JP2009529904A - 近接プローブを用いた検体検出法 - Google Patents
近接プローブを用いた検体検出法 Download PDFInfo
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Abstract
(a) 該サンプルを少なくとも1セットの少なくとも第1、第2および第3近接プローブに接触させ(各プローブは、検体結合ドメインおよび核酸ドメインを含み、同時に検体に結合することができ、該第3近接プローブの核酸ドメインは、該第1および第2近接プローブの核酸ドメインに少なくともハイブリダイズすることができるスプリントであり、ここで、少なくとも3個の近接プローブのすべてが該検体に結合するとき、該第1および第2近接プローブの核酸ドメインは、該第3近接プローブの該ハイブリダイズスプリントにより仲介される相互作用の手段により結合可能である);
(b) 該第1および第2近接プローブの核酸を結合させ;そして、
(c) 該結合を検出すること
を含む。また、そのような方法での使用のためのキットを提供する。
Description
(a) 該サンプルを少なくとも1セットの少なくとも第1、第2および第3近接プローブに接触させ(各プローブは、検体結合ドメインおよび核酸ドメインを含み、同時に検体に結合することができ、該第3近接プローブの核酸ドメインは、該第1および第2近接プローブの核酸ドメインに少なくともハイブリダイズすることができるスプリントであり、ここで、少なくとも3個の近接プローブのすべてが該検体に結合するとき、該第1および第2近接プローブの核酸ドメインは、該第3近接プローブの該ハイブリダイズスプリントにより仲介される相互作用の手段により結合可能である);
(b) 該第1および第2近接プローブの核酸を結合させ;そして、
(c) 該結合を検出すること
を含む。
(a) 該サンプルを少なくとも1セットの少なくとも第1、第2および第3近接プローブに接触させ(各プローブは、検体結合ドメインおよび核酸ドメインを含み、同時に検体に結合することができ、該第3近接プローブの核酸ドメインは、該第1および第2近接プローブの核酸ドメインに少なくともハイブリダイズすることができるスプリントであり、ここで、少なくとも3個の近接プローブのすべてが、該検体に結合するとき、該第1および第2近接プローブの核酸ドメインは、該ハイブリダイズスプリントにより仲介される相互作用の手段により結合可能である);
(b) 所望により、該第1および第2近接プローブの核酸を結合させるための手段;そして、
(c) 所望により、該結合を検出するための手段
を含む。
図1: 結合スプリントアッセイにおける異なる工程のための一般のスキーム。競合オリゴヌクレオチドでの近接プローブの妨害(A)。タンパク質の添加後、インキュベーション時間の間、該タンパク質は、近接プローブにより認識され、鋳型近接プローブは、妨害オリゴヌクレオチド(B)およびライゲーションならびに新規配列が形成され、q-PCRにより検出される検出反応(C)に競合する。
本発明の方法を、異なるタンパク質を検出するために使用した。
血管内皮細胞増殖因子(VEGF)は、低濃度で血管の成長を刺激するホモダイマーの42 kDaサイトカインである。VEGFの検出は、2個または3個のアリコートを、それぞれ、2つのプローブの、遊離スプリントアッセイおよび本発明の結合スプリントアッセイのために分割し、そして適当なオリゴヌクレオチドに結合する親和性精製ポリクローナル抗血清を用いて行った(表1)。
VEGFおよびVEGFに特異的な親和性精製ポリクローナル抗体を、R&D Systemsから取得し、一方で、PSA-ACTアッセイのための試薬は、Ulf Hakan Stenma, Helsinkiにより提供され、トロポニンアッセイは、Kim Pettersson (Turku)により提供された。遊離PSAを検出するためのモノクローナルPSA抗体は、Biodesignから購入した。オリゴヌクレオチドは、全PSAを認識するポリクローナル抗体の2つのアリコートのそれらの3'または5'末端に結合し、他の2つにハイブリダイズすることが可能な第3オリゴヌクレオチドは、遊離PSAを認識する抗体に結合した。共有結合オリゴヌクレオチドは、Olink (Sweden)と共同で、Solulink, USA(結合)およびTrilink, USA(オリゴヌクレオチド)から購入した。オリゴヌクレオチドの配列を、表1に示す。
遊離スプリントアッセイは、以前、商業的に入手可能なサンドウィッチELISA試験よりも約5000倍低いVEGFを検出することが示された。しかしながら、本発明の方法はさらに、VEGFのわずか60分子が、バックグラウンドよりも有意に大きいシグナルを生じるとき、感度の100倍の増加を示した(図2A)。両アッセイでは、1 μlのサンプルを5 μlの近接プローブミックスと合わせた。我々は、結合スプリントアッセイで、標的タンパク質の量を増やすことへの特徴のある二層性応答を観察した(低い濃度範囲では、標的濃度を増やすことへの予期される応答よりも低い)。二層性応答は、おそらく、短いインキュベーション時間のために(この場合には、2時間、37℃)、動力学的反応を反映し得る。
Claims (29)
- サンプル中の検体を検出するための方法であって、
(a) 該サンプルを少なくとも1セットの少なくとも第1、第2および第3近接プローブに接触させ(各プローブは、検体結合ドメインおよび核酸ドメインを含み、同時に検体に結合することができ、該第3近接プローブの核酸ドメインは、該第1および第2近接プローブの核酸ドメインに少なくともハイブリダイズすることができるスプリントであり、ここで、少なくとも3個の近接プローブのすべてが該検体に結合するとき、該第1および第2近接プローブの核酸ドメインは、該第3近接プローブの該ハイブリダイズスプリントにより仲介される相互作用の手段により結合可能である);
(b) 該第1および第2近接プローブの核酸を結合させ;そして、
(c) 該結合を検出すること
を含む、方法。 - 該検出が、定量的である、請求項1に記載の方法。
- 該検出が、定性的である、請求項1に記載の方法。
- 検体が、完全にまたは部分的にタンパク様分子である、請求項1から3のいずれか1項に記載の方法。
- 検体が、2個またはそれ以上の分子サブユニットの複合体である、請求項1から4のいずれか1項に記載の方法。
- 該複合体が、核酸およびタンパク様分子を含む、請求項5に記載の方法。
- 少なくとも該第1、第2および第3近接プローブの少なくとも1個の検体結合ドメインが、抗体、またはその結合断片もしくは誘導体である、請求項1から6のいずれか1項に記載の方法。
- 少なくとも1個の少なくとも該第1、第2および第3近接プローブが、直接標的検体に結合する媒介分子のための親和性を有する各検体結合ドメインのために、間接的に該検体に結合する、請求項1から7のいずれか1項に記載の方法。
- 該第1および第2近接プローブの核酸が、核酸ライゲーションにより結合する、請求項1から8のいずれか1項に記載の方法。
- 該第1および第2近接プローブの核酸ドメインが、互いにすぐ隣接した該第3近接プローブの核酸ドメインにハイブリダイズする、請求項1から9のいずれか1項に記載の方法。
- 該第1および第2近接プローブの核酸ドメインが、互いにすぐ隣接していない第3近接プローブの核酸ドメインにハイブリダイズし、その結果、該第3近接プローブの核酸ドメインにハイブリダイズするとき、該第1および第2近接プローブの核酸ドメインが、ギャップにより分離する、請求項1から9のいずれか1項に記載の方法。
- カセットオリゴヌクレオチドが、該第3近接プローブの核酸ドメインにハイブリダイズした該第1および第2近接プローブの核酸ドメインの各末端間のギャップにおいて、該第3近接プローブの核酸ドメインにハイブリダイズする、請求項11に記載の方法。
- 該第1および第2近接プローブの核酸ドメインの結合が、間接的にカセットオリゴヌクレオチドを介して起こる、請求項12に記載の方法。
- 該第1および第2近接プローブの核酸ドメイン間の該ギャップが、該第1および第2近接プローブ核酸ドメインの1個の末端の酵素学的伸張により埋められる、請求項11に記載の方法。
- 2個またはそれ以上のセットの少なくとも第1、第2および第3近接プローブを、2個またはそれ以上の検体を検出するために使用する、請求項1から14のいずれか1項に記載の方法。
- 一本鎖結合タンパク質が、反応混合物中に存在する、請求項1から15のいずれか1項に記載の方法。
- 該第1および第2近接プローブの核酸ドメインの遊離末端に結合可能な妨害オリゴヌクレオチドが、反応混合物中に存在する、請求項1から16のいずれか1項に記載の方法。
- 該妨害オリゴヌクレオチドが、少なくとも該第1および第2近接プローブでプレインキュベートされる、請求項17に記載の方法。
- 少なくとも該第1、第2および第3近接プローブの少なくとも1個が、固相に固定されているか、または固定することができる、請求項1から18のいずれか1項に記載の方法。
- さらに、該サンプルを、少なくとも1セットの少なくとも第1、第2および第3近接プローブに接触させる前に、検体結合ドメインを含む固定または固定可能捕捉プローブの手段により、検体を固相に結合させることを含む、請求項1から18のいずれか1項に記載の方法。
- 検体が、直接的に、固相に固定されるか、または固定することができる、請求項1から18のいずれか1項に記載の方法。
- 検体および少なくとも第1、第2および第3近接プローブの少なくとも1セットが、溶液中で遊離している、請求項1から18のいずれか1項に記載の方法。
- 結合を検出する工程が、結合産物を検出することを含む、請求項1から22のいずれか1項に記載の方法。
- 結合産物を検出することが、結合産物を増幅させ、そして増幅産物を検出することを含む、請求項23に記載の方法。
- 請求項1で定義した少なくとも第1、第2および第3近接プローブの少なくとも1セットを含む、サンプル中の検体を検出するためのキット。
- さらに、
(a) 該第1および第2近接プローブの核酸ドメインを結合させるための手段;および/または、
(b) 該結合を検出するための手段
を含む、請求項25に記載のキット。 - 該第1および第2近接プローブの核酸ドメインを結合させるための該手段が、核酸リガーゼを含む、請求項26に記載のキット。
- 該結合を検出するための該手段が、標識であるか、または増幅および/またはその検出のための手段である、請求項26に記載のキット。
- さらに、1個またはそれ以上の
(a) カッセトオリゴヌクレオチド;
(b) 該第1および第2近接プローブの核酸ドメインのための妨害オリゴヌクレオチド;
(c) 一本鎖結合タンパク質;
(d) 検体結合ドメインを含む固定または固定可能捕捉プローブ;そして、
(e) 該検体、捕捉プローブまたは少なくとも1個の少なくとも該第1、第2および第3近接プローブの固定化のための固相
を含む、請求項25から28のいずれか1項に記載のキット。
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US20100021890A1 (en) | 2010-01-28 |
EP1996941A1 (en) | 2008-12-03 |
JP5144639B2 (ja) | 2013-02-13 |
ATE457065T1 (de) | 2010-02-15 |
WO2007107743A1 (en) | 2007-09-27 |
DE602007004666D1 (de) | 2010-03-25 |
US8268554B2 (en) | 2012-09-18 |
GB0605584D0 (en) | 2006-04-26 |
EP1996941B1 (en) | 2010-02-03 |
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