JP4010474B2 - Glycated protein ratio measurement method - Google Patents

Description

【００２０】
＜操作＞
プロテアーゼ溶液を10〜20PU/ml になるように酵素希釈溶液にて溶解し、この液1ml を試験管に取り30℃に加温する。あらかじめ30℃に加温しておいた基質溶液5ml を加え正確に10分後反応停止液5ml を添加し反応を停止する。そのまま30℃30分加温を続け沈殿を凝集させ、東洋ろ紙N0.131(9cm) でろ過を行い、ろ液を得る。ブランク測定はプロテアーゼ溶液 1mlを試験管に取り30℃に加温し、まず反応停止液5ml を添加し続いて基質溶液 5mlを添加後同様に凝集、ろ過を行う。ろ液 2mlを0.55M 炭酸ナトリウム溶液 5ml、3 倍希釈フォリン試薬 1mlを加え30℃、30分反応後 660nmの吸光度を測定する。酵素作用を行った吸光度からブランク測定の吸光度を差し引いた吸光度変化、ΔAを求め、別に作成した作用標準曲線より酵素活性を求める。
【００２１】
＜標準作用曲線作成法＞
約50PU/ml に調整した酵素溶液を希釈し2 〜50PU/ml の一連の希釈倍率を持った酵素溶液を作成し上記操作を行い、得られたΔAを縦軸に希釈倍数を横軸にプロットする。一方L-チロシンを0.2N塩酸に0.01% の濃度に溶解しその1ml に0.2N塩酸10ml加えたものを標準チロシン溶液とする（チロシン濃度9.09μg/ml）。標準チロシン溶液2ml と0.2N塩酸2ml についてそれぞれ上記測定操作を行い、得られたΔAがチロシン18.2μg に相当する。このΔA を前記グラフ上にとり、その点から横軸に垂線を下ろし横軸との交点が10PU/ml に相当する。
【００２２】
本発明に使用しうる糖化アミノ酸に作用する酵素としては、前記プロテアーゼの作用により、被検液に含まれる糖化タンパク質から生成される糖化アミノ酸若しくは糖化ペプチドに有効に作用し、実質的に糖化タンパク質が測定できる酵素であれば如何なるものを用いても良い。例えば、糖化アルブミンを測定対象とする場合には、ε-アミノ基が糖化された糖化アミノ酸若しくはペプチドに作用する酵素が好ましく、また糖化ヘモグロビンを測定対象とする場合には、α-アミノ基が糖化された糖化アミノ酸若しくはペプチドに作用する酵素が好ましい。
【００２３】
ε-アミノ基が糖化された糖化アミノ酸に作用する酵素の例としては、ギベレラ（Gibberella）属またはアスペルギルス（Aspergillus)属（例えばIFO-6365、-4242 、-5710 等）由来フルクトサミンオキシダーゼ、カンジダ（Candida)属由来フルクトシルアミンデグリカーゼ、ペニシリウム（Penicillium)属（例えばIFO-4651、-6581 、-7905 、-5748 、-7994 、-4897 、-5337 等）由来フルクトシルアミノ酸分解酵素、フサリウム（Fusarium）属（例えばIFO-4468、-4471 、-6384 、-7706 、-9964 、-9971 、-31180、-9972 等）由来、アクレモニウム（Acremonium）属由来又はデバリオマイゼス（Debaryomyces）属由来ケトアミンオキシダーゼ等が挙げられる。
【００２４】
α-アミノ基が糖化された糖化アミノ酸若しくはペプチドに作用する酵素の例としては、上記ε-アミノ基が糖化された糖化アミノ酸若しくはペプチドに作用する酵素及びコリネバクテリウム（Corynebacterium)由来の酵素が挙げられ、α-アミノ基が糖化された糖化アミノ酸若しくはペプチドに特異的に作用する酵素の例としてはコリネバクテリウム属由来の酵素が挙げられる。
【００２５】
さらに、α-アミノ基及びε-アミノ基が糖化された糖化アミノ酸若しくはペプチドに作用し、プロテアーゼと共存させた状態でも充分な活性を有する酵素の例としては、遺伝子組み替え型フルクトサミンオキシダーゼ（R-FOD ；旭化成工業社製）が挙げられる。
【００２６】
糖化アミノ酸に作用する酵素の活性は下記の方法にて測定する。
＜＜糖化アミノ酸に作用する酵素の活性測定法＞＞
＜反応液の組成＞
50mM トリス緩衝液 pH7.5
0.03% 4-アミノアンチピリン（以下4-AAと略す。；和光純薬社製）
0.02% フェノール（和光純薬社製）
4.5U/ml パーオキシダーゼ（以下 PODと略す。；シグマ社製）
1.0mM α- カルボベンズオキシ- ε-D- フルクトシル-L- リジン若しくはフルクトシルバリン（ハシバらの方法に基づき合成、精製した。Hashiba H 、J.Agric.Food Chem.24：70、1976。以下 ZFLと略す。）
【００２７】
上記の反応液1ml を小試験管に入れ、37℃−5 分間予備加温した後、適当に希釈した酵素液0.02mlを添加して攪拌し、反応を開始する。正確に10分間反応の後に0.5%のSDSを2ml 添加して反応を停止し、波長500nm の吸光度を測定する(As)。またブランクとして酵素液のかわりに蒸留水0.02mlを用いて同一の操作を行って吸光度を測定する(Ab)。この酵素作用の吸光度(As)と盲検の吸光度(Ab)の吸光度差（As−Ab）より酵素活性を求める。別にあらかじめ過酸化水素の標準溶液を用いて吸光度と生成した過酸化水素との関係を調べ、37℃−1 分間に 1μM の過酸化水素を生成する酵素量を1Uと定義する。計算式を下記に示す。
酵素活性（U/ml）＝（As−Ab）×2.32×酵素の希釈率
【００２８】
本発明に使用しうるグロブリン成分選択的なプロテアーゼ阻害剤は、例えば血液成分のごとき、グロブリン成分及びグロブリン成分以外のタンパク質を含有する被検液に、プロテアーゼをグロブリン成分選択的なプロテアーゼ阻害剤存在下作用せしめ、主にグロブリン成分以外のタンパク質から、糖化アミノ酸に作用する酵素の基質を生じうるグロブリン成分選択的なプロテアーゼ阻害剤であれば、如何なるものを用いても良い。その例として金属イオン、プロテインＡ、プロテインＧなどが挙げられる。
【００２９】
金属イオンとしては、例えば、遷移金属、III 族、IV族の金属イオンが好ましく、遷移金属イオンとしては亜鉛、ニッケル、鉄、銅、コバルトイオンがより好ましく、III 族金属イオンとしてはアルミニウム、ガリウムイオンがより好ましく、IV族金属イオンとしては錫、鉛イオンがより好ましい。さらに金属の毒性、血清との相互作用による沈殿の生成等を考慮すると、アルミニウム若しくはニッケルイオンが最も好ましい。尚、これらの金属イオンは単独若しくは組み合わせて用いても良く、また金属イオンを添加するには、例えば、その金属イオン放出能力のある塩の水溶液を用いれば良い。
【００３０】
本発明の糖化タンパク質割合の定量方法に於ける液組成については、タンパク質定量試薬、プロテアーゼを用いたタンパク質の分解試薬、生成した糖化アミノ酸若しくはペプチドの定量を行う糖化アミノ酸定量試薬を同一反応槽中で使用できるように適宜組み合わせれば良い。
【００３１】
本発明に使用しうるタンパク質定量試薬組成は、公知のタンパク質定量試薬を用いれば良い。例えばアルブミンを定量する目的では、前記BCG 、BCP 、BPB 、MO、チバクロンブルー誘導体若しくはHABA等のアルブミン定量色素を用いることが出来、例えばHABAを用いる場合には pH3.0〜10.0、好ましくは pH4.0〜9.0 に於いて、0.001 〜10% 、好ましくは0.01〜1%の濃度で使用すれば良く、480 〜550nm の吸光度変化を測定し既知の濃度の標準品の発色と比較すればよい。また同様にBCP を用いる場合には、pH 4.0〜8.0 、好ましくはpH 4.5〜7.5 に於いて着色を押さえる界面活性剤、例えばBriJi35 等を0.01〜5%、好ましくは0.05〜1%の共存下、0.0001〜0.2%、好ましくは0.0005〜0.1%で使用すれば良く、600nm 付近の吸光度変化を測定し既知濃度の標準品の発色と比較すれば良い。
【００３２】
また例えばヘモグロビンを定量する目的には、公知のヘモグロビン定量法、例えば、前記メトへモグロビン法、シアンメトへモグロビン法、アザイドメトへモグロビン法、緑色発色団形成法またはオキシへモグロビン法等を用いることが出来、メトヘモグロビン法を用いる場合には、例えばフェリシアン化カリウム等の酸化剤を、シアンメトヘモグロビン法を用いる場合には例えばシアン化カリウム等のシアンイオンを、アザイドメトヘモグロビン法を用いる場合には例えばアジ化ナトリウム等のアザイドを、緑色発色団形成法を用いる場合には例えば非イオン性界面活性剤等の緑色発色団形成試薬を、公知の方法で添加するれば良く、またオキシヘモグロビン法を用いる場合には、例えば検体を蒸留水で希釈しオキシへモグロビンに変換後540nm の吸収を測定ば良い。またヘモグロビンの変性を防ぐ目的で界面活性剤、例えば少なくとも硫酸基を有する界面活性剤、及び／または非イオン性界面活性剤、及び／又は両イオン性界面活性剤を好ましくは0.001 〜10% の濃度で添加すると好ましい。
【００３３】
本発明に使用しうるタンパク質の分解試薬組成としては、使用するプロテアーゼの至適pHを考慮し、反応が効率よく進行するようにpH及びプロテアーゼ濃度を決定し、必要であればその後グロブリン成分選択的なプロテアーゼ阻害剤を有効な濃度になるよう適宜調製して添加すればよい。
【００３４】
例えば前記プロテアーゼタイプXXVII(シグマ社製）はpHが 7〜10付近でタンパク質分解活性が強く、反応のｐＨは7 〜10を選択できる。またプロテアーゼ添加濃度は実際に使用される反応時間中に被検液中の糖化タンパク質を十分に分解し得る濃度で有れば良く、500 〜50万PU/ml が好ましく、1000〜10万PU/ml がより好ましい。さらにグロブリン成分選択的なプロテアーゼ阻害剤として例えばアルミニウムイオンを使用する場合、好ましくは0.05mM以上、実質的にグロブリン成分へのプロテアーゼ作用が無くなる0.3mM 以上がさらに好ましく、また被検液添加時の濁りが2mM 以上から顕著になることから、0.05mM〜2mM が好ましく
、0.1mM 〜1mM がさらに好ましい。
【００３５】
本発明に使用しうる糖化アミノ酸定量試薬組成については、使用する糖化アミノ酸に作用する酵素の至適pHを考慮し、反応が効率よく進行するようにpHを選択しその後、糖化アミノ酸に作用する酵素量を決定すればよい。
例えばR-FOD （旭化成工業社製）を使用する場合、活性を有する領域が pH5.0〜11と広く、反応のpHは 5.0〜11を選択できる。また酵素添加濃度は、使用される反応液中で糖化アミノ酸を十分に検出し得る濃度で有れば良く、0.01〜1000U/mlが好ましく、0.1 〜500U/ml がより好ましい。
【００３６】
また本発明に使用しうる糖化アミノ酸に作用する酵素作用の検出は、例えばデヒドロゲナーゼを用いた場合には補酵素の変化量を、例えば補酵素としてニコチンアミドアデニンジヌクレオチド（以下NAD と略す。）を用た場合には、還元型補酵素である還元型NAD をその極大吸収波長域である340nm 付近の波長にて比色計で直接定量するか、若しくは生じた還元型補酵素を各種ジアフォラーゼ、またはフェナジンメトサルフェート（以下PMS と略す。）、メトキシPMS 、ジメチルアミノベンゾフェノキサジニウムクロライド（メルドラブルー）等の電子キャリアー及びニトロテトラゾリウム、2- (4-インドフェニル)-3- (4-ニトロフェニル)-5-(2、4 ジスルフォフェニル）-2H-テトラゾリウム、モノナトリウム塩（WST-1)〜2-(2- メトキシ-4- ニトロフェニル)-3-(4- ニトロフェニル)-5-(2- メトキシ-4- ニトロフェニル)-3-(4- ニトロフェニル)-5-(2,4- ジスルフォフェニル) -2H-テトラゾリウム、１ナトリウム塩(WST-8)(水溶性テトラゾリウム塩シリーズ１〜８)(以上、同人化学研究所社製) に代表される各種テトラゾリウム塩等の還元系発色試薬を用い間接的に定量してもよく、またこれ以外の公知の方法により直接、間接的に測定してもよい。
【００３７】
また例えばオキシダーゼを用いた場合には、酸素の消費量または反応生成物の量を測定することが好ましい。反応生成物として、例えばフルクトサミンオキシダーゼを用いた場合には反応により過酸化水素及びグルコソンが生成し、過酸化水素及びグルコソン共に公知の方法により直接、間接的に測定する事が出来る。上記過酸化水素の量は、例えばPOD 等を用いて色素等を生成し、発色、発光、蛍光等により定量しても良く、カタラーゼ等を用いてアルコールからアルデヒドを生成せしめて、生じたアルデヒドの量を定量しても良い。
【００３８】
過酸化水素の発色系は、POD の存在下で4-AA若しくは3-メチル-2- ベンゾチアゾリノンヒドラゾン（以下MBTHと略す。）等のカップラーとフェノール等の色原体との酸化縮合により色素を生成するトリンダー試薬、POD の存在下で直接酸化呈色するロイコ型試薬等を用いることが出来る。
トリンダー型試薬の色原体としては、フェノール誘導体、アニリン誘導体、トルイジン誘導体等が使用可能であり、具体例としてN,N ジメチルアニリン、N,N-ジエチルアニリン、2,4-ジクロロフェノール、N-エチル-N- （2-ヒドロキシ-3- スルホプロピル）-3, 5-ジメトキシアニリン（DAOS) 、N-エチル-N- スルホプロピル-3,5ジメチルアニリン（MAPS）、N-エチル-N- （2-ヒドロキシ-3- スルホプロピル）-3,5- ジメチルアニリン（MAOS）、N-エチル-N-(2-ヒドロキシ-3- スルホプロピル)-m-トルイジン（TOOS）、N-エチル-N- スルホプロピル-m- アニシジン（ADPS）、N-エチル-N- スルホプロピルアニリン（ALPS）、N-エチル-N- スルホプロピル-3,5- ジメトキシアニリン（DAPS）、N-スルホプロピル-3,5- ジメトキシアニリン（HDAPS)、N-エチル-N- スルホプロピル-m- トルイジン（TOPS) 、N-エチル-N-(2-ヒドロキシ-3- スルホプロピル）-m- アニシジン（ADOS）、N-エチル-N-(2-ヒドロキシ-3- スルホプロピル）アニリン（ALOS）、N-（2-ヒドロキシ-3- スルホプロピル）-3,5- ジメトキシアニリン（HDAOS)、N-スルホプロピル−アニリン（HALPS)（以上、同人化学研究所社製）等が挙げられる。
【００３９】
またロイコ型試薬の具体例としては、o-ジアニシジン、o-トリジン、3,3 ジアミノベンジジン、3,3,5,5-テトラメチルベンジジン (以上、同人化学研究所社製) 、N-（カルボキシメチルアミノカルボニル）-4,4- ビス（ジメチルアミノ）ビフェニルアミン（DA64）、10-(カルボキシメチルアミノカルボニル）-3,7- ビス（ジメチルアミノ）フェノチアジン(DA67) (以上、和光純薬社製) 等が挙げられる。
【００４０】
さらに蛍光法には、酸化によって蛍光を発する化合物、例えばホモバニリン酸、4-ヒドロキシフェニル酢酸、チラミン、パラクレゾール、ジアセチルフルオレスシン誘導体等を、化学発光法には、触媒としてルミノール、ルシゲニン、イソルミノール、ピロガロール等を用いることが出来る。
カタラーゼ等を用いてアルコールからアルデヒドを生成せしめて、生じたアルデヒドを定量する方法としては、ハンチ反応を用いる方法や、MBTHとの縮合反応により発色させる方法、若しくはアルデヒドデヒドロゲナーゼを用いる方法等が挙げられる。
グルコソンの定量はジフェニルアミン等の公知のアルドース試薬を用いて定量すればよい。
【００４１】
タンパク質定量試薬、タンパク質分解試薬及び糖化アミノ酸発色試薬を組み合わせる手順としては、例えば、タンパク質定量試薬と糖化アミノ酸定量用試薬の検出波長が等しくなるように発色試薬の種類を選択し、続いて、タンパク質定量のシグナルが糖化アミノ酸定量のシグナルに影響を与えないように、プロテアーゼの濃度、タンパク質分解及び糖化アミノ酸発色のpHを決定すれば良い。
タンパク質定量のシグナルが糖化アミノ酸定量のシグナルに影響を与えないように条件を調整するには、十分量のプロテアーゼを添加することにより、糖化アミノ酸定量試薬を添加する前にタンパク質分解反応を終了させ、タンパク質分解反応に伴うタンパク質定量試薬のシグナル変化が一定になるように調整すれば良い。また例えば、BCP 、BCG 、BPB 及びMO等のアルブミン定量試薬は、アルカリ性で激しく着色する色素がある為に、タンパク質定量試薬が糖化タンパク質定量に影響を及ぼさないように注意して反応のpHを選択する必要がある。
【００４２】
また、タンパク質定量試薬と糖化アミノ酸定量試薬の検出波長が等しくなるように設定するには、タンパク質定量色素及び糖化アミノ酸定量色素の吸収極大波長が、同一波長若しくはその近傍であるとよい。近傍とは測定波長において生体成分中のタンパク質及び糖化タンパク質が十分な感度で定量出来る範囲であれば良く、例えばUV〜可視領域で検出可能な色素を用いる場合には、健常者の試料( アルブミン量 3.5〜5.5/dl、ヘモグロビン12〜18g/dl、糖化アルブミン11.6〜16.3％、糖化ヘモグロビン 4.3％〜5.8 ％) から得られる吸光度が、それぞれの試薬において50mAbs以上であればよい。
例えばアルブミン糖化割合を定量する場合、アルブミン定量試薬としてHABAを、タンパク質の分解試薬としてアルブミンに高い分解活性を示すプロテアーゼタイプ XXVII (シグマ社製）を、糖化アミノ酸定量用試薬の主成分としてR-FOD(旭化成工業社製）を選択する事が出来、HABAは480 〜550nm 付近でアルブミンの定量が可能であるから、R-FOD により生じた過酸化水素を比色定量するには、例えば 480〜550nm 付近で十分な感度を有する色素、例えば 400〜630nm に吸収極大をもつ色素 (4-AAとTOOS等；λmax=555nm)の組み合わせを選択すれば良い。またHABAを用いる場合には pH4.0〜9.0 で使用可能であり、この間のpH変動により着色等が見られないために、続くタンパク質の分解試薬は何れのpHに選択しても良く、糖化アミノ酸定量用試薬はR-FOD の作用が強い、 pH5.0〜10.0の範囲で設定可能である。プロテアーゼ濃度としては 500〜50万PU/ml が好ましく、1000〜10万PU/ml がより好ましい。
【００４３】
またHABAの代わりにBCP を用いる場合には、BCP-アルブミンの発色は吸収極大が600nm 付近であり、 550〜630 nmでアルブミンの定量が可能であることから、例えば 550〜630 nm付近で十分な感度を有する色素、例えば 480〜700nm に吸収極大をもつ色素、MBTHとHALP等の組み合わせ (λmax=582nm)を選択すれば良い。また BCPは中性以上で激しく着色するために pH4.5〜7.5 で使用し、続くタンパク質の分解試薬、糖化アミノ酸定量試薬はpH7.5 以下を選択する必要があり、例えば糖化アミノ酸定量試薬は R-FODの至適pHを考慮すると pH5.0〜7.5 の範囲で設定可能である。
またHABAの代わりにBCG を用いる場合には、BCG-アルブミンの吸収極大が630 nm付近であり、530 〜670 nmでアルブミンの定量が可能であるから、例えば 530〜670nm 付近で十分な感度を有する色素、例えば 450〜750 nmに吸収極大をもつ色素、例えば4-AAとMAOS等の組み合わせ (λmax=630nm)を選択すればよい。また BCGはpH5.6 以上で激しく着色するために、続くタンパク質の分解試薬、糖化アミノ酸定量試薬はpH5.5 以下を選択する必要があり、例えば糖化アミノ酸定量試薬はR-FOD の至適pHを考慮すると pH5.0〜5.5 の範囲で設定可能である。
【００４４】
さらに、例えばヘモグロビン糖化割合を定量する場合には、ヘモグロビン定量方法としてオキシヘモグロビン法を、タンパク質の分解試薬としてヘモグロビンに高い分解活性を示すプロテアーゼタイプ XIV（シグマ社製）を、糖化アミノ酸定量用試薬の主成分として R-FOD（旭化成工業社製）を選択する事が出来、オキシへモグロビンは540nm 付近に吸収極大を持つことから、R-FOD により生じた過酸化水素を比色定量するには、例えば 540nm付近で十分な感度を有する色素、例えば 570〜610 nmに吸収極大を持つ色素、4AA とTOOS等の組み合わせ (λm=555)を用いれば良い。またオキシヘモグロビンはアルカリ性ではメト化が起こり吸収が変化することから、界面活性剤の存在下、酸性〜中性付近での測定が好ましく、例えばTritonX-100 0.01〜10% の存在下、pH5.0 〜9.5 付近でヘモグロビンの定量を行えば良く、続くプロテアーゼ反応及び糖化アミノ酸の検出反応も5.0 〜9.5 付近で行うと良い。またプロテアーゼ濃度としては 500〜10万PU/ml が好ましく、1000〜５万PU/ml がより好ましい。
タンパク質定量試薬、タンパク質分解試薬、糖化アミノ酸定量試薬のpHは液状であればそのまま、液状凍結品であれば溶解後、凍結乾燥品であれば蒸留水等に溶解後、市販のpHメーターで測定すればよい。
【００４５】
以上のことから、本発明に於ける糖化タンパク質割合定量用組成物としては、タンパク質定量用試薬、プロテアーゼ、糖化アミノ酸に作用する酵素を含有するものとして調製すれば良く、また、グロブリン成分の影響を受けない糖化タンパク質割合定量用組成物としては、タンパク質定量試薬、プロテアーゼ、グロブリン成分選択的なプロテアーゼ阻害剤及び糖化アミノ酸に作用する酵素を含有するものとして調製すれば良く、例えば液状品及び液状品の凍結物あるいは凍結乾燥品として提供できる。
【００４６】
さらに本発明に基づく糖化タンパク質割合定量試薬組成には、例えば界面活性剤、塩類、緩衝剤、pH調製剤や防腐剤などを適宜選択して添加しても良い。
適宜な添加物において、界面活性剤としてはポリオキシエチレンアルキルエーテル類〔例えばトリトンX-100 、ポリオキシエチレン（23）ラウリルエーテル（以上、ナカライテスク社製) 〕、ポリオキシエチレンソルビタン脂肪酸エステル類（ツイーン20、ツイーン40、ツイーン60、ツイーン80、ツイーン85；以上、関東化学社製）、ポリオキシエチレングリセリン脂肪酸エステル類、ポリオキシエチレンソルビット脂肪酸エステル類、ポリオキシエチレンアルキルフェニルホルムアルデヒド縮合物、ポリオキシエチレンひまし油、ポリオキシエチレンステロール類、ポリオキシエチレンポリオキシプロピレンアルキルエーテル類、ポリオキシエチレンラノリン類、ポリオキシエチレンアルキルアミン・脂肪酸アミド類、ポリオキシエチレンアルキルエーテルリン酸・リン酸塩類、ポリオキシエチレンアルキルエーテル硫酸塩類、ポリグリセリン脂肪酸エステル類、グリセリン脂肪酸エステル類、プロピレングリコール脂肪酸エステル類、ソルビタン脂肪酸エステル類、Ｎアシルアミノ酸塩類、アルキルエーテルカルボン酸塩類、アルキルリン酸塩、Ｎアシルタウリン酸塩、スルホン酸塩、アルキル硫酸、酢酸ベタイン型両性界面活性剤、イミダゾリン型両性界面活性剤、レシチン誘導体（以上、日光ケミカルズ社製）、アデカトール720N、アデカトールB-795、アデカトールSO-120、アデカノールB-795 （以上、旭電化工業社製）、ポリエチレングリコール類、ポリエチレングリコールラウリルエーテル、ポリエチレングリコールイソオクチルフェニルエーテル、ポリプロピレングリコール、ポリビニルアルコール、トリトンX-305 、トリトンX-114 、トリトンX-405 、トリトンWR-1339 （以上、ナカライテスク社製）等の0.01〜10% 、好適には0.05〜5%、各種金属塩類、例えば塩化リチウム、塩化ナトリウム、塩化カリウム、塩化マンガン、塩化コバルト、塩化亜鉛、塩化カルシウム等の1mM 〜5M、好適には10mM〜1M、各種緩衝液、例えばトリス−塩酸緩衝液、グリシン-NaOH 緩衝液、燐酸緩衝液、グッドの緩衝液等の10mM〜2M、好適には20mM〜1M、各種防腐剤、例えばアジ化ナトリウムの0.01〜10% 、好適には0.05〜1%を適宜添加すればよい。
【００４７】
このようにして調製された本発明の糖化タンパク質割合定量用組成物 (試薬) によって、被検液中の糖化タンパク質割合を定量するには、タンパク質定量用試薬に被検液 0.001〜1.0ml を加え、37℃にて反応させ、一定時間後の変化した吸光度を測定し、次いでタンパク質分解試薬 0.001〜1.0ml を加え37℃にてタンパク質分解反応を行い、次いで糖化アミノ酸定量試薬 0.001〜1.0ml を加え37℃にて吸光度変化を測定すれば良い。またタンパク質定量反応は、タンパク質分解反応に比して極めて早いことから、タンパク質定量試薬とタンパク質分解試薬を同時に添加し、素早くタンパク質の定量を行い、同時にタンパク質分解反応を進行させても良く、またタンパク質分解試薬と糖化アミノ酸定量試薬を同時に添加してタンパク質分解反応と糖化アミノ酸定量反応を同時に行っても良い。この場合、既知濃度の対象となるタンパク質濃度及び糖化タンパク質濃度の標準タンパク質を測定した場合の吸光度変化と比較すれば、被検液中の対象となるタンパク質濃度及び対象となる糖化タンパク質濃度を求めることが出来、この割合を取ることにより糖化タンパク質割合を算出する事が出来る。
【００４８】
【発明の実施の形態】
ついで、本発明の実施例を詳しく述べるが、本発明は何らこれにより限定されるものではない。
【実施例１】
アルブミン定量色素に及ぼすｐＨの影響

【００４９】
＜基質溶液＞
HSA 基質溶液；Albumin Human ；Essentially Globulin Free ; 25mg/ml 、糖 化アルブミン率=31.9%、フルクトサミン値＝265 μmol/L 〔シグマ社製；基質溶液中のアルブミンの濃度はアルブミン測定キット（アルブミンII -HA テストワコー；和光純薬社製）にて定量し、糖化アルブミン率は糖化アルブミン測定計（GAA-2000；京都第一科学社製）にて測定し、フルクトサミン値はフルクトサミン測定キット（オートワコー フルクトサミン （和光純薬社製）にて測定した。〕
【００５０】
＜操作＞
反応液1.2ml を試験管にとり0.06mlのHSA 基質溶液若しくは蒸留水を添加する。
攪拌後室温で１分以上放置し分光光度計にて吸収スペクトルを測定する。結果は図１〜３に記載した。
図 1〜3 から分かるように、HABAは pH4.0〜9.0(図１) で、BCG は pH5.5以下（図２）で、BCP は pH4.5〜7.5 （図３）にて使用可能である。また試料の代わりに蒸留水を加えたブランクの吸光度もこの範囲で低いことから、タンパク質定量試薬に直接添加される糖化タンパク質定量試薬のpHも、混合後に、HABAを用いた場合にはpH4.0 〜9.0 に、BCG を用いた場合には pH5.5以下に、BCP を用いた場合には pH4.5〜7.5 になるように調整すればよい。
さらに同様に界面活性剤の存在下ヘモグロビンの540nm の発色を様々なpHにて測定した結果、pH5.0 〜9.5 に於いて使用可能であった。
【００５１】
【実施例２】
アルブミン定量発色に及ぼすプロテアーゼ濃度の影響
＜試薬組成＞
100mM トリス緩衝液 pH8.5
0.017% HABA
166mg/dl HSA （実施例１記載のHSA)
【００５２】
＜操作＞
上記反応液 1.0mlを吸光光度計セルに分注し、37℃に加温する。温度が一定になったところで 545nmの測光を開始し、測光開始後1 分後に濃度の異なるプロテアーゼ溶液（プロテアーゼタイプXXVII ；シグマ社製）0.1ml を添加し継続して545nm の吸光度を測定した。結果を図４に示す。
【００５３】
図４から分かるようにプロテアーゼ最終濃度500PU/ml以上で 600秒 (10分) 以内にアルブミン発色のプロテアーゼ作用による変化が終了し、プロテアーゼ最終濃度500PU/ml以上にて遅くとも10分間後に続く糖化タンパク質測定を行えば良いことが明白であった。
【００５４】
【実施例３】
アルブミン糖化割合の測定１

【００５５】

【００５６】
＜基質溶液＞
実施例１記載のHSA 基質溶液（2.5g/dl)を調製し、0.2 、0.4 、0.6 、0.8 、1.0 倍濃度の試料を作成した。
【００５７】
＜操作＞
上記R-1 （タンパク質定量試薬、タンパク質分解試薬)270μl をセルにとり、37℃にインキュベートし、試料 9μl を添加、攪拌し37℃にて反応を開始した。反応開始後10秒後にタンパク質定量値を求める目的で545nm の吸光度（A1）を測定し、引き続き37℃にてタンパク質分解反応を継続した。反応開始後 270秒(4.5分) 後に545nm の吸光度（A2）を測定し、反応開始後 300秒(5分) 後に上記R-2 を90μl添加、攪拌し、さらに37℃ 300秒 (５分) 間反応を行い、545nm の吸光度（A3）を測定した。同様にブランク試料の測定を行い、A1ブランク、A2ブランク、A3ブランクを測定した。アルブミン定量の吸光度変化 (ΔA(Alb)) はA1−A1ブランク、糖化タンパク質定量の吸光度変化 (ΔA(GA))は（A3−A2) − (A3ブランク−A2ブランク）により計算した。HSA 濃度1.0 倍、0.6 倍及びブランクの反応曲線を図５に、測定結果を図６に示す。
【００５８】
図５から分かるように反応開始直後にアルブミン定量試薬の反応により発色が確認され、その後プロテアーゼの反応によりアルブミンが分解され200 秒近辺ではほぼ試料添加前のレベルに落ち着いていることから、反応開始直後にアルブミン定量を行えば良く、200 秒以降に続く糖化アミノ酸定量を行うことにより、アルブミン定量に由来する吸光度変化の影響を受けないことが明らかであった。また図６から分かるように、HSA 標準品を希釈したサンプルはアルブミン定量 (□) 、糖化アルブミン酸定量（○）共に良好な直線性を示し、連続で同一反応槽中で測定しても問題なく定量が行えることが明確となった。さらに同一の試薬を用いてヒトグロブリン（シグマ社製；コーンフラクションII、III)をヒト血清濃度（1.69mg/ml)になるように溶かし、同様の測定を行った結果、アルブミン定量、糖化アルブミン酸定量共に検出限界以下であり、糖化アルブミンのみを選択的に測定していることが明白であった。
【００５９】
【実施例４】
アルブミン糖化割合の測定２

【００６０】

【００６１】
＜基質溶液＞
実施例１記載のHSA 基質溶液（2.5g/dl)を調整し、0.0 、0.25、0.5 、0.75、1.0 倍濃度の試料を作成した。
【００６２】
＜操作＞
上記R-1 （タンパク質定量試薬、タンパク質分解試薬)270μl をセルにとり、37℃にインキュベートし、試料 9μl を添加、攪拌し37℃にて反応を開始した。反応開始後10秒後にタンパク質定量値を求める目的で600nm の吸光度(A1)を測定し、引き続き37℃にてタンパク質分解反応を継続した。反応開始後4.5 分後に600nm の吸光度(A2)を測定し、反応開始後5 分後に上記R-2 を90μl 添加、攪拌し、さらに37℃５分間反応を行い、600nm の吸光度(A3)を測定した。同様にブランク試料の測定を行い、A1ブランク、A2ブランク、A3ブランクを測定した。アルブミン定量の吸光度変化はA1−A1ブランク、糖化タンパク質定量の吸光度変化は（A3−A2) − (A3ブランク−A2ブランク）により計算した。測定結果を図７に示す。
【００６３】
図７から分かるように、HABA同様 BCPを用いてもHSA 標準品を希釈したサンプルはアルブミン定量（□）、糖化アルブミン酸定量（○）共に良好な直線性を示し、同一反応槽中で測定しても問題なく定量が行えることが明確となった。
【００６４】
【実施例５】
ヘモグロビン糖化割合の測定

【００６５】

【００６６】
＜基質溶液＞
Hb基質溶液；Hemoglobin Human；5.5g/dl 、糖化ヘモグロビン率；HbA1c ＝ 4.5%〔シグマ社製；HbA1c 値は糖化ヘモグロビン計（ハイオートエーワンシーHA-8150 ；京都第一科学社製）にて測定した。〕
Hb基質溶液（5.5g/dl)を調整し、0.2 、0.4 、0.6 、0.8 、1.0 倍濃度の試料を作成した。
【００６７】
＜操作＞
上記R-1 （タンパク質定量試薬、タンパク質分解試薬）540μl をセルにとり、37℃にインキュベートし、試料18μlを添加、攪拌し37℃にて反応を開始した。反応開始後50分後にヘモグロビン定量値を求める目的で545nm の吸光度（A1）を測定し、反応開始後60分後に反応液を排除分子量１万の膜（ウルトラフリーＭＣ；ミリポア社製）で濾過した。糖化アミノ酸の定量は、濾液279μl の吸光度（A2）を測定後、上記R-2 を90μl添加、攪拌し、さらに37℃５分間反応を行い、545nm の吸光度（A3）を測定した。同様にブランク試料の測定を行い、A1ブランク、A2ブランク、A3ブランクを測定した。ヘモグロビン定量の吸光度変化はA1−A1ブランク、糖化タンパク質定量の吸光度変化は（A3−A2）−（A3ブランク−A2ブランク）により計算した。測定結果を図８（ヘモグロビン定量値は吸光度変化に1/10を乗じて表示した。）に示す。
図８から分かるように、アルブミン同様、ヘモグロビン定量（□）、糖化ヘモグロビン定量（○）共に良好な直線性を示し、同一反応槽中で測定しても問題なく定量が行えることが明確となった。
【００６８】
さらに同一の試薬を用いてヒトグロブリン（シグマ社製；コーンフラクションII、III ）をヒト血清濃度（1.69mg/ml ）になるように溶かし、同様の測定を行った結果、ヘモグロビン定量、糖化ヘモグロビン定量共に検出限界以下であり、血清中のグロブリン成分の影響を回避してヘモグロビンが測定されている事が明白であった。
【００６９】
【実施例６】
糖化アルブミンHPLC法と酵素法（本発明）の相関性

【００７０】
健常者及び糖尿病患者血清20検体を用い本発明に基づく酵素法と、公知のHPLC法の相関を確認した。尚HPLC法の測定は、糖化アルブミン計(GAA-2000;京都第一科学社製）にて糖化アルブミン率を測定した。アルブミン糖化割合 は実施例１記載のHSA をキャリブレーターとし、糖化アルブミン定量値／アルブミン定量値から算出した。結果本発明に基づく定量方法から得られる糖化アルブミン割合は、HPLC法糖化アルブミン率と、相関係数ｒ＝0.957 と非常によい相関を示し、本発明に基づく定量方法は糖化アルブミンを正確に定量していることが明らかとなった。
【図面の簡単な説明】
【図１】本発明の実施例１に基づくHABA発色のpH依存性を示す。
【図２】本発明の実施例１に基づくBCG 発色のpH依存性示す。
【図３】本発明の実施例１に基づくBCP 発色のpH依存性示す。
【図４】本発明の実施例２に基づくアルブミン発色変化に及ぼすプロテアーゼ濃度の影響示す。。
【図５】本発明の実施例２に基づく糖化アルブミン測定反応のタイムコース示す。
【図６】本発明の実施例３に基づく糖化アルブミン測定反応のタイムコース示す。
【図７】本発明の実施例４に基づくアルブミン、糖化アルブミンの定量曲線示す。
【図８】本発明の実施例５に基づくヘモグロビン、糖化ヘモグロビンの定量曲線を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for quantifying the ratio of glycated protein to protein and a composition for quantification. More specifically, the present invention relates to a method for quantitatively determining a ratio of a glycated protein to a protein and a composition for quantification, which uses an enzyme, which is simple, rapid, and useful in the field of clinical biochemistry.
[0002]
[Prior art]
In the diagnosis and management of diabetes, measurement of glycated protein is very important, glycated hemoglobin reflecting the average blood glucose level in the past about 1 to 2 months, glycated albumin reflecting the average blood glucose level in the past about 2 weeks, and Fructosamine, which is a general term for glycated proteins that exhibit reducing ability in serum, is routinely measured. Among them, glycated albumin and glycated hemoglobin are shown in terms of the ratio of glycated protein per protein, so there are few individual differences and they are not affected by protein concentration. Measurements are being made.
[0003]
As methods for quantifying glycated proteins, the following methods (a) to (e) and enzyme methods are known.
(a) Chromatographic method [J. Clin. Chem. Clin. Biochem. 19: 81-87 (1981)].
(b) Electrophoresis [Clin.chem. 26: 1958-1602 (1980)].
(c) Immunization [JCCLA 18: 620 (1993)].
(d) A method for measuring fructosamine using the reducing property of glycated protein in alkalinity [Clin. Chem. Acta 127: 87-95 (1982)].
(e) A method using thiobarbituric acid [Clin. Chem. Acta 112: 197-204 (1981)].
The methods (a) and (b) have many problems such as operability, accuracy, and the need for expensive dedicated devices.The method (c) does not necessarily have good accuracy, and the methods (d), (d), The method (e) was affected by the coexisting substances in the specimen and had a problem in specificity.
[0004]
As an accurate, simple and inexpensive quantitative method, there is an enzyme method, and the following methods (f) to (i) are known.
(f) Pronase treatment: a method for measuring fructosamine with fructosylamine deglycase (Japanese Patent Laid-Open No. 6-46846).
(g) Protease treatment and detection with fructosyl amino acid oxidase (Japanese Patent Laid-Open No. 5-192193).
(h) A method of detecting with a lysine residue free reagent-ε-alkyl lysinase (Japanese Patent Laid-Open No. 2-195900).
(i) A lysine residue free reagent-a method for detection with an oxidoreductase using a CH-OH group as a hydrogen donor and NAD and / or NADP as a hydrogen donor (Japanese Patent Laid-Open No. 2-195999).
[0005]
The group of the present inventors also created a substantially pure transformed microorganism that produces an enzyme that acts on glycated amino acids, and efficiently produces fructosylamine oxidase with high thermal stability and reactivity ( JP-A-10-201473) has been developed.
However, while the enzymatic method is simple, inexpensive and accurate, it is necessary to calculate the ratio of glycated protein by separately quantifying the total amount of the protein and dividing the quantitative value obtained by the enzymatic method. There was no example in which quantification and glycated protein quantification were performed in the same reaction vessel.
Furthermore, since there is a large amount of globulin components that are known to vary greatly in the blood due to various diseases, the inventors have avoided the effects of globulin components and other than globulin components. A method (Japanese Patent Application No. 11-231259) for selectively measuring glycated proteins in proteins has been developed.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for quantitatively determining a ratio of glycated protein to a protein useful in clinical biochemical tests, and a quantitative composition for use in the quantitative determination. More specifically, the object of the present invention is to provide a method for easily quantifying the ratio of glycated protein to protein by quantifying the protein and quantifying the glycated product of the protein using an enzymatic method in the same reaction tank, and its The object is to provide a quantitative composition used for quantitative determination.
[0007]
[Means for Solving the Problems]
In order to achieve the above object, protein quantification, protein protease treatment, and glycated amino acid quantification may be performed in the same reaction vessel.
However, the conditions for protein quantification and glycated protein quantification (for example, pH and wavelength for measurement) are different. When a protein is allowed to act on a protein quantification solution, the color of the protein quantification dye changes, and so on. In order to quantitatively determine the proportion of glycated albumin or glycated hemoglobin, the amount of glycated protein that causes a significant effect on the measurement of glycated protein, the coloration depending on the glycated protein quantification condition depending on the protein quantification reagent, and the glycated product in albumin or hemoglobin in the blood From the point that it is necessary to selectively measure only, it is difficult to accurately measure both in the same reaction tank only by a combination of known techniques.
[0008]
Therefore, as a result of intensive studies, the present inventors have made it possible to perform one-wavelength continuous measurement by optimizing the combination of the dye used for protein quantification and the dye used for glycated protein quantification, and adjusting the conditions under which proteases act It is possible to avoid the color change in protein concentration measurement due to protease action, and to avoid abnormal coloration of protein color reagent by optimizing the type of dye for protein quantification and the pH of glycated protein detection I found it. Furthermore, by combining with a method (Japanese Patent Application No. 11-231259) that has been developed by the present inventors to avoid the influence of the globulin component, the glycation ratio of albumin and hemoglobin in the blood is reduced. As a result, the present invention was completed.
[0009]
That is, the present invention has been made to achieve such an object, and is used as a quantitative method and quantitative composition useful for measuring the ratio of glycated protein to protein in clinical biochemical tests.
The present invention relates to a method for measuring a glycated protein ratio, characterized in that the following steps 1) to 3) are performed in the same reaction vessel;
1) Quantification of protein in test solution,
2) Protease treatment of the protein
3) Determination of glycated amino acids using enzymes that act on glycated amino acids
In the present invention, the glycated protein quantitative value may be divided by the protein quantified value to calculate the ratio of glycated protein to protein.
The present invention also relates to a composition for quantitative determination of a glycated protein comprising a protein quantification reagent, a protease, and an enzyme that acts on glycated amino acids.
[0010]
The configuration and preferred embodiments of the present invention will be described in more detail.
As the test solution to be measured in the present invention, any test solution containing at least a glycated protein may be used. Preferably, blood components such as whole blood, blood cells, red blood cells, and hemolyzed blood are used. Serum, plasma or urine.
The protein to be measured in the present invention may be any protein as long as it is useful for clinical examination, but is preferably a protein present in blood, such as albumin or hemoglobin.
[0011]
As a method for quantifying the protein of interest of the present invention, any method may be used as long as it can accurately measure the protein of interest. For example, when the target protein is albumin, any method may be used as long as it is a known method for measuring albumin. Examples of such methods include bromcresol green (hereinafter abbreviated as BCG), bromcresol purple (hereinafter abbreviated as BCP), bromophenol blue (hereinafter abbreviated as BPB), and methyl orange (hereinafter abbreviated as MO). Or a method using an albumin-specific dye such as 2- (4′-hydroxybenzeneazo) benzoic acid (hereinafter abbreviated as HABA). Furthermore, when the target protein is hemoglobin, any method may be used as long as it is a known method for measuring hemoglobin. Examples of such a method include a methemoglobin method, a cyan methemoglobin method, an azide methemoglobin method, a green chromophore formation method, and an oxyhemoglobin method. The green chromophore formation method is a method in which a green chromophore forming reagent and hemoglobin are reacted to form a stable product (green chromophore). The green chromophore is an alkaline substance described in British Patent Publication No. 2052056. It has an absorption spectrum similar to that of hematin D-575.
[0012]
As the protease that can be used in the present invention, any protease can be used as long as it effectively acts on the target protein contained in the test solution, and examples thereof include proteases derived from animals, plants, and microorganisms. Specific examples are shown below. However, these are only examples and are not limited at all.
Examples of animal-derived proteases include elastase, trypsin, chymotripsin, pepsin, bovine pancreatic protease, cathepsin, calpain, protease type-I, -XX (Above, Sigma), aminopeptidase M (Aminopeptidase M), carboxypeptidase A (Carboxypeptidase A) (above, manufactured by Boehringer Mannheim), pancreatin (Pancreatin: manufactured by Wako Pure Chemical Industries, Ltd.) and the like.
Examples of plant-derived proteases include Kallikrein, Ficin, Papain, Chimopapain, Bromelain (above, Sigma), Papain W-40, Bromelain F (above) And Amano Pharmaceutical Co., Ltd.).
[0013]
Examples of the microorganism-derived protease include the following (1) to (14).
(1) Bacillus ( Bacillus ) Genus-derived protease; Subtilisin, Protease-type-VIII, -IX, -X, -XV, -XXIV, -XXVII, -XXXI (above, manufactured by Sigma), thermolysin (above, Jun Wako) Yakuhin Co., Ltd.), Orientase-90N, -10NL, -22BF, -Y, -5BL, Nucleicin (above, made by Hankyu Bioindustry), Pro Leather, Protease-N, -NL, -S "Amano" (above , Amano Pharmaceutical Co., Ltd.), GODO-BNP, -BAP (Purchased by Joint Sake Seisha Co., Ltd.), Protin-A, -P, Deskin, Depirais, Biosoak, Samoaze (Previous: Daiwa Kasei Co., Ltd.), Toyoteam NEP ), Neutase, Esperase, Sabinase, Durazyme, Biofeed Pro, Alcalase, NUE, Pyrase, Clear Lens Pro, Everase, Novozyme-FM, Novoran (above, Novo Nordisk Bioindus Lee), Entilon-NBS, -SA (above, manufactured by Toto Kasei Kogyo Co., Ltd.), Alkaline Protease GL440, Opticlean-M375 Plus, -L1000, -ALP440 (above, manufactured by Kyowa Hakko), Nagase Biolase APL-30, SP-4FG, XL-416F, AL-15FG (above, manufactured by Nagase Seikagaku Corporation), Aroase AP-10, Protease YB, (above, manufactured by Yakult Pharmaceutical Co., Ltd.), Corolase-N, -7089, Belon W (above, manufactured by Higuchi Trading Company), Kirazyme P-1 (Roche).
[0014]
(2) Aspergillus ( Aspergillus) Protease type: Protease types -XIII, -XIX, -XXIII (SIGMA), Sumiteam -MP, -AP, -LP, -FP, -LPL, Enzyme P-3 (Shin Nippon Chemical Industry Co., Ltd.) ), Orientase-20A, -ONS, -ON5, Tetolase S (above, manufactured by Hankyu Bioindustry), Neurase A, Protease-A, -P, -M "Amano" (above, Amano Pharmaceutical) IP enzyme, Morsin F, AO protease (above, Kikkoman), Protin-F, -FN, -FA (above, Daiwa Kasei), Denapsin 2P, Denateam-SA-7, -AP, Denozyme AP (above , Nagase Seikagaku Corporation), Protease YP-SS, Pandidase-NP-2, -P (Yakult), Sakanase (Kaken Pharma), Flavorzyme (Novo Nordisk Bioindustry), Belon PS (made by Higuchi Trading Company) etc.
[0015]
(3) Rhizopath ( Rhizopus ) Protease: Protease type XVIII (manufactured by Sigma), peptidase R, Newase F (manufactured by Amano Pharmaceutical Co., Ltd.), XP-415 (manufactured by Nagase Seikagaku Corporation) and the like.
(4) Penicillium ( Penicillum ) Derived protease; PD enzyme (manufactured by Kikkoman Corporation) and the like.
(5) Streptomyces ( Streptomyces Protease type XIV; also known as Pronase, -XXI (above, Sigma), actinase-AS, -AF (above, Kaken Pharma), Tasinase (Kyowa Hakko), alkalofilicproteinase (Toyobo) etc.
(6) Staphylococcus ( Staphylococcus ) Derived protease; protease type XVII (manufactured by Sigma) and the like.
[0016]
(7) Clostridium ( Clostridium) Derived proteases: Clostripain, nonspesific nutoral proteinase (manufactured by Sigma), etc.
(8) Resolver ( Lysobacter ) Derived protease; endoproteinase Lys-C (manufactured by Sigma) and the like.
(9) Grifola ( Grifola) Derived protease; metalloendopeptidase (manufactured by Sigma), etc.
(10) Yeast ( Yeast) Proteinase A (proteinase A; manufactured by Sigma), carboxypeptidase Y (Carboxypeputidase Y; manufactured by Boehringer Mannheim), etc.
[0017]
(11) Tritylatium ( Tritirachium ) Derived protein; proteinase K (Proteinase K; manufactured by Sigma), etc.
(12) Thermus ( Thermus) Derived protease; aminopeptidase T (AminopeputidaseT; manufactured by Boehringer Mannheim) and the like.
(13) Pseudomonas ( Pseudomonus) Derived protease; endoproteinase Asp-N (Endoproteinase Asp-N; manufactured by Wako Pure Chemical Industries, Ltd.)
(14) Achromobacter ( Achromobacter) Origin protease: Lysylendopeputidase, achromopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.), etc.
[0018]
In addition, when the protein to be measured is albumin, a microorganism-derived protease belonging to the genus Bacillus and Streptomyces is more preferable because it has a large effect on human albumin, and when the protein to be measured is hemoglobin, Bacillus is preferred. Proteases derived from the genera, Aspergillus, Streptomyces, and Trityratium are more preferable because they have a large effect on human hemoglobin.
[0019]
A method for measuring the activity of a protease that can be used in the present invention is shown below.
<< Method for measuring protease activity >>
Proteolytic activity indicating a color corresponding to 1 μg of tyrosine per minute at 30 ° C. under the following measurement conditions is expressed as 1 PU (proteolytic Unit).

[0020]
<Operation>
Dissolve the protease solution in the enzyme dilution solution so as to be 10 to 20 PU / ml, take 1 ml of this solution in a test tube and heat to 30 ° C. Add 5 ml of substrate solution preheated to 30 ° C, and after 10 minutes, add 5 ml of stop solution to stop the reaction. Continue to heat at 30 ° C for 30 minutes to agglomerate the precipitate, and filter with Toyo filter paper N0.131 (9cm) to obtain a filtrate. In the blank measurement, 1 ml of protease solution is placed in a test tube and heated to 30 ° C. First, 5 ml of reaction stop solution is added, then 5 ml of substrate solution is added, and aggregation and filtration are performed in the same manner. Add 2 ml of the filtrate to 5 ml of 0.55 M sodium carbonate solution and 1 ml of 3-fold diluted forin reagent, and react at 30 ° C for 30 minutes. Then, measure the absorbance at 660 nm. The absorbance change, ΔA, obtained by subtracting the absorbance of the blank measurement from the absorbance at which the enzyme action was performed, is obtained, and the enzyme activity is obtained from a separately prepared action standard curve.
[0021]
<Standard action curve creation method>
Dilute the enzyme solution adjusted to about 50 PU / ml to prepare an enzyme solution with a series of dilution ratios of 2 to 50 PU / ml, perform the above operation, and plot the obtained ΔA on the vertical axis and the dilution factor on the horizontal axis To do. On the other hand, L-tyrosine is dissolved in 0.2N hydrochloric acid at a concentration of 0.01%, and 1 ml of this is added with 10 ml of 0.2N hydrochloric acid to obtain a standard tyrosine solution (tyrosine concentration: 9.09 μg / ml). The above measurement procedure is performed for 2 ml of standard tyrosine solution and 2 ml of 0.2N hydrochloric acid, and the obtained ΔA corresponds to 18.2 μg of tyrosine. Taking this ΔA on the graph, a perpendicular line is drawn from the point to the horizontal axis, and the intersection with the horizontal axis corresponds to 10 PU / ml.
[0022]
As an enzyme that acts on a glycated amino acid that can be used in the present invention, by the action of the protease, it effectively acts on a glycated amino acid or a glycated peptide produced from a glycated protein contained in a test solution. Any enzyme that can be measured may be used. For example, when glycated albumin is to be measured, an enzyme that acts on a glycated amino acid or peptide in which the ε-amino group is glycated is preferable, and when glycated hemoglobin is to be measured, the α-amino group is glycated. Enzymes that act on the resulting glycated amino acids or peptides are preferred.
[0023]
Examples of enzymes that act on glycated amino acids whose ε-amino group is glycated include Gibberella ( Gibberella ) Genus or Aspergillus ( Aspergillus) Fructosamine oxidase, Candida (eg, IFO-6365, -4242, -5710 etc.) Candida) Genus fructosylamine deglycase, penicillium ( Penicillium) Fructosyl amino acid-degrading enzyme derived from genus (eg IFO-4651, -6581, -7905, -5748, -7994, -4897, -5337, etc.) Fusarium ) Genus (eg IFO-4468, -4471, -6384, -7706, -9964, -9971, -31180, -9972, etc.), Acremonium ( Acremonium ) Genus or Debariomyces ( Debaryomyces ) Genus-derived ketoamine oxidase and the like.
[0024]
Examples of an enzyme that acts on a glycated amino acid or peptide in which an α-amino group is glycated include an enzyme that acts on a glycated amino acid or peptide in which the ε-amino group is glycated, and Corynebacterium ( Corynebacterium) Examples of enzymes that specifically act on glycated amino acids or peptides in which the α-amino group is glycated include enzymes derived from the genus Corynebacterium.
[0025]
Furthermore, as an example of an enzyme that acts on a glycated amino acid or peptide in which an α-amino group and an ε-amino group are glycated and has sufficient activity even in the presence of a protease, a recombinant fructosamine oxidase (R-FOD) Asahi Kasei Kogyo Co., Ltd.).
[0026]
The activity of an enzyme that acts on a glycated amino acid is measured by the following method.
<< Method for measuring activity of enzyme acting on glycated amino acid >>
<Composition of reaction solution>
50 mM Tris buffer pH 7.5
0.03% 4-aminoantipyrine (hereinafter abbreviated as 4-AA; manufactured by Wako Pure Chemical Industries, Ltd.)
0.02% Phenol (Wako Pure Chemical Industries)
4.5U / ml peroxidase (hereinafter abbreviated as POD; manufactured by Sigma)
1.0 mM α-carbobenzoxy-ε-D-fructosyl-L-lysine or fructosyl valine (synthesized and purified based on the method of Hashiba et al. Hashiba H, J. Agric. Food Chem. 24:70, 1976. (Hereinafter abbreviated as ZFL)
[0027]
Place 1 ml of the above reaction solution in a small test tube, preheat at 37 ° C for 5 minutes, add 0.02 ml of an appropriately diluted enzyme solution, and stir to start the reaction. After exactly 10 minutes of reaction, 2 ml of 0.5% SDS is added to stop the reaction, and the absorbance at a wavelength of 500 nm is measured (As). Further, the absorbance is measured by performing the same operation using 0.02 ml of distilled water instead of the enzyme solution as a blank (Ab). The enzyme activity is determined from the difference in absorbance (As-Ab) between the absorbance of this enzyme action (As) and the absorbance of the blind (Ab). Separately, using a standard solution of hydrogen peroxide in advance, the relationship between the absorbance and the generated hydrogen peroxide is examined, and the amount of enzyme that produces 1 μM hydrogen peroxide per minute at 37 ° C is defined as 1 U. The calculation formula is shown below.
Enzyme activity (U / ml) = (As-Ab) x 2.32 x enzyme dilution rate
[0028]
The globulin component-selective protease inhibitor that can be used in the present invention is, for example, a blood component such as a blood component, and a protease containing a globulin component and a protein other than the globulin component in the presence of a globulin component-selective protease inhibitor. Any protease inhibitor can be used as long as it is a globulin component-selective protease inhibitor that can act and produce a substrate of an enzyme that acts on glycated amino acids mainly from proteins other than the globulin component. Examples thereof include metal ions, protein A, and protein G.
[0029]
As the metal ions, for example, transition metal, group III, and group IV metal ions are preferable. As the transition metal ions, zinc, nickel, iron, copper, and cobalt ions are more preferable. As the group III metal ions, aluminum and gallium ions are preferable. Are more preferable, and the group IV metal ions are more preferably tin and lead ions. Furthermore, aluminum or nickel ions are most preferable in consideration of metal toxicity, precipitate formation due to interaction with serum, and the like. These metal ions may be used alone or in combination, and for adding metal ions, for example, an aqueous solution of a salt capable of releasing metal ions may be used.
[0030]
Regarding the liquid composition in the glycated protein ratio quantification method of the present invention, a protein quantification reagent, a protein degradation reagent using a protease, and a glycated amino acid quantification reagent for quantifying the generated glycated amino acid or peptide are contained in the same reaction tank. What is necessary is just to combine suitably so that it can be used.
[0031]
The protein quantification reagent composition that can be used in the present invention may be a known protein quantification reagent. For example, for the purpose of quantifying albumin, albumin quantification dyes such as BCG, BCP, BPB, MO, cibacron blue derivative or HABA can be used. For example, when HABA is used, pH 3.0 to 10.0, preferably pH 4 In the range of 0.0 to 9.0, it may be used at a concentration of 0.001 to 10%, preferably 0.01 to 1%, and the change in absorbance at 480 to 550 nm may be measured and compared with the color development of a standard product having a known concentration. Similarly, when BCP is used, a surfactant that suppresses coloring at pH 4.0 to 8.0, preferably pH 4.5 to 7.5, such as BriJi35, is used in the presence of 0.01 to 5%, preferably 0.05 to 1%. It may be used at 0.0001 to 0.2%, preferably 0.0005 to 0.1%, and the absorbance change near 600 nm may be measured and compared with the color development of a standard product of known concentration.
[0032]
For example, for the purpose of quantifying hemoglobin, a known hemoglobin quantification method such as the methemoglobin method, cyan methemoglobin method, azide methemoglobin method, green chromophore formation method or oxyhemoglobin method can be used. When using the methemoglobin method, an oxidizing agent such as potassium ferricyanide is used. When using the cyanmethemoglobin method, cyan ion such as potassium cyanide is used. When using the azide methemoglobin method, sodium azide is used. When a green chromophore forming method is used, a green chromophore forming reagent such as a nonionic surfactant may be added by a known method, and when an oxyhemoglobin method is used. For example, after diluting the specimen with distilled water and converting it to oxyhemoglobin 5 What is necessary is to measure the absorption at 40nm. Further, for the purpose of preventing hemoglobin denaturation, a surfactant, for example, a surfactant having at least a sulfate group, and / or a nonionic surfactant, and / or a zwitterionic surfactant is preferably added at a concentration of 0.001 to 10%. Is preferably added.
[0033]
The composition of the protein degradation reagent that can be used in the present invention is determined in consideration of the optimum pH of the protease to be used, and the pH and protease concentration are determined so that the reaction proceeds efficiently. A suitable protease inhibitor may be appropriately prepared and added to an effective concentration.
[0034]
For example, the protease type XXVII (manufactured by Sigma) has a strong proteolytic activity around pH 7 to 10, and the pH of the reaction can be selected from 7 to 10. Further, the protease addition concentration may be a concentration that can sufficiently decompose the glycated protein in the test solution during the reaction time actually used, preferably 500 to 500,000 PU / ml, and 1,000 to 100,000 PU / ml. More preferred is ml. Furthermore, when using, for example, aluminum ions as a globulin component-selective protease inhibitor, it is preferably 0.05 mM or more, more preferably 0.3 mM or more, which substantially eliminates the protease action on the globulin component, and turbidity when a test solution is added. From 2 mM or more, 0.05 mM to 2 mM is preferable.
0.1 mM to 1 mM is more preferable.
[0035]
Regarding the glycated amino acid quantification reagent composition that can be used in the present invention, considering the optimum pH of the enzyme that acts on the glycated amino acid to be used, the pH is selected so that the reaction proceeds efficiently, and then the enzyme that acts on the glycated amino acid. The amount can be determined.
For example, when R-FOD (manufactured by Asahi Kasei Kogyo Co., Ltd.) is used, the active region is wide as pH 5.0 to 11, and the reaction pH can be selected from 5.0 to 11. Moreover, the enzyme addition concentration should just be a density | concentration which can fully detect a glycated amino acid in the reaction liquid used, 0.01-1000 U / ml is preferable and 0.1-500 U / ml is more preferable.
[0036]
The detection of the enzyme action acting on the glycated amino acid that can be used in the present invention is, for example, when dehydrogenase is used, for example, the amount of coenzyme change, for example, nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) as a coenzyme. When used, reduced NAD, which is a reduced coenzyme, is directly quantified with a colorimeter at a wavelength near its maximum absorption wavelength region of 340 nm, or the resulting reduced coenzyme is various diaphorases, or Electronic carriers such as phenazine methosulfate (hereinafter abbreviated as PMS), methoxy PMS, dimethylaminobenzophenoxazinium chloride (Meldra Blue), and nitrotetrazolium, 2- (4-indophenyl) -3- (4-nitrophenyl) ) -5- (2,4 Disulfophenyl) -2H-tetrazolium, monosodium salt (WST-1) to 2- (2-methoxy-4-nitrophenyl) -3- (4- Trophenyl) -5- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, monosodium salt (WST-8) (Water-soluble tetrazolium salt series 1 to 8) (above, manufactured by Dojin Chemical Laboratories Co., Ltd.) You may measure directly and indirectly by the method.
[0037]
For example, when oxidase is used, it is preferable to measure the amount of oxygen consumed or the amount of reaction products. For example, when fructosamine oxidase is used as the reaction product, hydrogen peroxide and glucosone are produced by the reaction, and both hydrogen peroxide and glucosone can be measured directly or indirectly by a known method. The amount of hydrogen peroxide may be determined by, for example, producing dyes using POD or the like, and quantifying by color development, luminescence, fluorescence, or the like, and generating aldehyde from alcohol using catalase or the like. The amount may be quantified.
[0038]
In the presence of POD, the hydrogen peroxide coloring system is formed by oxidative condensation between a coupler such as 4-AA or 3-methyl-2-benzothiazolinone hydrazone (hereinafter abbreviated as MBTH) and a chromogen such as phenol. A Trinder reagent that produces a dye, a leuco-type reagent that directly oxidizes in the presence of POD, and the like can be used.
As the chromogen of the Trinder type reagent, phenol derivatives, aniline derivatives, toluidine derivatives, etc. can be used. Specific examples include N, N dimethylaniline, N, N-diethylaniline, 2,4-dichlorophenol, N- Ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N-sulfopropyl-3,5 dimethylaniline (MAPS), N-ethyl-N- ( 2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS), N-ethyl-N- Sulfopropyl-m-anisidine (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline (DAPS), N-sulfopropyl-3,5 -Dimethoxyaniline (HDAPS), N-ethyl-N-sulfopropyl-m-toluidine (TOPS), N- Tyl-N- (2-hydroxy-3-sulfopropyl) -m-anisidine (ADOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline (ALOS), N- (2-hydroxy- 3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N-sulfopropyl-aniline (HALPS) (manufactured by Doujin Chemical Laboratory Co., Ltd.) and the like.
[0039]
Specific examples of the leuco-type reagent include o-dianisidine, o-tolidine, 3,3 diaminobenzidine, 3,3,5,5-tetramethylbenzidine (above, manufactured by Dojindo Laboratories), N- (carboxy Methylaminocarbonyl) -4,4-bis (dimethylamino) biphenylamine (DA64), 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67) (above, manufactured by Wako Pure Chemical Industries, Ltd.) ) Etc.
[0040]
Furthermore, for fluorescence methods, compounds that emit fluorescence upon oxidation, such as homovanillic acid, 4-hydroxyphenylacetic acid, tyramine, paracresol, diacetylfluorescin derivatives, etc., are used for chemiluminescence methods, and luminol, lucigenin, isoluminol as catalysts. Pyrogallol or the like can be used.
Examples of a method for producing aldehyde from alcohol using catalase or the like and quantifying the generated aldehyde include a method using a hunch reaction, a method of developing a color by a condensation reaction with MBTH, or a method using an aldehyde dehydrogenase. .
The glucosone may be quantified using a known aldose reagent such as diphenylamine.
[0041]
The procedure for combining the protein quantification reagent, proteolysis reagent, and glycated amino acid chromogenic reagent is, for example, selecting the type of chromogenic reagent so that the detection wavelengths of the protein quantification reagent and glycated amino acid quantification reagent are equal, and then protein quantification. The concentration of protease, proteolysis, and pH of glycated amino acid color development may be determined so that the above signal does not affect the glycated amino acid quantification signal.
To adjust the conditions so that the protein quantification signal does not affect the glycated amino acid quantification signal, the proteolytic reaction is terminated before adding the glycated amino acid quantification reagent by adding a sufficient amount of protease, What is necessary is just to adjust so that the signal change of the protein quantitative reagent accompanying a proteolytic reaction may become fixed. In addition, for example, albumin quantification reagents such as BCP, BCG, BPB and MO have alkaline and intensely colored dyes, so the pH of the reaction should be selected carefully so that the protein quantification reagent does not affect glycated protein quantification. There is a need to.
[0042]
In order to set the detection wavelengths of the protein quantification reagent and the glycated amino acid quantification reagent to be equal, the absorption maximum wavelength of the protein quantification dye and the glycated amino acid quantification dye may be the same wavelength or the vicinity thereof. The vicinity means a range in which the protein and glycated protein in biological components can be quantified with sufficient sensitivity at the measurement wavelength.For example, when using a dye that can be detected in the UV to visible region, a sample of a healthy person (albumin content) The absorbance obtained from 3.5 to 5.5 / dl, hemoglobin 12 to 18 g / dl, glycated albumin 11.6 to 16.3%, glycated hemoglobin 4.3% to 5.8%) may be 50 mAbs or more in each reagent.
For example, when quantifying the saccharification rate of albumin, HABA is used as an albumin quantification reagent, protease type XXVII (manufactured by Sigma) showing high degradation activity on albumin as a protein degradation reagent, and R-FOD as a main component of a glycated amino acid quantification reagent. (Asahi Kasei Kogyo Co., Ltd.) can be selected, and HABA can quantitate albumin in the vicinity of 480 to 550 nm. For colorimetric determination of hydrogen peroxide generated by R-FOD, for example, 480 to 550 nm A combination of dyes having sufficient sensitivity in the vicinity, such as a dye having an absorption maximum at 400 to 630 nm (4-AA and TOOS, etc .; λmax = 555 nm) may be selected. In addition, when HABA is used, it can be used at pH 4.0 to 9.0, and since coloring or the like is not observed due to pH fluctuations during this period, the subsequent protein degradation reagent may be selected at any pH. The quantification reagent has a strong R-FOD action and can be set within the pH range of 5.0 to 10.0. The protease concentration is preferably 500 to 500,000 PU / ml, more preferably 1,000 to 100,000 PU / ml.
[0043]
When BCP is used instead of HABA, BCP-albumin has a color absorption maximum of around 600 nm, and albumin can be quantified at 550 to 630 nm. A dye having sensitivity, for example, a dye having an absorption maximum at 480 to 700 nm, a combination of MBTH and HALP (λmax = 582 nm) may be selected. In addition, BCP should be used at pH 4.5 to 7.5 to neutrally and vigorously color, and the subsequent protein degradation reagent and glycated amino acid quantification reagent should be selected to have pH 7.5 or less. -Considering the optimum pH of FOD, it can be set in the range of pH 5.0 to 7.5.
When using BCG instead of HABA, the absorption maximum of BCG-albumin is around 630 nm, and albumin can be quantified at 530 to 670 nm. For example, it has sufficient sensitivity around 530 to 670 nm. A dye, for example, a dye having an absorption maximum at 450 to 750 nm, such as a combination of 4-AA and MAOS (λmax = 630 nm) may be selected. In addition, since BCG is intensely colored at pH 5.6 or higher, it is necessary to select pH 5.5 or lower for the subsequent protein degradation reagent and glycated amino acid quantification reagent.For example, the glycated amino acid quantification reagent has the optimum pH for R-FOD. Considering this, it can be set in the range of pH 5.0 to 5.5.
[0044]
Furthermore, for example, when quantifying the hemoglobin saccharification ratio, the oxyhemoglobin method is used as a hemoglobin quantification method, protease type XIV (manufactured by Sigma) showing high degrading activity for hemoglobin as a protein degradation reagent, and a glycated amino acid quantification reagent. R-FOD (manufactured by Asahi Kasei Kogyo Co., Ltd.) can be selected as the main component, and oxyhemoglobin has an absorption maximum near 540 nm, so the colorimetric determination of hydrogen peroxide produced by R-FOD For example, a dye having sufficient sensitivity near 540 nm, for example, a dye having an absorption maximum at 570 to 610 nm, a combination of 4AA and TOOS (λm = 555) may be used. In addition, since oxyhemoglobin undergoes methemolysis and absorption changes when it is alkaline, it is preferably measured in the presence of a surfactant in the vicinity of acidity to neutrality.For example, in the presence of TritonX-100 0.01 to 10%, pH 5.0 Hemoglobin may be quantified in the vicinity of ˜9.5, and the subsequent protease reaction and glycated amino acid detection reaction may be carried out in the vicinity of 5.0 to 9.5. The protease concentration is preferably 500 to 100,000 PU / ml, more preferably 1,000 to 50,000 PU / ml.
The pH of the protein quantification reagent, proteolysis reagent, and glycated amino acid quantification reagent should be measured with a commercially available pH meter as they are if they are liquid, dissolved if they are liquid frozen products, or dissolved in distilled water if lyophilized products. That's fine.
[0045]
From the above, the composition for quantifying glycated protein in the present invention may be prepared as containing a protein quantifying reagent, a protease, and an enzyme that acts on glycated amino acids. The composition for quantifying the proportion of glycated protein that is not affected may be prepared as containing a protein quantification reagent, a protease, a globulin component-selective protease inhibitor, and an enzyme that acts on glycated amino acids. It can be provided as a frozen product or a lyophilized product.
[0046]
Further, for example, surfactants, salts, buffers, pH adjusters, preservatives, and the like may be appropriately selected and added to the glycated protein ratio determination reagent composition based on the present invention.
In appropriate additives, surfactants include polyoxyethylene alkyl ethers [eg, Triton X-100, polyoxyethylene (23) lauryl ether (manufactured by Nacalai Tesque)], polyoxyethylene sorbitan fatty acid esters ( Tween 20, tween 40, tween 60, tween 80, tween 85; manufactured by Kanto Chemical Co., Inc.), polyoxyethylene glycerin fatty acid esters, polyoxyethylene sorbite fatty acid esters, polyoxyethylene alkylphenyl formaldehyde condensate, polyoxy Ethylene castor oil, polyoxyethylene sterols, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene lanolins, polyoxyethylene alkylamine / fatty acid amides, polyoxyethylene alkyl Ether phosphates / phosphates, polyoxyethylene alkyl ether sulfates, polyglycerol fatty acid esters, glycerol fatty acid esters, propylene glycol fatty acid esters, sorbitan fatty acid esters, N acyl amino acid salts, alkyl ether carboxylates, alkyl Phosphate, N acyl taurate, sulfonate, alkyl sulfuric acid, betaine acetate type amphoteric surfactant, imidazoline type amphoteric surfactant, lecithin derivative (manufactured by Nikko Chemicals), Adekatol 720N, Adecator B-795 , Adecatol SO-120, Adecanol B-795 (above, manufactured by Asahi Denka Kogyo Co., Ltd.), polyethylene glycols, polyethylene glycol lauryl ether, polyethylene glycol isooctyl phenyl ether, polypropylene glycol, poly 0.01-10%, preferably 0.05-5%, various metal salts such as vinyl alcohol, Triton X-305, Triton X-114, Triton X-405, Triton WR-1339 (manufactured by Nacalai Tesque) Lithium chloride, sodium chloride, potassium chloride, manganese chloride, cobalt chloride, zinc chloride, calcium chloride, etc. 1 mM to 5 M, preferably 10 mM to 1 M, various buffers such as Tris-HCl buffer, glycine-NaOH buffer, 10 mM to 2 M, preferably 20 mM to 1 M, such as phosphate buffer and Good's buffer, and various preservatives such as 0.01 to 10%, preferably 0.05 to 1% of sodium azide may be added as appropriate.
[0047]
In order to quantify the glycated protein ratio in the test liquid using the glycated protein ratio quantifying composition (reagent) of the present invention thus prepared, 0.001 to 1.0 ml of the test liquid is added to the protein quantifying reagent. , Measure the changed absorbance after a certain period of time, add 0.001-1.0 ml of proteolytic reagent, perform the proteolytic reaction at 37 ° C, then add 0.001-1.0 ml of glycated amino acid quantification reagent The change in absorbance may be measured at 37 ° C. In addition, protein quantification reaction is extremely fast compared to proteolysis reaction, so protein quantification reagent and proteolysis reagent can be added at the same time, protein quantification can be performed quickly, and protein degradation reaction can proceed at the same time. A proteolytic reaction and a glycated amino acid quantitative reaction may be simultaneously performed by simultaneously adding a degradation reagent and a glycated amino acid quantitative reagent. In this case, the target protein concentration and the target glycated protein concentration in the test solution can be obtained by comparing the change in absorbance when measuring the target protein concentration of known concentration and the standard protein of glycated protein concentration. The ratio of glycated protein can be calculated by taking this ratio.
[0048]
DETAILED DESCRIPTION OF THE INVENTION
Next, examples of the present invention will be described in detail, but the present invention is not limited thereto.
[Example 1]
Effect of pH on albumin quantification dye

[0049]
<Substrate solution>
HSA Substrate Solution; Albumin Human; Essentially Globulin Free; 25 mg / ml, glycated albumin ratio = 31.9%, fructosamine value = 265 μmol / L [manufactured by Sigma; albumin concentration in the substrate solution is determined by albumin measurement kit (albumin II − HA test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) was quantified, the glycated albumin rate was measured with a glycated albumin meter (GAA-2000; manufactured by Kyoto Daiichi Kagaku), and the fructosamine value was measured using a fructosamine measurement kit (Autowaco Fructosamine (Measured by Wako Pure Chemical Industries, Ltd.)]
[0050]
<Operation>
Add 1.2 ml of the reaction solution to a test tube and add 0.06 ml of HSA substrate solution or distilled water.
After stirring, leave at room temperature for 1 minute or longer and measure the absorption spectrum with a spectrophotometer. The results are shown in FIGS.
As can be seen from Figs. 1-3, HABA can be used at pH 4.0-9.0 (Fig. 1), BCG at pH 5.5 or lower (Fig. 2), and BCP at pH 4.5-7.5 (Fig. 3). is there. In addition, since the absorbance of the blank added with distilled water instead of the sample is also low in this range, the pH of the glycated protein quantification reagent added directly to the protein quantification reagent is pH 4.0 when HABA is used after mixing. The pH should be adjusted to ~ 9.0 when BCG is used, and to pH 4.5 or less when BCP is used.
Similarly, the color development of hemoglobin at 540 nm was measured at various pH values in the presence of a surfactant. As a result, it could be used at pH 5.0 to 9.5.
[0051]
[Example 2]
Effect of protease concentration on albumin quantitative color development
<Reagent composition>
100 mM Tris buffer pH 8.5
0.017% HABA
166 mg / dl HSA (HSA described in Example 1)
[0052]
<Operation>
Dispense 1.0 ml of the above reaction solution into an absorptiometer cell and warm to 37 ° C. When the temperature became constant, photometry at 545 nm was started. One minute after the start of photometry, 0.1 ml of protease solutions having different concentrations (protease type XXVII; manufactured by Sigma) were added, and the absorbance at 545 nm was measured continuously. The results are shown in FIG.
[0053]
As can be seen from Fig. 4, the change in albumin coloration due to the protease action is completed within 600 seconds (10 minutes) at a final protease concentration of 500 PU / ml or more, and the glycated protein measurement continues after 10 minutes at the latest at a final protease concentration of 500 PU / ml or more. It was clear that it should be done.
[0054]
[Example 3]
Measurement of saccharification rate of albumin 1

[0055]

[0056]
<Substrate solution>
The HSA substrate solution (2.5 g / dl) described in Example 1 was prepared, and samples with 0.2, 0.4, 0.6, 0.8, and 1.0-fold concentrations were prepared.
[0057]
<Operation>
270 μl of the above R-1 (protein quantification reagent, proteolysis reagent) was placed in a cell, incubated at 37 ° C., 9 μl of sample was added, stirred and the reaction was started at 37 ° C. Absorbance (A1) at 545 nm was measured for the purpose of obtaining a quantitative protein value 10 seconds after the start of the reaction, and the proteolytic reaction was continued at 37 ° C. Absorbance (A2) at 545 nm was measured 270 seconds (4.5 minutes) after the start of the reaction. After 300 seconds (5 minutes) after the start of the reaction, 90 μl of the above R-2 was added, stirred, and further 37 ° C for 300 seconds (5 minutes) The reaction was performed and the absorbance (A3) at 545 nm was measured. Similarly, blank samples were measured, and A1 blank, A2 blank, and A3 blank were measured. Absorbance change (ΔA (Alb)) for albumin quantification was calculated using A1-A1 blank, and absorbance change (ΔA (GA)) for glycated protein quantification was calculated using (A3-A2)-(A3 blank-A2 blank). FIG. 5 shows the response curves of HSA concentrations of 1.0 times, 0.6 times and blanks, and FIG. 6 shows the measurement results.
[0058]
As can be seen from FIG. 5, color development was confirmed by the reaction of the albumin quantification reagent immediately after the start of the reaction, and then albumin was decomposed by the reaction of the protease and settled at a level almost before the sample addition in about 200 seconds. It was clear that albumin quantification could be performed, and that glycated amino acid quantification continued after 200 seconds was not affected by changes in absorbance derived from albumin quantification. In addition, as can be seen from Fig. 6, the HSA standard diluted sample shows good linearity for both albumin quantification (□) and glycated albumin acid quantification (○). It became clear that quantification was possible. Furthermore, human globulin (manufactured by Sigma; Corn Fraction II, III) was dissolved to the human serum concentration (1.69 mg / ml) using the same reagent, and the same measurement was performed. As a result, albumin quantification, glycated albumin acid Both quantitative values were below the detection limit, and it was clear that only glycated albumin was selectively measured.
[0059]
[Example 4]
Measurement of saccharification rate of albumin 2

[0060]

[0061]
<Substrate solution>
The HSA substrate solution (2.5 g / dl) described in Example 1 was prepared, and samples having 0.0, 0.25, 0.5, 0.75, and 1.0-fold concentrations were prepared.
[0062]
<Operation>
270 μl of the above R-1 (protein quantification reagent, proteolysis reagent) was placed in a cell, incubated at 37 ° C., 9 μl of sample was added, stirred and the reaction was started at 37 ° C. Absorbance (A1) at 600 nm was measured for the purpose of obtaining a quantitative protein value 10 seconds after the start of the reaction, and the proteolytic reaction was continued at 37 ° C. Absorbance (A2) at 600 nm is measured 4.5 minutes after the start of the reaction, 90 μl of the above R-2 is added and stirred 5 minutes after the start of the reaction, and further reacted at 37 ° C. for 5 minutes to measure the absorbance (A3) at 600 nm. did. Similarly, blank samples were measured, and A1 blank, A2 blank, and A3 blank were measured. The absorbance change for albumin quantification was calculated using A1-A1 blank, and the absorbance change for glycated protein quantification was calculated using (A3-A2)-(A3 blank-A2 blank). The measurement results are shown in FIG.
[0063]
As can be seen from Fig. 7, the sample diluted with HSA standard product using BCP as well as HABA showed good linearity for both albumin quantification (□) and glycated albumin acid quantification (○). However, it became clear that quantification was possible without problems.
[0064]
[Example 5]
Measurement of glycation rate of hemoglobin

[0065]

[0066]
<Substrate solution>
Hb substrate solution; Hemoglobin Human; 5.5 g / dl, glycated hemoglobin rate; HbA1c = 4.5% [manufactured by Sigma; did. ]
The Hb substrate solution (5.5 g / dl) was prepared, and samples with 0.2, 0.4, 0.6, 0.8, and 1.0 times concentration were prepared.
[0067]
<Operation>
540 μl of the above R-1 (protein quantification reagent, proteolysis reagent) was placed in a cell, incubated at 37 ° C., 18 μl of sample was added, stirred, and the reaction was started at 37 ° C. Absorbance (A1) at 545 nm was measured 50 minutes after the start of the reaction to obtain a hemoglobin quantitative value, and after 60 minutes from the start of the reaction, the reaction solution was filtered through a membrane having an excluded molecular weight of 10,000 (Ultrafree MC; manufactured by Millipore). . The glycated amino acid was quantified by measuring the absorbance (A2) of 279 μl of the filtrate, adding 90 μl of the above R-2, stirring, and further reacting at 37 ° C. for 5 minutes, and measuring the absorbance (A3) at 545 nm. Similarly, blank samples were measured, and A1 blank, A2 blank, and A3 blank were measured. The change in absorbance for hemoglobin quantification was calculated by A1-A1 blank, and the change in absorbance for glycated protein quantification was calculated by (A3-A2)-(A3 blank-A2 blank). The measurement results are shown in FIG. 8 (the hemoglobin quantitative value is displayed by multiplying the change in absorbance by 1/10).
As can be seen from FIG. 8, as with albumin, both hemoglobin quantification (□) and glycated hemoglobin quantification (◯) showed good linearity, and it was clarified that quantification can be performed without problems even when measured in the same reaction tank. .
[0068]
Furthermore, human globulin (Sigma; Corn Fraction II, III) was dissolved to the human serum concentration (1.69 mg / ml) using the same reagent, and the same measurement was performed. As a result, hemoglobin quantification and glycated hemoglobin quantification Both were below the detection limit, and it was clear that hemoglobin was measured avoiding the influence of the serum globulin component.
[0069]
[Example 6]
Correlation between glycated albumin HPLC method and enzyme method (present invention)

[0070]
Correlation between the enzymatic method based on the present invention and a known HPLC method was confirmed using 20 samples of serum from healthy and diabetic patients. The HPLC method was performed by measuring the glycated albumin ratio with a glycated albumin meter (GAA-2000; manufactured by Kyoto Daiichi Kagaku). The saccharification rate of albumin was calculated from the glycated albumin quantitative value / albumin quantitative value using the HSA described in Example 1 as a calibrator. Results The glycated albumin ratio obtained from the quantification method based on the present invention shows a very good correlation with the HPLC glycated albumin rate and the correlation coefficient r = 0.957. The quantification method based on the present invention accurately quantifies glycated albumin. It became clear that.
[Brief description of the drawings]
FIG. 1 shows the pH dependence of HABA color development according to Example 1 of the present invention.
FIG. 2 shows the pH dependence of BCG color development based on Example 1 of the present invention.
FIG. 3 shows the pH dependence of BCP color development according to Example 1 of the present invention.
FIG. 4 shows the effect of protease concentration on albumin color change based on Example 2 of the present invention. .
FIG. 5 shows a time course of glycated albumin measurement reaction based on Example 2 of the present invention.
FIG. 6 shows a time course of a glycated albumin measurement reaction based on Example 3 of the present invention.
FIG. 7 shows quantitative curves of albumin and glycated albumin based on Example 4 of the present invention.
FIG. 8 shows quantitative curves of hemoglobin and glycated hemoglobin based on Example 5 of the present invention.

Claims (16)

A method for measuring a ratio of glycated albumin to albumin, wherein the following steps 1) to 3) are carried out in the same reaction vessel.
1) Step of quantifying albumin in the test solution using 2- (4′-hydroxybenzoazo) benzoic acid within the range of pH 4.0 to 9.0 2) 500 to 500,000 PU / m1 in the test solution Step 3) The signal change in Step 1) is made constant, and the reaction solution in Step 2) is reacted with ketoamine oxidase in the range of pH 5.0 to 10.0 to glycated albumin. Quantifying process The quantification of albumin in step 1) is performed at 480 to 550 nm, and the glycated albumin in step 3) is further quantified with hydrogen peroxide generated by reacting glycated albumin with ketoamine oxidase at 480 to 550 nm. It has a such sensitivity, and carried out by quantifying using a dye that have a maximum absorption at 400～630Nm, the ratio method of measurement for albumin glycated albumin according to claim 1. The method for measuring the ratio of glycated albumin to albumin, wherein the following steps 1) to 3) are carried out in the same reaction vessel:
1) Step of quantifying albumin in the test solution using bromocresol green within a pH range of 5.5 or lower 2) Addition of 500 to 500,000 PU / m1 protease to the test solution, signal change in step 1) 3) A step of quantifying glycated albumin by causing ketoamine oxidase to act on the reaction solution of 2) above in the range of pH 5.0 to 5.5. The quantification of albumin in step 1) is carried out at 530 to 670 nm, and the glycated albumin in step 3) is further quantified with hydrogen peroxide generated by reacting glycated albumin with ketoamine oxidase at 530 to 670 nm. It has a sensitivity, and with a dye to have a maximum absorption at 450~750nm performed by quantitatively measuring method of percentage of albumin glycated albumin according to claim 3. A method for measuring a ratio of glycated albumin to albumin, wherein the following steps 1) to 3) are carried out in the same reaction vessel.
1) Step of quantifying albumin in a test solution using bromocresol purple within a range of pH 4.5 to 7.5 2) Add 500 to 500,000 PU / ml brothase to the test solution, and step 1 Step 3) of making signal change constant in step 3) Step of quantifying glycated albumin by allowing ketoamine oxidase to act on the reaction solution in 2) above in the range of pH 5.0 to 7.5 The quantification of albumin in step 1) is performed at 550 to 630 nm. Further, the quantification of glycated albumin in step 3) is sufficient for hydrogen peroxide generated by reacting glycated albumin with ketoamine oxidase at 550 to 630 nm. It has a sensitivity, and with a dye to have a maximum absorption at 480~700nm performed by quantitatively measuring method of percentage of albumin glycated albumin according to claim 5.   The measurement method according to claim 1, wherein the determination of albumin and the determination of glycated albumin are performed at the same wavelength. The method for measuring the ratio of glycated hemoglobin to hemoglobin, wherein the following steps 1) to 3) are carried out in the same reaction vessel.
1) the hemoglobin in the test solution, surface active agent having at least sulfuric acid group, and / or non-ionic surfactants, and / or the presence of a zwitterionic detergent, with o alkoxy hemoglobin method, Step 2) Quantifying in the range of pH 5.0 to 9.5 Step 2) Adding 500 to 500,000 PU / ml protease to the test solution to make the signal change in step 1) constant 3) Above 2 ) Quantifying glycated hemoglobin by allowing ketoamine oxidase to act on the reaction solution in the range of pH 5.0 to 9.5.   The method according to claim 8, wherein the quantification of hemoglobin and the glycated hemoglobin are quantified at the same wavelength. 1) 2- (4′-hydroxybenzoazo) benzoic acid, and a buffer solution having a pH of 4.0 to 9.0 at the time of determination ;
2) 500 to 500,000 PU / ml protease;
3) Ketoamine oxidase, a buffer solution having a pH of 5.0 to 10.0 at the time of quantification , and a dye for glycated albumin quantification;
A composition for quantifying albumin and glycated albumin in the same reaction tank. Glycated albumin quantitative dye, it has sufficient sensitivity 480～550N m, and the composition of claim 10 which is a dye that have a maximum absorption at 400～630Nm. 1) Bromocresol green and a buffer solution with a pH of 5.5 or less at the time of determination ;
2) 500 to 500,000 PU / ml protease;
3) Ketoamine oxidase, a buffer solution having a pH of 5.0 to 5.5 at the time of quantification , and a dye for glycated albumin quantification;
A composition for quantifying albumin and glycated albumin in the same reaction tank. Glycated albumin quantitative dye, it has sufficient sensitivity 530～670N m, and the composition of claim 12 is a dye that have a maximum absorption at 450 to 750 nm. 1) Bromocresol purple and a buffer solution having a pH of 4.5 to 7.5 at the time of determination ;
2) 500 to 500,000 PU / ml protease;
3) Ketoamine oxidase, a buffer solution having a pH of 5.0 to 7.5 at the time of quantification , and a dye for glycated albumin quantification;
A composition for quantifying albumin and glycated albumin in the same reaction tank. Glycated albumin quantitative dye, it has sufficient sensitivity 550～630N m, and the composition of claim 14 which is a dye that have a maximum absorption at 480 to 700 nm. 1) O carboxymethyl hemoglobin quantitation dye consisting of coloring agent used in the hemoglobin method, surface active agent having at least sulfuric acid group, and / or non-ionic surfactants, and / or zwitterionic surfactants, and during quantification A buffer solution with a pH of 5.0 to 9.5;
2) 500 to 500,000 PU / ml protease;
3) a ketoamine oxidase, buffer pH during quantification is 5.0 to 9.5, sugar hemoglobin quantitative dye, and a surfactant having at least sulfuric acid group, and / or non-ionic surfactant, And / or zwitterionic surfactants ;
A composition for quantifying hemoglobin and glycated hemoglobin in the same reaction vessel containing

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