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RU2010140478A - Иммунологические анализы активности ботулинического токсина серотипа а - Google Patents

Иммунологические анализы активности ботулинического токсина серотипа а Download PDF

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RU2010140478A
RU2010140478A RU2010140478/10A RU2010140478A RU2010140478A RU 2010140478 A RU2010140478 A RU 2010140478A RU 2010140478/10 A RU2010140478/10 A RU 2010140478/10A RU 2010140478 A RU2010140478 A RU 2010140478A RU 2010140478 A RU2010140478 A RU 2010140478A
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Эстер ФЕРНАНДЕЗ-САЛАС (US)
Эстер ФЕРНАНДЕЗ-САЛАС
Джоанн ВАНГ (US)
Джоанн ВАНГ
Паттон Е. ГЭРЭЙ (US)
Паттон Е. ГЭРЭЙ
Лина С. ВОНГ (US)
Лина С. ВОНГ
Д. Дайанн ХОДЖЕС (US)
Д. Дайанн ХОДЖЕС
Кей Роджер АОКИ (US)
Кей Роджер АОКИ
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Abstract

1. Композиция, содержащая носитель, гибкий линкер и антиген SNAP-25, где гибкий линкер содержит по меньшей мере три аминокислоты, и антиген SNAP-25 содержит SEQ ID NO:148. ! 2. Композиция по п.2, где аминокислотная последовательность гибкого линкера и антигена SNAP-25 представляет собой SEQ ID NO:38. ! 3. Выделенное антитело α-SNAP-25, ! где выделенные антитело α-SNAP-25 связывается с эпитопом, содержащим C-концевой глутамин расщепляемой связи сайта расщепления BoNT/A продукта расщепления SNAP-25; ! где антитело α-SNAP-25 имеет константу скорости ассоциации для эпитопа, не содержащего C-концевой глутамин расщепляемой связи сайта расщепления BoNT/A продукта расщепления SNAP-25, составляющую менее 1·101 М-1 с1; и ! где антитело α-SNAP-25 имеет равновесную константу диссоциации для эпитопа, составляющую менее 0,450 нМ. ! 4. Выделенные антитело α-SNAP-25 по п.3, где эпитоп представляет собой SEQ ID NO:32. ! 5. Выделенное антитело α-SNAP-25, содержащее: ! а. вариабельную область тяжелой цепи, содержащую SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:80 или SEQ ID NO:82; и ! б. вариабельную область легкой цепи, содержащую SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90 или SEQ ID NO:92. ! 6. Выделенное антитело α-SNAP-25, содержащее: ! а. вариабельную область тяжелой цепи, содержащую SEQ ID NO:93, SEQ ID NO:121 или SEQ ID NO:100; и ! б. вариабельную область легкой цепи, содержащую SEQ ID NO:105, SEQ ID NO:110 или SEQ ID NO:115. ! 7. Выделенное антитело α-SNAP-25, содержащее по меньшей мере VH CDR3 из SEQ ID NO:100, VH CDR3 из SEQ ID NO:101 или VH CDR3 из SEQ ID NO:102. ! 8. Способ обнаружения активности BoNT/A, включающий стадии: ! а. обработки клетки из стабильной клеточной линии образцом, содержащим BoNT/A, где клетка из стабильной клеточной линии чувствительна к интоксикации BoNT/A в концентрации BoNT/A приблизительно 500 пМ или менее

Claims (15)

1. Композиция, содержащая носитель, гибкий линкер и антиген SNAP-25, где гибкий линкер содержит по меньшей мере три аминокислоты, и антиген SNAP-25 содержит SEQ ID NO:148.
2. Композиция по п.2, где аминокислотная последовательность гибкого линкера и антигена SNAP-25 представляет собой SEQ ID NO:38.
3. Выделенное антитело α-SNAP-25,
где выделенные антитело α-SNAP-25 связывается с эпитопом, содержащим C-концевой глутамин расщепляемой связи сайта расщепления BoNT/A продукта расщепления SNAP-25;
где антитело α-SNAP-25 имеет константу скорости ассоциации для эпитопа, не содержащего C-концевой глутамин расщепляемой связи сайта расщепления BoNT/A продукта расщепления SNAP-25, составляющую менее 1·101 М-1 с1; и
где антитело α-SNAP-25 имеет равновесную константу диссоциации для эпитопа, составляющую менее 0,450 нМ.
4. Выделенные антитело α-SNAP-25 по п.3, где эпитоп представляет собой SEQ ID NO:32.
5. Выделенное антитело α-SNAP-25, содержащее:
а. вариабельную область тяжелой цепи, содержащую SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:80 или SEQ ID NO:82; и
б. вариабельную область легкой цепи, содержащую SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90 или SEQ ID NO:92.
6. Выделенное антитело α-SNAP-25, содержащее:
а. вариабельную область тяжелой цепи, содержащую SEQ ID NO:93, SEQ ID NO:121 или SEQ ID NO:100; и
б. вариабельную область легкой цепи, содержащую SEQ ID NO:105, SEQ ID NO:110 или SEQ ID NO:115.
7. Выделенное антитело α-SNAP-25, содержащее по меньшей мере VH CDR3 из SEQ ID NO:100, VH CDR3 из SEQ ID NO:101 или VH CDR3 из SEQ ID NO:102.
8. Способ обнаружения активности BoNT/A, включающий стадии:
а. обработки клетки из стабильной клеточной линии образцом, содержащим BoNT/A, где клетка из стабильной клеточной линии чувствительна к интоксикации BoNT/A в концентрации BoNT/A приблизительно 500 пМ или менее;
б. выделения из обработанной клетки компонента SNAP-25, содержащего продукт расщепления SNAP-25, имеющий С-концевой глутамин расщепляемой связи сайта расщепления BoNT/A;
в. приведения в контакт компонента SNAP-25 с антителом α-SNAP-25, связанным с твердофазным носителем,
где антитело α-SNAP-25 связывается с эпитопом, содержащим С-концевой глутамин расщепляемой связи сайта расщепления BoNT/A продукта расщепления SNAP-25, где антитело α-SNAP-25 имеет константу скорости ассоциации для эпитопа из SNAP-25, не содержащего С-концевой глутамин расщепляемой связи сайта расщепления BoNT/A продукта расщепления SNAP-25, составляющую менее 1·101 М-1 с-1; и антитело α-SNAP-25 имеет равновесную константу диссоциации для эпитопа, составляющую мене 0,450 нМ;
г. обнаружения наличия комплекса антиген-антитело, содержащего антитело α-SNAP-25 и продукт расщепления SNAP-25, имеющий С-концевой глутамин расщепляемой связи сайта расщепления BoNT/A;
где обнаружение при помощи комплекса антиген-антитело является показателем активности BoNT/A.
9. Способ по п.8, где продукт расщепления SNAP-25 представляет собой SNAP-25197.
10. Способ по п.8, где присутствие комплекса антиген-антитело обнаруживают с использованием сэндвич ИФА.
11. Способ по п.8, где способ имеет отношение сигнала к шуму на нижней асимптоте по меньшей мере 3:1 и отношение сигнала к шуму на верхней асимптоте по меньшей мере 10:1.
12. Способ по п.8, где образец содержит максимально 100 пМ BoNT/A.
13. Способ по п.8, где клетка из стабильной клеточной линии чувствительна к интоксикации BoNT/A в концентрации BoNT/A приблизительно 100 пМ или менее.
14. Способ по п.8, где способ осуществляют в моноплексном варианте или мультиплексном варианте.
15. Способ определения иммунорезистентности к BoNT/A у млекопитающего, включающий стадии:
а. добавления BoNT/A к тестируемому образцу, полученному у млекопитающего, тестируемого на наличие или отсутствие антител, нейтрализующих α-BoNT/А;
б. обработки клетки из стабильной клеточной линии тестируемым образцом, где клетка из стабильной клеточной линии чувствительна к интоксикации BoNT/A;
в. выделения из обработанных клеток компонента SNAP-25, содержащего продукт расщепления SNAP-25, имеющего C-концевой глутамин расщепляемой связи сайта расщепления BoNT/A;
г. приведения в контакт компонента SNAP-25 с антителом α-SNAP-25, связанным с твердофазным носителем;
где антитело α-SNAP-25 связывается с эпитопом, содержащим C-концевой глутамин расщепляемой связи сайта расщепления BoNT/A продукта расщепления SNAP-25, где антитело α-SNAP-25 имеет константу скорости ассоциации для эпитопа из SNAP-25, не содержащего C-концевой глутамин расщепляемой связи сайта расщепления BoNT/A продукта расщепления SNAP-25, составляющую менее 1·101 М-1 с-1; и антитело α-SNAP-25 имеет равновесную константу диссоциации для эпитопа, составляющую менее 0,450 нМ;
д. обнаружения наличия комплекса антиген-антитело, содержащего антитело α-SNAP-25 и продукт расщепления SNAP-25, имеющий C-концевой глутамин расщепляемой связи сайта расщепления BoNT/A;
е. повторения стадий б-д с отрицательным контрольным образцом вместо тестируемого образца, где отрицательный контрольный образец содержит BoNT/A и сыворотку, о которой известно, что она не содержит нейтрализующие антитела α-BoNT/A; и
ж. сравнения количества комплекса антиген-антитело, обнаруженного на стадии д, с количеством комплекса антиген-антитело, обнаруженного на стадии е,
где обнаружение меньшего количества комплекса антиген-антитело, обнаруженного на стадии д, по сравнению с количеством комплекса антиген-антитело, обнаруженного на стадии е, является показателем присутствия нейтрализующих антител α-BoNT/A.
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