biopreparatБИОпрепараты. Профилактика, диагностика, лечениеBiological Products. Prevention, Diagnosis, Treatment2221-996X2619-1156Scientific Centre for Expert Evaluation of Medicinal Products10.30895/2221-996X-2025-632biopreparat-632Research ArticleРАЗРАБОТКА И ВАЛИДАЦИЯ МЕТОДИКDEVELOPMENT AND VALIDATION OF METHODSРазработка количественной иммуноферментной тест-системы для определения концентрации Е2 антигена вируса Чикунгунья и расчета массы цельновирионного антигена в культуральных образцахDevelopment of an enzyme-linked immunosorbent assay test system for quantifying Chikungunya virus E2 protein and calculating the mass of whole-virion antigen in culture fluid sampleshttps://orcid.org/0009-0006-9604-0139ТаханБ.TahhanB.

Тахан Бана

просп. Вернадского, д. 78, Москва, 119454, Российская Федерация; г. Алеппо, 12212, Сирийская Арабская Республика

Bana Tahhan

78 Vernadsky Ave, Moscow 119454, Russian Federation; 12212 Aleppo, Syrian Arab Republic

banatahan.sy@gmail.comhttps://orcid.org/0000-0002-2997-8892ПритвороваЛ. Н.PritvorovaL. N.

Притворова Людмила Николаевна, канд. мед. наук

Малый Казенный пер., д. 5а, Москва, 105064

Lyudmila N. Pritvorova, Cand. Sci. (Med.)

5A Maly Kazenny Ln., Moscow 105064

lexx294@yandex.ruhttps://orcid.org/0000-0003-3264-6722СамарцеваТ. Г.SamartsevaT. G.

Самарцева Татьяна Геннадьевна

Малый Казенный пер., д. 5а, Москва, 105064

Tatiana G. Samartseva

5A Maly Kazenny Ln., Moscow 105064

samartseva08020@mail.ruhttps://orcid.org/0000-0003-2610-8493КедикС. А.KedikS. A.

Кедик Станислав Анатольевич, д-р техн. наук, проф.

просп. Вернадского, д. 78, Москва, 119454

Stanislav A. Kedik, Dr. Sci. (Tech.), Prof.

78 Vernadsky Ave, Moscow 119454

doctorkedik@yandex.ruhttps://orcid.org/0000-0002-8600-7347ОксаничА. С.OksanichA. S.

Оксанич Алексей Сергеевич, канд. биол. наук

Малый Казенный пер., д. 5а, Москва, 105064

Alexey S. Oksanich, Cand. Sci. (Biol.)

5A Maly Kazenny Ln., Moscow 105064

oksanich@yahoo.comФедеральное государственное бюджетное образовательное учреждение высшего образования «МИРЭА — Российский технологический университет»; Университет Алеппо, факультет естественных наукСирияMIREA — Russian Technological University; University of Aleppo, Faculty of Science, Department of ChemistrySyrian Arab RepublicФедеральное государственное бюджетное научное учреждение «Научно-исследовательский институт вакцин и сывороток им. И.И. Мечникова»РоссияI. Mechnikov Research Institute of Vaccines and SeraRussian FederationФедеральное государственное бюджетное образовательное учреждение высшего образования «МИРЭА — Российский технологический университет»РоссияMIREA — Russian Technological UniversityRussian Federation202525062025252214225Copyright © Тахан Б., Притворова Л.Н., Самарцева Т.Г., Кедик С.А., Оксанич А.С., 20252025Тахан Б., Притворова Л.Н., Самарцева Т.Г., Кедик С.А., Оксанич А.С.Tahhan B., Pritvorova L.N., Samartseva T.G., Kedik S.A., Oksanich A.S.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.biopreparations.ru/jour/article/view/632ВВЕДЕНИЕ

ВВЕДЕНИЕ. Вирус Чикунгунья (ВЧИК) за последние годы широко распространился во многих частях мира, вызывая крупномасштабные вспышки с серьезными экономическими и социальными последствиями. Для повышения эффективности борьбы с вирусом необходимо разрабатывать и совершенствовать методы диагностики ВЧИК не только в сыворотке крови пациентов, но и в полевых материалах — для выявления и уничтожения очагов инфекции. Определение антигенов ВЧИК также важно проводить в образцах культуральной жидкости на разных этапах разработки и производства вакцин.

ЦЕЛЬ

ЦЕЛЬ. Разработка количественной тест-системы на основе одностадийного сэндвич-варианта иммуноферментного анализа для выявления Е2 антигена вируса Чикунгунья в культуральных жидкостях, а также методики расчета массы цельновирионного антигена в образце.

МАТЕРИАЛЫ И МЕТОДЫ

МАТЕРИАЛЫ И МЕТОДЫ. В работе использовали два мышиных моноклональных антитела к ВЧИК; очищенный вирус Чикунгунья (штамм Nika21, GenBank ID PQ673601) и рекомбинантный Е2 антиген ВЧИК. Для сравнения чувствительности методов иммуноферментного анализа (ИФА) и обратной транскрипции с последующей количественной ПЦР в режиме реального времени (ОТ-кПЦР-РВ) использовали образцы культуральной жидкости, которые собирали в разные временные точки (18, 24, 46, 72 ч) после заражения ВЧИК клеток Vero. Основные аналитические и технические характеристики разработанной ИФА тест-системы определяли в соответствии с требованиями ГОСТ 51352–2013.

РЕЗУЛЬТАТЫ

РЕЗУЛЬТАТЫ. Аналитическая чувствительность метода составила не менее 0,625 нг/мл, значение коэффициента вариации — не более 3,56%, тест на «открытие» — 100%, результаты теста на «линейность» в диапазоне концентраций 1,5–16 нг/мл были в пределах 90–110%. Специфичность метода составила 100%; перекрестная реактивность не наблюдалась с образцами, содержащими вирусы денге, желтой лихорадки, Синдбис, краснухи и вирус Западного Нила. Разработанная ИФА тест-система и метод ОТ-кПЦР-РВ продемонстрировали схожие результаты при определении массы цельновирионного антигена в вируссодержащей жидкости — 1,06 и 1,09 мкг/мл соответственно при использовании коэффициента пересчета.

ВЫВОДЫ

ВЫВОДЫ. Для количественного определения E2 антигена ВЧИК в культуральных образцах была разработана простая, специфичная и чувствительная ИФА тест-система, которую также можно использовать для быстрого анализа полевых образцов. Предложен метод расчета массы цельновирионного антигена по количеству Е2 белка (ИФА) и геномным эквивалентам (ОТ-кПЦР-РВ). Между оптической плотностью в ИФА и пороговым циклом в ПЦР-РВ установлена сильная обратная корреляция.

 

 

INTRODUCTION

INTRODUCTION. In recent years, Chikungunya virus (CHIKV) has spread in many parts of the world and has caused large-scale outbreaks with serious economic and social consequences. To improve the effectiveness of CHIKV control measures, it is necessary to develop and optimise diagnostic methods applicable not only to patient serum samples but also to mosquito samples (to identify and eliminate the foci of infection). In addition, it is important to determine antigens in culture fluid samples taken at various stages in the development and production of CHIKV vaccines.

AIM

AIM. This study aimed to develop a quantitative one-step sandwich enzyme-linked immunosorbent assay (ELISA) test system for detecting CHIKV E2 protein and a procedure for calculating the mass of whole-virion antigen in culture fluid samples.

MATERIALS AND METHODS

MATERIALS AND METHODS. The study focused on two mouse monoclonal antibodies, purified CHIKV (Nika21 strain, GenBank ID: PQ673601), and recombinant CHIKV E2 protein. The sensitivity of ELISA was compared with that of real-time quantitative reverse transcription polymerase chain reaction (real-time RT-qPCR). The comparison used culture fluid samples collected at different time points after infection of Vero cells with CHIKV (18, 24, 46, and 72 h). The main analytical and technical characteristics of the ELISA test system developed were determined in accordance with GOST 51352-2013.

RESULTS

RESULTS. The sensitivity of the assay was not less than 0.625 ng/mL, and its coefficient of variation was not more than 3.56%. The recovery of the assay was 100%. The assay demonstrated an acceptable linearity of 90–110% in the concentration range of 1.5–16 ng/mL. The specificity of the assay was 100%, as no cross-reactivity was observed with samples containing dengue, yellow fever, Sindbis, rubella, and West Nile viruses. The ELISA test system developed in this study and real-time RT-qPCR showed similar results (1.06 and 1.09 μg/mL, respectively) in calculating the mass of whole-virion antigen in culture fluid samples with the use of a conversion factor.

CONCLUSIONS

CONCLUSIONS. A simple, specific, and sensitive ELISA test system was developed for the quantitative determination of CHIKV E2 protein in culture fluid samples (and for rapid testing of mosquito samples). The authors offered a method for calculating the mass of whole-virion antigen from the amount of E2 protein (ELISA) and the quantity of genomic equivalents (real-time RT-qPCR). The study demonstrated a strong negative correlation between optical density values obtained using ELISA and cycle threshold values derived from real-time RT-qPCR.

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