Detection of Hepatitis B Virus X Antigen and Anti-X Antibody in Sera of HBV Infected Patients by ELISA using Recombinant X Proteins Expressed in E. coli and Anti-HBx Monoclonal Antibodies / 대한임상병리학회지

ABSTRACT

BACKGROUND: The X gene is the smallest coding region of the hepatitis B virus (HBV) genome. Several studies reported that X gene-encoded protein may be related to viral replication, and possibly used as a new marker indicative of HBV infection. However, its practical application as a diagnostic reagent remains limited. In this study, we developed anti-X monoclonal antibodies using recombinant hepatitis B virus X (HBx) proteins and investigated the humoral immune responses against HBx in sera of HBV-infected patients by enzyme-linked immunosorbent assay (ELISA). METHODS: Sera of 47 HBV-associated patients and 12 normal controls were studied. Using recombinant HBx expressed in Escherichia coli, seven clones of monoclonal anti-HBx antibodies were developed. The binding site and activity of each monoclonal antibody were determined by ELISA and Western blot analysis, and antibodies that gave the best signals in both assays were selected for the detection of HBx antigen. An ELISA to detect anti-X was also constructed by using recombinant HBx proteins. RESULTS: Clinical samples from patients with liver cirrhosis and hepatocellular carcinoma (HCC) were more than 60% positive for anti-HBx antibody. The positive rate of X antigen in patients with liver cirrhosis and HCC was 27% and 33%, respectively. None of acute hepatitis patients and chronic asymptomatic carriers were positive for HBx antigen or anti-X antibody. The present ELISA system detected circulating HBx with a dynamic range from 5 to 1000 ng per milliliter and the specificity of the assay was also acceptable. The analysis of binding site and activity of monoclonal antibodies performed by ELISA were in agreement with Western blotting results. CONCLUSIONS: ELISA using recombinant HBx and monoclonal antibodies showed good sensitivity and corresponded well with immunoblotting results. For the clinical application of this assay, however, further study is needed on the relationship between HBx and the progression of the disease.