title: Quality enhancement of the non-immune phage scFv library to isolate effective antibodies.
abstract: The non-immune phage antibody library system is one of the most attractive technologies available to current therapeutic, diagnostic and basic scientific research. This system allows the rapid isolation of antibodies of interest that could subsequently be applied directly to drug delivery systems and antibody therapy. Previously, we reported the primer sets to encompass the antibody repertoire and thus improve library quality. However, a wide number of varying primer sets cause to decrease the amplification efficiency of antibody genes. In the present study, we re-generated the library primer sets newly and constructed an improved library from non-immune mice that was far superior in terms of variety and quality. This new library contained 2.4 billion independent clones. In addition, we optimized the selection step from this library to isolate high-affinity antibodies. The optimization of an affinity panning protocol by the incorporation of an automated Microfluidics instrument led to the successful isolation of three different monoclonal antibodies for human vascular endothelial growth factor receptor 2 (KDR). These antibodies were demonstrated to exhibit high specificity and were able to detect a mere 0.6 fmol of KDR by dot blot analysis. Previously reported antibodies for luciferase were also isolated successfully from this library. Our results clearly demonstrate the importance of the improved protocol for the library preparation of antibodies and the resulting isolation of antibodies for clinical and research applications.
authors.affiliation: Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation, Osaka, Japan.
authors.forename: Sunao
authors.initials: S
authors.lastname: Imai
authors.forename: Yohei
authors.initials: Y
authors.lastname: Mukai
authors.forename: Kazuya
authors.initials: K
authors.lastname: Nagano
authors.forename: Hiroko
authors.initials: H
authors.lastname: Shibata
authors.forename: Toshiki
authors.initials: T
authors.lastname: Sugita
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Abe
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Nomura
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Tsutsumi
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Kamada
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Nakagawa
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Tsunoda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D006801:Humans; D007135:Immunoglobulin Variable Region; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D019151:Peptide Library