Related Experiment Video
Updated: Aug 20, 2026
A Protocol for Analyzing Hepatitis C Virus Replication
Published on: June 26, 2014
Diagnosis of hepatitis C virus (HCV) infection by antigen-capturing ELISA
1Molecular Genetics Center, Institute of Biotechnology, Academy of Military Medical Sciences, 20 Dongdajie, Fengtai District, Beijing 100071, People's Republic of China.
Insights
A new antigen-capturing ELISA offers a more specific and sensitive method for diagnosing Hepatitis C virus (HCV) infection. This improved diagnostic approach utilizes recombinant chimeric polyproteins for enhanced accuracy in detecting HCV.
Area of Science:
- Virology
- Immunology
- Biotechnology
Background:
- Hepatitis C virus (HCV) is a primary cause of non-A, non-B hepatitis.
- Enzyme-linked immunosorbent assay (ELISA) detecting antibodies against HCV is the standard diagnostic method.
- Current ELISA kits often use a mix of HCV antigens and goat anti-human IgG conjugate.
Purpose of the Study:
- To develop an antigen-capturing ELISA system for HCV diagnosis.
- To utilize a recombinant chimeric polyprotein containing four HCV antigenic components as the antigen.
Main Methods:
- HCV antigens were expressed in Escherichia coli as chimeric polyproteins in both soluble and inclusion body forms.
- Inclusion body protein served as the solid-phase antigen.
- Soluble protein labeled with horseradish peroxidase (HRP) was used as the enzyme conjugate.
Main Results:
- Chimeric polyproteins containing core, NS3, NS4, and NS5 components were successfully expressed and retained antigenicity.
- The developed antigen-capturing ELISA demonstrated high sensitivity (97.9%) and specificity (100%).
- This assay showed high concordance (98.9%) with commercial HCV enzyme immunoassays.
Conclusions:
- Antigen-capturing ELISA is a sensitive and specific method for HCV diagnosis.
- This approach offers a convenient and improved diagnostic strategy for hepatitis C virus infection.
Background:
Hepatitis C virus (HCV) is a major cause of non-A non-B hepatitis. Detection of circulating antibodies against HCV by enzyme-linked immunosorbent assay (ELISA) has provided the main approach for the diagnosis of HCV infection. Most ELISA kits use a mixture of core, NS3, NS4 and NS5 antigen as capture antigens and enzyme-labeled goat anti-human IgG as conjugate.
Objectives:
To establish an ELISA system based on the antigen-capturing principle, using a recombinant chimeric polyprotein containing four HCV antigenic components as antigen.
Study Design:
HCV antigens were expressed in Escherichia coli as chimeric polyprotein either in inclusion bodies or in soluble form. Protein expressed in inclusion bodies was used as solid-phase antigen, and the antigen expressed in a soluble form was used as enzyme conjugate after being labeled with horseradish peroxidase (HRP).
Results:
Genes coding HCV antigens were cloned and sequenced, chimeric polyproteins containing four immunodominant components (core, NS3, NS4 and NS5) were expressed in E. coli both in soluble and in inclusion body form. These two chimeric proteins retained the antigenicity of HCV antigens. Antibody-capturing ELISA using the chimeric antigens showed a sensitivity of 97% (97/100) and a specificity of 98% (97/99) using the reference panel from the National Institute for the Control of Pharmaceutic and Biological Products of China (NICPBC); the same assay showed a sensitivity of 97.9% (48/49) and a specificity of 100% (43/43) using the self-established reference panel. Antigen-capturing ELISA was set up using the antigen labeled with horseradish peroxidase as conjugate, and was shown to be as sensitive as (97.9%) and more specific than (100%) antibody-capturing ELISA using the reference panel in this work. The antigen-capturing ELISA also showed a high accordance (98.9%) with UBI HCV enzyme immunoassay (EIA) 4.0 kits (United Biomedical Inc. USA).
Conclusion:
Antigen-capturing ELISA provided a convenient, sensitive and more specific approach for the diagnosis of hepatitis C virus infection.
Related Concept Videos
01:25Hepatitis
01:33Enzyme-Linked Immunosorbent Assay
There are many different types of ELISAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. Enzyme-substrate reaction allows the antigen to be visualized or quantified.