JP6289604B2 - 改善されたアッセイ方法 - Google Patents
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- JP6289604B2 JP6289604B2 JP2016502025A JP2016502025A JP6289604B2 JP 6289604 B2 JP6289604 B2 JP 6289604B2 JP 2016502025 A JP2016502025 A JP 2016502025A JP 2016502025 A JP2016502025 A JP 2016502025A JP 6289604 B2 JP6289604 B2 JP 6289604B2
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Description
2013年3月13日出願の米国仮出願第61/779,050号、および2013年12月23日出願の米国仮出願第61/919,887号に対する参照がなされ、それぞれ、各々の開示は参照によりその全体が本明細書に組み入れられる。
さらなる実施形態を図7に図示する。パネル(a)のサンドイッチ免疫アッセイ複合体における各近接プローブの一部分が、各セグメントにハイブリダイズしたRNAの短鎖により一時的に保護される。RNA鎖は酵素で除去され、近接プローブの各々が互いにハイブリダイズし、鎖はビオチン化dNTPを使用して、ポリメラーゼ伸長により伸長する(パネル(b))。鎖に組み込まれた各ビオチン化塩基はそれぞれ、検出可能な標識で標識したストレプトアビジンに結合する(パネル(c))。
標的分析物に対する一対の検出抗体を、近接プローブ1および2を追加して以下のように修飾した:100uLバッファー中200ugの第1の検出抗体に、1.74uL、23mMのsulfo−SMCCを追加し、150mMリン酸緩衝液で希釈し、30分間室温でインキュベートした。遊離したsulfo SMCCはサイズ排除クロマトグラフィーにより除去した。検出抗体の最終濃度は2mg/mLまたはこれよりわずかに低かった。300uMのチオール修飾オリゴヌクレオチド(近接プローブ1および2)95uLを、100mMリン酸緩衝液中1mM DTTの5uL、0.5mM EDTA、pH8.4で室温で1時間還元した。近接プローブ1および2の配列は次の通りである:
実施例2に記述したアッセイを繰り返したが、ここでは環状テンプレートを形成するために2つの別個のライゲーション部位を有する2つのコネクターオリゴヌクレオチドを使用する代わりに、ライゲーション部位を1つ有する単一の線状コネクターオリゴヌクレオチドを使用した。図11(a)に示すように、ライゲーション部位1またはライゲーション部位2で開いた単一の線状コネクターオリゴヌクレオチドを準備した。単一の線状コネクターオリゴヌクレオチドを両方とも、実施例1と2で使用したオリゴヌクレオチド、Circ−1とCirc−2との組合せと並行してテストした。実施例2に記述したプロトコルを用いて、さらに、単一の線状コネクターオリゴヌクレオチドを3種類の濃度、125nM、62.5nMおよび31nMでテストし、一方、Circ−1とCirc−2のオリゴヌクレオチドの組合せを使用する標準アッセイを125nMでテストした。図11(b)に示すように、2つの単一線状コネクターオリゴヌクレオチドは、2種のコネクターオリゴヌクレオチド混合物(Circ−1とCirc−2)とおよそ同一の効率でRCA増幅産物に成功裡に組み込まれた。ライゲーション部位1で開いていた単一の線状コネクターオリゴヌクレオチドは、信号強度、非特異的バックグラウンドおよび総合的な感度に基づいて、2種のコネクター混合物に匹敵する性能を有していた。予想した通り、ライゲーション部位2で開いていた単一の線状コネクターオリゴヌクレオチドは、非特異的なバックグラウンドがより高く、感度はより低かった。この後者のケースでは、ライゲーションとプライミングの両方が、近接プローブ#1の存在にのみ依存しており;したがって、近接増幅アプローチの特定の利点のうちのいくつかが損なわれた。
以下の試薬の代替セットを用いて、実施例1で概説したプロトコルを使用してアッセイを実施した:
(a)コード化粒子を使用する、ビーズベースの免疫アッセイフォーマット
アッセイ工程はすべて96ウェルフィルタプレートで実施した。真空マニホールドでプレートから液体を除去した(Hg10インチを超過せず)。プレートは反転させない。目詰まりが生じた場合、コニカルチューブ15mlの先端を使用して目詰まりしたウェルの下の区域をやさしく押し、次に、1mlのパスツールピペットのゴム球を使用するか、目詰まりしたウェルを親指でやさしく押して、詰まりを取り除く。最終の吸引工程後、ペーパータオルを重ねた上でプレートの底を軽く叩き、次に、フィルタプレートの底面をKimwipeで拭いて残留液/液滴を除去した。
(1)10xキャプチャービーズストックをボルテックス(30秒)および超音波分解(30秒)する。ホイルにくるんだチューブで、10xキャプチャービーズストック(ウェル当たり2.5μl)を洗浄溶液に希釈する(ウェル当たり25μl 約2,000〜5,000ビーズ/アッセイ)。より高度な多重化には、保持した10xキャプチャービーズストックの追加分の容積を調製できるよう洗浄溶液の容積を調整する。
(6)蛍光標識した検出抗体の1x混合物の調整:希釈剤(ウェル当たり100μl)で10x検出抗体混合物(ウェル当たり10μl)を希釈した。混合物は目的の分析物に特異的な一対の検出抗体を含み、1つはAlexa Fluor350(青色蛍光標識)で標識し、もう1つはAlexa Fluor594(赤色蛍光標識)で標識した(これらの各々の蛍光標識はLife Technologies, Grand Island、NY、www.lifetechnologies.comから入手可能)。より高度な多重化は、必要な10x抗体混合物の追加の容積を調製できるよう希釈剤の容積を調整する。吸引し洗浄溶液200μLで2回アッセイウェルを洗浄した。100μlの希釈検出抗体混合物を各アッセイウェルに追加した。プレートをカバーし、プレート振とう機で1時間インキュベートした(500〜600rpm)。
(8)吸引し洗浄溶液200μLで3回アッセイウェルを洗浄した。清潔なペーパータオルでフィルタプレートの底を乾かし、完全に全残留液滴を除去した。100μl洗浄溶液を各アッセイウェルに追加し、2〜3分間プレート振とう機(500〜600rpm)上にプレートを載せた。
実施例5(a)で概説したように、アッセイ工程はすべて96−ウェルフィルタプレートで実施した。実施例5(a)で記述したように、洗浄溶液およびアッセイの標準試料を調製し、実施例1で記述したように、標的分析物に対する一対の検出抗体を、近接プローブ1および2を追加して修飾した。分析物を実施例5(a)で記述したように捕捉ビーズ上で捕捉した。捕捉ビーズは固定部分を含むが、この固定部分は、BSA−オリゴヌクレオチド複合体としてビーズ表面に固定化され、オリゴヌクレオチドはローリングサークルのアンプリコンに特異的であるよう選択される。使用した固定オリゴヌクレオチドの配列は配列番号3である。
試料を25%ウシ血清(2〜4倍希釈)100ulで調製し、捕捉抗体でコートした500Kビーズ(常磁性2.7μm、場合によって蛍光コード化)を、試料に追加した。試料を23℃で約2時間インキュベートした。試料をPBS(5X、0.1%Tween−20)で3回洗浄し、標識した検出抗体の混合物を追加した(第1ビオチン化検出抗体およびハプテンコンジュゲート抗体を含む混合物)。混合物を23℃で約1時間インキュベートした。混合物をPBS(5X、0.1%Tween20)で3回洗浄し、酵素標識を追加し、ストレプトアビジン−β‐ガラクトシダーゼ(40pM)、抗ハプテンコンジュゲート酵素も追加し、混合物を23℃で約30分間インキュベートした(またはSimoaアナライザーに3分間かけた)。この混合物を、PBS(5X、0.1%Tween 20)で7回洗浄し、酵素基質、レゾルフィン−ベータ−d−ガラクトピラノシド 15ul(ローディングバッファー内100uM)を追加した。
第1のインキュベーション:試料10ul、ビオチン化モノクローナル分析物特異的捕捉抗体(希釈標準溶液2.6mg/l)、およびそれぞれオリゴヌクレオチドにコンジュゲートさせたモノクローナル分析物特異抗体の混合物(0.3mg/lの希釈標準溶液)を反応させてサンドイッチ複合体を形成した。モノクローナル分析物特異抗体の混合物は実施例1の記述通りに調製し、混合物には、実施例1で上述したように、近接プローブ1および2にコンジュゲートした一対の抗体が挙げられる。
材料、方法および結果:
実施例1に記述した手順をHIV−1 p24を検出するために使用した。HIV−1 mixed titer performance panel(Seracare Life Sciences、www.seracarecatalog.comから入手可能)、HIV−1セロコンバージョンパネル(これもSeracare Life Sciencesから入手可能)、HIV抗体陽性試料(ProMedDx、LLC、www.promeddx.comから入手可能)および正常な対応試料(Bioreclamation、www.bioreclamation.comから入手可能)から入手した約64の血清または血漿試料を試験した。上述の手順に従って導出されたHIV−1 p24アッセイの検量線を図12に示す。アッセイのLODは、1.3fg/mLであることが見出され、LLOQは3.0fg/mL、ULOQは37、500fg/mLだった。25uL試料の検出限界1.3fg/mLはp24分子約650個に相当し、ウィルス粒子(分子)はそれぞれ、p24タンパク質の複製を約2000個生成する。
HIVに感染して間もない患者は、この疾病の伝播の一因となる傾向がある。ウィルス負荷は感染後最初の数週間高く、新たに感染した患者は、自らの感染とともに、他者へ感染を拡大し得ることを自覚していない可能性が高い。したがって、急性HIV感染の早期発見は、公衆衛生上非常に重要である。PCR法は確立された感受性を有し;血清または血漿mL当たりわずか60のHIV RNA複製を検出できる(mL当たりウィルス粒子30個)。しかしながら、PCR法は複雑で費用が高く、したがって、すべての状況で適切とはならない。免疫アッセイはより単純でより安価であるが、現行の検出限界は、第四世代のp24免疫アッセイにおいてわずか約10pg/mL、または1mL当たり約2億5000万個のカプシドタンパク質となっている。ウィルス当たり約2,000個のp24カプシドタンパク質が存在するという事実にもかかわらず、これらの免疫アッセイは、ウイルスベースでPCR検査の数千倍分の一の感度である。
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Claims (8)
- 試料中の目的の分析物を検出する方法であって、
a.該分析物を、(i)該分析物に関する捕捉試薬を含む表面上の該捕捉試薬、およびアンカーオリゴヌクレオチド配列を含むアンカー試薬;(ii)第1の核酸プローブに連結された、該分析物に関する第1の検出試薬;ならびに(iii)第2の核酸プローブに連結された、該分析物に関する第2の検出試薬に結合させ;それにより、結合試薬、該分析物ならびに該第1および第2の検出試薬を含む該表面上の複合体を形成させること;
b.該第1および第2のプローブが近接されることを必要とする伸長プロセスを用いて、該第2のプローブを伸長して、該アンカー配列に対して相補的であるアンカー配列相補体を含む伸長配列を形成させること;
c.該アンカー配列を該アンカー配列相補体にハイブリダイズさせること;ならびに
d.該表面に結合した伸長配列の量を測定すること
を含み、
該分析物は抗菌抵抗性マーカーである、前記方法。 - マーカーはPBP2a/mecA(S.aureus、グラム陽性)、またはTEM1(E.coli、グラム陰性)から選択される、請求項1に記載の方法。
- 分析物はバンコマイシン、ベータラクタム、カルバペネム、アミノ配糖体および/またはマクロライド抗生物質に対する抗菌抵抗性と関連するタンパク質から選択される、請求項1に記載の方法。
- 分析物はermファミリー、vanA、vanB、aac(6')−aph(2'')、KPC、NDM、OXA−48、VIM、OXA−23like、OXA−40like、OXA−58like、CTX−M−1およびCTX−M−9ファミリーのESBL遺伝子、およびその組み合わせから選択される、請求項3に記載の方法。
- 試料中の目的の分析物の検出のためのキットであって、1つまたはそれ以上のバイアル、容器、またはコンパートメントにおいて、
a.(i)分析物に関する捕捉試薬、および(ii)アンカーオリゴヌクレオチド配列を含むアンカー試薬を含む表面;
b.第1の核酸プローブに連結された、該分析物に関する第1の検出試薬;ならびに
c.第2の核酸プローブに連結された、該分析物に関する第2の検出試薬
を含み、
該分析物は抗菌抵抗性マーカーである、前記キット。 - マーカーはPBP2a/mecA(S.aureus、グラム陽性)、またはTEM1(E.coli、グラム陰性)から選択される、請求項5に記載のキット。
- 分析物はバンコマイシン、ベータラクタム、カルバペネム、アミノ配糖体および/またはマクロライド抗生物質に対する抗菌抵抗性と関連するタンパク質から選択される、請求項5に記載のキット。
- 分析物はermファミリー、vanA、vanB、aac(6')−aph(2'')、KPC、NDM、OXA−48、VIM、OXA−23like、OXA−40like、OXA−58like、CTX−M−1およびCTX−M−9ファミリーのESBL遺伝子、およびその組み合わせから選択される、請求項7に記載のキット。
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