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Журнал микробиологии, эпидемиологии и иммунобиологии. 2021; 98: 253-265

Адаптация МТТ-теста для определения нейтрализующих антител к вирусу SARS-CoV-2

Грачёва А. В., Корчевая Е. Р., Кудряшова А. М., Борисова О. В., Петруша О. А., Смирнова Д. И., Чернышова И. Н., Свитич О. А., Зверев В. В., Файзулоев Е. Б.

https://doi.org/10.36233/0372-9311-136

Аннотация

Введение. Основным показателем специфической активности антител к вирусу SARS-CoV-2 является их способность нейтрализовать вирус. Тест на вируснейтрализующие антитела (ВНА) широко востребован в различных направлениях биомедицинских исследований.

Целью работы являлся подбор оптимальных условий для определения ВНА к вирусу SARS-CoV-2 по ингибированию цитопатогенного действия (ЦПД) в культуре клеток с возможностью как микроскопического, так и спектрофотометрического учёта результата.

Материалы и методы. Сыворотку крови реконвалесцентов COVID-19 и здоровых лиц (n = 96) изучали методом ИФА. Коронавирус SARS-CoV-2, штамм Dubrovka (номер GenBank: MW514307.1) выращивали в культуре клеток Vero CCL81 (ATСС). Идентификацию вируса проводили методами ОТ-ПЦР-РВ, ИФА и секвенирования по Сэнгеру. Результаты реакции нейтрализации (РН) учитывали по ЦПД микроскопически и в метилтетразолиевом (МТТ) тесте.

Результаты. От больного COVID-19 изолирован коронавирус SARS-CoV-2 и адаптирован к выращиванию в культуре клеток. При заражении низкой дозой (MOI = 0,00001) вирус вызывал выраженное ЦПД с выживаемостью клеток менее 3%, что позволяло учитывать результаты РН по ингибированию ЦПД. Сравни- тельный анализ сывороток в РН и методом ИФА показал достоверную корреляцию между титрами ВНА и титрами антител к RBD-домену S-белка (Спирмен r = 0,714; р < 0,001). Результаты определения ВНА с микроскопической и спектрофотометрической детекцией (тест МТТ) также достоверно коррелировали (Спирмен r = 0,963; р < 0,05).

Заключение. На основе адаптированного к культуре клеток Vero вируса SARS-CoV-2 разработана система оценки титра ВНА, позволяющая учитывать результат как с помощью микроскопического исследования, так и спектрофотометрически в МТТ-тесте. Применение теста МТТ позволяет автоматизировать учёт результатов РН, проводить статистическую обработку получаемых данных, снижает субъективизм при оценке результата. Являясь витальным красителем, МТТ выявляет только живые клетки, что повышает надёжность получаемых результатов по сравнению с другими красителями.

Список литературы

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21. Müller J.A., Harms M., Schubert A., Mayer B., Jansen S., Herbeuval J.P., et al. Development of a high-throughput colorimetric Zika virus infection assay. Med. Microbiol. Immunol. 2017; 206(2): 175–85. https://doi.org/10.1007/s00430-017-0493-2

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23. Feoktistova M., Geserick P., Leverkus M. Crystal violet assay for determining viability of cultured cells. Cold Spring Harb. Protoc. 2016; 2016(4): pdb.prot087379. https://doi.org/10.1101/pdb.prot087379

Journal of microbiology, epidemiology and immunobiology. 2021; 98: 253-265

Adaptation of the MTT assay for detection of neutralizing antibodies against the SARS-CoV-2 virus

Gracheva A. V., Korchevaya E. R., Kudryashova A. M., Borisova O. V., Petrusha O. A., Smirnova D. I., Chernyshova I. N., Svitich O. A., Zverev V. V., Faizuloev E. B.

https://doi.org/10.36233/0372-9311-136

Abstract

Introduction. The ability of SARS-CoV-2 antibodies to neutralize the virus is the primary indicator of their specific activity. The test for virus neutralizing antibodies (NAbs) is much needed in different biomedical studies.

The aim of the study is to find optimum conditions for microscopic and spectrophotometric detection of SARSCoV-2 NAbs by inhibition of cytopathic effect (CPE) in cell cultures.

Materials and methods. Blood sera collected from COVID-19 convalescent patients and healthy individuals (n = 96) were tested using the ELISA method. The SARS-CoV-2 coronavirus, Dubrovka strain (GenBank accession no. MW514307.1) was grown in culture medium of Vero cell line CCL-81 (ATCC). Real-time RT-PCR, ELISA, and Sanger sequencing were used for detection of the virus. The results of the neutralization test (NT) were assessed through the microscopic examination for CPE and by the methyl thiazolyl tetrazolium (MTT) assay.

Results. SARS-CoV-2 was isolated from a COVID-19 patient and adapted to grow in cell culture. At a low dose of infection (MOI = 0.00001), the virus caused a pronounced CPE with the cell viability less than 3%, thus making it possible to assess NT results by CPE inhibition. The NT and ELISA-based comparative study of sera showed positive correlation between virus NAb titers and Nab titers to S-protein RBD (Spearman’s r = 0.714; p < 0.001). The results of NAbs microscopic and spectrophotometric detection (the MTT assay) also demonstrated positive correlation (Spearman’s r = 0.963; p < 0.05).

Conclusion. The SARS-CoV-2 virus adapted to Vero cell culture served to develop a NAb titer assessment system, which can be used both in microscopic studies and for an MTT assay in spectrophotometric studies. The MTT assay provides automated reading of NT results, optimizes the statistical analysis of the obtained data, and minimizes subjectivity in assessment of results. Being a vital dye, MTT penetrates only viable cells, thus contributing to the reliability of the obtained results compared to other dyes.

References

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2. Mekonnen D., Mengist H.M., Derbie A., Nibret E., Munshea A., He H., et al. Diagnostic accuracy of serological tests and kinetics of severe acute respiratory syndrome coronavirus 2 antibody: A sys- tematic review and meta-analysis. Rev. Med. Virol. 2020; e2181. https://doi.org/10.1002/rmv.2181

3. Zost S.J., Gilchuk P., Case J.B., Binshtein E., Chen R.E., Nkolola J.P., et al. Potently neutralizing and protective human antibodies against SARS-CoV-2. Nature. 2020; 584(7821): 443–9. https://doi.org/10.1038/s41586-020-2548-6

4. Rogers T.F., Zhao F., Huang D., Beutler N., Burns A., He W.T., et al. Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model. Science. 2020; 369(6506): 956–63. https://doi.org/10.1126/science.abc7520

5. Chi X., Yan R., Zhang J., Zhang G., Zhang Y., Hao M., et al. A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. Science. 2020; 369(6504): 650–5. https://doi.org/10.1126/science.abc6952

6. Brown B.L., McCullough J. Treatment for emerging viruses: Convalescent plasma and COVID-19. Transfus. Apher. Sci. 2020; 59(3): 102790. https://doi.org/10.1016/j.transci.2020.102790

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9. Tan C.W., Chia W.N., Qin X., Liu P., Chen M.I., Tiu C., et al. A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction. Nat. Biotechnol. 2020; 38(9): 1073–8. https://doi.org/10.1038/s41587-020-0631-z

10. Meyer B., Reimerink J., Torriani G., Brouwer F., Godeke G.J., Yerly S., et al. Validation and clinical evaluation of a SARSCoV-2 surrogate virus neutralisation test (sVNT). Emerg. Microbes Infect. 2020; 9(1): 2394–403. https://doi.org/10.1080/22221751.2020.1835448

11. Nie J., Li Q., Wu J., Zhao C., Hao H., Liu H., et al. Establishment and validation of a pseudovirus neutralization assay for SARSCoV-2. Emerg. Microbes Infect. 2020; 9(1): 680–6. https://doi.org/10.1080/22221751.2020.1743767

12. Schmidt F., Weisblum Y., Muecksch F., Hoffmann H.H., Michailidis E., Lorenzi J.C.C., et al. Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses. J. Exp. Med. 2020; 217(11): e20201181. https://doi.org/10.1084/jem.20201181

13. Brouwer P.J.M., Caniels T.G., van der Straten K., Snitselaar J.L., Aldon Y., Bangaru S., et al. Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability. Science. 2020; 369(6504): 643–50. https://doi.org/10.1126/science.abc5902

14. Liu L., Wang P., Nair M.S., Yu J., Rapp M., Wang Q., et al. Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike. Nature. 2020; 584(7821): 450–6. https://doi.org/10.1038/s41586-020-2571-7

15. Chan J.F., Yip C.C., To K.K., Tang T.H., Wong S.C., Leung K.H., et al. Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel real-time reverse transcription-PCR assay validated in vitro and with clinical specimens. J. Clin. Microbiol. 2020; 58(5): e00310-20. https://doi.org/10.1128/JCM.00310-20

16. Ramakrishnan M.A. Determination of 50% endpoint titer using a simple formula. World J. Virol. 2016; 5(2): 85–6. https://doi.org/10.5501/wjv.v5.i2.85

17. Gao Q., Bao L., Mao H., Wang L., Xu K., Yang M., et al. Development of an inactivated vaccine candidate for SARSCoV-2. Science. 2020; 369(6499): 77–81. https://doi.org/10.1126/science.abc1932

18. Logunov D.Y., Dolzhikova I.V., Zubkova O.V., Tukhvatulin A.I., Shcheblyakov D.V., Dzharullaeva A.S., et al. Safety and immunogenicity of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised phase 1/2 studies from Russia. Lancet. 2020; 396(10255): 887–97. https://doi.org/10.1016/S0140-6736(20)31866-3

19. Chan J.F., Zhang A.J., Yuan S., Poon V.K., Chan C.C., Lee A.C., et al. Simulation of the clinical and pathological manifestations of coronavirus disease 2019 (COVID-19) in a golden Syrian hamster model: implications for disease pathogenesis and transmissibility. Clin. Infect. Dis. 2020; 71(9): 2428–46. https://doi.org/10.1093/cid/ciaa325

20. Haddad E.E., Whitfill C.E., Ricks C.A., Fredericksen T., Rowe D., Owen L., et al. Adaptation of the MTT (3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide) assay for the determination of virus-neutralizing antibodies using the virus-neutralization assay. Avian Dis. 1994; 38(4): 755–61.

21. Müller J.A., Harms M., Schubert A., Mayer B., Jansen S., Herbeuval J.P., et al. Development of a high-throughput colorimetric Zika virus infection assay. Med. Microbiol. Immunol. 2017; 206(2): 175–85. https://doi.org/10.1007/s00430-017-0493-2

22. Heldt C.L., Hernandez R., Mudiganti U., Gurgel P.V., Brown D.T., Carbonell R.G. A colorimetric assay for viral agents that produce cytopathic effects. J. Virol. Methods. 2006; 135(1): 56–65. https://doi.org/10.1016/j.jviromet.2006.01.022

23. Feoktistova M., Geserick P., Leverkus M. Crystal violet assay for determining viability of cultured cells. Cold Spring Harb. Protoc. 2016; 2016(4): pdb.prot087379. https://doi.org/10.1101/pdb.prot087379