Abstract:Aim. The development of the hepatitis E virus (HEV) genotype 3 recombinant capsid protein.Materials and methods. E.coli strains, plasmid vectors, serological and clinical samples, ELISA reagent kits, molecular biological, bioinformatic, biotechnological, biochemical and serological methods.Results. Using viruscontaining material from pigs of Belgorod region (Russian Federation) we made E.coli strains producing recombinant capsid protein, containing C-terminal of viral ORF2 protein fragment fused to E.coli β-ga… Show more
Viral hepatitis E infection affects up to 80-100 % of domestic pigs worldwide and is characterized by high seroprevalence among domestic pigs in temperate climate countries. Epizootic monitoring of HEV infection is insufficient in the Republic of Belarus due to lack of the required number of available and inexpensive diagnostic ELISA kits with good sensitivity and specificity. In this regard, research on development of domestic ELISA kit for semi-quantitative detection of antibodies to hepatitis E virus in pigs with subsequent assessment of seroprevalence to HEV in pig population in the Republic of Belarus is relevant. The results of studies on development of enzyme-linked immunosorbent assay kit for semi-quantitative determination of class G immunoglobulins to HEV in pig blood serum using recombinant proteins, including immunodominant amino acid sequences corresponding to the ORF2 and ORF3 proteins of HEV genotype 3 are presented in the paper. The optimal concentration for sorption of ORF2 and ORF3 proteins has been determined, which is 2 μg/ml and 1 μg/ml, respectively. The diagnostic sensitivity of this test kit makes 94.8 %, and the diagnostic specificity makes 100 %. Coefficients of variation being the criterion for assessing the intra-serial and inter-serial reproducibility of this test kit, make 3.5 % and 12.4 %, respectively, which allows to obtain reproducible results and identify specific anti-HEV antibodies in all positive samples of pig blood serum. When studying 1235 pig sera samples from various pig farms of Brest, Vitebsk, Gomel, Grodno, Minsk and Mogilev regions, seroprevalence of anti-HEV antibodies has been determined in 168 or 13.6 % of animals. The described diagnostic method can be widely used in science and practice for the further study of seroprevalence of anti-HEV. Acknowledgments. The research was carried out as part of the Interstate program of innovation cooperation of States - participants of the CIS up to 2020, with the financial support of the Ministry of education and science of the Russian Federation, the project ID RFMEFI61316X0 061, and the State Committee for Science and Technology of the Republic of Belarus.
Viral hepatitis E infection affects up to 80-100 % of domestic pigs worldwide and is characterized by high seroprevalence among domestic pigs in temperate climate countries. Epizootic monitoring of HEV infection is insufficient in the Republic of Belarus due to lack of the required number of available and inexpensive diagnostic ELISA kits with good sensitivity and specificity. In this regard, research on development of domestic ELISA kit for semi-quantitative detection of antibodies to hepatitis E virus in pigs with subsequent assessment of seroprevalence to HEV in pig population in the Republic of Belarus is relevant. The results of studies on development of enzyme-linked immunosorbent assay kit for semi-quantitative determination of class G immunoglobulins to HEV in pig blood serum using recombinant proteins, including immunodominant amino acid sequences corresponding to the ORF2 and ORF3 proteins of HEV genotype 3 are presented in the paper. The optimal concentration for sorption of ORF2 and ORF3 proteins has been determined, which is 2 μg/ml and 1 μg/ml, respectively. The diagnostic sensitivity of this test kit makes 94.8 %, and the diagnostic specificity makes 100 %. Coefficients of variation being the criterion for assessing the intra-serial and inter-serial reproducibility of this test kit, make 3.5 % and 12.4 %, respectively, which allows to obtain reproducible results and identify specific anti-HEV antibodies in all positive samples of pig blood serum. When studying 1235 pig sera samples from various pig farms of Brest, Vitebsk, Gomel, Grodno, Minsk and Mogilev regions, seroprevalence of anti-HEV antibodies has been determined in 168 or 13.6 % of animals. The described diagnostic method can be widely used in science and practice for the further study of seroprevalence of anti-HEV. Acknowledgments. The research was carried out as part of the Interstate program of innovation cooperation of States - participants of the CIS up to 2020, with the financial support of the Ministry of education and science of the Russian Federation, the project ID RFMEFI61316X0 061, and the State Committee for Science and Technology of the Republic of Belarus.
Introduction. The diagnostic efficacy of methods for hepatitis E serodiagnostic varies over a wide range; therefore, the combined use of tests of various formats is recommended. The aim of the research was to develop a test system for the detection of IgG antibodies to hepatitis E virus (HEV) in human serum by linear immunoassay (LIA). Material and methods. Serum samples from patients with hepatitis and healthy individuals were tested using commercial enzyme-linked immunosorbent assay systems for the presence of IgG antibodies to viral agents causing hepatitis and other infections associated with liver pathology. Recombinant antigens ORF2 and ORF3 of HEV genotypes 1 and 3 were used. The “RecomLine HEV IgG/IgM” reagent kit (Mikrogen GmbH, Germany) was used as a comparison test system. Results. The first Russian diagnostic kit “Blot-HEV”, designed to detect IgG antibodies to individual HEV proteins in human serum using LIA, was developed. The antigenic base is represented by strips of a nitrocellulose membrane with immobilized recombinant antigens ORF2 (aa 406–660) and ORF3 (aa 1–113) of HEV genotypes 1 and 3, and control antigens in the form of discrete lines. The conjugate was mouse monoclonal antibodies to human class G immunoglobulins labeled with horseradish peroxidase. The chromogen solution contained the 3,3’,5,5’-tetramethylbenzidine. A visual and digital recording of results was provided. The analytical sensitivity of the test kit was 0.625 IU/ml for ORF2 antigens and 2.5 IU/ml for ORF3 antigens. The absence of the influence of endogenous interfering substances on the results of the analysis and the absence of cross-reactions with antibodies to hepatitis pathogens of the other etiologies had been shown. The sensitivity of the test system compared to the “RecomLine HEV IgG/IgM” kit was 92%, specificity 97%. Shelf life in condition of storage was determined to be 12 months. Conclusions. The developed test can be used to confirm the results of ELISA in laboratory diagnosis of hepatitis E.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.