AbstractHepatitis C virus (HCV) infection can be detected by using immunoassay techniques that measure reactivity to viral protein antigens. In this study seven discreet proteins derived from HCV genomic coding sequences have been expressed, purified and characterized. Six proteins represent the structural regions of the core (C22‐3), the envelope (E1 and E2), and the non‐structural regions NS3 (C33C), NS3–NS4 (C100‐3) and NS5. The seventh, C25, is a chimeric fusion protein containing C33C, C100‐3 and C22‐3 regions. Using these recombinant proteins, multi‐antigen radioimmunoassays and enzyme immunoassays (EIA) were designed. The fusion protein, C25, was demonstrated to be an improved antigen for serodiagnosis of HCV antibody. Use of the C25 protein accelerated HCV antibody detection by 3–46 weeks in non‐A, non‐B hepatitis seroconversion cases and significantly increased the rate of detection in a paid donor population by 20%. The C25 assay also demonstrated excellent specificity in 2446 randomly selected low prevalence samples. The repeated reactive rate in this group of samples was 0.5%.Samples from volunteer blood donors pre‐selected for repeat reactivity with the first generation C100‐3‐based HCV antibody tests (n= 175) were tested using the C25 assay. The C25 assay detected 37.7% samples as reactive and 53.1% samples as non‐reactive. This result was in agreement with all other supplementary tests that include RIBATM, multi‐antigen assay, Abbott neutralization and peptide assay. The other 9.2% samples were classified as ‘indeterminant’ because these samples were only partially in agreement with some of the above supplementary tests.The C25 enzyme‐linked immunosorbent assay (ELISA), with its improved assay sensitivity, can identify additional HCV antibody reactive cases in both hepatocellular carcinoma and cryptogenic cirrhosis patients. The C25 and multi‐antigen EIA assays were used to investigate the vertical transmission of HCV. It was observed that these improved assays are able to detect antibodies in a vertical transmission case. The C25 ELISA was also compared with a synthetic peptide assay. The C25 assay was found to be superior to the peptide assay that performed poorly in the detect