G. Esposito et al., A HUMAN-ANTIBODY SPECIFIC FOR HEPATITIS-C VIRUS CORE PROTEIN - SYNTHESIS IN A BACTERIAL SYSTEM AND CHARACTERIZATION, Gene, 164(2), 1995, pp. 203-209
The cDNA coding for the Fab fragment of the human B12.F8 antibody (Ab)
, directed against the putative nucleocapsid component (core protein)
of hepatitis C virus (HCV), was cloned in the prokaryotic phagemid vec
tor, pHEN-1, to obtain its expression in Escherichia coli. The functio
nality and specificity of the recombinant Ab, called B12Fab, were exam
ined by Western blot and ELISA using recombinant HCV core protein as a
ntigen. The specificity of B12Fab was further confirmed by ELISA with
the 33-mer peptide epitope recognized by the original whole B12.F8 Ab.
By immunofluorescence, the recombinant B12Fab was shown to recognize
HCV core protein produced in cells transfected with HCV cDNA, indicati
ng that the recombinant B12Fab is suitable as a diagnostic tool for ti
ssue localization of the virus. The B12Fab also functioned when displa
yed on phage particles, providing the basis for future experiments of
in vitro affinity maturation and selection of mutants. The variable ch
ain coding regions of the recombinant B12Fab clone were sequenced and
the V-gene usage was determined by comparison with the V(k)appa and V-
H germline sequences. The B12Fab V-kappa chain belongs to the subgroup
II and shows the highest degree of homology with the A(3) germline ge
ne, whereas the sequence of the V-H chain is strictly related to that
of the Humhv3019b18 gene of the V(H)3 family. These results are, to ou
r knowledge, the first report of molecular cloning and characterizatio
n of a functional human Ab specific for an HCV antigen.