Enzyme-linked immunosorbent assay for antibodies against the capsid protein of hepatitis C virus with a synthetic oligopeptide

Abstract

An enzyme-linked immunosorbent assay was developed for the determination of antibodies against the putative capsid protein of hepatitis C virus (HCV). A 36-mer oligopeptide with a sequence of RRGPRLGVRATRKTSERSQPRGRRQPIPKVRRPEGR (CP9) was synthesized; it was selected on the translation product of the presumptive HCV core gene, because of a high local hydrophilicity and excellent conservation by different HCV strains. The synthetic peptide was immobilized on a solid-support to capture antibodies directed to CP9 (anti-CP9) in test sera, which were detected by Fab' fragments of monoclonal anti-human IgG/gamma labeled with horseradish peroxidase. The specificity of anti-CP9 was confirmed by absorption tests. Anti-CP9 was detected in 13 (68%) of 19 patients with sporadic acute non-A, non-B (NANB) hepatitis and in 15 (83%) of 18 patients with post-transfusion acute NANB hepatitis. In 7 cases of acute NANB hepatitis who were followed, anti-CP9 developed earlier than antibodies against HCV (anti-HCV) detectable by a commercial assay kit. Among patients with chronic NANB liver diseases, anti-CP9 was detected in 103 (77%) of 133 with chronic hepatitis, 70 (62%) of 113 with liver cirrhosis and 31 (76%) of 41 with hepatocellular carcinoma. Anti-CP9 and anti-HCV overlapped in 175 (54%) among 324 cases of acute or chronic NANB liver diseases; 58 (18%) were positive only for anti-CP9 while 49 (15%) were positive only for anti-HCV. HCV RNA was detected, by amplifying HCV cDNA with polymerase chain reaction, in 10 of 11 sera positive only for anti-CP9. Among sera from 606 blood donors, 21 were positive only for anti-CP9. HCV RNA was detected in 5 (24%) of them, all of which had A492 values greater than 0.600 in ELISA for anti-CP9. Based on these results, anti-CP9 would complement anti-HCV for the diagnosis of HCV infection and contribute toward further decreasing posttransfusion NANB hepatitis.