Journal for Veterinary Medicine, Biotechnology and Biosafety
Volume 2, Issue 1, May 2016, Pages 11–15
ISSN 2411-3174 (print version) ISSN 2411-0388 (online version)
THE VALIDATION OF ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DIAGNOSIS LEPTOSPIROSIS AMONG DOGS, PIGS AND CATTLE
Pyskun A. V. 1, Spiridonov V. G. 2, Ukhovskyi V. V. 1, Rybalchenko D. Y. 2, Homenko Y. V. 2
1 Institute of Veterinary Medicine, National Academy of Agrarian Sciences of Ukraine, Kyiv, Ukraine, e-mail: email@example.com
2 Ukrainian Laboratory of Quality and Safety of Agricultural Products, Kyiv, Ukraine
Download PDF (print version)
Citation for print version: Pyskun, A. V., Spiridonov, V. G., Ukhovskyi, V. V., Rybalchenko, D. Y. and Homenko, Y. V. (2016) ‘The validation of enzyme-linked immunosorbent assay for diagnosis Leptospirosis among dogs, pigs and cattle’, Journal for Veterinary Medicine, Biotechnology and Biosafety, 2(1), pp. 11–15.
Download PDF (online version)
Citation for online version: Pyskun, A. V., Spiridonov, V. G., Ukhovskyi, V. V., Rybalchenko, D. Y. and Homenko, Y. V. (2016) ‘The validation of enzyme-linked immunosorbent assay for diagnosis Leptospirosis among dogs, pigs and cattle’, Journal for Veterinary Medicine, Biotechnology and Biosafety. [Online] 2(1), pp. 11–15. Available at: http://jvmbbs.kharkov.ua/archive/2016/volume2/issue1/oJVMBBS_2016021_011-015.pdf
Summary. This study was aimed 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 to all serogroups of Leptospira that are recommended for the diagnosis of this zoonoses in Ukraine (high sensitivity and specificity); 2) minimization of the risk of laboratory-acquired infection at the preparation of reagents for ELISA and its conducting. The article presents the results of validation of ELISA for the diagnosis of leptospirosis among dogs, pigs, and cattle. During the validation, several technological stages were conducted. The first stage was carried out by selection and determination of the optimal concentration and titers of the main components of ELISA (antigen, enzyme conjugate, enzyme substrate, and blocking substance), and the second stage — by statistical analysis of the results after testing ELISA on the reference panel of sera blood samples. Established that the sensitivity of the developed ELISA test is less than its specificity (89.8% against 96.7%), but the general efficiency of this method is high and equals 93.6%.
Keywords: leptospirosis, microscopic agglutination test, enzyme-linked immunosorbent assay, validation, titer
Adler, B. and la Peсa Moctezuma, A. de (2010) ‘Leptospira and Leptospirosis’, Veterinary Microbiology, 140(3–4), pp. 287–296. http://dx.doi.org/10.1016/j.vetmic.2009.03.012
Bolin, C. A. (2008) Chapter 2.1.9. Leptospirosis. In: Manual of diagnostic tests and vaccines for terrestrial animals. 6th ed. Vol. 2. Available at: http://web.oie.int/eng/normes/MMANUAL/2008/pdf/2.01.09_LEPTO.pdf
Bomfim, M. R. Q., Ko, A. and Koury, M. C. (2005) ‘Evaluation of the recombinant LipL32 in enzyme-linked immunosorbent assay for the serodiagnosis of Bovine Leptospirosis’, Veterinary Microbiology, 109(1–2), pp. 89–94. http://dx.doi.org/10.1016/j.vetmic.2005.05.002
Ivanska, N. V., Kucheriavenko, O. O.,
Kucheriavenko, O. O., Rezunenko, Y. V. and
Hanova, L. O. (2003) Practical guide to work with the ELISA test
system for the detection of antibodies against Leptospira
‘ELISA-Leptospirosis-cattle’ [Praktychnyi posibnyk po roboti z
imunofermentnoiu test-systemoiu dlia vyiavlennia antytil proty leptospir
‘IFA-leptospiroz-VRKh’]. Edited by
M. Ya. Spivak. Kyiv:
Available at: http://diaproph.com.ua/pdf/metodichky/ua/10_ifa_leptospiroz_kpc.pdf. [in Ukrainian].
Kurstak, E. (1985) ‘Progress in enzyme immunoassays: Production of reagents, experimental design, and interpretation’, Bulletin of the World Health Organization, 63(4), pp. 793–811. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2536367/pdf/bullwho00087-0162.pdf
Levett, P. N. (2001) ‘Leptospirosis’, Clinical Microbiology Reviews, 14(2), pp. 296–326. http://dx.doi.org/10.1128/cmr.14.2.296-326.2001
Murray, G. L., Srikram, A., Hoke, D. E., Wunder, E. A., Henry, R., Lo, M., Zhang, K., Sermswan, R. W., Ko, A. I. and Adler, B. (2008) ‘Major surface protein LipL32 is not required for either acute or chronic infection with Leptospira interrogans’, Infection and Immunity, 77(3), pp. 952–958. http://dx.doi.org/10.1128/iai.01370-08
Pyskun, A. V. (2015) ‘Selection of optimal conjugate for the enzyme-linked immunosorbent assay against Leptospirosis’ [Pidbir optymalnoho koniuhatu dlia postanovky imunofermentnoho analizu na leptospiroz], Veterinary Biotechnology [Veterynarna Biotekhnolohiia], 26, pp. 156–163. Available at: http://vetbiotech.kiev.ua/volumes/JRN26/23.pdf [in Ukrainian].
Sharma, R., Tuteja, U., Khushiramani, R., Shukla, J. and Batra, H. V. (2007) ‘Application of rapid dot-ELISA for antibody detection of Leptospirosis’, Journal of Medical Microbiology, 56(6), pp. 873–874. http://dx.doi.org/10.1099/jmm.0.46926-0
Tokuda, H. (2009) ‘Biogenesis of outer membranes in Gram-negative bacteria’, Bioscience, Biotechnology and Biochemistry, 73(3), pp. 465–473. http://dx.doi.org/10.1271/bbb.80778
Wautkins, S. A. and Zochowski, W. J. (1990) ‘Leptospira’, in Wreghitt, T. G. and Morgan-Capner, P. (eds.) ELISA in the clinical microbiology laboratory. London: Public Health Laboratory Service, pp. 225–237. ISBN: 9780901144249