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Competitive Antigen Panning for Selection of HIV-1 Neutralizing Human Monoclonal Antibodies Specific for gp41

  • Mei-Yun Zhang
  • Dimiter S. Dimitrov
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 525)

Abstract

HIV envelope glycoprotein (Env) is composed of two non-covalently associated subunits: gp120 and gp41. Panning of phage-displayed antibody libraries against gp140 (covalently linked gp120 and the extracellular portion of gp41) has resulted mostly in selection of anti-gp120 antibodies. Native gp41 in the absence of gp120 is unstable. The use of gp41 fragments as antigens has resulted in selection of antibodies with only relatively modest neutralizing activity. To enhance selection of antibodies specific for gp41 in the context of the whole Env we developed a methodology termed competitive antigen panning (CAP). Using CAP, we identified a panel of gp41-specific human monoclonal antibodies from an HIV-1 immune library derived from long-term nonprogressors. These antibodies recognize conformational epitopes in gp41 and exhibited, to various extents, neutralization activity in assays based on spreading infection in peripheral blood mononuclear cells. The CAP methodology is generally applicable for selection of antibodies specific for any epitope that is not a dominant epitope in the antigen. It is superior to a traditional pre-depletion method in avoiding potential loss of target-specific antibodies.

Key words

Phage display antibody HIV envelop gp41 methodology 

References

  1. 1.
    Marks, J. D., Hoogenboom, H. R., Bonnert, T. P., McCafferty, J., Griffiths, A. D., and Winter, G. (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222, 581–97.PubMedCrossRefGoogle Scholar
  2. 2.
    Hoogenboom, H. R., de Bruine, A. P., Hufton, S. E., Hoet, R. M., Arends, J. W., and Roovers, R. C. (1998) Antibody phage display technology and its applications. Immunotechnology 4, 1–20.PubMedCrossRefGoogle Scholar
  3. 3.
    Hoogenboom, H. R. (2002) Overview of antibody phage-display technology and its applications. Methods Mol. Biol. 178, 1–37.PubMedGoogle Scholar
  4. 4.
    Hoogenboom, H. R. (1997) Designing and optimizing library selection strategies for generating high-affinity antibodies. Trends Biotechnol. 15, 62–70.PubMedCrossRefGoogle Scholar
  5. 5.
    Zhang, M. Y., Choudhry, V., Sidorov, I. A., Tenev, V., Vu, B. K., Choudhary, A., Lu, H., Stiegler, G. M., Katinger, H. W., Jiang, S., Broder, C. C., and Dimitrov, D. S. (2006) Selection of a novel gp41-specific HIV-1 neutralizing human antibody by competitive antigen panning. J. Immunol. Methods 317, 21–30.PubMedCrossRefGoogle Scholar
  6. 6.
    Zhang, M. Y., and Dimitrov, D. S. (2007) Novel approaches for identification of broadly cross-reactive HIV-1 neutralizing human monoclonal antibodies and improvement of their potency. Curr. Pharm. Des. 13, 203–12.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Mei-Yun Zhang
    • 1
  • Dimiter S. Dimitrov
    • 1
  1. 1.NCI-Frederick, National Institutes of HealthFrederickUSA

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