Double Antigen Sandwich Format In The Detection Of Anti Hepatitis C Virus With Improved Specificity

Hepatitis C Virus (HCV), a major causative agent of posttransfusional hepatitis, usually results in liver disease, even liver cirrhosis and hepatocellular carcinoma. The overall worldwide prevalence of HCV infection is approximately 3%, and the prevalence in the developing countries is much higher than the developed countries. According to the data provided by a national study conducted in 2006, HCV prevalence was reported to be 0.6% in China overall. With a characteristic of extremely high rate of chronicity and less than 50 percentage responses to interferon therapy, till now, there is still no preventive vaccine applied for the control of HCV infection. The optimal method to prevent HCV infection is to promote the sensitivity and specificity of virological testing, which is widely used in the diagnosis and manage of hepatitis c virus infection.Testing for the presence of antibody to hepatitis C virus(anti-HCV) is recommended for initially identifying persons with hepatitis C virus infection. Testing for anti-HCV includes screening tests based on enzyme immunoassays(EIAs) and supplemental assays based on immunoblot testing. Because of relative cost-effectiveness, ease of use, and low variability, anti-HCV antibody immunoassay of indirect format can be applied for blood screening and clinical testing. Anti-HCV antibody immunoassays have now progressed to the third generation. Although these assays have better sensitivity and specificity, there is still a high rate of false-positive among a population with a low prevalence of infection. According to the Guidelines for Laboratory Testing and Result Reporting of Antibody to Hepatitis C Virus provided by American CDC, among immunocompetent population with anti-HCV prevalence<10%, even a specificity of 99% does provide a desired predictive value for a positive test.; and the proportion of false-positive results with HCV EIA2.0 or HCV version 3.0 ELISA averages approximately 35%(range:15%-60%). So that, the establishment of a novel format instead of indirect format with better specificity without depressing sensitivity is so necessary. Based on the successful application of double antigen sandwich immunoassay in the detection of anti-HIV antibody and anti-Treponema pallidum antibodies, it has been proved that the double-antigen sandwich(DAS) format can substantially improve the assay's specificity. Furthermore, DAS format can not only detect lgG antibodies, but also lgM and other classess or subclasses of antibodies. As CORE is one of the most important epitopes, which contains a vast number of basic amino acid. If the protein is labeled directly, the CORE antigen will be blocked, resulting in lower sensitivity. So we improve the traditional DAS format. By genetic engineering methods, HCV antigen and bridging protein(TRX) were recombined and connected. TRX can react with anti-HCV mAb conjugated with enzyme, then HCV antigen can combine to labeled-mAbs to produce HCV antigen-bridging protein TRX-anti-TRX mAb-enzyme compounds. Hence the HCV antigen was marked with enzyme indirectly, and combined with the coating antigen, it can be used to detect HCV antibody in serum.HCV antigen-expressing vector pETlld-C44P, covering B-cell epitopes of CORE, NS3 and NS4, was constructed and kept by our laboratory. The HCV antigen coding sequence amplified by PCR from pETlld-C44P is subcloned into TRX-expressing vector pET43.1a-TRX to create recombinant pET43.1a-TRX-C44P. pETlld-C44P and pET43.1a-TRX-C44P were transformed into Escherichia coli BL21 cells for protein expression respectively. After induced by IPTG for 5h, Western blotting was used to identified the immunoreactivity of the two proteins. The two proteins were purified with ammonium sulfate precipitation, ion exchange chromatograph and gel filtration step by step, ultimately, both of them were proved to be 90% purity.Indirect format and double antigen sandwich format were established with the two proteins. A total of 756 samples, including 156 samples from Beijing Blood Center and 600 samples from Suijing of Sichun province, were detected with indirect format, double antigen sandwich format and another two commercialized EIA(approved by China FDA), and the discordant samples were tested by Real-time PCR. The sensitivity of established double antigen sandwich format is 100%, the specificity is 99.86%, the concordance rate is 99.87%; positive predictive value is 98.1%, negative predictive value is 100%. The sensitivity of established indirect format is 100%, the specificity is 96.59%, the concordance rate is 96.83%; Positive predictive value is 68.4%, negative predictive value is 100%.In conclusion, the established double antigen sandwich format has equal sensitivity and specificity with commercialized EIA of anti-HCV. From the view of methodology, the double antigen sandwich format has a better specificity than the indirect format with the same sensitivity.