Construction and characterization of the chimeric antibody 8C11 to the hepatitis E virus.
Homodimers of the truncated hepatitis E virus (HEV) capsid proteins, E2 and p239, were conformed to model the dominant antigenic determinants of HEV. Using E2 as an immunogen, two neutralizing monoclonal antibodies (mAbs), namely 8C11 and 8H3, were produced. We constructed a mouse-human chimeric antibody derived from 8C11 and its expression in Chinese hamster ovary (CHO) cells. cDNAs encoding variable regions of heavy and light chains were isolated from hybridoma cells and inserted into mammalian expression vectors containing cDNA of human gamma-1 and kappa constant regions, respectively. The vectors were then cotransfected into CHO cells, and a stable cell line was established. Results from indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the chimeric antibody was assembled correctly to the native IgG molecule and could be secreted from the cells. Similar to the original mAb, the expressed chimeric antibody displayed HEV antigen-binding activity and an enhancement effect on 8H3 binding to HEV antigen. The chimeric antibody could specifically inhibit the binding of p239 to HepG2 cells and compete with HEV IgG in positive serum by antibody-competitive ELISA. The chimeric antibody is expected to be less immunogenic in human and more suitable for antibody therapy of hepatitis E.
Luo
Wenxin
W
National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, Xiamen University, Xiamen, China.
Chen
Yingwei
Y
Li
Lifeng
L
Xu
Chenyu
C
Miao
Ji
J
Shih
James Wai-Kou
JW
Zhang
Jun
J
Xia
Ningshao
N
D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D006508:Hepatitis Antibodies / Q000096:biosynthesis* / Q000276:immunology; D016752:Hepatitis E virus / Q000276:immunology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology
D006508:Hepatitis Antibodies; D011993:Recombinant Fusion Proteins