Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library.
OBJECTIVE
To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library.


METHODS
A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DNA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in E.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation.


RESULTS
Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M(r) value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells.


CONCLUSIONS
The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.
Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
Bing
B
Yu
Ming
M
Ni
Wen-Han
WH
Li
Ping
P
Lei
Wei
W
Xing
Dai-Wen
DW
Xiao
Yu
Y
Huang
Zhen-Jie
ZJ
Tang
Hui-Fen
HF
Zhu
Guan-Xin
GX
Shen
D006528:Carcinoma, Hepatocellular / Q000276:immunology* / Q000473:pathology; D002455:Cell Division / Q000276:immunology; D045744:Cell Line, Tumor; D003090:Coliphages / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000821:virology; D006801:Humans; D007135:Immunoglobulin Variable Region; D008113:Liver Neoplasms / Q000276:immunology* / Q000473:pathology; D008969:Molecular Sequence Data; D019151:Peptide Library
D007135:Immunoglobulin Variable Region; D019151:Peptide Library