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 nonmurine 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.
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Acknowledgment
This work was supported in parts by the National Research Foundation of Korea (NRF) grant for Medical Bioconvergence Research Center (NRF-2013M3A6A4044991), the Bio & Medical Technology Development Program of the NRF funded by the Korean government, MSIP (NRF-2015M3A9B6029138), and the small and medium business convergence R&D program funded by the Small and Medium Business Administration (S2373145) to H.S.
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Nguyen, T.T., Lee, J.S., Shim, H. (2018). Construction of Rabbit Immune Antibody Libraries. In: Hust, M., Lim, T. (eds) Phage Display. Methods in Molecular Biology, vol 1701. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7447-4_7
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DOI: https://doi.org/10.1007/978-1-4939-7447-4_7
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