JP5814344B2 - 体液からの標的分析物の単離 - Google Patents
体液からの標的分析物の単離 Download PDFInfo
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Description
本願は、2010年8月4日に出願された米国特許出願第12/850,203号(この米国特許出願は、2010年4月21日に出願された米国仮特許出願第61/326,588号の利益および優先権を主張する)の利益および優先権を主張し、この米国仮特許出願の全体の内容は、本明細書中に参考として援用される。
本発明は、体液試料から標的分析物を単離するための磁気粒子および磁石の使用に関する。
血液由来病原体は、健康管理上重要な問題である。細菌感染症の診断の遅れまたは不適切な診断は、感染への重大でしばしば致命的な炎症性応答である敗血症をもたらすことがある。敗血症は、米国で10番目の主要な死因である。血液中の細菌感染症の早期発見は、敗血症の開始の阻止での鍵である。血液由来感染症の伝統的な検出および同定の方法には、血液培養および抗生物質感受性アッセイが含まれる。それらの方法は細胞を培養することを一般に必要とし、それは費用がかかり、72時間という長い時間を要することがある。細胞培養結果が得られる前に敗血症性ショックが起こることがしばしばある。
本発明は、生物試料中の病原体を単離するための方法およびデバイスを提供する。本発明は、試料中の非常に低いレベルでの病原体の急速な検出を可能にし、したがって、病原体の早期発見および正確な検出および同定を可能にする。本発明は、標的特異的結合部分を有する磁気粒子を用いて実施される。本発明の方法は、標的特異的結合部分を含む磁気粒子を体液試料に導入して混合物を作製すること、この混合物をインキュベートして上記粒子を標的に結合させること、磁場を加えて表面上に標的/磁気粒子複合体を捕捉すること、および粒子凝集を低減する洗浄溶液で洗浄し、それによって標的/磁気粒子複合体を単離することを含む。本発明の方法の特別な利点は、多くの臨床試料に存在する低い濃度(血液試料に1CFU/mlという低い濃度の細菌)での、血液試料からの細菌および真菌の直接的な捕捉および単離についてのものである。
本発明は、例えば以下の項目を提供する。
(項目1)
体液試料から標的分析物を単離するための方法であって、
標的特異的結合部分を含む磁気粒子を体液試料に導入して、混合物を作製する工程と、
前記混合物をインキュベートして前記粒子を標的に結合させる工程と、
磁場を加えて、表面上に標的/磁気粒子複合体を捕捉する工程と、
粒子凝集を低減する洗浄溶液で洗浄し、それによって標的/磁気粒子複合体を単離する工程と
を含む、方法。
(項目2)
前記標的特異的結合部分が抗体である、項目1に記載の方法。
(項目3)
前記抗体が細菌特異的である、項目2に記載の方法。
(項目4)
前記粒子が超常磁性ビーズである、項目1に記載の方法。
(項目5)
前記インキュベーション工程が、細胞溶解を阻害するバッファー中で前記混合物をインキュベートすることを含む、項目1に記載の方法。
(項目6)
前記体液が、血液、痰(sputum)、尿、唾液、汗および脳脊髄液から選択される、項目1に記載の方法。
(項目7)
前記バッファーが約75mMの濃度のトリス(ヒドロキシメチル)−アミノメタン塩酸塩を含む、項目5に記載の方法。
(項目8)
前記磁気粒子が少なくとも70重量%の超常磁性ビーズを含む、項目4に記載の方法。
(項目9)
前記超常磁性ビーズの直径が約100nmから約250nmである、項目4に記載の方法。
(項目10)
前記洗浄工程の間に前記磁気粒子を磁場に保持する工程をさらに含む、項目1に記載の方法。
(項目11)
洗浄する工程が、
前記磁場を除去し、それによって前記標的/磁気粒子複合体を再懸濁させる工程と、
前記洗浄溶液を導入する工程と、
再び加えられた磁場の存在下で再懸濁された標的/磁気粒子複合体を前記表面上に流し、それによって前記表面上に標的/磁気粒子複合体を再捕捉する工程と
を含む、項目1に記載の方法。
(項目12)
前記抗体が真菌に特異的である、項目2に記載の方法。
(項目13)
前記細菌が病原性である、項目3に記載の方法。
(項目14)
前記細菌がグラム陽性細菌である、項目3に記載の方法。
(項目15)
前記細菌がグラム陰性細菌である、項目3に記載の方法。
(項目16)
前記細菌が、E.coli、Listeria、Clostridium、Mycobacterium、Shigella、Borrelia、Campylobacter、Bacillus、Salmonella、Staphylococcus、Enterococcus、Pneumococcus、Streptococcusおよびそれらの組合せからなる群より選択される、項目3に記載の方法。
(項目17)
前記磁気粒子が鉄含有磁気粒子である、項目1に記載の方法。
(項目18)
前記磁気粒子が酸化鉄または白金鉄を含む、項目17に記載の方法。
(項目19)
体液試料から標的微生物を単離するための方法であって、
標的特異的結合部分を含む磁気粒子を体液試料に導入して、混合物を作製する工程と、
前記混合物をインキュベートして前記粒子を標的に結合させる工程と、
磁場を加えて、標的が結合している磁気粒子を表面上に単離する工程と、
粒子凝集を低減する洗浄溶液で洗浄する工程と、
捕捉された細菌を溶解して、PCR、マイクロアレイハイブリダイゼーションまたは配列決定によるさらなる分析のためにDNAを抽出する工程と
を含む、方法。
(項目20)
体液試料から標的分析物を単離するための方法であって、
70重量%の超常磁性粒子を含み、標的特異的結合部分を有する磁気ビーズを体液試料に導入して、混合物を作製する工程と、
前記混合物をインキュベートして前記粒子を標的に結合させる工程と、
磁場を加えて、標的が結合している磁気粒子を表面上に単離する工程と、
粒子凝集を低減する洗浄溶液で洗浄する工程と
を含む、方法。
(項目21)
体液試料から標的分析物を単離するための方法であって、
約100nmから約250nmの直径を有し、標的特異的結合部分を含む超常磁性粒子を体液試料に導入して、混合物を作製する工程と、
前記混合物をインキュベートして前記粒子を標的に結合させる工程と、
磁場を加えて、表面上に標的/磁気粒子複合体を単離する工程と、
粒子凝集を低減する洗浄溶液で洗浄する工程と
を含む、方法。
(項目22)
血液試料中の1CFU/mlという低い濃度の細菌を単離するための方法であって、
約100nmから約250nmの直径を有し、細菌特異的結合部分を含む超常磁性粒子を体液試料に導入して、混合物を作製する工程と、
前記混合物をインキュベートして前記粒子を細菌に結合させる工程と、
磁場を加えて、表面上に細菌/磁気粒子複合体を単離する工程と、
粒子凝集を低減する洗浄溶液で洗浄し、それによって前記血液試料中の1CFU/mlという低い濃度の細菌を単離する工程と
を含む、方法。
本発明は、体液試料中の標的病原体を捕捉する磁気粒子、および標的を単離するための磁石の使用に一般に関する。本発明の方法は、標的特異的結合部分を含む磁気粒子を体液試料に導入して混合物を作製すること、この混合物をインキュベートして上記粒子を標的に結合させること、磁場を加えて表面上に標的/磁気粒子複合体を捕捉し、それによって標的/磁気粒子複合体を単離すること、ならびに粒子凝集を低減する洗浄溶液中で上記混合物を洗浄することを含む。本発明の方法は、粒子凝集を低減する洗浄溶液中で混合物を洗浄することをさらに含むことができる。磁性材料を標的実体に結合させ、その後磁場および勾配の利用によって分離することには、特定の基盤技術および原理が関連する。そのような基盤技術および原理は当該分野で公知であり、その内容は参照により完全に本明細書に援用されるJaneway(Immunobiology、6版、Garland Science Publishing)に記載されるものなど、以前に記載されている。
この開示全体で、特許、特許出願、特許公報、雑誌、本、論文、ウェブコンテンツなどの他の文書が参照および引用されている。全てのそのような文書は、全ての目的のためにここに参照により完全に本明細書に組み込まれる。
本明細書で示され、記載されるものに加えて、本発明およびその多くのさらなる実施形態の様々な変更は、本明細書で引用される科学文献および特許文献への参照を含むこの文書の全内容から、当業者に明らかになる。本明細書の対象物は、その様々な実施形態および同等物での本発明の実施へ適合させることができる、重要な情報、例証および指針を含む。
健康なボランティアからの血液試料に、血流感染症で最も頻繁に見出される細菌種の研究所株および臨床分離物の両方を含む細菌の臨床上の適切な濃度(1〜10CFU/mL)を混入させた。
ポリクローナルの汎グラム陽性細菌特異的IgGを生み出すために、最初に完全フロイントアジュバントに懸濁させた細菌抗原をリンパ節内に投与し、続いて不完全フロイントアジュバント中の細菌抗原の皮下注射を2週間隔で行うことによってヤギを免疫化した。細菌を指数増殖期(OD600=0.4〜0.8)まで増殖させることによって、抗原を抗体産生のために調製した。遠心による細菌の集菌の後、37℃で4時間の4%ホルムアルデヒドでのホルマリン固定を用いて、細菌を不活性化した。PBSで細菌を3回洗浄した後(15分間洗浄、4000rpmで20分間の遠心)、BCAアッセイを用いて抗原濃度を測定し、免疫化のために抗原を1mg/mLで用いた。グラム陽性細菌特異的IgGを生み出すために、接種のためにいくつかの細菌種を用いた:Staphylococcus aureus、Staphylococcus epidermidis、Enterococcus faeciumおよびEnterococcus fecalis。
超常磁性ビーズは、酸化鉄ナノ粒子(直径5〜15nm)をラテックスコアに封入し、ヤギIgGで標識することによって合成された。有機溶媒にナノ粒子を含む強磁性流体をエタノールで沈殿させ、スチレンおよび界面活性剤Hitenol BC−10の水溶液にナノ粒子を再懸濁させ、超音波処理を用いて乳化した。混合液を撹拌させながら一晩平衡させ、1.2および0.45μmフィルターによって濾過して、均一なミセルサイズを達成した。スチレン、アクリル酸およびジビニルベンゼンを、pH9.6の炭酸バッファーに加えた。K2S2O8の添加により70℃の混合液で重合を開始させ、反応は一晩完了させた。合成された粒子は、磁気捕捉を用いて0.1%SDSで3回洗浄し、1.2、0.8および0.45μmフィルターによって濾過し、抗体コンジュゲーションのために用いた。
血流感染の間に血液に存在する細菌を、上の実施例3で調製される超常磁性ビーズを用いて磁気標識した。実施例1に記載の混入された試料をトリスベースの結合バッファーおよび標的特異的ビーズで3倍に希釈し、その後37℃の振盪プラットホーム上で最大2時間インキュベートした。インキュベーションの後、標識標的を磁気によって分離し、その後血液生成物を除去するように設計された洗浄工程を行った。下の実施例5を参照されたい。
磁気標識標的細菌および過量の遊離ビーズを含む血液を、試料の流れに直角に置かれたいくつかの強力な希土類棒磁石を有するフロースルー捕捉セルに注入した。流路断面0.5mm×20mm(h×w)および7個のNdFeB棒磁石を有するフローチャンバーを用いて、5mL/分という速さの流速が達成された。磁石の存在下で混合液をチャンネル内に流した後に、ヘパリンを含む洗浄溶液をチャンネル内に流した。血液成分を除去して磁気粒子の凝集体の形成を低減するために、結合した標的をヘパリン含有バッファーで1回洗浄した。結合した標的を効果的に洗浄するために、磁石を取り出し、捕捉された磁性材料を洗浄バッファーに再懸濁させ、その後磁場を再び加え、同じフロースルー捕捉セルで磁性材料を捕捉した。
Claims (20)
- 体液試料から標的を単離するための方法であって、
1:1の比で体液試料とバッファーとを混合し、それによって希釈された体液試料を形成する工程であって、前記バッファーは、血液細胞の溶解を実質的に防止する、工程と、
標的特異的結合部分を含む磁気粒子を前記希釈された体液試料に導入して、混合物を作製する工程と、
前記混合物をインキュベートして前記粒子を標的に結合させる工程と、
前記混合物をチャネルを通って流す工程と、
磁場を加えて、前記チャネルの表面上に標的/磁気粒子複合体を捕捉する工程と、
粒子凝集を低減する洗浄溶液で洗浄する工程と、
前記磁場を除去し、それによって前記標的/磁気粒子複合体を再懸濁させる工程と、
前記洗浄溶液を再導入して、それによって前記標的/磁気粒子複合体を再懸濁させる工程と、
再び加えられた磁場の存在下で再懸濁された前記標的/磁気粒子複合体を前記表面上に流し、それによって標的/磁気粒子複合体を捕捉する工程と
を含む、方法。 - 前記標的特異的結合部分が抗体である、請求項1に記載の方法。
- 前記抗体が細菌特異的である、請求項2に記載の方法。
- 前記粒子が超常磁性ビーズである、請求項1に記載の方法。
- 前記インキュベーション工程が、細胞溶解を阻害するバッファー中で前記混合物をインキュベートすることを含む、請求項1に記載の方法。
- 前記体液が、血液、痰(sputum)、尿、唾液、汗および脳脊髄液から選択される、請求項1に記載の方法。
- 前記バッファーが75mMの濃度のトリス(ヒドロキシメチル)−アミノメタン塩酸塩を含む、請求項5に記載の方法。
- 前記磁気粒子が少なくとも70重量%の超常磁性ビーズを含む、請求項4に記載の方法。
- 前記超常磁性ビーズの直径が100nmから250nmである、請求項4に記載の方法。
- 粒子凝集を低減する前記洗浄溶液で洗浄する前記洗浄工程の間に前記磁気粒子を磁場に保持する工程をさらに含む、請求項1に記載の方法。
- 前記細菌が病原性である、請求項3に記載の方法。
- 前記細菌がグラム陽性細菌である、請求項3に記載の方法。
- 前記細菌がグラム陰性細菌である、請求項3に記載の方法。
- 前記細菌が、E.coli、Listeria、Clostridium、Mycobacterium、Shigella、Borrelia、Campylobacter、Bacillus、Salmonella、Staphylococcus、Enterococcus、Pneumococcus、Streptococcusおよびそれらの組合せからなる群より選択される、請求項3に記載の方法。
- 前記磁気粒子が鉄含有磁気粒子である、請求項1に記載の方法。
- 前記磁気粒子が酸化鉄または白金鉄を含む、請求項15に記載の方法。
- 体液試料から標的微生物を単離するための方法であって、
1:1の比で体液試料とバッファーとを混合し、それによって希釈された体液試料を形成する工程であって、前記バッファーは、血液細胞の溶解を実質的に防止する、工程と、
標的特異的結合部分を含む磁気粒子を前記希釈された体液試料に導入して、混合物を作製する工程と、
前記混合物をインキュベートして前記粒子を前記試料中の標的微生物に結合させる工程と、
前記混合物をチャネルを通って流す工程と、
磁場を加えて、前記標的微生物が結合している磁気粒子を前記チャネルの表面上に単離する工程と、
粒子凝集を低減する洗浄溶液で洗浄する工程と、
前記磁場を除去し、それによって前記標的/磁気粒子複合体を再懸濁させる工程と、
前記洗浄溶液を再導入して、それによって前記標的/磁気粒子複合体を再懸濁させる工程と、
再び加えられた磁場の存在下で再懸濁された前記標的/磁気粒子複合体を前記表面上に流し、それによって前記表面上で前記粒子を捕捉する工程と
捕捉された微生物を溶解して、PCR、マイクロアレイハイブリダイゼーションまたは配列決定によるさらなる分析のためにDNAを抽出する工程と
を含む、方法。 - 体液試料から標的を単離するための方法であって、
1:1の比で体液試料とバッファーとを混合し、それによって希釈された体液試料を形成する工程であって、前記バッファーは、血液細胞の溶解を実質的に防止する、工程と、
70重量%の超常磁性粒子を含み、標的特異的結合部分を有する磁気ビーズを希釈された体液試料に導入して、混合物を作製する工程と、
前記混合物をインキュベートして前記磁気ビーズを標的に結合させる工程と、
前記混合物をチャネルを通って流す工程と、
磁場を加えて、標的が結合している磁気ビーズを前記チャネルの表面上に単離する工程と、
粒子凝集を低減する洗浄溶液で洗浄する工程と、
前記磁場を除去し、それによって前記標的/磁気ビーズ複合体を再懸濁させる工程と、
前記洗浄溶液を再導入して、それによって前記標的/磁気ビーズ複合体を再懸濁させる工程と、
再び加えられた磁場の存在下で再懸濁された前記標的/磁気ビーズ複合体を前記表面上に流し、それによって標的/磁気ビーズ複合体を捕捉する工程と
を含む、方法。 - 体液試料から標的を単離するための方法であって、
1:1の比で体液試料とバッファーとを混合し、それによって希釈された体液試料を形成する工程であって、前記バッファーは、血液細胞の溶解を実質的に防止する、工程と、
100nmから250nmの直径を有し、標的特異的結合部分を含む超常磁性粒子を希釈された体液試料に導入して、混合物を作製する工程と、
前記混合物をインキュベートして前記超常磁性粒子を標的に結合させる工程と、
前記混合物をチャネルを通って流す工程と、
磁場を加えて、前記チャネルの表面上に標的/超常磁性粒子複合体を単離する工程と、
粒子凝集を低減する洗浄溶液で洗浄する工程と、
前記磁場を除去し、それによって前記標的/超常磁性粒子複合体を再懸濁させる工程と、
前記洗浄溶液を再導入して、それによって前記標的/超常磁性粒子複合体を再懸濁させる工程と、
再び加えられた磁場の存在下で再懸濁された前記標的/超常磁性粒子複合体を前記表面上に流し、それによって標的/超常磁性粒子複合体を捕捉する工程と
を含む、方法。 - 血液試料中の1CFU/mlという低い濃度の細菌を単離するための方法であって、
1:1の比で体液試料とバッファーとを混合し、それによって希釈された体液試料を形成する工程であって、前記バッファーは、血液細胞の溶解を実質的に防止する、工程と、
100nmから250nmの直径を有し、細菌特異的結合部分を含む超常磁性粒子を希釈された体液試料に導入して、混合物を作製する工程と、
前記混合物をインキュベートして前記超常磁性粒子を細菌に結合させる工程と、
前記混合物をチャネルを通って流す工程と、
磁場を加えて、前記チャネルの表面上に細菌/超常磁性粒子複合体を単離する工程と、
粒子凝集を低減する洗浄溶液で洗浄する工程と、
前記磁場を除去し、それによって前記標的/超常磁性粒子複合体を再懸濁させる工程と、
前記洗浄溶液を再導入して、それによって前記標的/超常磁性粒子複合体を再懸濁させる工程と、
再び加えられた磁場の存在下で再懸濁された前記標的/超常磁性粒子複合体を前記表面上に流し、それによって前記血液試料中の1CFU/mlという低い濃度の細菌を捕捉する工程と
を含む、方法。
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