RU2016103262A - Способы идентификации иммунных связующих веществ поверхностных антигенов клетки - Google Patents

Способы идентификации иммунных связующих веществ поверхностных антигенов клетки Download PDF

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RU2016103262A
RU2016103262A RU2016103262A RU2016103262A RU2016103262A RU 2016103262 A RU2016103262 A RU 2016103262A RU 2016103262 A RU2016103262 A RU 2016103262A RU 2016103262 A RU2016103262 A RU 2016103262A RU 2016103262 A RU2016103262 A RU 2016103262A
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Валери ХУЛЬМАН-КОТЬЕ
Дэвид УРЕХ
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ИЭсБиЭйТЕК, ЭН АЛЬКОН БАЙОМЕДИКАЛ РИСЕРЧ ЮНИТ ЭлЭлСи
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Claims (25)


     1. Способ идентификации В-клеточного клона, который специфически связывается с мишеневым антигеном, предусматривающий:
(a) наличие множества В-клеток;
(b) взаимодействие множества В-клеток с мишеневым антигеном, где мишеневый антиген содержит первую сортируемую метку, и окрашивание множества В-клеток анти-IgG антителами, содержащими вторую сортируемую метку, и анти-IgM антителами, содержащими третью сортируемую метку;
(c) отделение от множества В-клеток одной или нескольких В-клеток, которые могут специфически связываться с мишеневым антигеном, используя клеточный сортер, где присутствие первой и второй сортируемой метки, но не третьей сортируемой метки в комплексе является индикатором связывания В-клетки с мишеневым антигеном, тем самым идентифицируя В-клеточный клон, который связывается с мишеневым антигеном.

     2. Способ по п. 1, где множество В-клеток представляет собой множество лимфоцитов.

     3. Способ по п. 1, где множество В-клеток окрашивается перед ее взаимодействием с мишеневым антигеном на стадии (b).

     4. Способ по п. 1, где множество В-клеток окрашивается после ее взаимодействия с мишеневым антигеном на стадии (b).

     5. Способ по п. 1, где идентификация В-клеточного клона включает клональное выделение В-клеток, полученных на стадии (c), с последующей необязательной клональной экспансией из клонально выделенных клеток.

     6. Способ по п. 5, дополнительно включающий выделение молекулы нуклеиновой кислоты, кодирующей иммунное связующее вещество из В-клеточного клона, которое связывается с мишеневым антигеном.

     7. Способ по п. 6, дополнительно включающий продукцию иммунного связующего вещества, способного связываться с мишеневым антигеном, причем иммунное связующее вещество получают путем введения последовательности нуклеиновой кислоты в экспрессионную среду так, чтобы происходил синтез кодируемого иммунного связующего вещества.

     8. Способ по п. 1, где одна или несколько отдельных B-клеток культивируют в подходящих условиях так, чтобы происходила секреция антител в культуральную среду.

     9. Способ по п. 8, где антитела тестируют на специфическое связывание с мишеневым антигеном.

     10. Способ по п. 8, где антитела представляют собой антитела мыши, кролика, крысы, хомячка, овцы, курицы, верблюда, козы, человека.

     11. Способ по п. 1, где сортируемые метки представляют собой флуорисцентные метки.

     12. Способ по п. 1, где мишеневый антиген представляет собой растворимый антиген.

     13. Способ по п. 1, где В-клетки представляют собой В-клетки кролика.

     14. Способ по п. 13, где кролик был иммунизирован мишеневым антигеном.

     15. Способ идентификации В-клеточного клона, который специфически связывается с мишеневым антигеном, предусматривающий:
(а) иммунизирование животного мишеневым антигеном;
(b) выделение множества В-клеток из иммунизированного животного;
(c) взаимодействие множества В-клеток с мишеневым антигеном, где мишеневый антиген содержит первую сортируемую метку, и окрашивание множества В-клеток анти-IgG антителами, содержащими вторую сортируемую метку, и анти-IgM антителами, содержащими третью сортируемую метку;
(d) отделение от множества В-клеток одной или нескольких В-клеток, которые могут специфически связываться с мишеневым антигеном, используя клеточный сортер, где присутствие первой и второй сортируемой метки, но не третьей сортируемой метки в комплексе является индикатором связывания В-клетки с мишеневым антигеном, тем самым идентифицируя В-клеточный клон, который связывается с мишеневым антигеном.

     16. Способ по п. 15, где сортируемые метки представляют собой флуорисцентные метки.

     17. Способ по п. 15, где мишеневый антиген представляет собой растворимый антиген.

     18. Способ по п. 15, где животным является кролик.
RU2016103262A 2009-02-24 2016-02-02 Способы идентификации иммунных связующих веществ поверхностных антигенов клетки RU2635186C2 (ru)

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