JP2004347480A - Method for measuring lysate reagent reactive substance - Google Patents
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Abstract
Description
【０００１】
【発明の属する技術分野】
本発明は、ライセート試薬を用いたライセート試薬反応性物質の測定方法に関するものである。
【０００２】
【従来の技術】
経時的に反応生成物量、基質量等の酵素反応パラメータ値の変化を計測する酵素反応の測定は、特に医療分野等や生活環境分野等において、その有用性が高く、広い濃度範囲の検体を簡便にかつ精度良く測定する方法が従来より様々検討されてきており、その測定方法としてはエンドポイント法やカイネティック法等がある。
【０００３】
例えば、酵素反応のある１点におけるデータを基に分析するエンドポイント法を用いたＥＬＩＳＡ系での測定方法において検量線がＳ字状カーブになる場合には、Ｌｏｇｉｔ解析法を用いるとＳ字状カーブが直線化され、更にロジット（Ｌｏｇｉｔ）解析法によりＳ字状カーブを直線化すると、測定範囲が広範囲にわたり測定可能となり非常に有用であると考えられていたが、このＬｏｇｉｔ解析の為の演算式の組み立ては非常に複雑である為、殆ど用いられておらず、ＥＬＩＳＡ系等での測定に於いてはＳ字状カーブを示す検量線をそのまま用いているのが現状である。つまり、これら報告ではＬｏｇｉｔ解析法が有用であると述べているにも拘わらず、検量線作成の汎用解析法としては用いられていないのが現状である。
【０００４】
一方、経時的な反応量の変化を基に分析するカイネティック法を用いた生物活性測定法に関しては、例えば、カイネティック比色法を用いたエンドトキシン測定法において、測定範囲を低濃度と高濃度に分割し、２本の検量線を作成することにより広範囲にわたり測定可能であるとの報告がなされている（非特許文献）。この方法によると精度良く広範囲において測定が可能ではあるが、やはり低濃度範囲及び高濃度範囲２範囲に分け、検量線も２本、且つ、解析条件も２条件必要であり煩雑である。また、エンドトキシンの測定に於いてＬｏｇｉｔ解析法を用い、エンドトキシン測定がなされたという事は、現在、エンドポイント法、カイネティック法ともに報告は無い。
【０００５】
また、エンドトキシン（内毒素）はグラム陰性細菌の菌体に存在する発熱因子であり、細菌感染や医薬品に混入して血中に存在した状態では、発熱、悪寒、血圧低下等を引き起こすため、エンドトキシンと特異的に反応するカブトガニ血球成分（アメボサイト・ライセート）を利用した試薬（以下、ライセート試薬という。）を用いて医薬品や生体内のエンドトキシンの定量が行われている。同様に、（１→３）−β−Ｄ−グルカン（以下、β−グルカンともいう）は真菌の細胞壁多糖で、ライセート試薬と特異的に反応するため、これを用いて血中等に存在するβ−グルカンの定量が行われている。この（１→３）−β−Ｄ−グルカンの測定においてＬｏｇｉｔ解析法が用いられた報告はない。尚、本発明においては、エンドトキシン、β−グルカン等のライセート試薬と特異的に反応する物質を「ライセート試薬反応性物質」と称する。
【０００６】
【非特許文献】日本血液浄化技術研究会会誌 Ｖｏｌ．１９、Ｎｏ．２、ｐ．１０９−１１７、２００２ Ｏｃｔ．
【０００７】
【発明が解決しようとする課題】
現在、カイネティック法によるライセート試薬反応性物質の測定では、単一の検量線により広範囲の濃度を測定するには誤差が大きすぎる為、精度が悪く、少なくとも低濃度範囲と高濃度範囲に１本ずつ計２本の検量線を作成しなければ、精度良く測定することは不可能とされていた。また、作成した検量線の濃度範囲に対応させる為に検体濃度を希釈する等の作業を行う必要がある場合もあった。
【０００８】
【課題を解決するための手段】
本発明者らは、カイネティック法を用いるライセート試薬反応性物質の測定において、検量線作成の解析法（演算法）としてＬｏｇｉｔ解析法を用いたところ、作成された単一の検量線により、広い濃度範囲にわたり測定可能であり、精度においても従来法と同等又はそれ以上により高精度で測定出来る事を見出し、本発明を完成するに到った。
【０００９】
すなわち、本発明は以下の通りである。
（１） カイネティック法によりライセート試薬反応性物質を測定するに際し、検量線の作成に於いてロジット（Ｌｏｇｉｔ）解析法を用いることを特徴とするライセート試薬反応性物質の測定方法。
（２） 少なくとも３種類の異なる濃度のライセート試薬反応性物質の標準溶液を用いて作成した単一の検量線を用いる（１）記載のライセート試薬反応性物質の測定方法。
【００１０】
【発明の実施の形態】
以下、本発明を発明の実施の形態により詳説する。
本発明方法のカイネティック法を用いるライセート試薬反応性物質の測定方法（以下、本発明測定方法ともいう）は、ライセート試薬等を用いて当該ライセート試薬に反応する物質（ライセート試薬反応性物質）を定量する方法であって、検量線の作成の際の測定データの解析処理においてＬｏｇｉｔ解析法を用いる方法であれば、反応条件、測定条件、用いるライセート試薬の種類等には特に限定はされない。
【００１１】
ライセート試薬を用いるライセート試薬反応性物質の測定方法とは、ライセート試薬とエンドトキシン、β−グルカン等ライセート試薬反応性物質とが反応することにより、ライセート試薬に含有されている複数の因子により構成されているカスケード反応（本明細書において、この一連のカスケード反応をリムルス反応と称する。）が惹起され最終的に反応液が凝固（ゲル化）する現象又はライセート試薬に添加したペプチド合成基質が水解される現象を利用し、ライセート試薬反応性物質を測定する方法である。
【００１２】
リムルス反応の反応条件については、リムルス反応が進行し得る条件であれば良く、当業者が適宜選択及び設定する事が可能であり特に限定されない。
【００１３】
カイネティック法を用いた本発明測定方法では、反応測定データの処理において反応量と関連づける指標の違いにより反応時間法及び反応速度法での測定が可能である。反応時間法とは、反応開始直後から予め定めた反応量に達するまでの経過時間と、その間の予め定めた反応量とを関連づけることにより測定対象物質の濃度を算出する方法である。また、反応速度法とは、予め定めた一定時間内に起こる反応において、反応量の一定の時間当たりの変化率を求め、当該値を反応量と結びつけることにより測定対象物質の濃度を算出する方法である。
【００１４】
また、反応時間法及び反応速度法各々について、反応量を測定するパラメーターとしてライセート試薬が示す上述の現象のどちらを利用するかにより、ゲル化に伴う濁度の変化を利用する比濁法とペプチド合成基質を水解する現象を利用する比色法とに大別される。各々、濁度や吸光度などを測定することにより反応量の測定が行われ、例えば、比色反応速度法では検体濃度と生成する色素量（具体的には吸光度）の変化率との関係で表される検量線を、また、比濁反応時間法では検体濃度と一定のゲル化度（具体的には一定の濁度）に到達する時間との関係で表される検量線などが用いられる。
【００１５】
本発明測定方法においては、カイネティック法である限り何れにも用いる事が可能であり、比濁反応時間法、比濁反応速度法、比色反応時間法及び比色反応速度法何れかに限定されることは無いが、比色反応速度法、比濁反応時間法が好ましい。
【００１６】
ライセート試薬とは、カブトガニ血リンパ液から得られる血球抽出物を利用したリムルス試薬反応性物質測定用の試薬である。本発明方法に用いることが可能なライセート試薬は、カブトガニ血リンパ液から得られた血球抽出物（ライセート）から成るものであれば特に限定されず、当該ライセートを加工したものでも良い。つまり、目的とする測定対象物質との反応に最適な様にライセート試薬を適宜調製又は加工しても良く、酸、塩基、緩衝剤等を添加しｐＨを調製したり、エンドトキシンを特異的に測定する際にはβ−グルカンと反応するＧ因子を除去又は不活化させたライセート試薬を用いることも可能であり、逆に、β−グルカンを特異的に測定する際にはエンドトキシンと反応するＣ因子を除去又は不活化させたライセート試薬を用いる事も可能である。
【００１７】
更に、リムルス反応にはその適当量が必須であると知られているカルシウムやマグネシウムの様な２価金属イオンの適当量を添加したり、また、カスケード反応により生成するクロッティングエンザイムの基質となるペプチド合成基質、例えば、ｔ−ブトキシカルボニル−ロイシル−グリシル−アルギニン−パラニトロアニリド（Ｂｏｃ−Ｌｅｕ−Ｇｌｙ−Ａｒｇ−ｐＮＡ）等を添加したライセート試薬を用いる事も可能である。市販されているリムルス試薬を用いる事も可能であり、上記２価金属塩やペプチド合成基質を使用時に添加する様なキット化されたものも用いることが可能である。
【００１８】
なお、合成基質ｔ−ブトキシカルボニル−ロイシル−グリシル−アルギニン−パラニトロアニリド（Ｂｏｃ−Ｌｅｕ−Ｇｌｙ−Ａｒｇ−ｐＮＡ）を添加したライセート試薬を用いた場合、反応量は、リムルス反応により生成するクロッティングエンザイムが当該合成基質のアミド結合を水解して遊離させたパラニトロアニリン（以下、「ｐＮＡ」とも記載する。発色物質）の吸光度を測定することにより定量することが出来る。また、他にも合成基質として、ｐＮＡ等の発色性残基を有する上記合成基質とクロッティングエンザイムの作用において類似するアミノ酸配列を有するＡｃ−Ｉｌｅ−Ｇｌｕ−Ａｌａ−Ａｒｇ、Ｂｏｃ−Ｖａｌ−Ｐｒｏ−ＡｒｇやＢｏｃ−Ｖａｌ−Ｓｅｒ−Ｇｌｙ−Ａｒｇなどのペプチド合成基質、又は、これと同一若しくは類似の配列のペプチドであって、Ｃ末端のアミノ酸のカルボキシル基に発色性残基の代わりに公知の発蛍光性残基、発光性残基、アンモニアなどがアミド結合により置換した基質を用いることも出来る。
【００１９】
リムルス反応の生成物の量の変化を正確に反映し、反応の進行度合い（反応量）を測定するのに用いる反応パラメーター値としては、反応溶液の吸光度、濁度、透過光量、蛍光偏光、蛍光消光又は散乱光量などの他、反応の結果変化したｐＨ、伝導度、比電離及び比抵抗などの測定値を用いることが出来、更に、磁気又は電気信号に変換、増幅しうる反応パラメーター値も利用可能である。中でも本発明方法においては、吸光度、濁度又は透過光量を測定することがより好ましい。
【００２０】
当該反応パラメーター値を測定する方法としては、反応パラメーター値を測定することが出来る測定装置であれば特に限定されないが、分光光度計、吸光光度計、蛍光光度計、化学発光測定装置、濁度測定装置、粘度測定装置等があげられる。
【００２１】
反応パラメーター値の測定条件については、反応の進行度合い（反応量）の測定が適切に行えさえすれば良く、当業者が適宜選択可能である。
本発明測定方法で測定可能な物質は、ライセート試薬に反応性を有する物質であれば特に限定されないが、エンドトキシン、β−グルカン等があげられる。
【００２２】
なお、本発明測定方法は、エンドトキシン、β−グルカン等のリムルス試薬反応性物質を測定する方法である限り、ライセート試薬を用いるリムルス反応に基づく測定法には限定されない。
【００２３】
本発明測定方法では、検量線作成の条件（計算式）としてＬｏｇｉｔ解析法を用いる。本発明方法に用いるＬｏｇｉｔ解析法とは、各濃度の標準液の測定データを下記計算式によりＬｏｇｉｔ変換し、直線のグラフを作成する方法であり、計算式は、
▲１▼Ｌｏｇｉｔ（Ｙ）＝Ｌｏｇ（Ｙ／（Ｋ−Ｙ））＝Ａ（Ｌｏｇ（Ｘ））＋Ｂ、又は、Ｌｏｇｉｔ（Ｙ）＝Ｌｏｇ（Ｙ／（Ｋ−Ｙ））＝Ａ（Ｌｏｇ（Ｘ＊１０００））＋Ｂ
▲２▼Ｌｏｇｉｔ（Ｌｏｇ（Ｙ））＝Ｌｏｇ（Ｌｎ（Ｙ＊１０００）／（Ｋ−Ｌｎ（Ｙ＊１０００）））＝Ａ（Ｌｏｇ（Ｘ））＋Ｂ、又は、Ｌｏｇｉｔ（Ｌｏｇ（Ｙ））＝Ｌｏｇ（Ｌｏｇ（Ｙ＊１０００）／（Ｋ−Ｌｏｇ（Ｙ＊１０００）））＝ＡＬｏｇ（Ｘ＊１０００）＋Ｂ、
▲３▼Ｌｏｇｉｔ（Ｌｏｇ（Ｌｏｇ（Ｙ））＝Ｌｏｇ（Ｌｎ（Ｌｏｇ（Ｙ＊１０００））／（Ｋ−（Ｌｎ（ＬｏｇＹ＊１０００）））＝ＡＬｏｇ（Ｘ＊１０００）＋Ｂ、又は、Ｌｏｇｉｔ（Ｌｏｇ（Ｌｏｇ（Ｙ））＝Ｌｏｇ（Ｌｎ（Ｌｏｇ（Ｙ＊１０００））／Ｋ−（Ｌｎ（ＬｏｇＹ＊１０００）））＝Ａ（Ｌｏｇ（Ｘ））＋Ｂ
等で表される。式中、Ｘは濃度、Ｙは測定値（反応値）、Ｋは定数、Ａは定数（検量線の勾配）、Ｂは定数（Ｙ軸との切片）を示し、Ｌｏｇは常用対数、Ｌｎは自然対数を示し、式中＊は掛け算を意味する。
【００２４】
なお、一般式としては、Ｌｏｇｉｔ（Ｙ）＝Ｌｏｇ（Ｙ／（Ｋ−Ｙ））＝Ａ（Ｌｏｇ（Ｘ））＋Ｂで表され、Ｙは測定値又は測定値を基に算出した変換値、Ｘは濃度を示す。
【００２５】
本発明測定方法ではＬｏｇｉｔ解析法を用いて作成される検量線の２つの変数の直線的な関係の強さを表す相関係数（ｒ）は０．９９９０〜１．００００であり、非常に精度が高い。
【００２６】
本発明測定方法は、反応速度法、反応時間法、又は、比色法、比濁法の違いに拘わらず、Ｌｏｇｉｔ解析法による単一の直線の検量線を作成するだけで、エンドトキシンでは０．０００２〜１００ＥＵ／ｍＬ、より好ましくは０．００１〜５０ＥＵ／ｍＬ、また一方、β−グルカンでは０．５〜８，０００ｐｇ／ｍＬ、より好ましくは１〜４，０００ｐｇ／ｍＬの広範囲に渡る濃度のライセート試薬反応性物質の測定に用いることが可能である。
【００２７】
また、検量線作成には、ライセート試薬反応性物質（標準物質）の少なくとも３種類の異なる濃度の標準液による測定データがあれば良く、検体を希釈する等の煩雑な手間も不要となる。例えば、リムルス反応を用いる測定法の測定データ処理方法として信頼性が高い反応速度法に関しては、従来は感度と定量性に優れる反面、測定範囲が限定され、検体によっては適宜希釈を要するなど煩雑な面もあった。しかし、本発明測定方法を用いることにより、測定範囲は大幅に広がり、精度も高く、検体の希釈等の手間も不要となる。その為、操作時間の短縮とコストの低減となり、かつ希釈による失活や容器への吸着も含めた誤差も無くなり、精度の向上にもつながるものである。
【００２８】
反応測定データの処理条件や測定データの演算範囲の設定は、目的対象物質の種類や検体の種類に従い当業者が適宜設定可能である。例えば、一定の反応量に達する反応経過時間と反応量の関係がＳ字状を示すことが知られているカスケード反応において、反応開始付近の立ち上がり部分と反応終了付近の頭打ちの部分のデータによる誤差を考慮する為、上記の立ち上がり部分と頭打ち部分のデータを除いて解析する特開平１１―２９００９５記載の方法等を用いることも可能である。
【００２９】
また、本発明方法であるＬｏｇｉｔ解析法（Ｌｏｇｉｔ−Ｌｏｇ解析法とも呼ばれている）や各種条件設定などをソフトウェア化し、かかるソフトウェアが搭載された一般的な酵素反応測定装置を用いる事も可能である。
【００３０】
本発明測定方法で測定可能な検体は、ライセート反応性物質が含まれる可能性があり、本発明測定方法で測定可能な物質を含有する溶液であれば特に限定されないが、水、臨床透析液、輸液、注射液、血液（血清、血漿）、尿、髄液、その他医薬や医療用具等があげらる。これら検体溶液のエンドトキシンやβ−グルカン等の濃度管理や品質確認、これらの物質を定量することによるエンドトキシン血症、敗血症、真菌症等の診断に非常に有用である。
【００３１】
【実施例】
以下、実施例により本発明をより具体的に説明する。
実施例１ 比色反応速度法
ブランク液、０．０００２−１００ＥＵ／ｍＬの範囲における、全１３種類の異なる濃度（０．０００２４、０．０００４７、０．０００９４、０．００１８８、０．００３７５、０．００７５、０．０１５、０．０３、０．０６、０．１２、１．０、１０．０、１００．０ＥＵ／ｍＬ）のエンドトキシン標準液（エンドトキシン１００標準品；Ｅ． ｃｏｌｉ ＵＫＴ−Ｂ由来）の各５０μＬをトキシペットプレート（生化学工業（株）販売）のそれぞれ所定のウェルに分注し、エンドトキシン特異的ライセート試薬（エンドスペシーＥＳ−５０Ｍ、生化学工業（株）販売）を５０μＬずつ添加して蓋をかぶせ、Ｗｅｌｌｒｅａｄｅｒ ＳＫ６０３（生化学工業（株）販売）に装着し、リムルス反応による反応量（吸光度の変化）をＫｉｎｅｔｉｃ Ａｓｓａｙモード（反応速度法；Ｒａｔｅ Ａｓｓａｙ）、２波長（４０５−４９２ｎｍ）で３７℃、３０分間自動測定を行った。その測定値（吸光度変化率；ｍＡｂｓ／ｍｉｎ）を本発明のＬｏｇｉｔ解析法と従来のＳｉｍｐｌｅ法でそれぞれ解析し検量線を作成した。
【００３２】
尚、Ｗｅｌｌｒｅａｄｅｒ ＳＫ６０３におけるＬｏｇｉｔ法及びＳｉｍｐｌｅ法での解析パラメーターは下記の通り設定した。
Ｂｌａｎｋ（ｎｅｇａｔｉｖｅ ｃｏｎｔｒｏｌ） Ｓｕｂｔｒａｔｉｏｎ：Ｏｎとは、ブランク液による測定値を標準液や検体による測定値より差し引くことを示し、また、Ｃａｌｃ Ｔｉｍｅとは、連続する吸光度データを用いた処理を行うときの計算対象となる範囲の設定に用い、上記の場合、吸光度測定開始後４分４５秒〜３０分に測定された吸光度データを用いることを示す。Ｓｌｉｃｅ Ｌｏｗ、Ｓｌｉｃｅ Ｈｉｇｈとは連続する吸光度データを用いた処理を行う時に計算対象となる吸光度の下限及び上限を設定する。
【００３４】
本発明のＬｏｇｉｔ解析法と従来のＳｉｍｐｌｅ法による検量線をそれぞれ図１、図２に示した。本発明のＬｏｇｉｔ解析法では相関係数ｒ＝０．９９９８と非常に良好な直線性を示した。一方、従来のＳｉｍｐｌｅ法ではｒ＝０．９７６８と極めて悪く、検体を定量することは出来なかった。尚、Ｓｉｍｐｌｅ法による検量線解析とは、各濃度の標準液の測定データを基に最小二乗法により直線化する解析法であり、下記計算式により表される。
【００３５】
Ｙ＝ＡＸ＋Ｂ
【００３６】
実施例１（比色反応速度法）において、Ｘは標準液の濃度で、Ｙは測定値（吸光度変化率；ｍＡｂｓ／ｍｉｎ）であり、Ａは定数（検量線の勾配）であり、Ｂは定数（検量線におけるＹ軸との切片）である。
【００３７】
更に、上述の全１３種類の異なる濃度のエンドトキシン標準液の内、０．０００９４、０．１２、１０ＥＵ／ｍＬの３つの濃度の測定値をＬｏｇｉｔ法解析し検量線を得、その他の１０種類の濃度のエンドトキシン標準液を検体として定量を行った。結果を図３及び表１に示した。３つの異なる濃度のエンドトキシン標準液の測定値を基に作成した検量線はｒ＝１．００００と極めて良好な直線となった。また、その検量線から算出された他の１０種類の異なる濃度の検体のエンドトキシン濃度は、その理論値に対する相対活性が８６．１％〜１０４．６％の間となり、０．０００２〜１００ＥＵ／ｍＬを高精度で定量出来る事が判明した。
【００３８】
【表１】
［表１］
（相対活性は、検量線より算出した検体のエンドトキシン濃度÷検体のエンドトキシン濃度理論値Ｘ１００により算出した。）
【００４０】
実施例２：比濁反応時間法
ブランク液、０．００２〜１００ＥＵ／ｍＬの範囲における全１２種類の異なる濃度（０．００１９５３１、０．００３９０６２、０．００７８１２５、０．０１５６２５、０．０３１２５、０．０６２５、０．１２５、０．２５、０．５、１．０、１０．０、１００．０ＥＵ／ｍＬ）のエンドトキシン標準液（エンドトキシン１００標準品；Ｅ． ｃｏｌｉ ＵＫＴ−Ｂ由来）の各１００μＬをトキシペットプレート（生化学工業（株）販売）のそれぞれ所定のウェルに分注し、エンドトキシン特異的ライセート試薬（Ｐｙｒｏｔｕｒｂ−ＥＳ、生化学工業（株）販売）を１００μＬずつ添加して蓋をかぶせ、Ｗｅｌｌｒｅａｄｅｒ ＳＫ６０３に装着し、リムルス反応による反応量（吸光度の変化）をＫｉｎｅｔｉｃ Ａｓｓａｙモード（反応時間法；Ｄｅｌｔａ−Ｔ Ａｓｓａｙ）、２波長（４０５−６６０ｎｍ）で３７℃、６０分間自動測定した。その測定値（分；ｍｉｎ）を本発明のＬｏｇｉｔ解析法と従来のＳｉｍｐｌｅ法でそれぞれ解析し検量線を作成した。
【００４１】
尚、実施例１と同様にＷｅｌｌｒｅａｄｅｒ ＳＫ６０３における解析パラメーターを、Ｌｏｇｉｔ法及びＳｉｍｐｌｅ法共に下記の通り設定した。
本発明のＬｏｇｉｔ法と従来法であるＳｉｍｐｌｅ法による検量線をそれぞれ図４、図５に示した。本発明のＬｏｇｉｔ解析法では相関係数ｒ＝−０．９９９４と高精度で良好な直線性を示し、０．００２〜１００ＥＵ／ｍＬの広い濃度範囲においてエンドトキシンが定量出来ることが明らかである。一方、従来法であるＳｉｍｐｌｅ法では相関係数ｒ＝−０．９９４７とかなり精度が低く、この様な広い範囲のエンドトキシンの定量は難しい。
【００４３】
尚、Ｓｉｍｐｌｅ法による検量線は、Ｙ＝ＡＸ＋Ｂで表され、実施例２（比濁反応時間法）において、Ｘはエンドトキシン標準液の濃度で、Ｙは測定値（分；ｍｉｎ）であり、Ｂは定数（検量線におけるＹ軸との切片）である。
【００４４】
実施例３
８，０００〜０．５ｐｇ／ｍＬの２倍段階希釈シリーズの１５種類の異なる濃度のβ−グルカン標準液（ＣＳＢＧ；Ｃａｎｄｉｄａ ａｌｂｉｃａｎｓ ＩＦＯ１３８５の細胞壁由来の（１→３）−β−Ｄ−グルカン）を調製した。調製したβ−グルカン標準液、ブランク液の各２５μＬをトキシペットプレートのそれぞれ所定のウェルに分注し、β−グルカン特異的ライセート試薬（ファンギテックＧテストＭＫ、生化学工業（株）販売）を１００μＬずつ添加して蓋をかぶせ、Ｗｅｌｌｒｅａｄｅｒ ＳＫ６０３に装着し、リムルス反応による反応量（吸光度の変化）をＫｉｎｅｔｉｃ Ａｓｓａｙモード（反応速度法；Ｒａｔｅ Ａｓｓａｙ）、２波長（４０５−４９２ｎｍ）で３７℃、４０分間、自動測定した。調製したβ−グルカン標準液の０．５、２．０、７．８、３１．３、１２５．０、５００．０、２０００．０、８０００．０ｐｇ／ｍＬの４倍段階希釈シリーズの８種類の異なる濃度による測定値を本発明のＬｏｇｉｔ法と従来のＳｉｍｐｌｅ法でそれぞれ解析し検量線を作成した。また、その他の７種類の異なる濃度のβ−グルカン標準液を検体として定量を行った。
【００４５】
尚、実施例１同様にＷｅｌｌｒｅａｄｅｒにおける解析パラメーターは、Ｌｏｇｉｔ解析法及びＳｉｍｐｌｅ法共に下記の通り設定した。
本発明のＬｏｇｉｔ法及び従来のＳｉｍｐｌｅ法による検量線をそれぞれ図６、図７に示した。本発明のＬｏｇｉｔ解析法では、８種類の異なる濃度のβ−グルカン標準液により作成した検量線の相関係数はｒ＝０．９９９９で高精度で非常に良好な直線性を示したが、従来のＳｉｍｐｌｅ法ではｒ＝０．９１４１とかなり精度が低かった。
【００４７】
これらの検量線から検量線作成に用いなかった７種類の異なる濃度の標準液（検体）のβ−グルカン濃度を算出した結果をそれぞれ表２−１（Ｌｏｇｉｔ法）、表２−２（Ｓｉｍｐｌｅ法）に示した。この表から明らかな様にＬｏｇｉｔ法では、検体の理論値に対する相対活性は９２．６〜１０６．６％の間であり、０．５〜８，０００ｐｇ／ｍＬまで高い精度で定量出来ることが判明したが、従来法（Ｓｉｍｐｌｅ法）では、その相対活性は１５．２〜４８９．３％という大きなバラツキを示し、全く定量することが出来なかった。
【００４８】
【表２】
［表２−１］
（尚、相対活性は、検量線より算出した検体のβ−グルカン濃度÷検体のβ−グルカン濃度の理論値Ｘ１００にて算出した。）
【００５０】
【表３】
［表２−２］
（尚、相対活性は、検量線より算出した検体のβ−グルカン濃度÷検体のβ−グルカン濃度の理論値Ｘ１００にて算出した。）
【００５２】
【発明の効果】
カイネティック法を用いるライセート試薬反応性物質の測定において、検量線作成の解析法（演算法）としてＬｏｇｉｔ解析法を用いる本発明測定方法は、Ｌｏｇｉｔ解析法により作成された単一の検量線により、広い濃度範囲にわたる検体濃度の測定が高精度で可能である。また、本発明測定方法における検量線の作成には、少なくとも検量線作成用として３種類の異なる標準液による測定データがあれば良く、標準液の調製や希釈にかかる手間やその希釈操作による容器への吸着並びに失活による誤差や標準液と反応させる試薬等が従来より大幅に削減される為、ライセート試薬反応性物質であるエンドトキシンやβ−グルカン等の濃度管理や品質確認等に用いるのに簡便で且つ有用である。
【００５３】
【図面の簡単な説明】
【図１】図１は、実施例１において全１３種類の異なる濃度のエンドトキシン標準液の測定データに基づきＬｏｇｉｔ解析法により作成した検量線であり、縦軸は１分間あたりの吸光度変化率（ｍＡｂｓ／ｍｉｎ）、横軸はエンドトキシン濃度（ＥＵ／ｍＬ）を示す。
【図２】図２は、実施例１において全１３種類の異なる濃度のエンドトキシン標準液の測定データに基づきＳｉｍｐｌｅ法により作成した検量線であり、縦軸は１分間あたりの吸光度変化率（ｍＡｂｓ／ｍｉｎ）、横軸はエンドトキシン濃度（ＥＵ／ｍＬ）を示す。
【図３】図３は、実施例１において０．０００９４、０．１２、１０ＥＵ／ｍＬの３種類のエンドトキシン標準液の測定データに基づきＬｏｇｉｔ解析法により作成した検量線であり、縦軸は１分間あたりの吸光度変化率（ｍＡｂｓ／ｍｉｎ）、横軸はエンドトキシン濃度（ＥＵ／ｍＬ）を示す。
【図４】図４は、実施例２において全１２種類の異なる濃度のエンドトキシン標準液の測定データに基づきＬｏｇｉｔ解析法により作成した検量線であり、縦軸は活性化時間（ｍｉｎ）、横軸はエンドトキシン濃度（ＥＵ／ｍＬ）を示す。
【図５】図５は、実施例２において全１２種類の異なる濃度のエンドトキシン標準液の測定データに基づきＳｉｍｐｌｅ法により作成した検量線であり、縦軸は活性化時間（ｍｉｎ）、横軸はエンドトキシン濃度（ＥＵ／ｍＬ）を示す。
【図６】図６は、実施例３において全８種類の異なるβ−グルカン標準液の測定データに基づきＬｏｇｉｔ解析法により作成した検量線であり、縦軸は１分間あたりの吸光度変化率（ｍＡｂｓ／ｍｉｎ）、横軸はβ−グルカン濃度（ｐｇ／ｍＬ）を示す。
【図７】図７は、実施例３において全８種類の異なるβ−グルカン標準液の測定データに基づきＳｉｍｐｌｅ法により作成した検量線であり、縦軸は１分間あたりの吸光度変化率（ｍＡｂｓ／ｍｉｎ）、横軸はβ−グルカン濃度（ｐｇ／ｍＬ）を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for measuring a lysate reagent-reactive substance using a lysate reagent.
[0002]
[Prior art]
Enzymatic reaction measurement, which measures changes in enzymatic reaction parameters such as the amount of reaction products and the amount of substrate over time, is highly useful, especially in the medical field, living environment field, etc. Conventionally, various methods for accurate and accurate measurement have been studied, and the measurement methods include an endpoint method and a kinetic method.
[0003]
For example, if the calibration curve is an S-shaped curve in an ELISA-based measurement method using an endpoint method that analyzes based on data at a certain point in an enzymatic reaction, when the Logit analysis method is used, an S-shaped curve is obtained. If the curve was linearized and the S-shaped curve was further linearized by the Logit analysis method, the measurement range could be measured over a wide range, which was considered to be very useful. However, the calculation for this Logit analysis was considered. Since the assembly of the formula is very complicated, it is hardly used, and at present, a calibration curve showing an S-shaped curve is used as it is in the measurement with an ELISA system or the like. That is, although these reports state that the Logit analysis method is useful, at present it is not used as a general-purpose analysis method for preparing a calibration curve.
[0004]
On the other hand, with respect to a biological activity measurement method using a kinetic method that analyzes based on a change in a reaction amount over time, for example, in an endotoxin measurement method using a kinetic colorimetric method, the measurement range is set to a low concentration and a high concentration. It has been reported that measurement can be performed over a wide range by creating two calibration curves (Non-Patent Document). According to this method, measurement can be performed over a wide range with high accuracy, but it is also divided into a low concentration range and a high concentration range, and two calibration curves and two analysis conditions are required, which is complicated. At the present time, there is no report that the endotoxin was measured using the Logit analysis method in the measurement of endotoxin in both the endpoint method and the kinetic method.
[0005]
Endotoxin (endotoxin) is a heat-generating factor present in the cells of gram-negative bacteria, and if it is present in the blood after being contaminated with bacterial infection or pharmaceuticals, it causes fever, chills, and a decrease in blood pressure. Quantification of endotoxin in pharmaceuticals and living organisms has been performed using a reagent (hereinafter, referred to as a lysate reagent) utilizing a horseshoe crab blood cell component (amebosite lysate) that specifically reacts with liposomes. Similarly, (1 → 3) -β-D-glucan (hereinafter also referred to as β-glucan) is a fungal cell wall polysaccharide, which specifically reacts with a lysate reagent. -Glucan has been quantified. There is no report using Logit analysis in the measurement of (1 → 3) -β-D-glucan. In the present invention, a substance that specifically reacts with a lysate reagent such as endotoxin or β-glucan is referred to as a “lysate reagent-reactive substance”.
[0006]
[Non-Patent Document] Journal of the Japanese Society for Blood Purification Technology Vol. 19, no. 2, p. 109-117, 2002 Oct.
[0007]
[Problems to be solved by the invention]
At present, in the measurement of lysate reagent-reactive substances by the kinetic method, the accuracy is poor because the error is too large to measure a wide range of concentrations using a single calibration curve. Unless two calibration curves were prepared, it was impossible to measure accurately. In some cases, it is necessary to perform an operation such as diluting the sample concentration to correspond to the concentration range of the prepared calibration curve.
[0008]
[Means for Solving the Problems]
The present inventors have used Logit analysis as an analysis method (calculation method) for preparing a calibration curve in the measurement of a lysate reagent-reactive substance using the kinetic method. The inventors have found that the measurement can be performed over the concentration range, and that the measurement can be performed with high accuracy by the same level or higher than the conventional method, and the present invention has been completed.
[0009]
That is, the present invention is as follows.
(1) A method for measuring a lysate reagent-reactive substance, which comprises using a logit analysis method in preparing a calibration curve when measuring a lysate reagent-reactive substance by a kinetic method.
(2) The method for measuring a lysate reagent-reactive substance according to (1), wherein a single calibration curve prepared using standard solutions of at least three different concentrations of the lysate reagent-reactive substance is used.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail by embodiments of the present invention.
The method for measuring a lysate reagent-reactive substance using the kinetic method of the method of the present invention (hereinafter also referred to as the measurement method of the present invention) is a method for measuring a substance that reacts with the lysate reagent using a lysate reagent or the like (a lysate reagent-reactive substance). There is no particular limitation on the reaction conditions, measurement conditions, types of lysate reagents, and the like, as long as the method is a quantification method that uses the Logit analysis method in the analysis of the measurement data at the time of creating the calibration curve.
[0011]
The method for measuring a lysate reagent-reactive substance using a lysate reagent means that the lysate reagent reacts with a lysate reagent-reactive substance such as endotoxin or β-glucan, and is constituted by a plurality of factors contained in the lysate reagent. A cascade reaction (this series of cascade reactions is referred to as a Limulus reaction in the present specification) is induced, and the reaction solution eventually coagulates (gels) or the peptide synthesis substrate added to the lysate reagent is hydrolyzed. This is a method of measuring a lysate reagent-reactive substance using the phenomenon.
[0012]
The reaction conditions for the Limulus reaction may be any conditions under which the Limulus reaction can proceed, and can be appropriately selected and set by those skilled in the art, and are not particularly limited.
[0013]
In the measurement method of the present invention using the kinetic method, the measurement by the reaction time method and the reaction rate method can be performed depending on the difference in the index associated with the reaction amount in the processing of the reaction measurement data. The reaction time method is a method of calculating the concentration of a substance to be measured by associating an elapsed time from immediately after the start of the reaction to reaching a predetermined reaction amount with a predetermined reaction amount during the period. In addition, the reaction rate method is a method of calculating the rate of change of a reaction amount per fixed time in a reaction occurring within a predetermined time, and calculating the concentration of the substance to be measured by relating the value to the reaction amount. It is.
[0014]
In addition, for each of the reaction time method and the reaction rate method, depending on which of the above-mentioned phenomena exhibited by the lysate reagent is used as a parameter for measuring the reaction amount, a turbidimetric method utilizing a change in turbidity due to gelation and a peptide It is broadly divided into colorimetric methods that utilize the phenomenon of hydrolysis of synthetic substrates. The reaction amount is measured by measuring turbidity, absorbance, etc., respectively. For example, in the colorimetric reaction rate method, a table is expressed by the relationship between the analyte concentration and the rate of change in the amount of generated dye (specifically, absorbance). For the turbidimetric reaction time method, a calibration curve expressed by the relationship between the analyte concentration and the time required to reach a certain gelation degree (specifically, a certain turbidity) is used.
[0015]
In the measurement method of the present invention, any method can be used as long as it is a kinetic method, and is limited to any of the turbidimetric reaction time method, the turbidimetric reaction rate method, the colorimetric reaction time method and the colorimetric reaction rate method. However, the colorimetric reaction rate method and the turbidimetric reaction time method are preferred.
[0016]
The lysate reagent is a reagent for measuring a Limulus reagent-reactive substance using a blood cell extract obtained from horseshoe crab hemolymph. The lysate reagent that can be used in the method of the present invention is not particularly limited as long as it comprises a blood cell extract (lysate) obtained from horseshoe crab hemolymph, and may be a processed lysate. In other words, the lysate reagent may be appropriately prepared or processed so as to be optimal for the reaction with the target substance to be measured.The pH is adjusted by adding an acid, a base, a buffer, or the like, or the endotoxin is specifically measured. It is also possible to use a lysate reagent from which factor G, which reacts with β-glucan, has been removed or inactivated. Conversely, when β-glucan is specifically measured, a factor C, which reacts with endotoxin, can be used. It is also possible to use a lysate reagent from which is removed or inactivated.
[0017]
Further, it is known that an appropriate amount is required for the Limulus reaction, and an appropriate amount of a divalent metal ion such as calcium or magnesium is added thereto, or it becomes a substrate for a clotting enzyme generated by a cascade reaction. It is also possible to use a lysate reagent to which a peptide synthesis substrate, for example, t-butoxycarbonyl-leucyl-glycyl-arginine-paranitroanilide (Boc-Leu-Gly-Arg-pNA) is added. It is also possible to use a commercially available Limulus reagent, and it is also possible to use a kit that adds the above divalent metal salt or peptide synthesis substrate at the time of use.
[0018]
In the case of using a lysate reagent to which a synthetic substrate t-butoxycarbonyl-leucyl-glycyl-arginine-paranitroanilide (Boc-Leu-Gly-Arg-pNA) is added, the reaction amount is the clotting generated by the Limulus reaction. The enzyme can be quantified by measuring the absorbance of paranitroaniline (hereinafter also referred to as “pNA”, a coloring substance) released by hydrolysis of an amide bond of the synthetic substrate. In addition, as other synthetic substrates, Ac-Ile-Glu-Ala-Arg, Boc-Val-Pro- having an amino acid sequence similar to the above-mentioned synthetic substrate having a chromogenic residue such as pNA in the action of the clotting enzyme. A peptide synthesis substrate such as Arg or Boc-Val-Ser-Gly-Arg, or a peptide having the same or similar sequence as this, and a known carboxyl group at the carboxyl group of the amino acid at the C-terminus instead of a chromogenic residue. A substrate in which a fluorescent residue, a luminescent residue, ammonia or the like is substituted by an amide bond can also be used.
[0019]
The reaction parameter values used to accurately reflect the change in the amount of the product of the Limulus reaction and to measure the degree of reaction progress (reaction amount) include the absorbance, turbidity, transmitted light amount, fluorescence polarization, and fluorescence of the reaction solution. In addition to quenching or scattered light, measured values such as pH, conductivity, specific ionization and specific resistance that have changed as a result of the reaction can be used, and reaction parameter values that can be converted and amplified into magnetic or electric signals can also be used. It is possible. Above all, in the method of the present invention, it is more preferable to measure absorbance, turbidity or transmitted light amount.
[0020]
The method for measuring the reaction parameter value is not particularly limited as long as it is a measuring device capable of measuring the reaction parameter value, but a spectrophotometer, an absorption photometer, a fluorometer, a chemiluminescence measurement device, a turbidity measurement And a viscosity measuring device.
[0021]
The measurement conditions of the reaction parameter value only need to appropriately measure the degree of progress of the reaction (reaction amount), and can be appropriately selected by those skilled in the art.
The substance that can be measured by the measurement method of the present invention is not particularly limited as long as it is a substance having reactivity with the lysate reagent, and examples thereof include endotoxin and β-glucan.
[0022]
The measuring method of the present invention is not limited to a measuring method based on a Limulus reaction using a lysate reagent, as long as it is a method for measuring a Limulus reagent-reactive substance such as endotoxin and β-glucan.
[0023]
In the measurement method of the present invention, Logit analysis is used as a condition (calculation formula) for preparing a calibration curve. The Logit analysis method used in the method of the present invention is a method in which measured data of a standard solution of each concentration is Logit-converted by the following calculation formula to create a linear graph, and the calculation formula is:
(1) Logit (Y) = Log (Y / (KY)) = A (Log (X)) + B or Logit (Y) = Log (Y / (KY)) = A (Log ( X * 1000)) + B
(2) Logit (Log (Y)) = Log (Ln (Y * 1000) / (K-Ln (Y * 1000))) = A (Log (X)) + B or Logit (Log (Y)) = Log (Log (Y * 1000) / (K-Log (Y * 1000))) = ALLog (X * 1000) + B,
{Circle around (3)} Logit (Log (Log (Y)) = Log (Ln (Log (Y * 1000)) / (K− (Ln (LogY * 1000))) = ALLog (X * 1000) + B or Logit ( Log (Log (Y)) = Log (Ln (Log (Y * 1000)) / K- (Ln (LogY * 1000))) = A (Log (X)) + B
And so on. In the formula, X is a concentration, Y is a measured value (reaction value), K is a constant, A is a constant (gradient of a calibration curve), B is a constant (intercept with the Y axis), Log is a common logarithm, and Ln is a common logarithm. Shows natural logarithm, where * means multiplication.
[0024]
Note that the general formula is represented by Logit (Y) = Log (Y / (KY)) = A (Log (X)) + B, where Y is a measured value or a conversion value calculated based on the measured value, X indicates the concentration.
[0025]
In the measurement method of the present invention, the correlation coefficient (r) representing the strength of the linear relationship between the two variables of the calibration curve created using the Logit analysis method is 0.9990 to 1.0000, which is extremely accurate. Is high.
[0026]
The measurement method of the present invention merely prepares a single linear calibration curve by the Logit analysis method regardless of the difference between the reaction rate method, the reaction time method, and the colorimetric method and the turbidimetric method. 0002-100 EU / mL, more preferably 0.001-50 EU / mL, while β-glucan has a concentration over a wide range of 0.5-8,000 pg / mL, more preferably 1-4,000 pg / mL. It can be used for measurement of a lysate reagent-reactive substance.
[0027]
Further, in preparing the calibration curve, it is sufficient to use measurement data with at least three different concentrations of the lysate reagent-reactive substance (standard substance), and complicated work such as diluting the specimen is not required. For example, regarding a highly reliable reaction rate method as a measurement data processing method of a measurement method using the Limulus reaction, conventionally, although excellent in sensitivity and quantification, the measurement range is limited, and depending on the sample, complicated measurement such as appropriate dilution is required. There was also a side. However, by using the measurement method of the present invention, the measurement range is significantly widened, the accuracy is high, and labor such as dilution of the sample is not required. Therefore, the operation time is shortened and the cost is reduced, and errors including deactivation due to dilution and adsorption to a container are eliminated, which leads to improvement in accuracy.
[0028]
The setting of the processing conditions of the reaction measurement data and the calculation range of the measurement data can be appropriately set by those skilled in the art according to the type of the target substance and the type of the sample. For example, in a cascade reaction in which the relationship between the reaction elapsed time to reach a certain reaction amount and the reaction amount shows an S-shape, an error due to data of a rising portion near the reaction start and a peak portion near the reaction end. In consideration of the above, it is also possible to use a method described in Japanese Patent Application Laid-Open No. H11-290095, which analyzes the data except for the data of the rising portion and the crest portion.
[0029]
It is also possible to use software for the Logit analysis method (also referred to as Logit-Log analysis method) or various conditions to be set as the method of the present invention, and to use a general enzyme reaction measurement device equipped with such software. is there.
[0030]
The sample that can be measured by the measurement method of the present invention may contain a lysate-reactive substance, and is not particularly limited as long as it is a solution containing the substance that can be measured by the measurement method of the present invention. Examples include infusions, injections, blood (serum, plasma), urine, cerebrospinal fluid, other medicines and medical devices. It is very useful for controlling the concentration and quality of endotoxin and β-glucan in these sample solutions, and diagnosing endotoxinemia, sepsis, mycosis and the like by quantifying these substances.
[0031]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
Example 1 Colorimetric reaction rate method
Blank solution, all 13 different concentrations (0.00024, 0.00047, 0.00094, 0.00188, 0.00375, 0.0075, 0.015, 0) in the range of 0.0002-100 EU / mL. .03, 0.06, 0.12, 1.0, 10.0, 100.0 EU / mL) of endotoxin standard solution (
[0032]
The analysis parameters in the Logit method and the Simple method in Wellreader SK603 were set as follows.
Blank (negative control) Subtraction: "On" indicates that the measured value of the blank solution is subtracted from the measured value of the standard solution or the sample, and "Calc Time" is a calculation when performing a process using continuous absorbance data. It is used for setting the target range, and in the above case, the use of the absorbance data measured from 4 minutes 45 seconds to 30 minutes after the start of the absorbance measurement is shown. Slice Low and Slice High are used to set the lower and upper limits of the absorbance to be calculated when processing using continuous absorbance data is performed.
[0034]
Calibration curves by the Logit analysis method of the present invention and the conventional Simple method are shown in FIGS. 1 and 2, respectively. The Logit analysis method of the present invention showed a very good linearity with a correlation coefficient r = 0.9998. On the other hand, in the conventional Simple method, r = 0.9768, which was extremely poor, and the sample could not be quantified. Note that the calibration curve analysis by the Simple method is an analysis method in which the data is standardized by the least square method based on the measurement data of the standard solution of each concentration, and is represented by the following formula.
[0035]
Y = AX + B
[0036]
In Example 1 (colorimetric reaction rate method), X is the concentration of the standard solution, Y is the measured value (absorbance change rate: mAbs / min), A is a constant (gradient of the calibration curve), and B is It is a constant (intercept with the Y axis in the calibration curve).
[0037]
Further, among the above-mentioned 13 types of endotoxin standard solutions having different concentrations, the measured values of three concentrations of 0.00094, 0.12 and 10 EU / mL were analyzed by the Logit method to obtain a calibration curve, and the other 10 types were used. Quantification was performed using the endotoxin standard solution of the concentration as a specimen. The results are shown in FIG. The calibration curve created based on the measured values of the endotoxin standard solutions at three different concentrations was a very good straight line with r = 1.0000. In addition, the endotoxin concentration of the other 10 different concentrations of the sample calculated from the calibration curve is such that the relative activity with respect to the theoretical value is between 86.1% to 104.6%, and 0.0002 to 100 EU / mL. Was determined to be highly accurate.
[0038]
[Table 1]
[Table 1]
(The relative activity was calculated from the endotoxin concentration of the sample calculated from the calibration curve / the theoretical endotoxin concentration of the sample X100).
[0040]
Example 2: Turbidity reaction time method
Blank solution, all 12 different concentrations in the range of 0.002 to 100 EU / mL (0.0019531, 0.0039062, 0.0078125, 0.015625, 0.03125, 0.0625, 0.125, 0.125. 25, 0.5, 1.0, 10.0, 100.0 EU / mL) of endotoxin standard solution (
[0041]
In addition, the analysis parameters in Wellreader SK603 were set as follows in both the Logit method and the Simple method as in Example 1.
Calibration curves by the Logit method of the present invention and the Simple method, which is a conventional method, are shown in FIGS. 4 and 5, respectively. The Logit analysis method of the present invention shows good linearity with a high accuracy of correlation coefficient r = -0.9994, and it is clear that endotoxin can be quantified in a wide concentration range of 0.002 to 100 EU / mL. On the other hand, in the Simple method, which is a conventional method, the correlation coefficient r is -0.9947, which is considerably low precision, and it is difficult to quantify such a wide range of endotoxin.
[0043]
The calibration curve by the Simple method is represented by Y = AX + B. In Example 2 (turbidimetric reaction time method), X is the concentration of the endotoxin standard solution, Y is the measured value (minute; min), and B is Is a constant (intercept with the Y axis in the calibration curve).
[0044]
Example 3
A β-glucan standard solution (CSBG; (1 → 3) -β-D-glucan derived from the cell wall of Candida albicans IFO1385) at 15 different concentrations in a 8,000-0.5 pg / mL 2-fold serial dilution series was prepared. Prepared. 25 μL of each of the prepared β-glucan standard solution and blank solution was dispensed into predetermined wells of a Toxipet plate, and a β-glucan-specific lysate reagent (Fangitech G-test MK, manufactured by Seikagaku Corporation) was added. Add 100 μL each, cover with lid, attach to Wellreader SK603, measure reaction amount (change of absorbance) by Limulus reaction in Kinetic Assay mode (reaction rate method; Rate Assay), two wavelengths (405-492 nm) at 37 ° C., 40 Automatically measured for minutes. Eight kinds of prepared 4-fold serial dilution series of β-glucan standard solution 0.5, 2.0, 7.8, 31.3, 125.0, 500.0, 2000.0, 8000.0 pg / mL The measured values at different concentrations were analyzed by the Logit method of the present invention and the conventional Simple method, respectively, to prepare a calibration curve. In addition, quantification was performed using β-glucan standard solutions at seven different concentrations as samples.
[0045]
As in Example 1, the analysis parameters in Wellreader were set as follows for both the Logit analysis method and the Simple method.
Calibration curves by the Logit method of the present invention and the conventional Simple method are shown in FIGS. 6 and 7, respectively. According to the Logit analysis method of the present invention, the correlation coefficient of a calibration curve prepared with eight different concentrations of β-glucan standard solution showed very good linearity with high accuracy at r = 0.9999. In the Simple method, r = 0.9141 and the accuracy was considerably low.
[0047]
Table 2-1 (Logit method) and Table 2-2 (Simple method) show the results of calculating the β-glucan concentrations of seven different concentrations of standard solutions (samples) that were not used for preparing the calibration curves from these calibration curves. )Pointing out toungue. As is clear from this table, in the Logit method, the relative activity of the sample with respect to the theoretical value is between 92.6 to 106.6%, and it can be determined with high accuracy from 0.5 to 8,000 pg / mL. However, in the conventional method (Simple method), the relative activity showed a large variation of 15.2 to 489.3% and could not be quantified at all.
[0048]
[Table 2]
[Table 2-1]
(Note that the relative activity was calculated by the theoretical value of the β-glucan concentration of the sample calculated from the calibration curve divided by the theoretical value of the β-glucan concentration of the sample X100.)
[0050]
[Table 3]
[Table 2-2]
(Note that the relative activity was calculated by the theoretical value of the β-glucan concentration of the sample calculated from the calibration curve divided by the theoretical value of the β-glucan concentration of the sample X100.)
[0052]
【The invention's effect】
In the measurement of the lysate reagent-reactive substance using the kinetic method, the measurement method of the present invention using the Logit analysis method as an analysis method (calculation method) for preparing a calibration curve uses a single calibration curve created by the Logit analysis method. Measurement of analyte concentration over a wide concentration range is possible with high accuracy. Further, in preparing the calibration curve in the measurement method of the present invention, it is sufficient that there is at least measurement data of three different standard solutions for preparing the calibration curve. It is easy to use for concentration control and quality check of endotoxin and β-glucan, which are reactive substances of lysate reagent, because errors due to adsorption and deactivation of lysate and reagents that react with standard solutions are greatly reduced. And useful.
[0053]
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a calibration curve prepared by Logit analysis based on measurement data of endotoxin standard solutions of all 13 different concentrations in Example 1, and the vertical axis represents the rate of change in absorbance per minute (mAbs). / Min), and the horizontal axis indicates endotoxin concentration (EU / mL).
FIG. 2 is a calibration curve prepared by the Simple method based on measurement data of endotoxin standard solutions having all 13 different concentrations in Example 1, and the vertical axis represents the rate of change in absorbance per minute (mAbs / min), and the horizontal axis indicates endotoxin concentration (EU / mL).
FIG. 3 is a calibration curve prepared by Logit analysis based on measurement data of three types of endotoxin standard solutions of 0.00094, 0.12, and 10 EU / mL in Example 1; The absorbance change rate per minute (mAbs / min), and the horizontal axis indicates endotoxin concentration (EU / mL).
FIG. 4 is a calibration curve prepared by Logit analysis based on measurement data of endotoxin standard solutions having a total of 12 different concentrations in Example 2, the vertical axis is activation time (min), and the horizontal axis is Indicates endotoxin concentration (EU / mL).
FIG. 5 is a calibration curve prepared by the Simple method based on measurement data of endotoxin standard solutions having a total of 12 different concentrations in Example 2, the vertical axis represents activation time (min), and the horizontal axis represents Shows endotoxin concentration (EU / mL).
FIG. 6 is a calibration curve prepared by the Logit analysis method based on the measurement data of all eight different β-glucan standard solutions in Example 3, and the vertical axis indicates the rate of change in absorbance per minute (mAbs). / Min), and the horizontal axis indicates β-glucan concentration (pg / mL).
FIG. 7 is a calibration curve created by the Simple method based on the measurement data of all eight different β-glucan standard solutions in Example 3, and the vertical axis represents the rate of change in absorbance per minute (mAbs / min), and the horizontal axis indicates β-glucan concentration (pg / mL).
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| US10256385B2 (en) | 2007-10-31 | 2019-04-09 | Cree, Inc. | Light emitting die (LED) packages and related methods |
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