Non-A, non-B (NANB) hepatitis viruses are now classified as hepatitis E (enterically transmitted) and hepatitis C (parenterally transmitted). India experiences a large number of epidemics of the enteric disease every year. In addition, about 70% of the sporadic cases among adults are also due to NANB hepatitis. With the availability of an immunoblot assay for the detection of anti-HEV-IgM and the polymerase chain reaction (PCR) for the detection of HCV-RNA, serum samples from epidemic and sporadic NANB patients were screened for these markers. We found that a large number of cases from the epidemics were HEV, though a few remained undiagnosed, while of the sporadic cases only a few could be diagnosed as HCV or HEV; a large proportion remained undiagnosed.
Progress in studying pathogenesis and increasing the reliability of hepatitis C diagnosis can be achieved by analysis of different forms of virus particles circulating in blood of both patients and infected persons. Detection of hepatitis C virus (HCV) proteins faces two basic difficulties: low concentration of HCV proteins, and their blocking by antibodies. The aim of this work was to develop a method for the detection of nucleocapsid (core) protein in the plasma of HCV-infected persons using monoclonal antibodies (MABs). Twenty-seven anti-HCV-positive donor plasmas were studied of which 21 contained HCV RNA and 6 were negative. The plasmas were centrifuged for 3 hr at 143,000 g and the antigenic activity of core-protein was studied in the pellets by EIA using four MABs able to recognize four nonoverlapping determinants, two at N-terminus and two at C-terminus of recombinant core (1-150 aa). The determinants detected were present in the natural core protein of at least two genotypes (1b and 3a). Maximal efficiency of recombinant protein detection was achieved with 2 MABs, whereas a combination of 4 MABs was necessary for optimal detection of natural core protein. This is indicative of different conformational structures of natural protein and its gene-engineered analog. The sensitivity of core detection by monoclonal sandwich assay was 1 ng/ml in the pellet or 5 pg/ml after normalization to the initial plasma volume. To dissociate immune complexes, the pellet was treated with 2.5 M KBr after first treating the pellet with the nonionic detergent Tween 80 to remove the virus lipid envelope. Using this treatment protocol, core protein was found in 19 of 21 RNA positive plasmas.
Monoclonal antibodies (mAbs) were raised against hepatitis B virus core produced by a recombinant clone of Escherichia coli (rHBc). The three mAbs recognized rHBc by Western blot, suggesting that they reacted with non-conformational epitopes. Competition experiments between mAbs and human anti-HBc sera confirmed the existence of an immunodominant HBc epitope within the viral antigen. A monoclonal competition enzyme immunoassay using an IgM mAb conjugated to biotin and streptavidin-peroxidase as the detection system yielded 99% sensitivity and 100% specificity, when compared to other commercial assays.
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