title: Construction, expression, purification and biotin labeling of a single recombinant multi-epitope antigen for double-antigen sandwich ELISA to detect hepatitis C virus antibody.
abstract: Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.
authors: Department of Bio-diagnosis, Beijing Institute of Basic Medical Sciences, Beijing, China.
authors: Jing
authors: J
authors: He
authors: Bingshui
authors: B
authors: Xiu
authors: Guohua
authors: G
authors: Wang
authors: Kun
authors: K
authors: Chen
authors: Xiaoyan
authors: X
authors: Feng
authors: Xiaoguo
authors: X
authors: Song
authors: Cuixia
authors: C
authors: Zhu
authors: Xiqin
authors: X
authors: Yang
authors: Guanzhong
authors: G
authors: Bai
authors: Shigan
authors: S
authors: Ling
authors: Heqiu
authors: H
authors: Zhang
mesh_terms: D001710:Biotin / Q000737:chemistry; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018985:Epitopes, B-Lymphocyte / Q000276:immunology; D004926:Escherichia coli; D018937:Hepatitis C Antibodies / Q000097:blood*; D018936:Hepatitis C Antigens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012698:Serologic Tests / Q000379:methods; D014773:Virology / Q000379:methods
chemical_list: D018985:Epitopes, B-Lymphocyte; D018937:Hepatitis C Antibodies; D018936:Hepatitis C Antigens; D011993:Recombinant Fusion Proteins; D001710:Biotin