| Chapter title |
Construction of Rabbit Immune Antibody Libraries
|
|---|---|
| Chapter number | 7 |
| Book title |
Phage Display
|
| Published in |
Methods in molecular biology, January 2018
|
| DOI | 10.1007/978-1-4939-7447-4_7 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-7446-7, 978-1-4939-7447-4
|
| Authors |
Thi Thu Ha Nguyen, Jong Seo Lee, Hyunbo Shim, Nguyen, Thi Thu Ha, Lee, Jong Seo, Shim, Hyunbo |
| 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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