Разработка и сравнительная характеристика систем экспрессного иммунохимического определения микотоксинов тема диссертации и автореферата по ВАК РФ 03.01.04, кандидат химических наук Урусов, Александр Евгеньевич

• Урусов, Александр Евгеньевич
• кандидат химических наук
• 2012

Оглавление диссертации кандидат химических наук Урусов, Александр Евгеньевич

Оглавление

СПИСОК СОКРАЩЕНИЙ

ВВЕДЕНИЕ

Глава 1. Обзор литературы

1.1. Разнообразие микотоксинов и их распространение

1.2. Методы определения микотоксинов

1.3. Характеристика иммунохимических методов анализа микотоксинов

1.4. Иммуноаффинные колонки

1.5. Иммуноферментный анализ

1.6. Иммунофильтрация

1.7. Иммунохроматографические тесты

1.8. Поляризационный флуоресцентный анализ

1.9. Иммуносенсоры на основе поверхностного плазмонного резонанса

1.10. Электрохимические иммуносенсоры

1.11. Пьезоэлектрические сенсоры

1.12. Коммерческие иммуноаналитические системы

Глава 2. Материалы и методы —.—---------------------------

2.1. Материалы

2.2. Биотинилирование антител

2.3. Иммуноферментный анализ микотоксинов

2.4. Характеристика иммунохимического взаимодействия

с использованием проточной сенсорной системы Biacore X

2.5. Синтез конъюгатов коллоидного золота с антителами

2.6. Изготовление и хранение иммунохроматографических тест-полосок

2.7. Проведение иммунохроматографического анализа охратоксина А

и афлатоксина В1

2.8. Усиление сигнала коллоидным золотом в проточной сенсорной системе Biacore X

2.9. Иммунохроматографический анализ афлатоксина Ble усилением сигнала

РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ

Глава 3. Изучение свойств реагентов,

используемых в иммуноаналитических системах

3.1. Характеристика антител против микотоксинов методом

иммуноферментного анализа

3.2. Сравнение способов введения метки в иммуноферментных

системах определения микотоксинов

3.3. Оптимизация продолжительности стадий иммуноферментного анализа

3.4. Оптимизация концентрации метанола дня иммуноферментного

анализа проб, содержащих микотоксины

3.5. Исследование иммунохимических взаимодействий с использованием оптического сенсора 1&асоге

Глава 4. Синтез и характеристика конъшгатов специфических антител

с коллоидным золотом

4.1. Характеристика состава конъюгатов антитела - коллоидное золото

4.2. Характеристика иммунохимических свойств конъюгатов антитело-коллоидное золото, основанная на определении эквивалентного количества свободных антител

4.3. Сравнение конъюгатов антитела - коллоидное золото разного состава

в иммунохроматографическом анализе

Глава 5. Разработка иммунохроматографических тест-систем

определения охратоксина А и афлатоксина В1

5.1. Выбор оптимальных рабочих мембран для иммунохроматографического определения микотоксинов

5.2. Сравнение и выбор мембран для тестируемого образца

5.3. Апробация иммунохроматографических тест-систем

для определения микотоксинов в растительных экстрактах

Глава 6. Усиление сигнала в иммуноанализе коллоидным золотом

6.1. Применение коллоидного золота для усиления сигнала

в иммуносенсорном анализе

6.2. Иммунохроматографичеекий анализ с разделением стадий специфического взаимодействия и введения коллоидного маркера

ВЫВОДЫ

СПИСОК ЛИТЕРАТУРЫ

АГ

антиАТ

АТ

АФВ1

БСА

ВЭЖХ

ГХ

ДОН

жх/мс зхвк

ИФА

ИХА

КЗ

ОТА

о.е.

ППР

ТБСА

ТМБ

тех

ФБС

ФБСТ

ЕБС

Ш8

ОБ

БШ

СПИСОК СОКРАЩЕНИЙ

антиген

антивидовые антитела антитело афлатоксин В1

бычий сывороточный альбумин высокоэффективная жидкостная хроматография газовая хроматография дезоксиниваленол

жидкостная хроматография с масс-спектрометрией

золотохлористоводородная кислота

иммуноферментный анализ

иммунохроматографический анализ

коллоидное золото

охратоксин А

относительная единица

поверхностный плазмонный резонанс

10 мМ трис-буфер, рН 8.5, с 1% БСА и 1% сахарозы

3,3',5,5 '-тетраметилбензидин

тонкослойная хроматография

50 мМ К-фосфатный буфер, рН 7,4, содержащий 0,1 М №С1

ФБС, содержащий 0,05% Тритона Х-100

1-этил-3-(3-димеггиламинопропил) карбодшшида гидрохлорид

иммуноглобулин класса в

И-гидроксисукцинимид

оптическая плотность

резонансная единица

Введение диссертации (часть автореферата) на тему «Разработка и сравнительная характеристика систем экспрессного иммунохимического определения микотоксинов»

ВВЕДЕНИЕ

Развитие нормативной базы пищевой безопасности - как на национальном, так и на международном уровне - расширяет ряд потенциально опасных контролируемых соединений и предъявляет ужесточающиеся требования к их предельно допустимому содержанию [1]. Среди объектов контроля особое место занимают микотоксины -низкомолекулярные вторичные метаболиты плесневых грибов. Контаминация продуктов микотоксинами является серьезной проблемой для сельского хозяйства и здравоохранения практически всех стран мира. Обладая высокой токсичностью, данные соединения при попадании в продукты питания и корма могут причинить значительный вред здоровью человека и сельскохозяйственных животных. Их деконтаминация крайне затруднена - микотоксины стабильны при нагревании и обработке химическими агентами. Попадая в сырье, они не разлагаются в ходе технологических процессов и сохраняются до конечного продукта производства. Мониторинг содержания данных соединений на всех стадиях производства, транспортировки и хранения является необходимой и сложной задачей, решение которой требует наличия достоверных, чувствительных и производительных методов. Традиционные физико-химические методы анализа (прежде всего хроматографические), хотя и обеспечивают высокую чувствительность, сопряжены с использованием дорогого стационарного оборудования и длительными процедурами пробоподготовки. В связи с этим крайне актуально создание иммунохимических методов определения микотоксинов, сочетающих экспрессность, низкую стоимость и простоту применения. Однако, так как допустимые уровни содержания данных соединений в пищевых продуктах крайне низки, известные иммуно-химические методы их определения не всегда удовлетворяют практическим требованиям.

В связи с этим данное исследование направлено на разработку новых подходов с использованием конъюгатов антител и наночастиц коллоидного золота для создания двух высокочувствительных и экспрессных систем определения микотоксинов - им-мунохроматографического анализа и иммуносенсорного анализа на основе эффекта поверхностного плазменного резонанса.

Несмотря на широкое применение конъюгатов антител с коллоидным золотом в аналитических целях, вопросы, связанные с влиянием состава конъюгатов и реакци-

онной способности антител в них на характеристики иммуноаналитических систем, остаются малоизученными. Проведенное в работе исследование этих взаимосвязей позволяет направленно оптимизировать иммуноаналигические системы с применением коллоидного золота и расширить возможности их использования для детектирования как микотоксинов, так и других биологически активных соединений.

Целые настоящей работы являлось изучение взаимодействий нативных и иммобилизованных на коллоидном золоте антител с микотоксинами для разработки универсальных подходов, позволяющих снизить пределы обнаружения иммуноаналитических систем.

Достижение поставленной цели включало решение следующих задач:

• изучить взаимосвязь между составом и свойствами конъюгатов антител с коллоидным золотом;

• разработать и охарактеризовать методы усиления сигнала в проточных аналитических системах;

• провести сравнительную оценку пределов обнаружения микотоксинов в различных форматах иммуноанализа;

• апробировать разработанные тест-системы для анализа растительных проб, содержащих микотоксины.

Заключение диссертации по теме «Биохимия», Урусов, Александр Евгеньевич

выводы

1. Предложен метод характеристики иммунохимических свойств конъюгатов антитела - коллоидное золото, основанный на определении эквивалентного количества свободных антител. Установлен характер зависимости реакционной способности синтезируемых конъюгатов от количества специфических иммуноглобулинов.

2. Разработан высокочувствительный метод определения микотоксинов в иммуно-сенсорной системе с оптической регистрацией поверхностного плазмонного резонанса с использованием конъюгатов антивидовые антитела - коллоидное золото. Показано существенное усиление сигнала и снижение предела обнаружения до 40 пг/мл по сравнению с регистрацией взаимодействия с немечеными специфическими антителами.

3. Определены условия проведения иммунохроматографического анализа для контроля содержания микотоксинов в пробах с высоким содержанием органического растворителя (до 35% метанола). Пределы обнаружения в растительных экстрактах при визуальной детекции результатов составляют 10 нг/мл для афлатоксина В1 и 50 нг/мл для охратоксина А, а при видеоцифровой регистрации - 0,2 и 2 нг/мл, соответственно.

4. Предложен новый метод иммунохроматографического анализа, основанный на разделении стадий специфического взаимодействия и введения коллоидного маркера. Предел обнаружения афлатоксина В1 данным методом составляет 100 пг/мл, что в 10 раз ниже по сравнению с традиционным форматом анализа.

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