Вопросы вирусологии. 2022; 67: 331-340
Тест-система на основе конкурентного иммуноферментного анализа для выявления антител к вирусу бешенства животных (Rhabdoviridae: Lyssavirus)
https://doi.org/10.36233/0507-4088-127Аннотация
Введение. Основным направлением профилактики бешенства является вакцинация домашних и диких плотоядных животных. Для рутинной оценки эффективности антирабической вакцинации Международным эпизоотическим бюро (МЭБ) рекомендован иммуноферментный анализ (ИФА). Актуальной задачей остаётся разработка модификаций ИФА с большей диагностической специфичностью (ДС) и чувствительностью (ДЧ).
Цель работы – конструирование и валидация тест-системы на основе конкурентного ИФА (кИФА) для выявления антител к вирусу бешенства (ВБ).
Материалы и методы. Разработку тест-системы проводили в соответствии с рекомендациями МЭБ. Определяли дозу ВБ для сенсибилизации планшетов, состав блокирующего буфера; оптимальные разведения компонентов; порядок интерпретации результатов ИФА.
Результаты. Повторяемость результатов кИФА в рамках одной лаборатории была удовлетворительной: коэффициент вариации – 7,95–13,61%, коэффициент детерминации (КД) между результатами реакции нейтрализации (FAVN) и кИФА – 0,988, p < 0,001. Нижний порог обнаружения антител составил менее 0,02 МЕ/мл. Разработанная тест-система не демонстрировала кросс-реактивности в отношении антител к вирусу чумы плотоядных, вирусу парагриппа, парвовирусу, коронавирусу, аденовирусу собак (I и II серотипа). При исследовании 137 сывороток крови собак ДС и ДЧ кИФА составили 83,1 и 94,9% соответственно, КД между результатами кИФА и FAVN – 0,968, p < 0,001. Обсуждение. Диагностикумы для определения уровня антител к ВБ на основе непрямого ИФА недостаточно чувствительны по сравнению с референс-тестами. Оптимальным решением являются варианты кИФА с применением биотинилированных антител. Разработанная тест-система по ДС и ДЧ не уступает коммерческой тест-системе кИФА BioPro ELISA Rabies Ab: ДС 66,7%, ДЧ 94,4%.
Заключение. Таким образом, разработанная тест-система на основе кИФА может быть использована для выявления антител к ВБ в сыворотке крови собак при оценке эффективности программ массовой вакцинации.
Список литературы
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16. Fontana D., Rodriguez M.C., Garay E., Russo S., Prieto C. Optimization and validation of a blocking ELISA for quantitation of anti-rabies immunoglobulins in multispecies sera. Appl. Microbiol. Biotechnol. 2020; 104(9): 4127–39. https://doi.org/10.1007/s00253-020-10490-6
17. Zhang S., Liu Y., Zhang F., Hu R. Competitive ELISA using a rabies glycoprotein-transformed cell line to semi-quantify rabies neutralizing-related antibodies in dogs. Vaccine. 2009; 27(15): 2108–13. https://doi.org/10.1016/j.vaccine.2009.01.126
18. Korimbocus J., Dehay N., Tordo N., Cano F., Morgeaux S. Development and validation of a quantitative competitive ELISA for potency testing of equine anti rabies sera with other potential use. Vaccine. 2016; 34(28): 3310–6. https://doi.org/10.1016/j.vaccine.2016.04.086
19. Muhamuda K., Madhusudana S.N., Ravi V. Development and evaluation of a competitive ELISA for estimation of rabies neutralizing antibodies after post-exposure rabies vaccination in humans. Int. J. Infect. Dis. 2007; 11(5): 441–5. https://doi.org/10.1016/j.ijid.2006.09.013
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21. Wasniewski M., Labbe A., Tribout L., Rieder J., Labadie A., Schereffer J.L., et al. Evaluation of a rabies ELISA as an alternative method to seroneutralisation tests in the context of international trade of domestic carnivores. J. Virol. Methods. 2014; 195: 211–20. https://doi.org/10.1016/j.jviromet.2013.10.021
22. Feyssaguet M., Dacheux L., Audry L., Compoint A., Morize J.L., Blanchard I., et al. Multicenter comparative study of a new ELISA, PLATELIA RABIES II, for the detection and titration of anti-rabies glycoprotein antibodies and comparison with the rapid fluorescent focus inhibition test (RFFIT) on human samples from vaccinated and non-vaccinated people. Vaccine. 2007; 25(12): 2244–51. https://doi.org/10.1016/j.vaccine.2006.12.012
23. Welch R.J., Anderson B.L., Litwin C.M. An evaluation of two commercially available ELISAs and one in-house reference laboratory ELISA for the determination of human anti-rabies virus antibodies. J. Med. Microbiol. 2009; 58(Pt. 6): 806–10. https://doi.org/10.1099/jmm.0.006064-0
24. Cliquet F., McElhinney L.M., Servat A., Boucher J.M., Lowings J.P., Goddard T., et al. Development of a qualitative indirect ELISA for the measurement of rabies virus-specific antibodies from vaccinated dogs and cats. J. Virol. Methods. 2004; 117(1): 1–8. https://doi.org/10.1016/j.jviromet.2003.12.001
Problems of Virology. 2022; 67: 331-340
Competitive ELISA test system for the detection of antibodies to the rabies virus in animals (Rhabdoviridae: Lyssavirus)
https://doi.org/10.36233/0507-4088-127Abstract
Introduction. The main approach to the rabies prevention is the vaccination of domestic and wild carnivores. For the routine evaluation the anti-rabies vaccination effectiveness, World Organization for Animal Health (OIE) recommends various enzyme-linked immunosorbent assays (ELISA).
The aim of the study was to design and validate a competitive ELISA (cELISA) test system for the detection of antibodies to the rabies virus (RABV).
Materials and methods. The development of the cELISA was carried out following the OIE recommendations.
Results. The repeatability of the cELISA results within one laboratory was satisfactory (coefficient of variation 7.95–13.61%). The coefficient of determination (CD) between the results of the virus neutralization reaction (FAVN) and cELISA was 0.988, p < 0.001. The lower threshold for antibody detection was less than 0.02 IU/ml. The cELISA did not demonstrate cross-reactivity against antibodies to canine distemper virus, parainfluenza virus, parvovirus, coronavirus, and canine adenovirus (types I and II). During the study of 137 dog blood sera, diagnostic specificity (DSp) and diagnostic sensitivity (DSe) for the cELISA were 83.1% and 94.9%, respectively, and CD between the cELISA and FAVN results was 0.968, p < 0.001.
Discussion. Indirect ELISA test systems for determining the level of antibodies to RABV are not sensitive enough compared to reference tests, unlike cELISA. The developed test system is not inferior for its DSp and DSe to the commercial cELISA BioPro ELISA Rabies Ab (DSp 66.7%, DSe 94.4%).
Conclusion. The developed cELISA test system can be used to detect antibodies to RABV in the blood serum of dogs for evaluating the effectiveness of mass vaccination programs.
References
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6. Mojžiš M., Korytár P., Jerg S. Development and validation of ELISA test for detection of rabies anti-glycoprotein antibodies. In: Proceedings of the International Conference on Rabies in the Americas (RITA XIX). Atlanta; 2008: 48–9.
7. Wasniewski M., Cliquet F. Evaluation of ELISA for detection of rabies antibodies in domestic carnivores. J. Virol. Methods. 2012; 179(1): 166–75. https://doi.org/10.1016/j.jviromet.2011.10.019
8. Wasniewski M., Almeida I., Baur A., Bedekovic T., Boncea D., Chaves L.B., et al. First international collaborative study to evaluate rabies antibody detection method for use in monitoring the effectiveness of oral vaccination programmes in fox and raccoon dog in Europe. J. Virol. Methods. 2016; 238: 77–85. https://doi.org/10.1016/j.jviromet.2016.10.006
9. Lobanova V.A., Klyukina V.I. Poluchenie komponentov test-sistemy na osnove konkurentnogo immunofermentnogo analiza dlya vyyavleniya antitel k virusu beshenstva. Veterinarnyi vrach. 2022; (2): 29–39. https://doi.org/10.33632/1998-698X.2022_29_39
10. Burns R., ed. Immunochemical Protocols. Totowa, NJ: Humana Press; 2005.
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16. Fontana D., Rodriguez M.C., Garay E., Russo S., Prieto C. Optimization and validation of a blocking ELISA for quantitation of anti-rabies immunoglobulins in multispecies sera. Appl. Microbiol. Biotechnol. 2020; 104(9): 4127–39. https://doi.org/10.1007/s00253-020-10490-6
17. Zhang S., Liu Y., Zhang F., Hu R. Competitive ELISA using a rabies glycoprotein-transformed cell line to semi-quantify rabies neutralizing-related antibodies in dogs. Vaccine. 2009; 27(15): 2108–13. https://doi.org/10.1016/j.vaccine.2009.01.126
18. Korimbocus J., Dehay N., Tordo N., Cano F., Morgeaux S. Development and validation of a quantitative competitive ELISA for potency testing of equine anti rabies sera with other potential use. Vaccine. 2016; 34(28): 3310–6. https://doi.org/10.1016/j.vaccine.2016.04.086
19. Muhamuda K., Madhusudana S.N., Ravi V. Development and evaluation of a competitive ELISA for estimation of rabies neutralizing antibodies after post-exposure rabies vaccination in humans. Int. J. Infect. Dis. 2007; 11(5): 441–5. https://doi.org/10.1016/j.ijid.2006.09.013
20. Servat A., Cliquet F. OIE Reference Laboratory for Rabies; WHO. Collaborative study to evaluate a new ELISA test to monitor the effectiveness of rabies vaccination in domestic carnivores. Virus. Res. 2006; 120(1-2): 17–27. https://doi.org/10.1016/j.virusres.2006.02.011
21. Wasniewski M., Labbe A., Tribout L., Rieder J., Labadie A., Schereffer J.L., et al. Evaluation of a rabies ELISA as an alternative method to seroneutralisation tests in the context of international trade of domestic carnivores. J. Virol. Methods. 2014; 195: 211–20. https://doi.org/10.1016/j.jviromet.2013.10.021
22. Feyssaguet M., Dacheux L., Audry L., Compoint A., Morize J.L., Blanchard I., et al. Multicenter comparative study of a new ELISA, PLATELIA RABIES II, for the detection and titration of anti-rabies glycoprotein antibodies and comparison with the rapid fluorescent focus inhibition test (RFFIT) on human samples from vaccinated and non-vaccinated people. Vaccine. 2007; 25(12): 2244–51. https://doi.org/10.1016/j.vaccine.2006.12.012
23. Welch R.J., Anderson B.L., Litwin C.M. An evaluation of two commercially available ELISAs and one in-house reference laboratory ELISA for the determination of human anti-rabies virus antibodies. J. Med. Microbiol. 2009; 58(Pt. 6): 806–10. https://doi.org/10.1099/jmm.0.006064-0
24. Cliquet F., McElhinney L.M., Servat A., Boucher J.M., Lowings J.P., Goddard T., et al. Development of a qualitative indirect ELISA for the measurement of rabies virus-specific antibodies from vaccinated dogs and cats. J. Virol. Methods. 2004; 117(1): 1–8. https://doi.org/10.1016/j.jviromet.2003.12.001
