Advertisement

Applied Biochemistry and Biotechnology

, Volume 75, Issue 1, pp 129–137 | Cite as

Production and characterization of monoclonal antibodies against urea derivatives

  • Manuela Rohde
  • Jörg A. Schenk
  • Stephan Heymann
  • Olaf Behrsing
  • Gudrun Scharte
  • Gerhard Kempter
  • Jochen Woller
  • Wolfgang E. Höhne
  • Axel Warsinke
  • Burkhard Micheel
Original Articles

Abstract

A panel of monoclonal antibodies was generated against the ureabased haptenN-(2-N-chloroacetylaminobenzyl)-N′-4-chlorophenylurea as a tool for building up sensitive immune assays to detect urea derivatives and to screen them for catalytic antibodies (Abs). Eleven hybridomas were obtained that produced Abs reactive to the hapten. All Abs were of IgG class. Crossreactivities of the Abs to different haptens were examined, especially to a possible transition-state analog. Only four of the hybridomas (R2-DA10/F7, R2-GE7/H2, R2-HC2/A5, R2-HD6/F7) produced Abs crossreactive with the transition-state analog. From the 11 hybridomas, hybridoma B76-BF5 was chosen for further characterization. Compared to the other Abs, B76-BF5 showed the strongest binding and had a rather restricted specificity. These Abs could be used to build up a sensitive enzyme immunoassay for the detection of the hapten. All Abs were screened for crossreactivity with the pesticides monuron and diuron. No reactivity could be detected. In addition, the nucleotide sequences of the variable light and heavy chain genes of the similarly reactive Abs B76-BF5, B76-BB3, R2-DA10/F7, and R2-GA6/G3 were determined to clarify whether structure and binding specificity of these Abs showed any correlation.

Index entries

N-(2-N-chloroacetylaminobenzyl)-N′-4-chlorophenylurea pesticides catalytic antibodies hybridomas KABAT database 

Nomenclature

BSA

bovine serum albumin

PBS

phosphate buffered saline

NCS

neonatal calf serum

M-MLV-RT

Moloney murine leukemia virus-reverse transcriptase

Ig

immunoglobulin

EDCI

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Schlaeppi, J. M., Fory, W., and Ramsteiner, K. (1989),J. Agric. Food Chem. 37, 1532–1538.CrossRefGoogle Scholar
  2. 2.
    Dunbar, R., Riggle, B., and Niswender, G. (1990),J. Agric. Food Chem. 38, 433–477.CrossRefGoogle Scholar
  3. 3.
    Thurman, E. M., Meyer, M., Pomes, M., Perry, C. A., and Schwab P. (1990),Anal. Chem. 62, 2043–2048.CrossRefGoogle Scholar
  4. 4.
    Graham, B. M., Porter, A. J. and Harris, W. J. (1995),J. Chem. Tech. Biotech. 63, 279–289.CrossRefGoogle Scholar
  5. 5.
    Byrne, F. R., Grant, S. D., Porter, A. J., and Harris, W. J. (1996),Food Agricultural Immunol. 8, 19–29.CrossRefGoogle Scholar
  6. 6.
    Jencks, W. P., ed. (1969), inCatalysis in Chemistry and Enzymology, McGraw-Hill, New York, 288.Google Scholar
  7. 7.
    Lerner, R. A., Kang, A. S., Bain, J. D., Burton, D. R., and Barbas, C. R., III (1992),Science 258, 1313–1314.CrossRefGoogle Scholar
  8. 8.
    Lindner, W. and Robey, F. A. (1987),Int. J. Pept. Protein Res. 30, 794–800.CrossRefGoogle Scholar
  9. 9.
    Maloy, W. L. and Coligan, J. E. (1991), inCurrent Protocols in Immunology, vol. 2, (Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M., and Strober, W., eds.) Unit Greene and Wiley Interscience, New York, pp. 9.4.3.-9.4.4.Google Scholar
  10. 10.
    Kearney, J. F., Radbruch, A., Liesgang, B., and Rajewski, K. (1979),J. Immunol. 123, 1548–1550.Google Scholar
  11. 11.
    Köhler, G. and Milstein, C. (1975),Nature 256, 495–497.CrossRefGoogle Scholar
  12. 12.
    Behrsing, O., Kaiser, G., Karawajew, L., and Micheel, B. (1992),J. Immunol. Methods 156, 69–77.CrossRefGoogle Scholar
  13. 13.
    Micheel, B. and Scharte, G. (1993),Hybridoma 12, 227–229.Google Scholar
  14. 14.
    Chomczynski, P. and Sacchi, N. (1987),Anal. Biochem. 162, 156–159.CrossRefGoogle Scholar
  15. 15.
    Schenk, J. A., Hillebrand, T., Heymann, S., Peters, L. E., Mazaheri, R., Micheel, B., and Bendzko, P. (1995),Biotech. Prod. Int. 7, 30.Google Scholar
  16. 16.
    Schenk, J. A., Hillebrand, T., Lübbe, L., Heymann, S., Böttger, M., Micheel, B., and Bendzko P. (1997),J. Clin. Lab. Anal. 11, 340–342.CrossRefGoogle Scholar
  17. 17.
    Dübel, S., Breitling, F., Fuchs, P., Zewe, M., Gotter, S., Welschof, M., Moldenhauer, G., and Little, M. (1994),J. Immunol. Methods 175, 89–95.CrossRefGoogle Scholar
  18. 18.
    Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C. (1987), inSequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, U.S. Government Printing Office, Washington, D.C.Google Scholar
  19. 19.
    Pearson, W. R. and Lipman, D. J. (1988),Proc. Natl. Acad. Sci. USA 85, 2444–2448.CrossRefGoogle Scholar
  20. 20.
    Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990),J. Mol. Biol. 215, 403–410.Google Scholar
  21. 21.
    Jarvis, C. D., Cannon, L. E., and Stavnezer, J. (1989),J. Immunol. 143, 4213–4220.Google Scholar
  22. 22.
    McKean, D. J., Bell, M., and Potter, M. (1978),Proc. Natl. Acad. Sci. USA 75, 3913–3917.CrossRefGoogle Scholar
  23. 23.
    Berek, C., Griffiths, G. M., and Milstein C. (1985),Nature 316, 412–418.CrossRefGoogle Scholar
  24. 24.
    Pennell, C. A., Arnold, L. W., Haughton, G., and Clarke, S. H. (1988),J. Immunol. 141, 2788–2796.Google Scholar
  25. 25.
    Levy, S., Campbell, M. J., and Levy, R. J. (1989),J. Exp. Med. 170, 1–13.CrossRefGoogle Scholar
  26. 26.
    Bell, C. W., Scholthof, K.-B., Zhang, G., and Karu, A. E. (1995),Gene 165, 323–324.CrossRefGoogle Scholar
  27. 27.
    Stöcklein, W. F. M., Warsinke, A., Micheel, B., Kempfer, G., Höhne, W., and Scheller, E. W. (1978),Anal. Chim. Acta 362, 101–111.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc 1998

Authors and Affiliations

  • Manuela Rohde
    • 1
  • Jörg A. Schenk
    • 2
  • Stephan Heymann
    • 2
  • Olaf Behrsing
    • 1
  • Gudrun Scharte
    • 2
  • Gerhard Kempter
    • 3
  • Jochen Woller
    • 3
  • Wolfgang E. Höhne
    • 4
  • Axel Warsinke
    • 1
  • Burkhard Micheel
    • 1
    • 2
  1. 1.Institute of Biochemistry and Molecular PhysiologyPotsdam UniversityGermany
  2. 2.Max Delbrück Center for Molecular Medicine (MDC) Berlin-BuchGermany
  3. 3.Institute of Organic ChemistryPotsdam UniversityGermany
  4. 4.Institute of BiochemistryHumboldt UniversityBerlin

Personalised recommendations