Preparation And Identification Of Monoclonal Antibody Against Core Antigen Of Hepatitis C Virus

Hepatitis C Virus(HCV)is one of the major globally prevalent pathogen.It is a kind of positive-strand RNA virus that are mainly transmitted through blood and blood products.,leading to acute and chronic hepatitis,cirrhosis and liver cancer.It is currently estimated there would be more than 180 million people suffer from HCV chronic infections worldwide by 2002.And China,as a HCV prevalence country,shows a prevalence of HCV infection of 3.2%.The results highlight the need for more robust surveillance studies and effective strategies to address the HCV disease burden.HCV infection is usually diagnosed by means of the detection of antibody against HCV(anti-HCV)and HCV RNA.As for the anti-HCV assay,residual false negative results are expected because of a long window period of 10 days between HCV infection and seroconversion,causing great damage to the blood donors.In contrast,HCV RNA is a more reliable marker for the diagnosis of active HCV infection by reducing the window period.However,these HCV RNA assays involve considerable technical skill and are subject to contamination and false positive results.They are also very expensive and labor-intensive for routine use.Recent studies estimated that HCV core protein would directly bind with some HCV RNA to form the viral capsid in the viral life cycle.And because HCV core antigen levels correlate well with HCV RNA levels,it would be an alternative approach to predict HCV RNA replication in simplifying the detecting operation and reducing the window period.Besides,HCV core antigen level reflected a HCV viremia and might have clinical implications in the patients accepting antiviral therapy.Therefore,aim of the study was to screen HCV core antibodies with high sensitivity and specificity to detect the HCV core antigen in serum and lay the foundation for the establishment of HCV core Ag diagnostic assay.We analyzed the gene sequence and protein structure of HCV core protein and found that residues 1-120 contains most of the conserved B-cell epitopes.Furthermore,studies indicates that residues 21-40 is the major antigenic segment of core while residues 25-34 are recognized as an antibody binding site of the patient sera.In the experiment,because subtype 1b is the dominant HCV genotype in China,we synthesis the HCV core protein residues 21-40 as the target protein and conjugate keyhole limpet hemocyanin(KLH)to its C-terminal hydrophobic domain to immune the mice.Finally we screened out 11 monoclonal antibody strains with high reactivity to HCV core antigen by the conventional hybridoma cell cloning method and indirect ELISA method.Firstly,Elisa assay shows a 100%reactivity of these McAbs to both truncated recombinant core 1-120aa region and to synthetic peptide core 21-40aa.Secondly,we transfected the Huh 7.5.1 cells with full-length HCV RNA to produce the JFH-1 virus,which was isolated from genotype 2a HCV.Immunofluorescence assay indicates that all of these 11 McAbs can detect the JFH-1 core antigen in culture cells.Furthermore,we use these McAbs to detect the natural core antigen from three patients sera with high viral titer infection by western blot method,and screen out 2 McAbs(110F8 and 14B5)that have the best specificity and sensitivity to the core antigen.Finally,we analyze 60 patients sera with anti-HCV ELISA assay,nest PCR,quantitative PCR assays and these 2 McAbs.The result from nest PCR assay and western blot assay detecting HCV core antigen show no statistical difference.The result from Elisa assay detecing HCV antibody and western blot assay detecting HCV core antigen show no statistical difference.The results showed that these monoclonal antibodies had good specificity and sensitivity to natural core antigen,which laid the foundation for establishment of a diagnosis method for discriminating between acute and chronic HCV infections.