JP4363980B2 - 複製細胞の迅速な検出 - Google Patents
複製細胞の迅速な検出 Download PDFInfo
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Description
本発明は、医学、工業、および環境試料における複製細胞、特に微生物細胞(例えば、細菌、酵母、およびカビ)の検出、計数、および同定に関する。微生物培養は、その多くの魅力的な特徴のために、これらの市場において広く用いられている方法論である。本発明は、方法の有用な属性を維持しながら、微生物培養の主な欠点、すなわち結果を得るまでに必要な時間が長いことに取り組む。
19世紀から20世紀の間に、感染症を引き起こす際の、そして食品および飲料の品質を決定する際の細菌、酵母、およびカビの役割に関する理解が進んだ。強力な方法である微生物培養は、少数の微生物を検出するために早くから開発された。微生物培養は、急速に多数に繁殖するというその性向を利用することによって微生物の単純な肉眼的検出を可能にする。例えば、細菌細胞1個は、あまりにも小さすぎて肉眼では見えないが(約100万分の1メートル)、これを栄養ブロスに入れておくと、24時間までにブロスを目で見えるように混濁させることができる。
より迅速に微生物を計数するための微生物培養法が多数開発されている。一つの迅速な微生物培養法は、栄養培地をコーティングした顕微鏡スライドガラス上に細菌細胞を載せる。顕微鏡による検査を用いれば、顕微鏡は少数の細胞***に起因するマイクロコロニーを検出できることから、裸眼よりはるかに速やかに微生物の増殖を検出することができる。しかし、この方法は、顕微鏡の視野で観察することができるのは試料のごく少量に過ぎないことから、少数の微生物細胞を含む大きい試料を調べるためには有効ではない。顕微鏡的方法は感度が低いために、一般的に、1ミリリットルあたり10000個を超える細菌細胞を含む試料にその有用性が限られており、これらの方法は、従来の微生物培養よりはるかに感度が悪い。
微生物計数のための最も迅速な方法は、微生物培養を行わずにすませることである。医学および工業的微生物学者は一般的に、生存微生物、すなわち微生物培養の際に複製することができる生きた微生物の計数のみに関心がある。したがって、最も有効であるためには、細胞の複製に依存せずに個々の細胞を検出する方法は、細胞複製の生理的代用物を用いることによって、生きている微生物と死んだ微生物とを区別しなければならない(例えば、Nebe-von-Caron, G.ら、J. Microbiol. Methods 42:97〜114(2000);Mignon-Godefroy, K.ら、Cytometry 27:336〜44、1997)。細胞は、複製能に一般的に相関する生化学性質(例えば、エステラーゼ活性または生化学的呼吸)を測定する色素によって染色する。規制標準を満たすことが知られており、従来のプレート培養法を用いて無菌的であると評価された試料が、代用生化学活性に関して陽性であると評価される何千もの細胞を有することから、代用法を確認して制定することは問題がある。
その分子構成成分に基づいて微生物を検出および同定する多数の方法が、過去半世紀の間に開発されている。これらの方法のいくつかは、微生物培養より実質的に迅速であるが、いずれも微生物学者にとって重要である培養の特徴の全てを提供しない。例えば、微生物に関する多数のイムノアッセイが市販されているが、この技術は、本来定量的ではなく、微生物培養よりはるかに感度が低く、1回の試験で多くのタイプの微生物を検出することに関して培養ほど強力ではない。または、もう一つの例として、核酸増幅法は、微生物培養と同程度の感受性となりうるが、それらは生きた細胞と生きていない細胞とを区別することができず、抗生物質の感受性試験を行うための純粋な培養物を生成することができない。電気泳動、質量分析、およびクロマトグラフィーを用いる生化学分析法(例えば、脂肪酸、核酸、またはタンパク質の)は、微生物の同定にとって強力となりうるが、そのような方法は通常、微生物の計数には不適切であり、一般的に日常的な微生物診断にとって高価で複雑すぎる。
要約すると、現在の微生物計数試験は、微生物培養が主流である。微生物培養は、単純、超感受性、安価で定量的であるという重要な利点を有するが、遅いという重要な欠点を有する。結果が出るまでの時間が長いことは、健康管理および製造において大きい損害を有する。より迅速な方法が開発されているが、それらは、結果を得るまでの時間を改善しても、微生物培養の一つまたは複数の重要な利点を犠牲にしている。
本発明は、大領域の撮像を用いてインサイチューにおける細胞***に由来する顕微鏡コロニーを検出することによって、生きている細胞の効率的で迅速かつ感度のよい計数を可能にする。本発明に基づく微生物計数試験は、微生物培養に基づく従来の方法の重要な利点を保持しながら、臨床および工業的微生物学における重要な問題、すなわち従来の試験の検出のに必要な期間が長いことに取り組む。本発明の態様には、標識試薬を用いないで細胞マイクロコロニーを検出する非破壊的な無菌的方法が含まれる。これらの方法によって、微生物の同定および抗菌耐性の決定のために用いることができる純粋な培養物を作製することができる。
本発明の様々な態様のいくつかの利点を表2に記載する。
発明の概要
本発明は、増殖しつつある細胞に関する最少に処理された試料を迅速かつ費用高価が高いように分析する。本発明と従来の細菌培養法はいずれも、細菌「コロニー」、すなわち単一の細胞から連続的な細胞***によって生じた関連細胞の集団の形成を検出することによって細胞増殖を測定する(図1)。しかし、本発明は、従来の微生物培養によって検出された肉眼的に観察されるマクロコロニーより早い段階で出現するマイクロコロニーを検出することから、従来の微生物培養より迅速に細胞増殖を検出する(図2、図8)。微生物培養と同じ方法原理を用いることによって、本発明は、結果を得るまでの時間をなおも有意に改善しながら従来の方法の利点を保持することができる。
段階1:試験の疑問の作製、試料、検出すべき細胞のカテゴリー、増殖条件、およびシグナル伝達特徴の選択
段階2:細胞標的を検出領域に沈着させる
段階3:細胞を複製させてマイクロコロニーを形成させる
段階4:マイクロコロニーを選択的に標識する
段階5:マイクロコロニーを計数する
標的細胞の分子構成成分に結合する染料またはプローブを用いることは、幅広い診断上の疑問を問う応用において用いることができる。広範囲の標的細胞(例えば、全ての好気性菌)を検出するために用いることができる染料の例には、核酸染料(例えば、ヨウ化プロピジウムまたはサイバーグリーン(Molecular Probes)、および酵素活性の染料(例えば、蛍光原エステラーゼ染料)が含まれる。標的細胞のより狭いカテゴリーを標識するために、標的特異的分子成分に結合する標識プローブを用いることができる。例えば、食品病原菌大腸菌O157:H7の表面に限って存在する分子に特異的に結合する蛍光標識抗体を用いて、食品試料中の病原性マイクロコロニーを検出することができる。
混合物における区別可能な標識(またはシグナル伝達部分)の数は、シグナル複雑度と呼ばれる。高度な多重試験の場合、時に高いシグナル複雑度を有するシグナル伝達部分を用いることが都合がよい。高いシグナル複雑度を得るために本発明について用いることができる三つの一般的アプローチは、(1)別個の標識、(2)組み合わせ標識、および(3)比の標識である。
必要なシグナル強度のレベルは、当然、シグナル特徴および検出法/機器のタイプに依存する(下記参照)。
本発明は、当業者に既知である様々な方法を用いて、カテゴリー結合分子に直接結合させることができる多数のタイプのシグナル伝達部分を組み入れることができる(例えば、Hermanson, G.、Bioconjugate Techniques(アカデミック出版)、1996、および下記の特定の実施例を参照されたい)。例えば、抗体またはオリゴヌクレオチドカテゴリー結合分子は、蛍光体または量子ドットシグナル伝達部分に直接結合させることができる。または、抗体またはオリゴヌクレオチドカテゴリー結合分子を用いて、蛍光微粒子に基づくまたは光散乱ナノ粒子に基づくシグナル伝達部分をコーティングすることができる。シグナル伝達部分はカテゴリー結合分子に間接的に結合させることができる。例えば、アビジンを多数のシグナル要素に直接結合させて、シグナル伝達部分を構成することができる。次に、標識されたアビジン分子をビオチン化カテゴリー特異的抗体に結合させることができる。シグナル伝達部分は、結合段階の前、間、または後にカテゴリー結合分子に結合させることができる。例えば、本発明の一つの態様において、ジゴキシゲニン標識核酸プローブをカテゴリー結合分子として用いる。試料中の標的細胞におけるカテゴリー特異的結合部位にカテゴリー結合分子を結合させた後、未結合のプローブを洗浄して除去する。次に、抗ジゴキシゲニン抗体:アルカリホスファターゼ結合体(シグナル伝達部分)を、結合したジゴキシゲニン標識プローブに結合させる。次に、アルカリホスファターゼ基質(例えば、化学発光基質CDP-スター;NEN)を結合したアルカリホスファターゼに加えてシグナルを生成する。
一般的に、本発明に基づく応用において次の段階は、検出域に試料中の標的細胞を載せることである。本質的に平面の検出域がしばしば用いられるが、その理由の一部は、例えば、マイクロコロニーが栄養寒天の表面上または栄養寒天プレートの表面上に存在する膜において増殖すると、光学撮像システムが薄い検出域から効率よく集光できるためである。これらの場合、検出域の深さは、検出域の側方寸法と比較すると無視することができる。この段階はまた、特定の標的細胞を選択的に沈着させるために、細胞増殖を阻害する可能性がある物質を除去するために、または標的細胞を標識試薬に接触させるために用いることができる。
この段階において、標的細胞は、検出域においてその場で***することによってマイクロコロニーを形成する。マイクロコロニーの増殖は、栄養を含む増殖培地に細胞を曝露して、それらを細胞増殖および***を促進する条件でインキュベートすることによって支持される(これらのパラメータは上記の段階1において選択される)。典型的な態様において、細胞を多孔性のメンブレンフィルターに沈着させる。フィルターをペトリ皿において固化栄養寒天増殖培地の表面上に置き、次にこれに蓋をして、適当な温度に設定したインキュベータ内に入れる。この方法は、栄養は、前駆細胞から娘細胞の移動を引き起こさずにメンブレンの中を自由に拡散できるために、従来の微生物培養を用いるコロニー増殖を支持するために現在広く用いられている。または、マイクロコロニーは、栄養寒天培地または同等物の表面上で直接増殖させることができる。
この選択的段階において、カテゴリー結合分子および会合するシグナル伝達部分(同様にプローブ、標識または染色とも呼ばれる)を、特異的結合を促進する条件で試料中の標的細胞に接触させる。例えば、カテゴリー特異的核酸配列の集合体をこの段階において試料中の相補的標的配列にハイブリダイズさせる。同様に、試料中のカテゴリー特異的抗原を、対応するカテゴリー特異的抗体に結合させる。
試料中の標的細胞の計数は、本発明に基づいて試験応用の最終段階において行う。計数段階そのものは、撮像、画像解析、および報告書作成段階を含む。
背景および目的:微生物増殖の検出は、臨床微生物学(例えば細菌の同定および抗菌感受性試験)と工業微生物学(例えば、委任滅菌試験)の双方の中心部分であるが、共通して用いられる方法は遅い。その結果、分析の遅れは、臨床状況では不必要な死および疾患状態を引き起こし、工業では大きい出費となる。
背景および目的:微生物増殖を検出する方法の重要性と、現行の問題の限界は、背景の章において考察する。本実施例は、細菌マイクロコロニーの増殖を迅速に検出する、本発明に基づく非常に単純でなお強力な方法を証明する。方法は、検出可能なシグナルを産生するために標的細胞の内因性の蛍光(自己蛍光)に依存する。このように、本発明の方法は、顕微鏡的標的細胞の非拡大大領域撮像を得るためにカテゴリー結合分子または外因性のシグナル伝達部分を利用しない。無試薬の非破壊的計数の利点には、純粋培養の作製(微生物の同定および抗生物質感受性試験のため、方法の改善の確認、および微生物増殖を経時的に追跡できること(物体の識別および増殖動態に関して))が含まれる。
背景および目的:新しい微生物試験が「最も標準的な」方法と同等であることを証明することは、新規方法の開発者およびその顧客の双方にとって必須の作業である。公式の確認必要条件は、一般的に工業および健康管理における新しい微生物学的方法の導入の指針となる政府の規則に成文化されている。製薬業における微生物学試験の新しい方法は、許容された方法に対する同等性の証明が難しいことから時に難しい。本実施例の目標は、従来の微生物培養試験に対する本発明に基づく試験の同等性を提供する単純な方法を証明することである。
背景および目的:少数の微生物の正確な検出は、健康管理および工業微生物学の双方において重要である。例えば、おそらく致死的な血液感染症患者に存在する細菌細胞は、血液試料10ml中に1個のみであるかも知れない。同様に、医薬品の製造における注射用薬剤の滅菌試験は、試料中に生存微生物細胞1個を検出しなければならない。いずれの場合においても、偽陰性結果および偽陽性結果は重度の結末となりうる。偽陽性および偽陰性である試験結果の分画は、試験法の正確度を定義する。
背景および目的:本実施例の目標は、大領域撮像を用いてマイクロコロニーの自己蛍光の無試薬検出の感度および速度を証明することである。微生物増殖の迅速な検出は、本発明が、細胞数が少ない初期段階でマイクロコロニーを検出できる結果である。実施例における実験は、非拡大CCD撮像によって検出されたマイクロコロニーにおける細菌細胞数を決定する。
背景および目的:本実施例は、栄養的にストレスを受けている可能性がある無名の環境微生物に適用した場合の、微生物増殖の迅速な検出に関する本発明の効力を示すことをねらいとする。
背景および目的:本実施例の目的は、マイクロコロニーの迅速な検出に関して、本発明を用いて得られた結果と、より遅い従来の微生物培養を用いて得られた結果との数値的相関を決定することである。
背景および目的:新しい微生物試験法の二つの確認基準は、新しい方法の範囲と直線性である。範囲は、新しい試験法を用いる精度、正確度、および直線性で決定されることが証明された微生物の上のレベルおよび下のレベルの間の間隔である。微生物試験法の直線性は、所定の範囲内の試料に存在する微生物濃度に比例する結果の誘発能である。
背景および目的:抗菌保存剤は、製造プロセスによって導入される可能性がある、または個々の投与をやめた際に消費者によって導入される可能性がある微生物の増殖に対して保護するために、多用量容器において包装された製品に添加される。抗菌有効性は、内因性の抗菌活性を含む薬学的製剤または抗菌保存剤を含む製品に関して証明しなければならない。分析しなければならない試料希釈液が多数であるために、試験は、実施するために非常に労力と費用がかかる。典型的に、抗菌保存剤の有効性試験は、何百もの微生物培養プレートの分析を必要とする。本実施例の重要な目標は、試料の希釈の必要性を回避することによって、本発明が試験の労力のほとんどをなくす可能性があることを証明することである。
背景および目的:本実施例の目標は、生物学的指標としての熱耐性胞子を用いる応用に関して本発明を応用できる可能性を示すことである。一つの重要な応用は、薬学および医学装置の製造ならびに臨床検査における滅菌技法の有効性を保証するための滅菌剤定量法である。
背景および目的:本実施例は、本発明が、コンペンディアル(compendial)方法と比較して牛ひき肉における細菌マイクロコロニーの検出までの時間を短縮できることを示す。生肉における総生存細菌数の決定は、初期食物腐敗を予防するために必須である。現在の方法は2日を要し、試験結果を得る前にしばしば生産者は肉を輸送する。微生物を検出するまでの時間を短縮することは、食物伝染疾患の発生、非効率製造、および費用のかかる回収の発生を予防する。
目的:本実施例は、複合試料から個々の細菌細胞を非特異的に選択した後に、非拡大大領域撮像を用いて増殖しつつあるマイクロコロニーの迅速な検出を行うイムノアッセイ法を示す。より詳しくは、本実施例は、食物から効率よく広範囲の細菌を選択して、その後増殖直接法を用いて細菌の増殖を検出できることを証明する。本実施例は、結合物質の混合物によってコーティングした磁気ビーズが、複合試料から多様な細菌種を選択できることを示す。
背景および目的:適当な治療を決定するための抗菌感受性試験の重要性は、背景の章において考察する。固体媒体における微生物増殖のモニタリングは一般的であり、液体培養での増殖と比較していくつかの有意な利点を有する。これは、機器を用いることなく(例えば、ディスク拡散アッセイ法)、いくつかの抗体に対する細菌株の感受性を安価に、同時に、そして定量的に決定することができるが、現行の方法は、精製したコロニーを必要とし、このように、通常、患者の試料を処理した後1〜2日で行うことができない。そのような遅れは生命を脅かしうる。さらに、抗菌感受性試験の結果を検出および分析するために、一般的にさらに1〜2日を必要とする。
目的:ディスク拡散法を用いて細菌株の抗生物質感受性を迅速に決定するために本発明を用いることを証明する。既知濃度の抗生物質に含浸したディスクを、精製微生物培養物からの多数の細胞を含むプレートに載せる。抗生物質はディスクから拡散して、ディスクを中心とする放射状の抗生物質濃度勾配を作製する(すなわち、ディスクにより近ければ、抗生物質の濃度がより高い)。高度耐性菌は、ディスクの存在下で抗生物質濃度が最も高い端部近傍でも増殖することができる。より弱い耐性菌は、ディスク周辺の阻害域の外側で増殖する。阻害域の幅は、株の抗生物質耐性レベルと相関する。
目的:本実施例は、E-テスト(商標)抗生物質試験片を用いて細菌の抗生物質感受性を迅速に決定するために本発明を用いることを証明する。E-テスト(商標)片を、多様な濃度範囲のテトラサイクリンに含浸させ、ユーザーは、試験細菌の増殖を阻害するために必要な抗生物質の最低濃度を決定することができる。最少阻止濃度は、阻害域と呼ばれる増殖を示さない域の可視化に基づく。阻害域は、従来、一晩増殖後の裸眼によって測定する。本実施例は、非拡大大領域撮像を用いてマイクロコロニーの増殖を検出することによって、数時間で阻害域を決定できることを証明する。
本出願において参照した全ての特許、特許出願、および出版物は、参照として本明細書に組み入れられる。本発明の他の態様は、本明細書に開示の本発明の明細書および実践の検討によって当業者には明らかであると思われる。本明細書および実施例は、例として見なされるに過ぎず、本発明の真の範囲および趣旨は、特許請求の範囲によって示されるものである。本明細書に記述の方法に適合させてもよい他の態様の例は、そのそれぞれが参照として本明細書に組み入れられる、2002年9月6日に提出された「RAPID AND SENSITIVE DETECTION OF CELLS AND VIRUSES」と題する米国特許出願第____号および2002年9月6日に提出された「RAPID AND SENSITIVE DETECTION OF MOLECULES」に認められる。
Claims (111)
- (a)検出域に細胞が無作為に分散されて固定される、検出領域1mm2あたり標的細胞100個未満の密度で、試料中に存在する生存標的細胞を検出領域を含む検出域に提供する段階;
(b)インサイチューにおける複製によって該標的細胞の一つまたは複数のマイクロコロニーを形成させる段階;
(c)一つまたは複数のマイクロコロニーを検出する段階;
を含む、試料中の生存標的細胞を検出する方法であって、
該検出領域の最も長い直線寸法が1mmより大きく;一つまたは複数の該マイクロコロニーが少なくとも二つの直交寸法において50ミクロン未満の平均測定値を有する;該検出が5倍より大きい拡大を必要とせず;該検出が、シグナル伝達部分またはカテゴリー結合分子の添加に依存しない一つまたは複数のマイクロコロニーの性質を検出する;および一つまたは複数の該マイクロコロニーにおける該細胞が、検出後もなお複製コンピテントである方法。 - 標的細胞が、検出領域1mm2あたり標的細胞10個未満の密度で検出領域に無作為に分散される、請求項1記載の方法。
- 標的細胞が、検出領域1mm2あたり標的細胞1個未満の密度で検出領域に無作為に分散される、請求項1記載の方法。
- 検出段階が、検出領域においてマイクロコロニー1個を検出する、請求項1記載の方法。
- 検出段階が、重なり合うまたは連続したマイクロコロニーを検出する、請求項1記載の方法。
- 検出段階が、2倍より大きい拡大を必要としない、請求項1記載の方法。
- 検出段階が、1倍より大きい拡大を必要としない、請求項1記載の方法。
- 検出段階が、0.2倍より大きい拡大を必要としない、請求項1記載の方法。
- 一つまたは複数のマイクロコロニーにおける細胞数の平均値が細胞50,000個未満である、請求項1記載の方法。
- 一つまたは複数のマイクロコロニーが細胞10,000個未満を含む、請求項9記載の方法。
- 一つまたは複数のマイクロコロニーにおける細胞数の平均値が1000個未満である、請求項9記載の方法。
- 一つまたは複数のマイクロコロニーにおける細胞数の平均値が100個未満である、請求項9記載の方法。
- 一つまたは複数のマイクロコロニーにおける細胞数の平均値が10個未満である、請求項9記載の方法。
- 一つまたは複数のマイクロコロニーが、最も長い直線寸法において25ミクロン未満の平均測定値を有する、請求項9記載の方法。
- 一つまたは複数のマイクロコロニーが、最も長い直線寸法において10ミクロン未満の平均測定値を有する、請求項9記載の方法。
- 標的細胞が細菌である、請求項1記載の方法。
- 標的細胞が真核細胞である、請求項1記載の方法。
- 標的細胞がカビまたは真菌細胞である、請求項17記載の方法。
- 標的細胞がヒト、動物、または植物細胞である、請求項17記載の方法。
- 標的細胞がヒト、動物、または植物の寄生虫である、請求項1記載の方法。
- 検出段階が、一つより多い重なり合わないカテゴリーの細胞からのマイクロコロニーを検出および同定する、請求項1記載の方法。
- 試料が、多細胞生物から得た液体または組織を含む、請求項1記載の方法。
- 試料が、動物の体液または組織を含む、請求項1記載の方法。
- 試料がヒトに由来する、請求項1記載の方法。
- 試料がヒト以外の脊椎動物に由来する、請求項1記載の方法。
- 試料が、呼吸器、泌尿器、生殖器、中枢神経系、尿、血液、皮膚、血漿、血清、唾液、創傷組織、創傷浸出物、生検、便、および固体組織試料、ならびにその誘導体からなる群より選択される、請求項1記載の方法。
- 試料が血液または尿試料である、請求項1記載の方法。
- 試料が植物に由来する、請求項1記載の方法。
- 試料が環境の空気もしくは水、または環境に曝露された表面、物体、もしくは生物を採取することによって得られる、請求項1記載の方法。
- 試料が、ヒトもしくは動物において局所もしくは内服利用のための、医薬品、化粧品、血液、または他の製剤の製造における原料、最終材料、または加工途中の材料;食品または飲料の製造における原料、加工途中材料、または最終材料;医学またはインビトロ診断装置の製造における原料、加工途中材料、または最終材料;化学物質;工業用表面;機器;および機械からなる群より選択される材料から得られる、請求項1記載の方法。
- 検出域を、標的細胞の複製を促進する一つまたは複数の物質を含む液体培地に接触させる、請求項1記載の方法。
- 細胞が固体または半固体増殖培地に直接沈着される、請求項1記載の方法。
- 段階(a)の前に、選択法を用いて、一つまたは複数の標的細胞と選択部分との複合体を検出域に沈着させ、選択法が磁気選択、遠心、沈降、および濾過からなる群より選択される、請求項1記載の方法。
- 段階(a)の前に、標的細胞が標的細胞特異的磁気選択部分に接触し、一つまたは複数の該標的細胞と該選択部分との複合体がその後、磁力を用いて検出表面に沈着される、請求項33記載の方法。
- 標的細胞特異的磁気選択部分が、カテゴリー結合分子に結合された磁気粒子を含む、請求項34記載の方法。
- 標的細胞が、体液中で、液体の平均密度より大きい平均密度を有する標的細胞特異的磁気選択部分に接触し、一つまたは複数の該標的細胞と該選択部分との複合体がその後、重力、遠心、または求心力を用いて検出表面上に沈着される、請求項33記載の方法。
- 標的細胞が、磁気選択、遠心、沈降、および濾過からなる群より選択される選択法を用いて検出域に沈着され、選択部分が用いられない、請求項1記載の方法。
- 段階(a)の前に、試料が液化されるおよび/またはホモジナイズされるように処理される、請求項1記載の方法。
- 段階(a)の前に、試料が標的細胞以外の物質または物体を除去するように処理される、請求項1記載の方法。
- 標的細胞の一つまたは複数の属性に及ぼす一つまたは複数の物質または処理の影響を決定する段階をさらに含む、請求項1記載の方法。
- 属性が細胞の複製を受ける能力である、請求項40記載の方法。
- 一つまたは複数の物質が、標的細胞の複製を支持するために用いられる培地に存在する、請求項40記載の方法。
- 属性が、滅菌処理後の標的細胞の複製能である、請求項40記載の方法。
- 属性が、一つまたは複数の可能性がある複製阻害剤の存在下での標的細胞の複製能である、請求項40記載の方法。
- 標的細胞が、細菌、真菌、寄生虫、または培養細胞であり、物質が抗菌剤、物質、抗真菌剤、または抗寄生虫剤である、請求項40記載の方法。
- 一つまたは複数の阻害剤が抗菌化合物である、請求項44記載の方法。
- 一つまたは複数の阻害剤が抗腫瘍化合物である、請求項44記載の方法。
- 属性が、標的細胞の生存率、光学的性質の変化、代謝もしくは酵素活性、または生化学的構成である、請求項40記載の方法。
- 以下の段階をさらに含む、請求項34記載の方法:
(d)標的細胞を一つもしくは複数の物質に接触させる、または細胞を一つもしくは複数の処理によって処理する段階;および
(e)標的細胞の一つもしくは複数の属性に及ぼす、一つもしくは複数の物質または一つもしくは複数の処理の影響を決定する段階。 - 検出域が、ガラス、プラスチック、マイクロタイタープレートのウェル表面、水分を吸収しやすいメンブレン、プラスチック片、毛細管表面、微小流動小室の表面、および表面または微小流動チャンネルからなる群より選択される材料を含む、請求項1記載の方法。
- マイクロコロニーにおける細胞の複製が検出後も継続する、請求項1記載の方法。
- 段階(c)が少なくとも2サイクルを含み、そのそれぞれが、細胞を複製させた後に検出段階を行う期間を含む、請求項1記載の方法。
- 反復が自動化される、一つまたは複数のさらなる試料に段階(a)〜(c)を繰り返す段階をさらに含む、請求項1記載の方法。
- 試料が、検出器を含む機器に自動的にローディングされる、請求項53記載の方法。
- 試料が、物理的に会合して、検出器を含む機器に自動的かつ連続的にローディングされる一連の検出域に自動的に沈着される、請求項53記載の方法。
- 検出段階が、一つまたは複数のマイクロコロニーに光を照射して検出可能なシグナルを生成する段階を含む、請求項1記載の方法。
- 検出段階が、一つまたは複数のマイクロコロニーの照明の結果として、放射、散乱、反射、または吸収された光を検出する、請求項56記載の方法。
- 検出段階が蛍光を検出する、請求項1記載の方法。
- 蛍光がマイクロコロニーによって放射された自己蛍光である、請求項58記載の方法。
- 照明段階が一つまたは複数のレーザーを用いる、請求項56記載の方法。
- 照明段階が一つまたは複数の発光ダイオードを用いる、請求項56記載の方法。
- 照明段階が、白色光源を用いる、請求項56記載の方法。
- 照明段階が、選択された波長の光のみを通過させる一つまたは複数の光学フィルターを通して行われる、請求項56記載の方法。
- 以下の段階を含む、標的細胞のマイクロコロニーを検出する方法:
(a)検出領域内で、細胞が無作為に分散および固定されている、検出域中の標的細胞を提供する段階;
(b)該マイクロコロニーの少なくとも一つが標的細胞100個未満を含む、インサイチューにおける複製によって該標的細胞の一つまたは複数のマイクロコロニーを形成させる段階、および
(c)5倍未満の倍率を用いて、一つまたは複数の該マイクロコロニーの一つまたは複数の天然に存在する光学的性質を検出する段階。 - 光学的性質または複数の性質が自己蛍光を含む、請求項64記載の方法。
- 光学的性質または複数の性質が熱放射を含む、請求項64記載の方法。
- 光学的性質または複数の性質が吸光度を含む、請求項64記載の方法。
- 吸光度が赤外線領域においてである、請求項67記載の方法。
- 光学的性質または複数の性質が蛍光偏光を含む、請求項64記載の方法。
- 光学的性質または複数の性質が光反射率を含む、請求項64記載の方法。
- 光学的性質または複数の性質が光の散乱を含む、請求項64記載の方法。
- 段階(c)の前または間に、標的細胞と直接または間接的に会合するシグナル伝達部分に試料を接触させる段階をさらに含む、請求項1記載の方法。
- シグナル伝達部分がカテゴリー結合分子に会合している、請求項72記載の方法。
- 段階(c)の前または間に、カテゴリー結合分子と、一つまたは複数の標的細胞上の一つまたは複数のカテゴリー特異的結合部位との一つまたは複数の複合体を形成させる条件で、試料をカテゴリー結合分子に接触させる段階をさらに含む、請求項1記載の方法。
- カテゴリー結合分子が、抗体、アプタマー、またはリガンドを含む、請求項74記載の方法。
- カテゴリー結合分子が、一つまたは複数のシグナル伝達部分によって直接または間接的に標識される、請求項74記載の方法。
- 段階(c)の前に、一つまたは複数の複合体から任意の未結合のカテゴリー結合分子を除去する段階をさらに含む、請求項76記載の方法。
- カテゴリー結合分子が、カテゴリー結合分子の集合体のメンバーであり、該集合体が、検出される標的細胞のそれぞれの重なり合わないカテゴリーに対して特異的なカテゴリー結合分子の一つのファミリーを含む、請求項74記載の方法。
- カテゴリー結合分子のファミリーのそれぞれが、別個のシグナルクラスまたはシグナルサインのシグナルを放射するシグナル伝達部分によって標識される、請求項78記載の方法。
- シグナル伝達部分が粒子である、または粒子に物理的に会合している、請求項73記載の方法。
- シグナル伝達部分が蛍光シグナル伝達特徴を有する、請求項72記載の方法。
- シグナル伝達部分が、有機蛍光体、アップレギュレートされた燐、ランタノイド、量子ドット、非蛍光基質から蛍光産物を生成する酵素、および蛍光色素粒子からなる群より選択される、請求項81記載の方法。
- シグナル伝達部分が細胞の蛍光染色である、請求項81記載の方法。
- シグナル伝達部分が色素産生シグナル伝達特徴である、請求項72記載の方法。
- シグナル伝達部分が化学発光シグナル伝達特徴を有する、請求項72記載の方法。
- シグナル伝達部分が光散乱シグナル伝達特徴を有する、請求項72記載の方法。
- シグナル伝達部分が共鳴光散乱粒子またはプラズモン共鳴粒子である、請求項86記載の方法。
- シグナル伝達部分が、生存細胞の染色するための生存染料である、請求項72記載の方法。
- カテゴリー結合分子の集合体がファミリーの複雑度1を有する、請求項78記載の方法。
- カテゴリー結合分子の集合体のファミリーの複雑度が1より大きい、請求項78記載の方法。
- 集合体のファミリーの複雑度が≧5である、請求項90記載の方法。
- シグナル伝達部分が、標的細胞と会合するまでは検出可能でない一つまたは複数の化合物を含み、シグナル伝達部分が、標的細胞の構成成分によって、または標的細胞の生理学的、物理的もしくは微小環境状態によって作用する、請求項72記載の方法。
- 複製および検出段階が、さらに別の細胞が入らないように、または試料中の細胞が出て行かないように構成された容器において起こる、請求項1記載の方法。
- 複製および検出段階が、試料を自動的に追跡するためのバーコードまたは同等の標識を有する容器において起こる、請求項1記載の方法。
- 複製および検出段階が、同じ表面の多数の画像の配置を容易にするためにレジストレーションマークを有する表面上で起こる、請求項1記載の方法。
- 検出段階が、検出域の特定の領域における対照マークまたは対照細胞を検出する、請求項1記載の方法。
- 検出段階が、標的細胞の照明に由来するシグナルを検出するように適合された光学フィルターを用いる、請求項1記載の方法。
- 検出段階が光電検出器を用いる、請求項1記載の方法。
- 検出段階が、光電アレイ検出器を用いる、請求項1記載の方法。
- 光電検出器がCCD検出器を含む、請求項99記載の方法。
- 検出段階が画像増幅器を用いない、請求項1記載の方法。
- 検出段階が光電子増倍管を用いる、請求項1記載の方法。
- 検出段階がフォトダイオード検出器を用いる、請求項1記載の方法。
- 検出段階が光感受性被膜を用いる、請求項1記載の方法。
- 段階(c)の間または後に、マイクロコロニーの数を定量する段階をさらに含む、請求項1記載の方法。
- 段階(c)の間または後に、画像解析ソフトウェアを用いて、検出領域の画像を分析することによって、標的細胞のカテゴリーを決定する段階をさらに含む、請求項1記載の方法。
- 段階(c)の間または後に、画像解析ソフトウェアを用いて、検出領域の画像を分析することによって一つまたは複数のマイクロコロニーの検出域での位置を決定する段階をさらに含む、請求項1記載の方法。
- 段階(c)の間または後に、個々のマイクロコロニーの検出域での位置をマイクロコロニーの既に決定された位置と比較する段階をさらに含む、請求項107記載の方法。
- 画像解析ソフトウェアが、経時的に大きさが変化する物体を経時的に大きさが変化しない物体と区別するためのアルゴリズムを含む、請求項108記載の方法。
- 決定する段階が、検出領域の画像を分析することを含む、請求項105記載の方法。
- 滅菌処理が、熱滅菌、放射線照射、毒性ガス曝露、および消毒処理からなる群より選択される、請求項43記載の方法。
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