animalЭкология и животный мирEcology and Animal World2224-1647Институт экспериментальной ветеринарии им. С.Н. Вышелесскогоanimal-157Research ArticleСтатьиСоздание непрямого метода ИФА для выявления антител к вирусу чумы собакEstablishment of an indirect ELISA method for detecting canine distemper virus antibodiesЛайцинЛиLaiqingLi

Chairman of the board

Minsk

Guangzhou

ЦуйцуйЧенCuicuiChen

Project manager

Minsk

ХуанкуньЛянHuankunLiang

Quality manager

Minsk

Guangzhou

ЧжунШухайZhongShuhai

Head of research and development department

Minsk

Guangzhou

ЛеонтьевВ. Н.LeontievV. N.

Candidate of technical sciences

Minsk

ВойтовИ. В.VoitovI. V.

Doctor of technical sciences, professor

Minsk

Belarusian State Technological University ; Guangzhou Youdi Bio-technology Co., Ltd.BelarusBelarusian State Technological UniversityBelarus202411082024015964Copyright © Лайцин Л., Цуйцуй Ч., Хуанкунь Л., Чжун Ш., Леонтьев В.Н., Войтов И.В., 20242024Лайцин Л., Цуйцуй Ч., Хуанкунь Л., Чжун Ш., Леонтьев В.Н., Войтов И.В.Laiqing L., Cuicui C., Huankun L., Zhong S., Leontiev V.N., Voitov I.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://animal.belal.by/jour/article/view/157

Вирус чумы собак (CDV) может вызывать острые инфекционные заболевания у различных плотоядных животных. Целью данного исследования является разработка непрямого метода ИФА для обнаружения антител к CDV.

Используя очищенные рекомбинантные антигены CDV в качестве покрывающих агентов и после оптимизации условий, был разработан предварительный метод ELISA-детектирования антител, специфичных к CDV. Оптимальная концентрация рекомбинантного антигена CDV в покрытии при 2,0 мкг/мл, 100 мкл/лунка, нанесение покрытия на ночь при 4 °C, блокирование при температуре 4 °C в течение 4 часов, разведение сыворотки 1:200 и инкубация при температуре 37 °C в течение 1,5 часов или 2 часов либо 4 °C в течение ночи. Перекрестной реактивности с сывороткой, положительной по антителам к CPIV, CAV-1 и CCV, не наблюдалось, что указывает на хорошую специфичность. Коэффициент вариации как внутри, так и между партиями был ниже 5 %, а чувствительность обнаружения достигала 1:500.

Canine distemper virus (CDV) can cause acute infectious diseases in various carnivorous animals. This study aims to develop an indirect ELISA method for CDV antibody detecting.

Using the purified recombinant CDV antigens as coating agents, and after optimizing the conditions, a preliminary ELISA detection method for CDV specific antibodies was established. The optimal coating concentration of recombinant CDV antigen at 2,0 μg/mL 100 μL/well, overnight coating at 4 °C, blocking at 4 °C for 4 h, serum dilution at 1: 200 and incubation at 37 °C for 1,5 h or 2 h, or 4 °C overnight. No cross-reactivity was observed with CPIV, CAV-1, and CCV antibody-positive serum, indicating good specificity. Both intra-batch and inter-batch CVs were below 5 %, and the detection sensitivity reached 1:500.

непрямой ИФАвирус чумы собакантителометодинфекционные болезни животныхindirect ELISAcanine distemper virusantibodymethodanimal infectious diseasesReferencesMartella V., Elia G., Buonavoglia C.. Canine distemper virus. Vet Clin North Am Small Anim Pract. 2008;38(4):787-97, vii-viii. doi: 10.1016/j.cvsm.2008.02.007.Martella V., Elia G., Buonavoglia C.. Canine distemper virus. Vet Clin North Am Small Anim Pract. 2008;38(4):787-97, vii-viii. doi: 10.1016/j.cvsm.2008.02.007.Rendon-Marin S., da Fontoura Budaszewski R., Canal C.W., Ruiz-Saenz J. Tropism and molecular pathogenesis of canine distemper virus. Virol J. 2019;16(1):30. doi: 10.1186/s12985-019-1136-6.Rendon-Marin S., da Fontoura Budaszewski R., Canal C.W., Ruiz-Saenz J. Tropism and molecular pathogenesis of canine distemper virus. Virol J. 2019;16(1):30. doi: 10.1186/s12985-019-1136-6.Loots A.K., Mitchell E., Dalton D.L., Kotzé A., Venter E.H. Advances in canine distemper virus pathogenesis research: a wildlife perspective. J Gen Virol. 2017;98(3):311-321. doi: 10.1099/jgv.0.000666.Loots A.K., Mitchell E., Dalton D.L., Kotzé A., Venter E.H. Advances in canine distemper virus pathogenesis research: a wildlife perspective. J Gen Virol. 2017;98(3):311-321. doi: 10.1099/jgv.0.000666.Woo G.H., Jho Y.S., Bak E.J. Canine distemper virus infection in fennec fox (Vulpes zerda). J Vet Med Sci. 2010 Aug;72(8):1075-9. doi: 10.1292/jvms.09-0510.4.Woo G.H., Jho Y.S., Bak E.J. Canine distemper virus infection in fennec fox (Vulpes zerda). J Vet Med Sci. 2010 Aug;72(8):1075-9. doi: 10.1292/jvms.09-0510.4.Stancu A.C., Voia O.S., Boldura O.M., Pasca S.A., Luca I., Hulea A.S., Ivan O.R., Dragoescu A.A., Lungu B.C., Hutu I. Unusual Canine Distemper Virus Infection in Captive Raccoons (Procyon lotor). Viruses. 2023;15(7):1536. doi: 10.3390/v15071536.Stancu A.C., Voia O.S., Boldura O.M., Pasca S.A., Luca I., Hulea A.S., Ivan O.R., Dragoescu A.A., Lungu B.C., Hutu I. Unusual Canine Distemper Virus Infection in Captive Raccoons (Procyon lotor). Viruses. 2023;15(7):1536. doi: 10.3390/v15071536.Feng N., Yu Y., Wang T., Wilker P., Wang J., Li Y., Sun Z., Gao Y., Xia X. Fatal canine distemper virus infection of giant pandas in China. Sci Rep. 2016;6:27518. doi: 10.1038/srep27518.5.Feng N., Yu Y., Wang T., Wilker P., Wang J., Li Y., Sun Z., Gao Y., Xia X. Fatal canine distemper virus infection of giant pandas in China. Sci Rep. 2016;6:27518. doi: 10.1038/srep27518.5.Lin A.V. Direct ELISA. Methods Mol Biol. 2015;1318:61-7. doi: 10.1007/978-1-4939-2742-5_6.Lin A.V. Direct ELISA. Methods Mol Biol. 2015;1318:61-7. doi: 10.1007/978-1-4939-2742-5_6.Chen C.C., Lai H.R., Liang H.K. [et al.]. Establishment and preliminary application of timeresolved immunofluorescence analysis method for canine distemper virus. Chinese Journal of Preventive Veterinary Medicine, 2020, 42(3): 258-262.Chen C.C., Lai H.R., Liang H.K. [et al.]. Establishment and preliminary application of timeresolved immunofluorescence analysis method for canine distemper virus. Chinese Journal of Preventive Veterinary Medicine, 2020, 42(3): 258-262.Beineke A., Puff C., Seehusen F., Baumgärtner W. Pathogenesis and immunopathology of systemic and nervous canine distemper. Vet Immunol Immunopathol. 2009;127(1-2):1-18. doi: 10.1016/j.vetimm.2008.09.023.Beineke A., Puff C., Seehusen F., Baumgärtner W. Pathogenesis and immunopathology of systemic and nervous canine distemper. Vet Immunol Immunopathol. 2009;127(1-2):1-18. doi: 10.1016/j.vetimm.2008.09.023.Wang J., Wang J., Li R., Liu L., Yuan W. Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification. BMC Vet Res. 2017;13(1):241. doi: 10.1186/s12917-017-1180-7.Wang J., Wang J., Li R., Liu L., Yuan W. Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification. BMC Vet Res. 2017;13(1):241. doi: 10.1186/s12917-017-1180-7.Sarchahi A. A., Arbabi M., Mohebalian H. Detection of canine distemper virus in cerebrospinal fluid, whole blood and mucosal specimens of dogs with distemper using RT-PCR and immunochromatographic assays. Vet Med Sci. 2022;8(4):1390-1399. doi: 10.1002/vms3.790.Sarchahi A. A., Arbabi M., Mohebalian H. Detection of canine distemper virus in cerebrospinal fluid, whole blood and mucosal specimens of dogs with distemper using RT-PCR and immunochromatographic assays. Vet Med Sci. 2022;8(4):1390-1399. doi: 10.1002/vms3.790.Knight A.R., Taylor E.L., Lukaszewski R., Jensen K.T., Jones H.E., Carré J.E., Isupov M.N., Littlechild J.A., Bailey S.J., Brewer E., McDonald T.J., Pitt A.R., Spickett C.M., Winyard P.G. A highsensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells. Free Radic Biol Med. 2018;120:246-254. doi: 10.1016/j.freeradbiomed.2018.03.026.Knight A.R., Taylor E.L., Lukaszewski R., Jensen K.T., Jones H.E., Carré J.E., Isupov M.N., Littlechild J.A., Bailey S.J., Brewer E., McDonald T.J., Pitt A.R., Spickett C.M., Winyard P.G. A highsensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells. Free Radic Biol Med. 2018;120:246-254. doi: 10.1016/j.freeradbiomed.2018.03.026.Tan Y., Pang W., Jia X., Zhao M.H. Comparison of the performance of a chemiluminescence assay and an ELISA for detection of anti-GBM antibodies. Ren Fail. 2020;42(1):48-53. doi: 10.1080/0886022X.2019.1702056.Tan Y., Pang W., Jia X., Zhao M.H. Comparison of the performance of a chemiluminescence assay and an ELISA for detection of anti-GBM antibodies. Ren Fail. 2020;42(1):48-53. doi: 10.1080/0886022X.2019.1702056.Hayrapetyan H., Tran T., Tellez-Corrales E., Madiraju C. Enzyme-Linked Immunosorbent Assay: Types and Applications. Methods Mol Biol. 2023;2612:1-17. doi: 10.1007/978-1-0716-2903-1_1.Hayrapetyan H., Tran T., Tellez-Corrales E., Madiraju C. Enzyme-Linked Immunosorbent Assay: Types and Applications. Methods Mol Biol. 2023;2612:1-17. doi: 10.1007/978-1-0716-2903-1_1.Kohl T.O., Ascoli C.A. Indirect Immunometric ELISA. Cold Spring Harb Protoc. 2017 May 1;2017(5). doi: 10.1101/pdb.prot093708.Kohl T.O., Ascoli C.A. Indirect Immunometric ELISA. Cold Spring Harb Protoc. 2017 May 1;2017(5). doi: 10.1101/pdb.prot093708.Li L., Qiao S., Liu J., Zhou Y., Tong W., Dong S., Liu C., Jiang Y., Guo Z., Zheng H., Zhao R., Tong G., Li G., Gao F. A highly efficient indirect ELISA and monoclonal antibody established against African swine fever virus pK205R. Front Immunol. 2023;13:1103166. doi: 10.3389/fimmu.2022.1103166.Li L., Qiao S., Liu J., Zhou Y., Tong W., Dong S., Liu C., Jiang Y., Guo Z., Zheng H., Zhao R., Tong G., Li G., Gao F. A highly efficient indirect ELISA and monoclonal antibody established against African swine fever virus pK205R. Front Immunol. 2023;13:1103166. doi: 10.3389/fimmu.2022.1103166.Li J., Cao Y., Wang M., Cheng A., Ou X., Mao S., Sun D., Liu M., Zhang S., Zhao X.X., Jia R., Yang Q., Wu Y., Zhu D., Chen S., Huang J., Gao Q., Tian B. Development of an indirect ELISA method based on the VP4 protein for detection antibody against duck hepatitis A virus type 1. J Virol Methods. 2022;300:114393. doi: 10.1016/j.jviromet.2021.114393.Li J., Cao Y., Wang M., Cheng A., Ou X., Mao S., Sun D., Liu M., Zhang S., Zhao X.X., Jia R., Yang Q., Wu Y., Zhu D., Chen S., Huang J., Gao Q., Tian B. Development of an indirect ELISA method based on the VP4 protein for detection antibody against duck hepatitis A virus type 1. J Virol Methods. 2022;300:114393. doi: 10.1016/j.jviromet.2021.114393.Cheng T.Y., Magtoto R., Henao-Díaz A., Poonsuk K., Buckley A., Van Geelen A., Lager K., Zimmerman J., Giménez-Lirola L. Detection of pseudorabies virus antibody in swine serum and oral fluid specimens using a recombinant gE glycoprotein dual-matrix indirect ELISA. J Vet Diagn Invest. 2021;33(6): 1106-114. doi: 10.1177/10406387211040755.Cheng T.Y., Magtoto R., Henao-Díaz A., Poonsuk K., Buckley A., Van Geelen A., Lager K., Zimmerman J., Giménez-Lirola L. Detection of pseudorabies virus antibody in swine serum and oral fluid specimens using a recombinant gE glycoprotein dual-matrix indirect ELISA. J Vet Diagn Invest. 2021;33(6): 1106-114. doi: 10.1177/10406387211040755.

The authors declare that there are no conflicts of interest present.