JP4920415B2 - プローブ複合体 - Google Patents
プローブ複合体 Download PDFInfo
- Publication number
- JP4920415B2 JP4920415B2 JP2006527838A JP2006527838A JP4920415B2 JP 4920415 B2 JP4920415 B2 JP 4920415B2 JP 2006527838 A JP2006527838 A JP 2006527838A JP 2006527838 A JP2006527838 A JP 2006527838A JP 4920415 B2 JP4920415 B2 JP 4920415B2
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- JP
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- Prior art keywords
- probe complex
- bound
- antibody
- biotin
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Images
Classifications
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/545—Synthetic resin
- G01N33/547—Synthetic resin with antigen or antibody attached to the carrier via a bridging agent
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
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- General Physics & Mathematics (AREA)
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- Peptides Or Proteins (AREA)
- Investigating Or Analyzing Materials By The Use Of Ultrasonic Waves (AREA)
- Analysing Materials By The Use Of Radiation (AREA)
Description
Harlow, E.およびLane, D.著,「Antibodies: A LABORATORY MANUAL」(米国),Cold Spring Harbor Laboratory, 1988年,p. 340-341.
Ficoll400(Amersham Bioscienses)44mgを秤量し、0.1Mリン酸緩衝液(pH7.0)0.8mLに溶解し、過ヨウ素酸ナトリウム溶液を0.4mL添加混合した。室温で2時間反応させた後、ゲルろ過(SephadexG25,Amersham Bioscienses)により余剰の過ヨウ素酸ナトリウムを除去し、Bovine Serum Albumin(BSA)溶液を添加して、室温で3時間反応させ、FicollにBSAを導入した。反応産物の安定化のため、Dimethylamine Borate(DMAB;生化学工業株式会社)を添加混合して室温で1時間反応させた後、Ficoll上の未反応のアルデヒド基をブロックするためにTris溶液を添加した。室温で1晩反応させた後、ゲルろ過(SephacrylS300,1.6*30)により反応産物を精製し、280nmの吸光度を測定して担体BSA結合物の濃度を算出した。この担体BSA結合物1mgをシステアミン塩酸塩で還元し、ゲルろ過(SephadexG25,Amersham Bioscienses)により余剰のシステアミン塩酸塩を除去し、Dimethylfolmamideに溶解した1,2−bismaleimideおよびSulfo−NHS−LC−Biotin(Pierce #21335)水溶液を添加混合し、室温で1.5時間反応させ、担体BSA結合物にマレイミド基およびビオチンを導入した。余剰の1,2−bismaleimideとSulfo−NHS−LC−Biotinはゲルろ過(SephadexG25,Amersham Bioscienses)により除去した。0.1Mリン酸緩衝液(pH6.0)中の抗HCVコア抗原モノクローナル抗体のF(ab)’2溶液(C11−14F(ab)’2とC11−9F(ab)’2を等量混合)に、0.15Mシステアミン塩酸塩を1/10容量添加し、37℃で1.5時間インキュベーションを行い、ゲルろ過(SephadexG25,Amersham Bioscienses)により余剰のシステアミン塩酸塩を除去し、抗HCVコア抗原モノクローナル抗体Fab’を得た。このFab’とマレイミドおよびビオチンを導入した担体BSA結合物を混合し、4℃で1晩反応させた後、ゲルろ過(Sephacryl S300,1.6x30)を行い、遊離のFab’を除去した。このとき、本発明の担体BSA−Fab’複合体は、ボイド分画近傍に出現し、Sephacryl S300の排除限界は分子量150万であることから、数十万以上の分子量であることが推測された。このようにして調製された担体BSA−Fab’複合体の280nmの吸光度を測定し、抗体の吸光度とみなして濃度を算出した。
Sulfo−NHS−LC−Biotin(Pierce #21335)の添付文書に記載の方法に従って行った。PBS中の抗HCVコア抗原モノクローナル抗体(C11−14 IgGとC11−9 IgGを等量混合)にSulfo−NHS−LC−Biotinを混合し、室温で1時間反応させた後、余剰のSulfo−NHS−LC−Biotinをゲルろ過(SephadexG25,Amersham Bioscienses)により除去した。調製したビオチン化抗HCVコア抗原モノクローナル抗体の280nmの吸光度を測定し、抗体濃度を算出した。
抗HCVコア抗原モノクローナル抗体を0.1M酢酸・0.1Mリン酸緩衝液(pH4.8)で4μg/mlに調整し、96穴マイクロプレートの各ウェルに250μlずつ加え、4℃で1晩インキュベーションを行った。PBSで洗浄後、0.5%カゼインを各ウェルに350μlずつ加え、室温で3時間インキュベーションを行った。組み換え体HCVコア抗原(c11)を0fmol/L,148fmol/L,444fmol/L,1333fmol/L,4000fmol/L,12000fmol/L,36000fmol/Lの濃度に調製したものをサンプルとして添加し、攪拌しながら室温で1時間インキュベーションを行った。0.05% Tween20を含む10mMリン酸緩衝液pH7.3(洗浄液)で6回洗浄後、2次抗体として、実施例1で調製したビオチン−抗HCVコア抗原モノクローナル抗体複合体および実施例2で既存の方法により調製したビオチン化抗HCVコア抗原モノクローナル抗体を1μg/mlの濃度に希釈して200μl加え、室温で1時間インキュベーションを行った。洗浄液で6回洗浄し、HRP結合アビジンを5000倍希釈液200μをl加え、室温で30分間インキュベーションを行った。さらに洗浄液で6回洗浄し、基質溶液(オルトフェニレンジアミン・過酸化水素混合液)を200μl加え、室温で30分間インキュベーションを行い、5N硫酸を50μlずつ加えて反応を停止させた。492nmの吸光度(リファレンス波長600nm)をマイクロプレートリーダー(MPRA4i,TOSOH)で測定した。各濃度の組換え体HCVコア抗原(c11)を加えたウェルの吸光度から0fmol/Lの吸光度の値を差し引いた値を表1に示した。
Claims (6)
- 水溶性の担体に親水性の分子量2,000以上の仲介物質が結合し、仲介物質にプローブおよび2個以上の分子量10,000以下の検出マーカーが結合したプローブ複合体。
- 検出マーカーがビオチンである請求項1に記載のプローブ複合体。
- 検出マーカーがハプテンである請求項1に記載のプローブ複合体。
- 検出マーカーが蛍光物質または発光物質である請求項1に記載のプローブ複合体。
- 検出マーカーが放射性同位体を有するものである請求項1に記載のプローブ複合体。
- 検出マーカーが抗体との結合能を有する物質である請求項1に記載のプローブ複合体。
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JP2009042209A (ja) * | 2007-07-13 | 2009-02-26 | Fujifilm Corp | 担体およびその製造方法並びにバイオリアクター |
US8710958B2 (en) | 2008-07-10 | 2014-04-29 | Abbott Laboratories | Containers having radio frequency identification tags and method of applying radio frequency identification tags to containers |
US9005910B2 (en) | 2010-04-14 | 2015-04-14 | Eiken Kagaku Kabushiki Kaisha | Complex of labeled probes and water-soluble carrier |
WO2013016653A1 (en) * | 2011-07-28 | 2013-01-31 | Cell Signaling Technology, Inc. | Multi component detection |
CN104502596A (zh) * | 2014-12-23 | 2015-04-08 | 温州医科大学 | 一种慢性肾病诊断试剂盒 |
CN105044324B (zh) * | 2015-07-14 | 2017-01-18 | 上海拜豪生物科技有限公司 | 一种砷螯合型免疫复合物及其制备方法和应用 |
CN105017430B (zh) * | 2015-07-14 | 2017-02-01 | 上海拜豪生物科技有限公司 | 一种镍螯合型免疫复合物及其制备方法和应用 |
CN105001310B (zh) * | 2015-07-14 | 2017-12-15 | 上海拜豪生物科技有限公司 | 一种铬螯合型免疫复合物及其制备方法和应用 |
CN105044369B (zh) * | 2015-07-14 | 2017-01-18 | 上海拜豪生物科技有限公司 | 一种铅螯合型免疫复合物及其制备方法和应用 |
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ES2349649T3 (es) | 2011-01-07 |
KR101158271B1 (ko) | 2012-06-19 |
CA2574683A1 (en) | 2006-02-02 |
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