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The expression of cytoplasmic and surface proteins in cultured human skin fibroblasts, human umbilical cord cells obtained after normal delivery on gestation week 38-40, and mesenchymal bone marrow stem cells was compared by the methods of immunocytochemistry and flow cytofluorometry. Bone marrow mesenchymal stem cells expressed a great variety of marker proteins typical of stem and progenitor cells and did not express proteins typical of differentiated cells. Fibroblast-like umbilical cord cells expressed markers of both stem cells and differentiated cells. Fibroblasts of dermal origin were characterized by intensive expression of proteins typical of differentiated cells.

Expression of markers, collagens, and HLA-1 by human skin fibroblasts and fibroblast-like cells isolated from the umbilical Wharton's jelly was compared. Skin fibroblasts express collagens (proteins characteristic of differentiated cells of this histogenetic series) and HLA-1, while umbilical cells express, in addition to collagens, juvenile surface markers and almost no HLA-1. This indicates that fibroblast-like cells isolated from different sources are different and can serve as sources for the creation of cell preparations with different characteristics in future.

Comparative study of hepatitis C virus (HCV) markers (core protein, RNA, and virus-specific antibodies) was carried out in plasma samples from 80 donors. A method based on sandwich ELISA with monoclonal antibodies to recombinant protein was developed for measuring core protein. Nucleocapsid protein was detected after various treatments of precipitates obtained after concentration of virus-containing material from plasma samples. These treatments allowed differentiation of core protein in virions, free nucleocapsids, and immune complexes circulating in peripheral blood. The minimal detectable concentration was 5 pg/ml, maximal 850 pg/ml. The detection of core protein virtually coincided with the detection of HCV RNA: 94.4% RNA-positive samples contained the virus protein. Other parameters (activities of antibodies to HCV in ELISA and level of SGPT) did not allow differentiation of plasma samples by the presence of actively replicating virus. Assay of nucleocapsid protein in the plasma of subjects infected with HCV in various populations of virus particles is important from practical (for blood service) and theoretical viewpoints (for studies of virus pathogenesis mechanisms).

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.

Peptide fragments of hepatitis C virus (HCV) nonstructural protein NS4 capable of reacting with anti-HCV in enzyme immunoassay are synthesized. Addition of synthetic peptides to recombinant nucleocapsid HCV antigen absorbed on solid phase notably improves the efficacy of detection of antibodies to HCV in the sera of patients with hepatitis C.

A method has been developed for preparing and purifying recombinant polypeptide--hepatitis C virus core protein (HCcoreAg) expressed in E. coli from pFC105-302 plasmid coding for 150 N-terminal amino acids of HCcoreAg in the hybrid from the C-terminal with beta-galactosidase. HCcoreAg was purified by ion-exchange chromatography on P-11 phosphocellulose. The bulk of protein carrying HCcoreAg antigenic determinants was found in two polypeptides: with molecular weights 26 and 136 kD. Antigenic and immunogenic properties of the resultant polypeptide were studied. The 26 kD protein can be used in enzyme immunoassay and immunoblotting as antigen for detecting antibodies to the HCV core protein. The results of immunization of rabbits indicate a high immunogenic activity of the protein. High diagnostic value of HCcoreAg preparation has been demonstrated, for the rapid variant of indirect solid-phase enzyme immunoassay among other tests.

Highly affine murine monoclonal antibodies (MAB) to recombinant nucleocapsid (core) protein of hepatitis C virus (rHCcAg) expressed in E. coli were obtained. The MABs were analyzed by solid-phase enzyme immunoassay (EIA), immunodot, immunoblotting, and competitive immunochemical analysis. For estimating the epitope specificity of MAB, several immunoreactive fragments of different length were cloned from the HCcAg region overlapping 160 N-terminal amino acid (a. a.) residues. Use of these fragments and the competitive EIA demonstrated that MAB recognize 4 non-overlapping epitopes, 2 of which are localized in the 1-80 a. a. and 2 other in the 80-150 a. a. regions. A protocol of EIA for detecting HCcAg using MABs to two nonoverlapping HCcAg epitopes has been designed. The sensitivity of double-site sandwich is 1 ng/ml for the recombinant protein.