RU2015155197A - Способ культивирования одиночной в-клетки - Google Patents
Способ культивирования одиночной в-клетки Download PDFInfo
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- RU2015155197A RU2015155197A RU2015155197A RU2015155197A RU2015155197A RU 2015155197 A RU2015155197 A RU 2015155197A RU 2015155197 A RU2015155197 A RU 2015155197A RU 2015155197 A RU2015155197 A RU 2015155197A RU 2015155197 A RU2015155197 A RU 2015155197A
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- 238000000034 method Methods 0.000 title claims 13
- 210000004027 cell Anatomy 0.000 claims 18
- 210000003719 b-lymphocyte Anatomy 0.000 claims 10
- 239000000203 mixture Substances 0.000 claims 5
- 238000002372 labelling Methods 0.000 claims 3
- 150000007523 nucleic acids Chemical class 0.000 claims 3
- 102000039446 nucleic acids Human genes 0.000 claims 3
- 108020004707 nucleic acids Proteins 0.000 claims 3
- 241000283973 Oryctolagus cuniculus Species 0.000 claims 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims 2
- 210000004369 blood Anatomy 0.000 claims 2
- 239000008280 blood Substances 0.000 claims 2
- 239000012228 culture supernatant Substances 0.000 claims 2
- LOGFVTREOLYCPF-KXNHARMFSA-N (2s,3r)-2-[[(2r)-1-[(2s)-2,6-diaminohexanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxybutanoic acid Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-KXNHARMFSA-N 0.000 claims 1
- KINMYBBFQRSVLL-UHFFFAOYSA-N 4-(4-phenoxybutoxy)furo[3,2-g]chromen-7-one Chemical compound C1=2C=COC=2C=C2OC(=O)C=CC2=C1OCCCCOC1=CC=CC=C1 KINMYBBFQRSVLL-UHFFFAOYSA-N 0.000 claims 1
- 230000003844 B-cell-activation Effects 0.000 claims 1
- 102000003777 Interleukin-1 beta Human genes 0.000 claims 1
- 108090000193 Interleukin-1 beta Proteins 0.000 claims 1
- 102000003814 Interleukin-10 Human genes 0.000 claims 1
- 108090000174 Interleukin-10 Proteins 0.000 claims 1
- 108010002350 Interleukin-2 Proteins 0.000 claims 1
- 102000000588 Interleukin-2 Human genes 0.000 claims 1
- 102100030704 Interleukin-21 Human genes 0.000 claims 1
- 102000004388 Interleukin-4 Human genes 0.000 claims 1
- 108090000978 Interleukin-4 Proteins 0.000 claims 1
- 102000004889 Interleukin-6 Human genes 0.000 claims 1
- 108090001005 Interleukin-6 Proteins 0.000 claims 1
- 241001529936 Murinae Species 0.000 claims 1
- 239000004793 Polystyrene Substances 0.000 claims 1
- 241000191967 Staphylococcus aureus Species 0.000 claims 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 239000000427 antigen Substances 0.000 claims 1
- 102000036639 antigens Human genes 0.000 claims 1
- 108091007433 antigens Proteins 0.000 claims 1
- 230000027455 binding Effects 0.000 claims 1
- 238000004113 cell culture Methods 0.000 claims 1
- 238000005119 centrifugation Methods 0.000 claims 1
- 238000003501 co-culture Methods 0.000 claims 1
- 230000003247 decreasing effect Effects 0.000 claims 1
- 239000007850 fluorescent dye Substances 0.000 claims 1
- 238000011534 incubation Methods 0.000 claims 1
- 229940076144 interleukin-10 Drugs 0.000 claims 1
- 108010074108 interleukin-21 Proteins 0.000 claims 1
- 229940028885 interleukin-4 Drugs 0.000 claims 1
- 229940100601 interleukin-6 Drugs 0.000 claims 1
- 210000003519 mature b lymphocyte Anatomy 0.000 claims 1
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 238000004091 panning Methods 0.000 claims 1
- 239000000088 plastic resin Substances 0.000 claims 1
- 229920000642 polymer Polymers 0.000 claims 1
- 229920002223 polystyrene Polymers 0.000 claims 1
- 230000002062 proliferating effect Effects 0.000 claims 1
- 238000010839 reverse transcription Methods 0.000 claims 1
- 238000012163 sequencing technique Methods 0.000 claims 1
- 230000009870 specific binding Effects 0.000 claims 1
- 230000004936 stimulating effect Effects 0.000 claims 1
- 239000000758 substrate Substances 0.000 claims 1
- 102000003390 tumor necrosis factor Human genes 0.000 claims 1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0635—B lymphocytes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Claims (24)
1. Способ получения B-клетки, включающий следующие этапы:
а) получение B-клеток из крови кролика,
б) мечение IgG+-B-клеток, и/или CD138+-B-клеток,
в) инкубация В-клеток при температуре 37°C в течение одного часа в среде для совместного культивирования перед размещением меченых B-клеток в виде одиночных клеток,
г) культивирование размещенных по отдельности клеток вместе с фидерными клетками в среде для совместного культивирования,
д) выбор B-клетки, пролиферирующей на этапе г) и, тем самым, получение B-клетки.
2. Способ по п. 1, отличающийся тем, что способ включает этап центрифугирования клеток, размещенных в виде одиночных клеток, перед совместным культивированием.
3. Способ по п. 1, отличающийся тем, что способ включает непосредственно перед этапом мечения следующий этап: аб) пэннинг B-клеток с иммобилизованным антигеном.
4. Способ по п. 1, отличающийся тем, что совместное культивирование осуществляют в полистироловых многолуночных планшетах, покрытых неволокнистым субстратом, изготовленным из смеси полимерной пластической смолы и амфипатических молекул.
5. Способ по п. 1, отличающийся тем, что B-клетки получают центрифугированием в градиенте плотности.
6. Способ по п. 1, отличающийся тем, что фидерные клетки являются мышиными клетками EL-4 B5.
7. Способ по любому из пп. 1-6, отличающийся тем, что среда для совместного культивирования содержит фидерную смесь.
8. Способ по п. 7, отличающийся тем, что указанная фидерная смесь представляет собой культуральный супернатант тимоцитов.
9. Способ по п. 7, отличающийся тем, что указанная фидерная смесь содержит интерлейкин-1 бета и фактор некроза опухоли альфа, а также по меньшей мере один компонент, выбранный из интерлейкина-2, интерлейкина-10, клеток золотистого стафилококка штамма Cowan, интерлейкина-21, B-клеточного фактора активации из семейства фактора некроза опухоли (BAFF), интерлейкина-6, интерлейкина-4, 5-(4-феноксибутокси)псоралена и других стимулирующих компонентов, увеличивающих способность B-клеточного клона к продукции IgG, не уменьшая число IgG+-клеток.
10. Способ получения антитела, включающий следующие этапы, при которых:
а) берут популяцию зрелых В-клеток, полученную из крови кролика,
б) осуществляют мечение IgG+-B-клеток, и/или CD138+-B-клеток по меньшей мере одной флуоресцентной меткой,
в) инкубируют B-клетки при температуре 37°C в течение одного часа в среде для совместного культивирования перед размещением одиночных клеток из популяции меченых B-клеток в отдельные контейнеры,
г) культивируют размещенные по отдельности В-клетки в присутствии фидерных клеток и фидерной смеси,
д) определяют специфичность связывания антител, секретируемых в среду для культивирования отдельных В-клеток,
е) определяют аминокислотную последовательность вариабельных доменов легкой и тяжелой цепей специфически связывающихся антител путем ПЦР с обратной транскрипцией и путем секвенирования нуклеотидов, тем самым получая нуклеиновую кислоту, кодирующую вариабельные домены легкой и тяжелой цепей моноклонального антитела,
ж) вводят нуклеиновую кислоту, кодирующую вариабельные домены легкой и тяжелой цепей моноклонального антитела, в экспрессионную кассету для экспрессии антитела,
з) вводят нуклеиновую кислоту в клетку,
и) культивируют клетку и выделяют антитело из клетки или клеточного культурального супернатанта, тем самым получая антитело.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP10005602.7A EP2400298B1 (en) | 2010-05-28 | 2010-05-28 | Single B-cell cultivation method and specific antibody production |
| EP10005602.7 | 2010-05-28 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| RU2012153785/15A Division RU2575569C2 (ru) | 2010-05-28 | 2011-05-26 | Способ культивирования одиночной в-клетки |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| RU2015155197A true RU2015155197A (ru) | 2019-01-16 |
| RU2015155197A3 RU2015155197A3 (ru) | 2019-07-17 |
| RU2709531C2 RU2709531C2 (ru) | 2019-12-18 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| RU2015155197A RU2709531C2 (ru) | 2010-05-28 | 2011-05-26 | Способ культивирования одиночной в-клетки |
Country Status (15)
| Country | Link |
|---|---|
| US (4) | US20130084637A1 (ru) |
| EP (4) | EP2400298B1 (ru) |
| JP (3) | JP5779239B2 (ru) |
| KR (1) | KR101495976B1 (ru) |
| CN (2) | CN102918395B (ru) |
| BR (1) | BR112012025695B1 (ru) |
| CA (2) | CA3008822C (ru) |
| DK (1) | DK2400298T3 (ru) |
| ES (3) | ES2434256T3 (ru) |
| HK (2) | HK1181112A1 (ru) |
| MX (2) | MX2012013437A (ru) |
| PL (2) | PL2400298T3 (ru) |
| RU (1) | RU2709531C2 (ru) |
| SI (1) | SI2400298T1 (ru) |
| WO (1) | WO2011147903A1 (ru) |
Families Citing this family (26)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2794652B1 (en) | 2011-12-21 | 2017-11-15 | F. Hoffmann-La Roche AG | Rapid method for cloning and expression of cognate antibody variable region gene segments |
| 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 |
| EP2727941A1 (en) | 2012-11-05 | 2014-05-07 | MAB Discovery GmbH | Method for the production of multispecific antibodies |
| EP2914629A1 (en) | 2012-11-05 | 2015-09-09 | MAB Discovery GmbH | Method for the production of multispecific antibodies |
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