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 murine tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor 1 (PAI-1), were developed using monoclonal antibodies raised against the autologous proteins 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, with intra-assay, inter-assay and inter-dilution coefficients of variation 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 extracts. Levels of t-PA and PAI-1 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) of PAI-1 levels. u-PA antigen levels in urine from wild-type mice ranged between 0.2 and 8.2 micrograms/ml (1.8 +/- 1.9 micrograms/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 remodeling, atherosclerosis, embryogenesis, etc., in established mouse models. Gene-inactivated mice may 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.
Declerck
P J
PJ
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
Verstreken
M
M
Collen
D
D
D000818:Animals; D000911:Antibodies, Monoclonal; D007118:Immunoassay / Q000379:methods; D051379:Mice; D018345:Mice, Knockout; D017395:Plasminogen Activator Inhibitor 1 / Q000032:analysis* / Q000276:immunology; D010959:Tissue Plasminogen Activator / Q000032:analysis* / Q000276:immunology; D014568:Urokinase-Type Plasminogen Activator / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D017395:Plasminogen Activator Inhibitor 1; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator