Enzyme Immunoassay for the Detection of Hepatitis C Virus Antibody
Introduction
Hepatitis C Virus (HCV) is a member of the Flaviviridae family and was first identified in 1989 (Choo et al., 1989). HCV is responsible for post-transfusion hepatitis, formerly known as non-A, non-B hepatitis. The viral genome consists of a single-stranded RNA (~10,000 nucleotides) that encodes a precursor polyprotein, which is cleaved into structural (Core, E1, E2) and non-structural (NS1, NS2, NS3, NS4, NS5) proteins (Shepard et al., 2005).
Genetically, HCV is classified into six major genotypes and at least 80 subtypes. Studies indicate that the most prevalent genotypes in countries such as Iran are 1a, 3a, 1b, and 4, with an estimated 1% of the population being infected (Mohd Hanafiah et al., 2013). The primary modes of transmission include blood transfusion, blood products, and contaminated injections. Other transmission routes include tattooing, ear piercing, circumcision, and hemodialysis. Vertical transmission (mother-to-child) and sexual transmission have also been reported but are less common (Alter, 2007).
Before the implementation of specific HCV virus screening in blood transfusion, the virus was responsible for approximately 90% of post-transfusion hepatitis cases (Alter et al., 1992). Many individuals with acute HCV infection are asymptomatic or experience mild symptoms such as fatigue, nausea, and jaundice. The incubation period varies from 15 to 150 days. At least 80% of infected individuals develop chronic infection, and among these, cirrhosis occurs in 20-50% over 20 years, with an annual hepatocellular carcinoma incidence of approximately 3% (Chisari, 2005).
Clinical Significance
The detection of HCV antibodies plays a crucial role in diagnosing infected individuals and screening blood donors. Various methods, including enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), and nucleic acid testing (NAT), are available for HCV detection. ELISA tests offer high sensitivity and specificity and are particularly effective in preliminary and screening diagnostics (Gretch, 1997). Positive ELISA results should be confirmed with additional tests such as Western blot or polymerase chain reaction (PCR).
This HCV Antibody ELISA Kit is a third-generation assay with enhanced sensitivity and specificity. It detects antibodies against multiple HCV antigens, including NS3, NS4, NS5, and Core proteins. The inclusion of these antigens improves early detection and minimizes false-positive results (Simmonds et al., 2005).
Test Principle
The assay is based on an indirect enzyme immunoassay principle. Microtiter wells are coated with recombinant HCV antigens, including NS3, NS4, NS5, and Core proteins. Serum samples are incubated in these wells, allowing HCV-specific antibodies (IgG and IgM) to bind to the antigens. After washing to remove unbound antibodies, an HRP-conjugated anti-human IgG/IgM is added. Following further incubation and washing, the substrate tetramethylbenzidine (TMB) is added, leading to a color change that is measured spectrophotometrically at 450 nm. The absorbance is proportional to the concentration of anti-HCV antibodies in the sample.
References
- Alter, M. J. (2007). Epidemiology of hepatitis C virus infection. World Journal of Gastroenterology, 13(17), 2436-2441.
- Alter, H. J., Purcell, R. H., Shih, J. W., Melpolder, J. C., Houghton, M., Choo, Q. L., & Kuo, G. (1992). Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. New England Journal of Medicine, 327(8), 555-559.
- Chisari, F. V. (2005). Unscrambling hepatitis C virus-host interactions. Nature, 436(7053), 930-932.
- Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W., & Houghton, M. (1989). Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science, 244(4902), 359-362.
- Gretch, D. R. (1997). Diagnostic tests for hepatitis C. Hepatology, 26(3 Suppl 1), 43S-47S.
- Mohd Hanafiah, K., Groeger, J., Flaxman, A. D., & Wiersma, S. T. (2013). Global epidemiology of hepatitis C virus infection: New estimates of age-specific antibody to HCV seroprevalence. Hepatology, 57(4), 1333-1342.
- Shepard, C. W., Finelli, L., & Alter, M. J. (2005). Global epidemiology of hepatitis C virus infection. The Lancet Infectious Diseases, 5(9), 558-567.
- Simmonds, P., Bukh, J., Combet, C., Deleage, G., Enomoto, N., Feinstone, S., … & Viazov, S. (2005). Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology, 42(4), 962-973.