JP5791535B2 - 1つの試料について複数の測定を実施する方法及び装置 - Google Patents
1つの試料について複数の測定を実施する方法及び装置 Download PDFInfo
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Description
(a)前記混合物を、その上に固定された1つ又は複数の脂質/タンパク質層を含む1つ又は複数のアッセイ電極と、好ましくは電極誘導発光アッセイ(好ましくは、電気化学発光アッセイ)に適合したマルチウェルプレートに前記混合物を添加することによって接触させるステップであって、プレートのウェルがアッセイ電極を備えるステップと、
(b)発光を誘発させるのに十分な電圧又は電流を電極に適用するステップと、
(c)放出された発光を測定するステップとを含む。
(a)好ましくは前記混合物を電極誘導発光(好ましくは、電気化学発光)アッセイに適合したマルチウェルプレート中に導入することによって、前記混合物を1つ又は複数のアッセイ電極に接触させるステップであって、前記プレートが1つ又は複数のアッセイ電極を備える複数のウェルを有するステップと、
(b)発光を誘発させるのに十分な電圧又は電流を電極に適用するステップと、
(c)放出された発光を測定するステップとを含む。
(a)好ましくは電極誘導発光(好ましくは、電気化学発光)アッセイに適合したマルチウェルプレート中に前記試料を導入することによって、前記試料をアッセイ電極と接触させるステップであって、前記プレートが1つ又は複数のアッセイ電極を備える複数のウェルを有するステップと、
(b)発光を誘発させるのに十分な電圧又は電流を電極に適用するステップと、
(c)放出された発光を測定するステップとを含む。
(a)多数の試験用化合物を選択するステップと、
(b)(上述したマルチウェルプレート及び/又は装置のどれか1つを用いて)前記多数の化合物を生物活性についてスクリーニングして、1つ又は複数の生物活性化合物を見出すステップと、
(c)前記1つ又は複数の生物活性化合物を改変して毒性を低減し、かつ/又は生物活性を高め、それによって1つ又は複数の改変生物活性化合物を形成するステップとを含む。
以下の実施例は、本発明の範囲内にある電極、プレート、キット及び方法のいくつかを説明するものである。これらの実施例が、決して本発明を限定するものと考えるべきでないことは言うまでもない。本発明に関する多数の変更形態及び改変形態が、当業者によって不必要な実験をすることなくなされ得る。
多層プレートの底部を、0.007”厚みのMylarポリエステルシート上に電極及び電気接点をスクリーン印刷することによって調製した。Mylarシートを、まずCO2レーザで切断して、導電性スルーホール(すなわち、その後導電性インクを充填して導電性にされる穴)、及びプレート底部をプレート上部と整列させるために使用した整列ホールを形成した。適切にパターン形成された銀インク層(Acheson 479ss)及びカーボンインク上層(Acheson 407c)をスクリーン印刷して、Mylarシートの底部に電気接点を形成させた。カーボンインク層を銀インク層よりもわずかに(0.03cm(0.01インチ))大きくして銀フィルムの縁部が露出するのを防止した。3層のカーボンインクを用いて確実に銀が露出しないようにした以外は同様にしてMylarフィルム上部に作用電極及び対電極を形成させた。導電性スルーホールは、これらのスクリーン印刷ステップ中に導電性インクで充たされた。続いて、誘電体インクを電極層上に印刷して、作用電極の活性露出表面を画成した。一般に、9つのプレートの底部を、18”×12”のMylarシート上に同時に印刷した。スクリーン印刷ステップ中の典型的な見当合わせ許容誤差(registrational tolerance)は、基板上部で+/−0.018〜0.020cm(0.007〜0.008インチ)であり、底部で+/−0.03cm(0.010インチ)であった。印刷された対電極帯と作用電極帯の間隔を>0.025cm(0.010インチ)に維持して短絡が形成されるのを防止した。場合によっては、パターン形成されたプレート底部を、大面積の平面電極で改変されたプラズマチャンバ(Series B、Advanced Plasma Systems、St.Petersburg、FL)中で酸素プラズマ(2000W、200mtorr)で5分間処理して、作用電極をコンディショニングした。
個々の正方形セクターに電気的に接触するように設計された機器でプレートを読み取った。セクターから放出されたECLを画像化するために用いた冷却CCDカメラ(VersArray:1300F、Princeton Instruments)に連結された(直径が4.1”のフロントエレメントを有する)テレセントリックレンズを用いて、機器と電気的に接触しているセクターの位置を合わせた。カメラには、寸法がほぼ2.6cm×2.6cmで1340×1300ピクセルのCCDチップを使用した。ピクセルサイズは、0.02mm×0.022mmであった。光路中に光学的バンドパスフィルターを用いて、ルテニウム−トリス−ビピリジンの放出プロファイルに一致する光を選択した。6つのセクターすべてを読み取りできるように平行移動テーブルを用いてプレートをテレセントリックレンズの下に平行移動させた。画像分析ソフトウエアを用いて、ウェル又はウェル内のアッセイドメインを確認し、特定のウェル又はドメインからのECLを定量した。特に示さない限り、およそ2〜5Vで3秒間の線形電圧スキャンを用いてスクリーン印刷炭素作用電極を有するプレートからECLを誘発させた。ECLは、(バックグラウンド光レベル及び検出器オフセットが補正された後)電圧スキャン期間にわたって測定された全積分光シグナルとして記録される。
多数の核酸プロセシング酵素が、核酸合成(例えば、ポリメラーゼ又はリガーゼ)活性とヌクレアーゼ活性の両方を有する。一例は、RNA依存性DNAポリメラーゼ(RDDP)活性とリボヌクレアーゼH活性の両方を有する酵素であるHIV逆転写酵素(RT)である。以下の例に、MDMWプレートの1つのウェル中で両方のHIV RT活性を測定するECLアッセイを示す。
ECL測定用に構成され、各ウェル中に露出した作用電極表面上に4つの流体封じ込め領域を有するMDMWプレート(実施例1のプレートC)を、4つの呼吸器疾患に特異的な抗体、すなわち、インフルエンザA、インフルエンザB、呼吸器合胞体ウイルス(RSV)、及びStreptococcus Pyogenes(Strep A)で被覆した。各抗体で被覆された1つのアッセイドメインを各ウェルが含むように、捕捉抗体溶液(リン酸緩衝食塩水PBS中50ug/ml)を、BioDotディスペンサを用いてウェル内の流体封じ込め領域に分注した(250nl/スポット)。溶液を乾燥させ、5%BSA溶液を添加し(200ul/ウェル)、プレートを終夜冷蔵した。プレートをPBSで洗浄してから使用した(4×250ul/ウェル)。
この実施例では、ECL測定用に構成され、各ウェル中に露出した作用電極表面上に4つの流体封じ込め領域を有するMDMWプレート(実施例1のプレートC)を用いた。以下の溶液、すなわち、0.0075%Tritonを含むPBS緩衝液に希釈した1mg/mlポリ−Glu:Tyr(4:1)(PGT)、0.0075%Tritonを含むPBS緩衝液に希釈した1mg/mlミエリン塩基性タンパク質(MBP)、0.0075%Tritonを含むPBS緩衝液に希釈した0.5mg/mlアビジン、5%BSAのPBS溶液のうち1つの250nLを各流体封じ込め領域に入れた。次いで、プレートを終夜乾燥させ、5%BSA溶液中4℃で2日間ブロックした。プレートを洗浄して遮断薬を除去したから使用した。
この実施例において、出願人らは、結合ドメイン面積の関数として、ビオチン及びRu(bpy)3のスルホン化誘導体で標識されたウシIgG(タンパク質1つ当たり約2.3標識)のECL測定の検出限界を測定した。結合ドメインを、電極の1つ又は複数の露出領域(流体封じ込め領域)上にアビジンを被覆することによって(電極表面にアビジン溶液を微量分注し、乾燥させることによって)形成させた。以下の5つのプレートタイプを準備した。
標準96:アビジンで被覆された単一の大きな結合ドメインを有する実施例1のプレートタイプB。
4−スポット−1:3つがアビジンで被覆されて結合ドメインを形成している4つの小さな流体封じ込め領域を有する実施例1のプレートタイプC。
4−スポット−3:1つのみがアビジンで被覆されて結合ドメインを形成している4つの小さな流体封じ込め領域を有する実施例1のプレートタイプC。
7−スポット−1:3つがアビジンで被覆されて結合ドメインを形成している7つのより小さな流体封じ込め領域を有する実施例1のプレートタイプD。
7−スポット−3:1つのみがアビジンで被覆されて結合ドメインを形成している7つのより小さな流体封じ込め領域を有する実施例1のプレートタイプD。
4種の異なるサイトカイン−インターロイキン1β(IL−1β)、インターロイキン6(IL−6)、インターフェロンγ(IFN−γ)及び腫瘍壊死因子α(TNF−α)−のサンドイッチイムノアッセイを、実施例1のプレートCに対して記載した設計及び手順に従って作製されたプレートのウェルにおいて、同時に実施した。各ウェル内の流体封じ込め領域上に抗体溶液を微量分注することによって別個のアッセイドメイン中に(目的分析物の1つにそれぞれ選択的である)4つの捕捉抗体をパターン形成させ(領域1つ当たり1つの抗体)、作用電極表面に抗体を吸着させた。(IL−1β及びTNF−αの場合32ug/mL、IL−6及びIFN−γの場合64ug/mLの濃度で)抗体を含有する溶液(0.25uL)及び0.1%BSAのリン酸緩衝食塩水溶液を、ソレノイドバルブ制御マイクロディスペンサ(Biodot Dispensor、Cartesian Technologies)を用いて流体封じ込め領域上に分注し、蒸発乾燥させた。この体積の抗体は、流体封じ込め領域内の露出電極表面全体に拡散するのに十分であるが、誘電体層によって形成される境界を超えて拡散しないのに十分な少なさであった。作用電極上の抗体溶液を乾燥後、プレート上部を取り付け、5%(w/v)ウシ血清アルブミン(BSA)を含有するリン酸緩衝食塩水(PBS)溶液をウェルに充填することによって、余分な未結合抗体を取り除いた(及び被覆されていない表面をブロックした)。プレートをブロッキング溶液と共に終夜4℃でインキュベートし、次いで、PBSで洗浄した。
この実施例では、MDMWプレートの1つのウェルにおいて全(リン酸化及び非リン酸化)EGF受容体(T−EGFR)及びリン酸化EGF受容体(P−EGFR)を測定するECLアッセイを示す。
1.A−431細胞を150mm組織培養皿で培養し、血清を終夜飢餓状態においた(1%ペニシリン−ストレプトマイシン及び1%ピルビン酸ナトリウムを補充したDMEM)。
2.無血清培地で2回すすいだ後、1枚の皿に、無血清培地中200nM EGFで15分間室温で刺激を与えた。刺激を与えないプレートには、無血清培地のみを加えた。
3.細胞を2体積のPBSですすいだ。
4.改変RIPA緩衝液(アッセイ初期に添加した新しいオルトバナジン酸ナトリウム)2mlを皿に添加した。RIPA緩衝液には、緩衝液10mL当たり1錠の新しいプロテアーゼ阻害錠剤を含む水中に、1mM純粋オルトバナジン酸ナトリウム、150mM NaCl、50mM Tris、6mMデオキシコレート、0.5%NP40が含まれていた)。細胞をRIPAと共に10分間氷上でインキュベートした。
5.上清を収集し、Pierce BCAタンパク質アッセイによって定量した。
1.(EGFRの細胞質ドメインに特異的な)T−EGFRのビオチン標識抗体及びP−EGFR(抗ホスホチロシン)を、予め1当量のアビジンと結合させ(1時間)、MDMWプレート(実施例1のプレートC)の各ウェルにおける4つの流体封じ込め領域のうちの2つに分注して(領域1つ当たり1つの抗体、0.5pmol/領域0.25uL)付着させた。残り2つの流体封じ込め領域を、非特異的結合及び交差反応の対照として用いた。1つの領域は、アビジンのみで被覆された。他の領域は、露出したままであったが、最終的にBSAでブロックした。
2.抗体を乾燥させた。次いで、ウェル1つ当たり200μlの5%BSA水溶液で1時間室温でウェルをブロックした。
3.プレートをdH20で4回洗浄した。
4.溶解物50μg/ウェルを96ウェルプレートの各ウェルに添加し、1時間断続的に振とうさせた。
5.プレートをdH20で4回洗浄した。
6.Sulfo−Tag(商標)標識α−EGFR抗体(33nM50uL)を添加し、結合反応を1時間室温で振とうさせながら進めた。プレートをdH20で4回洗浄した。
7.400mM gly−glyアッセイ緩衝液を含むウェル1つ当たり100μlの100mM TPAをECL分析直前に添加した。
8.ECL検出によってプレートを分析した。
この実施例では、非リン酸化EGF受容体とチロシン1173がリン酸化されているEGF受容体との両方をMDMWプレートの単一のウェルにおいて測定するECLアッセイを示す。
1.チロシン1173が自己リン酸化されているEGF受容体に特異的な抗体(pY1173)及びチロシン1173がリン酸化されていないEGF受容体に特異的な抗体(Y1173)を、MDMWプレート(実施例1のプレートC)の各ウェルにおける4つの流体封じ込め領域のうち2つに微量分注し(領域1つ当たり1つの抗体、0.2pmol/領域0.25uL)、受動吸着させて付着させた。残り2つの流体封じ込め領域を、非特異的結合及び交差反応の対照として用いた。これらの領域は露出したままであったが、最終的にBSAでブロックした。
2.抗体を終夜乾燥させた。次いで、ウェル1つ当たり200μlの5%BSA水溶液で1時間室温でウェルをブロックした。
3.プレートをPBSで洗浄した。
4.溶解物5μg/ウェルを96ウェルプレートの各ウェルに添加し、プレートを3時間断続的に振とうさせた。
5.プレートをPBSで洗浄した。
6.受容体の細胞外ドメインに対するSulfo−TAG標識α−EGFR抗体(33nM溶液50uL)を添加し、結合反応を1時間室温で振とうさせながら進めた。プレートをPBSで4回洗浄した。
7.400mM gly−glyアッセイ緩衝液を含む100mM TPAを、ECL分析直前にウェル1つ当たり150μl添加した。
8.Sector HTS(商標)プレートリーダー(Meso Scale Discovery)を用いたECL検出によってプレートを分析した。
この実施例では、ECL測定用に構成され、各ウェル中に露出した作用電極表面上に4つの流体封じ込め領域を有するMDMWプレート(実施例1のプレートC)を用いた。4つの以下の溶液、すなわち、(i)0.015%Tritonを含むPBS緩衝液に希釈した0.5mg/mlポリ−Glu:Tyr(4:1)(PGT)、(ii)0.015%Tritonを含むPBS緩衝液に希釈した0.2mg/mlミエリン塩基性タンパク質(MBP)、(iii)0.015%Tritonを含むPBS緩衝液に希釈した0.3mg/mlストレプトアビジン、(iv)0.15%Tritonを含むPBS緩衝液に希釈した0.3mg/mlBSA溶液のうち1つの250nLを4つの流体封じ込め領域の各々に入れた。次いで、プレートを、周囲条件で1〜1.5時間乾燥させ、0.1%Tritonを含有するPBSで強力に洗浄し、水で洗浄し、5%BSA溶液中で少なくとも2時間室温でブロックした。洗浄には、ウェル中に洗浄溶液の一定の流れを形成し、余分なペプチド/タンパク質を電極表面から極めて効率的に洗浄除去可能であるBiotechの96ウェルプレートウォッシャーを用いた底部洗浄が含まれる。Triton含有溶液を用いて洗浄した後に3回洗浄して痕跡量のTritonを除去した。ブロッキング後、プレートを再度洗浄して遮断薬を除去してから使用した。
Claims (5)
- 複数のアッセイドメインを含むマルチウェルプレートであって、プレート上部及びプレート底部を含み、
前記プレート底部は、複数のウェルの底部をさらに画成し、それによって
(a)個別に指定可能な複数の電極を含む上部表面、
(b)1つ又は複数の電気的接続表面を含む底部表面、及び
(c)前記1つ又は複数の電気的接続表面と前記複数の電極とを電気的に接続する1つ 又は複数の導電性穴
を有する基板を含み、
前記複数のアッセイドメインが、試料中の1つ又は複数の分析物と結合可能な1つ又は複数の結合試薬を有する1つ又は複数のアッセイドメインを含み、
前記複数のアッセイドメインが、前記試料に添加された対照分析物と結合可能な対照結合試薬を有する第1の対照ドメインと、遮断薬を有する第2の対照ドメインとをさらに含む、
上記マルチウェルプレート。 - 前記第1の対照ドメインが、(i)非特異的結合又は生化学活性、(ii)ECL発生に対する化学的干渉、(iii)光透過に対する光学的干渉、(iv)ピペット操作誤差、(v)タイミング誤差、(vi)混合の変動、(vii)温度変動、及び(viii)アッセイモジュールの変動の1つ又は複数の対照である、請求項1に記載のマルチウェルプレート。
- 前記第2の対照ドメインが非特異的結合の対照である、請求項1に記載のマルチウェルプレート。
- 前記マルチウェルプレートが、前記1つ又は複数の分析物及び前記対照分析物をサンドイッチイムノアッセイ形式で検出するように構成されている、請求項1に記載のマルチウェルプレート。
- 前記遮断薬が、前記アッセイにおいて使用される検出抗体と組み合わせられない対照抗体である、請求項1に記載のマルチウェルプレート。
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CA2459893A1 (en) | 2003-03-20 |
EP1451348A4 (en) | 2008-08-06 |
US7858321B2 (en) | 2010-12-28 |
US20190257823A1 (en) | 2019-08-22 |
JP2009142283A (ja) | 2009-07-02 |
CA2459893C (en) | 2014-01-21 |
JP4768224B2 (ja) | 2011-09-07 |
WO2003023360A3 (en) | 2003-07-10 |
AU2002341621A1 (en) | 2003-03-24 |
US20030207290A1 (en) | 2003-11-06 |
WO2003022028A2 (en) | 2003-03-20 |
JP2015156865A (ja) | 2015-09-03 |
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