Construction of Human Fab Library and Isolation of Monoclonal Fabs with Rabies Virus-Neutralizing Ability

• Published in: Microbiology and immunology 2005-01, Vol.49 (4), p.311-322
• Main Authors: Ando, Tadasuke, Yamashiro, Tetsu, Takita-Sonoda, Yoshiko, Mannen, Kazuaki, Nishizono, Akira
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antibodies, Viral - blood; Antibodies, Viral - chemistry; Antibodies, Viral - genetics; Antibodies, Viral - immunology; antibody; Cloning, Molecular; combinatorial library; Escherichia coli; Humans; Immunoglobulin Fab Fragments - chemistry; Immunoglobulin Fab Fragments - genetics; Immunoglobulin Fab Fragments - immunology; Lymphocytes - immunology; Molecular Sequence Data; Neutralization Tests; Peptide Library; phage display; Rabies virus; Rabies virus - immunology; RNA, Messenger - genetics; RNA, Messenger - isolation & purification; Sequence Analysis, DNA; Viral Plaque Assay
• ISSN: 0385-5600; 1348-0421
• DOI: 10.1111/j.1348-0421.2005.tb03735.x

A combinatorial human Fab library was constructed using RNAs from peripheral blood lymphocytes of 6 rabies vaccine‐boosted volunteers using pComb3X phagemid vector. The size of the constructed library was approximately 7.0×107 Escherichia coli transformants. The library was selected against purified rabies virus (RV) virion or purified RV glycoprotein for isolation of phages displaying RV‐neutralizing human Fab antibody. Among 132 selected clones, two Fab preparations revealed neutralizing activities against RV strain CVS when assayed in the rapid fluorescent focus inhibition test (RFFIT). The Fab preparation EP5G3 exhibited neutralizing activity with an infected cell count reduction of 76% at a dilution of 1:2, and of 20% at a dilution of 1:4. The Fab preparation GD2D12 also exhibited neutralizing activity with a 57% reduction at 1:2 and 41% reduction at 1:4. In the co‐immunoprecipitation using strain CVS, the RV glycoprotein was precipitated in reactions with both Fab preparations. The RV neutralizing ability of the Fab preparations described in the study were not directly correlated with their binding specificity for RV antigens detected by ELISA.