Construction of human Fab (gamma1/kappa) library and identification of human monoclonal Fab possessing neutralizing potency against Japanese encephalitis virus.
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 3.3x10(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 microg/ml (ca. 1,000 nM) 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 1.21x10(-8) M. Sequence analysis demonstrated that TJE12B02 used a VH sequence homologous to the VH3 family showing 88.8% homology to germline VH3-23, and used a Vkappa sequence homologous to the VkappaII subgroup showing 92.8% homology to germline A17.
Arakawa
Mitsue
M
Department of Infectious Diseases, Faculty of Medicine, Oita University, Japan.
Yamashiro
Tetsu
T
Uechi
Gen-ichiro
G
Tadano
Masayuki
M
Nishizono
Akira
A
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004664:Encephalitis Virus, Japanese / Q000276:immunology*; D004672:Encephalitis, Japanese / Q000097:blood / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D015678:Viral Structural Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D004279:DNA, Viral; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D019151:Peptide Library; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D012313:RNA