Development of enzyme immunoassay for detecting I and II types of shiga-like toxins

Cover Page

Cite item

Full Text

Abstract

Introduction. The aim of the work was development of enzyme immunoassay for detecting I and II types of shiga-like toxins and assessment of it diagnostic properties. 

Materials and methods. For the research, we used hybridomas producing monoclonal antibodies to shiga-like toxins of types I and II, obtained at the branch of the Federal State Budgetary Institution “48 Central Research Institute” of the Ministry of Defense of Russian Federation (Kirov); BALB/c mice; shiga-like toxins of types I and II. Hybridoma cells were cultured in culture flasks and in the peritoneal cavity of BALB/c mice. Monoclonal antibodies were isolated from ascitic fluids by precipitation with a saturated solution of ammonium sulfate, followed by purification by ion exchange chromatography. The obtained preparations of monoclonal antibodies were used to develop enzyme immunoassay for the detection of shiga-like toxins of types I and II. Specific components of enzyme immunoassay were freeze-dried in a protective environment.

Results. As a result of research, preparative quantities of monoclonal antibodies against I and II types of shiga-like toxins were obtained and purified; selection of monoclonal antibodies for sorption on the solid phase and for the synthesis of immunoperoxidase conjugates was carried out. 

Conclusion. Experimental enzyme immunoassay allowing to identify 1 ng/ml I and II types of shiga-like toxins in «sandwich»-ELISA was developed.

About the authors

Galina Viktorovna Kuklina

Affiliated Branch of the Federal State Budgetary Institution «48th Central Research Institute» of the Ministry of Defense of the Russian Federation

Author for correspondence.
Email: noemail@neicon.ru
ORCID iD: 0000-0002-5429-6295

Кандидат биологических наук, младший научный сотрудник научно-исследовательского
отдела Филиала ФГБУ 48 ЦНИИ Минобороны РФ, 610000, г. Киров, Россия

Russian Federation

Denis Valerievich Pechenkin

Affiliated Branch of the Federal State Budgetary Institution «48th Central Research Institute» of the Ministry of Defense of the Russian Federation

Email: noemail@neicon.ru
ORCID iD: 0000-0001-8277-3573

Ph.D. in Medicine, Head of Research Department Affiliated Branch of the Federal State Budgetary Institution «48th Central Research Institute» of the Ministry of Defense of the Russian Federation, Kirov, 610000, Russian Federation, 610000, Kirov, Russia.

e-mail: 23527@mil.ru

Russian Federation

Sergei Sergeevich Ipatov

Affiliated Branch of the Federal State Budgetary Institution «48th Central Research Institute» of the Ministry of Defense of the Russian Federation

Email: noemail@neicon.ru
ORCID iD: 0000-0002-2881-4730

Младший научный сотрудник научно-исследовательского отдела Филиала ФГБУ 48 ЦНИИ Минобороны РФ, 610000, г. Киров, Россия.

Russian Federation

Andrei Valentinovich Eremkin

Affiliated Branch of the Federal State Budgetary Institution «48th Central Research Institute» of the Ministry of Defense of the Russian Federation

Email: noemail@neicon.ru
ORCID iD: 0000-0003-3133-1921

Кандидат биологических наук, начальник группы – заместитель начальника научно-исследовательского отдела Филиала ФГБУ 48 ЦНИИ Минобороны РФ, 610000, г. Киров, Россия.

Russian Federation

Aleksei Aleksandrovich Kytmanov

Affiliated Branch of the Federal State Budgetary Institution «48th Central Research Institute» of the Ministry of Defense of the Russian Federation

Email: noemail@neicon.ru
ORCID iD: 0000-0003-0277-2226

Кандидат биологических наук, научный сотрудник научно-исследовательского отдела Филиала ФГБУ 48 ЦНИИ Минобороны РФ, 610000, г. Киров, Россия.

Russian Federation

Oksana Victorovna Tikhvinskaya

Affiliated Branch of the Federal State Budgetary Institution «48th Central Research Institute» of the Ministry of Defense of the Russian Federation

Email: noemail@neicon.ru
ORCID iD: 0000-0001-8368-9648

Кандидат биологических наук, младший научный сотрудник научно-исследовательского отдела Филиала ФГБУ 48 ЦНИИ Минобороны РФ, 610000, г. Киров, Россия.

Russian Federation

References

  1. Мelton-Celsa A.R. Shiga toxin (Stx). Classification, structure, and Function. Microbiol Spectr. 2014; 2(2): 23. https://doi.org/10.1128/9781555818791.ch3
  2. Joseph A., Cointe A., Mariani Kurkdjian P. Shiga Toxin-Associated hemolytic uremic syndrome: А narrative review. Toxins (Basel). 2020; 12: 67. https://doi.org/10.3390/toxins12020067
  3. E. coli: Rapid Responce in a crisis. European Food Safety Authority. Available at: https://www.efsa.europa.eu/en/press/news/120711.
  4. Onishenko G., Dyatlov I., Svetoch N. Molecular genetic characteristics of shiga-toxins released during a food outbreak in St.Petersburg in 2013. Vestnik RAMN. 2015; 1: 70-80. (in Russian)
  5. Gyles C.L. Shiga toxin-producing Escherichia coli: An overview. J. Anim. Sci. 2007; 85: 45-62.
  6. Paletta A.C., Castro V.S., Conte-Junior C.A. Shiga toxin-producing and enteroaggregative Echerichia coli in animal, foods and humans: pathogenicity, mechanisms, detection methods, and epidemiology. Curr. Microbiol. 2019: 1-9. https://doi.org/10.1007/s00284-019-01842-1.
  7. Taylor М.С. Enterohaemorrhagic Escherichia coli and Shigella dysenteriae type 1-induced haemolytic uraemic syndrome. Pediatr. Nephrol. 2008; 23: 1425-31.
  8. Guidelines 4.2.2963-11 for laboratory diagnosis of diseases caused by Escherichia coli producing shiga toxins (STEC-cultures) and the detection of pathogens STEC-infections in food products. Moscow, 2011. Available at: https://www.docs.cntd.ru/document/1200094087. (in Russian)
  9. Laemmli U.K. Сleavage of structural proteins durimg the assembly of the head of bacteriophage T4. Nature. 1970; 227 (5259): 680-85.
  10. Szewczak A.A., Moore P.B., Chan Y.L. The conformation of the sarcin/ricin loop from 28S ribosomal RNA. Proc. Natl. Acad. Sci. USA. 1993; 90: 9581-85.
  11. Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951; 193(1): 265-75.
  12. D. Kettyu, ed. Antibodies. Methods. Vol. 1 [Antitela. Metody. Kn. 1]. Translated from English. Moscow: Mirbeau; 1991. (in Russian)
  13. Nakane P.K., Kawaoi A. Peroxidase-labeled antibody. A new method of conjugated. J. Histochem. Cytochem. 1974; 22(12): 1084-91.

Supplementary files

Supplementary Files
Action
1. JATS XML
Download

Copyright (c) 2021 Kuklina G.V., Pechenkin D.V., Ipatov S.S., Eremkin A.V., Kytmanov A.A., Tikhvinskaya O.V.


СМИ зарегистрировано Федеральной службой по надзору в сфере связи, информационных технологий и массовых коммуникаций (Роскомнадзор).
Регистрационный номер и дата принятия решения о регистрации СМИ: серия ПИ № ФС 77 - 81728 от 11 декабря 2013.