[Preparation of human Fab antibodies against HAb18G by guided selection and chain shuffling technique].
OBJECTIVE
To prepare humanized Fab antibody by guided selection and chain shuffling technique.


METHODS
Human Fd and C(L) repertoire genes were amplified by RT-PCR from PBMCs of patients with hepatocellular carcinoma. Human-murine chimeric C(L) genes were paired with the human Fd repertoire genes to construct phage antibody library. After four rounds of panning against HAb18GE, chimeric antibodies containing humanized Fd gene and binding to HAb18GE were obtained. Then the selected Fd genes were paired with the human C(L) repertoire genes to construct a humanized Fab antibody gene library. After four rounds of panning, completely humanized Fab antibodies were obtained.


RESULTS
After six rounds of panning, 7 chimeric Fab genes were obtained from the Fab gene library with 2 x 10 (7) pfu. Using the selected seven Fd genes, the humanized Fab gene library of 0.8 x 10 (7) pfu was constructed. After four rounds of panning, 2 humanized Fab genes with the strongest reactivity to HAb18G were obtained. DNA sequencing showed that Fds of the two humanized Fabs had the same DNA sequence, which belonged to IgG2, just the same as the parent antibody. And the C(L) belonged to kappa type, and V kappa 3 family.


CONCLUSIONS
By guided-selection and chain shuffling technique, the humanized Fab antibody against HAb18G is obtained, which lays the foundation for further research.
Bao
Guo-qiang
GQ
Department of General Surgery, Tangdu Hospital,Fourth Military Medical University Xi'an 710038, China. guoqiang_bao@hotmail.com
Ma
Qing-jiu
QJ
Xing
Jin-liang
JL
Yang
Xiang-min
XM
Chen
Zhi-nan
ZN
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000276:immunology* / Q000473:pathology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics*; D007128:Immunoglobulin Fragments / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D008113:Liver Neoplasms / Q000276:immunology* / Q000473:pathology; D008969:Molecular Sequence Data; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007147:Immunoglobulin Light Chains; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C100277:immunoglobulins, Fd