Summary
Three enzyme-linked immunosorbent assays for the quantitation of murine tissue-type
plasminogen activator (t-PA), urokinase-type plasminogen aetivator (u-PA) and plasminogen
activator inhibitor I (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
μ;g/ml (1.8 ± 1.9 μg/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.
