JP3841559B2 - Immunological examination method and immunological examination kit - Google Patents
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Description
【０００１】
【発明の属する技術分野】
本発明は、簡便、迅速に且つ高精度、高感度で被検試料中の検査対象物の検出を行い得る免疫学的検査方法およびそのためのキットに関する。より詳細には、本発明は検出シグナルを増幅させる工程を含む免疫クロマトグラフ法に関する。
【０００２】
【従来の技術】
生体試料等の免疫学的分析法において、迅速且つ簡便にその検査を行う方法として免疫クロマトグラフ法が挙げられる。この方法は、一般に以下のような工程を含む。すなわち、検査対象物と特異的に結合し得る抗体を固定化した固定相を有する吸水性基材からなる検査片の一端より、該検査対象物に特異的に結合し得る標識された抗体と被検液との混合物を吸収させて展開すると、該混合物中で形成された標識抗体−検査対象物複合体は固定化された抗体と結合して固定相上に捕捉される。したがって、該固定相に結合した標識抗体を測定することにより被検液中の検査対象物を測定することができる。
【０００３】
また、上記免疫クロマトグラフ法の検出シグナルをより高感度で得るための方法として、特開平１０−０６２４１９号において、二種の標識抗体を用いる方法が開示されている。つまり、検査対象物と特異的に結合し得る抗体を標識した第一標識抗体と、該抗体に特異的に結合し得る二次抗体を標識した第二標識抗体とを、検査片中の被検試料滴下部と固定相（該検査対象物と特異的に結合し得る別の抗体が固定化されている）の間に標識相としてそれぞれ設置した（吸収させた）構成である。試料中の検査対象物は、第一標識抗体と複合体を形成した後、さらに第二標識抗体が第一標識抗体に結合して（被検物−第一標識抗体−第二標識抗体）の複合体を形成する。該免疫複合体は固定相上に固定化された抗体により捕捉される。したがって、固定相上で第二標識抗体により増幅させたシグナルが検出される。
【０００４】
【発明が解決しようとする課題】
しかしながら、上記のシグナル増幅免疫クロマトグラフ法によってもまだ十分な検出感度が得られていないのが現状である。さらに、被検試料が糞便、尿、血液等の場合、前処理として試料を適当な緩衝液に懸濁する工程、および／または被検試料中の夾雑物質を分離除去する部分精製工程等の付加的な操作が必要となり、迅速性に欠けるといった問題点もある。したがって、本発明の目的は、免疫クロマトグラフ法に関し、より迅速に、且つ高感度で検査対象物を検出することが可能な免疫学的検査方法およびそのためのキットを提供することである。
【０００５】
【課題を解決するための手段】
本発明者らは、上記の目的を達成すべく鋭意研究を重ねた結果、検査対象物と特異的に結合する第二の抗体とともに検査対象物とは結合しない抗体を標識物質に結合して第一標識体とし、さらに、検査対象物とは結合しない抗体と介在物質を介して特異的に結合し得る免疫化学的成分（具体的には、例えば、検査対象物とは結合しない抗体により認識される抗原を介して結合する、該抗原を認識する抗体）に標識物質を結合して第二標識体とし、吸水性基材の被検試料滴下部と固定相との間に、これら二種の標識体を水との接触によって脱離可能な形態で保持した標識相を設け、この吸水性基材に該介在物質と被検試料とを展開して免疫クロマトグラフ法を行い、該吸水性基材の固定相上において、（固定化された抗体−検査対象物−第一標識体−介在物質−第二標識体）の免疫複合体を形成させることにより、固定相上での検出シグナルが従来よりも効率よく増幅され、より高感度に検査対象物を検出できることを見出した。また、被検試料を予め吸水性基材の固定相と該固定相より標識相により近い方の一端との間に供給し、該吸水性基材の該一端から第二標識体および介在物質を展開させる（以下、被検試料および介在物質を滴下する吸水性基材の一端を、吸液部と称する場合もある）ことにより、被検試料を希釈する前処理を省略できることを見出して、測定時間を短縮することに成功した。さらに、吸液部と固定相との間に、特に吸液部と標識相との間に、被検試料中の検査対象物と他の物質とを分離するための分離相を設けることにより、被検試料の部分精製などの前処理を省略でき、より迅速で、高精度且つ高感度の測定を行うことに成功して本発明を完成するに至った。
【０００６】
すなわち、本発明は以下の通りである。
１．以下の工程を含むことを特徴とする免疫学的検査方法：
(1) 検査対象物と特異的に結合し得る第一の免疫化学的成分を固定化した固定相と、検査対象物と特異的に結合し得る第二の免疫化学的成分および検査対象物とは結合しない第三の免疫化学的成分に標識物質（特に着色粒子、就中、着色されたラテックス粒子または金コロイド粒子）が結合してなる標識体（第一標識体）並びに該第三の免疫化学的成分と介在物質を介して特異的に結合し得る第四の免疫化学的成分に標識物質（特に着色粒子、就中、着色されたラテックス粒子または金コロイド粒子）が結合してなる標識体（第二標識体）が水との接触によって脱離し得る形態で保持される標識相とを、表面上の任意の領域に設けた吸水性基材の、固定相よりも標識相により近い方の一端から、被検試料を含有する液と該介在物質を含有する液との混合物を展開し、
(2) 該吸水性基材上で形成される検査対象物、第一標識体、介在物質および第二標識体からなる免疫複合体を固定相に固定化された第一の免疫化学的成分に結合させて捕捉した後、
(3) 該固定相上の標識物質のシグナルを測定することにより該検査対象物を検出する。
２．以下の工程を含むことを特徴とする免疫学的検査方法：
(1) 検査対象物と特異的に結合し得る第一の免疫化学的成分を固定化した固定相と、検査対象物と特異的に結合し得る第二の免疫化学的成分および検査対象物とは結合しない第三の免疫化学的成分に標識物質（特に着色粒子、就中、着色されたラテックス粒子または金コロイド粒子）が結合してなる標識体（第一標識体）並びに該第三の免疫化学的成分と介在物質を介して特異的に結合し得る第四の免疫化学的成分に標識物質（特に着色粒子、就中、着色されたラテックス粒子または金コロイド粒子）が結合してなる標識体（第二標識体）が水との接触によって脱離し得る形態で保持される標識相とを、表面上の任意の領域に設けた吸水性基材の、固定相と該固定相よりも標識相により近い方の一端との間の任意の領域に被検試料を供給し、
(2) 該吸水性基材の該一端から、該介在物質を含有する液を展開し、
(3) 該吸水性基材上で形成される検査対象物、第一標識体、介在物質および第二標識体からなる免疫複合体を固定相に固定化された第一の免疫化学的成分に結合させて捕捉した後、
(4) 該固定相上の標識物質のシグナルを測定することにより該検査対象物を検出する。
３．該吸水性基材が、固定相と該固定相よりも標識相により近い方の一端との間の任意の領域（但し、被検試料を該吸水性基材の該一端と固定相との間の任意の領域に供給する場合は、その領域を含めてそれより固定相側の領域）、特に該一端と標識相との間の任意の領域に、検査対象物と被検試料中の他の物質とを分離し得る分離相をさらに設けたものであることを特徴とする上記１または２の方法。
４．該介在物質が抗原抗体反応により第三および第四の免疫化学的成分と特異的に結合する物質である上記１〜３のいずれかの方法。
５．検査対象物と特異的に結合し得る第一の免疫化学的成分を固定化した固定相と、検査対象物と特異的に結合し得る第二の免疫化学的成分および検査対象物とは結合しない第三の免疫化学的成分に標識物質（特に着色粒子、就中、着色されたラテックス粒子または金コロイド粒子）が結合してなる標識体（第一標識体）並びに該第三の免疫化学的成分と介在物質を介して特異的に結合し得る第四の免疫化学的成分に標識物質（特に着色粒子、就中、着色されたラテックス粒子または金コロイド粒子）が結合してなる標識体（第二標識体）が水との接触によって脱離し得る形態で保持される標識相とを、表面上の任意の領域に設けた吸水性基材、並びに第三および第四の免疫化学的成分の結合を仲介する介在物質を含み、以下の（ａ）および（ｂ）の免疫学的検査方法に使用され得る免疫学的検査キット：
方法（ａ）
(1) 該吸水性基材の固定相よりも標識相により近い方の一端から、被検試料を含有する液と該介在物質を含有する液との混合物を展開し、
(2) 該吸水性基材上で形成される検査対象物、第一標識体、介在物質および第二標識体からなる免疫複合体を固定相に固定化された第一の免疫化学的成分に結合させて捕捉した後、
(3) 該固定相上の標識物質のシグナルを測定することにより該検査対象物を検出する。
方法（ｂ）
(1) 該吸水性基材の固定相と、該固定相よりも標識相により近い方の一端との間の任意の領域に被検試料を供給し、
(2) 該吸水性基材の該一端から、該介在物質を含有する液を展開し、
(3) 該吸水性基材上で形成される検査対象物、第一標識体、介在物質および第二標識体からなる免疫複合体を固定相に固定化された第一の免疫化学的成分に結合させて捕捉した後、
(4) 該固定相上の標識物質のシグナルを測定することにより該検査対象物を検出する。
６．該吸水性基材が、固定相と該固定相よりも標識相により近い方の一端との間の任意の領域（但し、被検試料を該吸水性基材の該一端と固定相との間の任意の領域に供給する場合は、その領域を含めてそれより固定相側の領域）、特に該一端と標識相との間の任意の領域に、検査対象物と被検試料中の他の物質とを分離し得る分離相をさらに設けたものであることを特徴とする上記５のキット。
７．該介在物質が抗原抗体反応により第三および第四の免疫化学的成分と特異的に結合する物質である上記５または６のキット。
【０００７】
【発明の実施の形態】
本発明の方法により検出され得る検査対象物は、免疫化学的反応（すなわち抗原抗体反応）により第一の免疫化学的成分および第二の免疫化学的成分と結合してサンドイッチ免疫複合体を形成し得るものであれば特に制限されない。例えば、細菌（特に大腸菌Ｏ−１５７、メチシリン耐性黄色ブドウ球菌等の病原性細菌）、放線菌、酵母、かび、ウイルス（特に、ＨＩＶ、ＨＢＶ、ＨＣＶ等）などの微生物もしくは表面抗原等の該微生物の構成蛋白質、またはそれらに対する抗体、あるいは腫瘍マーカー抗原などの生体試料中の抗原性ペプチド等が挙げられる。
【０００８】
本発明の方法において、固定相に固定化される第一の免疫化学的成分と、第一標識体として用いる第二の免疫化学的成分は、いずれも抗原抗体反応により検査対象物と特異的に結合し得る物質であれば特に制限はない。検査対象物が抗原（例えば、蛋白質、ペプチド、ハプテンなど）であれば、第一および第二の免疫化学的成分は、該抗原と特異的に結合し得る抗体である。該抗体はモノクローナル抗体であってもポリクローナル抗体であってもよい。また本発明における抗体とは、検査対象物との特異的親和性を保持する抗体の断片物、例えばＨ鎖、Ｌ鎖、Ｆab、Ｆ(ab')2、ＶH 、ＶL 等も含むものとする。一方、検査対象物が抗体である場合には、第一および第二の免疫化学的成分は、該抗体と特異的に結合し得る抗原もしくは該抗体を抗原として特異的に結合し得る二次抗体である。第一の免疫化学的成分および第二の免疫化学的成分は、検査対象物に応じてサンドイッチ法などで用いられる自体公知のものを適宜選択すればよい。また、該免疫化学的成分が抗体であれば、単離された検査対象物を感作抗原として、公知の抗体作製技術を用いて調製することもできる。第一および第二の免疫化学的成分がいずれも抗体の場合、第一の抗体および第二の抗体は、同一のものであっても異なるものであってもよい。また、異なる抗体としては、同一の抗原決定基を認識する二種の抗体を用いることも、あるいは異なる抗原決定基を認識する二種の抗体を用いることもできる。
【０００９】
本発明において、第一標識体として用いる第三の免疫化学的成分、および第二標識体として用いる第四の免疫化学的成分は、介在物質を介して特異的に結合し合える抗体（モノクローナル抗体であってもポリクローナル抗体であってもよい。また、Ｈ鎖、Ｌ鎖、Ｆab、Ｆ(ab')2、ＶH 、ＶL 等の断片化された抗体であってもよい）または抗原（例えば、蛋白質、ペプチド、ハプテンなど）であり、且つ検査対象物および固定化された第一の免疫化学的成分とは特異的親和性を有しないものである。好ましくは、これらの免疫化学的成分は、さらに第二の免疫化学的成分とも特異的親和性を有しないものである。
【００１０】
第三および第四の免疫化学的成分の結合を仲介する本発明の介在物質は、第三および第四の免疫化学的成分に同時に結合して（第三の免疫化学的成分−介在物質−第四の免疫化学的成分）の複合体を形成し得る物質であれば特に限定されないが、好ましくは、抗原抗体反応により第三および第四の免疫化学的成分と特異的に結合し得る免疫化学的成分である。すなわち、第三の免疫化学的成分が抗体であれば、介在物質は該抗体により認識される抗原もしくは該抗体と特異的に結合し得る二次抗体であり、第四の免疫化学的成分は該抗原と特異的に結合し得る抗体もしくは該二次抗体により認識される抗原であることが好ましい。このとき、第三および第四の免疫化学的成分がともに抗体であれば、それらは同一の抗体であっても異なるものであってもよい。一方、第三の免疫化学的成分が抗原であれば、介在物質は該抗原と特異的に結合し得る抗体であり、第四の免疫化学的成分は該抗体により認識される抗原もしくは該抗体と特異的に結合し得る二次抗体である。第三および第四の免疫化学的成分がともに抗原であれば、それらは同一の抗原であっても、介在物質である抗体と交叉反応性のある異なる抗原であってもよい。第三および第四の免疫化学的成分、並びに介在物質は、サンドイッチ法等で用いられている公知のものを適宜選択して使用することができる。
【００１１】
本発明において用いられる吸水性基材は、検査対象物を含有する被検試料、例えば、食品から抽出した溶液やその培養上清、血清や血液、尿、便、唾液等、あるいはそれらを適当な希釈溶媒によって希釈してなる希釈液、第一標識体、第二標識体および介在物質をそれぞれ含有する液を吸収できるものであれば特に限定されない。本発明においては、被検試料中の検査対象物が第一標識体や固定相に固定化された第一の免疫化学的成分と十分な反応を行うための、並びに第一標識体が介在物質を介して第二標識体と十分な反応を行うための時間を確保できるような吸水性基材が好ましく用いられる。
【００１２】
吸水性基材が吸水性に劣る場合には、後述するように被検試料が固定相に到達するのに長時間を要し、その結果、迅速な測定を行うことができない。一方、吸水性基材の吸水性があまりに高すぎる場合には、被検試料中の検査対象物が標識体や固定相の第一免疫化学的成分と十分な反応を行うために必要な時間が不足するので、正確な測定を行うことが困難である。
【００１３】
したがって、本発明における吸水性基材の好ましい吸水性の程度は、５ｍｍ幅の短冊状に裁断した吸水性基材の片端部を水に浸漬し、１分間経過後の吸水距離が０．５〜５ｃｍ程度である。
【００１４】
本発明の吸水性基材の好ましい具体例としては、不織布、濾紙、ガラス繊維布、ガラスフィルター、ニトロセルロースフィルター、多孔質材料などが挙げられる。これらの基材は適度な吸水速度を有するとともに、標識物質が着色粒子の場合、着色粒子が結合して発色した際の目視確認性に優れるなどの利点を有するものである。
【００１５】
また、これらの基材の吸水性を調整するために、基材の表面に親水性重合体や界面活性剤を被覆し、あるいは含浸させることもできる。さらに、本発明においては吸水性基材として同一材料からなる基材を用いてもよいし、あるいは異種の材料からなるものを任意の接着手段によって接合して得られる連続した基材を用いることもできる。
【００１６】
本発明において、吸水性基材の形状は、被検試料を展開できる形状であれば特に限定されるものではなく、例えば、矩形のシート状（片状）やロッド状などが好ましい。
【００１７】
本発明において、固定相とは、検査対象物と特異的に結合し得る第一の免疫化学的成分が吸水性基材上に固定化された領域を意味する。第一の免疫化学的成分を吸水性基材上に固定化する方法（固定化相の作製方法）も特に限定されるものではないが、従来から知られている物理吸着法や共有結合法によるのが好適であり、特に、該免疫化学的成分が基材から脱離しにくいという点で共有結合法によるのが好ましい。吸水性基材が上記共有結合法のための官能基を有しないときは、例えば適当な官能基を有する重合体を用いて基材を作製し、吸水性基材の吸水性を阻害しない程度に付着させることができる。また、第一の免疫化学的成分および親水性重合体を含む溶液を吸水性基材に塗布した後、上記親水性重合体を凝固させる凝固溶剤に浸漬することで固定相を作製することもできる。上記親水性重合体としては、ヒドロキシプロピルメチルセルロース、ポリビニルアルコール、ヒドロキシエチルセルロースなどが用いられる。また、上記凝固溶剤としては、アセトン、エタノール、メタノール、エーテルなどを用いることができる。
【００１８】
本発明において、上記固定相と、吸水性基材の一端の、被検試料含有液、第二標識体含有液、介在物質含有液等の吸収が開始される部位（すなわち吸液部）との間の距離は特に限定されないが好ましくは１〜６ｃｍ、より好ましくは３〜４ｃｍ程度である。距離があまりに遠すぎると、固定相まで被検試料が到達しなかったり、検出シグナル感度が強すぎたり、あるいは測定に時間がかかる等の問題を生じるおそれがある。一方、距離が近すぎると固定相上に捕捉される標識物質が均一ではなくまばらになったり、検出シグナル感度が低すぎるという問題を生じるおそれがある。
【００１９】
吸液部としては、被検試料、標識体、並びに介在物質をそれぞれ含有する液の吸水性基材への移動を妨げるものでなければ特に限定されず、基材と兼用したものであっても、新たに不織布や織布等を該吸水性基材に接着させたものであってもよい。本発明において、検査対象物に特異的に結合し得る第一の免疫化学的成分が固定化された固定相、並びに吸液部を有する吸水性基材を、以下、本発明の免疫学的検査片または単に検査片という場合もある。
【００２０】
本発明の方法における第一標識体は、検査対象物に特異的に結合し得る免疫化学的成分（第二の免疫化学的成分）および検査対象物とは結合しない第三の免疫化学的成分に標識物質を結合させたものであり、また、第二標識体は、該第三の免疫化学的成分と介在物質を介して特異的に結合し得る免疫化学的成分（第四の免疫化学的成分）に標識物質を結合させたものである。ここで用いられる標識物質は、免疫化学的測定法において常套的に使用されるいかなる標識物質であってもよく、例えば着色粒子、酵素（アルカリホスファターゼ、ペルオキシダーゼ等）、蛍光物質（ＦＩＴＣ、ローダミン等）などが挙げられるが、効率のよい検出シグナルの増幅を達成するためには、第一および第二標識体に使用される標識物質は同一のものであることが好ましい。本発明の方法では、迅速な検出を行う意味で、標識物質として着色粒子が好ましく使用される。着色粒子は肉眼で検出可能なものであれば特に制限はなく、例えば、金、銀、銅などの金属からなるコロイド粒子、スダンブルーやスダンレッドIV、スダンIII 、オイルオレンジ、キニザリングリーン等に代表される顔料や染料などでラテックスを着色してなる着色ラテックスなどを用いることができる。目視確認性の点からは、金コロイドや青、赤、緑もしくはオレンジ色に着色した着色ラテックスの使用が好ましく、また、分散安定性や検査対象物の検出感度の調節が容易であるなどの点を考慮すると、青色、赤色等に着色された水分散型高分子重合体粒子からなる着色ラテックスを使用することがさらに望ましい。
【００２１】
着色粒子の粒径は、検出の際の発色がよく且つ吸水性基材の吸水性を低下させない程度の該基材中での移動性を有するものであれば特に制限はないが、保存安定性や調製が容易であるなどの点から、好ましくは０．０１〜５μｍ、より好ましくは０．０５〜３μｍの範囲が例示される。粒径があまりに小さすぎると、１粒子あたりの着色の程度が少ないので、固定相に結合しても発色の程度が悪く目視確認性に劣るようになる。また、粒径が大きすぎると、着色粒子わずかに凝集しただけで吸水性基材に目詰まりを起こして吸水性を低下させたり、非特異的発色を生じたりすることがある。
【００２２】
第二および第三の免疫化学的成分の両方、並びに第四の免疫化学的成分を、このような着色粒子で標識する方法としては、従来公知の方法、例えば共有結合法、物理的吸着法、イオン結合法等を使用することができるが、免疫化学的成分からの着色粒子の脱離がなく安定であるという点から共有結合法がより好ましく用いられる。
【００２３】
本発明の方法において、被検試料中の複数の検査対象物を検出するために、対応する複数の免疫化学的成分をそれぞれ別の着色粒子で標識することができるが、この際に用いられる着色粒子は同一色であっても異なる色であってもよい。同一色の着色粒子を用いる場合、各検査対象物にそれぞれ特異的に結合し得る免疫化学的成分を固定化した固定相を識別可能な程度に離して設置することが望ましい。
【００２４】
標識物質が酵素や蛍光物質の場合には、固定相の標識物質の検出は、ＥＩＡや蛍光抗体法（ＦＩＡ）で従来使用されている検出手段が適宜選択される。
【００２５】
本発明において、標識相とは、吸水性基材の吸液部と固定相との間に設けられた、第一標識体および第二標識体を水との接触によって脱離し得る形態で保持する領域を意味する。標識相の作製方法としては特に制限はないが、例えば、標識体を含有する液を吸水性基材の吸液部と固定相との間の任意の領域に塗布し、適当な条件下で乾燥させる（例えば、凍結乾燥）方法が挙げられる。また、水溶性重合体もしくはサッカロース溶液中に標識体を分散させ、該分散液を吸水性基材上に塗布して同様に乾燥させてもよい。この方法は、水溶性重合体またはサッカロースが容易に水溶化し、第一標識体および第二標識体が速やかに基材から脱離して、第一標識体が被検試料中の検査対象物および介在物質を介して第二標識体と反応し得るとともに、水溶性重合体やサッカロースの濃度を調整することにより吸水性基材の所定の領域に標識体を保持させるのに適当な粘度を得ることができ、さらに乾燥に際して標識体の凝集や変性を防ぎ、また乾燥後に標識体が吸水性基材から脱離しにくいという点で有利である。
【００２６】
上記水溶性重合体としては、例えば、ポリビニルピロリドン、ポリビニルアルコール、ポリエチレングリコール、セルロースエステル（例えば、メチルセルロース、エチルセルロース、カルボキシメチルセルロース、カルボキシエチルセルロース、オキシエチルセルロース、シアンエチルセルロース等）、ゼラチンなどが好ましく用いられる。
【００２７】
介在物質含有液は、介在物質を適当な分散媒（溶媒）中に分散（溶解）させることにより調製される。介在物質を分散させる分散媒は、検査対象物と第一標識体、第一標識体と介在物質および介在物質と第二標識体の特異的結合反応を阻害しないものであれば特に制限はないが、好ましくは該抗原抗体反応に適したｐＨおよび塩濃度の有する緩衝液、例えばリン酸緩衝液、酢酸緩衝液、ホウ酸緩衝液、トリス塩酸緩衝液等を適宜選択して使用することができる。
【００２８】
本発明の方法の一態様では、各工程において以下の反応が行われる。すなわち、吸水性基材の一端（すなわち吸液部）から、被検試料を含有する液と第三および第四の免疫化学的成分の結合を仲介する介在物質を含有する液との混合物を滴下して展開すると、各成分は液の移動とともに吸水性基材上を移動する。液が標識相に到達すると、そこに保持されていた第一および第二標識体は水との接触によって標識相から脱離する。さらに、第一標識体中の第二の免疫化学的成分が被検試料中の検査対象物と、また第三の免疫化学的成分が介在物質を介して第二標識体中の第四の免疫化学的成分とそれぞれ結合して複合体を形成し、さらに吸水性基材上を移動する。そして、形成された免疫複合体は、固定相に固定化された第一の免疫化学的成分と結合して固定相上に捕捉される。このようにして、第一および第二標識体を構成する標識物質が固定相上に集合、結合して検出シグナルが増幅され、それによってより高感度に検査対象物の存在を検出することが可能となる。
【００２９】
本発明の方法の別の態様としては、上記の方法において、被検試料を吸液部から滴下する代わりに、吸液部と固定相との間に滴下または塗布した後、介在物質を含有する液を吸液部に滴下して展開する方法が挙げられる。この場合、該混合液が被検試料を滴下または塗布した領域に達すると、被検試料中の検査対象物は介在物質とともに吸水性基材上を移動する。液が標識相に到達すると、そこに保持されていた第一および第二標識体は水との接触によって標識相から脱離する。さらに、第一標識体中の第二の免疫化学的成分が被検試料中の検査対象物と、また第三の免疫化学的成分が介在物質を介して第二標識体中の第四の免疫化学的成分とそれぞれ結合して複合体を形成し、さらに吸水性基材上を移動する。そして、形成された免疫複合体は、固定相に固定化された第一の免疫化学的成分と結合して固定相上に捕捉される。
【００３０】
本発明の免疫学的検査用キットは、本発明の免疫学的検査方法に好ましく用いることができる。該キットは、少なくとも下記の内容を含むものである。
(a) 検査対象物と特異的に結合し得る第一の免疫化学的成分を固定化した固定相と、検査対象物と特異的に結合し得る第二の免疫化学的成分および検査対象物とは結合しない第三の免疫化学的成分に標識物質が結合してなる標識体（第一標識体）並びに該第三の免疫化学的成分と介在物質を介して特異的に結合し得る第四の免疫化学的成分に標識物質が結合してなる標識体（第二標識体）が水との接触によって脱離し得る形態で保持される標識相とを、表面上の任意の領域に設けた吸水性基材
(b) 第三および第四の免疫化学的成分の結合を仲介する介在物質
該吸水性基材、第一および第二標識体、並びに介在物質の好ましい態様は、上記したような本発明の方法において好ましく用いられるものである。
【００３１】
本発明のキットに用いられる吸水性基材の好ましい態様の１つとして、その一端（すなわち吸液部）と固定相との間の任意の領域（但し、被検試料を該吸水性基材の一端と固定相との間の任意の領域に滴下または塗布する場合はその領域を含めてそれより固定相側の領域）、特に吸液部と標識相との間に、検査対象物と被検試料中の他の物質とを分離し得る分離相をさらに設けたものが挙げられる。
【００３２】
本発明において、分離相は、分離しようとする方向の孔径が検査対象物、各標識体、並びに介在物質よりも大きく、分離除去しようとする被検試料中の他の物質よりも小さいことが望ましい。また、分離方向としては、標識体が吸水性基材上を展開する方向であっても、あるいはその方向と垂直の方向であってもよく、さらに、検査対象物を被検試料中の他の物質から分離した後は、該分離相を除去して後の測定を行ってもよい。
【００３３】
分離相の材質としては、例えば、レーヨン、ポリエステル等の不織布、濾紙、ガラス繊維布、ガラスフィルター、ニトロセルロースフィルター、ポリスルホンフィルター、多孔質材料等が挙げられる。
【００３４】
本発明のキットは、上記の内容物以外に、本発明の免疫学的検査方法において好ましく使用され得る付加的な内容物を含んでいてもよい。例えば、介在物質を分散（溶解）させるのに好ましく使用される上記の緩衝液などが挙げられる。
【００３５】
【実施例】
以下に実施例を挙げて本発明をより詳細に説明するが、これら実施例は本発明の範囲を何ら限定するものではない。
【００３６】
実施例１ 免疫学的検査用キット構成物の作製
（１）水分散型高分子重合体粒子からなる着色ラテックスの作製
スチレンモノマー５０ｇ、アクリル酸０．５ｇ、トリエチレングリコールジメタクリレート０．２ｇ、及び蒸留水４４０ｇを窒素気流下で温度７５℃にて攪拌しながら、これに過硫酸カリウム０．２５ｇを水１０ｇに溶解した水溶液を加え、１０時間重合させて、平均粒子径０．２２μｍの水分散型高分子重合体粒子の水分散液を得た。この重合体粒子分散液をアルカリ、酸、蒸留水の順序にて遠心洗浄した後、固形分濃度１０重量％に調整した（担体粒子分散液）。スダンブルー０．２ｇをトルエン２０ｍｌに溶解し、これにドデシル硫酸ナトリウム０．２ｇ、及び蒸留水１００ｍｌを加え、超音波分散機でこの混合液を乳化した。この液に上記担体粒子分散液（固形分濃度１０重量％）３０ｍｌを加え、室温にて２４時間攪拌した。この液をエバポレータにてトルエンを除去した後、０．０１Ｍほう酸緩衝液（ｐＨ７．５）にて遠心洗浄を行い、固形分濃度５重量％となるように調整した。この液５０ｍｌに、１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド塩酸塩水溶液（１０ｍｇ／ｍｌ）５ｍｌ、及び０．０３Ｍｍ−キシレンジアミン水溶液５０ｍｌを加え、室温にて５時間反応させた。この液を７５℃にて５時間加熱処理した後、前記と同じ緩衝液にて遠心洗浄を行い、固形分濃度１重量％となるように調整した（スダンブルー染色キシレンジアミンスペーサ化粒子分散液）。
【００３７】
（２）第一標識体含有液の作製
上記（１）で調製したスダンブルー染色キシレンジアミンスペーサ化粒子分散液１０ｍｌにグルタルアルデヒド水溶液（０．１ｍｇ／ｍｌ）１ｍｌを加え、室温にて２時間反応させた後、前記と同じ緩衝液にて遠心洗浄し、固形分濃度１重量％の分散液に調整した。この分散液１０ｍｌに第三の免疫化学的成分として抗ヒトヘモグロビン抗体（ウサギＩｇＧ，１０ｍｇ／ｍｌ）を１ｍｌ、第二の免疫化学的成分として抗ヒトＨＢｓ抗体（ウサギＩｇＧ，５ｍｇ／ｍｌ）を１ｍｌ、それぞれ加え、１０℃にて２４時間攪拌した。これを前記と同じ緩衝液で遠心洗浄し、固形分濃度１重量％となるように再分散させ、共有結合で抗体を結合したスダンブルー染色粒子標識抗ヒトヘモグロビン抗体−抗ヒトＨＢｓ抗体（第一標識体）含有液を得た。
【００３８】
（３）第二標識体含有液の作製
上記（１）で調製したスダンブルー染色キシレンジアミンスペーサ化粒子分散液１０ｍｌにグルタルアルデヒド水溶液（０．１ｍｇ／ｍｌ）１ｍｌを加え、室温にて２時間反応させた後、前記と同じ緩衝液にて遠心洗浄し、固形分濃度１重量％の分散液に調整した。この分散液１０ｍｌに、第四の免疫化学的成分として抗ヒトヘモグロビン抗体（ウサギＩｇＧ，１０ｍｇ／ｍｌ）２ｍｌ、を加え、１０℃にて２４時間攪拌した。これを前記と同じ緩衝液で遠心洗浄し、固形分濃度１重量％となるように再分散させ、共有結合で抗体を結合したスダンブルー染色粒子標識抗ヒトヘモグロビン抗体（第二標識体）含有液を得た。
【００３９】
（４）免疫学的検査片の作製
第一の免疫化学的成分として抗ヒトＨＢｓ抗体（ウサギＩｇＧ）を、０．１Ｍリン酸緩衝液（ｐＨ７．４）にて希釈し、最終濃度１ｍｇ／ｍｌの水溶液に調整した。この水溶液に、ニトロセルロースメンブランフィルター（東洋ろ紙、５×１００ｍｍ）の一端から５０ｍｍ部位に１０μｌ塗布した後、直ちに３７℃で１時間静置した後、ニトロセルロースメンブランフィルターを取り出し、０．１％ウシ血清アルブミン、０．１％Ｔｗｅｅｎ２０の水溶液に１時間浸漬させた。その後、ニトロセルロースメンブランフィルターを取り出し、室温で３時間静置し、抗ヒトＨＢｓ抗体を有するニトロセルロースメンブランフィルターを得た。次に、上記の抗ヒトＨＢｓ抗体固定化ニトロセルロースメンブランフィルターの試験片の一端（吸液部）の近傍に血球分離相（東洋ろ紙Ｎｏ．２、５×１０ｍｍ）を設け、固定相および血球分離相を有するニトロセルロースメンブランフィルターからなる検査片を得た。
５重量％ポリビニルピロリドン（粘度平均分子量２５０００）水溶液１ｍｌに、実施例１の（２）で作製した第一標識体含有液および実施例１の（３）で作製した第二標識体含有液をそれぞれ０．１ｍｌ加えて十分混合した後、この溶液１０μｌを上記検査片の固定相の位置から４０〜５０ｍｍの位置に塗布し、これをデシケーター内で２日間乾燥して、標識相を設けた免疫学的検査片を得た。
【００４０】
実施例２ 免疫学的検査用キットによるヒトＨＢｓ抗原の検出
実施例１の（４）で作製した検査片の血球分離相に、ヒトＨＢｓ抗原を生理食塩水溶液に溶解させた被検液１０μｌを滴下した。その後、直ちにヘモグロビン抗原溶液（蛋白質濃度２００ｎｇ／ｍｌ）１００μｌを吸液部に滴下して展開した（図１Ａ）。１０分後の固定相上での発色を目視観察した（図１Ｂ）。その結果を表１に示す。
【００４１】
【表１】
比較例
実施例１の（４）で作製した検査片の血球分離相に、ヒトＨＢｓ抗原を生理食塩水溶液に溶解させた被検液１０μｌを滴下した。その後、直ちに実施例１の（２）で作製した第一標識体含有液（固形分濃度０．２重量％）５０μｌを吸液部に滴下して展開した。１０分後、ヘモグロビン抗原溶液（蛋白質濃度２００ｎｇ／ｍｌ）１００μｌを吸液部に滴下して展開した。さらに１０分後、実施例１の（３）で作製した第二標識体含有液（固形分濃度０．２重量％）５０μｌを吸液部に滴下して展開した。１０分後の固定相上での発色を目視観察した。その結果を表２に示す。
【００４３】
【表２】
【発明の効果】
本発明の方法（および本発明のキットの使用）は、シグナルを増幅する工程を含む免疫クロマトグラフ法において、介在物質を介して第一標識体と第二標識体を結合させることにより、従来より効率よく検出シグナルを増幅させることができる。また、本発明の方法の好ましい態様においては、便や血液等の検査において従来必要とされた被検試料の前処理の工程を省略することができるので、より迅速に検査対象物を測定することが可能となる。
【図面の簡単な説明】
【図１】本発明の免疫学的検査キットを用いたヒトＨＢｓ抗原の測定の各工程における抗原抗体反応の様子を示す模式図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an immunological test method capable of detecting a test object in a test sample simply, quickly, with high accuracy and high sensitivity, and a kit therefor. More specifically, the present invention relates to an immunochromatographic method including a step of amplifying a detection signal.
[0002]
[Prior art]
An immunochromatographic method is an example of a method for quickly and simply examining a biological sample or the like in an immunological analysis method. This method generally includes the following steps. That is, from one end of a test piece made of a water-absorbing substrate having a stationary phase on which an antibody that can specifically bind to the test object is immobilized, a labeled antibody that can specifically bind to the test object and the target When the mixture with the test solution is absorbed and developed, the labeled antibody-test object complex formed in the mixture binds to the immobilized antibody and is captured on the stationary phase. Therefore, the test object in the test liquid can be measured by measuring the labeled antibody bound to the stationary phase.
[0003]
Moreover, as a method for obtaining a detection signal of the above immunochromatography method with higher sensitivity, JP-A-10-062419 discloses a method using two kinds of labeled antibodies. That is, a first labeled antibody labeled with an antibody that can specifically bind to a test object, and a second labeled antibody labeled with a secondary antibody that can specifically bind to the antibody are tested in a test piece. This is a configuration in which each is placed (absorbed) as a labeled phase between the sample dropping part and the stationary phase (another antibody capable of specifically binding to the test object is immobilized). After the test object in the sample forms a complex with the first labeled antibody, the second labeled antibody is further bound to the first labeled antibody (test object-first labeled antibody-second labeled antibody). Form a complex. The immune complex is captured by the antibody immobilized on the stationary phase. Therefore, a signal amplified by the second labeled antibody on the stationary phase is detected.
[0004]
[Problems to be solved by the invention]
However, at present, sufficient detection sensitivity has not been obtained even by the signal amplification immunochromatography method. In addition, when the test sample is feces, urine, blood, etc., additional steps such as a step of suspending the sample in an appropriate buffer as a pretreatment and / or a partial purification step of separating and removing contaminants in the test sample Operation is required and there is a problem that it is not quick. Accordingly, an object of the present invention relates to an immunochromatographic method, and is to provide an immunological test method and a kit therefor capable of detecting a test object more rapidly and with high sensitivity.
[0005]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the present inventors bind an antibody that does not bind to the test object together with a second antibody that specifically binds to the test object to the labeling substance. Furthermore, an immunochemical component that can be specifically bound via an intervening substance and an antibody that does not bind to the test object (specifically, for example, an antibody that does not bind to the test object) A second labeled body by binding a labeling substance to an antibody that binds via an antigen that recognizes the antigen), and between these two kinds of samples between the test sample dropping part of the water-absorbing substrate and the stationary phase A labeled phase holding the labeled body in a form that can be detached by contact with water is provided, and the intervening substance and the test sample are developed on the water-absorbing substrate to perform immunochromatography, and the water-absorbing group On the stationary phase of the material, (immobilized antibody-test object-first standard Body - mediators - by forming an immune complex of a second label), the detection signal on the stationary phase are amplified more efficiently than the conventional found to be able to detect an inspection target object with a higher sensitivity. Further, the test sample is supplied in advance between the stationary phase of the water-absorbing substrate and one end closer to the labeling phase than the stationary phase, and the second labeled body and the intervening substance are introduced from the one end of the water-absorbing substrate. Measured by developing (hereinafter, one end of the water-absorbing substrate to which the test sample and the intervening substance are dropped may be referred to as a liquid-absorbing part), so that the pretreatment for diluting the test sample can be omitted. We succeeded in shortening time. Furthermore, by providing a separation phase between the liquid absorption part and the stationary phase, particularly between the liquid absorption part and the labeling phase, for separating the test object and other substances in the test sample, Pretreatment such as partial purification of the test sample can be omitted, and the present invention has been completed by succeeding in performing measurement more quickly, with high accuracy and high sensitivity.
[0006]
That is, the present invention is as follows.
1. An immunological test method comprising the following steps:
(1) a stationary phase in which a first immunochemical component that can specifically bind to a test object is immobilized; a second immunochemical component that can specifically bind to the test object; and a test object A labeled body (first labeled body) formed by binding a labeling substance (especially colored particles, especially colored latex particles or gold colloidal particles) to a third immunochemical component that does not bind Labeled substance formed by binding a labeling substance (particularly colored particles, especially colored latex particles or gold colloidal particles) to a fourth immunochemical component capable of specifically binding via a chemical component and an intervening substance The labeled phase retained in a form in which the (second labeled body) can be detached by contact with water, the water-absorbing substrate provided in an arbitrary region on the surface, closer to the labeled phase than the stationary phase From one end, a liquid containing the test sample and a liquid containing the intervening substance And expand the mixture with
(2) The first immunochemical component immobilized on the stationary phase is an immunocomplex comprising the test object, the first labeled body, the intervening substance, and the second labeled body formed on the water-absorbing substrate. After binding and capturing,
(3) The test object is detected by measuring the signal of the labeling substance on the stationary phase.
2. An immunological test method comprising the following steps:
(1) a stationary phase in which a first immunochemical component that can specifically bind to a test object is immobilized; a second immunochemical component that can specifically bind to the test object; and a test object A labeled body (first labeled body) formed by binding a labeling substance (especially colored particles, especially colored latex particles or gold colloidal particles) to a third immunochemical component that does not bind Labeled substance formed by binding a labeling substance (particularly colored particles, especially colored latex particles or gold colloidal particles) to a fourth immunochemical component capable of specifically binding via a chemical component and an intervening substance A stationary phase of a water-absorbing substrate provided in an arbitrary region on the surface with a labeled phase that is retained in a form in which the (second labeled body) can be detached by contact with water. Supply the test sample to an arbitrary area between one end closer to
(2) From the one end of the water-absorbing substrate, expand the liquid containing the intervening substance,
(3) The first immunochemical component immobilized on the stationary phase is an immune complex composed of the test object, the first labeled body, the intervening substance and the second labeled body formed on the water-absorbing substrate. After binding and capturing,
(4) The test object is detected by measuring the signal of the labeling substance on the stationary phase.
3. Arbitrary region between the stationary phase and one end closer to the labeling phase than the stationary phase (where the test sample is between the one end of the absorbent substrate and the stationary phase) In the case of supplying to any region, the region including that region and the region on the stationary phase side), especially in any region between the one end and the labeled phase, 3. The method according to 1 or 2 above, further comprising a separated phase capable of separating the substance.
4). 4. The method according to any one of 1 to 3 above, wherein the intervening substance is a substance that specifically binds to the third and fourth immunochemical components by an antigen-antibody reaction.
5). The stationary phase immobilizing the first immunochemical component that can specifically bind to the test object does not bind to the second immunochemical component and the test object that can specifically bind to the test object. Labeled substance (first labeled body) formed by binding a labeling substance (particularly colored particles, especially colored latex particles or gold colloidal particles) to the third immunochemical component, and the third immunochemical component And a fourth immunochemical component that can specifically bind to the intervening substance with a labeling substance (particularly colored particles, in particular, colored latex particles or gold colloidal particles). A labeled phase held in a form that can be detached by contact with water, a water-absorbing substrate provided in an arbitrary region on the surface, and binding of the third and fourth immunochemical components Including intervening mediators, the following (a) and (b Immunological test kit of may be used in immunoassay methods:
Method (a)
(1) From one end closer to the labeling phase than the stationary phase of the water-absorbing substrate, develop a mixture of the liquid containing the test sample and the liquid containing the intervening substance,
(2) The first immunochemical component immobilized on the stationary phase is an immunocomplex comprising the test object, the first labeled body, the intervening substance, and the second labeled body formed on the water-absorbing substrate. After binding and capturing,
(3) The test object is detected by measuring the signal of the labeling substance on the stationary phase.
Method (b)
(1) supplying a test sample to an arbitrary region between the stationary phase of the water-absorbing substrate and one end closer to the labeling phase than the stationary phase;
(2) From the one end of the water-absorbing substrate, expand the liquid containing the intervening substance,
(3) The first immunochemical component immobilized on the stationary phase is an immune complex composed of the test object, the first labeled body, the intervening substance and the second labeled body formed on the water-absorbing substrate. After binding and capturing,
(4) The test object is detected by measuring the signal of the labeling substance on the stationary phase.
6). Arbitrary region between the stationary phase and one end closer to the labeling phase than the stationary phase (where the test sample is between the one end of the absorbent substrate and the stationary phase) In the case of supplying to any region, the region including that region and the region on the stationary phase side), especially in any region between the one end and the labeled phase, 6. The kit according to 5 above, further comprising a separation phase capable of separating the substance.
7). 7. The kit according to 5 or 6 above, wherein the intervening substance is a substance that specifically binds to the third and fourth immunochemical components by an antigen-antibody reaction.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The test object that can be detected by the method of the present invention binds to the first immunochemical component and the second immunochemical component by an immunochemical reaction (ie, an antigen-antibody reaction) to form a sandwich immunocomplex. There is no particular limitation as long as it can be obtained. For example, microorganisms such as bacteria (especially pathogenic bacteria such as Escherichia coli O-157 and methicillin-resistant Staphylococcus aureus), actinomycetes, yeasts, fungi, viruses (especially HIV, HBV, HCV, etc.) or the like such as surface antigens Or antigenic peptides in biological samples such as tumor marker antigens.
[0008]
In the method of the present invention, both the first immunochemical component immobilized on the stationary phase and the second immunochemical component used as the first label are specifically detected by the antigen-antibody reaction with the test object. There is no particular limitation as long as it can bind. If the test object is an antigen (eg, protein, peptide, hapten, etc.), the first and second immunochemical components are antibodies that can specifically bind to the antigen. The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody in the present invention includes antibody fragments that retain specific affinity with the test object, for example, H chain, L chain, Fab, F (ab ′) 2 , V H , VL, and the like. . On the other hand, when the test object is an antibody, the first and second immunochemical components include an antigen that can specifically bind to the antibody or a secondary antibody that can specifically bind using the antibody as an antigen. It is. The first immunochemical component and the second immunochemical component may be appropriately selected from those known per se used in the sandwich method or the like according to the test object. If the immunochemical component is an antibody, it can also be prepared using a known antibody production technique using the isolated test subject as a sensitizing antigen. When both the first and second immunochemical components are antibodies, the first antibody and the second antibody may be the same or different. Further, as the different antibodies, two kinds of antibodies that recognize the same antigenic determinant can be used, or two kinds of antibodies that recognize different antigenic determinants can be used.
[0009]
In the present invention, the third immunochemical component used as the first label and the fourth immunochemical component used as the second label are antibodies (monoclonal antibodies) that can specifically bind to each other through an intervening substance. It may be a polyclonal antibody, or it may be a fragmented antibody such as H chain, L chain, Fab, F (ab ′) 2 , V H , V L ) or antigen (for example, , Protein, peptide, hapten, etc.) and has no specific affinity for the test object and the immobilized first immunochemical component. Preferably, these immunochemical components also have no specific affinity with the second immunochemical component.
[0010]
The mediator of the present invention that mediates the binding of the third and fourth immunochemical components binds simultaneously to the third and fourth immunochemical components (third immunochemical component-mediator- The substance is not particularly limited as long as it is a substance capable of forming a complex of (four immunochemical components), but preferably, an immunochemical compound that can specifically bind to the third and fourth immunochemical components by an antigen-antibody reaction. It is an ingredient. That is, if the third immunochemical component is an antibody, the mediator is an antigen recognized by the antibody or a secondary antibody that can specifically bind to the antibody, and the fourth immunochemical component is the antibody. An antibody that can specifically bind to an antigen or an antigen recognized by the secondary antibody is preferable. At this time, if the third and fourth immunochemical components are both antibodies, they may be the same antibody or different ones. On the other hand, if the third immunochemical component is an antigen, the intervening substance is an antibody capable of specifically binding to the antigen, and the fourth immunochemical component is an antigen recognized by the antibody or the antibody. It is a secondary antibody that can specifically bind. If both the third and fourth immunochemical components are antigens, they may be the same antigen or different antigens that are cross-reactive with the intervening antibody. As the third and fourth immunochemical components and the intervening substance, known substances used in the sandwich method or the like can be appropriately selected and used.
[0011]
The water-absorbing substrate used in the present invention is a test sample containing a test object, for example, a solution extracted from food, a culture supernatant thereof, serum, blood, urine, feces, saliva, etc. There is no particular limitation as long as it can absorb the diluted solution diluted with the diluent solvent, the first labeled body, the second labeled body, and the liquid containing the intervening substance. In the present invention, the test object in the test sample sufficiently reacts with the first label or the first immunochemical component immobilized on the stationary phase, and the first label is an intervening substance. A water-absorbing substrate that can secure a sufficient time for carrying out a sufficient reaction with the second labeled body via a slag is preferably used.
[0012]
When the water-absorbing substrate is inferior in water absorption, it takes a long time for the test sample to reach the stationary phase as described later, and as a result, rapid measurement cannot be performed. On the other hand, if the water absorption of the water-absorbing substrate is too high, the time required for the test object in the test sample to sufficiently react with the label and the first immunochemical component of the stationary phase. Since it is insufficient, it is difficult to perform accurate measurement.
[0013]
Therefore, the preferable degree of water absorption of the water-absorbing substrate in the present invention is that one end of the water-absorbing substrate cut into a strip having a width of 5 mm is immersed in water, and the water absorption distance after 1 minute is 0.5 to It is about 5 cm.
[0014]
Preferable specific examples of the water-absorbing substrate of the present invention include nonwoven fabric, filter paper, glass fiber cloth, glass filter, nitrocellulose filter, and porous material. These base materials have an appropriate water absorption rate and, when the labeling substance is colored particles, have an advantage such as excellent visual confirmation when the colored particles are combined and colored.
[0015]
Moreover, in order to adjust the water absorption of these base materials, the surface of the base material can be coated or impregnated with a hydrophilic polymer or a surfactant. Furthermore, in the present invention, a base material made of the same material may be used as the water-absorbing base material, or a continuous base material obtained by joining materials made of different materials by any bonding means may be used. it can.
[0016]
In the present invention, the shape of the water-absorbing substrate is not particularly limited as long as it allows the specimen to be developed. For example, a rectangular sheet shape (piece shape) or a rod shape is preferable.
[0017]
In the present invention, the stationary phase means a region where a first immunochemical component capable of specifically binding to a test object is immobilized on a water-absorbing substrate. The method for immobilizing the first immunochemical component on the water-absorbing substrate (method for preparing the immobilized phase) is not particularly limited, but is based on a conventionally known physical adsorption method or covalent bond method. In particular, the covalent bond method is preferred in that the immunochemical component is not easily detached from the substrate. When the water-absorbing substrate does not have a functional group for the covalent bonding method, for example, a substrate is prepared using a polymer having an appropriate functional group, so that the water-absorbing substrate does not impair the water-absorbing property. Can be attached. Alternatively, the stationary phase can be prepared by applying a solution containing the first immunochemical component and the hydrophilic polymer to the water-absorbing substrate and then immersing the solution in a coagulation solvent for coagulating the hydrophilic polymer. . As the hydrophilic polymer, hydroxypropylmethylcellulose, polyvinyl alcohol, hydroxyethylcellulose and the like are used. As the coagulation solvent, acetone, ethanol, methanol, ether or the like can be used.
[0018]
In the present invention, the stationary phase and a portion of one end of the water-absorbent substrate where absorption of the test sample-containing liquid, the second labeled body-containing liquid, the intervening substance-containing liquid, etc. is started (that is, the liquid-absorbing part). Although the distance between is not specifically limited, Preferably it is 1-6 cm, More preferably, it is about 3-4 cm. If the distance is too far, the test sample may not reach the stationary phase, the detection signal sensitivity may be too strong, or the measurement may take a long time. On the other hand, if the distance is too close, the labeling substance captured on the stationary phase may not be uniform and sparse, or the detection signal sensitivity may be too low.
[0019]
The liquid-absorbing part is not particularly limited as long as it does not hinder the movement of the liquid containing the test sample, the label, and the intervening substance to the water-absorbing base material. Further, a non-woven fabric or a woven fabric may be newly bonded to the water-absorbing substrate. In the present invention, a stationary phase on which a first immunochemical component capable of specifically binding to a test object is immobilized, and a water-absorbing substrate having a liquid-absorbing part are hereinafter referred to as an immunological test of the present invention. It may be a piece or simply a test piece.
[0020]
The first labeled body in the method of the present invention is an immunochemical component (second immunochemical component) that can specifically bind to the test object and a third immunochemical component that does not bind to the test object. A labeling substance is bound, and the second label is an immunochemical component (fourth immunochemical component) that can specifically bind to the third immunochemical component via an intervening substance. ) With a labeling substance. The labeling substance used here may be any labeling substance conventionally used in immunochemical measurement methods, such as colored particles, enzymes (alkaline phosphatase, peroxidase, etc.), fluorescent substances (FITC, rhodamine, etc.). In order to achieve efficient amplification of the detection signal, it is preferable that the labeling substances used for the first and second labeled bodies are the same. In the method of the present invention, colored particles are preferably used as a labeling substance in the sense of rapid detection. The colored particles are not particularly limited as long as they can be detected with the naked eye. For example, colloidal particles made of metals such as gold, silver, and copper, represented by Sudan Blue, Sudan Red IV, Sudan III, Oil Orange, Quinizarin Green, etc. Colored latex obtained by coloring latex with pigments or dyes can be used. From the viewpoint of visual confirmation, it is preferable to use colloidal gold or colored latex colored blue, red, green or orange, and it is easy to adjust the dispersion stability and detection sensitivity of the test object. In view of the above, it is more desirable to use a colored latex composed of water-dispersed polymer particles colored in blue, red or the like.
[0021]
The particle size of the colored particles is not particularly limited as long as it has good color development upon detection and has mobility in the base material that does not decrease the water absorption of the water-absorbent base material, but storage stability In view of ease of preparation and the like, a range of preferably 0.01 to 5 μm, more preferably 0.05 to 3 μm is exemplified. If the particle size is too small, the degree of coloring per particle is small, so that even if it is bonded to the stationary phase, the degree of color development is poor and the visual confirmation is poor. On the other hand, if the particle size is too large, a slight aggregation of the colored particles may cause clogging of the water-absorbent base material to reduce water absorption or cause non-specific color development.
[0022]
Methods for labeling both the second and third immunochemical components and the fourth immunochemical component with such colored particles include conventionally known methods such as covalent bonding, physical adsorption, An ion binding method or the like can be used, but the covalent bonding method is more preferably used from the viewpoint that the colored particles are not detached from the immunochemical component and are stable.
[0023]
In the method of the present invention, in order to detect a plurality of test objects in a test sample, a plurality of corresponding immunochemical components can be labeled with different colored particles. The particles may be the same color or different colors. When using colored particles of the same color, it is desirable that the stationary phase on which an immunochemical component capable of specifically binding to each test object is immobilized be separated so as to be distinguishable.
[0024]
When the labeling substance is an enzyme or a fluorescent substance, detection means conventionally used in EIA or fluorescent antibody method (FIA) are appropriately selected for detection of the stationary phase labeling substance.
[0025]
In the present invention, the labeled phase is held in such a form that the first labeled body and the second labeled body provided between the liquid-absorbing part of the water-absorbing substrate and the stationary phase can be detached by contact with water. Means an area. There are no particular restrictions on the method for producing the labeled phase, but for example, a liquid containing the labeled body is applied to an arbitrary region between the liquid-absorbing part of the water-absorbing substrate and the stationary phase, and dried under appropriate conditions. (For example, freeze-drying). Alternatively, the label may be dispersed in a water-soluble polymer or saccharose solution, and the dispersion may be applied on a water-absorbing substrate and dried in the same manner. In this method, the water-soluble polymer or saccharose is easily water-solubilized, and the first labeled body and the second labeled body are quickly detached from the substrate, and the first labeled body is inspected in the test sample and the intervening substance. It is possible to react with the second label through a substance, and to obtain an appropriate viscosity for holding the label in a predetermined region of the water-absorbing substrate by adjusting the concentration of the water-soluble polymer or saccharose. Further, it is advantageous in that the labeling body is prevented from aggregating and denaturing during drying, and the labeling body is less likely to be detached from the water-absorbing substrate after drying.
[0026]
As the water-soluble polymer, for example, polyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, cellulose ester (for example, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, oxyethyl cellulose, cyanethyl cellulose, etc.), gelatin and the like are preferably used.
[0027]
The intervening substance-containing liquid is prepared by dispersing (dissolving) the intervening substance in an appropriate dispersion medium (solvent). The dispersion medium for dispersing the intervening substance is not particularly limited as long as it does not inhibit the specific binding reaction between the test object and the first labeled body, the first labeled body and the intervening substance, and the intervening substance and the second labeled body. Preferably, a buffer solution having a pH and a salt concentration suitable for the antigen-antibody reaction, such as a phosphate buffer solution, an acetate buffer solution, a borate buffer solution, a Tris-HCl buffer solution, and the like can be appropriately selected and used.
[0028]
In one embodiment of the method of the present invention, the following reaction is performed in each step. That is, a mixture of a liquid containing a test sample and a liquid containing an intermediary that mediates the binding of the third and fourth immunochemical components is dropped from one end of the water-absorbing substrate (that is, the liquid absorption part). Then, each component moves on the water-absorbing substrate as the liquid moves. When the liquid reaches the labeled phase, the first and second labeled bodies held therein are detached from the labeled phase by contact with water. Further, the second immunochemical component in the first label is the test object in the test sample, and the third immunochemical component is the fourth immunity in the second label through the intervening substance. Each of them binds with a chemical component to form a complex, and further moves on the water-absorbing substrate. The formed immune complex binds to the first immunochemical component immobilized on the stationary phase and is captured on the stationary phase. In this way, the labeling substances constituting the first and second labeling bodies are assembled and bound on the stationary phase to amplify the detection signal, thereby detecting the presence of the test object with higher sensitivity. It becomes.
[0029]
As another aspect of the method of the present invention, in the above method, instead of dropping the test sample from the liquid absorption part, it contains an intervening substance after being dropped or applied between the liquid absorption part and the stationary phase. The method of dripping a liquid to a liquid absorption part and developing is mentioned. In this case, when the mixed solution reaches a region where the test sample is dropped or applied, the test object in the test sample moves on the water-absorbing substrate together with the intervening substance. When the liquid reaches the labeled phase, the first and second labeled bodies held therein are detached from the labeled phase by contact with water. Further, the second immunochemical component in the first label is the test object in the test sample, and the third immunochemical component is the fourth immunity in the second label through the intervening substance. Each of them binds with a chemical component to form a complex, and further moves on the water-absorbing substrate. The formed immune complex binds to the first immunochemical component immobilized on the stationary phase and is captured on the stationary phase.
[0030]
The immunological test kit of the present invention can be preferably used for the immunological test method of the present invention. The kit includes at least the following contents.
(a) a stationary phase in which a first immunochemical component that can specifically bind to a test object is immobilized, a second immunochemical component that can specifically bind to the test object, and the test object A label formed by binding a labeling substance to a third immunochemical component that does not bind (first labeled body), and a fourth that can specifically bind to the third immunochemical component via an intervening substance A water-absorbing property in which a labeled phase formed by binding a labeling substance to an immunochemical component (second labeled body) is held in a form that can be detached by contact with water in any region on the surface Base material
(b) Intervening substance that mediates the binding of the third and fourth immunochemical components The preferred embodiment of the water-absorbing substrate, the first and second labeled bodies, and the intervening substance is the method of the present invention as described above. Are preferably used.
[0031]
As one of the preferred embodiments of the water-absorbing substrate used in the kit of the present invention, an arbitrary region between one end (that is, the liquid-absorbing part) and the stationary phase (provided that the test sample is a sample of the water-absorbing substrate). When dropping or applying to any area between one end and the stationary phase, including the area, the area closer to the stationary phase), especially between the liquid absorption part and the labeled phase There may be mentioned those provided with a separate phase capable of separating from other substances in the sample.
[0032]
In the present invention, it is desirable that the separated phase has a pore size in the direction of separation larger than the test object, each labeled body, and the intervening substance, and smaller than other substances in the test sample to be separated and removed. . Further, the separation direction may be a direction in which the labeling body develops on the water-absorbing substrate, or a direction perpendicular to the direction. After separation from the substance, the separated phase may be removed for subsequent measurements.
[0033]
Examples of the material for the separated phase include non-woven fabric such as rayon and polyester, filter paper, glass fiber cloth, glass filter, nitrocellulose filter, polysulfone filter, and porous material.
[0034]
In addition to the above contents, the kit of the present invention may contain additional contents that can be preferably used in the immunological test method of the present invention. For example, the above-mentioned buffer solution preferably used for dispersing (dissolving) the intervening substance can be mentioned.
[0035]
【Example】
The present invention will be described in more detail with reference to the following examples, but these examples do not limit the scope of the present invention.
[0036]
Example 1 Preparation of immunological test kit composition (1) Preparation of colored latex comprising water-dispersed polymer particles 50 g of styrene monomer, 0.5 g of acrylic acid, 0.2 g of triethylene glycol dimethacrylate, and While stirring 440 g of distilled water at a temperature of 75 ° C. under a nitrogen stream, an aqueous solution in which 0.25 g of potassium persulfate is dissolved in 10 g of water is added thereto, and polymerized for 10 hours to obtain an aqueous dispersion having an average particle size of 0.22 μm. Type aqueous polymer particle dispersion in water was obtained. This polymer particle dispersion was centrifugally washed in the order of alkali, acid and distilled water, and then adjusted to a solid content concentration of 10% by weight (carrier particle dispersion). Sudan blue (0.2 g) was dissolved in toluene (20 ml), sodium dodecyl sulfate (0.2 g) and distilled water (100 ml) were added thereto, and the mixture was emulsified with an ultrasonic disperser. 30 ml of the above carrier particle dispersion (solid concentration: 10% by weight) was added to this liquid and stirred at room temperature for 24 hours. Toluene was removed from this solution with an evaporator, and then centrifugal washing was performed with 0.01 M borate buffer (pH 7.5) to adjust the solid concentration to 5% by weight. To 50 ml of this solution, 5 ml of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride aqueous solution (10 mg / ml) and 50 ml of 0.03 Mm-xylenediamine aqueous solution were added and reacted at room temperature for 5 hours. This solution was heat-treated at 75 ° C. for 5 hours, and then subjected to centrifugal washing with the same buffer solution as described above, so that the solid content concentration was adjusted to 1% by weight (Sudan blue dyed xylenediamine spacer particle dispersion).
[0037]
(2) Preparation of first labeled substance-containing liquid 1 ml of a glutaraldehyde aqueous solution (0.1 mg / ml) is added to 10 ml of the Sudan blue dyed xylenediamine spacer particle dispersion prepared in (1) above, and reacted at room temperature for 2 hours. After that, the mixture was centrifuged and washed with the same buffer as described above to prepare a dispersion having a solid concentration of 1% by weight. 10 ml of this dispersion was 1 ml of anti-human hemoglobin antibody (rabbit IgG, 10 mg / ml) as the third immunochemical component, and 1 ml of anti-human HBs antibody (rabbit IgG, 5 mg / ml) as the second immunochemical component. Each was added and stirred at 10 ° C. for 24 hours. This was centrifugally washed with the same buffer as described above, redispersed to a solid content concentration of 1% by weight, and a Sudan blue-stained particle-labeled anti-human hemoglobin antibody-anti-human HBs antibody (first label) to which an antibody was covalently bound. Body) -containing liquid was obtained.
[0038]
(3) Preparation of second labeled substance-containing liquid 1 ml of glutaraldehyde aqueous solution (0.1 mg / ml) is added to 10 ml of the Sudan blue dyed xylenediamine spacer particle dispersion prepared in (1) above, and reacted at room temperature for 2 hours. After that, the mixture was centrifuged and washed with the same buffer as described above to prepare a dispersion having a solid concentration of 1% by weight. To 10 ml of this dispersion, 2 ml of anti-human hemoglobin antibody (rabbit IgG, 10 mg / ml) was added as a fourth immunochemical component, and the mixture was stirred at 10 ° C. for 24 hours. This was centrifuged and washed with the same buffer as described above, redispersed to a solid content concentration of 1% by weight, and a Sudan blue-stained particle-labeled anti-human hemoglobin antibody (second labeled body) -containing solution in which the antibody was covalently bound. Obtained.
[0039]
(4) Preparation of immunological test strip Anti-human HBs antibody (rabbit IgG) as a first immunochemical component is diluted with 0.1 M phosphate buffer (pH 7.4) to a final concentration of 1 mg / ml. To an aqueous solution. To this aqueous solution, 10 μl was applied to a 50 mm site from one end of a nitrocellulose membrane filter (Toyo filter paper, 5 × 100 mm), and immediately left at 37 ° C. for 1 hour, and then the nitrocellulose membrane filter was taken out and 0.1% bovine was removed. It was immersed in an aqueous solution of serum albumin and 0.1% Tween 20 for 1 hour. Thereafter, the nitrocellulose membrane filter was taken out and allowed to stand at room temperature for 3 hours to obtain a nitrocellulose membrane filter having an anti-human HBs antibody. Next, a blood cell separation phase (Toyo Filter Paper No. 2, 5 × 10 mm) is provided in the vicinity of one end (liquid absorbing part) of the test piece of the above-mentioned anti-human HBs antibody-immobilized nitrocellulose membrane filter. A test piece comprising a nitrocellulose membrane filter having a phase was obtained.
The 1st labeled body containing liquid produced by (2) of Example 1 and the 2nd labeled body containing liquid produced by (3) of Example 1 were respectively added to 1 ml of 5 weight% polyvinylpyrrolidone (viscosity average molecular weight 25000) aqueous solution. After 0.1 ml was added and mixed well, 10 μl of this solution was applied to a position 40 to 50 mm from the stationary phase position of the test piece, and this was dried in a desiccator for 2 days to provide an immunology with a labeled phase. A test piece was obtained.
[0040]
Example 2 Detection of human HBs antigen using immunological test kit 10 μl of a test solution in which human HBs antigen is dissolved in a physiological saline solution is dropped into the blood cell separation phase of the test piece prepared in (4) of Example 1. did. Immediately thereafter, 100 μl of hemoglobin antigen solution (protein concentration 200 ng / ml) was dropped into the liquid absorption part and developed (FIG. 1A). The color development on the stationary phase after 10 minutes was visually observed (FIG. 1B). The results are shown in Table 1.
[0041]
[Table 1]
Comparative Example 10 μl of a test solution prepared by dissolving human HBs antigen in a physiological saline solution was added dropwise to the blood cell separation phase of the test piece prepared in (4) of Example 1. Immediately thereafter, 50 μl of the first labeled body-containing liquid (solid content concentration 0.2% by weight) prepared in (2) of Example 1 was dropped into the liquid absorption part and developed. Ten minutes later, 100 μl of hemoglobin antigen solution (protein concentration: 200 ng / ml) was dropped into the liquid absorption part and developed. After further 10 minutes, 50 μl of the second labeled body-containing liquid (solid content concentration 0.2% by weight) prepared in (3) of Example 1 was dropped into the liquid absorption part and developed. The color development on the stationary phase after 10 minutes was visually observed. The results are shown in Table 2.
[0043]
[Table 2]
【The invention's effect】
The method of the present invention (and use of the kit of the present invention) is a conventional method of immunochromatography including a step of amplifying a signal by binding a first label and a second label via an intervening substance. The detection signal can be amplified efficiently. In a preferred embodiment of the method of the present invention, the pretreatment step of the test sample that has been conventionally required in the examination of stool and blood can be omitted, so that the test object can be measured more quickly. Is possible.
[Brief description of the drawings]
FIG. 1 is a schematic diagram showing the state of an antigen-antibody reaction in each step of measurement of human HBs antigen using the immunological test kit of the present invention.
Claims (13)
(1) 検査対象物と特異的に結合し得る第一の免疫化学的成分を固定化した固定相と、検査対象物と特異的に結合し得る第二の免疫化学的成分および検査対象物とは結合しない第三の免疫化学的成分に標識物質が結合してなる標識体（第一標識体）並びに該第三の免疫化学的成分と介在物質を介して特異的に結合し得る第四の免疫化学的成分に標識物質が結合してなる標識体（第二標識体）が水との接触によって脱離し得る形態で保持される標識相とを、表面上の任意の領域に設けた吸水性基材の、固定相よりも標識相により近い方の一端から、被検試料を含有する液と該介在物質を含有する液との混合物を展開し、
(2) 該吸水性基材上で形成される検査対象物、第一標識体、介在物質および第二標識体からなる免疫複合体を固定相に固定化された第一の免疫化学的成分に結合させて捕捉した後、
(3) 該固定相上の標識物質のシグナルを測定することにより該検査対象物を検出する。An immunological test method comprising the following steps:
(1) a stationary phase in which a first immunochemical component that can specifically bind to a test object is immobilized; a second immunochemical component that can specifically bind to the test object; and a test object A label formed by binding a labeling substance to a third immunochemical component that does not bind (first labeled body), and a fourth that can specifically bind to the third immunochemical component via an intervening substance A water-absorbing property in which a labeled phase formed by binding a labeling substance to an immunochemical component (second labeled body) is held in a form that can be detached by contact with water in any region on the surface From one end of the substrate closer to the labeling phase than the stationary phase, develop a mixture of the liquid containing the test sample and the liquid containing the intervening substance,
(2) The first immunochemical component immobilized on the stationary phase is an immunocomplex comprising the test object, the first labeled body, the intervening substance, and the second labeled body formed on the water-absorbing substrate. After binding and capturing,
(3) The test object is detected by measuring the signal of the labeling substance on the stationary phase.
(1) 検査対象物と特異的に結合し得る第一の免疫化学的成分を固定化した固定相と、検査対象物と特異的に結合し得る第二の免疫化学的成分および検査対象物とは結合しない第三の免疫化学的成分に標識物質が結合してなる標識体（第一標識体）並びに該第三の免疫化学的成分と介在物質を介して特異的に結合し得る第四の免疫化学的成分に標識物質が結合してなる標識体（第二標識体）が水との接触によって脱離し得る形態で保持される標識相とを、表面上の任意の領域に設けた吸水性基材の、固定相と該固定相よりも標識相により近い方の一端との間の任意の領域に被検試料を供給し、
(2) 該吸水性基材の該一端から、該介在物質を含有する液を展開し、
(3) 該吸水性基材上で形成される検査対象物、第一標識体、介在物質および第二標識体からなる免疫複合体を固定相に固定化された第一の免疫化学的成分に結合させて捕捉した後、
(4) 該固定相上の標識物質のシグナルを測定することにより該検査対象物を検出する。An immunological test method comprising the following steps:
(1) a stationary phase in which a first immunochemical component that can specifically bind to a test object is immobilized; a second immunochemical component that can specifically bind to the test object; and a test object A label formed by binding a labeling substance to a third immunochemical component that does not bind (first labeled body), and a fourth that can specifically bind to the third immunochemical component via an intervening substance A water-absorbing property in which a labeled phase formed by binding a labeling substance to an immunochemical component (second labeled body) is held in a form that can be detached by contact with water in any region on the surface Supplying the test sample to an arbitrary region between the stationary phase and one end closer to the labeling phase than the stationary phase of the substrate;
(2) From the one end of the water-absorbing substrate, expand the liquid containing the intervening substance,
(3) The first immunochemical component immobilized on the stationary phase is an immune complex composed of the test object, the first labeled body, the intervening substance and the second labeled body formed on the water-absorbing substrate. After binding and capturing,
(4) The test object is detected by measuring the signal of the labeling substance on the stationary phase.
方法（ａ）
(1) 該吸水性基材の固定相よりも標識相により近い方の一端から、被検試料を含有する液と該介在物質を含有する液との混合物を展開し、
(2) 該吸水性基材上で形成される検査対象物、第一標識体、介在物質および第二標識体からなる免疫複合体を固定相に固定化された第一の免疫化学的成分に結合させて捕捉した後、
(3) 該固定相上の標識物質のシグナルを測定することにより該検査対象物を検出する。
方法（ｂ）
(1) 該吸水性基材の固定相と、該固定相よりも標識相により近い方の一端との間の任意の領域に被検試料を供給し、
(2) 該吸水性基材の該一端から、該介在物質を含有する液を展開し、
(3) 該吸水性基材上で形成される検査対象物、第一標識体、介在物質および第二標識体からなる免疫複合体を固定相に固定化された第一の免疫化学的成分に結合させて捕捉した後、
(4) 該固定相上の標識物質のシグナルを測定することにより該検査対象物を検出する。The stationary phase immobilizing the first immunochemical component that can specifically bind to the test object does not bind to the second immunochemical component and the test object that can specifically bind to the test object. A label formed by binding a labeling substance to a third immunochemical component (first label), and a fourth immunochemical that can specifically bind to the third immunochemical component via an intervening substance A water-absorbing substrate provided in any region on the surface with a labeling phase in which a labeling substance (second labeling substance) formed by binding a labeling substance to a component is held in a form that can be detached by contact with water; And an immunological test kit that includes an intermediary that mediates binding of the third and fourth immunochemical components and can be used in the following immunological test methods (a) and (b):
Method (a)
(1) From one end closer to the labeling phase than the stationary phase of the water-absorbing substrate, develop a mixture of the liquid containing the test sample and the liquid containing the intervening substance,
(2) The first immunochemical component immobilized on the stationary phase is an immunocomplex comprising the test object, the first labeled body, the intervening substance, and the second labeled body formed on the water-absorbing substrate. After binding and capturing,
(3) The test object is detected by measuring the signal of the labeling substance on the stationary phase.
Method (b)
(1) supplying a test sample to an arbitrary region between the stationary phase of the water-absorbing substrate and one end closer to the labeling phase than the stationary phase;
(2) From the one end of the water-absorbing substrate, expand the liquid containing the intervening substance,
(3) The first immunochemical component immobilized on the stationary phase is an immune complex composed of the test object, the first labeled body, the intervening substance and the second labeled body formed on the water-absorbing substrate. After binding and capturing,
(4) The test object is detected by measuring the signal of the labeling substance on the stationary phase.
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| JP19215198A JP3841559B2 (en) | 1998-07-07 | 1998-07-07 | Immunological examination method and immunological examination kit |
| EP99926868A EP1020726A4 (en) | 1998-07-01 | 1999-06-30 | Immunologic test method and immunologic test kit |
| PCT/JP1999/003539 WO2000002049A1 (en) | 1998-07-01 | 1999-06-30 | Immunologic test method and immunologic test kit |
| US09/486,640 US6753190B1 (en) | 1998-07-01 | 1999-06-30 | Immunologic test method and immunologic test kit |
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| JP19215198A JP3841559B2 (en) | 1998-07-07 | 1998-07-07 | Immunological examination method and immunological examination kit |
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| EP2957909B1 (en) | 2014-06-18 | 2019-01-30 | Ruprecht Keller | Method for identifying of a biological sample of a mammal and composition for use in this method |
| TWI691716B (en) | 2014-08-13 | 2020-04-21 | 美商艾巴希斯公司 | Signal amplification in plasmonic specific-binding partner assays |
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