JP4814103B2 - アレイフォーマットにおいて複数の結合反応を実行するためのシステムおよび方法 - Google Patents
アレイフォーマットにおいて複数の結合反応を実行するためのシステムおよび方法 Download PDFInfo
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Description
(a)第1結合要素を複数の複数のマイクロスポットにおいて表面に吸収させること
(b)各マイクロスポットにおける第1結合要素に第2結合要素を付与すること、ここで、複数のマイクロスポットの中で第1結合要素の表面密度と第2結合要素の密度の組み合わせには複数ある
(c)複数のマイクロスポットのそれぞれにおける第1および第2結合要素間の結合反応を示すデータをバイオセンサ検出法によって取得すること、
(d)第1および第2結合要素間の結合の1つ以上の動態パラメータを取得できるようにデータを処理すること
を具備する。
(a)表面の局地化領域を活性化すること
(b)局地化領域における1つ以上のマイクロスポットのそれぞれについて、分子種をマイクロスポットに吸収させること
(c)局地化領域を随意的に非活性にすること
を具備する。
(a)表面と、
(b)プローブ種がマイクロスポットに吸収されるようにマイクロスポットにプローブ種を施し且つ表面に吸収された各プローブ種に対してターゲットを付与することが可能なアプリケータと、
(c)表面からの反射光を受け取り且つターゲットのプローブへの結合を示す信号を生成する感光面と、
(d)感光面によって生成された信号を受け取り且つ結合の動態解析を生成できるように信号を解析するよう構成されたプロセッサと、
を具備する。
例1
結合アッセイが、図1に示すシステム10を用いて実行された。抗IL−4抗体(αIL−4)が、この実験でプローブとして用いられ、システム10の説明において上記したように、6個の長方形プローブ領域42(図1を参照)のそれぞれのSPR表面上で固定化された。プローブ領域は、(a)乃至(f)を付された。6個のプローブ領域、すなわわち「反応単位」(RU)内の抗体の密度は、表1に示されている。
6つの抗体プローブ(αIgG1、αIgG2b、αIgA、αIgG2a、αIgG3)の抗原(Ig1、IgG1、IgG2a、IgG2b、IgG3)への結合が、図1のシステム10を用いて研究された。プローブおよびターゲットの使用された濃度は、それぞれ表3、表4に示されている。得られた結合曲線は、図6に示されており、30個の結合反応のそれぞれの結合反応は表5に示されている。
5つのシトクロムーP450(CYP)酵素プローブ(3A4、2C19、1A2、2C9、2D6)の6個の異なるターゲット(ケトコナゾール、ニフェジピン、デキストロメトルファン、ジクロフェナク、サルファフェナゾール、プロプラノロール)への結合が、図1のシステム10を用いて実施された。ターゲットは、濃度1,000、500、250、125、62.5、31.25、15.5、7.8μMで付与された。親和性定数KDが各反応について求められた。この結果は、図7および表6に示されている。
表7は、図3に示す方法で用意された36個の独立したマイクロスポット上での、図1のシステム10を用いたウサギIgGおよびヤギIgGプローブの固定化を示している。各プローブ領域は順次活性化され、ウサギIgGおよびヤギIgGの6個のプローブが活性化されたプローブ領域に交互に吸収された。この結果、表7に示すように、36個のマイクロスポット(各表面領域で6個)において36の交互のプローブが固定化された。
Claims (13)
- 再生ステップを必要としないで、第1結合要素と第2結合要素との結合の1つ以上の動態パラメータを決定する方法において、
前記動態パラメータは結合定数、解離定数、または親和力から選択されており、
(a)前記第1結合要素を複数のマイクロスポットにおいて表面に吸収させ、
(b)前記マイクロスポットのそれぞれにおいて前記第1結合要素に複数の濃度で前記第2結合要素を同時に付与し、それにおいて複数のマイクロスポットにおいては前記第1結合要素の表面密度と前記第2結合要素の濃度とは複数の組合わせがあり、
(c)前記複数のマイクロスポットのそれぞれにおいて前記第1および第2結合要素間の結合反応を示すデータをバイオセンサ検出法によって同時に取得し、
(d)前記複数のマイクロスポットの2以上のマイクロスポットの間の表面に位置している複数のインタースポットから参照データを同時に取得し、
(e)前記第1および第2結合要素間の結合の1以上の動態パラメータを取得するように前記データを処理する、動態パラメータを決定する方法。 - 前記バイオセンサ検出法が、表面プラズモン共鳴(SPR)、臨界角度屈折率測定、全反射蛍光、全反射燐光、全反射光散乱、エバネセント波エリプソメトリ、ブルースター角反射率測定から選択される、請求項1記載の方法。
- 前記検出法が表面プラズモン共鳴(SPR)であって、
前記複数のマイクロスポットのそれぞれにおける第1および第2結合要素間の結合反応を示す前記データが、SPR共鳴角度、共鳴波長、反射率変化、位相変化から選択されたSPRパラメータである、請求項1または2記載の方法。 - 前記第1結合要素が分子種であり、
前記分子種を前記マイクロスポットに吸収させるステップにおいて、
(a)前記マイクロスポットを含んだ領域の周囲にチャネルを形成し、
(b)前記分子種を含んだ溶液を前記チャネル内に導入し、
(c)余剰の溶液を前記チャネルから除去する請求項1記載の方法。 - 前記マイクロスポットを活性化するステップにおいて、前記マイクロスポットの上方で電界を発生させる請求項1記載の方法。
- (a)前記表面のマイクロスポットに含まれていない部分を非活性化し、
(b)1つ以上の前記第1チャネルに垂直な1つ以上の第2チャネルを形成し、
(c)各第2チャネルについて、第2結合要素を前記第2チャネル内に導入するステップをさらに含んでいる請求項4乃至6のいずれか1項記載の方法。 - 前記表面のマイクロスポットに含まれていない領域から参照データを取得するステップをさらに含んでいる請求項1乃至6のいずれか1項記載の方法。
- 表面上の2以上のマイクロスポットのそれぞれにおける第1結合要素である分子種を局地化する方法において、
局地化される領域のそれぞれに対して、
(a)前記表面の局地化される領域を活性化し、
(b)前記局地化される領域における1つ以上のマイクロスポットのそれぞれに対して、分子種をそのマイクロスポットに吸収させ、
(c)各マイクロスポットにおいて前記分子種に複数の濃度で第2結合要素を同時に付与し、それらの第2結合要素の濃度と前記第1結合要素である分子種とは複数の組合わせを有していることを特徴とする分子種を局地化する方法。 - 前記表面を活性化するステップが、
(a)前記局地化領域の周囲に第1チャネルを形成し、
(b)活性化物質を含んだ溶液を前記チャネル内に導入し、
(c)余剰の活性化溶液を前記チャネルから除去するステップを含んでいる請求項8記載の方法。 - 前記マイクロスポットを活性化するステップが、前記マイクロスポットの上方に電界を発生するステップを含んでいる請求項8記載の方法。
- 分子種を前記マイクロスポットに吸収させるステップにおいて、
(a)前記局地化領域に垂直なチャネルを形成し、
(b)前記分子種を含んだ溶液を前記チャネル内に導入し、
(c)余剰な溶液を前記チャネルから除去する請求項8記載の方法。 - (a)前記表面の局地化領域に含まれていない部分を非活性化し、
(b)1つ以上の前記第1チャネルに垂直な1つ以上の第2チャネルを形成し、
(c)第2チャネルのそれぞれについて、第2結合要素を前記第2チャネル内に導入するステップをさらに含んでいる請求項11記載の方法。 - 請求項9乃至12のいずれか1項記載の分子種を局地化する方法によって形成されたプローブアレイ。
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US51887803P | 2003-11-12 | 2003-11-12 | |
US60/518,878 | 2003-11-12 | ||
PCT/IL2004/001043 WO2005046859A2 (en) | 2003-11-12 | 2004-11-14 | System and method for carrying out multiple binding reactions in an array format |
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US9175421B2 (en) | 2015-11-03 |
US8105845B2 (en) | 2012-01-31 |
US20120129723A1 (en) | 2012-05-24 |
EP1682261B1 (en) | 2012-08-15 |
EP1682261A2 (en) | 2006-07-26 |
US20070087348A1 (en) | 2007-04-19 |
WO2005046859A2 (en) | 2005-05-26 |
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