[Selection and characterization of human scFv antibodies against hepatocellular carcinoma].
OBJECTIVE
To construct a fully human anti-hepatoma single-chain phage antibody library, select the anti-hepatoma scFv from it and identify its characteristics.


METHODS
A fully human scFv-displaying phage library was constructed by using phage antibody library technique in combination with in vitro immunization and EBV transformation. The library was subjected to three rounds of positive and negative cell panning and enrichment and then it was selected by ELISA. The binding specificity of phage antibodies with hepatoma carcinoma cells was confirmed by immunohistochemistry.


RESULTS
After scFv genes being cloned into vector Fuse5 and transformed into E.coli MC1061 via electroporation, a phage antibody library containing 1.0 x 10(8) TU (transduced unit) was obtained through tetracycline-resistant screening. After panning, enrichment and testing by ELISA, 3 phage antibody clones reacting more strongly to HepG(2) than QSG-7701 were picked out of 2798 clones. One clone, SA3, was further analysed after DNA sequencing. The results of immunohistochemistry with cultured cells were similar to those of ELISA. SA3 reacted specifically to hepatoma cells in most human hepatoma tissue sections but in few human normal liver tissue sections. The distinction of positive rates is of a great statistical significance.


CONCLUSIONS
A fully human anti-hepatoma scFv fusion phage library has been constructed. ELISA and immunohistochemistry results conform SA3 specifically bind with hepatoma carcinoma cells. The scFv fragment against hepatoma may be further developed and applied in clinical diagnosis and therapy of liver cancer.
Huang
Jian
J
Laboratory of Tumor Immunobiology, Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, Changsha 410078, China. xyhuangjian@163.com
Xiao
Yan
Y
Tong
Yong-qing
YQ
Guo
Feng-jie
FJ
Zhou
Guo-hua
GH
Li
Yue-hui
YH
Hu
Jin-yue
JY
Li
Guan-cheng
GC
D006528:Carcinoma, Hepatocellular / Q000175:diagnosis* / Q000188:drug therapy*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology* / Q000627:therapeutic use*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008113:Liver Neoplasms / Q000175:diagnosis* / Q000188:drug therapy*; D019151:Peptide Library; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000627:therapeutic use*
D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D057127:Single-Chain Antibodies