Monoclonal Antibody Directed Against Heparin and Heparin-Fractions

  • Günter Huhle
  • Job Harenberg
  • Reinhard Malsch
  • Dieter L. Heene

Summary

A monoclonal heparin antibody H 1.18 has been raised against native heparin using a heparin-bovine serum albumin conjugate prepared by reductive amination. The H 1.18 antibody was purified by protein A/G affinity chromatography. For further analyses tyramine, which was covalently bound to low molecular mass heparin by endpoint attachment (Maisch R et al: Anal Biochem 1994; 217: 255–264), was labeled with 125-iodine at the aryl residue. The tracer antibody complex was immunoprecipitated by goat anti-mouse immunoglobuline IgG. H 1.18 recognized specifically intact heparin and heparin fractions. The lower detection limit of heparin preparations was 100 ng/ml. No cross reactivity of H 1.18 occurred with other glycosaminoglycans such as heparan sulfate, dermatan sulfate, chondroitin sulfate A and C. Oversulfated heparin showed lower affinity to the antibody H 1.18 than 2–0-and 6–0-desulfated heparin.

In conclusion, the present paper describes an IgG 1 monoclonal directed against heparin fractions between 4.500 and 6.500 dalton, which can be used for biological measurements.

Keywords

Lipase Polysaccharide Serine Sarcoma Hull 

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References

  1. 1.
    Casu, B. (1989) Structure of heparin and heparin fragments. Ann. N. Y. Acad. Sci. 556, 1–17PubMedCrossRefGoogle Scholar
  2. 2.
    Linhardt, R.J., Merchant, Z. M., Rice, K. G., Kim, Y. S., Fitzgerald, G. L., Grant, A. C. and Langer, R. Evidence of random structural features in the heparin polymer. Biochemistry 1985; 24: 7805–7810.PubMedCrossRefGoogle Scholar
  3. 3.
    Harenberg J, de Vries JX: Characterization of heparins by high performance size exclusion liquid chromatography. J Chromatogr 1983; 261: 287–292.CrossRefGoogle Scholar
  4. 4.
    Maisch R, Harenberg J: Considerations about the correct nomenclature of glycosaminoglycans (GAGs). Thromb Haemostas 1993; 70: 718–719.Google Scholar
  5. 5.
    Rosenberg RD: Role of heparin and heparin-like molecules in thrombosis and atherosclerosis. Fed Proc 1977; 36: 10–18.PubMedGoogle Scholar
  6. 6.
    Lane DA, Lindahl U (Eds.): Heparin. Chemical and biological properties, clinical application. Arnold 1989; London.Google Scholar
  7. 7.
    Linhardt RJ, Rice KG, Kin YS, Engelken J and Weiler J: Homogeneous structural defined heparin-oligosaccharides with low anticoagulant activity inhibit the generation of the amplification pathway C3 convertase in vitro. J Biol Chem 1988; 263: 13040–13046.Google Scholar
  8. 8.
    Merchant ZM, Erbe,EE, Eddy WP,Patel D, Linhardt RJ: Effect of very low molecular weight heparin-de-rived oligosaccharides on lipoprotein lipase release in rabbits. Atherosclerosis 1986; 62 (2): 151–158.PubMedCrossRefGoogle Scholar
  9. 9.
    Baba M, Pauwels R, Balzarini J, Arnout J, De Clerqu E: Mechanism of inhibitory effect of dextransulfate and heparin on replication of human immunodeficiency virus in vitro. Proc Natal Acad Sci USA 1988; 85: 6132–6136.CrossRefGoogle Scholar
  10. 10.
    Kakkar VV, Cohen A, Edmonson RA, Phillips MJ, Cooper DJ, Das SK, Maher KT, Sanderson RM; Ward VP, Kakkar S: Low molecular weight versus standard heparin for prevention of venous thromboembolism after major abdominal surgery. Lancet 1993; 341: 259–265.PubMedCrossRefGoogle Scholar
  11. 11.
    Nourmahomed MT, Rosendaal FR, BŸller HR, Dekker E, Hommes DW, Vandenbroucke JP, Briet E: Low molecular weight heparin versus standard heparin in general and orthopedic surgery: a meta-analysis. Lancet 1992; 340: 152–156.CrossRefGoogle Scholar
  12. 12.
    THRIFT Consensus Group: Risk of and prophylaxis for venous thromboembolism in hospital patients. Br Med J 1992; 305: 567–573.CrossRefGoogle Scholar
  13. 13.
    Harenberg J, Roebruck P, Heene DL and the HESIM-group: Comparison of subcutaneous low-molecular-weight heparin with unfractionated heparin for prophylaxis of thromboembolism in medical inpatients. Ann Hematol 1994; 68: 51 (abstract).CrossRefGoogle Scholar
  14. 14.
    Hull R, Raskob GE, Pineo GF Green D, Trowbridge A, Elliott CG, Lerner RG, Hall J, Sparling T, Brettell HR, Norton J, Carter DJ, George R, Merli G, Ward J, Mayo W, Rosenbloom D, Brant R: Subcutaneous low-molecular-weight heparin compared with continuous intravenous heparin in the treatment of proximal-vein thrombosis. N Engl J Med 1992; 326: 975–982.PubMedCrossRefGoogle Scholar
  15. 15.
    Prandoni P, Lensing AWA, BŸller HR, Carta M, Cogo A, Vigo M, Casara D, Ruol A, ten Cate JW: Comparison of subcutaneous standard heparin in proximal deep-vein thrombosis. Lancet 1992; 339: 441–445.PubMedCrossRefGoogle Scholar
  16. 16.
    Linhardt RJ. Heparin: An important drug enters into seventh decade. Chem Ind 1991; 2: 45–50.Google Scholar
  17. 17.
    Kure S, Yoshie O: A synergetic monoclonal antibody to murine with A sarcoma C (Hep SS-1) recognizes heparin sulfate glycosaminoglycans (HS-GAG): Cell density and transalteration in all surface HS-GAG defined by Hep SS-1. J Immunol 1986; 137: 3900–3908.PubMedGoogle Scholar
  18. 18.
    Born Van-den J, Heuvel Van-den LP, Bakker MA, Veerkamp JH, Assmann KJ; Berden JH: A monoclonal antibody against GBM heparan sulfate induces an acute selective proteinuria in rats. Kidney Int 1992; 41: 115–123.PubMedCrossRefGoogle Scholar
  19. 19.
    Yagamata M, Kimata K, Oike Y, Tani K, Maeda N, Yoshida K, Shimomura Y, Yoneda M, Suzuki S: A monoclonal antibody that specifically recognized a glucuronic acid 2-sulfate-containing determinant in intact chondroitin-sulfate chain. J Biol Chem 1987; 262: 4146–4152Google Scholar
  20. 20.
    Caterson B, Christner JE, Baker JR: Identification of a monoclonal antibody that specifically recognized corneal and skeletal keratan sulfate. J Biol Chem 1983; 258: 8848–8854.PubMedGoogle Scholar
  21. 21.
    Mehmet H, Scudder P, Tang PW, Hounsell EF, Caterson B, Feizi T: The antigenic determinants recognized by three monoclonal antibodies to keratan sulfate involve sulphated hepta-or larger oligosaccharides of the poly (N-acetyl-lactosamine) serines. Eur J Biochem 1986; 157: 385–391.PubMedCrossRefGoogle Scholar
  22. 22.
    Pejler G, Lindahl U, Larm O, Scholander E, Sandgren E, Lundblad A: Monoclonal antibodies for oligosaccharides prepared by partial nitrous acid deamination of heparin. J Biol Chem 1988; 263: 5197–5201.PubMedGoogle Scholar
  23. 23.
    Gitel SN, Medina VM, Wessler S: Preparation and identification of a population of antibodies that recognize carbodiimide-modified heparin. Blood 1985; 65: 902–911.PubMedGoogle Scholar
  24. 24.
    Strauss AH, Travassos LR, Takahashi HK: A monoclonal antibody (ST-1) directed to the native heparin chain. Anal Biochem 1992; 201: 1–8.CrossRefGoogle Scholar
  25. 25.
    Harenberg J, Maisch R; German Patent Nr. P 4217916.5–43.Google Scholar
  26. 26.
    Maisch R, Guerrini M, Torri G, Casu B, Harenberg J: Synthesis of a N«-Alkylamine anticoagulant active low-molecular-mass heparin for radioactive and fluorescent labeling. Anal Biochem 1994; 217: 255–264.CrossRefGoogle Scholar
  27. 27.
    Hunter WM, Greenwood FC: Preparation of iodine-131 labeled human growth hormone of high specific activity. Nature 1962; 194: 485–496.CrossRefGoogle Scholar
  28. 28.
    Harenberg J, Lshr G, Maisch R, Dempfle CE, Stehle G, Guerrini M, Torri G, Casu B, Heene DL: Magnetic bead protamine linked microtiter assay for detection of heparins using iodinated tyramine-heparin fragmin. Thromb Haemostas 1993; 69: 2012 (Abstract).Google Scholar
  29. 29.
    Harenberg J, Giese C, Knsdler A, Zimmermann R: Comparative study on a new one stage clotting assay for heparin and its low molecular weight derivates. Haemostasis 1989; 19: 13–20.PubMedGoogle Scholar
  30. 30.
    Casu B: Structure of heparin and heparin fragments. Heparin and related polysaccharides. Structure and activities. Ann New York Acad Sci 1989; 556: 1–18.CrossRefGoogle Scholar
  31. 31.
    Huhle G, Harenberg J, Maisch R, Heene DL: Comparison of three heparin bovine serum-ablumin binding methods for production of antiheparin antibodies. Semin Thromb Hemost 1994; 20: 193–204.PubMedCrossRefGoogle Scholar
  32. 32.
    Yin ET, Wessler S, Butler J: Plasma heparin: A unique, practical submicrogramm sensitive assay. J Lab Clin Med 1973; 81:298.PubMedGoogle Scholar
  33. 33.
    Bessler WG, Hauschild S: Bakterielle Lipopeptide als Immunadjuvantien. Forum Mikrobiol 1987; 4: 106–111.Google Scholar
  34. 34.
    Kshler G, Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495–497.CrossRefGoogle Scholar
  35. 35.
    Kearany JF, Radbruck A, Liesgang B, Rajewsky K: A new mouse myeloma cell line that has lost immunglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J Immunol 1979; 123: 1548–1550.Google Scholar
  36. 36.
    Goding GW: Monoclonal antibodies. Principles and practice. Academic Press London New York 1983; 67 (Ed).Google Scholar
  37. 37.
    Lietzke R, Unsicker K: A statistical approach to determine monoclonality after limiting cell plating of a hybridoma clone. J Immunol Meth 1985; 76: 223–228.CrossRefGoogle Scholar
  38. 38.
    Eliasson M, Olsson A, Palmcrantzm E, Wiberg K, Inganas M, Guss B, Lindberg M, Uhlen M: Chimeric IgG-binding receptors engineered from staphylococcal protein A and streptococcal protein G. J Biol Chem 1988; 263: 4323–4327.PubMedGoogle Scholar
  39. 39.
    Laemmli UK: Cleavage of structural proteine during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680–685.PubMedCrossRefGoogle Scholar
  40. 40.
    Oakley R: A simplified ultrasensitive silver stain for detecting proteins in Polyacrylamide gels. Analyt Biochem 1980; 105: 361–363.PubMedCrossRefGoogle Scholar
  41. 41.
    Stehle G, Friedrich EA, Sinn HJ, Wunder A, Harenberg J, Dempfle CE, Maier-Borst W, Heene DL: Hepatic uptake of a modified low molecular weight heparin in rats. J Clin Invest 1992; 90: 2110–2115.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1996

Authors and Affiliations

  • Günter Huhle
    • 1
  • Job Harenberg
    • 1
  • Reinhard Malsch
    • 1
  • Dieter L. Heene
    • 1
  1. 1.1st Department of Medicine, Faculty of Clinical Medicine MannheimUniversity of HeidelbergMannheimGermany

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