Applied Biochemistry and Biotechnology

, Volume 83, Issue 1–3, pp 287–295 | Cite as

A preliminary study for isolation of catalytic antibodies by histidine ligand affinity chromatography as an alternative to conventional protein A/G methods

  • Elsa Nedonchelle
  • Olivier Pitiot
  • Mookambeswaran A. Vijayalakshmi


Catalytic autoimmune antibodies from the sera of lupus patients were purified using histidyl-aminohexyl-Sepharose gel and compared with the antibodies purified with protein A and protein G affinity chromatography. The IgG preparations from the histidine affinity column had a much higher catalytic activity in hydrolyzing the peptide substrate Pro-Phe-Arg-methyl-coumarinamide compared to the antibodies obtained by the conventional protein A/G method. This preservation of catalytic activity is attributed to the gentle buffer conditions used in the histidine ligand method that allowed the integrity of three-dimensional structure of purified catalytic antibodies. Thus, histidine affinity offer a superior method for isolating autoimmune catalytic antibodies.

Index Entries

Catalytic autoimmune antibodies activity preservation immobilized histidine affinity chromatography 


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  1. 1.
    Paul, S. (1996), Mol. Biotechnol. 5, 197–207.PubMedGoogle Scholar
  2. 2.
    Sarath, G., De La Motte, R. S., and Wagner, F. W. (1989), Protease assy methods. In Proteolytic Enzymes: Apractical Approach. Beyron, R. J. and Bond, J. S., eds. IRL Press, Oxford, UK, pp. 25–55.Google Scholar
  3. 3.
    Shuster, A. M., Gololobov, G. V., Kvashuk, O. A., Bogomolova, A. E., Smirnov, I. V., and Gabibov, A. G. (1992), Science 256, 665–667.PubMedCrossRefADSGoogle Scholar
  4. 4.
    Li, L., Paul, S., Tyutyulkova, S., Kazatchkine, M., and Kaveri, S. (1995), J. Immunol. 154, 3328–3332.PubMedGoogle Scholar
  5. 5.
    Tyutyulkova, S. and Paul, S. (1994), Appl. Biochem. Biotechnol. 47, 191–198.PubMedGoogle Scholar
  6. 6.
    Tyutyulkova, S., Gao, Q. S., and Paul, S. (1995), in Methods in Molecular Biology Vol. 51: Antibody Engineering Protocols (Paul S., ed.) Humana, Totowa, NJ pp. 377–394.CrossRefGoogle Scholar
  7. 7.
    Buchner, J., Renner, M., Lilie, H., Hinz, H. J., and Jaenicke, R. (1991), Biochemistry 30, 6922–6929.PubMedCrossRefGoogle Scholar
  8. 8.
    Vijayalakshmi, M. A. (1999), Proceeding of the Catalytic Antibodies and Antibodies Engineering of Chantilly, Oct. 1996, in press.Google Scholar
  9. 9.
    Pitiot, O., Nedonchelle, E., Legallais, C., Coffinier, Y., and Vijayalakshmi, M. A. (1999), in preparation.Google Scholar
  10. 10.
    Bueno, S. M. A., Haupt, K., and Vijayalakshmi, M. A. (1995), J. Chromatogr. Biomed. Applications 667, 57–67.CrossRefGoogle Scholar
  11. 11.
    Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randell, R. J. (1951), J. Biol. Chem. 193, 265.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • Elsa Nedonchelle
    • 1
  • Olivier Pitiot
    • 1
  • Mookambeswaran A. Vijayalakshmi
    • 1
  1. 1.Laboratoire d'Interactions Moleculaires et de Technologie des Séparations (LIMTechS)Unité CNRS UPRES A 6022, Centre de Recherche de RoyallieuCompiègne cedexFrance

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