Preliminary Establishment Of HCV Core Antigen Detection System

Hepatitis C virus ( HCV) is an enveloped RNA virus that belongs to the family of Flaviviridae. HCV is mainly spread through blood, causes acute or chronic viral hepatitis, which often leads to chronic progressive liver disease (cirrhosis) or even liver cancer. It is estimated that 123-170millon people worldwide are living with HCV infection, of whom 40 million are in china. Therefore, the control of HCV spread has become a very urgent public health concern in china. The introduction of anti-HCV screening in blood donor have significantly decreased the risk of HCV infection through blood transfusion. However, HCV infection has a 7-10 weeks'serological window period which still threaten the safe use of blood. Although HCV RNA detection can effectively shorten the window period, it is difficult to be widely adopted because of cost and practicality. HCV core protein generated in patient before seroconversion, and the quantity of core protein in blood usually indicated the HCV viral load of a patient. Therefore, the detection of HCV core protein will effectively reduce the serological window period and evaluate the effect of antiviral therapy. The present study intended to construct a HCV core antigen detection system.We firstly constructed a non-fusion expression vector pTO-T7-C120 which contains 1-120aa of core sequence. When transformed into E.coli, the bacteria expressed a 13kD recombinant protein named c120. c120 mainly existed in the expression supernatant. c120 were purified through saturated ammonium sulfate deposition and cation exchange column chromatography and the purity of c120 is above 95% by SDS-PAGE analysis. As c120 deleted the C-terminal of core antigen which may lead to the conformational changes and epitope deficiencies, we constructed another expression vector pTrc-CKS-C173 which with contained 1-173aa sequence of core protein that fused with cks. The expression product is about 45kD, named cks-c173. cks-c173 mainly expressed in inclusion body which was dissolved in 8 M urea. cks-c173 was purified by electroelution and the final purity is above 97%. Both c120 and cks-c173 were identified to have good activity and specificity by using Western Blotting and indirect ELISA methods. The purified c120, cks-c173 and a currently existed HCV core protein (core-R) were used to immunize BALB/c mice separately, followed with establishment of 103 hybridoma cell lines secreting McAbs against core antigen. We found that all McAbs specifically reacted with c120 in Western Blotting. Meanwhile, we identified the epitope of 60 McAbs by using synthetic peptides and indirect ELISA, and these epitopes can be divided into 5 groups according to reaction site: 25 McAbs recognized 25-39aa, 21 McAbs recognized 90-119aa, 9 McAbs recognized 49-78aa, 4 McAbs recognized 17-31aa, and 1 McAb recognized 33-47aa of HCV core sequrence. These results clarified the dominant epitopes of the recombinant proteins, and indicated that exposed dominant epitopes of the three proteins are different. In addition, we also used synthetic peptides and indirect ELISA to analyze the epitope distribution of anti-HCV core antibody in HCV patient sera. Our result showed that the epitope distribution was much wider, and there were more conformational anti-core antibodies in the sera of patient, when compared with mice sera that was immunized by recombinant core protein. Further, from the results we speculate that 49-78aa may be an important linear epitope of nature core antigen.Finally, we examined 103 McAbs for their detecting ability on c120 protein by using a double-antibody sandwich ELISA. We successfully screened 6 pairs of McAbs combinations in which the sensitivity to c120 could achieve to 100pg/mL, and one pair 83-65HRP could reach to 50pg/mL. The screened out McAbs were then used to detect natural core antigen from 25 patients sera that both anti-HCV and HCV PCR were positive. We found 2 pairs (83/97-65HRP) combinations could successfully detect natural core antigen with a sensitivity of 8/25, and the 2 pairs also had good specificity.This study preliminary established a HCV core antigen detection system, which provides many useful information and experience to finally establish a HCV Ag/Ab assay. However, the system needs to be further evaluated with serological window period sera.