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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495 –497
Little M, Kipriyanov SM, Le Gall F, Moldenhauer G (2000) Of mice and men: hybridoma and recombinant antibodies. Immunol Today 21:364 –370
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
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
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
Keen MJ (1995) The culture of rat myeloma and rat hybridoma cells in a protein-free medium. Cytotechnology 17:193 –202
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
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
Shim H (2016) Therapeutic antibodies by phage display. Curr Pharm Des 22:6538
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
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
Hammers CM, Stanley JR (2014) Antibody phage display: technique and applications. J Invest Dermatol 134:1 –5
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
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
Menzel C, Schirrmann T, Konthur Z, Jostock T, Dubel S (2008) Human antibody RNase fusion protein targeting CD30+ lymphomas. Blood 111:3830 –3837
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
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
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
Angeletti RH (1999) Design of useful peptide antigens. J Biomol Techniq 10:2 –10
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
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
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
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
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