Measurement of Urokinase-Type Plasminogen Activator (u-PA) with an Enzyme-Linked Immunosorbent Assay (ELISA) Based on Three Murine Monoclonal Antibodies

V Darras; M Thienpont; D C Stump; D Collen

doi:10.1055/s-0038-1661693

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1986; 56(03): 411-414
DOI: 10.1055/s-0038-1661693

Original Article

Schattauer GmbH Stuttgart

Measurement of Urokinase-Type Plasminogen Activator (u-PA) with an Enzyme-Linked Immunosorbent Assay (ELISA) Based on Three Murine Monoclonal Antibodies

V Darras

The Center for Thrombosis and Vascular Research, University of Leuven, Belgium

,

M Thienpont

The Center for Thrombosis and Vascular Research, University of Leuven, Belgium

,

D C Stump

The Center for Thrombosis and Vascular Research, University of Leuven, Belgium

,

D Collen

The Center for Thrombosis and Vascular Research, University of Leuven, Belgium

› Author Affiliations

Abstract

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Summary

An enzyme-linked immunosorbent assay (ELISA) for the measurement of urokinase-type plasminogen activator (u-PA) was developed. Three murine monoclonal antibodies to single chain urokinase-type plasminogen activator (scu-PA) were isolated and shown to react with non-overlapping epitopes in scu-PA. Two of the three antibodies were coated onto microtiter plates and bound u-PA was quantitated with the third antibody conjugated to horseradish peroxidase. The assay was equally sensitive to M_r 54,000 u-PA in the single chain or two-chain form but did not respond to M_r 33,000 urokinase. The lower limit of sensitivity of the assay was 0.1 ng/ml in buffer and 1 ng/ml in plasma. Coefficients of variation of the assay at physiological levels of u-PA were 6.5 percent within assays and 13 percent between assays. The level of u-PA in normal resting plasma was 1.9 ± 0.66 ng/ml (mean ± SD, n = 54). The assay can be performed within one working day and provides an efficient, reproducible, and stable means for the measurement of u-PA in biological fluids. As such it may facilitate physiological and pharmacological studies of urokinase-type plasminogen activators in man.

Key words

ELISA - Urokinase-type plasminogen activator (u-PA) - Monoclonal antibodies - Fibrinolysis

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References

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