JP5200003B2 - 磁場を用いた試料中の標的分子の検出 - Google Patents
磁場を用いた試料中の標的分子の検出 Download PDFInfo
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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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
- G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
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Description
a)前記試料及び磁性粒子に付着する第1結合分子を、同時又はそれぞれ異なる時期に、固体支持体に付着する第2結合分子と接触させる手順であって、前記第1結合分子は前記第2結合分子と結合する能力を有し、前記標的はこの結合を妨害する能力を有する手順;及び、前記磁性粒子が前記固体支持体付近へ移動するように磁力を印加する手順;並びに
b)前記第1結合分子の前記第2結合分子への結合によって前記固体支持体と結合する磁性粒子数を検出する手順;
を有する。
(strep-tagII)(登録商標)/ストレプ-タクチン(strep-tactin)(登録商標)対がある。
A. 抗モルヒネ抗体(Ab)に付着する磁性粒子(MP)の調製
磁性粒子回収装置(MPC)では、MPsは、100μlの磁性粒子溶液(アデムテック(Ademtech)社製、200nmのタンパク質GでコーティングされたMPs、製造番号0433)から回収されて、100μlのPBS+0.65%のTween20(商標)内で溶解された。10μlの単クローン性のマウスの抗体(1mg/mlの原液)を加えた後、混合物は室温で1時間混合され、それに続いて100μlのPBS+0.65%のTween20(商標)内のMPCを用いて2回洗浄して100μlのトリエタノールアミン(0.2M,pH8)で1回洗浄された。MPsは1mlトリエタノールアミン(0.2M,pH8)中のDMP(20mM)で溶解し、その溶液は室温で30分間混合された。架橋反応は、50μlのトリス(1M、pH7.5、最終濃度50mM)を加え、室温で15分間混合を続けることによって止まった。MPsは、100μlのトリス(50mM、pH7.5)と共にMPCを1回用い、そしてMPC中の貯蔵緩衝剤を1回用いて分離及び洗浄し、最終的に100μlの貯蔵緩衝材中で溶解させた。
金のディスクが、コーティング緩衝材(15mMの炭酸ナトリウム、35mMの炭酸水素ナトリウム、0.05%のアジ化ナトリウム、pH〜9.6)中のBSA又はBSA-OPI(3μg/ml)によってエッチング及びコーティングされた。コーティングされたディスクは洗浄緩衝材(PBS中の0.05%Tween20(商標))によって3回洗浄された。希釈緩衝材には一連のモルヒネ希釈溶液が反応管内で調製された。事前に混合したMP溶液(緩衝材中で0.1%w/v=1mg/mlのMP)が管内の各希釈溶液に加えられた。50μlのこれらの各溶液はウエルに入れられ、かつMPsは30秒間の磁力の印加(6-24fN)によって作動し、又は30分間沈降可能となる。従って、未結合MPsが30秒間の磁力印加によって除去され、かつ100μlの抗マウスIgG-HRP(希釈緩衝材中で原液を1/3000に希釈)が各ウエルに加えられた。その後室温で60分間培養された。ディスクはウエル毎に200μlの洗浄緩衝材で4回洗浄され、洗浄緩衝材を含むウエルから白のマイクロタイタープレートへ移送された。洗浄緩衝材は除去された。100μlのAB混合物(ECL;A+Bは1:1で混合された)が加えられ、室温で5分間培養された。発光が読み取られた。
A. 金表面へのAbのコーティング
本発明の方法に係る代替実施例では、最初にAbを金表面上に設ける必要がある。図3に図示されているように、これは、表面上にタンパク質G(pG)が設けられるときに最も良く機能する。ここで金表面をコーティングするのに50μg/mlのタンパク質Gが用いられた。続いて様々な量のAbで表面を洗浄して培養した。その後、モルヒネ-HRPで表面を培養して、洗浄した。それに続いて、結合モルヒネの量がルミノール/発光を用いて決定された。得られた信号は表面上で機能するAbの量を反映する。図3に示された暗いグレーの棒は、タンパク質Gが取り除かれたときに、金表面上に直接結合(物理吸着)するAbを表す。白の棒は、非選択結合のチェックを行うためのBSA(Abもタンパク質Gも含まない)でコーティングされた金表面を表す。
競合アッセイ法は、上述したようにMPsと表面を用いてウエルプレート内で行われた。その結果は図5に示されている。
Claims (22)
- 小さな分子、薬、タンパク質、酵素、ホルモン、ペプチド、及び核酸からなる群から選ばれる標的を含んでいる疑いのある試料中での前記標的の検出方法であって:
a)前記試料及び磁性粒子に付着する複数の第1結合分子を、同時又はそれぞれ異なる時期に、固体支持体に付着する第2結合分子と接触させる手順であって、前記複数の第1結合分子は前記第2結合分子と結合する能力を有し、前記標的はこの結合を妨害する能力を有する手順;及び、前記磁性粒子が前記固体支持体付近へ移動するように磁力を印加する手順;
b)前記手順a)の間に、前記第2結合分子と結合していない第1結合分子を除去する手順;並びに、
c)前記第1結合分子の前記第2結合分子への結合によって前記固体支持体と結合する磁性粒子数を検出する手順;
を有し、
前記磁性粒子の数は、前記第1結合分子との選択的結合の可能な第3結合分子を、前記磁性粒子に付着する前記第1結合分子と結合可能にし、かつ前記第1結合分子と結合する前記第3結合分子を検出することによって、決定され、
前記第3結合分子が結合可能な前記第1結合分子は、前記第2結合分子との結合に関与しない前記第1結合分子で、かつ
前記検出する手順は、前記第3結合分子と結合する検出可能なラベルを介して行われ、又は、
前記検出する手順は、検出可能なラベルと結合し、かつ前記第3結合分子に対して選択的に結合できる薬剤と、前記第1結合分子と結合する前記第3結合分子との結合を可能にさせ、かつ前記検出可能なラベルを検出することにより行われる、
方法。 - 前記第1結合分子が前記の試料中での標的及び前記第2結合分子と選択的に結合する能力を有し、かつ
接触手順a)が、前記の第1結合分子との選択結合を行うため、前記第2結合分子と前記標的との競合を可能にする手順を有する、
請求項1に記載の方法。 - 前記第2結合分子が前記標的と同一であるか、又は標的ホモログである、請求項2に記載の方法。
- 前記第2結合分子が前記の試料中での標的及び前記第1結合分子と選択的に結合する能力を有し、かつ
接触手順a)が、前記の第2結合分子との選択結合を行うため、前記第1結合分子と前記標的との競合を可能にする手順を有する、
請求項1に記載の方法。 - 前記第1結合分子が前記標的と同一であるか、又は標的ホモログである、請求項4に記載の方法。
- 前記薬が、モルヒネ、コカイン、THC、アナボリック、又はアンフェタミン/メタアンフェタミン基の薬である、請求項1に記載の方法。
- 前記磁力は、前記第2結合分子への結合中、磁気ラベルに付着する前記標的と前記第1結合分子との間での移動度の差異の効果を緩和又は除去するように印加される、請求項1から6までのいずれか一項に記載の方法。
- 前記磁力は、電磁石、空心コイル、又は永久磁石によって印加される、請求項7に記載の方法。
- 前記第2結合分子と結合していない前記第1結合分子が磁力の印加によって除去される、請求項1に記載の方法。
- 前記第1結合分子が、スペーサ分子、タンパク質、又は強結合対を介して前記磁性粒子に付着する、請求項1から9までのいずれか一項に記載の方法。
- 前記スペーサ分子がアルキレンジアミン又はエチレンジアミンである、請求項10に記載の方法。
- 前記固体支持体が、センサ素子の表面である、請求項1から11までのいずれか一項に記載の方法。
- 前記センサ素子の表面が金の表面である、請求項12に記載の方法。
- 前記金の表面は、前記第2結合分子が結合するタンパク質でコーティングされる、請求項13に記載の方法。
- 前記タンパク質がタンパク質A又はタンパク質Gで、かつ
前記第2結合分子は、タンパク質A又はタンパク質Gによって結合されることのできる抗体である、
請求項14に記載の方法。 - 磁性粒子の数は、手順c)において磁気センサ素子によって決定される、請求項1から15までのいずれか一項に記載の方法。
- 前記検出可能なラベルは、蛍光ラベル、比色ラベル、化学発光ラベル、酵素ラベル、放射線ラベル、静電ラベル、及び供与体/受容体ラベルであるからなる群から選ばれる、請求項1に記載の方法。
- 前記試料が、尿試料、血液試料、汗、眼の流体、口の流体、若しくは髪からなり、又は、尿試料、血液試料、汗、眼の流体、口の流体、若しくは髪から得られる、請求項1から17までのいずれか一項に記載の方法。
- 請求項1から18までのいずれかに記載の方法において、前記磁力を印加することで、前記磁性粒子を前記固体支持体へ近づけるための磁石の使用。
- 前記磁石が電磁石、空心コイル、又は永久磁石である、請求項19に記載の使用。
- 前記磁性粒子は、磁性、反磁性、常磁性、超常磁性、フェリ磁性、又は強磁性粒子からなる群から選ばれる、請求項1から20までのいずれか一項に記載の方法又は使用。
- 前記磁性粒子は、10nmから10μmの大きさを有する、請求項1から21までのいずれか一項に記載の方法又は使用。
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