Characterisation of a panel of monoclonal antibodies raised against recombinant HCV core protein.
Hepatitis C virus (HCV) has as yet no practical culture system so any antigen or antibody studies must be carried out using recombinant antigens. In this study, HCV core sequence was amplified by PCR, inserted into pRSET, and expressed in E. coli. The resultant core protein was purified using nickel affinity chromatography which bound the six histidine tag attached to the N-terminus of the protein. After elution in imidazole buffer, the core protein was used to immunise Balb/c mice and monoclonal antibodies against HCV core were raised. Six monoclonals were examined in a variety of assays. All of them recognised the p27 kDa protein which they were raised against and 2D2 and 3D7 recognised the core component of an HCV Recombinant Immunoblot Assay (RIBA). None of the antibodies recognised the linear peptides in an Innolia HCV assay. 2D2 showed cytoplasmic granular staining in 1-5% of cells in frozen section of HCV infected livers. Cross-competition assays between themselves and human anti-HCV core positive sera divided the antibodies into two main groups (I and II), with a sub-division of group I into a and b. Group I antibodies were unable to be inhibited by human anti-HCV sera whereas group II antibodies were inhibited by these sera (up to 62%). Epitopes recognised by all the monoclonals were probably conformational with the group I epitope being located within the first 105 amino acids of the core sequence and the group II epitope between amino acids 105 and 160.
Ferns
R B
RB
Department of Virology, University College London Medical School, United Kingdom.
Tuke
P W
PW
Sweenie
C H
CH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D016174:Hepacivirus / Q000276:immunology*; D006526:Hepatitis C / Q000097:blood / Q000276:immunology*; D018937:Hepatitis C Antibodies / Q000276:immunology*; D018936:Hepatitis C Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014758:Viral Core Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D018937:Hepatitis C Antibodies; D018936:Hepatitis C Antigens; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus