Advertisement

Generation and Selection of Rabbit Antibody Libraries by Phage Display

  • Christoph Rader
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 525)

Abstract

The rabbit antibody repertoire is an exceptional source for both polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs) that combine high specificity with high avidity and affinity, respectively. In contrast to rabbit pAbs, which have been utilized for decades, rabbit mAbs are defined reagents with infinite supply, prompting considerable interest in recent years. Phage-display technology based on chimeric rabbit/human Fab libraries allows the selection of rabbit mAbs that cross-react with mouse and human antigens while maintaining high affinity and specificity.

Key words

Rabbit monoclonal antibodies phage display phagemid antibody library Fab panning 

Notes

Acknowledgments

I would like to thank Dr. Carlos F. Barbas III (The Scripps Research Institute, La Jolla, CA) for training me in antibody technologies including phage display, and members of my laboratory at the National Cancer Institute, in particular Drs. Thomas Hofer and Ka Yin Kwong, for their outstanding contributions to further evolving the methodology.

References

  1. 1.
    Ridder, R., Schmitz, R., Legay, F., and Gram, H. (1995) Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and their production in the yeast Pichia pastoris. Biotechnology 13, 255–260.PubMedCrossRefGoogle Scholar
  2. 2.
    Rader, C. (2001) Antibody libraries in drug and target discovery. Drug Discov. Today 6, 36–43.PubMedCrossRefGoogle Scholar
  3. 3.
    Hoogenboom, H. R. (2005) Selecting and screening recombinant antibody libraries. Nat. Biotechnol. 23, 1105–1116.PubMedCrossRefGoogle Scholar
  4. 4.
    Mage, R. G., Lanning, D., and Knight, K. L. (2006) B cell and antibody repertoire development in rabbits: the requirement of gut-associated lymphoid tissues. Dev. Comp. Immunol. 30, 137–153.PubMedCrossRefGoogle Scholar
  5. 5.
    Rader, C., Steinberger, P., and Barbas, C. F., III (2001) Selection from antibody libraries, in Phage Display: A Laboratory Manual (Barbas, C. F., III, Burton, D.R., Scott, J. K., and Silverman, G. J., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  6. 6.
    Rader, C., Ritter, G., Nathan, S., Elia, M., Gout, I., Jungbluth, A. A., Cohen, L. S., Welt, S., Old, L. J., and Barbas, C. F., III (2000) The rabbit antibody repertoire as a novel source for the generation of therapeutic human antibodies. J. Biol. Chem. 275, 13668–13676.PubMedCrossRefGoogle Scholar
  7. 7.
    Popkov, M., Mage, R. G., Alexander, C. B., Thundivalappil, S., Barbas, C. F., III, and Rader, C. (2003) Rabbit immune repertoires as sources for therapeutic monoclonal antibodies: the impact of kappa allotype-correlated variation in cysteine content on antibody libraries selected by phage display. J. Mol. Biol. 325, 325–335.PubMedCrossRefGoogle Scholar
  8. 8.
    Popkov, M., Jendreyko, N., Gonzalez-Sapienza, G., Mage, R. G., Rader, C., and Barbas, C. F., III (2004) Human/mouse cross-reactive anti-VEGF receptor 2 recombinant antibodies selected from an immune b9 allotype rabbit antibody library. J. Immunol. Methods 288, 149–164.PubMedCrossRefGoogle Scholar
  9. 9.
    Hofer, T., Tangkeangsirisin, W., Kennedy, M. G., Mage, R. G., Raiker, S. J., Venkatesh, K., Lee, H., Giger, R. J., and Rader, C. (2007) Chimeric rabbit/human Fab and IgG specific for members of the Nogo-66 receptor family selected for species cross-reactivity with an improved phage display vector. J. Immunol. Methods 318, 75–87.PubMedCrossRefGoogle Scholar
  10. 10.
    Steinberger, P., Sutton, J. K., Rader, C., Elia, M., and Barbas, C. F., III (2000) Generation and characterization of a recombinant human CCR5-specific antibody: A phage display approach for rabbit antibody humanization. J. Biol. Chem. 275, 36073–36078.PubMedCrossRefGoogle Scholar
  11. 11.
    Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C., and Barbas, C. F., III (2001) Generation of antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences, in Phage Display: A Laboratory Manual (Barbas, C. F., III, Burton, D.R., Scott, J. K., and Silverman, G. J., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  12. 12.
    Popkov, M., Rader, C., and Barbas, C. F., III (2004) Isolation of human prostate cancer cell reactive antibodies using phage display technology. J. Immunol. Methods 291, 137–151.PubMedCrossRefGoogle Scholar
  13. 13.
    Scott, J. K. and Barbas, C. F., III (2001) Phage-display vectors, in Phage Display: A Laboratory Manual (Barbas, C. F., III, Burton, D.R., Scott, J. K., and Silverman, G. J., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  14. 14.
    Barbas, C. F., III, Kang, A. S., Lerner, R. A., and Benkovic, S. J. (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc. Natl. Acad. Sci. USA 88, 7978–7982.Google Scholar

Copyright information

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

Authors and Affiliations

  • Christoph Rader
    • 1
  1. 1.Center for Cancer ResearchNational Cancer Institute, National Institutes of HealthBethesdaUSA

Personalised recommendations