JP4603894B2 - 抗体産生細胞を同定するためのアッセイ - Google Patents
抗体産生細胞を同定するためのアッセイ Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/243—Colony Stimulating Factors
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
- C07K16/245—IL-1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Description
a)抗体産生細胞集団を提供すること、
b)前記抗体産生細胞集団を選択した抗原及び標識抗抗体抗体と共にインキュベートすることであって、前記抗抗体抗体は前記選択した抗原に結合する抗体を産生する細胞とそうでない細胞とを区別することができること、及び
c)前記選択した抗原に結合する抗体を産生する抗体産生細胞を同定すること
を含む、選択した抗原に結合する抗体を産生する抗体産生細胞を同定するための均一系アッセイを提供する。
a)抗体産生細胞集団を提供すること、
b)前記抗体産生細胞集団を選択した抗原及び標識抗抗体抗体と共にインキュベートすることであって、前記抗抗体抗体は選択した抗原に結合する抗体を産生する細胞とそうでない細胞とを区別することができること、
c)選択した抗原に結合する抗体を産生する抗体産生細胞を同定すること、
d)抗体産生細胞を単離して同定すること、及び場合によっては、
e)それらから得られた抗体を合成すること
を含む選択した抗原に結合する抗体の産生方法を提供する。所望するならば、工程(d)及び(e)を複数回繰り返して、複数の抗体産生細胞を単離し、複数の抗体を合成することができる。したがって、本発明は、前記の方法によって同定された少なくとも一種類の抗体産生細胞及び前記細胞から合成された少なくとも一種類の抗体にも適用される。
ICM−免疫細胞培地(RPMI+10%牛胎児血清、2−β−メルカプトエタノール50μM、グルタミン2mM、hepes20mM並びに1×ペニシリン及びストレプトマイシン)
RPMI−Roswell Park Memorial Institute培地
PBS−リン酸緩衝生理食塩水
SRBCの抗原コーティング
(TCS Biosciencesから入手した)SRBCのコーティングは、ビオチン化抗原をビオチンコーティングSRBCの表面へ結合させることによって実施した。抗原コーティングSRBCは、使用日に調製し、免疫細胞培地中で5%(v/v)で保存した。
該測定混合物はICM中で調製し、ELISA陽性集団の10〜1000個のB細胞を含有するウサギB細胞10μl、抗原コーティングSRBC(5%v/v)10μl及び各実験について様々な濃度のヤギ抗ウサギIgGFc特異的FITC複合体(Jackson ImmunoResearch)(1:100、1:200、1:400及び1:800)20μlを含めた。該実験は、蛍光のバックグラウンドを高くせずに抗体産生細胞を同定するために必要なヤギ抗ウサギIgGFc特異的FITC複合体の最適濃度を決定するために設定した。
いずれの実験においても、ストレプトアビジンコーティング磁気ビーズ(New England Biolabs)1μMを使用した。
磁石を使用してビーズ保存物一定量をPBSで3回洗浄して保存剤を除去し、同量の免疫細胞培地、(ICM)、(RPMI+10%牛胎児血清、2−β−メルカプトエタノール50μM、グルタミン2mM、Hepes 20mM、及び1×ペニシリン及びストレプトマイシン)に再懸濁した。
ストレプトアビジンコーティングビーズを50μl量ずつ洗浄して、PBS 50μlに再懸濁した。50μl量それぞれを0.1μgから25μgまでの範囲の異なる量のビオチン化抗原保存物(1mg/ml)と共にインキュベートした。これらを室温で時々手動で振盪しながらインキュベートした。次に磁石を使用して該ビーズをPBSで洗浄し、ICM 50μlに再懸濁した。
COS−1細胞上における抗原の一時的発現
選択抗原を一時的に発現するCOS−1細胞を免疫細胞培地に懸濁した。細胞密度は、1ml当たり細胞2×107個に変更した。
該測定混合物はICM中で調製し、ELISA陽性B細胞40μl、1:400に希釈したヤギ抗ウサギIgG Fc特異的FITC複合体(Jackson ImmunoResearch)40μl及びCOS−1細胞懸濁液40μlを含めた。
CHO細胞上における抗原の一時的発現
選択抗原を一時的に発現するCHO細胞を免疫細胞培地に懸濁した。細胞密度は、1ml当たり細胞2×107個に変更した。
該測定混合物はICM中で調製し、ELISA陽性B細胞40μl、1:400に希釈したヤギ抗ウサギIgG Fc特異的FITC複合体(Jackson ImmunoResearch)40μl及びCHO細胞懸濁液40μlを含めた。
Claims (17)
- a)抗体産生細胞集団を提供すること、
b)前記抗体産生細胞集団を選択した抗原及び標識抗抗体抗体と共にインキュベートすることであって、前記抗抗体抗体は前記選択した抗原に結合する抗体を産生する細胞とそうでない細胞とを区別することができること、
c)前記選択した抗原に結合する抗体を産生することができる抗体産生細胞を同定すること、及び
d)工程c)で同定された抗体産生細胞を顕微操作技術を使用して単離すること
を含む、選択した抗原に結合する抗体を産生する抗体産生細胞を同定するためのin vitro均一系アッセイ。 - 赤血球に前記抗原を結合させる請求項1に記載のアッセイ。
- ビーズに前記抗原を結合させる請求項1に記載のアッセイ。
- ポリクローナル抗体を介して前記抗原をビーズに結合させる請求項3に記載のアッセイ。
- 前記ポリクローナル抗体が抗体断片である請求項4に記載のアッセイ。
- 前記ポリクローナル抗体は、抗体Fab、Fab’又はF(ab’)2断片である請求項5に記載のアッセイ。
- 前記抗原を細胞の表面上に発現させる請求項1に記載のアッセイ。
- 前記細胞はトランスフェクト細胞である請求項7に記載のアッセイ。
- 前記細胞は腫瘍細胞である請求項7に記載のアッセイ。
- 前記抗原は感染因子である請求項1〜9のいずれか一項に記載のアッセイ。
- 前記標識抗抗体抗体は抗Fc抗体である請求項1〜10のいずれか一項に記載のアッセイ。
- 前記標識抗抗体抗体は蛍光複合体で標識してある請求項1〜11のいずれか一項に記載のアッセイ。
- 前記蛍光標識抗抗体抗体はFITCで標識してある請求項12に記載のアッセイ。
- 前記FITC標識抗抗体抗体は抗Fc抗体である請求項13に記載のアッセイ。
- 前記抗体産生細胞は、B細胞、血漿細胞、形質芽細胞、活性化B細胞又は記憶B細胞である請求項1〜14のいずれか一項に記載のアッセイ。
- 前記抗体産生細胞は抗体を発現するように操作されたハイブリドーマ細胞又は哺乳類細胞である請求項1〜15のいずれか一項に記載のアッセイ。
- a)抗体産生細胞集団を提供すること、
b)前記抗体産生細胞集団を選択した抗原及び標識抗抗体抗体と共にインキュベートすることであって、前記抗抗体抗体は前記選択した抗原に結合する抗体を産生する細胞とそうでない細胞とを区別することができること、
c)前記選択した抗原に結合する抗体を産生する抗体産生細胞を同定すること、
d)前記同定した抗体産生細胞を顕微操作技術を使用して単離すること、及び、
e)抗体又はそれらの抗体断片を合成すること
を含む、選択した抗原に結合する抗体のin vitro産生方法。
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WO2021065766A1 (ja) | 2019-09-30 | 2021-04-08 | 東京応化工業株式会社 | 分泌物産生細胞のスクリーニング方法、及び分泌物産生細胞のスクリーニングキット |
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PT1570267E (pt) | 2012-01-03 |
WO2004051268A1 (en) | 2004-06-17 |
ATE528648T1 (de) | 2011-10-15 |
JP2006509217A (ja) | 2006-03-16 |
US20060148012A1 (en) | 2006-07-06 |
EP1570267A1 (en) | 2005-09-07 |
CA2507004A1 (en) | 2004-06-17 |
AU2003285578A1 (en) | 2004-06-23 |
AU2003285578B2 (en) | 2010-07-15 |
EP1570267B1 (en) | 2011-10-12 |
ES2374068T3 (es) | 2012-02-13 |
US7993864B2 (en) | 2011-08-09 |
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