Establishment of a sandwich ELISA method for detection of vascular endothelial growth factor in serum samples of hepatocellular carcinoma patients.
OBJECTIVE
To establish a sandwich ELISA method for detecting vascular endothelial growth factor (VEGF) in sera of population and the patients with hepatocellular carcinoma (HCC).


METHODS
Full length and two truncated human VEGF cDNA sequences were amplified from a commercial plasmid pBLAST49-hVEGF by PCR and inserted into the prokaryotic-expression plasmid pET-32a or pGEX-2T. Various VEGF proteins were expressed and purified from E. coli in His-Trx or GST fusion forms. The specific VEGF antibodies were elicited in experimental rabbits and mice by immunization of the full length VEGF fusion protein His-Trx-VEGF1-165. After purification of antibodies with chromatograph of Protein G, a sandwich ELISA technique was established. Serum VEGF levels were evaluated in 229 adults and 291 HCC patients.


RESULTS
SDS-PAGE displayed that the molecular weights of the expressed full length (His-Trx-VEGF1-165), N-terminal (His-Trx-VEGF1-100) and C-terminal (GST-VEGF100-165) human VEGF fusion proteins were about 38KD, 31KD, and 33KD, respectively. Western blots confirmed that the prepared antisera were able to recognize both prokaryoticly and eukaryoticly expressed recombinant VEGF proteins. Assays of serially diluted His-Trx-VEGF1-100 by the established sandwich ELISA method showed that the linear range of the standard curve was 0.625-320 ng/mL, with the squared correlation coefficient R2 = 0.991. Screening of a serum panel containing 291 serum samples of HCC patients and 229 health adults revealed that the average VEGF level in HCC patients was higher than that in healthy controls, with a statically significant difference.


CONCLUSIONS
The established sandwich ELISA reflects the level of serum VEGF and provide scientific basis for screening metastasis and recurrence of HCC using serum VEGF as an index.
Shan
Bing
B
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
Gao
Chen
C
Chen
Jian-Ming
JM
Bi
Xin-Yu
XY
Zhang
Bao-Yun
BY
Guo
Yan
Y
Dong
Chen-Fang
CF
An
Run
R
Shi
Qi
Q
Hu
Jing-Qun
JQ
Zhao
Ping
P
Han
Jun
J
Dong
Xiao-Ping
XP
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000097:blood*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007106:Immune Sera; D008113:Liver Neoplasms / Q000097:blood*; D016133:Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000097:blood*
D017931:DNA Primers; D007106:Immune Sera; D042461:Vascular Endothelial Growth Factor A