Bulletin "Veterinary biotechnology"

Veterynarna biotehnologija – Veterinary biotechnology, 2016, 28, 142-149 [in Ukrainian].

LOZHKINA O.V., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., MARCHUK O.T., e-mail:This email address is being protected from spambots. You need JavaScript enabled to view it.PAVLUNKO V.G., e-mail:This email address is being protected from spambots. You need JavaScript enabled to view it.

State Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise

VERIFICATION OF ELISA METHOD FOR DIAGNOSIS SPONGIFORM ENCEPHALOPATHY OF CATTLE

Introduction. At the end of the last century, humanity faced the problem of prion infections. Spongiform encephalopathy (SE) in cattle, which in 1985 began to spread massively in the UK, and and later in Europe, causing major economic losses and social disruption.

In 1996, it was found that the BSE pathogen can infect people, causing a new variant Creutzfeldt-Jakob disease. People infected by eating meat products from subclinical sick animals.

Today in the world there is a tendency of reducing the number of cases of SE cow.

For researches on SE of cow in Ukraine it was purchased test kit HerdChek BSE-Scrapie Antigene - for ELISA to detect antigen of bovine spongiform encephalopathy (BSE) - Skrepi (EIA). This test kit was validated in the IDEXX company laboratory.

To establish the possibility of using this test system in a laboratory of the State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise it was necessary to verify the method.

The goal of the work. Verification of ELISA method for the diagnosis of bovine spongiform encephalopathy.

Materials and methods of research. The work was carried out on the basis of research pathomorphological department of the State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise.

For the verification method we used following material:

  • 50 samples of bovine brain (obex), imported from Europe, which came to SSRILDVSE for diagnostic tests according to p. 7.8. Instructions for diagnosis, prevention and control of spongiform encephalopathy in cattle.
  • negative samples – 10 samples of bovine brain (obex) that previously tested at the Research Pathomorphologically Department immunoassay method.
  • positive samples (reference material) - brain homogenates cattle positive for TSE provided by the National Veterinary Laboratory of Lithuania, Vilnius.

Interpretation of the results was performed using the «Magellan for F50».

The verification method is performed using a test kit HerdChek BSE-Scrapie Antigen (IDEXX) and equipment for ELISA. The study was conducted according to the guidelines to the test kit.

After the test was carried out calculations of sensitivity, specificity and reproducibility of the method. Determine the coefficient of variation (CV) between holes one plates.

Results of research and discussion. At the first stage of sampling was performed, using disposable syringe 0.30 g were taken (± 0.05 g) of the nervous tissue of the left or right side of the brain stem (obex). Selected tissue was placed in a test tube for crushing and tissue homogenization was performed.

After homogenization of samples brought to the dies, and conducted trials under guidance instructions to the test kit. In the verification method obtained the following results:

  • the sensitivity of the method: M = 2 / (2 + 0) × 100% = 100%
  • the specificity of the method: C = 10 / (10 + 0) × 100% = 100%
  • the reproducibility of the method: σ = √ 0,00006806 / (9-1) = 0,00291
  • the coefficient of variation: CV = 0,00291 / 0,045 × 100% = 6.47%

In order to monitoring BSE of cattle in Ukraine during 2012, 2013 and 2014 studied 20,061 samples of bovine brain enzyme imunoadsorbtsiyi by using a test set HerdChek BSE-Scrapie Antigen (IDEXX). As a result of investigations the BSE have not found.

Conclusions and prospects for further research:

  1. Test kit HerdChek BSE-Scrapie Antigen (IDEXX) has high diagnostic sensitivity (100%) and specificity (100%).
  2. Recommend enzymatic immunoadsorption method when using test kits HerdChek BSE-Scrapie Antigen (IDEXX) for monitoring research on TSE of cattle.

Keywords: spongiform encephalopathy, cattle, ELISA method, verification.

REFERENCES

  1. Verbic'kij, P.І. (2005). Gubchastopodіbna encefalopatіja velikoї rogatoї hudobita іnshі prіonnі іnfekcії [Spongiformencephalopathyofcattleandotherprioninfection]. Kіiv: Vetіnform [in Ukrainian].
  2. Will R.G. Ironside J.W.; Zeidler M.; Cousens S.N.; Estibeiro K. & Alperovitch A. et al (1996). A new variant of Creutzfeldt-Jakob Disease in the UK. Lancet, 347 (9006), 921-925.
  3. Animal Health in the World. BSE situation in the world and annual incidence rate. www.oie.int.Retrieved from http://www.oie.int/animal-health-in-the-world/bse-specific-data/number-of-cases-in-the-united-kingdom/.
  4. Animal Health in the World. BSE situation in the world and annual incidence rate. www.oie.int. Retrieved from http://www.oie.int/animal-health-in-the-world/bse-situation-in-the-world-and-annual-incidence-rate/10-13-number-of-reported-cases-worldwide-excluding-the-united-kingdom-copy-1/.
  5. Іnstrukcіja shhodo dіagnostiki, profіlaktiki ta borot'bi z gubchastopodіbnoju encefalopatієju velikoї rogatoї hudobi [Instructions for diagnosis, prevention and control of spongiform encephalopathy in cattle]. www.rada.gov.ua. Retrieved from http://zakon2.rada.gov.ua/laws/show/z0897-02 [in Ukrainian].
  6. The Fitness for Purpose of Analytical Methods. – A Laboratory Guide to Method Validation and Related Topics. www.eurachem.org. Retrieved from https://www.eurachem.org/images/stories/Guides/pdf/MV_guide_2nd_ed_EN.pdf.
  7. The FEM Microbiology Action Team. FEM Document Number 2009-01 (2009). The EPA Forum on Environm ental Measurements (FEM). Method Validation of U.S. Environmental Protection Agency Microbiological Methods of Analysis. www.epa.gov. Retrieved from http://www.readbag.com/epa-fem-pdfs-final-microbiology-method-guidance-110409.

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