Monoclonal Antibodies Against Human High Molecular Weight Urinary Urokinase: Application for Affinity Purification of Urinary Prourokinase^[*]

Summary

Monoclonal antibodies against urinary urokinase were obtained by immunizing mice with purified human high molecular weight urokinase. Five antibodies were selected and denominated MPW1UK, MPW2UK, MPW3UK, MPW4UK, and MPW5UK, respectively. All selected antibodies reacted with high and low molecular weight urokinase. Cleavage of the low molecular weight paranitroanilide substrate pyro-Glu-Gly-Arg-pNA by urokinase was not inhibited by the antibodies and only one antibody (MPW5UK) inhibited plasminogen activation by urokinase. The ability of MPW5UK to bind to coated urokinase was 100-fold higher than that of the other antibodies. MPW5UK was used to prepare an immunosorbent for the purification of urokinase antigen from freshly voided crude urine. One-chain prourokinase was separated from two-chain urokinase by chromatography of the urokinase antigen containing mixture on agmatine Sepharose. As judged by SDS gel electrophoresis one-chain prourokinase as well as two-chain urokinase were purified to apparent homogeneity by this two-step procedure; the yields were 18% and 47% for single-chain prourokinase and two-chain urokinase, respectively, as calculated from total urokinase antigen contained in the starting material.

Keywords

Monoclonal antibodies - Two-chain urokinase - Single-chain prourokinase - Affinity purification

• References

• 1 Rijken DC, Wijngaards G, Welbergen J. Immunological characterization of plasminogen activator activities in human tissues and body fluids. J Lab Clin Med 1981; 97: 477-486
• 2 Wun TC, Schleuning WD, Reich E. Isolation and characterization of urokinase from human plasma. J Biol Chem 1982; 257: 3276-3283
• 3 Binder BR, Spragg J, Austen KF. Purification and characterization of human vascular plasminogen activator derived from blood vessel perfusates. J Biol Chem 1979; 254: 1998-2003
• 4 Bachmann F, Fletcher AP, Alkjaersig N. Partial purification and properties of the plasminogen activator from pig heart . Biochem 1964; 3: 1578-1585
• 5 Rijken DC, Collen D. Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture. J Biol Chem 1981; 256: 7035-7041
• 6 Wagner O, Wojta J, Binder BR. nalysis of the plasminogen activator production by two human tumor cell lines. In: Clinical Aspects of Fibrinolysis and Thrombolysis Jespersen J, Kluft C, Korsgaard O. (eds) 35-42 South Jutland University Press; Esbjerg, Denmark: 1983
• 7 Bachmann F, Kruithof EKO. Tissue plasminogen activator: Chemical and physiological aspects. Seminars in Thrombosis and Haemostasis 1984; 10: 6-17
• 8 Husain SS, Gurewich V, Lipinski B. Purification and partial characterization of a single-chain high-molecular-weight form of urokinase from human urine. Arch Biochem Biophys 1983; 220: 31-38
• 9 Herion P, Glineur C, Franssen JD, Urbain J, Bollen A. Monoclonal antibodies against urokinase. Bioscience Reports 1981; 1: 885-892
• 10 Kaltof K, Nielson LS, Zeuthe J, Dano K. Monoclonal antibody that specifically inhibits a human Mr 52,000 plasminogen-activating enzyme. Proc Natl Acad Sci USA 1982; 79: 3720-3723
• 11 Herion P, Bollen A. Purification of urokinase by monoclonal antibody affinity chromatography. Biosci Rep 1983; 3: 373-379
• 12 Vetterlein D, Calton GJ. Purification of urokinase from complex mixtures using immobilized monoclonal antibody against urokinase light chain. Thromb Haemostas 1983; 49: 24-27
• 13 Nielson LS, Hansen JG, Skriver L, Wilson EL, Kaltoft K, Zeuthen J, Dano K. Purification of zymogen to plasminogen activator from human glioblastoma cells by affinity chromatography with monoclonal antibody. Biochemistry 1982; 21: 6410-6415
• 14 Deutsch DG, Mertz ET. Plasminogen purification from human plasma by affinity chromatography. Science 1970; 170: 1095-1096
• 15 Huber K, Kirchheimer J, Binder BR. Rapid isolation of high molecular weight urokinase from native human urine. Thromb Haemostas 1982; 47: 197-202
• 16 Huber K, Kirchheimer J, Binder BR. Characterization of a specific anti-human urokinase antibody: development of a sensitive competitive radioimmunoassay for urokinase antigen. J Lab Clin Med 1984; 103: 684-694
• 17 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254
• 18 Lai CY. Detection of peptides by fluorescence methods. Methods in Enzymology 1977; 47: 236-243
• 19 Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495-497
• 20 Yelton DE, Diamond BA, Kwan SP, Scharff MD. Fusion of mouse myeloma and spleen cells. In: Lýmphocyte Hybridomas, Vol. 71 Melchers F, Potter M, Warner NL. (eds) 1-8 Springer Verlag, Berlin; Heidelberg New York: 1978
• 21 Galfre G, Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods in Enzymol 1981; 73: 43-46
• 22 Laemmli UK. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 1970; 227: 680-685
• 23 Laas T, Olsson I, Söderberg L. High voltage isoelectric focusing with Pharmalyte: Field strength and temperature distribution, zone sharpening, isoelectric spectra and pi determinations. Analyt Biochem 1980; 101: 449-461
• 24 Wray W, Bonlikas T, Wray VP, Hancock R. Silver staining of proteins in polyacrylamide gels. Analyt Biochem 1981; 118: 197-203
• 25 Geiger M, Binder BR. Plasminogen activation in diabetes mellitus. Kinetic analysis of plasmin formation using components isolated from the plasma of diabetic donors. J Biol Chem 1984; 259: 2976-2981
• 26 Wohl RC, Summaria L, Robbins KC. Kinetics of activation of human plasminogen by different activator species at pH 7.4 and 37° C. J Biol Chem 1980; 255: 2005-2013
• 27 Lowe CR, Dean PDG. Cyanogen bromide activation of agarose. Affinity Chromatography pp 250-253 John Wiley and Sons Ltd; London: 1974
• 28 Granelli-Piperno A, Reich E. A study of proteases and protease-inhibitor complexes in biological fluids. J Exp Med 1978; 148: 223-234
• 29 Lijnen HR, Zamarron C, Collen D. Pro-urokinase: Kinetics and mechanism of action. Thromb Haemostas 1985; 54: 118 (Abstr)
• 30 Pannell R, Gurewich V. Prourokinase (PUK) - characterization of its zymogenic properties and inherent amidolytic activity. Thromb Haemostas 1985; 54: 104 (Abstr)
• 31 Kirchheimer J, Wojta J, Resch I, Jörg M, Binder BR. Effect of FCB-2 fibrinogen fragments on activation and plasminogen activator activity of prourokinase (Pro-UK) isolated from urine. Thromb Haemostas 1985; 54: 160 (Abstr)