The Journal of surgical research · 2002
Construction and preliminary screening of a human phage single-chain antibody library associated with gastric cancer.
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He Jin, Zhou Ge, Liu Kang-Da, Qin Xin-Yu
Аннотация
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.
MATERIALS AND 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.
MATERIALS AND 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.