Establishment And Preliminary Application Of The Novel Bio-barcode Assay For HCV Core Antigen

Hepatitis C virus(HCV) is a major global health problem, and there are more than 170 million people chronically infected in the world. Estimates indicate that three to four million people are newly infected each year, 80% cases of acute hepatitis C progress to chronic infection, and 10~20% of them will develop into complications of liver cirrhosis and cancer. As a member of Hepacivirus genus within the Flaviviridae family, HCV is a 9.6 kb positive-strand RNA virus containing an open reading frame(ORF) that encodes a precursor polyprotein of about 3,010 amino acids. The precursor polyprotein eventually divides into several structural and nonstructural proteins. HCV core protein lying at the N-terminal end of the polyprotein appears in the serum of individuals during the window period, and the amino acid sequence is highly conserved compared with other HCV proteins. These characteristics have made the HCV core protein as a significant marker for diagnosing and monitoring HCV infection.Accurate measurement of HCV is essential for early disease diagnosis and tracking therapeutic efficacy. Tests for HCV antibodies have been used for routine screening of plasma, but they are indirect serological assays and have a long window period between HCV infection and seroconversion(45~68 days). Although sensitive detection of HCV-RNA has been used to confirm the presence of HCV infection, these methods are vulnerable to be blinded by the immune response and suitable for detecting HCV in the stage of active infection. Recently, based on the enzyme-linked immunosorbnent assay(ELISA), several HCV core antigen(HCVc Ag) assays are available. ELISA systems are not widely used for detection of HCVc Ag due to their low sensitivity. Thus, figuring out a sensitive, specific method based on immunoassay technology is necessary.Bio-barcode assay(BCA), a new immunoassay technology introduced by Mirkin in 2003, used short oligonucleotides as target strands in protein detection and offered an innovative approach having typical characteristic of ultrasensitivity to diagnostic test of antigens. In this study, the novel BCA was established by optimizing the steps of forming sandwich structures and gold nanoparticles probes(GNP), and further modifying the BCA with fluorescence-based quantitative real-time PCR(q PCR) using Taq Man probes. The study included 4 sections:1. The design of barcode DNA and preparation of its recombinant plasmids A specific plant sequence genetically distant from HCV was selected from the Gen Bank as the original version. Based on this sequence, barcode DNA, primers and Taq Man probes were designed for the novel BCA. The length of barcode DNA was optimized as well. After being amplified by PCR, the barcode DNA were cloned into p MD18-T vector, and the products were transformed into DH5α chemically competent Escherichia coli. Positive colonies were identified by ampicillin resistance and colony PCR; then they were amplified in the Amp+ liquid medium. Recombinant plasmids extracted from the positive clones were successfully confirmed with Sal I, Bam H I digestion and sequencing. After the reaction conditions of q PCR were optimized, recombinant plasmids were ten-fold serially diluted as quantitative standards to perform the calibration curves. Finally,the sensitivity of the q PCR for detection of barcode DNA was assessed.2.Preparation and identification of GNP and MMP The HCVc Ag antibody pairs optimized for high signal strength and minimal background were selected to establish the antibody sandwich ELISA. The lower limit of detection for this HCVc Ag assay was 2ng/m L. Testing HCVc Ag polyclonal antibody loading on gold nanoparticles would determine the reasonable antibody amount for probe generation. The gold nanoparticles were functionalized with HCVc Ag polyclonal antibody and 3’-thiol-modified barcode DNA, followed by incubation of DNase I to efficiently remove the unbound barcode DNA. The research identified antibody and barcode DNA on the GNP through transmission electron microscope(TEM), ultraviolet-visible spectrophotometry(UV-vis), Dot-ELISA and q PCR assay. Finally, the concentration of GNP was calculated to be 1.22×10-9M. HCVc Ag monoclonal antibody was covalently attached to the tosyl-functionalized magnetic microparticles(MMP) according to the manufacturer’s protocol. By measuring change before and after the reaction in absorbance at 280 nm the coupling efficiency was 75.6%. Dot-ELISA identified that coated antibodies had high activity.3. Optimization and evaluation of the novel BCA system This study showed that MMP had no significant effect on q PCR in a certain range of volume. After optimizing the reaction ratio between GNP and MMP, the two probes were mixed with recombinant HCVc Ag to form ‘GNP-HCVc Ag-MMP’ complexes. These sandwich structures were directly quantified by q PCR method without releasing the barcode strands. We evaluated the sensitivity, specificity, reproducibility of the novel BCA system respectively. The assay sensitivity: the detection sensitivity of the novel BCA is 1fg/m L, which is 6 orders of magnitude more sensitive than ELISA. The assay specificity: in order to determine the specificity, 100 specimens pre-screened as negative for HCV antibodies from a blood donor population and hospital patients were evaluated, providing a preliminary assay specificity of 99%. The assay reproducibility: the intra and inter group coefficient of variations(CV) were both <5%, showing reasonable repeatability.4. Preliminary application of the novel BCA The commercial HCVc Ag assay and HCV RT-PCR kit performed as reference assays for this study. Of 10 anti-HCV-positive serum specimens, 2 were positive by the novel BCA for HCVc Ag, showing worse detection than the commercial assays. Combining with the specificity evaluation, the result indicated that this assay could only be used to detect the samples prior to HCV antibody formation at present level.In conclusion, the novel BCA for HCVc Ag is established and initially performed the diagnosis of hepatitis C. Although there are some problems to be addressed, the increase in sensitivity of the novel BCA may provide a supplement for the earlier identification of HCV infection. Thereby, further developing the diagnostic approaches of viral protein will increase the safety of the blood supply.