DEVELOPMENT OF A DOMESTIC ENZYME IMMUNE TEST SYSTEM FOR DETECTION OF ANTI-HEV IGM IN BLOOD SERUM
Abstract
Background. Though the Republic of Belarus does not belong to countries endemic for viral hepatitis E, numerous studies have proved that hepatitis E virus (HEV) actively circulates among humans and animals. However, the circulation is latent that makes it difficult to diagnose the infection in a timely manner. Objective. To develop and evaluate the quality of a domestic ELISA test system for the detection of anti-HEI IgM. Material and methods. 96-well plates, recombinant proteins ORF2 and ORF3 of the 3rd HEV genotype, conjugate of monoclonal antibodies to human IgM with horseradish peroxidase, solutions for dilution of serums and conjugate containing bovine serum albumin. Results. The recommended concentrations of recombinant polypeptides ORF2 and ORF3 of hepatitis E virus genotype 3 for sorption are 2 and 1 μg/ml, respectively. The optimum dilution of the conjugate is 1:13 000. False positive and false negative results were not detected, sensitivity and specificity being at least 99%. The mean value for coefficient of variation within one plate is 6.4%, within two plates is 11.3%. Conclusions. The characteristics of the developed national test system comply with the recommended standards, thus indicating the possibility of its implementation into clinical laboratory diagnostics.
References
Kumar A, Beniwal M, Kar P, Sharma JB, Murthy NS. Hepatitis E in pregnancy. Int J Gynecol Obstet. 2004;85(3):240-244. https://doi.org/10.1016/j.ijgo.2003.11.018.
Nimgaonkar I, Ding Q, Schwartz RE, Ploss A. Hepatitis E virus: advances and challenges. Nat Rev Gastroenterol Hepatol. 2018;15(2):96-110. https://doi.org/10.1038/nrgastro.2017.150.
Gorski I, Babić I, Bingulac-Popović J, Topić-Šestan P, Jagnjić S, Jemeršić L, Prpić J, Jukić I. Prevalence of HEV RNA in Croatian blood donors. Transfus Clin Biol. 2023;30(2):244-248. https://doi.org/10.1016/j.tracli.2023.01.005.
Nagashima S, Takahashi M, Kobayashi T, Tanggis Nishizawa T, Nishiyama T, Primadharsini PP, Okamoto H. Characterization of the Quasi-Enveloped Hepatitis E Virus Particles Released by the Cellular Exosomal Pathway. J. Virol. 2017;91(22):e00822-17. https://doi.org/10.1128/JVI.00822-17.
Pallerla SR, Harms D, Johne R, Todt D, Steinmann E, Schemmerer M, Wenzel JJ, Hofmann J, Shih JWK, Wedemeyer H, C-Thomas Bock, Thirumalaisamy P Velavan. Hepatitis E virus infection: Circulation, molecular epidemiology, and impact on global health. Pathogens. 2020;9(10):856. https://doi.org/10.3390/pathogens9100856.
Le Blanc D, Poitras E, Gagné MJ, Ward P, Houde A. Hepatitis E virus load in swine organs and tissues at slaughterhouse determined by real-time RT-PCR. Int J Food Microbio. 2010;139(3):206-209. https://doi.org/10.1016/j.ijfoodmicro.2010.02.016.
Tsyrkunov VM, Davydov VV, Zhavoronok SV, CHernyak LK, Babenko AS, Marchuk SI, Gasich EL, Zadora IS. Kliniko-jepidemiologicheskaja i molekuljarno- geneticheskaja harakteristika pervogo sluchaja ostrogo gepatita E v Grodnenskom regione [Clinical-epidemiological and molecular-genetic characteristics of the first case of acute hepatitis Е in the Grodno region]. Gepatologija i Gastrojenterologija [Hepatology and Gastroenterology]. 2022;6(2):115-22. https://doi.org/10.25298/2616-5546-2022-6-2-115-122. https://elibrary.ru/ykoyzw. (Russian).
Alatortseva GI, Sidorov AV, Nesterenko LN, Lukhverchik LN, Amiantova II, Dotsenko VV, Vorobev DS, Ammur YI, Zhukina MV, Bajyzbekova DA, Mikhajlov MI, Potemkin IA, Kyuregyan KK, Nurmatov ZS, Nurmatov AZ, Kasymov OT, Zhavoronok SV, Krasochko PA, Zverev VV, inventors; Mechnikov Research Institute of Vaccines and Sera, assignee. Rekombinantnyj belok, soderzhashhij antigenno-znachimye fragmenty belkov virusa gepatita E, ispol’zuemyj v test-sistemah dlja serodiagnostiki gepatita E (varianty) [Recombinant protein containing antigen significant fragments of hepatitis E virus proteins, used in test systems for hepatitis E (embodiments)]. RU patent 2711907 C2. 2020 Jan 23. edn: NBFICC. (Russian).
Surjit M, Varshney B, Lal SK. The ORF2 glycoprotein of hepatitis E virus inhibits cellular NF-kappa B activity by blocking ubiquitination mediated proteasomal degradation of Ikappa Balpha in human hepatoma cells. BMC Biochem. 2012;13:7. https://doi.org/10.1186/1471-2091-13-7.
Chandra V, Holla P, Ghosh D, Chakrabarti D, Padigaru M, Jameel S. The hepatitis E virus ORF3 protein regulates the expression of liver-specific genes by modulating localization of hepatocyte nuclear factor 4. PLoS One. 2011;6(7):e22412. https://doi.org/10.1371/journal.pone.0022412.
