chemicallytechFine Chemical TechnologiesТонкие химические технологии2410-65932686-7575MIREA – Russian Technological University (RTU MIREA).10.32362/2410-6593-2025-20-6-565-581SCSQPAchemicallytech-2335Research ArticleBIOCHEMISTRY AND BIOTECHNOLOGYБИОХИМИЯ И БИОТЕХНОЛОГИЯDevelopment of immunochromatographic assay for simultaneous detection of tetracyclines and streptomycin in milkРазработка иммунохроматографического анализа для одновременного обнаружения тетрациклинов и стрептомицина в молокеhttps://orcid.org/0009-0007-9730-1465МаксинИ. В.MaksinI. V.

Максин Иван Владимирович, аспирант, кафедра биотехнологии и промышленной фармации; ученый-исследователь

119454, Москва, пр-т Вернадского, д. 78

 

Ivan V. Maksin, Postgraduate Student, Department of Biotechnology and Industrial Pharmacy; Research Scientist

78, Vernadskogo pr., Moscow, 119454

42/1, Bol’shoi bul., Moscow, 121205

maxinivanv@gmail.comhttps://orcid.org/0009-0007-8354-6883ПоляковаД. И.PolyakovaD. I.

Полякова Дарья Ивановна, студент, Институт фармации и биотехнологии; лаборант

121205, Москва, Большой бульвар, д. 42, стр. 1

117198, Москва, ул. Миклухо-Маклая, д. 6

 

Darya I. Polyakova, Student, Institute of Pharmacy and Biotechnology; Laboratory Assistant

42/1, Bol’shoi bul., Moscow, 121205

6, Miklukho-Maklaya ul., Moscow, 117198

dpolyakova@drdbiotech.ruhttps://orcid.org/0000-0001-6544-4057КесареваВ. А.KesarevaV. A.

Кесарева Виктория Александровна, студент; лаборант

121205, Москва, Большой бульвар, д. 42, стр. 1

117198, Москва, ул. Миклухо-Маклая, д. 6

 

Viktoriya A. Kesareva, Student, Institute of Pharmacy and Biotechnology; Laboratory Assistant

42/1, Bol’shoi bul., Moscow, 121205

6, Miklukho-Maklaya ul., Moscow, 117198

vkesareva@drdbiotech.ruhttps://orcid.org/0009-0007-8233-4221СысуевА. А.SysuevA. A.

Сысуев Александр Анатольевич, ассистент, кафедра биотехнологии и промышленной фармации

119454, Москва, пр-т Вернадского, д. 78

Alexander A. Sysuev, Assistant, Department of Biotechnology and Industrial Pharmacy

78, Vernadskogo pr., Moscow, 119454

sysuev@mirea.ruhttps://orcid.org/0000-0003-1025-2714ИвановВ. С.IvanovV. S.

Иванов Владислав Сергеевич, ведущий ученый-исследователь

121205, Москва, Большой бульвар, д. 42, стр. 1

Vladislav S. Ivanov, Leading Research Scientist

42/1, Bol’shoi bul., Moscow, 121205

vivanov@drdbiotech.ruСимоноваЕ. И.SimonovaE. I.

Симонова Евгения Игоревна, аспирант, Департамент ветеринарной медицины, Аграрно-технологический институт); директор по проектам

121205, Москва, Большой бульвар, д. 42, стр. 1

117198, Москва, ул. Миклухо-Маклая, д. 6

 

Evgeniya I. Simonova, Postgraduate Student, Department of Veterinary Medicine, Agro-Technological Institute; Project Director

Scopus Author ID 57222665772

42/1, Bol’shoi bul., Moscow, 121205

6, Miklukho-Maklaya ul., Moscow, 117198

simonova1999@yandex.ruhttps://orcid.org/0000-0001-8182-1836ХунтеевГ. А.KhunteevG. A.

Хунтеев Герман Анатольевич, к.м.н., директор по науке

Scopus Author ID 7801430884, ResearcherID AAC-4456-2019

121205, Москва, Большой бульвар, д. 42, стр. 1

German A. Khunteev, Can. Sci. (Med.), Director of Science

Scopus Author ID 7801430884, ResearcherID AAC-4456-2019

42/1, Bol’shoi bul., Moscow, 121205

gk@drdbiotech.ruhttps://orcid.org/0000-0002-0988-8515КирилловаЮ. Г.KirillovaYu. G.

Кириллова Юлия Геннадьевна, к.х.н., доцент, кафедра биотехнологии и промышленной фармации

Scopus Author ID 9332799900, ResearcherID F-5697-2016

119454, Москва, пр-т Вернадского, д. 78

Yulia G. Kirillova, Can. Sci. (Chem.), Associate Professor, Department of Biotechnology and Industrial Pharmacy

Scopus Author ID 9332799900, ResearсherID F-5697-2016

78, Vernadskogo pr., Moscow, 119454

dryulets@mail.ruМИРЭА – Российский технологический университет (Институт тонких химических технологий им. М.В. Ломоносова); ООО «Рапид Био»РоссияMIREA – Russian Technological University (Lomonosov Institute of Fine Chemical Technologies); Rapid BioRussian FederationООО «Рапид Био»; Российский университет дружбы народов им. П. ЛумумбыРоссияRapid Bio; Patrice Lumumba Peoples’ Friendship University of RussiaRussian FederationМИРЭА – Российский технологический университет (Институт тонких химических технологий им. М.В. Ломоносова)РоссияMIREA – Russian Technological University (Lomonosov Institute of Fine Chemical TechnologiesRussian FederationООО «Рапид Био»РоссияRapid BioRussian Federation202515012026206565581Copyright © Maksin I.V., Polyakova D.I., Kesareva V.A., Sysuev A.A., Ivanov V.S., Simonova E.I., Khunteev G.A., Kirillova Y.G., 20262026Максин И.В., Полякова Д.И., Кесарева В.А., Сысуев А.А., Иванов В.С., Симонова Е.И., Хунтеев Г.А., Кириллова Ю.Г.Maksin I.V., Polyakova D.I., Kesareva V.A., Sysuev A.A., Ivanov V.S., Simonova E.I., Khunteev G.A., Kirillova Y.G.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.finechem-mirea.ru/jour/article/view/2335Objectives

Objectives. To optimize indirect antibody immobilization on gold nanoparticles (GNPs) using anti-species antibodies for enhanced conjugate stability and to develop an immunochromatographic assay (ICA) for antibiotic detection in milk.

Methods

Methods. The GNPs were synthesized by reduction of tetrachloroauric acid in the presence of seed particles. The size of GNPs was determined spectrophotometrically according to literature data using a Thermo Fisher Scientific Varioskan LUX instrument. Monoclonal mouse antibodies to tetracycline and streptomycin were immobilized on the surface of the GNPs via anti-mouse antibodies. Conjugates of bovine serum albumin with tetracycline and streptomycin were obtained through Mannich reaction and click-reaction, respectively. The coupling ratio was determined by MALDI-TOF mass spectrometry on a Bruker RapifleX instrument. Immunoreagents were dispensed onto a nitrocellulose membrane using a BioDot ZX1010 dispenser. The assembled multi-membrane composite was cut into test strips using a KinBio ZQ4500 guillotine cutter. The test results were interpreted visually and using an Allsheng TSR-100 test strip reader.

Results

Results. Following conjugate formation via indirect immobilization using anti-species antibodies, it was necessary to block residual binding sites on the anti-species antibodies in order to enhance solution-phase conjugate stability. As a result of optimizing the concentrations of immunoreagents, an ICA was developed for the simultaneous detection of streptomycin and tetracyclines in milk. The detection limit of the optimized ICA for tetracyclines and streptomycin was 2–7.5 and 25 ng/mL, respectively, for visual result interpretation, and 0.29–2.15 and 1.34 ng/mL, respectively, when using a test strip reader.

Conclusions

Conclusions. It is shown that the stability of the resulting conjugates in solution can be enhanced by blocking the free binding sites of the anti-species antibodies to prevent cross-linking of the nanoparticles caused by anti-species antibody binding.

Цели

Цели. Оптимизация стабильности конъюгатов, полученных методом непрямой иммобилизации специфических антител на наночастицах золота (НЧЗ) при помощи антивидовых антител, и разработка на их основе иммунохроматографического анализа (ИХА) для определения антибиотиков в молоке.

Методы

Методы. НЧЗ синтезировали восстановлением золотохлористоводородной кислоты в присутствии зародышей. Размер определяли спектрофотометрически по литературным данным на приборе Thermo Fisher Scientific Varioskan LUX. Моноклональные мышиные антитела к тетрациклину и стрептомицину иммобилизовали на поверхности НЧЗ посредством анти-мышиных антител. Конъюгаты бычьего сывороточного альбумина с тетрациклином и стрептомицином получали реакцией Манниха и клик- реакцией соответственно. Степень конъюгации определяли при помощи MALDI-TOF масс-спектрометрии на приборе Bruker RapifleX. Иммунореагенты наносили на нитроцеллюлозную мембрану на диспенсере BioDot ZX1010. Собранный мультимембранный композит нарезали на тест-полоски на гильотинном резчике KinBio ZQ4500. Результаты ИХА интерпретировали визуально, а также при помощи считывателя тест-полосок Allsheng TSR-100.

Результаты

Результаты. После получения конъюгатов посредством непрямой иммобилизации при помощи антивидовых антител необходимо проводить блокировку оставшихся незанятыми сайтов связывания антивидовых антител, чтобы повысить стабильность конъюгатов в растворе. В результате оптимизации концентраций иммунореагентов был разработан ИХА для одновременного обнаружения стрептомицина и тетрациклинов в молоке. Предел обнаружения оптимизированного анализа тетрациклинов и стрептомицина составил 2–7.5 и 25 нг/мл при визуальной интерпретации результатов, 0.29–2.15 и 1.34 нг/мл при использовании считывателя тест-полосок соответственно.

Выводы

Выводы. Установлено, что блокировка свободных сайтов связывания вторичных антител повышает стабильность полученных конъюгатов антител в растворе, предотвращая кросс-сшивки наночастиц, вызываемые связыванием антивидовых антител.

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