Monoclonal Antibody-based Immunoassays for the Specific Quantitation of Rat PAI-1 Antigen and Activity in Biological Samples

Summary

Two enzyme-linked immunosorbent assays (ELISAs) for the quantitation of rat plasminogen activator inhibitor-1 (PAI-1) antigen and activity, respectively, in biological fluids were developed using monoclonal antibodies raised against recombinant rat PAI-1. These assays had a lower limit of sensitivity in plasma of 0.3 and 0.15 ng/ml, respectively. The intra-assay, inter-assay and inter-dilution coefficients of variation were 9, 14 and 9%, respectively, for the antigen assay and 8, 17 and 13%, respectively for the activity assay. Assay recoveries of recombinant rat PAI-1 (5 to 20 ng/ml) added to plasma were 73 to 88% and 89 to 93% for the antigen and the activity assay, respectively. The level of PAI-1 antigen in rat plasma was 1.8 ± 0.9 ng/ml (mean ± SD, n = 18), with a corresponding value of 1.0 ± 0.5 ng/ml for PAI-1 activity. In lysed platelet-rich rat plasma PAI-1 antigen was 6.0 ± 1.0 ng/ml (n = 8) and PAI-1 activity was 2.3 ± 0.4 ng/ml (n = 8). Endotoxin injection (0.5 mg/kg) induced a time-dependent increase of both PAI-1 antigen and PAI-1 activity levels in rat plasma, eventually resulting in a 100- to 200-fold increase (p <0.0001 vs. baseline). A linear correlation was found between PAI-1 antigen and activity levels in normal plasma (r = 0.63, n = 18, p <0.01) and in plasma from endotoxin-treated rats (r = 0.90, n = 35, p <0.001). Application of these assays for the analysis of gel filtration experiments of plasma from endotoxin-treated rats demonstrated that PAI-1 antigen eluted as two peaks (with corresponding Mr of ~430 kDa and 61 kDa) whereas PAI-1 activity eluted as a single peak corresponding with the high molecular weight antigen form.

Thus, these unique assays allowing the specific determination of rat PAI-1 antigen and rat PAI-1 activity may constitute important tools for further investigations on the pathophysiological role of PAI-1 in a variety of experimental rat models.

• References

• 1 Kruithof EKO, Tran-Thang C, Ransijn A, Bachmann F. Demonstration of a fast-acting inhibitor of plasminogen activators in human plasma. Blood 1984; 64: 907-13.
• 2 Declerck PJ, Juhan-Vague I, Felez J, Wiman B. Pathophysiology of fibrinolysis. J Int Med 1994; 236: 425-32.
• 3 Van Meijer M, Pannekoek H. Structure of plasminogen activator inhibitor 1 (PAI-1) and its function in fibrinolysis: an update. Fibrinolysis 1995; 9: 263-76.
• 4 Diéval J, Nguyen G, Gross S, Delobel J, Kruithof EKO. A lifelong bleeding disorder associated with a deficiency of plasminogen activator inhibitor type-1. Blood 1991; 77: 528-32.
• 5 Lee MH, Vosburgh E, Anderson K, McDonagh J. Deficiency of plasma plasminogen activator inhibitor 1 results in hyperfibrinolytic bleeding. Blood 1993; 81: 2357-62.
• 6 Schleef RR, Higgins DL, Pillemer E, Levitt LJ. Bleeding diathesis due to decreased functional activity of type 1 plasminogen activator inhibitor. J Clin Invest 1989; 83: 1747-52.
• 7 Fay WP, Shapiro AD, Shih JL, Schleef RR, Ginsburg D. Complete deficiency of plasminogen activator inhibitor type 1 due to a frame-shift mutation. N Engl J Med 1992; 327: 1729-33.
• 8 Hamdan AD, Quist WC, Gagne JB, Feener EP. Angiotensin-converting enzyme inhibition suppresses plasminogen activator inhibitor-1 expression in the neointima of balloon-injured rat aorta. Circulation 1996; 93: 1073-8.
• 9 Emeis JJ, Brower A, Barelds RJ, Horan MA, Durham SK, Kooistra T. On the fibrinolytic system in aged rats, and its reactivity to endotoxin and cytokines. Thromb Haemost 1992; 67: 679-701.
• 10 Gallimore MJ, Tyle HM, Shaw JTB. The measurement of fibrinolysis in the rat. Thromb Diath Haemorrh 1971; 26: 279-309.
• 11 Van Giezen JJJ. Modulation of fibrinolytic activity in rats and other species. PhD thesis. Universiteit Utrecht The Netherlands: 1993
• 12 Emeis JJ, Van Den Hoogen CM. Pharmacological modulation of the endotoxin-induced increase in plasminogen activator inhibitor activity in rats. Blood Coagulation and Fibrinolysis 1992; 3: 575-81.
• 13 Johnson MR, Bruzdzinski CJ, Winograd SS, Gelehrter TD. Regulatory sequences and protein-binding sites involved in the expression of the rat plasminogen activator inhibitor-1 gene. J Biol Chem 1992; 267: 12202-10.
• 14 Heaton JH, Kathju S, Gelehrter TD. Transcriptional and posttranscriptional regulation of type 1 plasminogen activator inhibitor and tissue-type plasminogen activator gene. Expression in HTC rat hepatoma cells by glucocorticoids and cyclic nucleotides. Mol Endocrinol 1992; 6: 53-60.
• 15 Newman MJ, Lane EA, Iannotti AM, Nugent MA, Pepinsky RB, Keski-Oja J. Characterization and purification of a secreted plasminogen activator inhibitor (PAI-1) induced by transforming growth factor-1 in normal rat kidney (NRK) cells: Decreased PAI-1 expression in transformed NRK cells. Endocrinology 1990; 126: 2936-46.
• 16 Samama M, Nguyen G, Szwarcer E, Conrad J. Problems of t-PA specific inhibitor determination in human plasma. Thromb Haemost 1985; 54: 726.
• 17 Galfré F, Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol 1981; 73: 3-46.
• 18 Ngo TH, Bijnens AP, Knockaert I, Declerck PJ. Expression, purification and characterization of recombinant rat plasminogen activator inhibitor-1. Fibrinolysis 1997; 11: 37-43.
• 19 Anderson PM, Potter M. Induction of plasma cell tumors in Balb/c mice with 2,6,10,4 tetramethyl-pentadecane (pristane). Nature 1969; 222: 994-5.
• 20 Ey PL, Prowse SJ, Jenkins CR. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulin from mouse serum using protein A-Sepharose. Immuno-chemistry 1978; 15: 429-36.
• 21 Nakane PA, Kawaoi A. Peroxidase labelled antibody. A new method for conjugation. J Histochem Cytochem 1974; 22: 1084-91.
• 22 Declerck PJ, Verstreken M, Collen D. An immunofunctional assay for active plasminogen activator inhibitor-1 (PAI-1). Fibrinolysis 1988; 2: 77-8.
• 23 Holvoet P, Cleemput H, Collen D. Assay of human tissue-type plasminogen activator (t-PA) with an enzyme-linked immunosorbent assay (ELISA) based on three murine monoclonal antibodies. Thromb Haemost 1985; 54: 684-7.
• 24 Verheijen JH, Chang GTG, Kluft C. Evidence for the occurrence of a fast-acting inhibitor for tissue-type plasminogen activator in human plasma. Thromb Haemost 1984; 51: 392-5.
• 25 Declerck PJ, Alessi MC, Verstreken M, Kruithof EKO, Juhan-Vague I, Collen D. Measurement of plasminogen activator inhibitor 1 in biologic fluids with a murine monoclonal antibody-based enzyme-linked immunosorbent assay. Blood 1988; 71: 220-5.
• 26 Booth NA, Simpson AJ, Croll A, Bennett B, MacGregor IR. Plasminogen activator inhibitor (PAI-1) in plasma and platelets. Br J Haematol 1988; 70: 327-33.
• 27 Kruithof EKO, Nicolosa G, Bachmann F. Plasminogen activator inhibitor 1: Development of a radioimmunoassay and observasions on its plasma concentration during venous occlusion and after platelet aggreation. Blood 1987; 70: 1645-53.
• 28 Declerck PJ, Verstreken M, Collen D. Immunoassay of murine t-PA, u-PA and PAI-1 using monoclonal antibodies raised in gene-inactivated mice. Thromb Haemost 1995; 74: 1305-9.
• 29 Steele P, Weily H, Davies H, Pappas G, Genton E. Platelet survival time following aortic valve replacement. Circulation 1975; 51: 358-62.
• 30 Steele P, Battock D, Genton E. Effects of clofibrate and sulfinpyrazone on platelet survival time in coronary artery disease. Circulation 1975; 52: 473-6.
• 31 Meuleman DG, Vogel GMT, Stulemeyer SM, Moelker HCT. Analysis of platelet survival curves in an arterial thrombosis model in rat. Thromb Res 1980; 20: 31-43.
• 32 Meuleman DG, Vogel GMT, van Delft AML. Effects of intra-arterial cannulation on blood platelet consumption in rats. Thromb Res 1980; 20: 45-55.
• 33 Declerck PJ, De Mol M, Alessi MC, Baudner S, Pâques EP, Preissner KT, Müller-Berghaus KT, Collen D. Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. J Biol Chem 1988; 263: 5454-61.
• 34 Wiman B, Lindahl T, Almquist Å. Evidence for a discrete binding protein of plasminogen activator inhibitor in plasma. Thromb Haemost 1988; 59: 392-5.