JP2004020344A

JP2004020344A - Method for measuring substance fixed on minute particle solid phase

Info

Publication number: JP2004020344A
Application number: JP2002174825A
Authority: JP
Inventors: Eiji Ishikawa; 石川　榮治; Takuya Odawara; 小田原　卓哉; Hisashi Takenaka; 竹中　久師
Original assignee: Sysmex Corp
Current assignee: Sysmex Corp
Priority date: 2002-06-14
Filing date: 2002-06-14
Publication date: 2004-01-22
Anticipated expiration: 2022-06-14
Also published as: JP4359416B2

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for accurately measuring a substance to be examined being fixed to a minute particle solid phase with superior reproducibility and high sensitivity. <P>SOLUTION: The minute-particle-like solid phase where a specimen is fixed is mixed with a solution (a reagent solution for measurement) containing a substance for enabling the measurement of the specimen having superior accuracy, reproducibility, and/or sensitivity to measure the specimen after a substance that disturbs more uniform dispersion in the solid phase and/or measurement having the superior accuracy, reproducibility, and/or sensitivity is dispersed in a liquid (a dispersion liquid) that is not contained in concentration for disturbing the dispersion and/or measurement in advance. <P>COPYRIGHT: (C)2004,JPO

Description

【０００１】
【発明が属する技術分野】
本発明は、ヒトの臨床検査、獣医検査、食品衛生検査など多くの分野で利用される技術に関する。詳しくは被検物質が固定された微小粒子固相を被検物質測定用試薬溶液と混合接触させる方法、該測定のために使用しうる少なくとも１の固相および／または試薬を含むキット並びに該測定法のために使用しうる固相、試薬および自動化ソフトを含む測定システムに関する。
【０００２】
【従来の技術】
従来、ヒトの臨床検査をはじめとして獣医検査、食品検査など多くの分野で、抗原、抗体、ＤＮＡ、生理作用物質、病原関連物質、腫瘍マーカーなど種々の物質がそれらに対する特異結合物質との複合体を固相上に形成、固定またはトラップし、固相上の複合体を測定することにより測定されてきた。固相上の複合体を測定するためには、抗原抗体結合、ＤＮＡハイブリッド結合、ビオチン−アビジン結合、イオン結合など様々な結合を介して固相上に固定された酵素、蛍光物質、発光物質、ラジオアイソトープなどの測定用標識物質が広く使われている。複合体を形成、固定またはトラップするための固相としてマイクロプレート、ポリチレン球、ガラス球、ポリスチレンチューブの内面の他、ラテックス粒子、磁性ビーズなどの微小粒子固相が使われている。固相上に固定された測定用標識物質の測定は、測定用試薬溶液と固相とを接触させた後、標識物質の種類に対応したシグナルを測定することにより実施されている。例えば、測定用標識物質が酵素の場合には、酵素が固定された固相と酵素基質を含む測定用試薬溶液とを接触させた後、測定用試薬溶液の吸光度、発光強度、蛍光強度、磁気共鳴度などが測定される。また、測定用標識物質が蛍光物質、発光物質の場合にはそれらの蛍光物質、発光物質が固相とそれらの蛍光測定、発光測定に最も適した組成の測定用試薬溶液と接触させて、蛍光強度の測定が行われる。この際、測定用標識物質が固定された固相と測定用試薬溶液との接触が瞬時に均一となることが正確で再現性のある高感度測定に必要である。殊に固相が微小粒子固相の場合には、測定用試薬溶液との均一な混合が微小粒子固相の凝集により妨げられることがあり、測定の正確度、再現性および／または感度を低下させることが問題である。
【０００３】
【発明が解決しょうとする課題】
微小粒子状の固相上に固定された被検物質の正確度、再現性および／または感度のより高い測定を可能にすることを課題とする。
【０００４】
【課題を解決するための手段】
被検物質が固定された微小粒子固相を、該固相のより均一な分散および／または正確度、再現性および／または感度のより高い被検物質の測定を妨害する物質を、該分散および／または該測定を妨害する濃度では含まない液（分散液）中にあらかじめ分散させた後、正確度、再現性および／または感度のより高い被検物質の測定を可能にする物質を含む溶液（測定用試薬溶液）と混合して被検物質を測定することにより課題を解決した。
つまり、本発明は
１．被検物質が固定化された微小粒子固相を、該固相のより均一な分散および／または正確度、再現性および／または感度のより高い被検物質の測定を妨害する物質を、該分散および／または該測定を妨害する濃度では含まない液（分散液）中にあらかじめ分散させた後、正確度、再現性および／または感度のより高い被検物質の測定を可能にする物質を含む溶液（測定用試薬溶液）と混合して、被検物質を測定することを特徴とする測定法。
２．微小粒子固相が直径０．１〜１０μｍの磁性ビーズまたはこれと同等である前記１の測定法。
３．微小粒子固相が直径０．１〜１０μｍのラテックス粒子またはこれと同等である前記１の測定法。
４．被検物質が酵素、発光物質または蛍光物質であることを特徴とする前記１〜３のいずれか１に記載の測定法。
５．被検物質が加水分解酵素であることを特徴とする前記１〜３のいずれか１に記載の測定法。
６．被検物質がアルカリホスファターゼであることを特徴とする前記１〜３のいずれか１に記載の測定法。
７．被検物質がβ−Ｄ−ガラクトシダーゼであることを特徴とする前記１〜３のいずれか１に記載の測定法。
８．酵素基質がジオキセタン誘導体であることを特徴とする前記４〜７のいずれか１に記載の測定方法。
９．被検物質の測定のために酵素基質の他にエンハンサーを使用することを特徴とする前記４〜８のいずれか１に記載の測定法。
１０．エンハンサーが界面活性剤であることを特徴とする前記９に記載の測定法。
１１．分散液がエンハンサーを、被検物質が固定化された微小粒子固相のより均一な分散および／または正確度、再現性および／または感度の高い被検物質の測定を妨害する濃度では含まないことを特徴とする前記８〜１０のいずれか１に記載の測定法。
１２．分散液が酵素基質を含まないことおよび測定用試薬溶液が酵素基質とエンハンサーを含むことを特徴とする前記１１に記載の測定法。
１３．分散液が酵素基質を含むことおよび測定用試薬溶液がエンハンサーを含むことを特徴とする前記１１に記載の測定法。
１４．前記１〜１３のいずれか１に記載の測定法に使用しうる試薬および／または固相の少なくとも１を含むキット。
１５．前記１〜１３のいずれか１に記載の測定法に使用しうる試薬、固相および自動化ソフトを含む測定システム。
からなる。
【０００５】
【発明の実施の形態】
以下に本発明を詳しく説明する。
本発明は、微小粒子状固相上に固定された被検物質を測定用試薬溶液と接触させた後シグナルを計測して測定する方法に適用することができる。
本発明において使用する固相は、好ましくは直径０．１〜１０μｍの微小粒子状固相であって、それらの材質、形状、製造方法などにより限定されない。例えば、市販のラテックス粒子、好ましくは磁性ビーズなどであるが、これらに限定されない。
【０００６】
本発明における被検物質は例示により限定されないが、酵素、蛍光物質、発光物質など、免疫測定、ＤＮＡ測定などにおいて測定用標識として使用される物質が例示される。酵素としてはアルカリホスファターゼ、ペルオキシダーゼ、β−Ｄ−ガラクトシダーゼなどが、蛍光物質としてはフルオレセイン、ローダミンなどまたはそれらの誘導体が、発光物質としてはアクリジニウム、エクオリンなどまたはそれらの誘導体などがそれぞれ例示される。
【０００７】
被検物質が微小粒子状固相に固定化される方法、固定された状態に制限はないので、被検物質は種々の方法で種々の状態に固定される。例示により限定されないが、例えば被検物質は種々の物質、種々の結合を介して微小粒子状固相上に固定される。つまり、被検物質は、抗原、抗体、ホルモン、病原関連物質、腫瘍マーカー、アビジン、ＤＮＡ、ストレプトアビジン、ビオチンこれらのフラグメント、これらの誘導体、抗原−抗体結合、イオン結合、ＤＮＡハイブリッド結合、ビオチン−アビジン結合、ビオチン−ストレプトアビジン結合、ハプテン−抗ハプテン抗体結合などを介して微小粒子状固相の上に固定される。これらは、種々の物質の免疫測定、ＤＮＡの測定などにおいて使用される物質、結合である。
【０００８】
上記のように種々の方法で微小粒子状固相に固定化された被検物質を正確に、しかも再現性よく測定するためには、微小粒子状固相が被検物質の測定用試薬溶液中に凝集することなく均一に分散されていることが必要である。しかし、被検物質の測定用試薬溶液の中には、被検物質測定のために種々の物質が含まれている。例えば、ジオキセタン誘導体を基質として測定するβ−Ｄ−ガラクトシダーゼ、アルカリホスファターゼの測定用試薬溶液の中には、界面活性剤または界面活性剤様作用物質がエンハンサーとして含まれる。これにより、いったん集積された微小粒子状固相の測定用試薬溶液中への均一な分散が妨げられ、正確度、再現性および／または感度の高い被検物質の測定が妨害される。
【０００９】
本発明においては、微小粒子状固相の均一な分散を妨げ、正確度、再現性および／または感度の高い被検物質の測定を妨害する物質を、該分散を妨げ、該測定を妨害する濃度では、含まない液（分散液）中に被検物質が固定化された微小粒子状固相を均一に分散させた後、被検物質の測定用試薬溶液と混合することにより、測定用試薬溶液中に含まれる微小粒子状固相の均一な分散を妨げる物質の効果を減弱することにより、正確度、再現性および／または感度の高い被検物質の測定を実施する。
被検物質を固定した微小粒子状固相の該分散液には、該固相が例えば凝集することなく均一に分散され、正確度、再現性または／および感度の高い被検物質の測定に支障がない限り、特に制約はない。種々の物質または物質群、例えば被検物質を測定するための酵素基質、各種のタンパク質、脂質、糖質、各種血清などを含むまたは含まない既知のまたは今後開発される液、溶液または緩衝液が例示されるが、例示に限定されるものではない。ｐＨ、イオン強度なども制限はなく、分散液は単なる水でも良い。
ただし、被検物質を固定化した微小粒子固相の均一な分散を妨げ、正確度、再現性または／および感度の高い被検物質の測定を妨害するエンハンサーを、該分散または／および該測定を妨害する濃度では、分散液が含まないことが重要である。
該分散液の使用方法にも、該固相が例えば凝集することなく分散され被検物質が正確に、しかも再現性よくまた高感度に測定しうる限り、特に制約ない。該固相を撹拌、振とうなど、どのようにして該分散液中に分散させてもよい。該分散液の体積は測定用試薬液の体積より小さいことが好ましいが、大きくてもよい。該分散時の温度も通常２０〜４０℃であるが、特に限定されない。
本発明における測定用試薬溶液は、正確度、再現性および／または感度の高い被検物質の測定のために、上記の分散液に分散させた微小粒子固相とシグナルを計測する前に混合、接触させる溶液であり、したがって、該測定を可能にする物質を該測定を可能にする濃度で含有する。例示により限定されないが、測定用試薬溶液が、酵素基質およびエンハンサーを含む場合が例示される。しかし、分散液中にすでに酵素基質が添加されている場合には、測定用試薬溶液の中には酵素基質が必ずしも含まれる必要はない。つまり、酵素基質は、いずれの段階で１回または２回以上添加してもよい。エンハンサーの添加でも同様であり、酵素基質、エンハンサーの濃度に制限はなく、酵素基質または／およびエンハンサーを含む測定用試薬の数、添加回数にも制限はない。上記の分散液中に分散させた微小粒子固相とエンハンサーを含む測定用試薬溶液を混合し、さらに酵素基質を含む測定用試薬溶液と混合してもよいが、酵素反応をより早く始めるためには、酵素基質はエンハンサーと同時かまたはエンハンサーより前に添加することが好ましい。
被検物質を固定化した微小粒子固相を分散液中に分散させ、測定用試薬溶液と混合した後のシグナルの測定は、公知の方法または今後開発される方法により実施することができる。該微小粒子固相の濃度が低く、シグナルの測定に支障がない場合には、そのままシグナルを測定することができるが、該微小粒子固相の濃度が高く、消光などによりシグナルの測定が困難な場合は、該微小粒子固相を集積した後に、シグナルを測定することもできる（特願２００１−１８９４１９）。
【００１０】
本発明は、上記に説明した測定方法の実施のための固相、試薬等におよび、また、該固相および緩衝液、ブロッキング液、洗浄液、基質液、抗体、ハプテン等に例示される本発明に使用する少なくとも１の固相および／または試薬を含む測定キットにもおよぶ。さらに、本発明は、上記に説明した測定方法の実施のための固相、試薬および自動化ソフトを含む測定システムにもおよぶ。
以上は例示により説明したが、本発明はこれらの例示により限定されるものではない。
【００１１】
【実施例】
以下実施例により本発明を具体的に説明するが、本発明は以下の実施例により限定されるものではない。
【００１２】
【実施例１】
この実施例では、磁性ビーズに固定されたアルカリホスファターゼ（ＡＬＰ）の発光測定に対する、該磁性ビーズを予めＡＬＰ活性測定用試薬溶液以外の緩衝液により分散することの効果を示す。
【００１３】
（材料と方法）
・磁性ビーズ
ＭＧ２１０（直径１．７μｍ、比重１．３）
ＪＳＲ　Ｃｏｒｐｏｒａｔｉｏｎ、Ｔｏｋｙｏ、Ｊａｐａｎ
・アルカリホスファターゼ（以下ＡＬＰと記載）
Ｏｒｉｅｎｔａｌ　Ｙｅａｔ　Ｃｏ．，Ｌｔｄ．，Ｔｏｋｙｏ，Ｊａｐａｎ
・モノクローナル抗体
抗ヒトＢ型肝炎ウイルス表面抗原（以下ＨＢｓＡｇと記載）ＩｇＧ−６４９および８５ならびに抗２，４−ジニトロフェニル基（以下ＤＮＰと記載）ＩｇＧ−１７５３
Ｉｎｔｅｒｎａｔｉｏｎａｌ　Ｒｅａｇｅｎｔｓ　Ｃｏｒｐｏｒａｔｉｏｎ、Ｋｏｂｅ、Ｊａｐａｎ
・ＤＮＰビオチン−抗ＨＢｓＡｇ　Ｆａｂ’とＡＬＰ−抗ＨＢｓＡｇ　Ｆａｂ’
ＤＮＰ化ビオチン化ウシ血清アルブミン−抗ＨＢｓＡｇ　Ｆａｂ’−６４９（ＤＮＰ−ビオチン−抗ＨＢｓＡｇ　Ｆａｂ’）とＡＬＰ−抗ＨＢｓＡｇ　Ｆａｂ’−８５（ＡＬＰ−抗ＨＢｓＡｇ　Ｆａｂ’）をマレイミド基とチオール基の反応を使う公知の方法（Ｅ．　Ｉｓｈｉｋａｗａ，　Ｕｌｔｒａｓｅｎｓｉｔｉｖｅ　ａｎｄ　Ｒａｐｉｄ　Ｅｎｚｙｍｅ　Ｉｍｍｕｎｏａｓｓａｙ，　Ｌａｂｏｒａｔｏｒｙ　Ｔｅｃｈｎｉｑｕｅｓ　ｉｎ　Ｂｉｏｃｈｅｍｉｓｔｒｙ　ａｎｄ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ　Ｖｏｌ．２７，　Ｓ．　Ｐｉｌｌａｉ，　Ｐ．Ｃ．　ｖａｎ　ｄｅｒ　Ｖｌｉｅｔ　ｅｄｓ．，Ｅｌｓｅｖｉｅｒ，　Ａｍｓｔｅｒｄａｍ，　ｐｐ．１４１−１７６，　１９９９）により調製した。
・ブロッキング液
０．１５Ｍ　ＮａＣｌ、２．５ｍＭ　ＥＤＴＡ、２．５ｇ／Ｌウシ血清アルブミン、１０ｇ／Ｌシュークロース、１ｇ／Ｌ　ＮａＮ_３を含む１０ｍＭ　リン酸ナトリウム緩衝液、ｐＨ７．０
・抗ＤＮＰＩｇＧ不溶化磁性ビーズ
磁性ビーズにモノクローナル抗ＤＮＰＩｇＧ−１７５３をＪＳＲ　Ｃｏｒｐｏｒａｔｉｏｎの指示書に従って不溶化し、ブロッキング液で洗浄した後、４℃で同液中に保存した。
・ＤＮＰ−ビオチン−抗ＨＢｓＡｇ　Ｆａｂ’、ＡＬＰ−抗ＨＢｓＡｇ　Ｆａｂ’、ＨＢｓＡｇ、抗ＤＮＰＩｇＧ不溶化磁性ビーズおよびストレプトアビジン不溶化磁性ビーズの希釈液
ＴＥＡ緩衝液
・ＴＥＡ緩衝液
１ｍＭ　ＭｇＣｌ_２、０．１ｍＭ　ＺｎＣｌ_２、１ｇ／Ｌ　ウシ血清アルブミン、０．５ｇ／Ｌ　ＮａＮ_３を含む０．１Ｍトリエタノラミン・ＨＣｌ緩衝液、ｐＨ７．６
・ＡＬＰ用洗浄液
０．１５Ｍ　ＮａＣｌ、０．１％　Ｔｗｅｅｎ２０、１ｇ／Ｌ　ＮａＮ_３を含む２０ｍＭ　Ｔｒｉｓ−ＨＣｌ緩衝液、ｐＨ７．４
・インキュベーション温度
非競合的転移固相測定法の全工程におけるインキュベーション温度は３７℃とした。
・ＤＮＰ−ビオチン−抗ＨＢｓＡｇ　Ｆａｂ’の抗ＤＮＰＩｇＧ不溶化磁性ビーズへの結合
１００ｐｍｏｌ／ｍＬのＤＮＰ−ビオチン−抗ＨＢｓＡｇ　Ｆａｂ’、２５μＬとＴＥＡ緩衝液で洗浄した抗ＤＮＰＩｇＧ不溶化磁性ビーズ、０．５ｍｇを１０分間インキュベートした後、磁性ビーズを１０秒間の磁気分離により分離し、上清を吸引除去した後、ＡＬＰ用洗浄液２００μＬで１０秒間の磁気分離により１回洗浄し、ＴＥＡ緩衝液８０μＬで懸濁した。
・ＨＢｓＡｇのＤＮＰ−ビオチン−抗ＨＢｓＡｇ　Ｆａｂ’結合抗ＤＮＰＩｇＧ不溶化磁性ビーズへの結合
２．５ＩＵ／ｍＬのＨＢｓＡｇ２０μＬと上記の洗浄したＤＮＰ−ビオチン−抗ＨＢｓＡｇ　Ｆａｂ’結合抗ＤＮＰＩｇＧ不溶化磁性ビーズを２分間インキュベートした後、該磁性ビーズをＡＬＰ用洗浄液２００μＬで１０秒間の磁気分離により１回洗浄した。
・ＡＬＰ−抗ＨＢｓＡｇ　Ｆａｂ’のＨＢｓＡｇ結合ＤＮＰ−ビオチン−抗ＨＢｓＡｇ　Ｆａｂ’結合抗ＤＮＰＩｇＧ不溶化磁性ビーズへの結合
４．８ｐｍｏｌ／ｍＬのＡＬＰ−抗ＨＢｓＡｇ　Ｆａｂ’、１００μＬと上記の洗浄したＨＢｓＡｇ結合ＤＮＰ−ビオチン−抗ＨＢｓＡｇ　Ｆａｂ’結合抗ＤＮＰＩｇＧ不溶化磁性ビーズを１分間インキュベートした後、ＡＬＰ用洗浄液２００μＬで１０秒間の磁気分離により１回洗浄した。
・リン緩衝液
０．５ｇ／Ｌ　ＮａＮ_３を含む０．１Ｍ　リン酸ナトリウム緩衝液、ｐＨ７．５
・ストレプトアビジン
ＴｙｐｅＩＩ、Ｗａｋｏ　Ｐｕｒｅ　Ｃｈｅｍｉｃａｌ　Ｉｎｄｕｓｔｒｉｅｓ，Ｌｔｄ．，　Ｏｓａｋａ，Ｊａｐａｎ
・ＤＮＰ−Ｌｙｓ液
３ｍＭ　εＮ−２．４−ジトロフェニル−Ｌ−リジンを含むＴＥＡ緩衝液
・ストレプトアビジン不溶化磁性ビーズ
ビオチン化ウシ血清アルブミンを、公知の方法（Ｅ．　Ｉｓｈｉｋａｗａ，　Ｕｌｔｒａｓｅｎｓｉｔｉｖｅａｎｄ　Ｒａｐｉｄ　Ｅｎｚｙｍｅ　Ｉｍｍｕｎｏａｓｓａｙ，　Ｌａｂｏｒａｔｏｒｙ　Ｔｅｃｈｎｉｑｕｅｓ　ｉｎ　Ｂｉｏｃｈｅｍｉｓｔｒｙ　ａｎｄＭｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ　Ｖｏｌ．２７，　Ｓ．　Ｐｉｌｌａｉ，　Ｐ．Ｃ．　ｖａｎ　ｄｅｒ　Ｖｌｉｅｔ　ｅｄｓ．，Ｅｌｓｅｖｉｅｒ，　Ａｍｓｔｅｒｄａｍ，　ｐｐ．１４１−１７６，　１９９９）により調製し、これをＪＳＲ　Ｃｏｒｐｏｒａｔｉｏｎの指示書にしたがって磁性ビーズに不溶化した後、３０μｇ／ｍＬのストレプトアビジンをリン酸緩衝液に溶解して反応させ、リン酸緩衝液で１回洗浄、ブロッキング液中に４℃で保存した。
・免疫複合体の抗ＤＮＰＩｇＧ不溶化磁性ビーズからの溶出
上記のＤＮＰ−ビオチン−抗ＨＢｓＡｇ　Ｆａｂ’、ＨＢｓＡｇ、ＡＬＰ−抗ＨＢｓＡｇ　Ｆａｂ’の３者からなる免疫複合体を結合させた抗ＤＮＰＩｇＧ不溶化磁性ビーズとＤＮＰ−Ｌｙｓ液１００μＬを０．５分間インキュベートした後、該磁性ビーズを磁石により１５秒間集積した上清を溶出液とした。
・免疫複合体のストレプトアビジン不溶化磁性ビーズヘの結合
上記溶出液とストレプトアビジン不溶化磁性ビーズ０．５ｍｇを１分間インキュベートした後、該磁性ビーズを磁石により１０秒間集積し、上清を除去した後、ＡＬＰ用洗浄液２００μＬ中に該磁注ビーズを分散させ、再び１０秒間磁石により集積して直ちに上清を除去することにより１回洗浄した。
・ＡＬＰ発光基質液
ＣＤＰ−ｓｔａｒ　ｒｅａｄｙ　ｔｏ　ｕｓｅ　ｗｉｔｈ　Ｓａｐｐｈｉｒｅ　ＩＩ、Ｔｒｏｐｉｘ，Ｉｎｃ．，Ｂｅｄｆｏｒｄ，ＭＡ
・ＡＬＰ分散液
１Ｍ　ジエタノラミン・ＨＣｌ緩衝液、ｐＨ１０．０
・発光測定装置
Ｌｕｍｉｃｏｕｎｔｅｒ２５００、Ｍｉｃｒｏｔｅｃ　Ｃｏ．Ｌｔｄ．、Ｃｈｉｂａ、Ｊａｐａｎ
・ＡＬＰ結合磁性ビーズのＡＬＰ活性の測定
測定１：上記の洗浄したＡＬＰ結合磁性ビーズを、３０μＬの分散液中に分散し、基質液１００μＬと混合し、３７℃、２．７５分間インキュベートした後、該磁性ビーズを磁石で集積した上清の０．１秒間の発光量を同時に５例測定した。
測定２：上記の洗浄したＡＬＰ結合磁性ビーズを、分散液３０μＬと基質液１００μＬの混合液１３０μＬ中に分散し、３７℃、２．７５分間インキュベートした後、該磁性ビーズを磁石で集積した上清の０．１秒間の発光量を同時に５例測定した。
【００１４】
（結果）
実施例１の結果を表１に示す。
ＡＬＰ結合磁性ビーズを基質とエンハンサーを含まない分散液中に予め分散させた後、基質とエンハンサーを含む基質液と混合してＡＬＰ活性を測定したときの発光強度は９２，８９２、再現性はＣＶ２．８％（５例）であった。基質液と分散液の混合液中に直接分散させてＡＬＰ活性を測定したときの発光強度は６３，２２８、再現性ＣＶは１９．４％と非常に高かった。つまり、ＡＬＰ結合磁性ビーズを、エンハンサーを含まない分散液で予め分散した後、基質とエンハンサーを含む測定用試薬溶液と混合して発光強度を測定することにより、シグナルとしての発光強度、したがって感度も再現性もともに、分散液を使用しないで該磁性ビーズを酵素基質とエンハンサーを含む測定用試薬溶液と直接混合した場合に比べて格段に改善された。
【００１５】
【実施例２】
この実施例では、磁性ビーズに固定されたアルカリホスファターゼの発光測定における、エンハンサーより前の酵素基質の添加のシグナルに対する効果を示す。
【００１６】
（材料と方法）
以下に示す材料と方法の他は、実施例１のそれらと同じである。
・基質液
基質としてのＣＤＰ−ｓｔａｒのみを含む溶液、トロピックス社。
・エンハンサー液
エンハンサーとしてのＳａｐｐｈｉｒｅＩＩのみを含む溶液、トロピックス社。
・ＡＬＰ結合磁性ビーズのＡＬＰ活性の測定
測定１：洗浄したＡＬＰ結合磁性ビーズを、分散液３０μＬと基質液９０μＬの混合液中に分散し、その１５秒後に実施例１で添加したＡＬＰ発光基質液（基質ＣＤＰ−ｓｔａｒとエンハンサーＳａｐｐｈｉｒｅＩＩを含む）の代わりにエンハンサー液１０μＬを添加し、実施例１と同様にインキュベートし、発光強度を測定した。
測定２：エンハンサー液１０μＬを測定１における添加より２分１５秒遅れで添加した以外は、測定１と同様にして測定した。
【００１７】
（結果）
実施例２の結果を表２に示す。
エンハンサーを基質の添加より遅れて添加し、エンハンサーの添加から発光測定までの時間を短縮することにより、発光強度は、１２１，５００から７９，０９０に低下した。この結果は、エンハンサーを可及的早期に添加することにより高いシグナル、つまり高い感度が得られることを示すと同時に、エンハンサー非存在下でも酵素反応が進むことを示しているので、エンハンサーを添加することができない早い段階で、例えば分散液添加の段階で基質を添加することにより、それだけ高いシグナルを得ることができることをも示している。
【００１８】
【表１】

【００１９】
【表２】

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a technique used in many fields such as a human clinical test, a veterinary test, and a food hygiene test. Specifically, a method of mixing and contacting a solid phase of microparticles on which a test substance is immobilized with a reagent solution for measuring a test substance, a kit containing at least one solid phase and / or a reagent that can be used for the measurement, and the measurement The present invention relates to a measurement system including a solid phase, reagents and automation software that can be used for the method.
[0002]
[Prior art]
Conventionally, in many fields such as human clinical tests, veterinary tests, and food tests, various substances such as antigens, antibodies, DNA, physiologically active substances, pathogenic substances, and tumor markers are complexed with specific binding substances to them. Has been measured by forming, fixing or trapping on a solid phase and measuring the complex on the solid phase. To measure the complex on the solid phase, antigen-antibody binding, DNA hybrid binding, biotin-avidin binding, enzymes immobilized on the solid phase through various bonds such as ionic bonds, fluorescent substances, luminescent substances, Measurement labeling substances such as radioisotopes are widely used. As a solid phase for forming, fixing or trapping a complex, a microparticle solid phase such as a latex particle or a magnetic bead is used in addition to the inner surface of a microplate, a polystyrene sphere, a glass sphere, or a polystyrene tube. The measurement of the labeling substance for measurement immobilized on the solid phase is performed by bringing the reagent solution for measurement into contact with the solid phase, and then measuring the signal corresponding to the type of the labeling substance. For example, when the labeling substance for measurement is an enzyme, after contacting the solid phase on which the enzyme is immobilized with the reagent solution for measurement containing the enzyme substrate, the absorbance, luminescence intensity, fluorescence intensity, magnetic intensity, The degree of resonance is measured. When the labeling substance for measurement is a fluorescent substance or a luminescent substance, the fluorescent substance or the luminescent substance is brought into contact with a solid phase and a measuring reagent solution having a composition most suitable for the fluorescence measurement and the luminescence measurement to obtain a fluorescent substance. An intensity measurement is taken. In this case, it is necessary for accurate and reproducible high-sensitivity measurement that the contact between the solid phase on which the measurement label is fixed and the measurement reagent solution is instantaneously uniform. In particular, when the solid phase is a fine particle solid phase, uniform mixing with the reagent solution for measurement may be hindered by aggregation of the fine particle solid phase, thereby reducing measurement accuracy, reproducibility, and / or sensitivity. Is a problem.
[0003]
[Problems to be solved by the invention]
An object of the present invention is to enable measurement of a test substance immobilized on a microparticle solid phase with higher accuracy, reproducibility, and / or sensitivity.
[0004]
[Means for Solving the Problems]
The microparticle solid phase to which the test substance is immobilized is dispersed in the solid phase and / or the substance that interferes with the measurement of the test substance with higher accuracy, reproducibility and / or sensitivity is dispersed and dispersed. And / or a solution containing a substance that enables measurement of a test substance with higher accuracy, reproducibility, and / or sensitivity after being previously dispersed in a liquid (dispersion liquid) that does not interfere with the measurement (dispersion liquid) The problem was solved by measuring the test substance by mixing with a test reagent solution).
That is, the present invention provides: The microparticle solid phase on which the test substance is immobilized is dispersed in the solid phase more uniformly and / or the substance that interferes with the measurement of the test substance with higher accuracy, reproducibility and / or sensitivity is dispersed. And / or a solution containing a substance capable of measuring a test substance with higher accuracy, reproducibility, and / or sensitivity after being previously dispersed in a liquid (dispersion liquid) not containing the substance at a concentration that interferes with the measurement. (Measurement reagent solution), and measuring a test substance.
2. 2. The measuring method according to the above 1, wherein the microparticle solid phase is a magnetic bead having a diameter of 0.1 to 10 μm or equivalent thereto.
3. The method according to the above 1, wherein the fine particle solid phase is latex particles having a diameter of 0.1 to 10 μm or equivalent thereto.
4. 4. The measuring method according to any one of the above items 1 to 3, wherein the test substance is an enzyme, a luminescent substance or a fluorescent substance.
5. 4. The method according to any one of the above items 1 to 3, wherein the test substance is a hydrolase.
6. 4. The method according to any one of the above items 1 to 3, wherein the test substance is alkaline phosphatase.
7. 4. The method according to any one of the above items 1 to 3, wherein the test substance is β-D-galactosidase.
8. The method according to any one of the above items 4 to 7, wherein the enzyme substrate is a dioxetane derivative.
9. 9. The measurement method according to any one of the above items 4 to 8, wherein an enhancer is used in addition to the enzyme substrate for the measurement of the test substance.
10. 10. The measuring method according to the above item 9, wherein the enhancer is a surfactant.
11. The dispersion does not contain the enhancer at a concentration that interferes with the more uniform dispersion of the analyte-immobilized microparticle solid phase and / or the more accurate, reproducible and / or sensitive determination of the analyte. The measurement method according to any one of the above items 8 to 10, wherein
12. 12. The measurement method according to the above 11, wherein the dispersion does not contain an enzyme substrate, and the reagent solution for measurement contains an enzyme substrate and an enhancer.
13. 12. The measuring method according to the above item 11, wherein the dispersion contains an enzyme substrate and the measuring reagent solution contains an enhancer.
14． A kit comprising at least one of a reagent and / or a solid phase that can be used in the measurement method according to any one of the above items 1 to 13.
15. 14. A measurement system including a reagent, a solid phase, and automation software that can be used in the measurement method according to any one of 1 to 13 above.
Consists of
[0005]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
INDUSTRIAL APPLICABILITY The present invention can be applied to a method in which a test substance immobilized on a fine particulate solid phase is brought into contact with a reagent solution for measurement, and then the signal is measured and measured.
The solid phase used in the present invention is preferably a fine particulate solid phase having a diameter of 0.1 to 10 μm, and is not limited by the material, shape, manufacturing method, and the like. Examples include, but are not limited to, commercially available latex particles, preferably magnetic beads.
[0006]
The test substance in the present invention is not limited by the examples, but examples include substances used as measurement labels in immunoassays, DNA measurements, and the like, such as enzymes, fluorescent substances, and luminescent substances. Examples of the enzyme include alkaline phosphatase, peroxidase, β-D-galactosidase and the like, examples of the fluorescent substance include fluorescein and rhodamine and derivatives thereof, and examples of the luminescent substance include acridinium and aequorin and derivatives thereof.
[0007]
There is no limitation on the method of immobilizing the test substance on the microparticulate solid phase and the immobilized state, so the test substance is immobilized in various states by various methods. For example, without limitation, the test substance is immobilized on the microparticulate solid phase through various substances and various bonds. That is, the test substance is an antigen, an antibody, a hormone, a pathogen-related substance, a tumor marker, avidin, DNA, streptavidin, biotin, a fragment thereof, a derivative thereof, an antigen-antibody bond, an ion bond, a DNA hybrid bond, a biotin- It is immobilized on the microparticulate solid phase via avidin bond, biotin-streptavidin bond, hapten-anti-hapten antibody bond and the like. These are substances and bonds used in immunoassay of various substances, measurement of DNA, and the like.
[0008]
In order to accurately and reproducibly measure a test substance immobilized on a fine particulate solid phase by various methods as described above, the fine particulate solid phase must be contained in a reagent solution for measuring the test substance. It is necessary that the particles are uniformly dispersed without agglomeration. However, various substances are contained in the reagent solution for measuring a test substance for measuring the test substance. For example, in a reagent solution for measurement of β-D-galactosidase or alkaline phosphatase using a dioxetane derivative as a substrate, a surfactant or a surfactant-like active substance is included as an enhancer. This prevents the once dispersed microparticulate solid phase from being evenly dispersed in the measurement reagent solution, and prevents measurement of the analyte with high accuracy, reproducibility and / or sensitivity.
[0009]
In the present invention, a substance that prevents uniform dispersion of the fine particulate solid phase and hinders measurement of a test substance with high accuracy, reproducibility and / or sensitivity, and a concentration that hinders the dispersion and hinders the measurement. Then, after uniformly dispersing the fine particulate solid phase on which the test substance is immobilized in a liquid (dispersion liquid) not containing, and mixing the same with the test reagent solution for the test substance, By attenuating the effect of the substance that hinders the uniform dispersion of the fine particulate solid phase contained therein, measurement of the test substance with high accuracy, reproducibility, and / or sensitivity is performed.
In the dispersion liquid of the microparticulate solid phase to which the test substance is fixed, the solid phase is uniformly dispersed without, for example, aggregation, which hinders the measurement of the test substance with high accuracy, reproducibility, and / or sensitivity. As long as there is no restriction. Known or later developed liquids, solutions or buffers containing or not containing various substances or substance groups, for example, enzyme substrates for measuring test substances, various proteins, lipids, carbohydrates, various sera, etc. Although illustrated, the invention is not limited to the example. The pH, ionic strength and the like are not limited, and the dispersion may be simply water.
However, an enhancer which prevents uniform dispersion of the solid phase of the microparticles on which the test substance is immobilized and prevents measurement of the test substance with high accuracy, reproducibility, and / or sensitivity, may be used for the dispersion or / and the measurement. At interfering concentrations, it is important that the dispersion be free.
The method of using the dispersion is not particularly limited as long as the solid phase is dispersed without aggregation, for example, and the test substance can be measured accurately, with good reproducibility, and with high sensitivity. The solid phase may be dispersed in the dispersion by stirring, shaking, or the like. The volume of the dispersion is preferably smaller than the volume of the reagent solution for measurement, but may be larger. The temperature at the time of the dispersion is also usually 20 to 40 ° C., but is not particularly limited.
The reagent solution for measurement in the present invention is mixed with a microparticle solid phase dispersed in the above-mentioned dispersion liquid before measuring a signal, in order to measure a test substance with high accuracy, reproducibility and / or sensitivity. The solution to be contacted, and thus contains the substance enabling the measurement at a concentration allowing the measurement. Although not limited by way of example, a case where the measurement reagent solution contains an enzyme substrate and an enhancer is exemplified. However, when the enzyme substrate has already been added to the dispersion, the enzyme substrate need not necessarily be contained in the reagent solution for measurement. That is, the enzyme substrate may be added once or twice or more at any stage. The same applies to the addition of an enhancer, and there is no limitation on the concentration of the enzyme substrate or enhancer, and there is no limitation on the number of enzyme substrates or / and reagents containing the enhancer and the number of additions. The measurement reagent solution containing the microparticle solid phase and the enhancer dispersed in the above dispersion liquid may be mixed, and further mixed with the measurement reagent solution containing the enzyme substrate. Preferably, the enzyme substrate is added at the same time as or before the enhancer.
The measurement of the signal after dispersing the solid phase of the microparticles on which the test substance is immobilized in the dispersion and mixing with the reagent solution for measurement can be performed by a known method or a method developed in the future. When the concentration of the microparticle solid phase is low and there is no problem in measuring the signal, the signal can be measured as it is. However, the concentration of the microparticle solid phase is high and the signal measurement is difficult due to quenching and the like. In such a case, the signal can be measured after accumulating the microparticle solid phase (Japanese Patent Application No. 2001-189419).
[0010]
The present invention extends to solid phases, reagents, and the like for carrying out the above-described measurement method, and also to the present invention exemplified by the solid phase and buffers, blocking solutions, washing solutions, substrate solutions, antibodies, haptens, and the like. The present invention also extends to a measurement kit containing at least one solid phase and / or reagent used for the above. Further, the present invention extends to a measurement system including a solid phase, reagents and automation software for performing the above-described measurement method.
Although the above has been described by way of examples, the present invention is not limited to these examples.
[0011]
【Example】
Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples.
[0012]
Embodiment 1
This example shows the effect of dispersing the magnetic beads in advance with a buffer solution other than the reagent solution for ALP activity measurement on the luminescence measurement of alkaline phosphatase (ALP) immobilized on the magnetic beads.
[0013]
(Materials and methods)
-Magnetic beads MG210 (diameter 1.7 µm, specific gravity 1.3)
JSR Corporation, Tokyo, Japan
・ Alkaline phosphatase (hereinafter referred to as ALP)
Oriental Yeat Co. , Ltd. , Tokyo, Japan
-Monoclonal antibody anti-human hepatitis B virus surface antigen (hereinafter referred to as HBsAg) IgG-649 and 85 and anti-2,4-dinitrophenyl group (hereinafter referred to as DNP) IgG-1753
International Reagents Corporation, Kobe, Japan
DNP biotin-anti-HBsAg Fab 'and ALP-anti-HBsAg Fab'
The reaction of DNP-biotinylated bovine serum albumin-anti-HBsAg Fab'-649 (DNP-biotin-anti-HBsAg Fab ') and ALP-anti-HBsAg Fab'-85 (ALP-anti-HBsAg Fab') was performed by reacting a maleimide group with a thiol group. known method using (E. Ishikawa, Ultrasensitive and Rapid Enzyme Immunoassay, Laboratory Techniques in Biochemistry and Molecular Biology Vol.27, S. Pillai, P.C. van der Vliet eds., Elsevier, Amsterdam, pp.141-176, 1999).
Blocking solution 0.15M NaCl, 2.5mM EDTA, 2.5g / L bovine serum albumin, 10 g / L sucrose, 10 mM sodium phosphate buffer containing 1g / L NaN _3, pH7.0
-Anti-DNP IgG Insolubilized Magnetic Beads Monoclonal anti-DNP IgG-1753 was insolubilized in magnetic beads according to the instructions of the JSR Corporation, washed with a blocking solution, and stored at 4 ° C in the same solution.
· DNP-biotin - anti HBsAg Fab ', ALP-anti-HBsAg Fab', HBsAg, anti DNPIgG insolubilized magnetic beads and streptavidin insolubilized diluent TEA buffer · TEA buffer 1 mM MgCl ₂ magnetic beads, 0.1 mM ZnCl _2, 0.1 M triethanolamine / HCl buffer containing 1 g / L bovine serum albumin and 0.5 g / L NaN ₃ , pH 7.6
・ ALP washing solution 20 mM Tris-HCl buffer containing 0.15 M NaCl, 0.1% Tween 20, 1 g / L NaN ₃ , pH 7.4
Incubation temperature The incubation temperature in all steps of the non-competitive transfer solid phase assay was 37 ° C.
-Binding of DNP-biotin-anti-HBsAg Fab 'to anti-DNP IgG insolubilized magnetic beads 100 pmol / mL DNP-biotin-anti-HBsAg Fab', 25 μL and anti-DNP IgG insolubilized magnetic beads washed with TEA buffer, 0.5 mg After incubation for 1 minute, the magnetic beads were separated by magnetic separation for 10 seconds, the supernatant was removed by suction, and then washed once with 200 μL of ALP washing solution by magnetic separation for 10 seconds, and suspended in 80 μL of TEA buffer.
-Binding of HBsAg to DNP-biotin-anti-HBsAg Fab'-conjugated anti-DNP IgG insolubilized magnetic beads 2.5 IU / mL of HBsAg 20 μL and the washed DNP-biotin-anti-HBsAg Fab'-conjugated anti-DNP IgG insolubilized magnetic beads were incubated for 2 minutes. After that, the magnetic beads were washed once with 200 μL of ALP washing solution by magnetic separation for 10 seconds.
-Binding of ALP-anti-HBsAg Fab 'to HBsAg-bound DNP-biotin-anti-HBsAg Fab'-bound anti-DNP IgG insolubilized magnetic beads 4.8 pmol / mL of ALP-anti-HBsAg Fab', 100 [mu] L and the washed HBsAg-bound DNP- After incubating the biotin-anti-HBsAg Fab'-conjugated anti-DNP IgG insolubilized magnetic beads for 1 minute, they were washed once with 200 μL of ALP washing solution by magnetic separation for 10 seconds.
Lin buffer 0.1M sodium phosphate buffer containing 0.5g / L NaN _3, pH7.5
Streptavidin Type II, Wako Pure Chemical Industries, Ltd. , Osaka, Japan
DNP-Lys solution TEA buffer containing 3 mM εN-2.4-ditrophenyl-L-lysineStreptavidin-insolubilized magnetic beads Biotinylated bovine serum albumin was obtained by a known method (E. Ishikawa, Ultrasensitivity and Rapid Energy Immunology Associates, Japan). in Biochemistry and Molecular Biology Vol.27, S.Pillai, PC van der Vlied eds., Elsevier, Amsterdam, pp. 141-176, 1999), and this was prepared according to the instructions in J.P. After insolubilization, 30 μg / mL streptavidin was added to phosphate buffer. The reaction was carried out by dissolving in a solution, washed once with a phosphate buffer, and stored at 4 ° C. in a blocking solution.
Elution of immune complex from anti-DNP IgG insolubilized magnetic beads Anti-DNP IgG insolubilized magnetic beads bound with an immune complex consisting of the above-mentioned DNP-biotin-anti-HBsAg Fab ', HBsAg, and ALP-anti-HBsAg Fab' After incubating 100 μL of the DNP-Lys solution for 0.5 minutes, the supernatant obtained by accumulating the magnetic beads with a magnet for 15 seconds was used as an eluate.
-Binding of immune complex to streptavidin-insolubilized magnetic beads After the above eluate and 0.5 mg of streptavidin-insolubilized magnetic beads were incubated for 1 minute, the magnetic beads were accumulated for 10 seconds with a magnet, and the supernatant was removed. The magnetic injection beads were dispersed in 200 μL of a washing solution for use, washed once by collecting again with a magnet for 10 seconds, and immediately removing the supernatant.
ALP luminescent substrate solution CDP-star ready to use with Sapphire II, Tropix, Inc. , Bedford, MA
-ALP dispersion 1M diethanolamine-HCl buffer, pH 10.0
Luminescence measuring device Lumicounter 2500, Microtec Co. Ltd. , Chiba, Japan
-Measurement of ALP activity of ALP-bound magnetic beads Measurement 1: The washed ALP-bound magnetic beads were dispersed in 30 µL of a dispersion, mixed with 100 µL of a substrate solution, and incubated at 37 ° C for 2.75 minutes. Five samples of the supernatant in which the magnetic beads were integrated with a magnet for 0.1 second were simultaneously measured.
Measurement 2: The washed ALP-bound magnetic beads were dispersed in 130 μL of a mixture of 30 μL of a dispersion and 100 μL of a substrate solution, and incubated at 37 ° C. for 2.75 minutes. Were measured at the same time for 5 seconds.
[0014]
(result)
Table 1 shows the results of Example 1.
The ALP-bound magnetic beads were previously dispersed in a dispersion containing no substrate and enhancer, and then mixed with a substrate solution containing the substrate and enhancer to measure the ALP activity. The luminescence intensity was 92,892, and the reproducibility was CV2. 0.8% (5 cases). When the ALP activity was measured by directly dispersing in a mixture of the substrate solution and the dispersion solution, the luminescence intensity was 63,228, and the reproducible CV was 19.4%, which was very high. That is, the ALP-bound magnetic beads are preliminarily dispersed in a dispersion liquid not containing an enhancer, and then mixed with a substrate and a measurement reagent solution containing the enhancer to measure the luminescence intensity. The reproducibility was significantly improved as compared with the case where the magnetic beads were directly mixed with a reagent solution for measurement containing an enzyme substrate and an enhancer without using a dispersion.
[0015]
Embodiment 2
This example shows the effect on the signal of the addition of an enzyme substrate before the enhancer in the luminescence measurement of alkaline phosphatase immobilized on magnetic beads.
[0016]
(Materials and methods)
Other than the materials and methods described below, they are the same as those of Example 1.
-Substrate solution A solution containing only CDP-star as a substrate, Tropics.
Enhancer solution A solution containing only SapfireII as an enhancer, Tropics.
Measurement of ALP activity of ALP-bound magnetic beads Measurement 1: Washed ALP-bound magnetic beads are dispersed in a mixture of 30 μL of a dispersion and 90 μL of a substrate solution, and 15 seconds later, the ALP luminescence substrate solution added in Example 1 Instead of (containing the substrate CDP-star and the enhancer SapphireII), 10 μL of an enhancer solution was added, incubated as in Example 1, and the luminescence intensity was measured.
Measurement 2: The measurement was performed in the same manner as in Measurement 1, except that 10 μL of the enhancer solution was added 2 minutes and 15 seconds later than the addition in Measurement 1.
[0017]
(result)
Table 2 shows the results of Example 2.
The luminescence intensity was reduced from 121,500 to 79,090 by adding the enhancer later than the addition of the substrate and shortening the time from the addition of the enhancer to the measurement of luminescence. This result indicates that a high signal, that is, high sensitivity can be obtained by adding the enhancer as early as possible, and that the enzymatic reaction proceeds even in the absence of the enhancer. It also shows that a higher signal can be obtained by adding the substrate at an earlier stage where it is not possible, for example at the stage of adding the dispersion.
[0018]
[Table 1]

[0019]
[Table 2]

Claims

The microparticle solid phase on which the test substance is immobilized may be dispersed in the solid phase in a more uniform manner and / or with a substance that interferes with the measurement of the test substance with higher accuracy, reproducibility and / or sensitivity. And / or a solution containing a substance capable of measuring a test substance with higher accuracy, reproducibility, and / or sensitivity after being previously dispersed in a liquid (dispersion liquid) not containing the substance at a concentration that interferes with the measurement. (Measurement reagent solution), and measuring the test substance.

2. The method according to claim 1, wherein the microparticle solid phase is a magnetic bead having a diameter of 0.1 to 10 [mu] m or equivalent thereto.

2. The method according to claim 1, wherein the fine particle solid phase is latex particles having a diameter of 0.1 to 10 [mu] m or equivalent thereto.

The measuring method according to any one of claims 1 to 3, wherein the test substance is an enzyme, a luminescent substance, or a fluorescent substance.

The method according to claim 1, wherein the test substance is a hydrolase.

The method according to any one of claims 1 to 3, wherein the test substance is alkaline phosphatase.

The method according to any one of claims 1 to 3, wherein the test substance is β-D-galactosidase.

The method according to any one of claims 4 to 7, wherein the enzyme substrate is a dioxetane derivative.

The method according to any one of claims 4 to 8, wherein an enhancer is used in addition to the enzyme substrate for the measurement of the test substance.

The method according to claim 9, wherein the enhancer is a surfactant.

The dispersion does not contain the enhancer at a concentration that interferes with the more uniform dispersion of the analyte-immobilized microparticle solid phase and / or the more accurate, reproducible and / or sensitive determination of the analyte. The method according to any one of claims 8 to 10, wherein:

The method according to claim 11, wherein the dispersion does not contain an enzyme substrate, and the reagent solution for measurement contains an enzyme substrate and an enhancer.

The method according to claim 11, wherein the dispersion contains an enzyme substrate, and the reagent solution for measurement contains an enhancer.

A kit comprising at least one of a reagent and / or a solid phase that can be used in the measurement method according to claim 1.

A measurement system comprising a reagent, a solid phase, and automation software that can be used in the measurement method according to claim 1.