JP6704882B2 - 結合要素を用いたアッセイのための装置及び方法 - Google Patents
結合要素を用いたアッセイのための装置及び方法 Download PDFInfo
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Description
本発明は、アッセイ、例えば、ポリヌクレオチドのためのアッセイに関する。
一態様において、装置は、堅固な基材と、該基材を少なくとも部分的に覆う柔軟なカバー要素と、前記基材中に形成され、液体を収容するように適合され、及び1つ又はそれ以上の細胞、芽胞又はウイルスの内容物を放出するように適合された第一の構造と(前記内容物は、標的分子(例えば、構造又はチャンバー又はウェル中の乾燥された緩衝液)を含む。)、前記基材中に形成され、液体を収容するように適合され、並びに標的分子を捕捉するように及び標的分子の存在及び/又は量の指標となる値を測定するように適合された少なくとも1つの結合要素を含む第二の構造(第一の構造とは異なり得る。)と、少なくとも前記第一の構造と前記第二の構造を相互接続する微小流体ネットワークと、並びに微小流体ネットワークの一部を選択的に閉鎖するために前記基材に対して前記柔軟なカバー要素を押圧することによって前記第一の構造と前記第二の構造の間に液体流を生じさせるように適合されたアクチュエータ要素とを含む。
第二の結合要素上の、標的核酸と複合体を形成していないレポーター化合物の前記量の残りの一部を捕捉すること;並びに第二の結合要素上に捕捉されたレポーター化合物の存在及び/又は量の指標となる1つ又はそれ以上の値を測定すること。
生物学的試料の分析は、1つ又はそれ以上のポリヌクレオチド(例えば、DNA、RNA、mRNA又はrRNA)が試料中に存在するかどうかを測定することを含み得る。例えば、特定の病原体の存在の指標となるポリヌクレオチドが存在するかどうかを測定するために、試料を分析することができる。
a)レポーター化合物のある量;捕捉分子のアンカー基を結合するように構成されている第一の結合要素;レポーター化合物を捕捉することができる第二の結合要素;前記レポーター化合物と複合体を形成することができる標的核酸のある量(レポーター化合物との複合体の形成は第二の結合要素によるレポーター化合物の補足を阻害する。);捕捉分子のある量(各捕捉分子は標的核酸のある領域に対して特異的な結合部分及びアンカー基を含む。)を提供すること;
b)それぞれ標的核酸及び捕捉分子を含む複合体を形成すること;
c)複合体を第一の結合要素に結合させるために、複合体を第一の結合要素と接触させること;
d)第一の結合要素から標的核酸の前記量の少なくとも一部を放出させること;
e)標的核酸の前記量の少なくとも一部とのレポーター化合物の前記量の一部の複合体を形成させること;
f)第二の結合要素上の、標的核酸と複合体を形成していないレポーター化合物の前記量の残りの一部を捕捉すること;並びに
g)第二の結合要素上に捕捉されたレポーター化合物の存在及び/又は量の指標となる値を測定すること。
−レポーター化合物のある量、レポーター化合物を結合することができる結合要素及びレポーター化合物を結合することができる標的核酸の量のある量を含む組成物を形成させること(レポーター化合物への標的核酸の結合は結合要素へのレポーター化合物の結合を阻害する。);
−標的核酸の前記量の少なくとも一部と、レポーター化合物の前記量の一部とを結合させること;
−結合要素上の、標的核酸と複合体を形成していないレポーター化合物の量の残りの一部とを結合させること;並びに
−結合要素に結合されたレポーター化合物の存在及び/又は量の指標となる値を測定すること
を含む。
−第一のレポーター化合物のある量と、
−第一のレポーター化合物と複合体を形成することができる第一の標的核酸の量と(第一のレポーター化合物との複合体の形成は結合要素による第一のレポーター化合物の捕捉を阻害する。)、
−第二のレポーター化合物、並びに
−第二のレポーター化合物と複合体を形成することができる第二の標的核酸のある量(第二のレポーター化合物との複合体の形成は結合要素による第二のレポーター化合物の捕捉を阻害する。)
を含む組成物を形成することを含む。
実施した競合的アッセイの原理は、図22中に模式的に示されている。適切な発現ベクター中(pCR(R)2.1−TOPO(R),Clontech,Inc.Palo Alto,CA,USA)にクローニングされたヒトポリオウイルス1単離株TCDCO1−861(GenBank受付番号AF538843)のDNAをDNAテンプレートとして使用した(本明細書では、「EV」(エンテロウイルス)DNAとも表記される。)。
フォワードPCRプライマー:
pr for EV 02:5’−CAAACCAGTGATTGGCCTGTCGTAACG−3’(AF538843のヌクレオチド位置492から518に対応する。)
リバースPCRプライマー:
pr rev EV01:5’−TTCACCGGATGGCCAATCCAATTCG−3’(AF538843のヌクレオチド位置617から641に対応する。)。
HP EV2 001:FAM−5’−ACCGACTACTTTGGGTGTCCGTGTTT−3’−TAMRA
(AF538843のヌクレオチド位置536から561に対応する。)。
EV2 02CY3:5’−ACCGACTACTTTGGGTGTCCGTGTTT−3’−CY3
(AF538843のヌクレオチド位置536から561に相当する。)の20nM(最終濃度)をさらに含有した。
実施した競合的アッセイの原理は、図24A中に模式的に示されている。発現ベクターpCR(R)2.1−TOPO(R)(Clontech,Inc.Palo Alto,CA,USA)のEcoRIエンドヌクレアーゼ制限部位中にクローニングされた合成HIV1gag/env融合構築物(EMBL受付番号A06258)のDNAをDNAテンプレートとして使用した。
フォワードPCRプライマー:cdia:5’−TGAAGGGTACTAGTAGTTCCTGCTATGTC−3’(A06258のヌクレオチド位置214から232に対応する。)
リバースプライマー:
cdis:5’−ATCAAGCAGCCATGCAAATGTT−3’(A06258のヌクレオチド位置384から405に対応する。)。
非特異的プローブ:
ara 54986 NH2:5’−ACCAGCTTTGAACCCAACAC−3’受容体特異的プローブ:
cdso29 NH2:5’−ACCATCAATGAGGAAGCTGCAGAATGGGA−3’。
Claims (8)
- 標的核酸を捕捉するように適合された第一の結合要素と、標的核酸の存在及び/又は量の指標となるレポーター分子を捕捉するように適合された第二の結合要素とを含む反応チェンバーを含み、
前記第一の結合要素は、捕捉核酸および標的核酸を含む複合体に捕捉核酸のアンカー基を介して結合するように構成された、合成粒子、ビーズまたは多孔性マトリックスを含み、当該捕捉核酸は標的核酸の配列領域に対して相補的な少なくとも1つの特異的配列領域を含み、および
前記第二の結合要素は、表面に配置されたレポーター特異的捕捉分子のマイクロアレイであり、
前記レポーター分子は、検出可能な標識を含み、および標的核酸に少なくとも部分的に相補的な領域であり、かつ、第二の結合要素上にも捕捉され得る少なくとも1つの特異的結合領域を含む一本鎖核酸であり、および
前記レポーター特異的捕捉分子は、レポーター分子の配列領域に対して相補的である少なくとも1つの特異的配列領域を含む一本鎖オリゴヌクレオチドであり、
第二の結合要素に隣接して配置された装置の少なくとも一部が、約1nmと約10μmの間の波長の範囲にある電磁波照射に対して透過性であり、これにより、第二の結合要素で捕捉された標識したレポーター分子の電磁波照射ベースの検出を可能とする、
装置。 - 前記ビーズは、磁性ビーズまたはラテックスビーズである請求項1に記載の装置。
- 少なくとも10の異なる種、少なくとも20の異なる種または少なくとも50の異なる種のレポーター特異的捕捉分子が前記第二の結合要素の表面上に配置されている請求項1または2に記載の装置。
- 前記反応チェンバーに配置され、かつ前記第一の結合要素が前記反応チェンバーから取り除かれることを防ぐように適合された少なくも1つのフィルターを含む、請求項1から3のいずれかに記載の装置。
- 前記少なくとも1つのフィルターは、少なくとも1つのフリットである、請求項4に記載の装置。
- 前記反応チェンバーが1μLから1mLの範囲の容積を有する請求項1から5のいずれかに記載の装置。
- 前記反応チェンバーが20μLから300μLの範囲の容積を有する請求項1から5のいずれかに記載の装置。
- 前記第二の結合要素に隣接して配置された装置の少なくとも一部が、400nmから800nmの波長の範囲の電磁波照射に対して透過性である、請求項1から7のいずれか一項に記載の装置。
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