Development of Enzyme-Immunoassay for Bacillus anthracis Detection
https://doi.org/10.21055/0370-1069-2018-3-78-82
Abstract
Objective of the study was to develop enzyme-immunoassay test-kit for the detection of Bacillus anthracis spores. Materials and methods. Microbial cultures from the State Collection of Microorganisms at the premises of Affiliated Branch of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation and BALB/c mice were used in the research. Hybridization of B-lymphocytes with SP2/0-Ag14 myeloma cells was performed according to G. Kohler and C. Milstein procedure in De St. Fazekas and P. Scheidegger modification. Hybridomas were cultured in the peritoneal cavity of BALB/c mice. Ascitic fluids were isolated from mice, precipitated with ammonium sulfate and purified by means of ion-exchange chromatography for preparation of monoclonal antibodies. Specific activity of hybridoma’s supernatants, ascitic fluids, purified monoclonal antibodies was studied by «sandwich» ELISA. Specific components of test-kit were lyophilized in suitable cryoprotective medium. Results and conclusions.We have obtained new hybrid cell lines producing specific monoclonal antibodies against Bacillus anthracisspore antigens and ascitic fluids from which immunoglobulins were isolated. Optimum combinations of monoclonal antibodies as a sensitizer and a component of immunoperoxidase conjugates have been selected. Monoclonal antibodies 272E10G1-272F7A10 provide the highest sensitivity of ELISA for the detection of anthrax microbe spore antigens. Our enzyme-immunoassay test allows for identification of Bacillus anthracis spores in concentrations up to 5,0·105 spores per milliliter. No cross reaction with closely related saprophytes and other heterologous microorganisms in concentrations of 1,0·108 CFU per milliliter is observed.
About the Authors
D. V. Pechenkin
Branch of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation, Kirov
Russian Federation
Russian Federation
O. O. Fomenkov
Branch of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation, Kirov
Russian Federation
Russian Federation
A. V. Eremkin
Branch of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation, Kirov
Russian Federation
Russian Federation
G. D. Elagin
Branch of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation, Kirov
Russian Federation
Russian Federation
G. V. Kuklina
Branch of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation, Kirov
Russian Federation
Russian Federation
G. V. Baramzina
Branch of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation, Kirov
Russian Federation
Russian Federation
S. S. Ipatov
Branch of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation, Kirov
Russian Federation
Russian Federation
References
1. Yeremenko E.I., Ryazanova A.G., Buravtseva N.P. [Current situation on anthrax in Russia and in the world. Main trends and features]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2017; 1:65–1. DOI: 10.21055/0370-1069-2017-1-65-71.
2. Ketti D., editor. [Antibodies. Methods]. Moscow: “Mir”, 1991. Vol. 1. 287 p.
3. Netrusov A.I, editor. [Workshop on microbiology: Textbook for students of Higher Educational Institutions]. Moscow: «Academia»; 2005. 608 p.
4. Onischenko G.G., Kozhukhov V.V., editors. [Anthrax: relevant problems of elaboration of medical means of protection]. Moscow: Open Joint Stock Company «Publishing house «Medicine»; 2010. 424 p.
5. Onishchenko G.G., Kutyrev V.V., editors. [Laboratory Diagnostics of Dangerous Infectious Diseases. Practice guidelines]. M.: CJSC «Shiko»; 2013. 560 p.
6. Pavlov D.L., Onuchina N.V., Kuznetsovsky A. V., Fomenkov O.O., Tumanov A. S. [Results of the study of biological and genetic properties of anthrax isolates obtained during epizooty, 2016 in the Yamal-Nenets Autonomous District]. Vestnik Voisk Radiotsionnoi, Khimicheskoi i Biologicheskoi Zashchity. 2017; 1(1):23–32.
7. Tereshkina N.E., Devdariani Z.L. [Present status of the anthrax pathogen immunodiagnosis]. Problemy Osobo Opasnykh Infektsii [Problems of Particularly Dangerous Infections]. 2009; 1(95): 44–8.
8. Bartholomew R.A., Ozanich R.M., Arce J.S., Engelmann H.E., Heredia-Langner A., Hofstad B.A., Hutchison J.R., Jarman K., Melville A.M., Victry K.D., Bruckner-Lea C.J. Evaluation of Immunoassays and General Biological Indicator Tests for Field Screening of Bacillus anthracis and Ricin. Health Secur. 2017; 15:81–95. DOI: 10.1089/hs.2016.0044.
9. Fazekas de St. Groth S., Scheidegger D. Production of monoclonal antibodies: strategy and tactics. J. Immunol. Meth. 1980; 35:1–21.
10. Köhler G., Milstein C. Continuous culture of fused cells secreting antibody of predefined specificity. Nature. 1975; 256(5517):495–7.
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. Nakane P.K., Kawaoi A. Peroxidase-labeled antibody a new method of conjugated. J. Histochem. Cytochem. 1974; 22(12):1084–1091.
13. Wang D.-B., Yang R., Zhang Z.-P., Bi L.-J., You X.Y., Wei H.P., Zhou Y.F., Yu Z., Zhang X.E. Detection of B. anthracisspores and vegetative cels with the same monoclonal antibodies. PLoS ONE. 2009; 4(11): e7810. DOI: 10.1371/journal.pone.0007810.
Review
For citations:
Pechenkin D.V., Fomenkov O.O., Eremkin A.V., Elagin G.D., Kuklina G.V., Baramzina G.V., Ipatov S.S. Development of Enzyme-Immunoassay for Bacillus anthracis Detection. Problems of Particularly Dangerous Infections. 2018;(3):78-82. (In Russ.) https://doi.org/10.21055/0370-1069-2018-3-78-82
Views:
1124
Email this article 