ES2632928T3 - Procedimiento de cultivo de linfocitos B simples - Google Patents

Procedimiento de cultivo de linfocitos B simples Download PDF

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ES2632928T3
ES2632928T3 ES11722402.2T ES11722402T ES2632928T3 ES 2632928 T3 ES2632928 T3 ES 2632928T3 ES 11722402 T ES11722402 T ES 11722402T ES 2632928 T3 ES2632928 T3 ES 2632928T3
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antibody
igg
mouse
fitc
lymphocytes
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Josef Endl
Natalie Schuhmacher
Sonja Offner
Josef Platzer
Basile Siewe
Irmgard Thorey
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F Hoffmann La Roche AG
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Abstract

Procedimiento de selección de un linfocito B que comprende las etapas siguientes: a) cocultivar cada uno de los linfocitos B de una población de linfocitos B, que se ha depositado como célula simple, con células EL-4 B5 murinas como células alimentadoras, b) seleccionar un clon de linfocitos B que haga proliferar y segregue anticuerpo en la etapa a), en la que el cocultivo es en presencia de una mezcla alimentadora sintética que comprende IL-1β, TNFα, IL-10, y uno o más seleccionados de IL-21, SAC, BAFF, IL-2, IL-4 e IL-6.

Description

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Se pueden marcar linfocitos B de ratón con el anticuerpo anti-CD27 235 o 236 (AB 235, AB 236), el anticuerpo anti-CD38 192 (AB 192), el anticuerpo anti-CD138 233 (AB 233) y el anticuerpo anti-CD268 246 (AB 246).
Tabla 5: Marcado por inmunofluorescencia para la determinación de linfocitos B de ratón (A-J), hámster (K) y conejo (L-N) maduros.
marcado
Marcado por inmunofluorescencia para separación linfocitos B de Porcentaje de todas las células viables %
A
IgG+CD19+ -AB 227 FITC, AB 218 PE 0,5 ± 0,2 n = 14
B
IgG+CD38+ -AB 227 FITC, AB 192 PE 0,8 ± 0,5 n = 9
C
IgG+CD138+ -AB 227 FITC, AB 233 PE 0,06 ± 0,07 n = 6
D
IgG+CD138+ -AB 227 FITC, AB 233 PE 0,6 ± 0,5 n = 6
E
IgG+CD27+ -AB 227 FITC, AB 235 PE 0,1 ± 0,1 n = 8
F
CD27+CD138+ -AB 236 A647, AB 233 PE 1,5 ± 0,5 n = 2
G
CD27+IgG+CD3 -AB 235 PE, AB 227 FITC, AB 241 A647 0,10 ± 0,04 n = 3
H
CD3 -CD27+ -AB 189 FITC, AB 235 PE 1,33 n = 1
I
IgG+CD268+ -AB 227 FITC, AB 246 A647 0,8 n = 1
J
CD38+ CD3+ -AB 192 A647, AB 189 PE 12 ± 7 n = 2
K
IgG+IgM -AB 213 A647, AB 224 FITC 0,6 ± 0,1 n = 15
L
IgG+ -AB 184 FITC 0,6 ± 0,2, n = 5
M
IgG+IgM -AB 184 FITC, AB 254 Biotina, SA 263 PE 0,4 ± 0,2, n = 2
N
IgG+CD138+ -AB 259, AB 256 PE 0,3 ± 0,1, n = 5
AB 184 -anticuerpo IgG anti-conejo de cabra AbD Serotec STAR121F AB 189 -anticuerpo CD3 anti-ratón de hámster Becton Dickinson 553062 AB 192 -anticuerpo CD38 anti-ratón de rata Becton Dickinson 553764 AB213 -anticuerpo IgG anti-hámster de ratón Becton Dickinson 554010 AB218 -anticuerpo CD19 anti-ratón de rata Abcam ab22480 AB224 -anticuerpo IgG anti-hámster de ratón Becton Dickinson 554033 AB227 -anticuerpo IgG anti-ratón de cabra Sigma F 8264 AB233 -anticuerpo CD138 anti-ratón de rata Becton Dickinson 553714 AB235 -anticuerpo CD27 anti-ratón de hámster Becton Dickinson 558754 AB236 -anticuerpo CD27 anti-ratón de hámster Becton Dickinson 558753 AB241 -anticuerpo CD3 anti-ratón de hámster Becton Dickinson 553060 AB246 -anticuerpo BAFF-R anti-ratón de rata eBioscience 51-5943 AB254 -anticuerpo IgM anti-conejo de ratón Becton Dickinson personalizado AB256 -anticuerpo IgG anti-rata de cabra Southern Biotech 3030-09 AB259 -anticuerpo CD138 anti-conejo de rata Roche Glycart AG SA 263 -Estreptavidina Invitrogen S866 A647: Alexa Fluor® 647 FITC: Isotiocianato de fluoresceína
En un modo de realización los procedimientos comprenden la etapa de reducir la población de linfocitos B de 10 macrófagos y enriquecer los linfocitos B de la población de linfocitos B que segregan anticuerpos que se unen de forma específica a un antígeno diana.
Depósito de células simples:
15 El procedimiento del que se informa en el presente documento comprende la etapa de depositar los linfocitos B de una población de linfocitos B como células simples. En un modo de realización de todos los procedimientos de los que se informa en el presente documento el depósito como células simples es por separación de células activadas
10
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Claims (1)

  1. imagen1
ES11722402.2T 2010-05-28 2011-05-26 Procedimiento de cultivo de linfocitos B simples Active ES2632928T3 (es)

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EP10005602.7A EP2400298B1 (en) 2010-05-28 2010-05-28 Single B-cell cultivation method and specific antibody production
EP10005602 2010-05-28
PCT/EP2011/058616 WO2011147903A1 (en) 2010-05-28 2011-05-26 Single b-cell cultivation method

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ES2632928T3 true ES2632928T3 (es) 2017-09-18

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ES10005602T Active ES2434256T3 (es) 2010-05-28 2010-05-28 Método de cultivo de células B individuales y producción de anticuerpos específicos
ES17169362T Active ES2858450T3 (es) 2010-05-28 2011-05-26 Procedimiento de cultivo de linfocitos B individuales
ES11722402.2T Active ES2632928T3 (es) 2010-05-28 2011-05-26 Procedimiento de cultivo de linfocitos B simples
ES20214670T Active ES3039652T3 (en) 2010-05-28 2011-05-26 Single b-cell cultivation method

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ES10005602T Active ES2434256T3 (es) 2010-05-28 2010-05-28 Método de cultivo de células B individuales y producción de anticuerpos específicos
ES17169362T Active ES2858450T3 (es) 2010-05-28 2011-05-26 Procedimiento de cultivo de linfocitos B individuales

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ES20214670T Active ES3039652T3 (en) 2010-05-28 2011-05-26 Single b-cell cultivation method

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EP (4) EP2400298B1 (es)
JP (3) JP5779239B2 (es)
KR (1) KR101495976B1 (es)
CN (2) CN102918395B (es)
BR (1) BR112012025695B1 (es)
CA (2) CA2798286C (es)
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MX (2) MX2012013437A (es)
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JP2015500655A (ja) * 2011-12-21 2015-01-08 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 同起源の抗体可変領域遺伝子セグメントをクローニングおよび発現させるための迅速な方法
US10017739B2 (en) 2012-09-06 2018-07-10 Duke University Methods of expanding and assessing B cells and using expanded B cells to treat disease
EP2914629A1 (en) 2012-11-05 2015-09-09 MAB Discovery GmbH Method for the production of multispecific antibodies
EP2727943A1 (en) 2012-11-05 2014-05-07 MAB Discovery GmbH Trispecific antibodies against human EGFR, HER2 and HER3
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