Construction and preliminary screening of a human phage single-chain antibody library associated with gastric cancer.
BACKGROUND
The aim of this study was to construct a phage library of human single-chain antibodies associated with gastric cancer and screen such a library for CEA binding scFv.


METHODS
The cDNA library of antibody variable regions was constructed using mRNA from metastatic lymph nodes or spleen of patients with stomach cancer by RT-PCR. These cDNA were assembled into a single-chain format and cloned into phagemid pCANTAB-5 and then transformed into Escherichia coli TG1. The scFv gene library was rescued by M13KO7 helper phage. CEA and the viable CEA-positive gastric cancer cell line MKN-28 were used to screen the phage antibody library. Indirect and tumor cell ELISA was used to determine the specificity of phage antibody. Fixed cell immunofluorescence and live cell FACS analysis were used to further characterize the binding of phage scFv.


RESULTS
After transformation into E. coli TG1, 2.5 x 10(7) cfu/microg ampicillin-resistant clones grew. Sequences of those positive insert clones showed that the V(H) genes were derived from the V(H) III subgroup, while the V(L) genes belonged to the V(kappa) III subgroup. After four rounds of panning, the titer of eluted binding phage increased 135- to 158-fold and ELISA results showed that 20/95 clones can bind CEA and 47/95 clones can bind fixed tumor cells. Immunofluorescence and FACS analysis results showed that these phage scFv fragments could bind CEA-positive cells.


CONCLUSIONS
We successfully constructed a human phage antibody library from lymph nodes of stomach cancer patients. Such kinds of library prove useful for generating tumor-antigen-specific human antibody fragments.
He
Jin
J
Department of Surgery, Fudan University, Shanghai 200032, People's Republic of China.
Zhou
Ge
G
Liu
Kang-Da
KD
Qin
Xin-Yu
XY
D000595:Amino Acid Sequence; D000912:Antibodies, Neoplasm / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D005434:Flow Cytometry; D005820:Genetic Testing; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D013274:Stomach Neoplasms / Q000235:genetics*; D014407:Tumor Cells, Cultured
D000912:Antibodies, Neoplasm; D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D019151:Peptide Library