title: Anti-HCV immunoassays based on a multiepitope antigen and fluorescent lanthanide chelate reporters.
abstract: There is a need for simple to produce immunoassays for hepatitis C virus (HCV) antibody capable of detecting all genotypes worldwide. Current commonly used third generation immunoassays use three to six separate recombinant proteins or synthetic peptides. We have developed and expressed in Escherichia coli a single recombinant antigen incorporating epitopes from different HCV proteins. This multiepitope protein (MEP) was used to develop two types of HCV antibody immunoassays: a traditional antibody immunoassay using a labeled secondary antibody (indirect assay) and a double-antigen assay with the same MEP used as capture binder and labeled binder. The secondary antibody assay was evaluated with 171 serum/plasma samples and double-antigen assay with 148 samples. These samples included an in-house patient sample panel, two panels of samples with different HCV genotypes and a seroconversion panel. The secondary antibody immunoassay showed 95.6% sensitivity and 100% specificity while the double-antigen assay showed 91.4% sensitivity and 100% specificity. Both assays detected samples from all six HCV genotypes. The results showed that combining a low-cost recombinant MEP binder antigen with a high sensitivity fluorescent lanthanide reporter can provide a sensitive and specific immunoassay for HCV serology. The results also showed that the sensitivity of HCV double-antigen assays may suffer from the low avidity immune response of acute infections.
authors.affiliation: Department of Biotechnology, University of Turku, Turku, Finland. Electronic address: tjsalm@utu.fi.
authors.forename: Teppo
authors.initials: T
authors.lastname: Salminen
authors.affiliation: Department of Biotechnology, University of Turku, Turku, Finland.
authors.forename: Etvi
authors.initials: E
authors.lastname: Juntunen
authors.affiliation: Recombinant Gene Products Group, International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi, India; Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, India; Department of Paediatrics, Emory University School of Medicine, Atlanta, GA, USA.
authors.forename: Navin
authors.initials: N
authors.lastname: Khanna
authors.affiliation: Department of Biotechnology, University of Turku, Turku, Finland.
authors.forename: Kim
authors.initials: K
authors.lastname: Pettersson
authors.affiliation: Department of Biotechnology, University of Turku, Turku, Finland.
authors.forename: Sheikh M
authors.initials: SM
authors.lastname: Talha
mesh_terms: D000915:Antibody Affinity; D000939:Epitopes / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D005063:Europium; D005456:Fluorescent Dyes; D016174:Hepacivirus / Q000235:genetics / Q000276:immunology; D006526:Hepatitis C / Q000175:diagnosis; D018937:Hepatitis C Antibodies / Q000097:blood*; D018936:Hepatitis C Antigens / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity
chemical_list: D000939:Epitopes; D005456:Fluorescent Dyes; D018937:Hepatitis C Antibodies; D018936:Hepatitis C Antigens; D011994:Recombinant Proteins; D005063:Europium
keywords: Avidity index; Double-antigen; Europium chelate; HCV; Immunoassay; Multiepitope protein