Bulletin "Veterinary biotechnology"

Veterynarna biotehnologija – Veterinary biotechnology, 2019, 34, 97-107 [in Ukrainian]. https://doi.org/10.31073/vet_biotech34-12

PYSKUN A.V.1, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., UKHOVSKYI V.V.2, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., ALIEKSEIEVA G.B.1, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., SPIRIDONOV V.G.2, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., PYSKUN O.O.1e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

1 State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise

2 Institute of Veterinary Medicine NAAS

THE DEVELOPMENT OF ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR DIAGNOSTICS OF LEPTOSPIROSIS IN ANIMALS

Introduction. Leptospirosis is the most wide spread zoonosis worldwide, which is present in all continents except Antarctica and evidence for the carriage of Leptospira has been found in virtually all mammalian species examined. The wide spectrum of symptoms confuses the clinical diagnosis and makes it undependable. The laboratory diagnosis of leptospirosis, a prerequisite for treatment, is usually achieved either by isolation of the causative organisms or by serological evidence indicating recent infection. The microscopic agglutination test (MAT) is the reference test for diagnosis and detects antibodies at serovar levels. MAT has many advantages, but there are significant deficiencies. The maintenance of stock cultures and use of live organisms creates a risk of laboratory-acquired infection. Therefore, several methods have been developed for use in diagnosis of leptospirosis as an alternative to MAT, of which IgM ELISA is the most promising and detects genus-specific antibodies.

The goal of the work was to develop a modification of ELISA for the diagnosis of leptospirosis in animals that would correspond to the following requirements: 1) ability to detect antibodies against serogroups of Leptospira, that are recommended for the diagnosis of this zoonoses in Ukraine (to have high sensitivity and specificity); 2) minimize the risk of laboratory-acquired infection at preparation of reagents for ELISA and its conducting.

Materials and methods of research. During the validation, we have conducted several technological stages. The first of them was performed by selection and determination the optimal concentration and titers of ELISA main components (antigen, enzyme conjugate, enzyme substrate and blocking substance) and the second was carried out a statistical analysis of the results after testing ELISA on the reference panel of sera blood samples. To perform this research, ELISA was carried out with field samples of blood sera from sick and healthy for Leptospirosis dogs, pigs and cattle and seven reference sera (OIE), that were obtained from the Royal Tropical Institute, Amsterdam.

Results of research and discussion. As the antigen was used recombinant protein LipL 32, which is a modified compound one of the major outer membrane lipoprotein of pathogenic Leptospira species. To determine the optimal dose of sensitizing antigen, conducted its sorption within 0.5–4 μl volume. At the same time we have tested substances that eliminate the not specific interaction of antibodies with antigen – edible gelatin and skimmed milk powder (respectively, 0.5% and 5% solutions). Analysis of titration showed, that the optimum amount of LipL 32 in both cases was 1 μl volume. However, was registered high background reaction in all investigated wells of microplate when dilution of blood sera samples by phosphate buffer with 0.5% gelatin solution. When a 5% solution of skimmed milk was used, the indicators of optical density in negative controls were much smaller, and correlation coefficients of positive and negative sera were more. By analyzing the results of conjugate titration, the highest correlation coefficients of optical density between positive and negative samples of blood sera were registered in its titer 1/500 and dilution of sera 1/2.5 (40 μl volume). A comparison enzyme substrates showed, that the indicators of optical density in positive samples of blood sera by MAT were significantly higher by using TMB solution, than indicators by using OFD chromogen, respectively, 0.836±0.089 o.d. against 0.601±0.066 o.d. The difference of optical values in both cases was significant (р˂0.05). At the same time, the optical indicators of negative blood sera samples by MAT significantly were not different in both cases (0.098±0.002 o.d. against 0.085±0.0017 o.d.). The last technological stage was carried out a statistical analysis of the results after testing ELISA on the reference panel of sera blood samples. Established that the sensitivity of the developed ELISA is less than its specificity (89.8% against 96.7%), but overall efficiency of this method is high and equals 93.6%.

Keywords: Leptospirosis, microscopic agglutination test, enzyme-linked immunosorbent assay, validation, titer.

REFERENCES

  1. Adler, B., & Moctezuma, A.P. (2010). Leptospira and leptospirosis. Veterinary Microbiology, 27, 287-296.
  2. Ukhovskyi, V.V., Kucheryavenko, O.O., & Pyskun, A.V. (2013). Porivnyalna harakterystyka suchasnyh metodiv laboratornoyi diahnostyky leptospirozu s/h tvaryn ta perspektyva rozrobky dot-IFA z metoyu yiyi udoskonalennya [Comparative characteristics of modern methods laboratory diagnosis of leptospirosis farm animals and development perspective dot-ELISA for this improvement]. Byulleten «Veterinarna biotehnologiia» – Bulletin of Veterinary biotechnology, 23, 259-260 [in Ukrainian].
  3. Korniyenko, L.E., Busol, V.O., Nedosekov, V.V. et al. (2009). Hronichni infekcijni hvoroby tvaryn [Chronical infectious diseases of animals]. Bila Tserkva [in Ukrainian].
  4. Cinco, M., Balanzin, D., Banji, E. (1992). Evaluation of an immunoenzymatic test (ELISA) for the diagnosis of leptospirosis in Italy. European Journal of Epidemiology, 8, 677-682.
  5. Wautkins, S.A., & Zochowski, W.J. (1990). Leptospira in ELISA in the Clinical Microbiology Laboratory. London: Public Health Laboratory Service, 225-237.
  6. Kurstak, E. (1985). Progress in enzyme immunoassys: production of reagents, experimental design, and interpretation. Bulletin of the World Health Organization, 63 (4), 793-811.
  7. Ivanska, N.V., Kucheryavenko, O-yO., Kucheryavenko, O-rO. et al. (2003). Praktychnyj posibnyk po roboti z imunofermentnoyu test-systemoyu dlya vyznachennya antytil proty leptospir «IFA-leptospiroz-VRH» [Practical guide to working with the enzyme-linked immunosorbent assay for the detection of antibodies against Leptospira "ELISA-Leptospirosis-cattle"]. Kyiv [in Ukrainian].
  8. Tokuda, H. (2009). Biogenesis of outer membranes in Gram-negative bacteria. Bioscience, Biotechnology and Biochemistry, 73(3), 465-473.
  9. Murray, G.L., Srikram, A., Hoke, D.E. et al. (2009). Major surface protein LipL32 is not required for either acute or chronic infection with Leptospira interrogans. Infection and Immunity, 77(3), 952-958.
  10. Sharma, R., Tuteja, U., Khushiramani, R. et al. (2007). Application of rapid dot-ELISA for antibody detection of leptospirosis. Journal of Medical Microbiology, 56(6), 873-874.
  11. Bomfim, M.R., & Koury, M.C. (2005). Evaluation of the recombinant LipL32 in enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis. Veterinary Microbiology, 109, 89-94.
  12. Pyskun, A.V. (2015). Pidbir optymalnoho konyuhatu dlya postanovky imunofermentnoho analizu na leptospiroz [Selection of optimal conjugate for the enzyme-linked immunosorbent assay against Leptospirosis]. Byulleten «Veterinarna biotehnologiia» – Bulletin of Veterinary biotechnology, 26, 156-163 [in Ukrainian].

Download full text in PDF