Construction and selection of human Fab antibody phage display library of liver cancer.
The aim of this study was to construct the fully humanized anti-hepatoma Fab fragment phage libraries and select antibodies against hepatoma specifically. PBMCs of liver cancer patients were immunized in vitro with HpeG(2) cells and were then transformed by Epstein-Barr virus (EBV). After total RNA was extracted, the heavy chain Fd and kappa/lambda light chain were amplified by RT-PCR and cloned into the vector pComb3 to construct the libraries of Fab fragments. The libraries were then panned by HpeG(2) cells. By means of ELISA and immunochemistry, the Fab phage antibodies binding with hepatoma were selected and identified. The Fd and light chain PCR products were subsequently inserted into pComb3, and the volume of Fab libraries reached 1.7 x 10(7). The libraries were enriched about 138-fold by three cycles of panning. 540 phage clones were picked randomly. Using cell ELISA and immunohistochemistry with cultured cells, one clone Fab phage antibody, which had binding activity with hepatoma, was picked out. Fully humanized anti-hepatoma Fab antibody phage display libraries were constructed. One phage clone was selected and confirmed to specifically bind to hepatoma cells. The selected Fab antibody may be further developed and applied to clinical diagnosis and therapy.
Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, Hunan Province, China.
Xuan
X
Shui
Jian
J
Huang
Yue-Hui
YH
Li
Ping-Li
PL
Xie
Guan-Cheng
GC
Li
D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D007150:Immunohistochemistry; D008099:Liver / Q000276:immunology; D008113:Liver Neoplasms / Q000276:immunology*; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments