JP2002511140A - 高処理量検定法 - Google Patents
高処理量検定法Info
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- JP2002511140A JP2002511140A JP50299599A JP50299599A JP2002511140A JP 2002511140 A JP2002511140 A JP 2002511140A JP 50299599 A JP50299599 A JP 50299599A JP 50299599 A JP50299599 A JP 50299599A JP 2002511140 A JP2002511140 A JP 2002511140A
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B30/00—Methods of screening libraries
- C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
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Abstract
Description
Claims (1)
- 【特許請求の範囲】 1. 化合物によるリガンド/受容体相互作用または酵素触媒反応の阻害の程度 を決定すること、又は標的分子への該化合物の結合の程度を決定することを目的 とする複数の該化合物の迅速スクリーニングのための高効率検定法であって、 (a)試験対象である該化合物を (1)該リガンド及び該受容体、 (2)該酵素及び該酵素の基質、又は (3)該標的分子、 と接触させる工程であって、そこでは一つ以上の該化合物による阻害 又は結合が該検定法中に存在する懸濁可能な細胞又は固体支持体に結 合した光学的に検出可能な標識の量の変化を惹起するものであり、 (b)個々の細胞又は固体支持体に結合した該光学的に検出可能なシグ ナルの量を顕微鏡の使用により測定し、該量を該細胞又は固体支持体 に結合していない該検定法におけるバックグラウンドシグナルの量と 比較し、そして該結合シグナルと該バックグラウンドシグナルの間の 差から阻害又は結合の該程度を決定することにより、阻害又は結合の 該程度を決定する工程、 を含んで成る方法。 2. 個々の細胞又は固体支持体に結合したシグナルの該測定が共焦点顕微鏡検 査により行われるものである、請求項1記載の検定法。 3. 酵素阻害剤に対する検定を含むものである請求項2記載の検定法。 4. 受容体結合の阻害剤に対する検定を含むものである請求項2記載の検定法 。 5. 該受容体が該リガンドに結合する受容体ドメインからなるものである請求 項4記載の検定法。 6. 該光学的に検出可能な標識が、化学発光標識、蛍光性標識、及び可視光の 変化を生じさせる酵素からなる群より選択されるものである、請求項2記載の検 定法。 7. 該リガンドが蛍光標識されているものである請求項2記載の検定法。 8. 該ライブラリー化合物が蛍光標識されているものである請求項2記載の検 定法。 9. 溶液中の酵素及びライブラリー化合物が、懸濁可能な固体支持体に付着し ている蛍光的に標識されている基質とインキュベートされる検定を含むものであ る請求項3記載の検定法。 10. 酵素及び蛍光的に標識された基質が、該酵素/基質反応の反応生産物に 特異的に結合する部分で被覆されている懸濁可能な固体支持体とインキュベート され、そして該反応生産物が蛍光的に標識されるものである請求項3記載の検定 法。 11. 該受容体が標識を付されそして該リガンドが該懸濁可能な固体支持体に 結合するものである請求項2記載の検定法。 12. 該共焦点顕微鏡検査が共焦点レーザー走査顕微鏡検査である請求項2記 載の検定法。 13. 該共焦点顕微鏡検査がニプコウディスク(Nipkow disc)を用いるもので ある請求項2記載の検定法。 14. 該化合物が酵素及び基質と接触し、そして該酵素への結合、該基質への 結合、酵素と基質の複合体への結合、及び酵素と生産物の複合体への結合より成 る群から選択される1機構により阻害するものである請求項1記載の検定法。 15. 懸濁可能な細胞又は固体支持体上に含まれる標的分子への蛍光標識され たリガンドの結合の阻害を目的として複数の化合物を迅速スクリーニングするた めの高効率検定法であって、 (a)該化合物の存在下に該蛍光標識されたリガンドを該懸濁可能な細胞 又は懸濁可能な固体支持体上に含まれる該標的分子と接触させる工程、 (b)個々の細胞又は固体支持体に結合する蛍光標識されたリガンドの量 を共焦点顕微鏡検査により測定することにより該結合の阻害の程度を決定する工 程、 (c)該量を、該細胞又は固体支持体と結合していない該検定中の蛍光と 比較する工程、及び (d)該相違を該阻害の程度と相関させる工程、 を含んで成る方法。 16. 該標的分子が受容体又は受容体ドメインである請求項15記載の方法。 17. 該受容体又は受容体ドメインが懸濁された細胞の表面に含まれるもので ある請求項16記載の方法。 18. 該標的分子が懸濁可能な固体支持体に付着しているものである請求項1 5記載の方法。 19. 該懸濁可能な固体支持体が約50μmより小さな直径をもつものである 請求項18記載の方法。 20. 該懸濁可能な固体支持体が約10μmより小さくそして約1μmより大 きな直径をもつものである請求項18記載の方法。 21. リガンドと細胞表面受容体又は受容体ドメインの間の結合の阻害を目的 として化合物ライブラリーをスクリーニングするための均一検定法であって、 (a)該細胞表面受容体又はドメインを発現する細胞の液体懸濁液を複数 の容器に加える工程、 (b)該阻害のためにスクリーニングされる化合物のライブラリーを該複 数の容器に添加すにる工程、 (c)該受容体に対する蛍光標識リガンドを該容器に添加する工程、 (d)該細胞、該化合物、及び該蛍光標識リガンドをインキュベートする 工程、 (e)共焦点顕微鏡検査により該容器中の該細胞と結合している蛍光の量 を個々に測定する工程、及び (f)一つ以上の化合物による該受容体への該リガンドの結合の阻害の程 度を決定する工程、 を含んで成る方法。 22. 該インキュベーションの後でそして該測定の前に、約75%より多くの 該細胞が該容器の容積の約25%より少ない場所に沈着するように、該細胞を該 容器中に沈着させるものである請求項21記載の方法。 23. 該インキュベーションの後でそして該測定の前に、約90%より多くの 該細胞が該容器の容積の約10%より少ない場所に沈着するように、該細胞を該 容器中に沈着させるものである請求項21記載の方法。 24. 該化合物が約600nmより上の波長で蛍光を発する官能基部分で蛍光 標識されているものである請求項21記載の方法。 25. 該容器が一つ以上のミクロタイタープレート中のウエルを含むもので ある請求項21記載の方法。 26. 該容器が約1μl以下の検定溶液を含むものである請求項21記載の方 法。 27. リガンドと懸濁可能な固体支持体に付着した標的分子の間の結合の阻害 を目的とする化合物ライブラリーのスクリーニングのための均一検定法であって 、 (a)該懸濁可能な固体支持体に付着した該標的分子の液体懸濁液を複 数の容器に添加する工程、 (b)該阻害についてスクリーニングすべき複数の化合物を該複数の容器 に添加する工程、 (c)蛍光的に標識されたリガンドを該容器に添加する工程、 (d)固体支持体に付着した該標的分子、該化合物、及び該蛍光的に標識 されたリガンドをインキュベートする工程、 (e)共焦点顕微鏡検査により該固体支持体と結合している蛍光の量を個 々に測定する工程、及び (f)該リガンドの該標的分子への結合の、該化合物の一つ以上による阻 害の程度を決定する工程、 を含んで成る方法。 28. 該インキュベーションの後でそして該測定の前に、該細胞の約75%よ り多くが該容器の容積の約25%より少ない場所に沈着するように該固体支持体 を該容器に沈着させるものである請求項27記載の方法。 29. 該インキュベーションの後でそして該測定の前に、該細胞の約90%よ り多くが該容器の容積の約10%より少ない場所に沈着するように該固体支持体 を該容器に沈着させるものである請求項27記載の方法。 30. 該化合物が約600nmより上で蛍光を発する官能基部分で蛍光的に標 識されているものである請求項27記載の方法。 31. 該容器が一つ以上のミクロタイタープレート中のウエルを含むものであ る請求項27記載の方法。 32. 該容器が約1μl以下の検定溶液を含むものである請求項27記載の方 法。 33. リガンドと懸濁し得ない固体支持体に付着した標的部分との間の結合の 阻害を目的として化合物のライブラリーをスクリーニングするための均一検定法 であって、 (a)該阻害についてスクリーニングすべき複数の化合物を該懸濁し得な い固体支持体を含む容器に添加する工程、 (b)蛍光的に標識したリガンドを該容器に添加する工程、 (c)該懸濁し得ない固体支持体に付着した該標的部分、該化合物、及び 該蛍光標識したリガンドをインキュベートする工程、 (d)共焦点顕微鏡検査により該固体支持体と結合している蛍光の量を測 定する工程、及び (e)該標的部分への該リガンドの結合の、該化合物の一つ以上による阻 害の程度を決定する工程、 を含んで成る方法。 34. 該容器がミクロタイタープレート中のウエルを含み、そして該懸濁し得 ない固体支持体が該プレート中のウエルの底を含むものである請求項33記載の 検定法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US868,280 | 1997-06-03 | ||
US08/868,280 US5876946A (en) | 1997-06-03 | 1997-06-03 | High-throughput assay |
PCT/US1998/011749 WO1998055866A1 (en) | 1997-06-03 | 1998-06-01 | High-throughput assay |
Publications (2)
Publication Number | Publication Date |
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JP2002511140A true JP2002511140A (ja) | 2002-04-09 |
JP4007459B2 JP4007459B2 (ja) | 2007-11-14 |
Family
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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JP50299599A Expired - Fee Related JP4007459B2 (ja) | 1997-06-03 | 1998-06-01 | 高処理量検定法 |
Country Status (8)
Country | Link |
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US (2) | US5876946A (ja) |
EP (1) | EP1002233B1 (ja) |
JP (1) | JP4007459B2 (ja) |
AT (1) | ATE294391T1 (ja) |
AU (1) | AU741376B2 (ja) |
CA (1) | CA2291621A1 (ja) |
DE (1) | DE69829973T2 (ja) |
WO (1) | WO1998055866A1 (ja) |
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CA2291621A1 (en) | 1998-12-10 |
DE69829973D1 (de) | 2005-06-02 |
ATE294391T1 (de) | 2005-05-15 |
AU741376B2 (en) | 2001-11-29 |
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