Quantitation of Recombinant Hirudin by Enzyme-Linked Immunosorbent Assay

효소면역측정법 (ELISA)을 이용한 유전자 재조합 히루딘의 정량

  • Choi, Yun-Joo (Korea Research Institute Bioscience and Biotechnology) ;
  • Hahn, Bum-Soo (Natural Products Research Institute Seoul National University) ;
  • Ahn, Mi-Young (Natural Products Research Institute Seoul National University) ;
  • Park, Pyung-Keun (Natural Products Research Institute Seoul National University) ;
  • Sohn, Jung-Hoon (Korea Research Institute Bioscience and Biotechnology) ;
  • Choi, Eui-Sung (Korea Research Institute Bioscience and Biotechnology) ;
  • Lee, Sang-Ki (Korea Research Institute Bioscience and Biotechnology) ;
  • Kim, Yeong-Shik (Natural Products Research Institute Seoul National University)
  • Published : 1997.02.01

Abstract

A polygonal antibody against recombinant hirudin was raised for the development of a ELISA in biological fluids. Recombinant hirudin was conjugated to maleimide activated carrie r protein, KLH and injected to a rabbit. The third booster collection of antiserum was used as primary antibody for the ELISA. The titer for the detection antibody was determined. The direct ELISA could determine the concentration of hirudin in the range of ~10ng/ml. Affinity pulified IgG was obtained and conjugated to horseradish peroxidase. Purified IgG and IgG-HRP could be used as capture and detection antibody, respectively. Although sandwich ELISA would not give the satisfactory results. it could apply for the detection of hirudin level in the range of ~20 ${\mu}$g/ml.

Keywords

References

  1. Hoppe-Seyler's Z. Physiol. Chem. v.308 Die isolierung and chemische charakterisierung des hirudins Markwardt, F.
  2. Sem. Thromb. Hemostas. v.15 Thrombin interactions with hirudin Fenton, J. W.
  3. Thromb. Haemost. v.66 Hirudin and derivatives as anticoagualnt agents Markwardt, F.
  4. DNA v.5 Cloning and expression in Escherichia coli of a synthetic DNAs for hirudin. the blood coagulation inhibitor in the leech Fortkamp, E.;Rieger, M.;Heisterberg-Moustes, G.;Schweitzer, S.;Sommer, R.
  5. Hirudo medicinalis. Proc. Natl. Acad. Sci. USA. v.83 Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech Harvey, R. P.;Degryse, E.;Stefani, L.;Schamber, F.;Cazena, J. P.;Courtney, M.;tolstochev, P.;Lecocq, J. -P.
  6. Sem. Thromb. Hemost. v.17 Recombinant hirudin: Genetic engineering and structure analysis Marki, W. E.;Grossenbacher, H.;Gr tter, M. G.;Liersch, M. H.;Meyhack, B.;Heim, J.
  7. Haemostasis v.21 Comparative study of three recombinant hirudins with heparin in an experimental venous thrombosis model Doutremepuich, C.;Lalanne, M. C.;Doutremepuich, F.;Walenga, F.;Farred, J;Breddin, H. K.
  8. Haemostasis v.19 Antithrombotic effects of three thrombin inhibitors in a rat model of laser-induced thrombosis Krupinski, K.;Breddin, H. K.;Markwardt, F.;Haarmann, W.
  9. Haemostasis v.21 no.SUP.1 Hirudin as an anticoagulant in experimental hemodialysis Markwardt, F.;Nowak, G.;Bucha, E.
  10. Thromb. Haemost. v.63 Antithrombotic effects of recombinat hirudin in exprimental angioplasty and intraveneous thrombolysis Kaiser, B.;Simon, A.;Markwardt, F.
  11. Haemostasis v.21 Hirudin in diseminated intravasular coagulation Nowak, G.;Markwardt, F.
  12. J. Appl. Pharmacol. v.2 Evaluation in anticoagulant activity of recombinant hirudin Kim, Y. S.;eom, E. M.;Jung, C. S.;Jung, K. H.;Sohn, J. H.;Choi, E. S.;Rhee, S. K.
  13. Haemostasis v.21 no.SUP.1 A fast photometric assay for the determination of hirudin Spannagal, M.;Bichler, H.;Lill, H.;Schramm, W.
  14. Haemostasis v.21 no.SUP.1 Laboratory assays for the evaluation of recombinant hirudin Walenga, J. M.;Hoppensteadt, D.;Koza, M.;Wallock, M.;Pifarre, R.;Fareed, J.
  15. Thromb. Res. no.SUP.VII Epitope mapping and quantitation of hirudin with polyclonal and monoclonal antibodies Stoeffler, G.;Spinner, S.;Scheffauer, F.;Hampl. H.;Maschler, R.;Fink, E.
  16. Thromb. Res. v.36 Monoclonal antibodies directed against human alpha-throumbin and thrombin-antithrombin Ⅲ Complex Dawes, J.;James, K.;Micklem, L. R.;Pepper, D. S.;Prowse, C. V.
  17. J. Immunol. Methods v.87 Quantitative enzyme-linked immunosorbent assay (ELISA) for hirudin Spinner, S.;Stoffler, G.;Fink, E.
  18. Thromb. Res. v.51 A hirudin catching ELISA for quantitating the anticoagulant in biological fluids Spinner, S.;Scheffauer, F.;Maschler, R.;Stoffler, G.
  19. Eur. J. Biochem. v.188 Preparation of monoclonal antibodies to hirudin and hirudin peptides Schlaeppi, J.M.;Vekemans, S;Chang, J. Y.
  20. Thromb. Res. v.62 Preparation of monoclonal antibodies to the thrombin/hirudin complex Schlaeppi, J. M.
  21. Current Protocols in Molecular Biology v.11 Winston, S. E.;Fuller, S. A.;Hurrell, G. R.
  22. J. Immunol. Methods v.120 Preparation of antibodies to a synthetic C terminus of hirudin and identification of an antigenic site Mao, S. J. T.;Yates, M. T.;Owen, T. J.;Krstenansky, J. L.
  23. Science v.249 The structure of a complex of recombinant hirudin and human α-thrombin Rydel, T. J.;Ravichandran, K. G.;Tulinski, A.;Bode, W.;Huber, R.;Roitsch, C.;Fenton II J. W.