JP6695899B2 - 標準物質として外部生体分子を用いた生体分子分析方法およびそのキット - Google Patents
標準物質として外部生体分子を用いた生体分子分析方法およびそのキット Download PDFInfo
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Description
外部標準物質の濃度×2−△Ct−n
外部標準物質の濃度×2−△Ct−cal
患者の急性心筋梗塞を正確に診断し、迅速な診断のための特異性と感度の高い診断用バイオマーカー、すなわち、急性心筋梗塞特異マーカーの発掘が求められている。
精度管理および測定用の標準物質がある試料における一つまたはそれ以上の標的生体分子を、これらに相応するリガンドおよび設計核酸を用いるリガンド−核酸分析方法で分析するために、本発明で提案する設計核酸の基本構成は、次の一般式(I)で表されることを特徴とする方法:
ここで、CGGAAGCGTGCTGGGCCは配列番号1、CATAACCCAGAGGTCGAは配列番号2でそれぞれ特定される。
標的生体分子のそれぞれ異なるエピトープ(epitope)に対する2種の単クローン抗体または標的生体分子に結合する多クローン抗体を2つのグループに分け、一つのグループは設計核酸を、もう一つのグループは磁性物質を結合させて、前者は分析リガンドと命名し、後者は収穫リガンドと命名した。また、標準物質の抗体である場合は、設計核酸が結合された抗体を標準分析リガンドと命名し、また磁性物質が結合された抗体を標準収穫リガンドと命名した。
精度管理および測定用の標準物質であるE. coli Enoyl−ACP Reductaseと標的生体分子は、TBS[Tris−buffered saline;150mM NaCI、20mM tris−CI(pH7.5)、および0.02%NaN3]中で最終濃度10ng/mlにてPCRチューブに準備した。
45μLの前記準備された分析試料溶液をマイクタイタープレート(Falcon 3911;Becton Dickinson)のウェルに入れた。前記分析試料溶液に50μLの0.45% nonfat dried milkとdenatured salmon sperm DNA(0.1mg/ml)のあるTETBS(0.1mM EDTAおよび0.1%Tween 20の含まれたTBS)を添加した。
45μLの前記で準備された分析試料をマイクタイタープレート(Falcon 3911;Becton Dickinson)のウェルに入れた。分析試料のあるプレートを4℃で約15時間処理して生体分子をプレートのウェル(well)の表面に固定させる。250μLのTBSでプレートのウェルを3回洗浄した。
設計核酸の塩基配列を用いた精度管理および測定用標準物質の添加された試料における、一つまたはそれ以上の標的生体分子の分析は、設計核酸の塩基配列に相応するTaqmanプローブを用いたマルチプレックスリアルタイムポリメラーゼ連鎖反応法(multiplex real−time PCR)で行った。
外部標準物質の濃度×2−△Ct−n
外部標準物質の濃度×2−△Ct−cal
設計核酸の長さを用いた、精度管理および測定用標準物質が添加された試料における一つまたはそれ以上の標的生体分子の分析は、設計核酸の長さを分析することができるキャピララリー電気泳動で行った。
生体試料である血液を用いて急性心筋梗塞または敗血症などの慢性疾患を検診しており、特に、一般的に、患者モニタリング用生体分子を3〜4個検査し、結果のcut−off値のみを参考することで診断を行っている。
Claims (7)
- (a)標的タンパク質が1種以上存在する試料を準備する段階と、
(b)前記試料に、外部標準物質として、その試料に存在しない一つ以上のタンパク質を定量して一定の濃度で添加する段階と、
(c)前記外部標準物質が添加された試料に、その試料中の標的タンパク質と前記外部標準物質にそれぞれ特異的に結合するリガンドと設計核酸との連結構造体を添加して、前記標的タンパク質とその特異的連結構造体との複合体および前記外部標準物質とその特異的連結構造体との複合体を形成する段階と、
(d)前記複合体を分離する段階と、
(e)前記複合体の設計核酸を同時に増幅する段階と、
(f)前記標的タンパク質に対する複合体の設計核酸の増幅物と前記外部標準物質に対する複合体の設計核酸の増幅物を定量し比較して前記標的タンパク質に対する定量情報を得る段階と、
(g)前記(f)段階の標的タンパク質の定量情報に、前記標的タンパク質とそれに対する連結構造体の結合親和度と、前記外部標準物質とそれに対する連結構造体の結合親和度との差を反映する段階とを含んでなり、
前記反映段階が以下の(i)乃至(vi)段階を含み、
(i)前記標的タンパク質と前記外部標準物質を定量して、同じ濃度で含む対照試料を準備する段階、
(ii)前記対照試料の定量された標的タンパク質または定量された外部標準物質に特異的に結合し、前記(c)段階で使用した連結構造体と同じ連結構造体を添加して、前記標的タンパク質とその特異的連結構造体との複合体、および前記外部標準物質とその特異的連結構造体との複合体を形成する段階、
(iii)前記複合体を分離する段階、
(iv)前記複合体の設計核酸を同時に増幅する段階、
(v)前記標的タンパク質に対する増幅物と前記外部標準物質に対する増幅物との差異を求める段階、および
(vi)前記差異を前記(f)段階の標的タンパク質の定量情報に反映する段階;
前記設計核酸は、順方向プライマーが結合する領域、逆方向プライマーが結合する領域、およびこれらの領域の間にリガンドが特異的に結合するタンパク質を指し示してそのタンパク質を識別することを可能にする領域を持つ設計核酸であり、
前記設計核酸の順方向プライマーが結合する領域と逆方向プライマーが結合する領域は、一つの順方向プライマーおよび逆方向プライマーセットですべての設計核酸を増幅することができるようにすべての設計核酸で共通した塩基配列を有する、外部標準物質を用いた試料中のタンパク質分析方法。 - 前記すべての増幅は、その増幅物をリアルタイムで検出することができるリアルタイムポリメラーゼ連鎖反応で行われ、
その増幅物の量がCt(Threshold cycle)値で算出され、
前記(f)段階の定量し比較した結果が△Ct−n(Ct 標的タンパク質 −Ct 外部標準物質 )で算出され、
前記(f)段階の標的タンパク質に対する定量情報が前記(b)段階の外部標準物質の濃度×2 −△Ct−n で算出され、
前記(v)段階の差異が△Ct−c(Ct 対照試料の標的タンパク質 −Ct 対照試料の外部標準物質 )で算出されることにより、
前記(vi)段階が、前記△Ct−nと△Ct−cとの差異値である△Ct−cal(△Ct−n−△Ct−c)を反映して、前記(f)段階の標的タンパク質の定量情報が前記(b)段階の外部標準物質の濃度×2 −△Ct−cal で決定されるように行われる、請求項1に記載の方法。 - 前記試料中の標的タンパク質は2種以上であり、
これにより、その標的タンパク質に特異的なリガンドと設計核酸との連結構造体も2種以上であり、
前記2種以上の連結構造体の設計核酸における、その順方向プライマーが結合する領域とその逆方向プライマーが結合する領域は、その種類を問わず、すべての連結構造体の設計核酸の間で共通した塩基配列を有する、請求項1に記載の方法。 - 前記リガンドは抗体である、請求項1に記載の方法。
- 前記設計核酸は一本鎖DNAまたは一本鎖RNAであり、
前記リガンドが特異的に結合するタンパク質を指し示す領域は塩基配列またはその長さで指し示す領域である、請求項1に記載の方法。 - 前記(e)段階の増幅は、ポリメラーゼ連鎖反応(PCR)、リアルタイムポリメラーゼ連鎖反応(real time PCR)、逆転写−ポリメラーゼ連鎖反応(RT−PCR)、リアルタイム逆転写−ポリメラーゼ連鎖反応(real time RT−PCR)、マルチプレックスポリメラーゼ連鎖反応(multiplex PCR)、リアルタイムマルチプレックスポリメラーゼ連鎖反応(real time multiplex PCR)およびマルチプレックス逆転写−ポリメラーゼ連鎖反応(multiplex RT−PCR)のうちのいずれか一つのポリメラーゼ連鎖反応によって行われる、請求項1に記載の方法。
- 前記(e)段階の増幅は、リアルタイムポリメラーゼ連鎖反応(real time PCR)、リアルタイム逆転写−ポリメラーゼ連鎖反応(real time RT−PCR)、リアルタイムマルチプレックスポリメラーゼ連鎖反応(real time multiplex PCR)およびリアルタイムマルチプレックス逆転写−ポリメラーゼ連鎖反応(real time multiplex RT−PCR)よりなる群から選ばれたポリメラーゼ連鎖反応によって行われ、
前記増幅の際にTaqman(登録商標)プローブ[Taqman(登録商標)probe]が添加され、そのTaqman(登録商標)プローブでその増幅物を定量することを特徴とする、請求項1に記載の方法。
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