Skip to main content
SpringerLink
Log in

Construction of Rabbit Immune Antibody Libraries

  • Protocol
  • First Online:
Phage Display

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2702))

Abstract

Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from non-murine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 229.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 299.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495 –497

    Article  CAS  PubMed  Google Scholar 

  2. Little M, Kipriyanov SM, Le Gall F, Moldenhauer G (2000) Of mice and men: hybridoma and recombinant antibodies. Immunol Today 21:364 –370

    Article  CAS  PubMed  Google Scholar 

  3. Shay JW (1985) Human hybridomas and monoclonal antibodies: the biology of cell fusion. In: Engleman EG, Foung SKH, Larrick JR, Raubitschek A (eds) Human hybridomas and monoclonal antibodies. Plenum Press, New York

    Chapter  Google Scholar 

  4. Spieker-Polet H, Sethupathi P, Yam PC, Knight KL (1995) Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit-rabbit hybridomas. Proc Natl Acad Sci U S A 92:9348 –9352

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Liguori MJ, Hoff-Velk JA, Ostrow DH (2001) Recombinant human interleukin-6 enhances the immunoglobulin secretion of a rabbit-rabbit hybridoma. Hybridoma 20:189 –198

    Article  CAS  PubMed  Google Scholar 

  6. Keen MJ (1995) The culture of rat myeloma and rat hybridoma cells in a protein-free medium. Cytotechnology 17:193 –202

    Article  CAS  PubMed  Google Scholar 

  7. Digneffe C, Cormont F, Platteau B, Bazin H (1990) Fusion cell lines. In: Bazin H (ed) Rat hybridomas and rat monoclonal antibodies. CRC Press, New York

    Google Scholar 

  8. Bystryn JC, Jacobsen JS, Liu P, Heaney-Kieras J (1982) Comparison of cell-surface human melanoma-associated antigens identified by rabbit and murine antibodies. Hybridoma 1:465 –472

    Article  CAS  PubMed  Google Scholar 

  9. Shim H (2016) Therapeutic antibodies by phage display. Curr Pharm Des 22:6538

    Article  CAS  PubMed  Google Scholar 

  10. Huse WD, Sastry L, Iverson SA, Kang AS, Alting-Mees M, Burton DR, Benkovic SJ, Lerner RA (1989) Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246:1275 –1281

    Article  CAS  PubMed  Google Scholar 

  11. Barbas CF 3rd, Kang AS, Lerner RA, Benkovic SJ (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A 88:7978 –7982

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hammers CM, Stanley JR (2014) Antibody phage display: technique and applications. J Invest Dermatol 134:1 –5

    Article  PubMed  Google Scholar 

  13. Andris-Widhopf J, Steinberger P, Fuller R, Rader C, Barbas CF 3rd (2001) Generation of antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences. In: Barbas CF 3rd, Burton DR, Scott JK, Silverman GJ (eds) Phage display: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  14. Steinwand M, Droste P, Frenzel A, Hust M, Dubel S, Schirrmann T (2014) The influence of antibody fragment format on phage display based affinity maturation of IgG. MAbs 6:204 –218

    Article  PubMed  Google Scholar 

  15. Menzel C, Schirrmann T, Konthur Z, Jostock T, Dubel S (2008) Human antibody RNase fusion protein targeting CD30+ lymphomas. Blood 111:3830 –3837

    Article  CAS  PubMed  Google Scholar 

  16. Rader C, Steinberger P, Barbas CF 3rd (2001) Selection from antibody libraries. In: Barbas CF 3rd, Burton DR, Scott JK, Silverman GJ (eds) Phage display: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  17. Andris-Widhopf J, Rader C, Barbas CF 3rd (2001) Generation of antibody libraries: immunization, RNA preparation, and cDNA synthesis. In: Barbas CF 3rd, Burton DR, Scott JK, Silverman GJ (eds) Phage display: a laboratory manual. Cold Spring Harbot Laboratory Press, Cold Spring Harbor

    Google Scholar 

  18. Yang HY, Kang KJ, Chung JE, Shim H (2009) Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity. Mol Cells 27:225 –235

    Article  CAS  PubMed  Google Scholar 

  19. Angeletti RH (1999) Design of useful peptide antigens. J Biomol Techniq 10:2 –10

    CAS  Google Scholar 

  20. Popkov M, Mage RG, Alexander CB, Thundivalappil S, Barbas CF 3rd, 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

    Article  CAS  PubMed  Google Scholar 

  21. Tang X, Nakata Y, Li HO, Zhang M, Gao H, Fujita A, Sakatsume O, Ohta T, Yokoyama K (1994) The optimization of preparations of competent cells for transformation of E. coli. Nucleic Acids Res 22:2857 –2858

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported in parts by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1A6A1A10039823) and by Korea Drug Development Fund (KDDF) funded by Ministry of Science and ICT; Ministry of Trade, Industry, and Energy; and Ministry of Health and Welfare (HN21C0317, Republic of Korea) to H.S.

Author information

Authors and Affiliations

  1. Imvastech Inc., Seoul, Korea

    Thi Thu Ha Nguyen

  2. AbClon Inc., Seoul, Korea

    Jong Seo Lee

  3. Department of Life Sciences, Ewha Womans Univesity, Seoul, Korea

    Hyunbo Shim

Authors
  1. Thi Thu Ha Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jong Seo Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hyunbo Shim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hyunbo Shim .

Editor information

Editors and Affiliations

  1. Institut für Biochemie, Biotechnologie und Bioinformatik, Technische Universität Braunschweig, Braunschweig, Germany

    Michael Hust

  2. Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia

    Theam Soon Lim

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Nguyen, T.T.H., Lee, J.S., Shim, H. (2023). Construction of Rabbit Immune Antibody Libraries. In: Hust, M., Lim, T.S. (eds) Phage Display. Methods in Molecular Biology, vol 2702. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3381-6_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3381-6_6

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3380-9

  • Online ISBN: 978-1-0716-3381-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation