Characterization of Two Monoclonal Antibodies Against Human Tissue-Type Plasminogen Activator

Summary

A series of hybridoma clones, each producing monoclonal antibodies to human tissue-type plasminogen activator (t-PA), were prepared from mice by standard procedures. Two of these clones were selected for further study. One HI72A₁, produced antibodies that bound to t-PA and strongly inhibited its activity, whereas another, LI72D₁, produced antibodies that bound to t-PA but did not affect its activity. The specificity of these antibodies was assessed in immunoabsorption experiments. Both immunoprecipitated ¹²⁵I-labeled t-PA, and both were specific since only t-PA was recognized in conditioned media collected from Bowes melanoma cells cultured in the presence of ³H-leucine. Neither antibody recognized urokinase. t-PA was desorbed from antibody HI72A₁-Sepharose columns with 0.5 M NaCl, consistent with its relatively low association constant (Ka = 9.37 × 10⁷ M^-1). In contrast, a strong chaotropic agent (i.e., 2 M KI) was required to elute t-PA from antibody LI72D₁ columns (Ka = 2.08 × 10⁹ M^-1). This latter high affinity antibody was employed to develop an immunoradiometric assay for t-PA having a sensitivity of 0.5 ng/ml.

Keywords

Plasminogen activators - Monoclonal antibodies - Inhibition - Immunoradiometric assay

• References

• 1 Collen D. On the regulation and control of fibrinolysis. Thromb Haemostas 1980; 43: 77-89
• 2 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
• 3 Levin EG, Loskutoff DJ. Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators. J Cell Biol 1982; 94: 631-636
• 4 Rijken DC, Hoylaerts M, Collen D. Fibrinolytic properties of one-chain and two-chain human extrinsic (tissue-type) plasminogen activator. J Biol Chem 1982; 257: 2920-2925
• 5 Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. J Biol Chem 1982; 257: 2912-2919
• 6 Weimar W, Stibbe J, van Seyen AJ, Billiau A, de Somer P. Specific lysis of an iliofemoral thrombus by administration of extrinsic (tissue-type) plasminogen activator. Lancet 1981; 2: 1018-1020
• 7 Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, Bennett WF, Yelverton E, Seeburg PH, Heyneker HL, Goeddel DV, Collen D. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature 1983; 301: 214-221
• 8 Bányai L, Váradi A, Pathy L. Common evolutionary origin of the fibrin-binding structures of fibronectin and tissue-type plasminogen activator. FEBS Lett 1983; 163: 37-41
• 9 Schleef R, Loskutoff DJ. Monoclonal antibodies to human tissue-type plasminogen activator (t-PA). Thromb Haemostas 1983; 50: 82 (Abstr.)
• 10 Kruithof EK O, Ransijn A, Bachmann F. Inhibition of tissue plasminogen activator by human plasma. In: Progress in Fibrinolysis. Davidson JF, Bachmann F, Bouvier CA, Kruithof EK O. (Eds) Churchill Livingstone; Edinburgh: 1983. 6 362-366
• 11 Loskutoff DJ, van Mourik JA, Erickson LA, Lawrence D. Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells. Proc Natl Acad Sci USA 1983; 80: 2956-2960
• 12 Wiman B, Chmielewska J, Rånby M. Inactivation of tissue plasminogen activator in plasma. Demonstration of a complex with a new rapid inhibitor J Biol Chem 1984; 259: 3644-3647
• 13 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
• 14 Holmberg L, Bladh B, Åstedt B. Purification of urokinase by affinity chromatography. Biochem Biophys Acta 1976; 445: 215-222
• 15 Loskutoff DJ, Edgington TS. Synthesis of a fibrinolytic activator and inhibitor by endothelial cells. Proc Natl Acad Sci USA 1977; 74: 3903-3907
• 16 Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495-497
• 17 Kearney JF, Radbruch A, Liesegang B, Rajewsky K. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J Immunol 1979; 123: 1548-1550
• 18 Hoogenraad N, Helman T, Hoogenraad J. The effect of pre-injection of mice with pristane on ascites tumor formation and monoclonal antibody production. J Immunol Methods 1983; 61: 317-320
• 19 Kaltoft K, Nielsen LS, Zeuthen J, Danø K. Monoclonal antibody that specifically inhibits a human M_r 52,000 plasminogen activating enzyme. Proc Natl Acad Sci USA 1982; 79: 3720-3723
• 20 Frankel ME, Gerhard W. The rapid determination of binding constants for antiviral antibodies by a radioimmunoassay. An analysis of the interaction between hybridoma proteins and influenza virus Mol Immunol 1979; 16: 101-106
• 21 Ploplis VA, Cummings HS, Castellino FJ. Monoclonal antibodies to discrete regions of human glu₁-plasminogen. Biochemistry 1982; 21: 5891-5897
• 22 Loskutoff DJ. Effects of acidified fetal bovine serum on the fibrinolytic activity and growth of cells in culture. J Cell Physiol 1978; 96: 361-369
• 23 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
• 24 Rijken DC, Juhan-Vague I, de Cock F, Collen D. Measurement of human tissue-type plasminogen activator by a two-site immunoradiometric assay. J Lab Clin Med 1983; 101: 274-284
• 25 MacGregor IR, Prowse CV. Tissue plasminogen activator in human plasma measured by radioimmunoassay. Thromb Res 1983; 31: 461-474
• 26 Angles-Cano E, Sultan Y. A solid-phase fibrin immunoassay for the specific detection of monoclonal antibodies against different epitopic determinants of tissue-plasminogen activators. J Immunol Methods 1984; 69: 115-127
• 27 Pettersson G, Brandt J, Wallen P. Monoclonal antibodies to plasminogen activator from human uterine tissue. In: Progress in Fibrinolysis. Davidson JF, Bachmann F, Bouvier CA, Kruithof EK O. (Eds) Churchill Livingstone; Edinburg: 1983. 6 191-194
• 28 Nielsen LS, Hansen JG, Andreasen PA, Skriver L, Danø K, Zeuthen J. Monoclonal antibody to human 66,000 molecular weight plasminogen activator from melanoma cells. Specific enzyme inhibition and one-step affinity purification EMBO J 1983; 2: 115-119