JP2010533872A5 - - Google Patents
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- JP2010533872A5 JP2010533872A5 JP2010517171A JP2010517171A JP2010533872A5 JP 2010533872 A5 JP2010533872 A5 JP 2010533872A5 JP 2010517171 A JP2010517171 A JP 2010517171A JP 2010517171 A JP2010517171 A JP 2010517171A JP 2010533872 A5 JP2010533872 A5 JP 2010533872A5
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Claims (28)
- 試験試料中に存在しうるターゲット分子を検出するための方法であって:
(a)第一のタグを含み、そしてターゲット分子に対する特異的アフィニティを有するアプタマーと、試験試料を接触させることによって、混合物を調製し、ここで、前記試験試料中に前記ターゲット分子が存在するならば、アプタマー・アフィニティ複合体が形成され;
(b)第一の捕捉要素を含む第一の固体支持体に混合物を曝露し、そして第一のタグが第一の捕捉要素と会合するのを可能にし;
(c)前記の第一の固体支持体と会合していない混合物の構成要素をすべて除去し;
(d)前記の第一の固体支持体からアプタマー・アフィニティ複合体を遊離させ;
(e)アプタマー・アフィニティ複合体中の前記ターゲット分子に第二のタグを付着させ;
(f)遊離したアプタマー・アフィニティ複合体を、第二の捕捉要素を含む第二の固体支持体に曝露し、そして第二のタグが前記の第二の捕捉要素と会合するのを可能にし;
(g)前記アプタマー・アフィニティ複合体から,複合体化されていないアプタマーを分配することによって、複合体化されていないアプタマーを前記混合物からすべて除去し;そして
(h)前記アプタマー・アフィニティ複合体のアプタマー部分を検出することによって、前記ターゲット分子を検出する
工程を含む、前記方法。 - (h)アプタマーを検出する前に、前記アプタマー・アフィニティ複合体からアプタマーを解離させる工程をさらに含む、請求項1の方法。
- a.第一のタグが第二のタグと同じであり、そして第二のタグが(b)の後および(f)の前の任意の時点でターゲット分子に添加され、そして第二のタグの添加前に第一の捕捉剤をブロッキングする工程を含む、または b.第一のタグが第二のタグと異なり、そして第二のタグが(f)の前の任意の時点でターゲット分子に添加される、
請求項1の方法。 - (a)の後および(d)の前の任意の時点で動力学的負荷を導入する工程をさらに含む、請求項1の方法。
- 前記動力学的負荷が、アプタマー・アフィニティ複合体を含有する混合物を希釈し、及び/又はアプタマー・アフィニティ複合体を含有する混合物に競合剤を添加し、そして以下のいずれかの時間:
a.約30秒間以上、1分間以上、2分間以上、3分間以上、4分間以上、5分間以上、10分間以上、30分間以上、および60分間以上からなる群より選択される時間;または
b.非特異的複合体の測定レベルに対するアプタマー・アフィニティ複合体の測定レベルの比が増加するような時間;
に渡って、アプタマー・アフィニティ複合体を含有する混合物をインキュベーションする工程を含む、請求項4の方法。 - 動力学的負荷が、競合剤分子の導入を含み、そして前記競合剤分子が、オリゴヌクレオチド、ヘパリン、ニシン精子DNA、サケ精子DNA、硫酸デキストラン、ポリアニオン、脱塩基ホスホジエステルポリマー、dNTP、およびピロホスフェートからなる群より選択される、請求項4の方法。
- 前記アプタマー・アフィニティ複合体が緩慢な解離速度を有する、請求項1の方法。
- 前記アプタマー・アフィニティ複合体の解離速度(t1/2)が、
a.約30分間以上である;
b.約30分間〜約240分間の間である;またはc.約30分間以上、約60分間以上、約90分間以上、約120分間以上、約150分間以上、約180分間以上、約210分間以上、および約240分間以上からなる群より選択される、
請求項7の方法。 - Q−PCR、MS、およびハイブリダイゼーションからなる群より選択される方法を用いて、前記アプタマーを検出し、そして場合によって定量化する、請求項1の方法。
- TaqMan(登録商標)PCR、PCRプロセス中の挿入蛍光色素、またはPCRプロセス中の分子ビーコンを用いて前記Q−PCRを行う、請求項9の方法。
- アプタマーに検出可能部分を添加する工程をさらに含む、請求項1の方法。
- 前記検出可能部分が、色素、量子ドット、放射標識、電気化学官能基、酵素、および酵素基質からなる群より選択される、請求項11の方法。
- a.前記色素が蛍光色素である、または
b.前記酵素がアルカリホスファターゼまたは西洋ワサビペルオキシダーゼである、
請求項12の方法。 - 前記アプタマーが一本鎖核酸または二本鎖核酸である、請求項1の方法。
- 前記アプタマーがDNA、RNA、またはDNAおよびRNA両方を含む、請求項1の方法。
- 前記アプタマーが、少なくとも1つの化学的修飾を含む、請求項1の方法。
- 前記の少なくとも1つの化学的修飾が、
a.リボース位、デオキシリボース位、リン酸位、および塩基位から独立に選択される1以上の位での化学的置換である、または
b.図(19)に列挙される群より独立に選択される、
請求項16の方法。 - 前記ターゲット分子が、タンパク質、ペプチド、炭水化物、多糖、糖タンパク質、ホルモン、受容体、抗原、抗体、ウイルス、基質、代謝物、遷移状態類似体、補因子、阻害剤、薬剤、色素、栄養物、増殖因子、組織、および規制物質からなる群より選択される、請求項1の方法。
- 前記ターゲット分子がタンパク質またはペプチドである、請求項1の方法。
- 前記試験試料が、
a.生物学的試料、環境試料、化学試料、薬学的試料、食品試料、農業試料、および獣医学的試料からなる群より選択される、
b.全血、白血球、末梢血単核細胞、血漿、血清、痰、息、尿、精液、唾液、髄膜液、羊水、腺液、リンパ液、乳頭吸引液、気管支吸引液、滑液、関節吸引液、細胞、細胞抽出物、糞便、組織、組織抽出物、組織生検、および脳脊髄液からなる群より選択される生物学的試料である、または
c.血漿または血清である、
請求項1の方法。 - a.前記の第一のタグおよび前記の第二のタグが各々、ポリヌクレオチド、ポリペプチド、ペプチド核酸、ロック核酸、オリゴ糖、多糖、抗体、アフィボディ、抗体模倣体、細胞受容体、リガンド、脂質、ビオチン、アビジン、ストレプアビジン(strepavidin)、Extravidin、ニュートラアビジン、金属、ヒスチジン、および任意のこれらの構造の任意の部分からなる群より独立に選択される、少なくとも1つの構成要素を含む、 b.前記の第一の捕捉要素および前記の第二の捕捉要素が各々、ポリヌクレオチド、ポリペプチド、ペプチド核酸、ロック核酸、オリゴ糖、多糖、抗体、アフィボディ、抗体模倣体、細胞受容体、リガンド、脂質、ビオチン、アビジン、ストレプアビジン、Extravidin、ニュートラアビジン、金属、ヒスチジン、および任意のこれらの構造の任意の部分から独立に選択される、少なくとも1つの構成要素を含む、
c.前記の第一の固体支持体および第二の固体支持体が各々、ポリマービーズ、アガロースビーズ、ポリスチレンビーズ、アクリルアミドビーズ、固体コアビーズ、多孔ビーズ、常磁性ビーズ、ガラスビーズ、制御孔ビーズ、マイクロタイターウェル、シクロオレフィン・コポリマー支持体、膜、プラスチック支持体、ナイロン、ラングミュア−ボジェット(Langmuir−Blodgett)膜、ガラス、ゲルマニウム支持体、シリコン支持体、シリコンウェハーチップ、フロースルーチップ、マイクロビーズ、ポリテトラフルオロエチレン支持体、ポリスチレン支持体、ガリウムヒ素支持体、金支持体、および銀支持体からなる群より独立に選択される、
請求項1の方法。 - 前記アプタマーを定量化することによって、前記ターゲットを定量化する工程をさらに含む、請求項1の方法。
- アプタマーの検出が、第三の固体支持体にアプタマーをハイブリダイズさせる工程を含み、ここで第三の固体支持体が、複数のアドレス可能特徴を含み、そして前記特徴の少なくとも1つが、アプタマー内に含有される任意の配列に相補的である、該特徴上に配置された捕捉要素を少なくとも含む、請求項1の方法。
- 試料中のターゲット分子の存在を検出するかまたは該分子の量を決定する方法であって:
(i)ターゲット分子に複数のアプタマーを提供し、ここでアプタマーは切断可能捕捉タグを含み;
(ii)ターゲット分子を含有する試料と、アプタマーを接触させて、アプタマー−ターゲット分子複合体を含有する混合物を形成し;
(iii)支持体表面に付着したプローブを有する固体支持体を提供し、ここでプローブは切断可能捕捉タグに結合可能であり;
(iv)アプタマー−ターゲット分子複合体が、切断可能捕捉タグおよびプローブの結合を通じて支持体に結合されるように、固体支持体と混合物を接触させ;
(v)固体支持体に結合したアプタマー−ターゲット分子複合体を、混合物の残りから分配し;
(vi)アプタマー−ターゲット分子複合体のターゲット分子構成要素に、第二の捕捉タグを導入し;
(vii)切断可能捕捉タグを切断することによって、固体支持体表面からアプタマー−ターゲット分子複合体を解離させ;
(viii)支持体表面に付着したプローブを有する固体支持体を提供し、ここでプローブはターゲット分子上の第二の捕捉タグに結合可能であり;
(ix)アプタマー−ターゲット分子複合体が、第二の捕捉タグおよびプローブの結合を通じて支持体に結合されるように、(viii)由来の固体支持体と、解離したアプタマー−ターゲット分子を接触させ;
(x)アプタマー−ターゲット分子複合体を解離させて、未結合(free)アプタマーおよび支持体に結合したターゲット分子を生じ;
(xi)未結合アプタマーを検出する
工程を含む、前記方法。 - 固体支持体表面からのアプタマー−ターゲット分子複合体解離後(vii)、解離したアプタマー−ターゲット分子複合体を、
a.過剰な競合剤分子と接触させる、及び/又は
b.希釈する、
請求項24の方法。 - 検出される未結合アプタマーの量を測定する工程をさらに含む、請求項24または25の方法。
- a)関心対象の1以上のターゲットに特異的な1以上のアプタマー;および
b)1以上の固体支持体;および
c)1以上の分配試薬;および
d)アフィニティ複合体からのアプタマーの遊離のための1以上の試薬
を含む、キット。 - a.関心対象の1以上のターゲットを誘導体化するための試薬、
b.前記の1以上のアプタマーにおいて、切断可能部分を切断する試薬、
c.動力学的負荷において使用するための試薬、
のいずれかをさらに含む、請求項27のキット。
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