Quality enhancement of the non-immune phage scFv library to isolate effective antibodies.
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.
Imai
Sunao
S
Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation, Osaka, Japan.
Mukai
Yohei
Y
Nagano
Kazuya
K
Shibata
Hiroko
H
Sugita
Toshiki
T
Abe
Yasuhiro
Y
Nomura
Tetsuya
T
Tsutsumi
Yasuo
Y
Kamada
Haruhiko
H
Nakagawa
Shinsaku
S
Tsunoda
Shin-ichi
S
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
D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D019151:Peptide Library