JP4260541B2 - Test piece for measuring glycated protein - Google Patents
Test piece for measuring glycated protein Download PDFInfo
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- JP4260541B2 JP4260541B2 JP2003133578A JP2003133578A JP4260541B2 JP 4260541 B2 JP4260541 B2 JP 4260541B2 JP 2003133578 A JP2003133578 A JP 2003133578A JP 2003133578 A JP2003133578 A JP 2003133578A JP 4260541 B2 JP4260541 B2 JP 4260541B2
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Description
【０００１】
【発明の属する技術分野】
本発明は、診療の現場で、正確、簡便かつ迅速に試料中の糖化タンパク質を測定するための試験片、試験片の製造方法、試験片を用いた試験用具、及びそれを用いた糖化タンパク質の測定方法に関する。
【０００２】
【従来の技術】
糖尿病の診断及び管理及びおよび予防を行う上で糖化タンパク質及び糖化脂質の測定は非常に重要である。中でも糖化ヘモグロビン、糖化アルブミンは血糖コントロール状態を正確に反映することから臨床の現場でなくてはならない指標として多用されている。これらの糖化タンパク質、糖化脂質の定量法としては、通常電気泳動法、イオン交換クロマトグラフィ法、アフィニティクロマトグラフィ法、免疫法及び酵素法などが知られている。近年では大量検体を迅速、大量、正確、安価に測定できることから酵素法が多用され始めている。酵素法としては糖化たんぱく質に存在するケトアミンを測定する方法がもっとも多く用いられており、本発明者等もケトアミンオキシダーゼを用いた糖化アルブミンの測定方法を開発してきた（特許文献1、2、3）。
【０００３】
これまで知られている高分子中のケトアミンを測定する方法は大型の生化学自動分析計を用いた方法が主流である。しかし生化学自動分析装置は高価であり、少量の検体しか分析する必要のない診療所や中小規模の病院では使用することは困難である。そこで安価、簡便に高分子中のケトアミンを測定する装置が望まれている。これまで糖化ヘモグロビン及び糖化アルブミンの診療現場で安価に測定できる酵素法を用いた装置は知られていない。
また、これまでプロテアーゼを試験片に保持させてタンパク質を分解し、その分解断片を測定した例もない。
【０００４】
特許文献1) 特開2001-54398号公報
特許文献2) 特開2001-204495公報
特許文献3) WO 02/061119公報
【０００５】
【発明が解決しようとする課題】
本発明の課題は、診療の現場で、酵素を用いて正確、簡便、安価に糖化タンパク質を測定するための少なくともプロテアーゼを含有する新規な試験片、試験片の製造方法、試験用具、及びそれを用いた糖化タンパク質の測定方法を提供することにある。さらに詳しくは、臨床生検査、特に糖化ヘモグロビン、糖化アルブミンの測定に有用な試験片、試験片の製造方法、試験用具、それを用いた糖化タンパク質の測定方法を提供することにある。
【０００６】
【課題を解決するための手段】
一般的な酵素例えばグルコースオキシダーゼ等は乾燥した試験片上に保持されていても、サンプル中の水分で溶解されて直ぐに反応を開始できる。これは基質が低分子であり、溶解直後でも比較的簡単に基質と結合し酵素反応を行えるからだと考えられる。一方プロテアーゼは基質がタンパク質等の高分子であり、溶解直後簡単に基質を断片化できるとは通常考えにくい。本発明者らの検討によると、やはり想像していたとおり溶解直後のプロテアーゼ反応はほとんど反応が進まないことが分かった。
【０００７】
そこで本発明者らはこのプロテアーゼ試験片の条件検討を鋭意検討の結果、意外にも、ある一定量以上のプロテアーゼ量が存在すれば液系とほぼ同じ速度で反応が進行することを見出した。更に本発明者らは、糖化アミノ酸に作用する酵素を同時に試験片に保持させても糖化アミノ酸に作用する酵素がプロテアーゼとの共存により失活することなく糖化タンパク質が定量できること、タンパク定量試薬を含ませた試験片と組み合わせることで糖化タンパク質割合が簡便に測定できること、試験片は特に溶媒等を用いなくとも液体の試料をしみこませるだけで酵素等が溶解し即座に反応が進行すること、試験片の発色は光学反射層を利用した反射光の測定により簡便に行えることを見出し本発明の完成に至った。
【０００８】
すなわち、本発明は、次の試験片、その製造方法、それを使用する試験用具及びそれを用いた糖化タンパク質の測定方法に関する；
1) 少なくともプロテアーゼを含有する糖化タンパク質測定用試験片。
2) 少なくともプロテアーゼ及び糖化アミノ酸に作用する酵素を含有する糖化タンパク質測定用試験片。
3) 試験片1cm2あたりのプロテアーゼ含有量が5U以上であることを特徴とする1)または2)の試験片。
4) 試験片1cm2あたりの糖化アミノ酸に作用する酵素の含有量が10mU以上であることを特徴とする2)または3)のいずれかに記載の試験片。
【０００９】
5) 少なくともプロテアーゼを含有する溶液に試験片を浸し、乾燥することを特徴とする糖化タンパク質測定用試験片の製造方法。
6) 少なくともプロテアーゼ及び糖化アミノ酸に作用する酵素を含有する溶液に試験片を浸し、乾燥することを特徴とする糖化タンパク質測定用試験片の製造方法。
7) 少なくともプロテアーゼを含有する試験片を使用した糖化タンパク質測定用試験用具。
8) 少なくともプロテアーゼ及び糖化アミノ酸に作用する酵素を含有する試験片を使用した糖化タンパク質測定用試験用具。
9) 少なくともプロテアーゼ及び糖化アミノ酸に作用する酵素を含有する試験片及びタンパク質発色試薬を含有する試験片を使用した糖化タンパク質測定用試験用具。
10) 光学測定用の孔を有することを特徴とする7)〜9) のいずれかに記載の試験用具。
【００１０】
11）真中のシートに溝が形成され、その溝が毛細管となるように積層された3層のシートよりなり、その最上のシートに測定孔が形成され、測定孔の下で真中のシートの溝の上にプロテアーゼまたはプロテアーゼと糖化アミノ酸に作用する酵素を含有させた試験片が存在している糖化蛋白質測定用検出器具。
12) 少なくともプロテアーゼを含有する試験片を用いた糖化タンパク質の測定方法。
13) 少なくともプロテアーゼ及び糖化アミノ酸に作用する酵素を含有する試験片を用いた糖化タンパク質の測定方法。
14) 少なくともプロテアーゼ及び糖化アミノ酸に作用する酵素を含有する試験片及びタンパク質発色試薬を含有する試験片を用いた糖化タンパク質の割合の測定方法。
15) 試験片の酵素反応を試料中の水分によって行うことを特徴とする12)〜14) のいずれかに記載の測定方法。
16) 試験片の発色を反射光を用いて測定することを特徴とする12)〜15) のいずれかに記載の測定方法。
17) 糖化タンパク質がアルブミン若しくはヘモグロビンの糖化物であることを特徴とする12)〜16) のいずれかに記載の測定方法。
【００１１】
本発明によると、診療の現場で、酵素を用いて正確、簡便、安価に糖化タンパク質を測定することができる。さらに詳しくは、臨床生検査において、特に糖化ヘモグロビン、糖化アルブミンの測定に有用な試験片、試験片の製造方法、試験用具、それを用いた測定方法を提供することができる。
【００１２】
以下、本発明の構成及び好ましい形態について更に詳しく説明する。
本発明に用いることができるプロテアーゼはタンパク質、例えばアルブミンやヘモグロビンに作用して糖化アミノ酸若しくは糖化アミノ酸を含むペプチドを切り出すプロテアーゼであればいかなるプロテアーゼを用いても良い。また、酵素は目的とする活性が発現すれば精製物であっても粗精製物であっても良い。
【００１３】
本発明に使用し得るプロテアーゼの好ましい例としては、例えばトリプシン(Tripsin)、キモトリプシン(Chymotripsin)等の動物由来のプロテアーゼ、パパイン(Papain)、ブロメライン(Bromelain)等の植物由来のプロテアーゼ、微生物由来のプロテアーゼ等が挙げられる。
微生物由来のプロテアーゼの例としては、ズブチリシン(Subtilisin)等に代表されるバチルス(Bacillus)属由来プロテアーゼ、プロテアーゼタイプ-XIII（シグマ社製）等に代表されるアスペルギルス(Aspergillus)由来プロテアーゼ、PD酵素（キッコーマン社製）等に代表されるペニシリウム(Penicillium)由来プロテアーゼ、プロナーゼ(Pronase) 等に代表されるストレプトマイセス(Streptomyces)由来プロテアーゼ、エンドプロテイナーゼLys-c（シグマ社製）等に代表されるリソバクター(Lysobacter)由来プロテアーゼ、プロテイナーゼA(Proteinase A;シグマ社製) 等に代表される酵母(Yeast)由来プロテアーゼ、プロテイナーゼK(Proteinase K;シグマ社製)等に代表されるトリチラチウム(Tritirachium)由来プロテアーゼ、アミノペプチダーゼT(Aminopeptidase T;ベーリンガー・マンハイム社製)等に代表されるサーマス(Thermus)由来プロテアーゼ、エンドプロテイナーゼAsp-N(EndoproteinaseAsp-N;和光純薬社製)等に代表されるシュードモナス(Pseudomonus)由来、リジルエンドペプチダーゼ(Lysylendopeputidase和光純薬社製)等に代表されるアクロモバクター(Achromobacter)由来プロテアーゼが挙げられる。これらの具体的な例は１例に過ぎず、なんら限定されるものではない。
【００１４】
また測定対象が糖化アルブミンある場合にはバチルス属及びストレプトマイセス属の微生物由来プロテアーゼがヒトアルブミンに対する作用が大きいためより好ましく、また測定対象が糖化ヘモグロビンである場合にはバチルス属、アスペルギルス属、ストレプトマイセス属、トリチラチウム属由来のプロテアーゼがヒトヘモグロビンに対する作用が大きいため好ましい。
【００１５】
プロテアーゼの活性測定法はカゼインフォリン法を用いた。活性の定義は、１分間−37℃において1μgのチロシンに相当する発色を1Uとした。
また本発明のプロテアーゼの使用に関しては、プロテアーゼを単独で使用することはもちろんであるが、他のエンドプロテアーゼ、または他のエキソプロテアーゼを同時に使用しても良い。
【００１６】
プロテアーゼを含む試薬の試験片への保持は、プロテアーゼを含む液状の試薬を作成し、その液に試験片を例えば室温好ましくは冷蔵にて、0.1分〜１日、好ましくは１分から８時間程度浸し、乾燥させればよい。乾燥方法は常圧、減圧条件で行えば良く、温度は例えば 4℃〜60℃で１分〜２日程度行えばよいが、好ましくは酵素類が失活しにくい 4℃〜40℃程度が好ましい。乾燥のスピードを早める方法としては風を当てる、湿度の低い環境を選ぶ等の方法があるが、一般的には湿度の低い冷暗所で乾燥させれば十分である。
【００１７】
プロテアーゼを含む試薬を試験片に保持させる場合の、プロテアーゼを含む液状試薬中のプロテアーゼ濃度は125U/ml以上の濃度の試薬1mlに5cm×5cmの試験片を浸す程度で良く、この場合全ての試薬が吸収されたとすると試験片1cm2あたりのプロテアーゼ含有量は5U以上となる。またプロテアーゼの濃度は試験片1cm2あたりのプロテアーゼ含有量は5U以上であればいくらでも良いが、バッククラウンドの上昇やコストを考えると試験片1cm2あたりのプロテアーゼ含有量は10KU以下が好ましい。
【００１８】
プロテアーゼを含む液状試薬の pH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を選択すればよい。例えばプロテアーゼにプロテアーゼタイプXXVII (シグマ社製)を用いた場合には、プロテアーゼタイプXXVIIは pH7〜10付近で蛋白質分解活性が強いことから反応のpHは7 〜10を選択できる。
【００１９】
本発明に使用しうる試験片としては、シート状の物であればどの様な試験片を用いても良いが、例えば紙、プラスチックシート、不織布等が用いることができる。またその性質としては吸水性に富み十分なプロテアーゼ量を保持できる物であれば何れの物を用いても良い。
また、試験片の厚みとしては0.1mmから2mm程度であれば良く、0.2mm〜1mm程度が好ましく、実際に１試料を測定するためには0.001cm2〜5cm2の面積があれば良く、より好ましく0.005cm2〜2cm2程度である。
【００２０】
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、糖化アミノ酸のケトアミン構造を認識して作用するデヒドロゲナーゼ、キナーゼ、オキシダーゼ等があげられるが、もっとも安価に大量に入手できるオキシダーゼが好ましい。
【００２１】
また、糖化アミノ酸に作用する酵素としては、糖化アミノ酸又は糖化アミノ酸を含むペプチドのごとき低分子糖化アミンに良好に作用する酵素であれば如何なるものを用いても良いが、目的とする測定対象がヘモグロビンA1cである場合はαアミノ基が糖化されたアミノ酸に効率的に作用する酵素が好ましく、αアミノ基が糖化されたアミノ酸に特異的に作用しεアミノ基が糖化されたアミノ酸には実質的に作用しない酵素が最も好ましい。
【００２２】
また一般にαアミノ基ミノ基が糖化されたアミノ酸に特異的に作用しεアミノ基が糖化されたアミノ酸には実質的に作用しない酵素は安定性が悪いことから、安定性の高いεアミノ基及びαアミノ基が糖化された糖化アミノ酸両方に良く作用する酵素を用いて測定しても良く、さらに安定性が高いεアミノ基が糖化されたアミノ酸に特異的に作用する酵素を用いてεアミノ基が糖化されたアミノ酸のみを消去し、安定性の高いεアミノ基及びαアミノ基が糖化された糖化アミノ酸両方に良く作用する酵素を用いてαアミノ基が糖化されたアミノ酸のみを測定しても良い。
【００２３】
一方目的とする測定対象が糖化アルブミンある場合はεアミノ基が糖化されたアミノ酸に効率的に作用する酵素が好ましく、εアミノ基が糖化されたアミノ酸に特異的に作用しαアミノ基が糖化されたアミノ酸には実質的に作用しない酵素が最も好ましい。
【００２４】
また安定性の高いεアミノ基及びαアミノ基が糖化された糖化アミノ酸両方に良く作用する酵素を用いて測定しても良く、さらにαアミノ基が糖化されたアミノ酸に特異的に作用する酵素を用いてαアミノ基が糖化されたアミノ酸のみを消去し、安定性の高いεアミノ基及びαアミノ基が糖化された糖化アミノ酸両方に良く作用する酵素を用いてαアミノ基が糖化されたアミノ酸のみを測定しても良い。
【００２５】
特異性の点から、最も好ましいケトアミン構造を認識する酵素の例としては、εアミノ基が糖化されたアミノ酸には作用しないαアミノ基糖化アミノ酸特異的な酵素、例えばフルクトシルアミノ酸オキシダーゼ（FAOD）：コリネバクテリウム（Corynebacterium） 属由来（FERM P-8245）があげられる。一方εアミノ基及びαアミノ基が糖化された糖化アミノ酸両方に良く作用する酵素であり安定性が高い酵素としてはギベレラ（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）属由来又はデブリオマイゼス（Debaryomyces）属由来ケトアミンオキシダーゼ等のケトアミン構造を認識する酵素が挙げられ、さらに好ましい例としてはプロテアーゼと共存した状態でも十分な活性を有する、遺伝子組み替えケトアミンオキシダーゼ（旭化成社製）が挙げられる。
【００２６】
αアミノ基が糖化されたアミノ酸には作用しないが、εアミノ基糖化アミノ酸特異的な酵素としては、遺伝子改変で作成された遺伝子改変ケトアミンオキシダーゼ（旭化成社製）が挙げられる。
【００２７】
糖化アミノ酸に作用する酵素の活性は糖化Ｚリジン若しくは糖化バリン（ハシバらの方法に従って合成、精製した。（Hashiba H,J.Agric.Food Chem.24:70,1976））より、37℃、1分間に1μmolの過酸化水素を生成する酵素量を1Ｕ定義した。
【００２８】
プロテアーゼ及び糖化アミノ酸に作用する酵素を含む試薬の試験片への保持は、前記プロテアーゼを含む試薬の試験片への保持と同じ方法を用いればよい。乾燥のスピードを早める方法も同様である。
【００２９】
また、プロテアーゼ及び糖化アミノ酸に作用する酵素を含む試薬を試験片に保持させる場合の、プロテアーゼ及び糖化アミノ酸に作用する酵素を含む試薬液中のプロテアーゼ濃度、糖化アミノ酸に作用する酵素の濃度はそれぞれ125U/ml以上、250mU/ml以上の濃度の試薬1mlに5cm×5cmの試験片を浸す程度で良く、この場合全ての試薬が吸収させたとすると試験片1cm2あたりのプロテアーゼ含有量は5U以上、糖化アミノ酸に作用する酵素の量は10mU以上となる。またプロテアーゼの濃度は試験片1cm2あたりのプロテアーゼ含有量は5U以上であればいくらでも良いが、バッククラウンドの上昇やコストを考えると試験片1cm2あたりのプロテアーゼ含有量は10KU以下が好ましい。糖化アミノ酸に作用する酵素の濃度は1cm2あたり10mU以上であればいくらでも良いが、コストを考えると試験片1cm2あたりのプロテアーゼ含有量は50U以下が好ましい。
【００３０】
プロテアーゼ及び糖化アミノ酸に作用する酵素を含む液状試薬のpHは、使用するプロテアーゼ及び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を選択すればよい。例えばプロテアーゼにプロテアーゼタイプXXVII (シグマ社製)を用いた場合には、プロテアーゼタイプXXVIIは pH7〜10付近で蛋白質分解活性が強いことから反応の pHは7 〜10が好ましく、糖化アミノ酸に作用する酵素として遺伝子改変ケトアミンオキシダーゼ（旭化成社製）を用いた場合には最大活性の50%以上の活性を示す領域が pH6.5〜10と広く、反応の pHは6.5〜10が好ましく、両者を比較すると pH7〜10が選択できる。
また、プロテアーゼ及び糖化アミノ酸に作用する酵素を含有する試験片には反応を色に変える発色系の試薬を同時に保持させておくと検出がしやすい。
【００３１】
検出を助ける発色系の試薬成分としては、例えば糖化アミノ酸に作用する酵素としてデヒドロゲナーゼを用いた場合には例えば補酵素であるNAD等を用いることができ、その場合は生成される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付近の波長で検出すればよい。また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等の電子キャリアー及びニトロテトラゾリウム、WST-1、WST-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発色試薬を用いる事もでき、生じた還元型補酵素を色に変換して検出してもよい。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。
またオキシダーゼを用いた場合、例えばケトア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を検出できる公知の成分を用いることができる。
【００３２】
上記過酸化水素の量を検出できる成分としては、例えばパーオキシダーゼ等を用いて色素等を生成し、比色、発光、蛍光等に変換し検出すればよい。
過酸化水素の発色系は、パーオキシダーゼの存在下で4-アミノアンチピリン(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(MBTH)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、パーオキシダーゼの存在下で直接酸化、呈色するロイコ型試薬(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)等を用いることができる。
【００３３】
本発明の少なくともプロテアーゼを含有する試験片の製造方法としては前記少なくともプロテアーゼを含む試薬の試験片への保持の方法を用いて試験片を少なくともプロテアーゼを含有する試薬に浸し、乾燥させればよい。また少なくともプロテアーゼ及び糖化アミノ酸に作用する酵素を含有する試験片の製造方法も同様に前記少なくともプロテアーゼ及び糖化アミノ酸に作用する酵素を含む試薬の試験片への保持の方法を用いて試験片を少なくともプロテアーゼ及び糖化アミノ酸に作用する酵素を含有する試薬に浸し、乾燥させればよい。
【００３４】
少なくともプロテアーゼを含有する試験片を使用した試験用具としては、少なくともプロテアーゼを含有する試験片が構成要素に含まれていればいかなる試験用具を用いても良いが、例えば試料を一定量吸引しうる吸引部、少なくともプロテアーゼを含有する試験片、試験片の発色を光学的に検出するための孔等を含む物であってもよい。また光学的に検出を行う場合には、反射光の測定が一般的であり、光学反射層を試験片に張り合わせて用いると検出の感度が上がり好ましい。
【００３５】
試料を一定量吸引しうる試験用具の構造としては、一定量を吸引して排出する仕組みを用いても良いが、最も簡便な方法はキャピラリーを用いて毛管現象で試料を吸引する方法である。試験用具へのキャピラリーの形成は公知の方法で行えば良いが、例えば厚さ0.01mm〜1mm、幅0.5mm〜10cm、長さ1mm〜10cm程度のフィルムを３枚使用し、真ん中のフィルムに１本の幅0.01mm〜1cmの溝を形成し、上下から溝の無いフィルムで挟むことによりキャピラリーを形成する方法などがある。フィルムはエタノールで希釈した５％蔗糖脂肪酸エステル等を塗布、全てを重ねた後に乾燥させれば簡単に貼り合わせる事ができる。
【００３６】
この時上のフィルムに直径0.1mm〜80mmの円形の測定孔を開け、その真下、真中の溝を形成したフィルムとの間に試験片を、試験片の真中にキャピラリーが来るように、かつ試験片の中心と測定孔との中心が一致するようにセットすればよい。
【００３７】
少なくともプロテアーゼ及び糖化アミノ酸に作用する酵素を含有する試験片を使用した試験用具、及び、少なくともプロテアーゼ及び糖化アミノ酸に作用する酵素を含有する試験片及びタンパク質発色試薬を含有する試験片を使用した試験用具についても少なくともプロテアーゼを含有する試験片を使用した試験用具と同様である。
【００３８】
タンパク質発色試薬としては公知のタンパク質を測定する方法を用いた試薬であれば如何なるタンパク質発色試薬を用いても良いが、例えばタンパク質がアルブミンの場合にはブロモクレゾールグリーン(BCG)、ブロモクレゾールパープル(BCP)、ブロモフェノールブルー、メチルオレンジ、又は２-（４-ヒドロキシベンゼンアゾ）安息香酸(HABA)等のアルブミン特異的な色素を用いるかアルブミン抗体を用いた発色試薬を用いればよい。
【００３９】
試験片に保持させる前の溶液状態の試薬の例としては、例えばHABAを用いる場合、選択できるpHはpH3〜10であり、好ましくはpH4〜9である。HABAの濃度としては、0.001〜10%、好ましくは0.01〜5%であれば良い。この場合検出に用いる波長は480〜550nm付近である。また同様にBCPを用いる場合には、選択できるpHはpH4〜8、好ましくは4.5〜7.5であり、好ましくは着色を抑える界面活性剤、例えばBrij35等を0.01〜5%好ましくは0.05〜3%共存させれば良い。BCPの濃度としては0.0001〜0.2%、好ましくは0.0005〜0.1%であり、検出に用いることができる波長は600nm付近である。
【００４０】
また例えばタンパク質がヘモグロビンの場合には、例えばメトヘモグロビン法、シアンメトヘモグロビン法、アザイドメトヘモグロビン法、緑色発色団形成法またはオキシヘモグロビン法を用いた発色試薬が挙げられる。緑色発色団形成法とは緑色発色団形成試薬とヘモグロビンを反応させ、安定な生成物（緑色発色団）を形成する方法であり、緑色発色団は英国特許公開第2052056号公報に記述されるアルカリ性ヘマチンD-575と同様な吸収スペクトルを有する。
【００４１】
試験片に保持させる前の溶液状態の試薬の例としては、オキシヘモグロビン法を用いる場合には、例えば界面活性剤、例えば少なくとも硫酸基を有する界面活性剤、及び／又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%の濃度で調整し、試験紙を作成し、540nm付近の吸収を測定すればよい。
【００４２】
本発明に用いることができる測定方法としては、少なくともプロテアーゼを含有する試験片を用いた糖化タンパク質の測定方法、少なくともプロテアーゼ及び糖化アミノ酸に作用する酵素を含有する試験片を用いた糖化タンパク質の測定方法、少なくともプロテアーゼ及び糖化アミノ酸に作用する酵素を含有する試験片及びタンパク質発色試薬を含有する試験片を用いた糖化タンパク質割合の測定方法であれば如何なる測定方法を用いても良い。
【００４３】
本発明に使用しうる試料としては、測定対象になる糖化タンパク質が血球中に存在する場合、たとえばヘモグロビンＡ１ｃ若しくは糖化ヘモグロビン等を測定する場合には、全血、血球もしくは溶血操作を行った全血、血球を使用することができ、測定対象になる糖化タンパク質が血清中に存在する場合、たとえば糖化アルブミン等を測定する場合には、全血、血清、血漿を用いることができるが、診療現場で迅速に測定する目的から、全血を用いることが好ましい。
【００４４】
また、血球中の糖化タンパク質を測定する場合に、試料として全血、血球を用いる場合には、測定をする前に、もしくは同時に効率的に溶血されることが望ましい。溶血の方法としては公知の方法、たとえば生理的浸透圧と異なる溶液と混合する方法や界面活性剤と混合するなどの方法を用いて行えばよい。
【００４５】
一方、血清中の糖化タンパク質を測定する場合に、試料として全血を用いる場合には、測定をする前に血球分離をしておくことが望ましい。血球分離の方法としては膜を用いて血球を分離する方法が一般的である。
本発明の試験用具を用いて糖化タンパク質の測定を行うには、試験用具のキャピラリーの先端を試料に付け試料を吸引し、試験片の発色を光学的に測定すればよい。試験片は通常乾燥しているが、試料の水分により試薬成分が溶解し反応が自動的に進行する。反応の温度は通常室温であるが、保温機能を持たせ一定温度で反応を行わせると再現性が良くなる。
【００４６】
反応の検出は試薬の発色した試験片に対して光をあて、その反射光を検出することが最も簡便であるが、これ以外の方法を用いても良い。例えば照射する光源としてはＵＶランプやハロゲンランプ等の通常の透過率測定に用いる光源はもちろんであるが、発行ダイオード、レーザーなどを使用することができる。光の照射角度は何れでも良いが、反射光の検出は検出面に対して垂直が好ましい。検出はフォトダイオードや市販の積分球等を用いれば簡便に行う事ができる。
【００４７】
検出された反射光は、濃度既知の糖化タンパク質のものと比較することにより糖化タンパク質濃度に換算すれば良いが、一般的には試験紙のロットにより感度は一定であるから、ロット毎に濃度既知の糖化タンパク質濃度における感度を測定して換算できるようにしておけば良い。
【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】
＜少なくともプロテアーゼを含有する試験片の作成＞
糖化タンパク質検出試薬溶液（糖化アルブミン測定用）
50mM トリス緩衝液 pH7.5
50,125,250,500,1000,2000U/ml
プロテアーゼタイプXXVII（シグマ社製）
4mM 4-アミノアンチピリン(4-AA;同仁化学研究所社製)
2mM 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;同人化学研究所社製)
100,250, 500, 1000,2000,4000mU/ml
ケトアミンオキシダーゼ (旭化成社製)
4U パーオキシダーゼ（ロシュ社製）
【００４９】
厚さ0.4mm、50mm×50mmのろ紙（ワットマン社製クロマトグラフィ用ろ紙）に上記酵素濃度を変化させた糖化タンパク質検出試薬を室温にて５分間浸し、37℃２時間風乾し少なくともプロテアーゼ及び糖化アミノ酸に作用する酵素を含有する試験片を作製した。尚プロテアーゼの濃度を変化させる場合はケトアミンオキシダーゼの濃度を4000mU/mlに固定し、ケトアミンオキシダーゼの濃度を変化させる場合にはプロテアーゼ濃度は2000U/mlに固定した。
【００５０】
【実施例２】
＜試験用具の作成＞
図１Ａに示すように実施例１で作成した試験片を直径4mmの円状(5)に切り出した。厚さ0.03mmのポリエステルフィルム(1)〜(3)4mm×40mm 3枚を使用し、真中のフィルムに１本の幅0.5mmの溝(6)を形成し、上下から溝の無いフィルム(1)、(3)で挟むことによりキャピラリーを形成させた。この時上のフィルム(1)に直径1.6mmの円形の測定孔(4)を開け、その真下、真中の溝を形成したフィルムとの間に前記の試験片(5)を、試験片の真中に溝が来るように、かつ試験片の中心と測定孔との中心が一致するようにセットした。フィルムはエタノールで希釈した5%蔗糖糖脂肪酸エステルを塗布、全てを重ねた後に乾燥させることにより貼り合わせた(図1Ｂ参照)。
【００５１】
【実施例３】
＜糖化タンパク質の測定に及ぼすプロテアーゼ及び糖化アミノ酸に作用する酵素の濃度の影響＞
＜操作方法＞
実施例２で作成した試験用具のキャピラリーの先端をルシカGA用キャリブレータH（旭化成社製）に浸し、室温にて反射光を20分間測定した。少なくともプロテアーゼ及び糖化アミノ酸に作用する酵素を含有する試験片を用いた試験用具を用いた場合の反射光の測定は積分球（日立U3010）を用い550nmの光を照射し、反射光を測定した。
プロテアーゼの濃度を変化させた場合の反応曲線を図２に、ケトアミンオキシダーゼ濃度を変化させた場合の反応曲線を図３に示す。
【００５２】
図２から分かるようにプロテアーゼ濃度が50U/mlつまり試験片1cm2あたりプロテアーゼ含有量が2Uである場合には感度が得られなかった。一方125U/ml以上つまり試験片1cm2あたりプロテアーゼ含有量が5U以上である場合には感度が得られ、1000U/ml以上つまり試験片1cm2あたりプロテアーゼ含有量が40U以上で20分間でエンドポイント到達している反応曲線が得られた。よって試験片1cm2あたりプロテアーゼ含有量が5U以上であればこの試験片及び試験片を用いた測定用具を用いて糖化タンパク質を測定できることが明白であった。
【００５３】
一方図３から分かるように糖化アミノ酸に作用する酵素であるケトアミンオキシダーゼ濃度が100mU/mlつまり試験片1cm2あたりプロテアーゼ含有量が4mUである場合には感度がえられなかったが、250mU/ml以上つまり試験片1cm2あたりのケトアミンオキシダーゼ含有量が10mU以上である場合には感度が得られ1000mU/ml以上つまり試験片1cm2あたりケトアミンオキシダーゼ含有量が40mU以上で20分間でエンドポイント到達している反応曲線が得られた。よって試験片1cm2あたり糖化アミノ酸に作用する酵素含有量が10mU以上であればこの試験片及び試験片を用いた測定用具を用いて糖化タンパク質を測定できることが明白であった。
【００５４】
【実施例４】
＜糖化アルブミンの測定＞
血清試料として健常者５検体、糖尿病患者５検体を用いて糖化アルブミン割合の測定を行った。糖化アルブミン濃度及びアルブミン濃度はルシカGA用のキャリブレーター（旭化成社製）を用いて換算した。糖化アルブミン比率のHPLCを用いた測定はグリコアルブミン計を(GAA-2000;アークレイ社製)を使用した。
糖化アルブミンを測定する試験用具は実施例１及び２の試験用具において、プロテアーゼ2000U/ml、ケトアミンオキシダーゼ4000mU/mlの条件で作成されたものを用いた。
【００５５】
アルブミン測定用試験片は下記の試薬を用い、実施例１と同じ方法で作成し、実施例２と同じ方法で試験用具とした。
＜アルブミン測定試薬＞
50mM クエン酸緩衝液 pH4.0
1% Briji35(和光純薬社製)
0.03% グロモクレゾールグリーン（和光純薬社製）
【００５６】
糖化アルブミン測定用の試験用具の操作方法、検出方法は実施例３と同じ方法で行った。但しアルブミン測定試薬を含有する試験片を用いた試験用具を用いた場合の反射光の測定は波長630nmで光を照射し、反射光量を測定した。
別途キャリブレーターを測定し試料の糖化アルブミン濃度、アルブミン濃度を求めその値から糖化アルブミン割合を計算した。尚健常者血清１検体は５回測定し再現性を確認した。結果を表１に示す。
【００５７】
【表１】
表１に示すように、本発明の試験片、及び試験片を使用した試験用具を用いて測定した糖化アルブミン割合の測定結果はHPLC法と良く一致しており、本発明の測定方法、試験片、試験用具を用いて、正確に糖化アルブミン濃度、アルブミン濃度及び糖化アルブミン割合が測定されていることが明白であった。また健常者血清の５重測定のCVは5.3%であり良好な結果であった。
【００５９】
【実施例５】
＜糖化ヘモグロビンの測定＞
1) 糖化ヘモグロビン濃度測定試験片、試験用具の作成
以下の試薬を用いて実施例１及び２に記載の方法を用いて試験片、試験用具を作成した。
【００６０】
糖化タンパク質検出試薬溶液（糖化ヘモグロビン測定用）
50mM トリス緩衝液 pH7.5
200U/ml プロテアーゼタイプXXVII(シグマ社製)
6000U/ml プロテアーゼタイプXIV(シグマ社製)
20μＭ DA64（N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamin e)- diphenylamine,sodium salt；同人化学研究所社製）
4U/ml ケトアミンオキシダーゼ (旭化成社製)
4U/ml パーオキシダーゼ（ロシュ社製）
【００６１】
2) ヘモグロビン濃度測定試験片、試験用具の作成
市販のヘモグロビン測定試薬、へモグロビンＢテストワコー（和光純薬社製）を用いて実施例１及び２に記載の方法を用いて試験片、試験用具を作成した。尚本試験片の検出は540nmにて行った。また、測定の試料には健常者全血、糖尿病患者全血を用い、HPLCを用いたヘモグロビンA1cの測定はグリコヘモグロビン計（HLC723G7;アークレイ社製）を使用した。
【００６２】
試料は以下の溶血試薬500μlおよび試料100μlを混合し37℃10分インキュベーションし溶血操作を行った。
R-1;溶血試薬
50mM トリス緩衝液 pH7.5
1% ポリオキシエチレンラウリルエーテル（和光純薬社製）糖化ヘモグロビン測定用の試験用具の操作方法、検出方法は実施例３と同じ方法で行ったが、糖化ヘモグロビン濃度を測定する試験用具の検出は730nmにて行った。
別途キャリブレーター（協和メデックス社）を測定し試料の糖化ヘモグロビン濃度、ヘモグロビン濃度を求めその値から糖化ヘモグロビン割合を計算した。尚健常者全血１検体は５回測定し再現性を確認した。結果を表２に示す。
【００６３】
【表２】
表２に示すように、本発明の試験片、及び試験片を使用した試験用具を用いて測定した糖化ヘモグロビン割合の測定結果はHPLC法と良く一致しており、本発明の測定方法、試験片、試験用具を用いて、正確に糖化ヘモグロビン濃度、ヘモグロビン濃度及び糖化ヘモグロビン割合が測定されていることが明白であった。また健常者全血の５重測定のCVは6.1%であり良好な結果であった。
【００６５】
【発明の効果】
本発明の試験片、試験用具、それを用いた測定方法を用いることにより、診療現場で、正確、簡便かつ安価に糖化タンパク質、特に糖化ヘモグロビン、糖化アルブミを測定することができる。
【図面の簡単な説明】
【図１】Ａ;実施例２の試験用具をそれぞれの部品に分解したときの部品の斜視図を示す。Ｂ;実施例２の試験用具の斜視図を示す。
【符号の説明】
(1)上フィルム
(2)真中の溝付きフィルム
(3)下フィルム
(4)測定孔
(5)試験片
(6)溝
【図２】本発明の実施例３に基づく糖化タンパク質の測定に於けるプロテアーゼ濃度の影響を示す。
【図３】本発明の実施例３に基づく糖化タンパク質の測定に於ける糖化アミノ酸に作用する酵素濃度の影響を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a test piece for measuring glycated protein in a sample accurately, simply and quickly at a clinical site, a method for producing the test piece, a test tool using the test piece, and a glycated protein using the test piece. It relates to a measurement method.
[0002]
[Prior art]
Measurement of glycated protein and glycated lipid is very important in the diagnosis, management and prevention of diabetes. Among them, glycated hemoglobin and glycated albumin are frequently used as indicators that must be used in the clinical field because they accurately reflect the blood glucose control state. As quantification methods for these glycated proteins and glycated lipids, electrophoretic methods, ion exchange chromatography methods, affinity chromatography methods, immunization methods, enzyme methods, and the like are generally known. In recent years, enzymatic methods have begun to be widely used because they can measure a large amount of samples quickly, in large quantities, accurately, and inexpensively. As an enzymatic method, a method for measuring ketoamine present in a glycated protein is most often used, and the present inventors have also developed a method for measuring glycated albumin using ketoamine oxidase (
[0003]
A known method for measuring ketoamine in a polymer is a method using a large biochemical automatic analyzer. However, biochemical automatic analyzers are expensive and difficult to use in clinics and small and medium-sized hospitals where only a small amount of sample needs to be analyzed. Therefore, an inexpensive and simple apparatus for measuring ketoamine in a polymer is desired. Until now, there has not been known an apparatus using an enzymatic method capable of measuring glycated hemoglobin and glycated albumin at a low cost in clinical practice.
In addition, there has been no example in which a protease is retained on a test piece to degrade a protein and its degradation fragment is measured.
[0004]
Patent Document 1) Japanese Patent Application Laid-Open No. 2001-54398
Patent Document 2) Japanese Patent Application Laid-Open No. 2001-204495
Patent Document 3) WO 02/061119
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide a novel test piece containing at least a protease, a method for producing a test piece, a test tool, and a test tool for measuring glycated protein accurately, simply and inexpensively using an enzyme at a clinical site. The object is to provide a method for measuring the glycated protein used. More specifically, an object of the present invention is to provide a test piece useful for clinical biopsy, in particular, measurement of glycated hemoglobin and glycated albumin, a method for producing the test piece, a test tool, and a method for measuring glycated protein using the test piece.
[0006]
[Means for Solving the Problems]
Even if a general enzyme such as glucose oxidase is held on a dried test piece, the reaction can be started as soon as it is dissolved by moisture in the sample. This is thought to be because the substrate is a small molecule, and even after dissolution, it can bind to the substrate relatively easily and carry out an enzymatic reaction. On the other hand, a protease is a polymer such as a protein, and it is usually difficult to think that the substrate can be easily fragmented immediately after dissolution. According to the study by the present inventors, it was found that the protease reaction immediately after dissolution hardly proceeded as expected.
[0007]
As a result of intensive studies on the conditions of the protease test strip, the present inventors have unexpectedly found that the reaction proceeds at almost the same rate as the liquid system if a certain amount or more of protease is present. Furthermore, the present inventors can quantify a glycated protein without causing the enzyme acting on a glycated amino acid to be inactivated by coexistence with a protease even if the enzyme acting on the glycated amino acid is simultaneously held in a test piece, and includes a protein quantification reagent. The ratio of glycated protein can be easily measured by combining it with the test piece, and the test piece dissolves the enzyme etc. just by soaking a liquid sample without using a solvent, etc., and the reaction proceeds immediately. It has been found that the color development of can be easily performed by measuring reflected light using an optical reflective layer, and the present invention has been completed.
[0008]
That is, this invention relates to the following test piece, its manufacturing method, the test tool using the same, and the measuring method of glycated protein using the same;
1) A test piece for measuring a glycated protein containing at least a protease.
2) Test piece for measuring glycated protein containing at least an enzyme that acts on protease and glycated amino acid.
3) Test piece 1cm 2 The test piece according to 1) or 2), wherein the per protease content is 5 U or more.
4) 1cm specimen 2 The test piece according to any one of 2) and 3), wherein the content of the enzyme that acts on the glycated amino acid is 10 mU or more.
[0009]
5) A method for producing a test piece for measuring glycated protein, which comprises immersing the test piece in a solution containing at least a protease and drying it.
6) A method for producing a test piece for measuring a glycated protein, which comprises immersing the test piece in a solution containing at least a protease and an enzyme that acts on a glycated amino acid and drying the solution.
7) A test tool for measuring glycated protein using a test piece containing at least a protease.
8) A test tool for measuring glycated protein using a test piece containing at least an enzyme that acts on protease and glycated amino acid.
9) A test tool for measuring glycated protein using a test piece containing at least an enzyme that acts on protease and glycated amino acid and a test piece containing a protein coloring reagent.
10) The test tool according to any one of 7) to 9), which has a hole for optical measurement.
[0010]
11) A groove is formed in the middle sheet, consisting of a three-layer sheet laminated so that the groove becomes a capillary, a measurement hole is formed in the uppermost sheet, and a groove in the middle sheet under the measurement hole A detection instrument for measuring glycated protein, on which a test piece containing protease or an enzyme that acts on a protease and a glycated amino acid is present.
12) A method for measuring a glycated protein using a test piece containing at least a protease.
13) A method for measuring a glycated protein using a test piece containing at least an enzyme that acts on a protease and a glycated amino acid.
14) A method for measuring the ratio of glycated protein using a test piece containing at least an enzyme that acts on proteases and glycated amino acids and a test piece containing a protein coloring reagent.
15) The measuring method according to any one of 12) to 14), wherein the enzyme reaction of the test piece is performed with moisture in the sample.
16) The measuring method according to any one of 12) to 15), wherein the color development of the test piece is measured using reflected light.
17) The measuring method according to any one of 12) to 16), wherein the glycated protein is a glycated product of albumin or hemoglobin.
[0011]
According to the present invention, glycated protein can be measured accurately, simply, and inexpensively using an enzyme at the clinical site. More specifically, in clinical biopsy, it is possible to provide a test piece particularly useful for measuring glycated hemoglobin and glycated albumin, a method for producing the test piece, a test tool, and a measurement method using the test piece.
[0012]
Hereinafter, the configuration and preferred embodiments of the present invention will be described in more detail.
As the protease that can be used in the present invention, any protease may be used as long as it is a protease that acts on a protein such as albumin or hemoglobin to cleave a glycated amino acid or a peptide containing a glycated amino acid. The enzyme may be a purified product or a crude product as long as the desired activity is expressed.
[0013]
Preferred examples of proteases that can be used in the present invention include, for example, proteases derived from animals such as trypsin and chymotripsin, plant-derived proteases such as papain and bromelain, and proteases derived from microorganisms. Etc.
Examples of microorganism-derived proteases include proteases derived from the genus Bacillus typified by Subtilisin, etc., proteases derived from Aspergillus typified by protease type-XIII (manufactured by Sigma), PD enzymes ( Penicillium-derived protease represented by Kikkoman Corp.), Streptomyces-derived protease represented by Pronase, etc., Lysobacter represented by endoproteinase Lys-c (Sigma), etc. (Lysobacter) derived protease, proteinase A (Proteinase A; manufactured by Sigma), etc.Yeast (Yeast) derived protease, proteinase K (Proteinase K; manufactured by Sigma) and the like, a protease derived from Tritirachium (Tritirachium), Aminopeptidase T (Boehringer Mannheim) Thermus-derived proteases typified by Pseudomonus, lysyl endopeptidase (Lysylendopeputidase made by Wako Pure Chemicals), etc. And a protease derived from Achromobacter. These specific examples are merely examples and are not limited in any way.
[0014]
In addition, when the measurement target is glycated albumin, a protease derived from microorganisms of the genus Bacillus and Streptomyces is more preferable because it has a large effect on human albumin, and when the measurement target is glycated hemoglobin, Proteases derived from the genus Myces and Trityratium are preferred because they have a large effect on human hemoglobin.
[0015]
The caseinfoline method was used as a method for measuring protease activity. The definition of activity was defined as 1 U for color development corresponding to 1 μg of tyrosine at −37 ° C. for 1 minute.
Further, regarding the use of the protease of the present invention, it is of course possible to use the protease alone, but other endoproteases or other exoproteases may be used simultaneously.
[0016]
The reagent containing the protease is retained on the test piece by preparing a liquid reagent containing the protease, and immersing the test piece in the solution, for example, at room temperature, preferably refrigerated, for 0.1 minute to 1 day, preferably 1 minute to 8 hours. What is necessary is just to dry. The drying method may be carried out under normal pressure and reduced pressure conditions, and the temperature may be, for example, about 4 ° C. to 60 ° C. for about 1 minute to 2 days, but preferably about 4 ° C. to 40 ° C., where enzymes are not easily deactivated. . There are methods for increasing the speed of drying, such as applying wind and selecting an environment with low humidity. Generally, it is sufficient to dry in a cool and dark place with low humidity.
[0017]
When holding a reagent containing protease on a test piece, the protease concentration in a liquid reagent containing protease should be soaked in a 5 cm x 5 cm test piece in 1 ml of a reagent with a concentration of 125 U / ml or more. Specimen 1cm 2 Per protease content is 5U or more. Protease concentration is 1cm. 2 Protease content per unit is not limited as long as it is 5 U or more, but considering the increase in background and cost, 1 cm specimen 2 The per protease content is preferably 10 KU or less.
[0018]
The pH of the liquid reagent containing the protease may be selected so that the reaction proceeds efficiently in consideration of the optimum pH of the protease to be used. For example, when protease type XXVII (manufactured by Sigma) is used as the protease, protease type XXVII has a strong proteolytic activity around pH 7-10, so the pH of the reaction can be selected from 7-10.
[0019]
As a test piece that can be used in the present invention, any test piece may be used as long as it is a sheet-like material. For example, paper, a plastic sheet, a nonwoven fabric, and the like can be used. In addition, as long as it has a high water absorption property and can maintain a sufficient amount of protease, any product may be used.
The thickness of the test piece may be about 0.1 mm to 2 mm, preferably about 0.2 mm to 1 mm, and 0.001 cm to actually measure one sample. 2 ~ 5cm 2 Is better, more preferably 0.005cm 2 ~ 2cm 2 Degree.
[0020]
Examples of enzymes that act on glycated amino acids that can be used in the present invention include dehydrogenases, kinases, oxidases, and the like that act by recognizing the ketoamine structure of glycated amino acids, but oxidases that are available in large quantities at the lowest cost are preferred.
[0021]
Any enzyme that acts on a glycated amino acid may be used as long as it acts on a low-molecular-weight glycated amine, such as a glycated amino acid or a peptide containing a glycated amino acid. In the case of A1c, an enzyme that efficiently acts on an amino acid in which the α-amino group is glycated is preferable, and an amino acid that specifically acts on the amino acid in which the α-amino group is glycated and the ε-amino group is glycated substantially Most preferred are enzymes that do not act.
[0022]
In general, an enzyme that specifically acts on an amino acid in which an α-amino group mino group is glycated and does not substantially act on an amino acid in which an ε-amino group is glycated has poor stability. It may be measured using an enzyme that works well on both glycated amino acids whose α-amino group is glycated, and ε-amino group using an enzyme that specifically acts on glycated amino acids whose ε-amino group is more stable Even if only amino acids with α-amino group glycated are measured using an enzyme that erases only glycated amino acids and works well with both highly stable ε-amino groups and glycated amino acids with α-amino groups glycated good.
[0023]
On the other hand, when the target analyte is glycated albumin, an enzyme that efficiently acts on amino acids whose ε-amino group is glycated is preferable, and the ε-amino group specifically acts on glycated amino acids and the α-amino group is glycated. Most preferred are enzymes that do not substantially act on amino acids.
[0024]
Alternatively, it may be measured using a highly stable enzyme that acts well on both ε-amino groups and α-amino group glycated amino acids, and an enzyme that specifically acts on amino acids on which α-amino groups are glycated. Only amino acids with α-amino group glycated using an enzyme that works well on both stable ε-amino group and glycated amino acid with α-amino group glycated. May be measured.
[0025]
Examples of enzymes that recognize the most preferred ketoamine structure in terms of specificity include α-amino group glycated amino acid-specific enzymes that do not act on glycated amino acids such as fructosyl amino acid oxidase (FAOD): Examples include the genus Corynebacterium (FERM P-8245). On the other hand, enzymes that work well on both ε-amino groups and α-glycosylated glycated amino acids and highly stable enzymes include the genus Gibberella or the genus Aspergillus (for example, IFO-6365, -4242,- 5710 etc.) derived fructosamine oxidase, Candida genus fructosylamine deglycase, Penicillium genus (eg IFO-4651, -6581, -7905, -5748, -7994, -4897, -5337 etc.) Derived from fructosyl amino acid degrading enzyme, derived from the genus Fusarium (eg IFO-4468, -4471, -6384, -7706, -9964, -9971, -31180, -9972, etc.), derived from the genus Acremonium or Enzymes that recognize ketoamine structures such as ketoamine oxidase derived from the genus Debaryomyces can be mentioned, and more preferable examples include sufficient activity even in the presence of proteases. To, genetically modified ketoamine oxidase (manufactured by Asahi Kasei Corporation).
[0026]
An enzyme specific to an ε-amino group glycated amino acid, which does not act on an amino acid whose α-amino group is glycated, includes a genetically modified ketoamine oxidase (manufactured by Asahi Kasei Co., Ltd.) prepared by genetic modification.
[0027]
The activity of the enzyme that acts on glycated amino acids was determined from glycated Z lysine or glycated valine (synthesized and purified according to the method of Hashiba et al. (Hashiba H, J. Agric. Food Chem. 24:70, 1976)) at 37 ° C., 1 The amount of enzyme that produces 1 μmol of hydrogen peroxide per minute was defined as 1 U.
[0028]
The retention of the reagent containing the protease and the enzyme acting on the glycated amino acid on the test piece may be performed using the same method as the retention of the reagent containing the protease on the test piece. The same applies to the method of increasing the drying speed.
[0029]
In addition, when a reagent containing an enzyme that acts on a protease and a glycated amino acid is held on a test piece, the concentration of the protease in the reagent solution containing the enzyme that acts on the protease and the glycated amino acid and the concentration of the enzyme that acts on the glycated amino acid are 125 U each. It is only necessary to immerse a 5 cm x 5 cm test piece in 1 ml of a reagent with a concentration of 250 mU / ml or more. In this case, if all reagents are absorbed, the test piece is 1 cm 2 The per protease content is 5 U or more, and the amount of enzyme acting on glycated amino acids is 10 mU or more. Protease concentration is 1cm. 2 Protease content per unit is not limited as long as it is 5 U or more, but considering the increase in background and cost, 1 cm specimen 2 The per protease content is preferably 10 KU or less. Enzyme concentration acting on glycated amino acids is 1cm 2 As long as it is 10mU or more per unit, any amount is acceptable, but considering the cost, 1cm specimen 2 The per protease content is preferably 50 U or less.
[0030]
The pH of the liquid reagent containing the protease and the enzyme acting on the glycated amino acid may be selected so that the reaction proceeds efficiently in consideration of the optimum pH of the protease used and the enzyme acting on the glycated amino acid. For example, when protease type XXVII (manufactured by Sigma) is used as the protease, protease type XXVII has a strong proteolytic activity around pH 7-10, so the pH of the reaction is preferably 7-10, and an enzyme that acts on glycated amino acids When genetically modified ketoamine oxidase (manufactured by Asahi Kasei Co., Ltd.) is used, the region showing an activity of 50% or more of the maximum activity is as wide as 6.5 to 10, and the pH of the reaction is preferably 6.5 to 10, comparing the two Then pH 7-10 can be selected.
In addition, a test piece containing protease and an enzyme that acts on a glycated amino acid can be easily detected by simultaneously holding a chromogenic reagent that changes the reaction into a color.
[0031]
For example, when a dehydrogenase is used as an enzyme that acts on a glycated amino acid, for example, NAD that is a coenzyme can be used as a reagent component of a chromogenic system that assists in detection. A certain reduced NAD may be detected at a wavelength around 340 nm which is the maximum absorption wavelength region. In addition, various diaphorases, or electron carriers such as phenazine methosulfate, and coloring reagents such as various tetrazolium salts represented by nitrotetrazolium, WST-1, WST-8 (above manufactured by Doujin Chemical Laboratory Co., Ltd.) can be used. The reduced coenzyme may be detected by converting it into a color. Moreover, you may measure directly and indirectly by other well-known methods.
When oxidase is used, for example, when ketoamine oxidase is used, hydrogen peroxide and glucosone are generated by the reaction, and known components that can detect hydrogen peroxide and glucosone can be used.
[0032]
As the component capable of detecting the amount of hydrogen peroxide, for example, a dye or the like may be generated using peroxidase or the like, and converted into colorimetric, luminescent, fluorescent, or the like and detected.
In the presence of peroxidase, the hydrogen peroxide coloring system is a combination of a coupler such as 4-aminoantipyrine (4-AA) or 3-methyl-2-benzothiazolinone hydrazone (MBTH) and a chromogen such as phenol. A Trinder reagent that produces a dye by oxidative condensation, a leuco-type reagent that directly oxidizes and colors in the presence of peroxidase (N- (carboxymethylaminocarbonyl) -4,4-bis (dimethylamino) biphenylamine (DA64), 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine (DA67); manufactured by Wako Pure Chemical Industries, Ltd.) and the like can be used.
[0033]
As a method for producing a test piece containing at least a protease according to the present invention, the test piece may be dipped in a reagent containing at least a protease and dried by using the method for holding a reagent containing at least a protease on the test piece. Similarly, a method for producing a test piece containing at least a protease and an enzyme that acts on a glycated amino acid similarly uses the above-described method for holding a reagent containing at least a protease and an enzyme that acts on a glycated amino acid on the test piece. And dipping in a reagent containing an enzyme that acts on glycated amino acids and drying.
[0034]
As a test tool using a test piece containing at least a protease, any test tool may be used as long as a test piece containing at least a protease is contained in the constituent elements. For example, suction capable of sucking a sample in a certain amount. Part, a test piece containing at least a protease, a hole for optically detecting the color of the test piece, and the like may be used. In the case of optical detection, reflected light is generally measured, and it is preferable to use an optical reflective layer attached to a test piece because the sensitivity of detection increases.
[0035]
As a structure of a test tool that can suck a certain amount of sample, a mechanism for sucking and discharging a certain amount may be used, but the simplest method is a method of sucking a sample by capillary action using a capillary. The capillary can be formed on the test tool by a known method. For example, three films having a thickness of 0.01 mm to 1 mm, a width of 0.5 mm to 10 cm, and a length of 1 mm to 10 cm are used. There is a method in which a capillary is formed by forming a groove having a width of 0.01 mm to 1 cm and sandwiching between a film having no groove from above and below. The film can be easily pasted by applying 5% sucrose fatty acid ester diluted with ethanol, etc., all of which are dried and then dried.
[0036]
At this time, a circular measurement hole with a diameter of 0.1 mm to 80 mm is opened in the upper film, and the test piece is placed between the film with the groove formed in the middle and the capillary is placed in the middle of the test piece. What is necessary is just to set so that the center of a piece and the center of a measurement hole may correspond.
[0037]
Test tool using a test piece containing at least an enzyme that acts on protease and glycated amino acid, and test tool using a test piece containing an enzyme that acts on at least protease and glycated amino acid, and a test piece containing a protein coloring reagent This is the same as the test tool using a test piece containing at least a protease.
[0038]
As the protein coloring reagent, any protein coloring reagent may be used as long as it is a reagent using a known protein measuring method.For example, when the protein is albumin, bromocresol green (BCG), bromocresol purple (BCP ), Bromophenol blue, methyl orange, 2- (4-hydroxybenzeneazo) benzoic acid (HABA) or other albumin-specific dyes or color developing reagents using albumin antibodies may be used.
[0039]
As an example of the reagent in a solution state before being held on the test piece, for example, when HABA is used, the pH that can be selected is
[0040]
For example, when the protein is hemoglobin, for example, a chromogenic reagent using a methemoglobin method, a cyan methemoglobin method, an azide methemoglobin method, a green chromophore formation method or an oxyhemoglobin method can be mentioned. The green chromophore forming 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.
[0041]
As an example of a reagent in a solution state before being held on a test piece, when using the oxyhemoglobin method, for example, a surfactant, for example, a surfactant having at least a sulfate group, and / or a nonionic surfactant, And / or the amphoteric surfactant is preferably adjusted to a concentration of 0.001 to 10%, a test paper is prepared, and the absorption near 540 nm may be measured.
[0042]
The measuring method that can be used in the present invention includes a method for measuring a glycated protein using a test piece containing at least a protease, and a method for measuring a glycated protein using a test piece containing an enzyme that acts on at least a protease and a glycated amino acid. Any measurement method may be used as long as it is a method for measuring the ratio of glycated protein using a test piece containing at least an enzyme that acts on protease and glycated amino acid and a test piece containing a protein coloring reagent.
[0043]
Samples that can be used in the present invention include whole blood, blood cells, or whole blood subjected to hemolysis when the glycated protein to be measured is present in blood cells, for example, when measuring hemoglobin A1c or glycated hemoglobin. When blood cells can be used and the glycated protein to be measured is present in the serum, for example, when measuring glycated albumin or the like, whole blood, serum, or plasma can be used. For the purpose of rapid measurement, it is preferable to use whole blood.
[0044]
Further, when measuring glycated protein in blood cells, when whole blood or blood cells are used as a sample, it is desirable that the blood be efficiently lysed before or simultaneously with the measurement. As a method of hemolysis, a known method such as a method of mixing with a solution different from physiological osmotic pressure or a method of mixing with a surfactant may be used.
[0045]
On the other hand, when measuring glycated protein in serum, when whole blood is used as a sample, it is desirable to separate blood cells before measurement. As a method for separating blood cells, a method of separating blood cells using a membrane is common.
In order to measure glycated protein using the test tool of the present invention, the tip of the capillary of the test tool is attached to the sample, the sample is sucked, and the color of the test piece is optically measured. Although the test piece is usually dry, the reagent component dissolves due to the moisture of the sample, and the reaction proceeds automatically. The temperature of the reaction is usually room temperature, but the reproducibility is improved by carrying out the reaction at a constant temperature with a heat retaining function.
[0046]
It is most convenient to detect the reaction by irradiating the test piece colored with the reagent and detecting the reflected light, but other methods may be used. For example, as a light source for irradiation, a light emitting diode, a laser, or the like can be used as well as a light source used for normal transmittance measurement such as a UV lamp and a halogen lamp. The light irradiation angle may be any, but the reflected light is preferably detected perpendicular to the detection surface. Detection can be easily performed by using a photodiode, a commercially available integrating sphere, or the like.
[0047]
The detected reflected light may be converted into the glycated protein concentration by comparing with the glycated protein with a known concentration. Generally, the sensitivity is constant depending on the lot of the test paper, so the concentration is known for each lot. It is only necessary to measure the sensitivity at the glycated protein concentration so that it can be converted.
[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]
<Preparation of a test piece containing at least protease>
Glycated protein detection reagent solution (for glycated albumin measurement)
50 mM Tris buffer pH 7.5
50,125,250,500,1000,2000U / ml
Protease type XXVII (Sigma)
4 mM 4-aminoantipyrine (4-AA; manufactured by Dojindo Laboratories)
2 mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (Toos; manufactured by Doujin Chemical Laboratory)
100,250, 500, 1000,2000,4000mU / ml
Ketoamine oxidase (Asahi Kasei Corporation)
4U peroxidase (Roche)
[0049]
Soak a glycated protein detection reagent with varying enzyme concentration in 0.4 mm, 50 mm x 50 mm filter paper (Whatman's chromatographic filter paper) at room temperature for 5 minutes and air dry at 37 ° C for 2 hours to at least protease and glycated amino acid. Test specimens containing the working enzyme were prepared. When changing the protease concentration, the ketoamine oxidase concentration was fixed at 4000 mU / ml, and when changing the ketoamine oxidase concentration, the protease concentration was fixed at 2000 U / ml.
[0050]
[Example 2]
<Creation of test tool>
As shown in FIG. 1A, the test piece prepared in Example 1 was cut into a circle (5) having a diameter of 4 mm. Using three polyester films (1) to (3) 4 mm x 40 mm in thickness of 0.03 mm, one groove (6) with a width of 0.5 mm is formed in the middle film, and there is no film (1 ) And (3) to form a capillary. At this time, a circular measurement hole (4) having a diameter of 1.6 mm was opened in the upper film (1), and the test piece (5) was placed between the film having a groove formed directly below and in the middle of the test piece. The center of the test piece was set so that the center of the measurement hole coincided with the groove. The film was laminated by applying 5% sucrose sugar fatty acid ester diluted with ethanol, drying all over, and then drying (see FIG. 1B).
[0051]
[Example 3]
<Effect of concentration of enzymes acting on protease and glycated amino acid on measurement of glycated protein>
<Operation method>
The tip of the capillary of the test tool created in Example 2 was immersed in a Lucika GA calibrator H (manufactured by Asahi Kasei Corporation), and the reflected light was measured at room temperature for 20 minutes. The reflected light was measured using a integrating sphere (Hitachi U3010) and the reflected light was measured using a test tool using a test piece containing at least a protease and an enzyme that acts on a glycated amino acid.
FIG. 2 shows a reaction curve when the protease concentration is changed, and FIG. 3 shows a reaction curve when the ketoamine oxidase concentration is changed.
[0052]
As can be seen from FIG. 2, the protease concentration is 50 U / ml, that is, a test piece of 1 cm. 2 Sensitivity was not obtained when the protease content was 2U. On the other hand, 125U / ml or more, that is, 1cm specimen 2 Sensitivity is obtained when the protease content per unit is 5 U or more, and 1000 U / ml or more, that is, a test piece of 1 cm 2 A reaction curve in which the end point was reached in 20 minutes at a protease content of 40 U or more was obtained. Therefore test piece 1cm 2 It was clear that the glycated protein could be measured using this test piece and a measuring tool using the test piece when the protease content per unit was 5 U or more.
[0053]
On the other hand, as can be seen from FIG. 3, the concentration of ketoamine oxidase, an enzyme that acts on glycated amino acids, is 100 mU / ml, that is, a test piece of 1 cm. 2 Sensitivity was not obtained when the per protease content was 4 mU, but 250 mU / ml or more, that is, a test piece of 1 cm. 2 When the ketoamine oxidase content per unit is 10 mU or more, sensitivity is obtained and 1000 mU / ml or more, that is, 1 cm of the test piece 2 A reaction curve in which the end point was reached in 20 minutes at a ketoamine oxidase content of 40 mU or more was obtained. Therefore test piece 1cm 2 When the content of the enzyme acting on the glycated amino acid was 10 mU or more, it was clear that the glycated protein could be measured using this test piece and a measuring tool using the test piece.
[0054]
[Example 4]
<Measurement of glycated albumin>
The glycated albumin ratio was measured using 5 healthy subjects and 5 diabetic patients as serum samples. The glycated albumin concentration and albumin concentration were converted using a calibrator for Lucika GA (manufactured by Asahi Kasei Corporation). The glycated albumin ratio was measured by HPLC using a glycoalbumin meter (GAA-2000; manufactured by ARKRAY).
As the test tool for measuring glycated albumin, the test tool of Examples 1 and 2 prepared under the conditions of
[0055]
The test piece for albumin measurement was prepared by the same method as in Example 1 using the following reagents, and used as a test tool by the same method as in Example 2.
<Reagent for measuring albumin>
50 mM citrate buffer pH 4.0
1% Briji35 (Wako Pure Chemical Industries)
0.03% Glomocresol Green (Wako Pure Chemical Industries)
[0056]
The operation method and detection method of the test tool for measuring glycated albumin were the same as those in Example 3. However, in the case of using a test tool using a test piece containing an albumin measuring reagent, the reflected light was measured by irradiating light at a wavelength of 630 nm and measuring the amount of reflected light.
A calibrator was separately measured to determine the glycated albumin concentration and albumin concentration of the sample, and the glycated albumin ratio was calculated from these values. In addition, one sample of healthy subject serum was measured 5 times to confirm reproducibility. The results are shown in Table 1.
[0057]
[Table 1]
As shown in Table 1, the measurement results of the glycated albumin ratio measured using the test piece of the present invention and the test tool using the test piece are in good agreement with the HPLC method, and the measurement method and test piece of the present invention It was clear that the glycated albumin concentration, the albumin concentration, and the glycated albumin ratio were accurately measured using the test tool. In addition, the CV of quintuple serum of healthy subjects was 5.3%, which was a good result.
[0059]
[Example 5]
<Measurement of glycated hemoglobin>
1) Preparation of glycated hemoglobin concentration measurement specimen and test tool
Using the following reagents, test pieces and test tools were prepared using the methods described in Examples 1 and 2.
[0060]
Glycated protein detection reagent solution (for glycated hemoglobin measurement)
50 mM Tris buffer pH 7.5
200 U / ml protease type XXVII (manufactured by Sigma)
6000 U / ml protease type XIV (manufactured by Sigma)
20μM DA64 (N- (carboxymethylaminocarbonyl) -4,4'-bis (dimethylamine) -diphenylamine, sodium salt; manufactured by Dojin Chemical Laboratory)
4U / ml ketoamine oxidase (Asahi Kasei Corporation)
4U / ml peroxidase (Roche)
[0061]
2) Preparation of hemoglobin concentration measurement specimen and test tool
Test pieces and test tools were prepared using the methods described in Examples 1 and 2 using a commercially available hemoglobin measuring reagent, Hemoglobin B Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The test piece was detected at 540 nm. Moreover, healthy human whole blood and diabetic whole blood were used as samples for measurement, and a hemoglobin A1c measurement using HPLC was performed using a glycohemoglobin meter (HLC723G7; manufactured by ARKRAY).
[0062]
The sample was mixed with 500 μl of the following hemolysis reagent and 100 μl of sample, and incubated at 37 ° C. for 10 minutes for hemolysis.
R-1; hemolysis reagent
50 mM Tris buffer pH 7.5
1% polyoxyethylene lauryl ether (manufactured by Wako Pure Chemical Industries, Ltd.) The operating method and detection method of the test tool for measuring glycated hemoglobin were the same as in Example 3, but the test tool for measuring the glycated hemoglobin concentration was detected. Performed at 730 nm.
A calibrator (Kyowa Medex Co., Ltd.) was separately measured to determine the glycated hemoglobin concentration and hemoglobin concentration of the sample, and the glycated hemoglobin ratio was calculated from the values. One sample of healthy human whole blood was measured 5 times to confirm reproducibility. The results are shown in Table 2.
[0063]
[Table 2]
As shown in Table 2, the measurement results of the glycated hemoglobin ratio measured using the test piece of the present invention and the test tool using the test piece are in good agreement with the HPLC method, and the measurement method and test piece of the present invention It was clear that the glycated hemoglobin concentration, the hemoglobin concentration and the glycated hemoglobin ratio were accurately measured using the test tool. In addition, the CV of 5-fold measurement of healthy whole blood was 6.1%, which was a good result.
[0065]
【The invention's effect】
By using the test piece, the test tool, and the measurement method using the test piece of the present invention, glycated proteins, particularly glycated hemoglobin and glycated arbumi can be measured accurately, simply and inexpensively at the clinical site.
[Brief description of the drawings]
FIG. 1A is a perspective view of parts when the test device of Example 2 is disassembled into respective parts. B: A perspective view of the test device of Example 2 is shown.
[Explanation of symbols]
(1) Upper film
(2) Middle grooved film
(3) Lower film
(4) Measurement hole
(5) Test piece
(6) Groove
FIG. 2 shows the effect of protease concentration in the measurement of glycated protein according to Example 3 of the present invention.
FIG. 3 shows the influence of the concentration of an enzyme acting on a glycated amino acid in measurement of a glycated protein based on Example 3 of the present invention.
Claims (12)
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| JP4260541B2 true JP4260541B2 (en) | 2009-04-30 |
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Families Citing this family (23)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP1878801B1 (en) | 2005-05-06 | 2011-01-26 | ARKRAY, Inc. | Protein cleavage method and use thereof |
| EP1883341B1 (en) | 2005-05-09 | 2020-08-12 | Labrador Diagnostics LLC | Point-of-care fluidic systems and uses thereof |
| US11287421B2 (en) | 2006-03-24 | 2022-03-29 | Labrador Diagnostics Llc | Systems and methods of sample processing and fluid control in a fluidic system |
| US8007999B2 (en) | 2006-05-10 | 2011-08-30 | Theranos, Inc. | Real-time detection of influenza virus |
| US8012744B2 (en) | 2006-10-13 | 2011-09-06 | Theranos, Inc. | Reducing optical interference in a fluidic device |
| US20080113391A1 (en) | 2006-11-14 | 2008-05-15 | Ian Gibbons | Detection and quantification of analytes in bodily fluids |
| US8158430B1 (en) | 2007-08-06 | 2012-04-17 | Theranos, Inc. | Systems and methods of fluidic sample processing |
| BR122020017678B1 (en) | 2007-10-02 | 2021-08-03 | Labrador Diagnostics Llc | SYSTEM FOR AUTOMATIC DETECTION OF AN ANALYTE IN A BODY FLUID SAMPLE |
| JP5726167B2 (en) * | 2009-04-13 | 2015-05-27 | マイクロニクス， インコーポレイテッド | Microfluidic clinical analyzer |
| EP2491499A4 (en) | 2009-10-19 | 2016-05-18 | Theranos Inc | INTEGRATED CAPTURE AND ANALYSIS SYSTEM FOR HEALTH DATA |
| CN102740976B (en) | 2010-01-29 | 2016-04-20 | 精密公司 | Sample-Response Microfluidic Cartridges |
| JP5870919B2 (en) * | 2010-04-09 | 2016-03-01 | 東洋紡株式会社 | Method for measuring glycated hemoglobin |
| TW201224432A (en) * | 2010-09-27 | 2012-06-16 | Toyo Boseki | Measuring apparatus and measuring method |
| TW202208825A (en) | 2011-01-21 | 2022-03-01 | 美商拉布拉多診斷有限責任公司 | Systems and methods for sample use maximization |
| TW201312118A (en) * | 2011-09-15 | 2013-03-16 | Toyo Boseki | Multilayer test film for measuring glycosylated hemoglobin and measuring method using it |
| CN104919191B (en) | 2012-12-21 | 2019-07-09 | 精密公司 | Fluid circuit and relevant manufacturing method |
| WO2014100725A1 (en) | 2012-12-21 | 2014-06-26 | Micronics, Inc. | Portable fluorescence detection system and microassay cartridge |
| EP3549674B1 (en) | 2012-12-21 | 2020-08-12 | PerkinElmer Health Sciences, Inc. | Low elasticity films for microfluidic use |
| US10386377B2 (en) | 2013-05-07 | 2019-08-20 | Micronics, Inc. | Microfluidic devices and methods for performing serum separation and blood cross-matching |
| CN105189750B (en) | 2013-05-07 | 2020-07-28 | 珀金埃尔默健康科学有限公司 | Method for preparing nucleic acid-containing samples using clay minerals and alkaline solutions |
| CN105189784B (en) | 2013-05-07 | 2020-06-30 | 珀金埃尔默健康科学有限公司 | Device for preparing and analyzing nucleic acids |
| CN114563575A (en) | 2020-11-27 | 2022-05-31 | 爱科来株式会社 | Test piece for measuring albumin |
| WO2023127707A1 (en) * | 2021-12-29 | 2023-07-06 | 株式会社Provigate | Method for measuring element of hemocyte component and element of non-hemocyte component in minute amount of blood, device, and pipette cartridge |
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