vestichИзвестия Национальной академии наук Беларуси. Серия химических наукProceedings of the National Academy of Sciences of Belarus, Chemical Series1561-83312524-2342The Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1561-8331-2020-56-3-318-332vestich-600Research ArticleБИООРГАНИЧЕСКАЯ ХИМИЯBIOORGANIC CHEMISTRYИммуноферментные системы и набор реагентов для определения бацитрацина в пищевых продуктахEnzyme immunoassay systems and a reagent kit for the determination of bacitracin in foodsВашкевичИ. И.VashkevichI. I.

Вашкевич Ирина Игнатьевна - кандидат химических наук, ведущий научный сотрудник.

Irina I. Vashkevich - Ph. D. (Chemistry), Leading Researcher.

vashkevich@iboch.byЯстребоваА. А.YastrebovaA. A.

Ястребова Анна Андреевна - научный сотрудник.

Anna A. Yastrebova - Researcher.

yastrebova@iboch.byКуприенкоО. С.KuprienkoO. S.

Куприенко Ольга Сергеевна - кандидат химических наук, старший научный сотрудник.

Olga S. Kuprienko - Ph. D. (Chemistry), Senior Researcher.

kuprienko@iboch.byСерченяТ. С.SerchenyaT. S.

Серченя Татьяна Сергеевна - кандидат химических наук, ведущий научный сотрудник.

Tatyana S. Serchenya - Ph. D. (Chemistry), Leading Researcher.

serchenya@iboch.byИванькоМ. В.Ivan’koM. V.

Иванько Мария Валерьевна - мл. научный сотрудник.

Партизанский просп., 172, 220075, Минск

Mariya V. Ivan’ko - Junior Researcher.

ivanko.m-a@yandex.ruШкиндероваВ. О.ShkinderovaV. O.

Шкиндерова Валерия Олеговна - инженер-микробиолог.

Valeriya O. Shkinderova - Microbiologist Engineer.

ric-2010@yandex.ruПыжикИ. П.PyzhikI. P.

Пыжик Инесса Павловна - зав. сектором.

Партизанский просп., 172, 220075, Минск

Inessa P. Pyzhik - Head of the Sector.

ric-2010@yandex.ruСмолякТ. М.SmolyakT. M.

Смоляк ТатьянаМихайловна - заведующий лабораторией.

Tatyana M. Smolyak - Head of the Laboratory.

ric-2010@yandex.ruМелещеняА. В.MeleshchenyaA. V.

Мелещеня Алексей Викторович - канд. эконом. наук, директор.

Alexey V. Meleshchenya - Ph. D. (Economics), director.

172, Partizansky Ave., 220075, Minsk

info@instmmp.byСвиридовО. В.SviridovO. V.

Свиридов Олег Васильевич - доктор химических наук, заведующий лабораторией.

Oleg V Sviridov - D. Sc. (Chemistry), Head of the Laboratory.

5/2, Academician Kuprevich Str., 220141, Minsk

sviridov@iboch.byИнститут биоорганической химии, Национальная академия наук БеларусиInstitute of Bioorganic Chemistry, National Academy of Sciences of BelarusИнститут мясо-молочной промышленностиInstitute of Meat and Dairy Industry202028082020563318332Copyright © Вашкевич И.И., Ястребова А.А., Куприенко О.С., Серченя Т.С., Иванько М.В., Шкиндерова В.О., Пыжик И.П., Смоляк Т.М., Мелещеня А.В., Свиридов О.В., 20202020Вашкевич И.И., Ястребова А.А., Куприенко О.С., Серченя Т.С., Иванько М.В., Шкиндерова В.О., Пыжик И.П., Смоляк Т.М., Мелещеня А.В., Свиридов О.В.Vashkevich I.I., Yastrebova A.A., Kuprienko O.S., Serchenya T.S., Ivan’ko M.V., Shkinderova V.O., Pyzhik I.P., Smolyak T.M., Meleshchenya A.V., Sviridov O.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestichem.belnauka.by/jour/article/view/600

Разработаны тест-системы для непрямого и прямого иммуноферментного анализа (ИФА) пептидного антибиотика бацитрацина (Бц). Поликлональные антитела для обеих тест-систем получены иммунизацией кроликов конъюгатом Бц и гемоцианина улитки, который синтезировали взаимодействием пептида с белком в присутствии 1-этил-3-(3-диметиламинопропил)-карбодиимида (EDC). В системе непрямого ИФА твердофазным конъюгированным антигеном служил продукт присоединения Бц к тироглобулину, предварительно активированному EDC и N-гидроксисульфосукцинимидом. Для прямого ИФА на поверхности лунок микропланшета были иммунохимически иммобилизованы антитела к Бц, а жидкая фаза содержала конъюгат Бц с пероксидазой из корней хрена, полученный путем последовательных реакций присоединения аминогрупп Бц к окисленной периодатом углеводной части фермента и восстановления образовавшегося основания Шиффа борогидридом натрия. В системах прямого ИФА и непрямого ИФА связывание конъюгированных антигенов с антителами к Бц обусловливало максимальные колориметрические сигналы около 2,0 и 1,2 единиц оптической плотности соответственно и зависело от содержания Бц в жидкой фазе. При этом концентрации антибиотика, вызывающие ингибирование связывания на 50 %, составили 2,6 нг/мл в прямом ИФА и 10,0 нг/мл в непрямом ИФА. Простая и чувствительная система прямого ИФА послужила прототипом для создания готового набора реагентов и методики выполнения измерений, технико-аналитические параметры и метрологические характеристики которых позволяют определять остаточные количества Бц в пищевой продукции животного происхождения расширенного перечня, включающего 14 наименований, в диапазоне от 9,0 до 405,0 мкг/кг с надлежащей правильностью и точностью.

Two test-systems for a direct and an indirect enzyme-linked immunosorbent assay (ELISA) of peptide antibiotic bacitracin (BC) were developed and studied. For the both systems, polyclonal antibodies were obtained by immunizing rabbits with a conjugate of BC with keyhole limpet hemocyanine synthesized using reaction between the peptide and the high molecular weight protein in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The product of BC linking to thyroglobulin which was activated with EDC and N-hydroxysulfosuccinimide served as conjugated antigen on a solid phase in the indirect ELISA. For the direct ELISA, the antibodies against BC were immunochemically immobilized onto microplate surface, while the liquid phase contained a conjugate of BC with horseradish peroxidase. This conjugate was obtained by successive reactions of antibiotic amino groups coupling to periodate oxidized carbohydrate chains of enzyme and the reducting of formed Shiff’s base with sodium borohydride. Conjugated antigens binding to anti-BC antibodies provided maximum colorimetric signals of 2.0 and 1.2 optical units for the direct and indirect ELISA, respectively, and depended on BC content in the liquid phase. Antibiotic concentration that caused the inhibition of binding by 50 % was 2.6 ng/ml in the direct ELISA and 10.0 ng/ml in the indirect ELISA. The simple and sensitive direct ELISA system was used as a prototype of the finished reagent kit and a method for measurements with technical-analytical parameters and metrological characteristics allowing the determination of BC residues in a variety of foods including 14 items in a concentration range of 9.0 to 405.0 pg/kg with proper accuracy and precision.

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