteins in gene-inactivated mice. Dose-response was linear for t-PA and PAI-1 between 5 and 0.1 ng/ml and for u-PA between 50 and 1 ng/ml, wi th intra-assay, inter-assay and inter-dilution coefficients of variati on of 6 to 14%. Assay recoveries of proteins (5 to 100 ng/ml) added to plasma were 73 to 95% for t-PA and PAI-1. Linear correlations (r = 0. 65, r = 0.91 and r = 0.92, for t-PA, u-PA and PAI-1 respectively) were found between antigen and activity in plasma, urine and tissue extrac ts. Levels of t-PA and PAI-I antigen in murine plasma were 2.5 +/- 1.0 ng/ml (mean +/- SD, n = 9) and 1.9 +/- 0.6 ng/ml (mean +/- SD, n = 8) , respectively, in wild-type mice and undetectable in gene-inactivated mice. Bradykinin injection in mice provoked a 12-fold increase (p <0. 0002) of t-PA and endotoxin injection an 80-fold increase (p <0.005) o f PAI-1 levels. u-PA antigen levels in urine from wild-type; mice rang ed between 0.2 and 8.2 mu g/ml (1.8 +/- 1.9 mu g/ml, mean +/- SD, n = 17) and were undetectable in gene-inactivated mice. Thus, these assays may be useful for studies on the role of these proteins in tissue rem odeling, atherosclerosis, embryogenesis, etc., in established mouse mo dels. Gene-inactivated mice mag' constitute a general approach for the generation of monoclonal antibodies against the deficient translation products and for the development of specific immunoassays for murine proteins.