JP4856759B2 - インフルエンザウィルスのリアルタイム検出 - Google Patents
インフルエンザウィルスのリアルタイム検出 Download PDFInfo
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- JP4856759B2 JP4856759B2 JP2009510174A JP2009510174A JP4856759B2 JP 4856759 B2 JP4856759 B2 JP 4856759B2 JP 2009510174 A JP2009510174 A JP 2009510174A JP 2009510174 A JP2009510174 A JP 2009510174A JP 4856759 B2 JP4856759 B2 JP 4856759B2
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- hemagglutinin
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Description
本願は、2006年5月10日に出願された米国仮特許出願番号60/799,442、および2006年5月16日に出願された米国仮特許出願番号60/800,939に対する優先権を主張する。これらの米国仮特許出願の各々は、その全体が全ての目的で本明細書中に参考として援用される。本願は、2006年3月24日に出願された出願番号11/389,409に関する。この出願は、その全体が本明細書中に参考として援用される。
インフルエンザ(「インフルエンザ(flu)」)は、鳥類および哺乳動物を含む多種多様の宿主を罹患させる可能性のある感染症である。インフルエンザは、オルソミクソウイルス科(A型、B型、およびC型のインフルエンザウィルスを一般に含む)のRNAウィルスによって引き起こされる。トリインフルエンザは、鳥類に適応したこの科のウィルスによって引き起こされ、したがって、鳥類インフルエンザ、トリインフルエンザ、またはバードインフルエンザとも命名される。現在の汎発性流行の脅威は、アジアおよびヨーロッパにおけるA型インフルエンザウィルスのH5N1株の前例のない大発生に起因する。この株は、突然変異する能力を有し、鳥類およびヒトを含む広範囲の宿主にそれ自身を適応させる。自国安全保障会議(The Homeland Security Council)は、現在の汎発性流行の脅威に応じて2005年11月に「汎発性流行インフルエンザに対する国家戦略(National Strategy for Pandemic Influenza)」(「戦略(The Strategy)」)を公布した。その発案の重要な部分は、患者および鳥類におけるトリインフルエンザの迅速な同定に焦点を当てている。その戦略は、トリインフルエンザのサーベイランスおよび検出の改善に努めるものである。
本発明は、被験体からの体液中のインフルエンザウィルス感染を示す分析物を検出するシステムを提供する。このシステムは典型的に、a)サンプル採集ユニットおよびアッセイ用アセンブリを備える流体装置であって、前記サンプル採集ユニットが、前記分析物を含有する疑いのある体液サンプルと、前記アッセイ用アセンブリ内に含まれた反応物質とを反応させ、前記分析物の存在を示す検出可能なシグナルを生じさせる流体装置;b)前記検出可能なシグナルを検出する検出用アセンブリを備える読取りアセンブリ;c)前記検出シグナルを前記外部装置に伝送するための通信用アセンブリ、を備える。このシステムは、A型、B型および/またはC型のインフルエンザウィルス感染を検出することができる。一般に、分析物は、赤血球凝集素(例えば、H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、およびH16)および/またはノイラミニダーゼ(例えば、N1、N2、N3、N4、およびN5)であり得るインフルエンザウィルスの表面糖タンパク質を含み得る。体液は、ヒト、ニワトリおよび野生鳥類よりなる群から選択される被験体から採取できる。
本発明は、例えば以下の項目を提供する。
(項目1)
被験体からの体液中のインフルエンザウィルス感染を示す分析物を検出するシステムであって、
a)サンプル採集ユニットおよびアッセイ用アセンブリを備える流体装置であって、前記サンプル採集ユニットが、前記分析物を含有する疑いのある体液サンプルと、前記アッセイ用アセンブリ内に含まれた反応物質とを反応させ、前記分析物の存在を示す検出可能なシグナルを生じさせる流体装置;
b)前記検出可能なシグナルを検出する検出用アセンブリを備える読取りアセンブリ;ならびに
c)検出された前記シグナルを外部装置に伝送するための通信用アセンブリ、
を備えるシステム。
(項目2)
前記分析物を含有する疑いのある前記体液サンプルが、前記外部装置から伝送されたプロトコルに基づいて、前記アッセイ用アセンブリ内に含まれた反応物質と反応する項目1に記載のシステム。
(項目3)
前記分析物が、A型インフルエンザウィルス感染を示す項目1に記載のシステム。
(項目4)
前記分析物が、B型インフルエンザウィルス感染を示す項目1に記載のシステム。
(項目5)
前記分析物が、C型インフルエンザウィルス感染を示す項目1に記載のシステム。
(項目6)
前記分析物が、インフルエンザウィルスの表面糖タンパク質を含む項目1に記載のシステム。
(項目7)
前記表面糖タンパク質が赤血球凝集素を含む項目6に記載のシステム。
(項目8)
前記赤血球凝集素が、H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、およびH16よりなる群から選択される項目7に記載のシステム。
(項目9)
前記赤血球凝集素がH5である項目7に記載のシステム。
(項目10)
前記表面糖タンパク質がノイラミニダーゼを含む項目6に記載のシステム。
(項目11)
前記ノイラミニダーゼが、N1、N2、N3、N4、およびN5よりなる群から選択される項目10に記載のシステム。
(項目12)
前記ノイラミニダーゼがN1である項目10に記載のシステム。
(項目13)
前記分析物が、インフルエンザウィルスの表面糖タンパク質に特異的な抗体である項目1に記載のシステム。
(項目14)
前記被験体がヒトである項目1に記載のシステム。
(項目15)
前記被験体が動物である項目1に記載のシステム。
(項目16)
前記動物が家禽である項目15に記載のシステム。
(項目17)
複数の分析物を検出するシステムであって、前記分析物のうちの少なくとも2つは、被験体からの体液中のインフルエンザウィルス感染を示し、前記システムは、
a)サンプル採集ユニットおよびアッセイ用アセンブリを備える流体装置であって、前記サンプル採集ユニットは、前記複数の分析物を含有する疑いのある体液サンプルと、前記アッセイ用アセンブリ内に含まれた反応物質とを反応させ、前記少なくとも2つの分析物の存在を示す1つまたは複数の検出可能なシグナルを生じさせる前記流体装置;
b)前記1つまたは複数の検出可能なシグナルを検出するための検出用アセンブリを備える読取りアセンブリ;ならびに
c)前記検出されたシグナルを外部装置に伝送するための通信用アセンブリ、
を備えるシステム。
(項目18)
前記複数の分析物を含有する疑いのある前記体液サンプルが、前記外部装置から伝送されたプロトコルに基づいて、前記アッセイ用アセンブリ内に含まれた反応物質と反応する項目17に記載のシステム。
(項目19)
前記複数の分析物が、A型インフルエンザウィルス感染を示す項目17に記載のシステム。
(項目20)
前記複数の分析物が、B型インフルエンザウィルス感染を示す項目17に記載のシステム。
(項目21)
前記複数の分析物が、C型インフルエンザウィルス感染を示す項目17に記載のシステム。
(項目22)
前記複数の分析物が、インフルエンザウィルスの複数の表面糖タンパク質を含む項目17に記載のシステム。
(項目23)
前記複数の表面糖タンパク質が、赤血球凝集素およびノイラミニダーゼを含む項目22に記載のシステム。
(項目24)
前記赤血球凝集素が、H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、およびH16よりなる群から選択され、前記ノイラミニダーゼが、N1、N2、N3、N4、およびN5よりなる群から選択される項目23に記載のシステム。
(項目25)
前記赤血球凝集素がH5であり、前記ノイラミニダーゼがN1である項目23に記載のシステム。
(項目26)
前記複数の分析物が、インフルエンザウィルスの表面糖タンパク質に特異的な複数の抗体を含む項目17に記載のシステム。
(項目27)
前記被験体がヒトである項目17に記載のシステム。
(項目28)
前記被験体が動物である項目17に記載のシステム。
(項目29)
前記動物が家禽である項目28に記載のシステム。
(項目30)
被験体の体液中のインフルエンザウィルス感染を示す分析物を検出する方法であって、
a)項目1に記載のシステムを提供すること;
b)体液サンプルと、前記アッセイ用アセンブリ内に含まれた反応物質とを反応させ、前記分析物の存在を示す検出可能なシグナルを生じさせること;および
c)前記検出可能なシグナルを検出すること、
を包含する方法。
(項目31)
被験体からの体液中のインフルエンザウィルス感染を示す分析物を検出する方法であって:
a)少なくとも1つのサンプル採集ユニット、免疫アッセイ用試薬を含有する免疫アッセイ用アセンブリ、前記サンプル採集ユニットおよび/または前記免疫アッセイ用アセンブリと流体連絡する複数のチャネルを含む流体装置を提供すること;
b)前記流体装置を作動させて前記流体装置内の前記免疫アッセイ用試薬を方向付けること;
c)前記分析物を含有する疑いのある体液サンプルと、前記アッセイの免疫アッセイ用アセンブリ内に含まれた前記免疫アッセイ用試薬とを反応させて、前記サンプル中でのインフルエンザウィルス感染を示す前記分析物の存在を示す検出可能なシグナルを生じさせること;ならびに
d)前記体液サンプル中で採集された前記分析物から生じた前記検出可能なシグナルを検出すること、
を包含する方法。
(項目32)
前記体液サンプルが、約500マイクロリットル未満である項目31に記載の方法。
(項目33)
前記分析物が、A型インフルエンザウィルス感染を示す項目31に記載の方法。
(項目34)
前記分析物が、B型インフルエンザウィルス感染を示す項目31に記載の方法。
(項目35)
前記分析物が、C型インフルエンザウィルス感染を示す項目31に記載の方法。
(項目36)
前記分析物が、インフルエンザウィルスの表面糖タンパク質を含む項目31に記載の方法。
(項目37)
前記表面糖タンパク質が、赤血球凝集素を含む項目36に記載の方法。
(項目38)
前記赤血球凝集素が、H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、およびH16よりなる群から選択される項目37に記載の方法。
(項目39)
前記赤血球凝集素がH5である項目37に記載の方法。
(項目40)
前記表面糖タンパク質が、ノイラミニダーゼを含む求項36に記載の方法。
(項目41)
前記ノイラミニダーゼが、N1、N2、N3、N4、およびN5よりなる群から選択される項目40に記載の方法。
(項目42)
前記ノイラミニダーゼがN1である項目40に記載の方法。
(項目43)
前記分析物が、インフルエンザウィルスの表面糖タンパク質に対する抗体である項目31に記載の方法。
(項目44)
前記被験体がヒトである項目31に記載の方法。
(項目45)
前記被験体が動物である項目31に記載の方法。
(項目46)
前記動物が家禽である項目45に記載の方法。
(項目47)
複数の分析物を検出する方法であって、前記分析物のうちの少なくとも2つは、被験体からの体液中のインフルエンザウィルス感染を示し、前記方法は、
a)少なくとも1つのサンプル採集ユニット、免疫アッセイ用試薬を含有する免疫アッセイ用アセンブリ、前記サンプル採集ユニットおよび/または前記免疫アッセイ用アセンブリと流体連絡する複数のチャネルを備える流体装置を提供すること;
b)前記流体装置を作動させて前記流体装置内の前記免疫アッセイ用試薬を方向付けること;
c)前記複数の分析物を含有する疑いのある体液サンプルと、前記アッセイの免疫アッセイ用アセンブリ内に含まれた前記免疫アッセイ用試薬とを反応させて、前記サンプル中での前記少なくとも2つの分析物の存在を示す1つまたは複数の検出可能なシグナルを生じさせること;ならびに
d)前記体液サンプル中で採集された前記複数の分析物から生じた前記1つまたは複数の検出可能なシグナルを検出すること、
とを包含する方法。
(項目48)
前記体液サンプルが、約500マイクロリットル未満である項目47に記載の方法。
(項目49)
前記複数の分析物が、A型インフルエンザウィルス感染を示す項目47に記載の方法。(項目50)
前記複数の分析物が、B型インフルエンザウィルス感染を示す項目47に記載の方法。(項目51)
前記複数の分析物が、C型インフルエンザウィルス感染を示す項目47に記載の方法。(項目52)
前記少なくとも2つの分析物が、複合体を形成する項目47に記載の方法。
(項目53)
前記複合体がH5N1を含む項目52に記載の方法。
(項目54)
前記分析物のうちの少なくとも1つが、インフルエンザウィルスの表面糖タンパク質であり、前記分析物のうちの少なくとも1つが、前記表面糖タンパク質に対するヒト抗体である項目47に記載の方法。
(項目55)
前記分析物のうちの少なくとも1つが、インフルエンザウィルスの表面糖タンパク質であり、前記分析物のうちの少なくとも1つが、前記ウィルス感染により人体に課せられたストレスを示す体液サンプル中のバイオマーカーである項目47に記載の方法。
(項目56)
前記複数の分析物が、インフルエンザウィルスの複数の表面糖タンパク質を含む項目47に記載の方法。
(項目57)
前記複数の表面糖タンパク質が、赤血球凝集素およびノイラミニダーゼを含む項目56に記載の方法。
(項目58)
前記赤血球凝集素が、H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、およびH16よりなる群から選択され、前記ノイラミニダーゼが、N1、N2、N3、N4、およびN5よりなる群から選択される項目57に記載の方法。
(項目59)
前記赤血球凝集素がH5であり、前記ノイラミニダーゼがN1である項目57に記載の方法。
(項目60)
前記複数の分析物が、インフルエンザウィルスの表面糖タンパク質に対する複数の抗体を含む項目47に記載の方法。
(項目61)
前記被験体がヒトである項目47に記載の方法。
(項目62)
前記被験体が動物である項目47に記載の方法。
(項目63)
前記動物が家禽である項目62に記載の方法。
(項目64)
インフルエンザウィルス感染の1タイプを検出するための流体装置であって、
複数種の反応物質を含むカートリッジを備え、前記反応物質のうちの少なくとも2種は、被験体からの体液中に存在する異なる分析物に反応性であり、前記異なる分析物が、インフルエンザ感染のタイプを示す流体装置。
(項目65)
前記少なくとも2種の反応物質の各々が、インフルエンザウィルスの異なる表面糖タン
パク質に結合する項目64に記載の流体装置。
(項目66)
前記異なる表面糖タンパク質が、赤血球凝集素およびノイラミニダーゼよりなる群から選択されるメンバーを含む項目65に記載の流体装置。
(項目67)
前記異なる表面糖タンパク質が、赤血球凝集素1、赤血球凝集素2、赤血球凝集素3、赤血球凝集素4、赤血球凝集素5、赤血球凝集素6、赤血球凝集素7、赤血球凝集素8、赤血球凝集素9、赤血球凝集素10、赤血球凝集素11、赤血球凝集素12、赤血球凝集素13、赤血球凝集素14、赤血球凝集素15、赤血球凝集素16、ノイラミニダーゼ1、ノイラミニダーゼ2、ノイラミニダーゼ3、ノイラミニダーゼ4、およびノイラミニダーゼ5よりなる群から選択されるメンバーを含む項目65に記載の流体装置。
(項目68)
前記少なくとも2種の反応物質のうちの1種がH5に結合し、前記少なくとも2種の反応物質のうちの1種がN1に結合する項目64に記載の流体装置。
(項目69)
前記カートリッジが、サンプル採集ユニットおよび前記反応物質を含むアッセイ用アセンブリをさらに備える項目64に記載の流体装置。
(項目70)
前記アッセイ用アセンブリが、免疫アッセイ用アセンブリを備える項目69に記載の流体装置。
本明細書に記載された刊行物および特許出願の全ては、個々の刊行物または特許出願の各々が具体的に個々に、参照として援用されることが示される場合と同じ範囲で参照として本明細書に援用されている。
本発明の一態様は、体液サンプル中に存在するインフルエンザウィルス感染を示す分析物を検出するシステムである。この分析物は、A型、B型および/またはC型のインフルエンザウィルス感染を示すことができる。分析物は、インフルエンザウィルスの少なくとも1つの表面糖タンパク質を含むことができる。代表的な表面糖タンパク質は、限定はしないが、赤血球凝集素およびノイラミニダーゼである。赤血球凝集素表面糖タンパク質としては、限定はしないが、H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、およびH16が挙げられる。非限定的ノイラミニダーゼ表面タンパク質としては、N1、N2、N3、N4、およびN5が挙げられる。分析物はまた、感染宿主により産生されるインフルエンザウィルスの表面糖タンパク質に対する抗体を含む。
(登録商標) X−100、およびTWEEN (登録商標) 20などの非変性界面活性剤 などを含む溶解剤を用いて実施することができる。幾つかの実施形態において、サンプルの前処理は、流体装置内で自動的に達成される。
mmunol.Methods 74:307頁に記載された手法を用いてクラススイッチ変異体を単離するために、同胞選択技法を用いることによって異なるアイソタイプのモノクローナル抗体を分泌する親のハイブリドーマから二次的に調製することができる。
本発明の装置およびシステムは、被験体からの体液中に存在するインフルエンザウィルス感染を示す分析物のリアルタイムの検出に有効な手段を提供する。
Claims (22)
- 被験体由来の体液中のインフルエンザウィルス粒子を検出する方法であって、
a)該インフルエンザウィルス粒子を含有する疑いがある該体液のサンプルを、第一の免疫アッセイ用試薬および第二の免疫アッセイ用試薬と反応させる工程であって、それらの両方は、カートリッジの免疫アッセイ用アセンブリ内に含有され、該第一の免疫アッセイ用試薬は、赤血球凝集素分子に結合して、該インフルエンザウィルス粒子上に第一の免疫複合体を形成し、該第二の免疫アッセイ用試薬は、ノイラミニダーゼ分子に結合して該インフルエンザウィルス粒子上に第二の免疫複合体を形成し、該免疫複合体は、一つまたは一つより多い検出可能なシグナルを生成し、該シグナルは、該インフルエンザウィルス粒子上の赤血球凝集素およびノイラミニダーゼの同時の存在を示し、該第一の免疫アッセイ用試薬または該第二の免疫アッセイ用試薬のいずれかは、固体支持体上に固定化される、工程;ならびに
b)該免疫複合体から生成された該一つまたは一つより多い検出可能なシグナルを検出する工程であって、赤血球凝集素およびノイラミニダーゼの該同時の存在は、該インフルエンザウィルス粒子の存在を示す、工程
を含む、方法。 - 前記体液のサンプルは、約500マイクロリットル未満である、請求項1に記載の方法。
- 前記赤血球凝集素および前記ノイラミニダーゼの検出は、A型インフルエンザウィルス感染を示す、請求項1に記載の方法。
- 前記赤血球凝集素および前記ノイラミニダーゼは、前記免疫アッセイ用アセンブリ内に含有される前記免疫アッセイ用試薬と免疫複合体を形成する、請求項1に記載の方法。
- 前記赤血球凝集素および前記ノイラミニダーゼは、赤血球凝集素5(H5)およびノイラミニダーゼ(N1)を含む、請求項4に記載の方法。
- 前記赤血球凝集素が、H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、およびH16よりなる群から選択され、前記ノイラミニダーゼが、N1、N2、N3、N4、およびN5よりなる群から選択される、請求項1に記載の方法。
- 前記赤血球凝集素がH5であり、前記ノイラミニダーゼがN1である、請求項1に記載の方法。
- 前記被験体がヒトである、請求項1に記載の方法。
- 前記被験体が動物である、請求項1に記載の方法。
- 前記動物が家禽である、請求項9に記載の方法。
- 前記カートリッジは、少なくとも1つのサンプル採集ユニット、免疫アッセイ用試薬を含有する免疫アッセイ用アセンブリ、ならびに該サンプル採集ユニットおよび/または該免疫アッセイ用アセンブリと流体連絡する複数のチャネルを含む、請求項1に記載の方法。
- 前記カートリッジを作動させて、該流カートリッジ内の前記免疫アッセイ用試薬を方向付ける工程をさらに含む、請求項1に記載の方法。
- 前記インフルエンザウィルス粒子を含有する疑いがある前記体液のサンプルが、外部装置から伝送されたプロトコルに基づいて、前記免疫アッセイ用アセンブリ内に含まれる前記免疫アッセイ用試薬と反応する請求項1に記載の方法。
- 前記検出可能なシグナルを外部装置に伝送する工程をさらに含む、請求項1に記載の方法。
- 前記カートリッジは、前記サンプル採集ユニットと前記免疫アッセイ用アセンブリとの間の流体連絡を提供する複数のチャネルを含む、請求項1に記載の方法。
- 前記外部装置はサーバーである、請求項13に記載の方法。
- 前記プロトコルは、前記外部装置からワイヤレスで伝送される、請求項13に記載の方法。
- A型インフルエンザウィルス粒子を含有する疑いがある体液のサンプル中において、該粒子を検出する方法であって:
a)免疫アッセイ用アセンブリを提供する工程であって、該アセンブリは、第一の抗原に特異的に結合する第一の抗体を含み、該第一の抗体は、該アセンブリの表面に結合されている、工程;
b)該第一の抗原とは異なる第二の抗原に特異的に結合する第二の抗体を含む免疫アッセイ用試薬の存在下で、該免疫アッセイ用アセンブリ中で該体液のサンプルを反応させる工程であって、該第二の抗体は、検出可能な標識で標識され、ここで、該第一の抗原が赤血球凝集素を含み、そして該第二の抗原がノイラミニダーゼを含むか、または該第一の抗原がノイラミニダーゼを含み、そして該第二の抗原が赤血球凝集素を含むかのいずれかであり、そして該反応は、該第一の抗原に結合した該第一の抗体、および該第二の抗原に結合した該第二の抗体を含む免疫複合体を生成する、工程;
c)該アセンブリを洗浄して、過剰な免疫アッセイ用試薬を除去する工程;
d)該検出可能な標識により、該免疫複合体から生成されたシグナルを検出する工程であって、それにより該粒子の存在が示される、工程
を含む、方法。 - 前記赤血球凝集素が、H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、およびH16よりなる群から選択され、前記ノイラミニダーゼが、N1、N2、N3、N4、およびN5よりなる群から選択される、請求項18に記載の方法。
- 前記赤血球凝集素がH5であり、前記ノイラミニダーゼがN1である、請求項18に記載の方法。
- 前記赤血球凝集素がH1であり、前記ノイラミニダーゼがN1である、請求項18に記載の方法。
- 前記カートリッジが識別子と、サンプル採集ユニットと、前記免疫アッセイ用試薬を含む免疫アッセイ用アセンブリとを含み;識別子用検出器は、該識別子を読み取り、外部装置へ該識別子の情報を伝送する通信用アセンブリに通信し;プロトコルは該識別子に関連させられており、該プトロコルは、該識別子の該情報の該外部装置への伝送に応答して、該外部装置から伝送され;ここで、該サンプル採集ユニット中で採集された前記体液のサンプルは、該プロトコルに基づいて該免疫アッセイ用試薬と反応し、前記ウィルス粒子の存在を示す前記検出可能なシグナルを生成する、請求項1に記載の方法。
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