Development and evaluation of an enzyme immunoassay for diagnosis of acute viral hepatitis E
I. S. Zadora,
S. V. Zhavoronok,
G. I. Alatortseva,
A. I. Shcherban,
L. N. Pritvorova,
L. N. Nesterenko,
V. V. Davydov,
L. A. Anisko,
T. A. Rogacheva,
N. V. Bayur,
N. V. Shchuka,
Yu. A. Mytko,
V. V. Simirsky,
M. I. Mikhailov,
V. V. Zverev https://doi.org/10.51523/2708-6011.2025-22-2-04
Abstract
Objective. To develop a national high-sensitivity and marked specifity enzyme immunoassay test system for determining class М immunoglobulins to the hepatitis E virus (HEV) in human blood serum “ELISA-anti-HEV IgМ“.
Materials and methods. The base for this research is City Clinical Hospital of Infectious Diseases in Minsk. Qualitative determination of anti-HEV IgМ in human blood serum samples was carried out by indirect enzyme immunoassay using reference kits – “ELISA-ANTI-HEV-IgМ“, Vecto hep E-IgМ. 96-well polystyrol strip plates were used to design a national test system for the qualitative detection of anti-HЕV-IgМ; recombinant antigens – analogues of the protein ORF2 and ORF3 HЕV of the genotype 3; antibodies against human IgM conjugated with horseradish peroxidase; post-closing solution; serum dilution solution and conjugate dilution solution.
Results. Optimal concentrations for sorption of ORF2 and ORF3 proteins were determined, amounting to 2 mg/ml and 1 mg/ml. The incubation time with TMB is set to 20 minutes. Diagnostic sensitivity of the test system was no less than 99%, and diagnostic specificity was no less than 99%. The index of in-series reproducibility was 92,3%, inter-series reproducibility was 86,1%. Technical specifications TU BY 100185093.093-2023 were developed and registered. Direction for use of the reagent kit of the test system “ELISA-anti-HEV-IgM human“ for determining markers of acute viral hepatitis E in human blood serum by enzyme immunoassay was approved.
Conclusion. The first national test system “ELISA-anti-HЕV-IgM” for the determination of class M immunoglobulins against hepatitis E virus in human blood serum by indirect enzyme immunoassay was developed. Laboratory and technical tests of the experimental sample of the test system were carried out, shown its high diagnostic sensitivity, specificity and reproducibility. The regulatory, technical and operational documentation for the test system was approved.
Supporting agencies: This research work was carried out within the funded activity 13 “To develop a technology for the industrial production of test systems for the detection of IgG and IgM class antibodies to the hepatitis E virus in humans and animals using the enzyme immunoassay method, and organize their production” subprogram 5 “Chemical products and molecular technologies” of the State Program “High technology and techniques” for 2021-2025, State Registration No. 20213143. The authors thank the chief physician Yurovsky N.N., the staff of the clinical diagnostic laboratory of the City Clinical Hospital of Infectious Diseases for assistance in organizing and conducting the research (Dolbuz O.N., Metlitskaya E.A., Malinovskaya N.V.).
About the Authors
I. S. Zadora
Belarusian State Medical University; Pilot Production of the Institute of Bioorganic Chemistry National Academy of Sciences of Belarus
Belarus
Belarus
Ilona S. Zadora - Senior Lecturer at the Department of Microbiology, Virology, Immunology, Belarusian State Medical University; Junior Researcher, Pilot Production of the Institute of Bioorganic Chemistry National Academy of Sciences of Belarus.
Minsk
S. V. Zhavoronok
Belarusian State Medical University
Belarus
Belarus
Sergey V. Zhavoronok - Doctor of Medical Sciences, Professor, Professor at the Department of Infectious Diseases, Belarusian State Medical University.
Minsk
G. I. Alatortseva
I.I. Mechnikov Vaccine and Serum Research Institute
Russian Federation
Russian Federation
Galina I. Alatortseva - Candidate of Biological Sciences, Head of the Laboratory of Cloning Viral Genomes, I.I. Mechnikov Vaccine and Serum Research Institute.
Moscow
A. I. Shcherban
Pilot Production of the Institute of Bioorganic Chemistry National Academy of Sciences of Belarus
Belarus
Belarus
Aleksandr I. Shcherban - Candidate of Biological Sciences, Head of the Scientific and Technical Department, Pilot Production of the Institute of Bioorganic Chemistry National Academy of Sciences of Belarus.
Minsk
L. N. Pritvorova
I.I. Mechnikov Vaccine and Serum Research Institute
Russian Federation
Russian Federation
Lyudmila N. Pritvorova - Candidate of Medical Sciences, Leading Researcher of the Laboratory of Cloning of Viral Genomes, I.I. Mechnikov Vaccine and Serum Research Institute.
Moscow
L. N. Nesterenko
I.I. Mechnikov Vaccine and Serum Research Institute
Russian Federation
Russian Federation
Lyubov N. Nesterenko - Candidate of Chemical Sciences, Leading Researcher of the Laboratory of Cloning of Viral Genomes, I.I. Mechnikov Vaccine and Serum Research Institute.
Moscow
V. V. Davydov
Belarusian State Medical University
Belarus
Belarus
Vladimir V. Davydov - Candidate of Biological Sciences, Associate Professor, Head of the Department of Biology, Belarusian State Medical University.
Minsk
L. A. Anisko
Belarusian State Medical University; City Clinical Hospital of Infectious Diseases
Belarus
Belarus
Lyudmila A. Anisko - Candidate of Medical Sciences, Associate professor, Associate professor at the Departments of Infectious Diseases, Belarusian State Medical University; Board Certified Physician, Head of the Laboratory, City Clinical Hospital of Infectious Diseases.
Minsk
T. A. Rogacheva
Belarusian State Medical University; City Clinical Hospital of Infectious Diseases
Belarus
Belarus
Tamara A. Rogacheva - Candidate of Medical Sciences, Associate professor, Associate Professor at the Departments of Infectious Diseases, Belarusian State Medical University; Board Certified in Laboratory Diagnostics, City Clinical Hospital of Infectious Diseases.
Minsk
N. V. Bayur
Belarusian State Medical University; City Clinical Hospital of Infectious Diseases
Belarus
Belarus
Nadzeya G. Bayur - External Part-timer of the Department of Infectious Diseases, Belarusian State Medical University; Biologists at the Clinical Diagnostic Laboratory, City Clinical Hospital of Infectious Diseases.
Minsk
N. V. Shchuka
Pilot Production of the Institute of Bioorganic Chemistry National Academy of Sciences of Belarus
Belarus
Belarus
Natalia V. Shchuka - Researcher, Pilot Production of the Institute of Bioorganic Chemistry National Academy of Sciences of Belarus.
Minsk
Yu. A. Mytko
Pilot Production of the Institute of Bioorganic Chemistry National Academy of Sciences of Belarus
Belarus
Belarus
Yulia A. Mytko - Researcher, Pilot Production of the Institute of Bioorganic Chemistry National Academy of Sciences of Belarus.
Minsk
V. V. Simirsky
Pilot Production of the Institute of Bioorganic Chemistry National Academy of Sciences of Belarus
Belarus
Belarus
Vladimir V. Simirsky - Candidate of Technical Sciences, Director of the Pilot Production of the Institute of Bioorganic Chemistry National Academy of Sciences of Belarus.
Minsk
M. I. Mikhailov
I.I. Mechnikov Vaccine and Serum Research Institute; Central Research Institute of Epidemiology of Rospotrebnadzor
Russian Federation
Russian Federation
Mikhail I. Mikhailov - Doctor of Medical Sciences, Professor, Corresponding Member of the Russian Academy of Sciences, Chief Researcher, Head of the Laboratory of Viral Hepatitis, I.I. Mechnikov Vaccine and Serum Research Institute; Chief Researcher of the Central Research Institute of Epidemiology of Rospotrebnadzor.
Moscow
V. V. Zverev
I.I. Mechnikov Vaccine and Serum Research Institute; I.M. Sechenov First Moscow State Medical University (Sechenovskiy University)
Russian Federation
Russian Federation
Vitaly V. Zverev - Doctor of Biological Sciences, Professor, Academician of the Russian Academy of Sciences, Head of the Department of Microbiology, Virology and Immunology, I.M. Sechenov First Moscow State Medical University (Sechenovskiy University); Academic Advisor, I.I. Mechnikov Vaccine and Serum Research Institute.
Moscow
References
1. Purdy MA, Drexler JF, Meng XJ, Norder H, Okamoto H, Van der Poel WHM, et al. ICTV Virus Taxonomy Profile: Hepeviridae 2022. J Gen Virol. 2022 Sep;103(9). DOI: https://doi.org/10.1099/jgv.0.001778
2. Rein DB, Stevens GA, Theaker J, Wittenborn JS, Wiersma ST. The global burden of hepatitis E virus genotypes 1 and 2 in 2005. Hepatology. 2012 Apr;55(4):988-997. DOI: https://doi.org/10.1002/hep.25505
3. Li B, Wu H, Miao Z, Hu L, Zhou L, Lu Y. Codon Usage of Hepatitis E Viruses: A Comprehensive Analysis. Front Microbiol. 2022 Jun 21;13:938651. DOI: https://doi.org/10.3389/fmicb.2022.938651
4. Marano G, Vaglio S, Pupella S, Facco G, Bianchi M, Calizzani G, et al. Hepatitis E: an old infection with new implications. Blood Transfus. 2015 Jan;13(1):6-17. DOI: https://doi.org/10.2450/2014.0063-14
5. Aslan AT, Balaban HY. Hepatitis E virus: Epidemiology, diagnosis, clinical manifestations, and treatment. World J Gastroenterol. 2020 Oct 7;26(37):5543-5560. DOI: https://doi.org/10.3748/wjg.v26.i37.5543
6. Meng XJ. Hepatitis E virus: animal reservoirs and zoonotic risk. Vet Microbiol. 2010 Jan 27;140(3-4):256-265. DOI: https://doi.org/10.1016/j.vetmic.2009.03.017
7. Pavio N, Meng XJ, Renou C. Zoonotic hepatitis E: animal reservoirs and emerging risks. Vet Res. 2010 Nov-Dec;41(6):46. DOI: https://doi.org/10.1051/vetres/2010018
8. Vina-Rodriguez A, Schlosser J, Becher D, Kaden V, Groschup MH, Eiden M. Hepatitis E virus genotype 3 diversity: phylogenetic analysis and presence of subtype 3b in wild boar in Europe. Viruses. 2015 May 22;7(5):2704-2726. DOI: https://doi.org/10.3390/v7052704
9. Zhavoronok SV, Karpov IA, Mikhaylov MI, Arabey AA, et al. Epidemiological and epizootic intensity of HEV in Belarus. Infectious Diseases: News, Opinions, Training. 2019;8(1):11-22. (In Russ.). DOI: https://doi.org/10.2441l/2305-3496-2019-11001
10. Davydov VV, Zhavoronok SV, Zadora IS, Novak LV, Karpenko FN, Levandovskaya OV, et al. Prevalence of antibodies to the hepatitis E virus in the blood of belarusian donors. Medical Journal. 2022;(4):53-59. (In Russ.). DOI: https://doi.org/10.51922/1818-426X.2022.4.53
11. Krumova-Valcheva GL, Di Bartolo I, Smith RP, Gyurova E, Mateva G, Milanov M, et al. Detection of HEV RNA Using One-Step Real-Time RT-PCR in Farrow-to-Finish Pig Farms in Bulgaria. Pathogens. 2023;12(5):673. DOI: https://doi.org/10.3390/pathogens12050673
12. Pischke S, Wedemeyer H. Hepatitis E virus infection: multiple faces of an underestimated problem. J Hepatol. 2013 May;58(5):1045-1046. DOI: https://doi.org/10.1016/j.jhep.2012.12.013
13. Alatortseva GI, Sidorov AV, Nesterenko LN, Luhverchik LN, Dotsenko VV, Amiantova II, et al. Development of hepatitis E 3 genotype recombinant protein capsid of: cloning, expression, purification, evaluation of the antigenic properties. Journal of microbiology, epidemiology and immunobiology. 2019;96(1):10-17. (in Russ.). DOI: http://doi.org/10.36233/0372-9311-2019-1-10-17
14. Alatortseva GI, Sidorov AV, Nesterenko LN, Luhverchik LN, Milovanova AV, Ammur YI, et al. Obtaining the recombinant ORF3 protein of hepatitis Е genotype 3 and evaluation of its antigenic properties. Journal of microbiology, epidemiology and immunobiology, 2018; 95(5):46-53. (in Russ.). DOI: http://doi.org/10.36233/0372-9311-2018-5-46-53
Supplementary files
Review
For citations:
Zadora I.S., Zhavoronok S.V., Alatortseva G.I., Shcherban A.I., Pritvorova L.N., Nesterenko L.N., Davydov V.V., Anisko L.A., Rogacheva T.A., Bayur N.V., Shchuka N.V., Mytko Yu.A., Simirsky V.V., Mikhailov M.I., Zverev V.V. Development and evaluation of an enzyme immunoassay for diagnosis of acute viral hepatitis E. Health and Ecology Issues. 2025;22(2):35-45. (In Russ.) https://doi.org/10.51523/2708-6011.2025-22-2-04
Views:
8
Email this article 