A novel recombinant multiepitope protein has been designed that consists of six linear, immunodominant, and phylogenetically conserved epitopes from hepatitis C virus. Five of these antigens (core, NS3, NS4I, NS4II, and NS5) are being used in many of the third-generation kits while sixth epitope (core3g) is an additional sequence from a newly identified Indian isolate. The genes for these epitopes have been joined together to code for a single multiepitope protein that has been evaluated for its diagnostic potential for the detection of anti-HCV antibodies in human plasma. Two separate synthetic genes have been designed, both encoding the same six epitopes in a single open reading frame along with spacers having additional amino acids to function as flexible (r-HCV-F-MEP) or rigid (r-HCV-R-MEP) linkers. High-level expression of hepatitis C multiepitope protein in Escherichia coli has been achieved. The protein has been purified using a single affinity step yielding > 25 mg pure protein/liter culture and used as the coating antigen in anti-HCV EIA. The use of this multiepitope protein eliminates the requirement for multiple diagnostic intermediates for the development of anti-HCV diagnostic kit. The sensitivity and specificity of the HCV multiepitope protein was evaluated by Boston Biomedica Worldwide Performance Panels, HCV Seroconversion Panels and Viral Co-infection Panels, and was found to be comparable with commercially available anti-HCV EIA kits. This analysis indicated its unequivocal performance as capture antigen in anti-HCV EIA. The high epitope density, careful choice of epitopes and use of E. coli system for expression, coupled with simple purification protocol provides the potential for the development of an inexpensive diagnostic test with high degree of sensitivity and specificity. (c) 2006 Elsevier Inc. All rights reserved.