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Construction of Human Fab ( Y 1/ K ) Library and Identification of Human Monoclonal Fab Possessing Neutralizing Potency against Japanese Encephalitis Virus
Author(s) -
Arakawa Mitsue,
Yamashiro Tetsu,
Uechi Genichiro,
Tadano Masayuki,
Nishizono Akira
Publication year - 2007
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.2007.tb03948.x
Subject(s) - japanese encephalitis , virology , phage display , microbiology and biotechnology , monoclonal antibody , biology , virus , antibody , neutralizing antibody , neutralization , phagemid , encephalitis , escherichia coli , biochemistry , bacteriophage , gene , genetics
A combinatorial human Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from Japanese encephalitis virus hyper‐immune volunteers on pComb3H phagemid vector. The size of the constructed Fab library was 3.3 × 10 8 Escherichia coli transformants. The library was panned 3 times on the purified Japanese encephalitis virus (JEV) virion, and phage clones displaying JEV antigen‐specific Fab were enriched. The enriched phage pool was then screened for clones producing Fab molecule with JEV neutralizing activity by the focus reduction‐neutralizing test. Among 188 randomly selected clones, 9 Fab preparations revealed neutralizing activities against JEV strain Nakayama. An E. coli transformed with TJE12B02 clone, which produced human monoclonal Fab with the highest neutralizing activity was cultured in a large scale, and the Fab molecule was purified using affinity chromatography. The purified FabTJE12B02 showed the 50% focus reduction endpoint at the concentration of 50.2 μg/ml (ca. 1,000 n M ) when JEV strain Nakayama was used. The FabTJE12B02 recognized E protein of JEV strain Nakayama, and the dissociation equilibrium constant ( Kd ) of the FabTJE12B02 against purified JEV antigen was calculated as 1.21 × 10 –8 M. Sequence analysis demonstrated that TJE12B02 used a V H sequence homologous to the V H 3 family showing 88.8% homology to germline VH3–23, and used a V K sequence homologous to the VκII subgroup showing 92.8% homology to germline A17.