RU2010140478A - Иммунологические анализы активности ботулинического токсина серотипа а - Google Patents
Иммунологические анализы активности ботулинического токсина серотипа а Download PDFInfo
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- 230000000694 effects Effects 0.000 title claims abstract 4
- 230000001900 immune effect Effects 0.000 title claims 2
- 239000003053 toxin Substances 0.000 title 1
- 231100000765 toxin Toxicity 0.000 title 1
- 108030001720 Bontoxilysin Proteins 0.000 claims abstract 28
- 102000004183 Synaptosomal-Associated Protein 25 Human genes 0.000 claims abstract 26
- 108010057722 Synaptosomal-Associated Protein 25 Proteins 0.000 claims abstract 26
- 238000003776 cleavage reaction Methods 0.000 claims abstract 25
- 230000007017 scission Effects 0.000 claims abstract 25
- 210000004027 cell Anatomy 0.000 claims abstract 16
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract 12
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 claims abstract 12
- 238000000034 method Methods 0.000 claims abstract 11
- 210000004899 c-terminal region Anatomy 0.000 claims abstract 10
- 239000000427 antigen Substances 0.000 claims abstract 6
- 102000036639 antigens Human genes 0.000 claims abstract 6
- 108091007433 antigens Proteins 0.000 claims abstract 6
- 238000010494 dissociation reaction Methods 0.000 claims abstract 4
- 230000005593 dissociations Effects 0.000 claims abstract 4
- 230000035987 intoxication Effects 0.000 claims abstract 4
- 231100000566 intoxication Toxicity 0.000 claims abstract 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 150000001413 amino acids Chemical class 0.000 claims abstract 2
- 239000000523 sample Substances 0.000 claims 7
- 241000124008 Mammalia Species 0.000 claims 2
- 238000001514 detection method Methods 0.000 claims 2
- 239000013642 negative control Substances 0.000 claims 2
- 230000003472 neutralizing effect Effects 0.000 claims 2
- 239000007790 solid phase Substances 0.000 claims 2
- 238000002965 ELISA Methods 0.000 claims 1
- 241000582786 Monoplex Species 0.000 claims 1
- 210000002966 serum Anatomy 0.000 claims 1
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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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