DETECTION OF HEPATITIS-C VIRUS CORE PROTEIN CIRCULATING WITHIN DIFFERENT VIRUS PARTICLE-POPULATIONS

Progress in studying pathogenesis and increasing the reliability of he patitis C diagnosis can be achieved by analysis of different forms of virus particles circulating in blood of both patients and infected per sons. Detection of hepatitis C virus (HCV) proteins faces two basic di fficulties: low concentration of HCV proteins, and their blocking by a ntibodies. The aim of this work was to develop a method for the detect ion of nucleocapsid (core) protein in the plasma of HCV-infected perso ns using monoclonal antibodies (MABs). Twenty-seven anti-HCV-positive donor plasmas were studied of which 21 contained HCV RNA and 6 were ne gative. The plasmas were centrifuged for 3 hr at 143,000 g and the ant igenic activity of core-protein was studied in the pellets by EIA usin g 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 l east two genotypes (Ib and 3a). Maximal efficiency of recombinant prot ein detection was achieved with 2 MABs, whereas a combination of 4 MAB s 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 m onoclonal sandwich assay was 1 ng/ml in the pellet or 5 pg/ml after no rmalization to the initial plasma volume. To dissociate immune complex es, the pellet was treated with 2.5 M KBr after first treating the pel let with the nonionic detergent Tween 80 to remove the virus lipid env elope. Using this treatment protocol, core protein was found in 19 of 21 RNA positive plasmas. (C) 1998 Wiley-Liss, Inc.