Quality Enhancement of the Non-immune Phage scFv Library to Isolate Effective Antibodies

Sunao Imai; Yohei Mukai; Kazuya Nagano; Hiroko Shibata; Toshiki Sugita; Yasuhiro Abe; Tetsuya Nomura; Yasuo Tsutsumi; Haruhiko Kamada; Shinsaku Nakagawa; Shin-ichi Tsunoda

doi:10.1248/bpb.29.1325

Regular Articles

Quality Enhancement of the Non-immune Phage scFv Library to Isolate Effective Antibodies

Sunao Imai, Yohei Mukai, Kazuya Nagano, Hiroko Shibata, Toshiki Sugita, Yasuhiro Abe, Tetsuya Nomura, Yasuo Tsutsumi, Haruhiko Kamada, Shinsaku Nakagawa, Shin-ichi Tsunoda

Author information

Sunao Imai
Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NiBio), Japan
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University
Yohei Mukai
Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NiBio), Japan
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University
Kazuya Nagano
Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NiBio), Japan
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University
Hiroko Shibata
Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NiBio), Japan
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University
Toshiki Sugita
Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NiBio), Japan
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University
Yasuhiro Abe
Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NiBio), Japan
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University
Tetsuya Nomura
Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NiBio), Japan
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University
Yasuo Tsutsumi
Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NiBio), Japan
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University
Haruhiko Kamada
Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NiBio), Japan
Shinsaku Nakagawa
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University
Shin-ichi Tsunoda
Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NiBio), Japan

Keywords: non-immune antibody library, phage display system, single-chain Fv, high-throughput screening, vascular endothelial growth factor receptor 2

JOURNAL FREE ACCESS

2006 Volume 29 Issue 7 Pages 1325-1330

DOI https://doi.org/10.1248/bpb.29.1325

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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.

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