JP4517066B2 - Enzyme activity measuring reagent containing a trace amount of azide and measuring method - Google Patents
Enzyme activity measuring reagent containing a trace amount of azide and measuring method Download PDFInfo
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- JP4517066B2 JP4517066B2 JP14216398A JP14216398A JP4517066B2 JP 4517066 B2 JP4517066 B2 JP 4517066B2 JP 14216398 A JP14216398 A JP 14216398A JP 14216398 A JP14216398 A JP 14216398A JP 4517066 B2 JP4517066 B2 JP 4517066B2
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- reagent
- azide
- enzyme activity
- measurement
- concentration
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- 150000001540 azides Chemical class 0.000 title claims description 55
- 238000000034 method Methods 0.000 title claims description 37
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- 238000006911 enzymatic reaction Methods 0.000 claims description 7
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- 238000005259 measurement Methods 0.000 description 60
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【０００１】
【発明の属する技術分野】
本発明は、血清や尿等の生体試料中の各種酵素活性を測定するための試薬及び方法に関し、４００〜５００ｎｍの波長域での酵素活性の測定における溶血した試料において生じる誤差を回避するものである。
【０００２】
【従来の技術】
血清や尿等の生体試料中のγ−グルタミルトランスペプチダーゼ、α−アミラーゼやロイシンアミノペプチダーゼ等の酵素活性を測定するには、ニトロフェニル基又はニトロアニリン基等を有する基質にこれらの酵素が反応して生ずる４−ニトロフェノール、２−クロロ−４−ニトロフェノール、４−ニトロアニリン又は３−カルボキシ−４−ニトロアニリン等の４００〜５００ｎｍに吸収極大（λmax ）を持つ物の波長４００〜５００ｎｍでの時間経過による吸光度の増加速度を求め、その測定結果から酵素活性を求めるカイネティックアッセイ（レートアッセイ）が一般的である。
【０００３】
ところで、このような方法では、溶血作用により生成したヘモグロビンが試料中に含まれていると、測定中にそれが分解して、測定波長域における吸光度の経時的な減少を引き起こし、酵素活性測定の波長域での吸光度の増加速度を低下させてしまい、負の誤差が生じて、正確な測定値が得られないという問題があった。
【０００４】
そこで、溶血作用により生成したヘモグロビンが測定中に分解するのを防止するため、従来、酵素活性測定試薬にチオ尿素を含有させる方法（特公平６−１２９９８号）や、ピリジン類、イミダゾール類、ヒスタミン類を含有させる方法（特公平３−５６４２５号）が知られているが十分ではなかった。
【０００５】
なお、アジ化ナトリウムは、微生物の繁殖を抑える防腐剤として広く知られており、防腐能を有する０．１％以上の濃度で防腐剤としてのアジ化ナトリウムを含有する生体試料中の酵素活性測定試薬は存在した。
しかし、０．０５％以下の濃度でアジ化物を含有する生体試料中の酵素活性測定試薬、及び溶血した試料における誤差発生の抑制剤としてのアジ化物を含有する生体試料中の酵素活性測定試薬は、知られていなかった。
【０００６】
【発明が解決しようとする課題】
前記のように従来の生体試料中の酵素活性測定試薬及び酵素活性測定方法は、溶血した試料中のヘモグロビンの影響を受け、測定値に負誤差を生じてしまうものであった。
本発明者は、この問題点の解決を目指して鋭意検討を行った結果、酵素活性測定試薬中にアジ化物が０．００００７％〜０．０５％存在するとこの誤差が生じるのを抑制できることを見い出し、溶血した試料であっても、試料中の酵素活性を正確に測定することができる測定試薬及び測定方法を完成するに至った。
【０００７】
【課題を解決するための手段】
本発明は、溶血した試料における誤差発生の抑制剤として、０．００００７％〜０．０５％の濃度のアジ化物を含有する生体試料中の酵素活性測定試薬（第１の酵素活性測定試薬）である。
本発明の第１の酵素活性測定試薬においては、アジ化物の濃度が、０．０００１％〜０．０１％であることが好適である。
本発明の第１の酵素活性測定試薬においては、アジ化物の濃度が、０．０００２５％〜０．００５％であることが特に好適である。
【０００８】
また、本発明は、０．００００７％〜０．０５％の濃度のアジ化物を含有する酵素活性測定試薬と生体試料を混合し、微量のアジ化物の存在下で酵素反応を行わせ、４００〜５００ｎｍの波長域の吸光度を測定する、生体試料中の酵素活性測定方法（第１の酵素活性測定方法）である。
本発明の第１の酵素活性測定方法においては、アジ化物の濃度が、０．０００１％〜０．０１％であることが好適である。
本発明の第１の酵素活性測定方法においては、アジ化物の濃度が、０．０００２５％〜０．００５％であることが特に好適である。
本発明の第１の酵素活性測定方法は、アジ化物を、溶血した試料における誤差発生の抑制剤として含有する、生体試料中の酵素活性測定方法である。
【０００９】
更に、本発明は、第１試薬及び第２試薬の２試薬よりなる測定試薬であって、第１試薬と第２試薬を測定時の添加量の比で混合した際に、この混合後の測定試薬中のアジ化物の濃度が０．００００７％〜０．０５％となるように、溶血した試料における誤差発生の抑制剤として、アジ化物を第１試薬及び／又は第２試薬に含有する生体試料中の酵素活性測定試薬（第２の酵素活性測定試薬）である。
本発明の第２の酵素活性測定試薬においては、アジ化物の濃度が、０．０００１％〜０．０１％であることが好適である。
本発明の第２の酵素活性測定試薬においては、アジ化物の濃度が、０．０００２５％〜０．００５％であることが特に好適である。
【００１０】
そして、本発明は、第１試薬と第２試薬を測定時の添加量の比で混合した際にこの混合後の測定試薬中のアジ化物の濃度が０．００００７％〜０．０５％となるようにアジ化物を第１試薬及び／又は第２試薬に含有する酵素活性測定試薬の第１試薬並びに第２試薬及び生体試料を混合し、微量のアジ化物の存在下で酵素反応を行わせ、４００〜５００ｎｍの波長域の吸光度を測定する、生体試料中の酵素活性測定方法（第２の酵素活性測定方法）である。
本発明の第２の酵素活性測定方法においては、アジ化物の濃度が、０．０００１％〜０．０１％であることが好適である。
本発明の第２の酵素活性測定方法においては、アジ化物の濃度が、０．０００２５％〜０．００５％であることが特に好適である。
本発明の第２の酵素活性測定方法は、アジ化物を、溶血した試料における誤差発生の抑制剤として含有する、生体試料中の酵素活性測定方法である。
また、本発明は、０．００００７％〜０．０５％の濃度のアジ化物よりなる、溶血した試料における誤差発生の抑制剤である。
本発明の溶血した試料における誤差発生の抑制剤においては、アジ化物の濃度が、０．０００１％〜０．０１％であることが好適である。
本発明の溶血した試料における誤差発生の抑制剤においては、アジ化物の濃度が、０．０００２５％〜０．００５％であることが特に好適である。
更に、本発明は、生体試料中の酵素活性測定方法において、０．００００７％〜０．０５％の濃度のアジ化物の存在下で酵素反応を行わせることによる、溶血した試料における誤差発生の抑制方法である。
本発明の溶血した試料における誤差発生の抑制方法においては、アジ化物の濃度が、０．０００１％〜０．０１％であることが好適である。
本発明の溶血した試料における誤差発生の抑制方法においては、アジ化物の濃度が、０．０００２５％〜０．００５％であることが特に好適である。
【００１１】
【発明の実施の形態】
本発明において、アジ化物とは、アジ化水素（ＨＮ3 ）の水素が金属で置換されたものである。
このアジ化物の具体例として、アジ化ナトリウム〔ＮａＮ3 〕、アジ化カリウム〔ＫＮ3 〕、アジ化バリウム〔Ｂａ（Ｎ3）2〕、アジ化リチウム〔ＬｉＮ3 〕等を挙げることができる。
【００１２】
なお、本発明において、生体試料中の酵素活性測定試薬に含有させるアジ化物は、従来０．１％以上の濃度で用いられていた防腐剤としてではなく、溶血した試料における誤差発生の抑制剤として使用するものである。
【００１３】
上記アジ化物は、一種又は二種以上を組み合わせて用いてもよい。
【００１４】
本発明において、溶血した試料における負誤差を回避するために、上記アジ化物を生体試料中の酵素活性測定試薬に含有させる濃度は、０．００００７％〜０．０５％であればよく、好ましくは０．０００１％〜０．０１％であり、特に好ましくは０．０００２５％〜０．００５％である。
【００１５】
本発明の生体試料中の酵素活性測定試薬は、１試薬からなるものでもよく、また２試薬等の複数の試薬から構成されるものであってもよい。
【００１６】
上記酵素活性測定試薬が第１試薬及び第２試薬の２試薬よりなる測定試薬の場合は、この第１試薬と第２試薬を生体試料中の酵素活性を測定する時の各々の添加量の比で混合した際に、この混合後の測定試薬中のアジ化物の濃度が０．００００７％〜０．０５％（好ましくは０．０００１％〜０．０１％、特に好ましくは０．０００２５％〜０．００５％）となるように、アジ化物を第１試薬及び第２試薬に含有させるか、又はアジ化物を第１試薬若しくは第２試薬のいずれかに含有させる必要がある。
【００１７】
混合後の測定試薬中のアジ化物の濃度が上記濃度範囲に入るのであれば、アジ化物は第１試薬のみに含有させてもよく、又は第２試薬のみに含有させてもよく、あるいは第１試薬と第２試薬の両方に含有させてもよい。
【００１８】
この第１試薬と第２試薬を測定時の添加量の比で混合した後の測定試薬中のアジ化物の濃度と第１試薬及び第２試薬に含有させるアジ化物の濃度の関係の式を下記の数式１に示した。
【００１９】
【数式１】
【００２０】
測定時の第１試薬と第２試薬の添加量の比（Ｖ１、Ｖ２）が定まっている場合、混合後の測定試薬中のアジ化物濃度（ＣＭ）が、０．００００７％〜０．０５％（好ましくは０．０００１％〜０．０１％、特に好ましくは０．０００２５％〜０．００５％）の範囲内の特定の値となるよう数式１に従って、第１試薬及び第２試薬に含有させるアジ化物の濃度を決めればよい。
【００２１】
混合後のアジ化物濃度が０．００００７％〜０．０５％となる、第１試薬及び第２試薬に含有させるアジ化物濃度の例を表１、表２、表３、表４、表５、及び表６に示した。
【００２２】
【表１】
【表２】
【表３】
【表４】
【表５】
【表６】
なお、上記酵素活性測定試薬が３試薬以上の試薬から構成される場合も同様である。
【００２９】
ところで、本発明においては、酵素活性測定試薬中の濃度、又は第１試薬、第２試薬等の測定試薬を構成する試薬の混合後の測定試薬中での濃度で、アジ化物の濃度を０．００００７％〜０．０５％（好ましくは０．０００１％〜０．０１％、特に好ましくは０．０００２５％〜０．００５％）と規定しているが、これを酵素活性測定試薬と生体試料の混合後の濃度、又は第１試薬、第２試薬等の測定試薬を構成する試薬と生体試料の混合後の濃度、つまり酵素活性測定反応の最終反応液中の濃度に置き換えても支障はない。
【００３０】
それは、現在行われている生体試料中の酵素活性の測定において、試料の添加量を酵素活性測定試薬の添加量で割った時の比率は、測定試薬の添加量に対する試料の添加量が大変小さいため通常１〜５％の範囲内に収まるものであり、試料の添加量を考慮してもしなくても大差はないからである。
【００３１】
なお、後に実施例で示すが、酵素活性測定試薬に含有するアジ化物の濃度は、溶血した試料における誤差の発生を抑制する効果からすると、０．００００７％〜０．０５％の範囲にあることが至適であり、これより高くても、また低くても溶血試料における誤差の発生を抑制する効果は小さくなるので好ましくない。
【００３２】
本発明において、生体試料とは、酵素活性を測定しようとする酵素が存在する可能性がある液状のものをいう。
例えば、ヒト又は動物の血液、血清、血漿、尿、精液、髄液、唾液、汗、涙、腹水、羊水等の体液；ヒト若しくは動物の脳、肝臓等の臓器、毛髪、皮膚、爪、筋肉、又は神経組織等の抽出液、ヒト又は動物の糞便の抽出液又は懸濁液；細胞或いは菌体の抽出液；植物の抽出液等が挙げられる。
【００３３】
本発明において、酵素活性の測定を行う酵素とは、４００〜５００ｎｍの波長域の吸光度を測定することにより酵素活性の測定を行うことができるものである。
つまり、この活性の測定を行おうとする酵素が反応を触媒して生成する物、又はこの生成物より導かれる物質が４００〜５００ｎｍの波長域に吸収を持つような化合物を、基質とすることができる酵素である。
【００３４】
例えば、γ−グルタミルトランスペプチダーゼ（γ−グルタミルトランスフェラーゼ、γ−ＧＴＰ、γ−ＧＴ）、ロイシンアミノペプチダーゼ（ＬＡＰ）、アリルアミダーゼ（ＡＡ）、Ｌ−シスチンアミノトランスペプチダーゼ（ＣＡＰ）、α−アミラーゼ（ＡＭＹ）、アルカリ性ホスファターゼ（ＡＬＰ）、コリンエステラーゼ（Ｃｈ−Ｅ、ＣＨＥ）、トロンビン、トロンボプラスチン、アンチトロンビンIII（ＡＴIII）、プラスミノーゲン、α2 −プラスミンインヒビター、第II凝固因子、第VIII凝固因子、第Ｘ凝固因子、第Ｘa 因子、又はプロテインＣ等を挙げることができる。
【００３５】
また、上記の酵素のアイソザイムの活性測定に用いることもできる。
この場合、例えば特定のアイソザイムを阻害する抗体等の阻害剤を生体試料中の酵素に作用させ、酵素活性を測定することにより行うことができる。
【００３６】
本発明の生体試料中の酵素活性測定方法（第１の酵素活性測定方法）においては、０．００００７％〜０．０５％の濃度のアジ化物を含有する酵素活性測定試薬と生体試料を混合し、微量のアジ化物の存在下で酵素反応を行わせ、酵素が基質と反応して生成する物又はこの生成物より導かれる物質の４００〜５００ｎｍの波長域の吸光度を測定し、この測定値より生体試料中の酵素活性を算出する。
【００３７】
本発明の生体試料中の酵素活性測定方法（第２の酵素活性測定方法）においては、第１試薬と第２試薬を測定時の添加量の比で混合した際にこの混合後の測定試薬中のアジ化物の濃度が０．００００７％〜０．０５％となるようにアジ化物を第１試薬及び／又は第２試薬に含有する酵素活性測定試薬の第１試薬並びに第２試薬及び生体試料を混合し、微量のアジ化物の存在下で酵素反応を行わせ、酵素が基質と反応して生成する物又はこの生成物より導かれる物質の４００〜５００ｎｍの波長域の吸光度を測定し、この測定値より生体試料中の酵素活性を算出する。
【００３８】
なお、先に述べたように、試料の添加量を酵素活性測定試薬の添加量で割った時の比率は、通常１〜５％の範囲内に収まるものであり、試料の添加量はほとんど無視できるものであるので、酵素反応を行わせる時の微量のアジ化物の濃度は、０．００００７％〜０．０５％（好ましくは０．０００１％〜０．０１％、特に好ましくは０．０００２５％〜０．００５％）とほぼ同じである。
【００３９】
本発明の生体試料中の酵素活性測定方法は、酵素が基質と反応して生成する物又はこの生成物より導かれる物質の生成速度を４００〜５００ｎｍの波長域の吸光度の１分間当たりの変化量より算出するか、酵素が基質と反応して生成する物又はこの生成物より導かれる物質の生成量を４００〜５００ｎｍの波長域の吸光度を測ることにより求める測定原理のものであれば、いかなる方法にも用いることができる。例えば、反応速度法（カイネティック法、レート法）又は終点法（エンドポイント法）に適用できる。
このような測定方法において、それ自体公知の方法によって、用手法又は自動分析装置による方法により酵素活性を測定することができる。
【００４０】
例えば、γ−グルタミルトランスペプチダーゼの測定用基質としては、Ｌ−γ−グルタミル−４−ニトロアニリド、Ｌ−γ−グルタミル−３−カルボキシ−４−ニトロアニリド等が用いられ；ロイシンアミノペプチダーゼの測定用基質としては、Ｌ−ロイシル−４−ニトロアニリド等が用いられ；Ｌ−シスチンアミノトランスペプチダーゼの測定用基質としては、Ｓ−ベンジル−Ｌ−システイン−４−ニトロアニリド等が用いられ；α−アミラーゼの測定用基質としては、４−ニトロフェニル−α−Ｄ−マルトヘプタオシド、４，６−ベンジリデン−４−ニトロフェニル−α−Ｄ−マルトヘプタオシド、４5 −Ｏ−β−Ｄ−ガラクトピラノシル−４−ニトロフェニル−α−Ｄ−マルトペンタオシド、２−クロロ−４−ニトロフェニル−β−Ｄ−マルトペンタオシド等が用いられ；アルカリ性ホスファターゼの測定用基質としては、４−ニトロフェニルリン酸等が用いられ；コリンエステラーゼの測定用基質としては、ブチリルチオコリン等が用いられる。
これらの基質に酵素が作用して遊離してくる４−ニトロフェノール、２−クロロ−４−ニトロフェノール、４−ニトロアニリン若しくは３−カルボキシ−４−ニトロアニリン等の４００〜５００ｎｍに吸収極大（λmax ）を持つ物の４００〜５００ｎｍの波長域の吸光度を経時的に測定して、その吸光度の増加より酵素活性を算出する。
【００４１】
または、これらの基質に酵素が作用して遊離してくるチオコリン等の物に、ＳＨ基と反応して定量的に４００〜５００ｎｍの波長域の吸光度の変化を生ずるＤＴＮＢ〔５，５’−ジチオビス（２−ニトロ安息香酸）〕等を加えて反応させ、生成してきた４００〜５００ｎｍに吸収極大（λmax ）を持つ物質の４００〜５００ｎｍの波長域の吸光度を経時的に測定して、その吸光度の増加より酵素活性を算出する。
この場合、本発明によれば、生体試料中のヘモグロビンの分解により生じる誤差を抑制することができる。
【００４２】
【実施例】
以下実施例を挙げて本発明を説明するが、本発明はこれらによって限定されるものではない。
【００４３】
〔実施例１〕
（血清試料中のγ−グルタミルトランスペプチダーゼ［γ−ＧＴ］活性の測定）
【００４４】
（１）試薬の調製
▲１▼γ−ＧＴ・第１試薬の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、1N水酸化ナトリウムを加えてpHを8.2（20℃）に調整して、アジ化ナトリウム濃度が異なる１３種類のγ−ＧＴ・第１試薬を調製した。
【００４５】
グリシルグリシン 225mM
アジ化ナトリウム
0%、0.0001%、0.0002%、0.0004%、0.0006%、0.0008%、0.001%、0.0015%、
0.002%、0.003%、0.005%、0.01%、又は0.1%
【００４６】
▲２▼γ−ＧＴ・第２試薬の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、１N水酸化ナトリウムを加えてpHを5.25（20℃）に調整して、γ−ＧＴ・第２試薬を調製した。
【００４７】
酢酸 20mM
Ｌ−γ−グルタミル−３−カルボキシ−４−ニトロアニリド 18mM
【００４８】
（２）血清試料の調製
γ−ＧＴ活性値70U/lのヒト血清試料の９容と、ヘモグロビンを含まない水溶液又はヒト赤血球を溶血させて調製したヘモグロビン濃度が、5,000又は10,000mg/dlの水溶液の１容をそれぞれ混合して、ヘモグロビン濃度が各々0、500、又は1,000mg/dlである血清試料を調製した。
【００４９】
（３）血清試料中のγ−ＧＴ活性の測定
日立7150形自動分析装置を用いて測定した。
上記（２）で調製した血清試料10μlに、上記（１）で調製したγ−ＧＴ・第１試薬250μlを添加し、37℃で5分間反応させた後、これに上記（１）で調製したγ−ＧＴ・第２試薬125μlを添加して37℃で反応させ、主波長405nm、副波長505nmにおける、γ−ＧＴ・第２試薬添加1分24秒後（32ポイント）から5分後（50ポイント）までの1分間当たりの吸光度変化量よりγ−ＧＴ活性値を下記の式により算出した。この式におけるＫ値である4,153は、5-アミノ-2-ニトロ安息香酸のモル吸光係数（ε）を9.27として算出したものである。
【００５０】
なお、対照として、血清試料の代わりに生理食塩水を試料として同様に測定を行い、得られた吸光度変化量を試薬盲験値とし、これを上記血清試料測定時の吸光度変化量から差し引いた値をγ−ＧＴ活性値の算出に用いた。
【００５１】
γ−ＧＴ活性値（U/l）＝（１分間当たりの吸光度変化量）× 4,153
【００５２】
ところで、上記の１３種類のγ−ＧＴ・第１試薬と上記γ−ＧＴ・第２試薬とを上記の添加量で混合した後の測定試薬中のアジ化ナトリウムの濃度は、各々、下記の様になる。
0%、0.000067%、0.00013%、0.00027%、0.0004%、0.00053%、0.00067%、
0.001%、0.0013%、0.002%、0.0033%、0.0067%、又は0.067%
【００５３】
（４）測定結果
測定の結果を表７及び図１に示す。
なお、この表７に示した数値は、各試薬における、ヘモグロビン濃度0mg/dlの血清試料のγ−ＧＴ活性測定値を100%とした時のヘモグロビン濃度500mg/dl、又は1,000mg/dlの血清試料のγ−ＧＴ活性測定値を相対比率（%）で表したものである。
【００５４】
【表７】
また、図１において、横軸はγ−ＧＴ・第１試薬とγ−ＧＴ・第２試薬を混合した後の測定試薬中でのアジ化ナトリウム濃度を、縦軸は各試薬における、ヘモグロビン濃度0mg/dlの血清試料のγ−ＧＴ活性測定値を100%とした時のヘモグロビン濃度500mg/dl、又は1,000mg/dlの血清試料のγ−ＧＴ活性測定値を相対比率（%）で表したものである。
ここで、「○」はヘモグロビン濃度500mg/dlの血清試料のγ−ＧＴ活性測定値を、「●」はヘモグロビン濃度1,000mg/dlの血清試料のγ−ＧＴ活性測定値を示す。
【００５６】
【図１】
【００５７】
表７及び図１から明らかなように、γ−ＧＴ活性測定試薬による測定では、混合後の測定試薬中のアジ化ナトリウム濃度が0.00007%以上及び0.05%以下において溶血試料において生じる誤差を抑制できることが分かる。特にアジ化ナトリウム濃度が0.0001%以上及び0.01%以下において、更に0.00025%以上及び0.005%以下においては、効果が顕著である。
よって、本発明の酵素活性測定試薬及び測定方法は、溶血した試料においても、誤差を生じずに正確な酵素活性値を得ることができることが確かめられた。
【００５８】
〔実施例２〕
（血清試料中のロイシンアミノペプチダーゼ［ＬＡＰ］活性の測定）
【００５９】
（１）試薬の調製
▲１▼ＬＡＰ・第１試薬の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、1N水酸化ナトリウムを加えてpHを8.0（20℃）に調整して、アジ化ナトリウム濃度が異なる５種類のＬＡＰ・第１試薬を調製した。
【００６０】
トリス（ヒドロキシメチル）アミノメタン 75mM
塩化ナトリウム 376mM
アジ化ナトリウム
0%、0.0001%、0.001%、0.01%、又は0.1%
【００６１】
▲２▼ＬＡＰ・第２試薬の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、１N水酸化ナトリウムを加えてpHを4.1（20℃）に調整して、ＬＡＰ・第２試薬を調製した。
【００６２】
酢酸 20mM
Ｌ−ロイシン−ｐ−ニトロアニリド 12.2mM
【００６３】
（２）血清試料の調製
ＬＡＰ活性値49U/lのヒト血清試料（血清試料Ａ）又はＬＡＰ活性値41U/lのヒト血清試料（血清試料Ｂ）の９容と、ヘモグロビンを含まない水溶液又はヒト赤血球を溶血させて調製したヘモグロビン濃度が、2,500、5,000、7,500又は10,000mg/dlの水溶液の１容をそれぞれ混合して、ヘモグロビン濃度が各々0、250、500、750又は1,000mg/dlである血清試料（血清試料Ａ及び血清試料Ｂ）を調製した。
【００６４】
（３）血清試料中のＬＡＰ活性の測定
日立7150形自動分析装置を用いて測定した。
上記（２）で調製した血清試料8μlに、上記（１）で調製したＬＡＰ・第１試薬260μlを添加し、37℃で5分間反応させた後、これに上記（１）で調製したＬＡＰ・第２試薬130μlを添加して37℃で反応させ、主波長405nm、副波長480nmにおける、ＬＡＰ・第２試薬添加1分24秒後（32ポイント）から5分後（50ポイント）までの1分間当たりの吸光度変化量よりＬＡＰ活性値を下記の式により算出した。この式におけるＫ値である5,097は、4-ニトロアニリンのモル吸光係数（ε）を9.76として算出したものである。
【００６５】
なお、対照として、血清試料の代わりに生理食塩水を試料として同様に測定を行い、得られた吸光度変化量を試薬盲験値とし、これを上記血清試料測定時の吸光度変化量から差し引いた値をＬＡＰ活性値の算出に用いた。
【００６６】
ＬＡＰ活性値（U/l）＝（１分間当たりの吸光度変化量）× 5,097
【００６７】
また、上記の５種類のＬＡＰ・第１試薬と上記ＬＡＰ・第２試薬とを上記の添加量で混合した後の測定試薬中のアジ化ナトリウムの濃度は、各々、下記の様になる。
0%、0.000067%、0.00067%、0.0067%、又は0.067%
【００６８】
（４）測定結果
測定の結果を表８に示す。
なお、この表８に示した数値は、各試薬における、ヘモグロビン濃度0mg/dlの血清試料のＬＡＰ活性測定値を100%とした時のヘモグロビン濃度250mg/dl、500mg/dl、750mg/dl、又は1,000mg/dlの血清試料のＬＡＰ活性測定値を相対比率（%）で表したものである。
【００６９】
【表８】
表８から明らかなように、本発明のＬＡＰ活性測定試薬による測定においても、混合後の測定試薬中のアジ化ナトリウム濃度が0.00007%以上及び0.05%以下において溶血試料において生じる誤差を抑制できることがわかる。特に0.0001%以上及び0.01%以下において効果が顕著である。
よって、本発明の酵素活性測定試薬及び測定方法では、測定項目が異なっても、溶血した試料において誤差を生じずに正確な酵素活性値を得ることができることが確かめられた。
【００７１】
〔実施例３〕
（血清試料中のα−アミラーゼ［ＡＭＹ］活性の測定）
【００７２】
（１）試薬の調製
ＡＭＹ活性測定試薬の調製
下記の試薬成分をそれぞれ記載の濃度になるように純水に溶解し、1N水酸化ナトリウムを加えてpHを6.3（37℃）に調整して、アジ化ナトリウム濃度が異なる２種類のＡＭＹ活性測定試薬を調製した。
【００７３】
2-（N-モルフォリノ）エタンスルホン酸・一水塩 50mM
チオシアン酸カリウム 900mM
塩化ナトリウム 300mM
酢酸カルシウム 5mM
2-クロロ-4-ニトロフェニル-α-D-マルトトリオシド 2.25mM
アジ化ナトリウム 0%、又は0.01%
【００７４】
（２）血清試料の調製
ＡＭＹ活性値64U/lのヒト血清試料の8容と、ヘモグロビンを含まない水溶液又はヒト赤血球を溶血させて調製したヘモグロビン濃度が、2,500、又は5,000mg/dlの水溶液の2容をそれぞれ混合して、ヘモグロビン濃度が各々0、500、又は1,000mg/dlである血清試料を調製した。
【００７５】
（３）血清試料中のＡＭＹ活性の測定
日立7150形自動分析装置を用いて測定した。
上記（２）で調製した血清試料5μlに、上記（１）で調製したＡＭＹ試薬300μlを添加して37℃で反応させ、主波長405nm、副波長546nmにおける、ＡＭＹ活性測定試薬添加2分12秒後（11ポイント）から4分12秒後（21ポイント）までの1分間当たりの吸光度変化量よりＡＭＹ活性値を下記の式により算出した。この式におけるＫ値である3,885は、2-クロロ-4-ニトロフェノールのモル吸光係数（ε）を15.7として算出したものである。
【００７６】
なお、対照として、血清試料の代わりに生理食塩水を試料として同様に測定を行い、得られた吸光度変化量を試薬盲験値とし、これを上記血清試料測定時の吸光度変化量から差し引いた値をＡＭＹ活性値の算出に用いた。
【００７７】
ＡＭＹ活性値（U/l）＝（１分間当たりの吸光度変化量）× 3,885
【００７８】
（４）測定結果
測定の結果を表９に示す。
なお、この表９に示した数値は、各試薬における、ヘモグロビン濃度0mg/dlの血清試料のＡＭＹ活性測定値を100%とした時のヘモグロビン濃度500mg/dl、又は1,000mg/dlの血清試料のＡＭＹ活性測定値を相対比率（%）で表したものである。
【００７９】
【表９】
表９から明らかなように、本発明のＡＭＹ活性測定試薬による測定においても、測定試薬中のアジ化ナトリウム濃度が0.00007%以上及び0.05%以下にある場合には、溶血試料において生じる誤差を抑制できることがわかる。
よって、本発明の酵素活性測定試薬及び測定方法では、測定項目にかかわらず、溶血した試料において誤差を生じずに正確な酵素活性値を得ることができることが確かめられた。
【００８１】
【発明の効果】
本発明の酵素活性測定試薬及び酵素活性測定方法は、酵素活性測定試薬中にアジ化物を０．００００７％〜０．０５％含有させることにより、溶血した試料であっても、測定値に誤差が生じるのを抑制し、生体試料中の酵素活性を正確に測定することができるものである。
【図面の簡単な説明】
【図１】本発明のγ−ＧＴ活性測定試薬での、試料の溶血による測定値への影響を検討した図である。
【数式１】
BACKGROUND OF THE INVENTION
The present invention relates to a reagent and method for measuring various enzyme activities in biological samples such as serum and urine, and avoids errors that occur in hemolyzed samples in the measurement of enzyme activity in the wavelength range of 400 to 500 nm. is there.
[0002]
[Prior art]
In order to measure enzyme activities such as γ-glutamyltranspeptidase, α-amylase and leucine aminopeptidase in biological samples such as serum and urine, these enzymes react with a substrate having nitrophenyl group or nitroaniline group. Of 4-nitrophenol, 2-chloro-4-nitrophenol, 4-nitroaniline, 3-carboxy-4-nitroaniline and the like having an absorption maximum (λmax) at 400 to 500 nm at a wavelength of 400 to 500 nm A kinetic assay (rate assay) is generally used to determine the rate of increase in absorbance over time and to determine enzyme activity from the measurement results.
[0003]
By the way, in such a method, if hemoglobin generated by hemolysis is contained in the sample, it is decomposed during the measurement, causing a decrease in absorbance over time in the measurement wavelength region, and measuring the enzyme activity. There is a problem that the rate of increase in absorbance in the wavelength region is reduced, a negative error occurs, and an accurate measurement value cannot be obtained.
[0004]
Therefore, in order to prevent the hemoglobin produced by hemolysis from being decomposed during the measurement, a conventional method for containing thiourea in an enzyme activity measurement reagent (Japanese Patent Publication No. 6-12998), pyridines, imidazoles, histamine Although a method of containing a kind (Japanese Patent Publication No. 3-56425) is known, it was not sufficient.
[0005]
Sodium azide is widely known as a preservative that suppresses the growth of microorganisms, and measures enzyme activity in biological samples containing sodium azide as a preservative at a concentration of 0.1% or more that has antiseptic properties. Reagent was present.
However, a reagent for measuring enzyme activity in a biological sample containing azide at a concentration of 0.05% or less, and a reagent for measuring enzyme activity in a biological sample containing azide as an inhibitor of error occurrence in a hemolyzed sample are: Was not known.
[0006]
[Problems to be solved by the invention]
As described above, the conventional enzyme activity measuring reagent and enzyme activity measuring method in a biological sample are affected by hemoglobin in the hemolyzed sample and cause a negative error in the measured value.
As a result of intensive studies aimed at solving this problem, the present inventor found that the presence of 0.00007% to 0.05% azide in the enzyme activity measurement reagent can suppress the occurrence of this error. Thus, even for a hemolyzed sample, a measuring reagent and a measuring method capable of accurately measuring the enzyme activity in the sample have been completed.
[0007]
[Means for Solving the Problems]
The present inventionAs an inhibitor of error generation in hemolyzed samples,It is an enzyme activity measuring reagent (first enzyme activity measuring reagent) in a biological sample containing azide at a concentration of 0.00007% to 0.05%.
In the first enzyme activity measurement reagent of the present invention, it is preferable that the concentration of azide is 0.0001% to 0.01%.
In the first enzyme activity measurement reagent of the present invention, it is particularly preferred that the concentration of the azide is 0.00025% to 0.005%.
[0008]
Further, the present invention mixes a biological sample with an enzyme activity measurement reagent containing a concentration of azide of 0.00007% to 0.05%, and allows the enzyme reaction to be carried out in the presence of a trace amount of azide. It is the enzyme activity measuring method (1st enzyme activity measuring method) in a biological sample which measures the light absorbency of a wavelength range of 500 nm.
In the first enzyme activity measurement method of the present invention, it is preferable that the azide concentration is 0.0001% to 0.01%.
In the first enzyme activity measurement method of the present invention, it is particularly preferable that the concentration of the azide is 0.00025% to 0.005%.
The first enzyme activity measurement method of the present invention is a method for measuring enzyme activity in a biological sample, which contains azide as an inhibitor of error generation in a hemolyzed sample.
[0009]
Furthermore, the present invention is a measurement reagent comprising two reagents, a first reagent and a second reagent, and when the first reagent and the second reagent are mixed at a ratio of the addition amount at the time of measurement, the measurement after mixing is performed. So that the concentration of azide in the reagent is 0.00007% to 0.05%,As an inhibitor of error generation in hemolyzed samples,It is an enzyme activity measuring reagent (second enzyme activity measuring reagent) in a biological sample containing an azide in the first reagent and / or the second reagent.
In the second enzyme activity measurement reagent of the present invention, it is preferable that the concentration of azide is 0.0001% to 0.01%.
In the second enzyme activity measurement reagent of the present invention, it is particularly preferable that the concentration of the azide is 0.00025% to 0.005%.
[0010]
In the present invention, when the first reagent and the second reagent are mixed at the ratio of the addition amount at the time of measurement, the concentration of the azide in the measurement reagent after mixing is 0.00007% to 0.05%. As described above, the first reagent and the second reagent of the enzyme activity measurement reagent containing the azide in the first reagent and / or the second reagent and the biological sample are mixed, and the enzyme reaction is performed in the presence of a trace amount of azide, It is the enzyme activity measuring method (2nd enzyme activity measuring method) in a biological sample which measures the light absorbency of a 400-500 nm wavelength range.
In the second enzyme activity measurement method of the present invention, the azide concentration is preferably 0.0001% to 0.01%.
In the second enzyme activity measurement method of the present invention, it is particularly preferable that the concentration of the azide is 0.00025% to 0.005%.
The second enzyme activity measurement method of the present invention is a method for measuring enzyme activity in a biological sample, which contains azide as an inhibitor of error generation in a hemolyzed sample.
In addition, the present invention is an error generation inhibitor in a hemolyzed sample made of azide at a concentration of 0.00007% to 0.05%.
In the inhibitor of occurrence of errors in the hemolyzed sample of the present invention, the concentration of azide is preferably 0.0001% to 0.01%.
In the inhibitor for error occurrence in the hemolyzed sample of the present invention, it is particularly preferable that the concentration of azide is 0.00025% to 0.005%.
Furthermore, the present invention provides a method for measuring enzyme activity in a biological sample, which suppresses the occurrence of errors in a hemolyzed sample by allowing an enzyme reaction to be carried out in the presence of an azide at a concentration of 0.00007% to 0.05%. Is the method.
In the method for suppressing error generation in a hemolyzed sample of the present invention, it is preferable that the concentration of azide is 0.0001% to 0.01%.
In the method for suppressing the occurrence of errors in the hemolyzed sample of the present invention, it is particularly preferable that the concentration of azide is 0.00025% to 0.005%.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, azide means hydrogen azide (HNThree) In which hydrogen is substituted with a metal.
As a specific example of this azide, sodium azide [NaNThree], Potassium azide [KNThree], Barium azide [Ba (NThree)2], Lithium azide [LiNThreeAnd the like.
[0012]
In the present invention, the azide contained in the enzyme activity measurement reagent in the biological sample is not used as a preservative that has been conventionally used at a concentration of 0.1% or more, but as an error generation inhibitor in a hemolyzed sample. It is what you use.
[0013]
The azide may be used alone or in combination of two or more.
[0014]
In the present invention, in order to avoid a negative error in the hemolyzed sample, the concentration of the azide contained in the enzyme activity measurement reagent in the biological sample may be 0.00007% to 0.05%, preferably It is 0.0001% to 0.01%, particularly preferably 0.00025% to 0.005%.
[0015]
The reagent for measuring enzyme activity in the biological sample of the present invention may be composed of one reagent, or may be composed of a plurality of reagents such as two reagents.
[0016]
In the case where the enzyme activity measuring reagent is a measuring reagent composed of two reagents, the first reagent and the second reagent, the ratio of the respective addition amounts when the enzyme activity in the biological sample is measured using the first reagent and the second reagent. The azide concentration in the measuring reagent after mixing is 0.00007% to 0.05% (preferably 0.0001% to 0.01%, particularly preferably 0.00025% to 0). 0.005%), the azide must be contained in the first reagent and the second reagent, or the azide must be contained in either the first reagent or the second reagent.
[0017]
If the concentration of the azide in the measurement reagent after mixing falls within the above concentration range, the azide may be contained only in the first reagent, or may be contained only in the second reagent, or the first You may make it contain in both a reagent and a 2nd reagent.
[0018]
An expression of the relationship between the concentration of the azide in the measurement reagent after mixing the first reagent and the second reagent in the ratio of the addition amount at the time of measurement and the concentration of the azide contained in the first reagent and the second reagent is as follows. This is shown in Formula 1.
[0019]
[Formula 1]
[0020]
When the ratio (V1, V2) of the addition amount of the first reagent and the second reagent at the time of measurement is determined, the azide concentration (CM) in the measurement reagent after mixing is 0.00007% to 0.05%. (Preferably 0.0001% to 0.01%, particularly preferably 0.00025% to 0.005%) According to Formula 1, the first reagent and the second reagent contain the specific value within the range. The concentration of azide may be determined.
[0021]
Table 1, Table 2, Table 3, Table 4, Table 5, Examples of azide concentration to be contained in the first reagent and the second reagent, where the azide concentration after mixing is 0.00007% to 0.05%, And in Table 6.
[0022]
[Table 1]
[Table 2]
[Table 3]
[Table 4]
[Table 5]
[Table 6]
The same applies when the enzyme activity measurement reagent is composed of three or more reagents.
[0029]
By the way, in this invention, the density | concentration of an azide is set to 0 by the density | concentration in an enzyme activity measuring reagent or the density | concentration in the measuring reagent after mixing the reagent which comprises measuring reagents, such as a 1st reagent and a 2nd reagent. It is defined as 00007% to 0.05% (preferably 0.0001% to 0.01%, particularly preferably 0.00025% to 0.005%). There is no problem even if it is replaced with the concentration after mixing, or the concentration after mixing the reagent constituting the measurement reagent such as the first reagent and the second reagent and the biological sample, that is, the concentration in the final reaction solution of the enzyme activity measurement reaction.
[0030]
The ratio of the amount of sample added to the amount of the reagent for measuring the enzyme activity is very small when the amount of sample added is divided by the amount of the reagent for measuring enzyme activity. Therefore, it is usually within the range of 1 to 5%, and there is no great difference whether or not the amount of sample added is taken into consideration.
[0031]
As will be shown later in the examples, the concentration of azide contained in the enzyme activity measurement reagent is in the range of 0.00007% to 0.05% in terms of the effect of suppressing the occurrence of errors in the hemolyzed sample. It is not preferable because the effect of suppressing the occurrence of errors in the hemolyzed sample is small, even if it is higher or lower than this.
[0032]
In the present invention, the biological sample refers to a liquid sample in which an enzyme whose enzyme activity is to be measured may exist.
For example, human or animal blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tears, ascites, amniotic fluid, etc .; human or animal brain, liver or other organs, hair, skin, nails, muscles Or an extract of neuronal tissue, an extract or suspension of human or animal feces, an extract of cells or cells, an extract of plants, and the like.
[0033]
In the present invention, an enzyme that measures enzyme activity is one that can measure enzyme activity by measuring absorbance in the wavelength region of 400 to 500 nm.
In other words, a substance produced by catalyzing the reaction of the enzyme whose activity is to be measured or a compound derived from this product having absorption in the wavelength range of 400 to 500 nm may be used as a substrate. It can be an enzyme.
[0034]
For example, γ-glutamyltranspeptidase (γ-glutamyltransferase, γ-GTP, γ-GT), leucine aminopeptidase (LAP), allylamidase (AA), L-cystine aminotranspeptidase (CAP), α-amylase (AMY) ), Alkaline phosphatase (ALP), cholinesterase (Ch-E, CHE), thrombin, thromboplastin, antithrombin III (ATIII), plasminogen, α2 -Plasmin inhibitor, factor II coagulation factor, factor VIII coagulation factor, factor X coagulation factor, factor Xa, protein C and the like.
[0035]
It can also be used to measure the activity of the above enzyme isozymes.
In this case, for example, an inhibitor such as an antibody that inhibits a specific isozyme is allowed to act on an enzyme in a biological sample, and the enzyme activity is measured.
[0036]
In the method for measuring enzyme activity in a biological sample of the present invention (first enzyme activity measuring method), an enzyme activity measuring reagent containing an azide at a concentration of 0.00007% to 0.05% is mixed with the biological sample. Then, the enzyme reaction is carried out in the presence of a trace amount of azide, and the absorbance in the wavelength range of 400 to 500 nm of the substance produced by the reaction of the enzyme with the substrate or the substance derived from this product is measured. The enzyme activity in the biological sample is calculated.
[0037]
In the enzyme activity measuring method in the biological sample of the present invention (second enzyme activity measuring method), when the first reagent and the second reagent are mixed in the ratio of the addition amount at the time of measurement, The first reagent, the second reagent, and the biological sample of the enzyme activity measurement reagent containing the azide in the first reagent and / or the second reagent so that the concentration of the azide in the solution is 0.00007% to 0.05%. Mix, let the enzyme reaction occur in the presence of a small amount of azide, measure the absorbance in the wavelength range of 400 to 500 nm of the product produced by the reaction of the enzyme with the substrate or the substance derived from this product, this measurement The enzyme activity in the biological sample is calculated from the value.
[0038]
As mentioned above, the ratio when the amount of sample added is divided by the amount of enzyme activity measuring reagent is usually within the range of 1 to 5%, and the amount of sample added is almost negligible. Therefore, the concentration of a trace amount of azide during the enzyme reaction is 0.00007% to 0.05% (preferably 0.0001% to 0.01%, particularly preferably 0.00025%). ~ 0.005%).
[0039]
In the method for measuring enzyme activity in a biological sample of the present invention, the amount of change per minute in the absorbance in the wavelength region of 400 to 500 nm is determined by the production rate of a product produced by reacting an enzyme with a substrate or a substance derived from this product. Any method can be used as long as it is based on the measurement principle calculated by measuring the absorbance in the wavelength range of 400 to 500 nm or the amount of the product produced by the reaction of the enzyme with the substrate or the substance derived from the product. Can also be used. For example, it can be applied to a reaction rate method (kinetic method, rate method) or an end point method (end point method).
In such a measuring method, the enzyme activity can be measured by a method known per se or a method using an automatic analyzer.
[0040]
For example, L-γ-glutamyl-4-nitroanilide, L-γ-glutamyl-3-carboxy-4-nitroanilide or the like is used as a substrate for measuring γ-glutamyltranspeptidase; for measuring leucine aminopeptidase L-Leucyl-4-nitroanilide or the like is used as a substrate; S-benzyl-L-cysteine-4-nitroanilide or the like is used as a substrate for measuring L-cystine aminotranspeptidase; α-amylase As the measurement substrate, 4-nitrophenyl-α-D-maltoheptaoside, 4,6-benzylidene-4-nitrophenyl-α-D-maltoheptaoside, 4Five -O-β-D-galactopyranosyl-4-nitrophenyl-α-D-maltopentaoside, 2-chloro-4-nitrophenyl-β-D-maltopentaoside, etc. are used; alkaline phosphatase As the measurement substrate, 4-nitrophenyl phosphate or the like is used; as the measurement substrate for cholinesterase, butyrylthiocholine or the like is used.
Absorption maximum at 400 to 500 nm such as 4-nitrophenol, 2-chloro-4-nitrophenol, 4-nitroaniline or 3-carboxy-4-nitroaniline released by the action of enzyme on these substrates (λmax ) Is measured over time, and the enzyme activity is calculated from the increase in absorbance.
[0041]
Alternatively, DTNB [5,5′-dithiobis which produces a change in absorbance in the wavelength region of 400 to 500 nm quantitatively by reacting with an SH group on a substance such as thiocholine released by the action of an enzyme on these substrates. (2-nitrobenzoic acid)] or the like is added and reacted, and the absorbance of the substance having an absorption maximum (λmax) at 400 to 500 nm is measured over time to measure the absorbance. The enzyme activity is calculated from the increase.
In this case, according to the present invention, it is possible to suppress errors caused by the decomposition of hemoglobin in the biological sample.
[0042]
【Example】
Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto.
[0043]
[Example 1]
(Measurement of γ-glutamyltranspeptidase [γ-GT] activity in serum samples)
[0044]
(1) Preparation of reagents
(1) Preparation of γ-GT / first reagent
The following reagent components are dissolved in pure water to the respective concentrations described above, and 1N sodium hydroxide is added to adjust the pH to 8.2 (20 ° C.) to obtain 13 types of γ-GT having different sodium azide concentrations. A first reagent was prepared.
[0045]
Glycylglycine 225mM
Sodium azide
0%, 0.0001%, 0.0002%, 0.0004%, 0.0006%, 0.0008%, 0.001%, 0.0015%,
0.002%, 0.003%, 0.005%, 0.01%, or 0.1%
[0046]
(2) Preparation of γ-GT / second reagent
The following reagent components were dissolved in pure water so as to have the respective concentrations described above, and 1N sodium hydroxide was added to adjust the pH to 5.25 (20 ° C.) to prepare γ-GT · second reagent.
[0047]
Acetic acid 20mM
L-γ-glutamyl-3-carboxy-4-nitroanilide 18 mM
[0048]
(2) Preparation of serum sample
Nine volumes of a human serum sample with a γ-GT activity value of 70 U / l are mixed with one volume of an aqueous solution containing no hemoglobin or hemoglobin prepared by lysing human erythrocytes with a hemoglobin concentration of 5,000 or 10,000 mg / dl. Serum samples with hemoglobin concentrations of 0, 500, or 1,000 mg / dl, respectively, were prepared.
[0049]
(3) Measurement of γ-GT activity in serum samples
Measurements were made using a Hitachi 7150 automatic analyzer.
To 10 μl of the serum sample prepared in (2) above, 250 μl of the γ-GT / first reagent prepared in (1) above was added and reacted at 37 ° C. for 5 minutes, and then prepared in (1) above. 125 μl of γ-GT · second reagent was added and allowed to react at 37 ° C., and 1 minute 24 seconds (32 points) after addition of γ-GT · second reagent at the main wavelength of 405 nm and subwavelength of 505 nm (32 points) to 5 minutes later (50 From the amount of change in absorbance per minute until (point), the γ-GT activity value was calculated by the following formula. The K value of 4,153 in this equation is calculated with the molar extinction coefficient (ε) of 5-amino-2-nitrobenzoic acid as 9.27.
[0050]
As a control, the same measurement was performed using physiological saline instead of a serum sample, and the obtained change in absorbance was used as a reagent blind test value, which was subtracted from the change in absorbance during the measurement of the serum sample. Was used to calculate the γ-GT activity value.
[0051]
γ-GT activity value (U / l) = (Absorbance change per minute) × 4,153
[0052]
By the way, the concentration of sodium azide in the measurement reagent after mixing the above-mentioned 13 kinds of γ-GT · first reagent and the above γ-GT · second reagent in the above addition amounts is as follows. become.
0%, 0.000067%, 0.00013%, 0.00027%, 0.0004%, 0.00053%, 0.00067%,
0.001%, 0.0013%, 0.002%, 0.0033%, 0.0067%, or 0.067%
[0053]
(4) Measurement results
The measurement results are shown in Table 7 and FIG.
In addition, the numerical value shown in Table 7 is the serum having a hemoglobin concentration of 500 mg / dl or 1,000 mg / dl when the γ-GT activity measurement value of a serum sample having a hemoglobin concentration of 0 mg / dl is 100%. The measured value of γ-GT activity of a sample is expressed as a relative ratio (%).
[0054]
[Table 7]
In FIG. 1, the horizontal axis represents the sodium azide concentration in the measurement reagent after mixing the γ-GT · first reagent and the γ-GT · second reagent, and the vertical axis represents the hemoglobin concentration of 0 mg in each reagent. γ-GT activity measurement value of a serum sample with a hemoglobin concentration of 500 mg / dl or 1,000 mg / dl when the measurement value of γ-GT activity of a serum sample of / dl is 100%, expressed as a relative ratio (%) It is.
Here, “◯” represents a measured value of γ-GT activity of a serum sample having a hemoglobin concentration of 500 mg / dl, and “●” represents a measured value of γ-GT activity of a serum sample having a hemoglobin concentration of 1,000 mg / dl.
[0056]
[Figure 1]
[0057]
As is apparent from Table 7 and FIG. 1, in the measurement using the γ-GT activity measuring reagent, it is possible to suppress errors occurring in the hemolyzed sample when the sodium azide concentration in the measuring reagent after mixing is 0.00007% or more and 0.05% or less. I understand. In particular, when the sodium azide concentration is 0.0001% or more and 0.01% or less, and further 0.00025% or more and 0.005% or less, the effect is remarkable.
Therefore, it was confirmed that the enzyme activity measuring reagent and measuring method of the present invention can obtain an accurate enzyme activity value without causing an error even in a hemolyzed sample.
[0058]
[Example 2]
(Measurement of leucine aminopeptidase [LAP] activity in serum samples)
[0059]
(1) Preparation of reagents
(1) Preparation of LAP / first reagent
Dissolve the following reagent components in pure water to the indicated concentrations, add 1N sodium hydroxide to adjust the pH to 8.0 (20 ° C), and adjust the pH to 8.0 (20 ° C). One reagent was prepared.
[0060]
Tris (hydroxymethyl) aminomethane 75mM
Sodium chloride 376mM
Sodium azide
0%, 0.0001%, 0.001%, 0.01%, or 0.1%
[0061]
(2) Preparation of LAP / second reagent
The following reagent components were dissolved in pure water to the respective concentrations described above, and 1N sodium hydroxide was added to adjust the pH to 4.1 (20 ° C.) to prepare LAP · second reagent.
[0062]
Acetic acid 20mM
L-Leucine-p-nitroanilide 12.2mM
[0063]
(2) Preparation of serum sample
Prepared by hemolyzing 9 volumes of a human serum sample (serum sample A) having a LAP activity value of 49 U / l or a human serum sample (serum sample B) having a LAP activity value of 41 U / l and an aqueous solution or human erythrocytes not containing hemoglobin Serum samples (serum samples A and A) having a hemoglobin concentration of 0, 250, 500, 750, or 1,000 mg / dl, respectively, by mixing one volume of an aqueous solution having a hemoglobin concentration of 2,500, 5,000, 7,500, or 10,000 mg / dl. Serum sample B) was prepared.
[0064]
(3) Measurement of LAP activity in serum samples
Measurements were made using a Hitachi 7150 automatic analyzer.
To 8 μl of the serum sample prepared in (2) above, 260 μl of the LAP • first reagent prepared in (1) above was added and reacted at 37 ° C. for 5 minutes, and then the LAP • prepared in (1) above was added thereto. Add 130 µl of the second reagent, react at 37 ° C, and 1 minute 24 seconds (32 points) to 5 minutes (50 points) after adding LAP / second reagent at the main wavelength of 405 nm and sub wavelength of 480 nm for 1 minute The LAP activity value was calculated by the following formula from the amount of change in absorbance per unit. The K value of 5,097 in this equation is calculated with the molar extinction coefficient (ε) of 4-nitroaniline as 9.76.
[0065]
As a control, the same measurement was performed using physiological saline instead of a serum sample, and the obtained change in absorbance was used as a reagent blind test value, which was subtracted from the change in absorbance during the measurement of the serum sample. Was used to calculate the LAP activity value.
[0066]
LAP activity value (U / l) = (Absorbance change per minute) × 5,097
[0067]
In addition, the concentration of sodium azide in the measurement reagent after mixing the above five types of LAP · first reagent and the above LAP · second reagent in the above addition amounts is as follows.
0%, 0.000067%, 0.00067%, 0.0067%, or 0.067%
[0068]
(4) Measurement results
Table 8 shows the measurement results.
The numerical values shown in Table 8 are the hemoglobin concentrations of 250 mg / dl, 500 mg / dl, 750 mg / dl when the LAP activity measurement value of a serum sample having a hemoglobin concentration of 0 mg / dl is 100%. The LAP activity measurement value of a 1,000 mg / dl serum sample is expressed as a relative ratio (%).
[0069]
[Table 8]
As is apparent from Table 8, even in the measurement using the LAP activity measurement reagent of the present invention, it is understood that errors occurring in the hemolyzed sample can be suppressed when the sodium azide concentration in the measurement reagent after mixing is 0.00007% or more and 0.05% or less. . In particular, the effect is remarkable at 0.0001% or more and 0.01% or less.
Therefore, it was confirmed that the enzyme activity measuring reagent and measuring method of the present invention can obtain an accurate enzyme activity value without causing an error in a hemolyzed sample even if the measurement items are different.
[0071]
Example 3
(Measurement of α-amylase [AMY] activity in serum samples)
[0072]
(1) Preparation of reagents
Preparation of AMY activity measuring reagent
Dissolve the following reagent components in pure water to the indicated concentrations, adjust the pH to 6.3 (37 ° C) by adding 1N sodium hydroxide, and measure two types of AMY activity with different sodium azide concentrations Reagents were prepared.
[0073]
2- (N-morpholino) ethanesulfonic acid monohydrate 50 mM
Potassium thiocyanate 900 mM
Sodium chloride 300mM
Calcium acetate 5mM
2-Chloro-4-nitrophenyl-α-D-maltotrioside 2.25 mM
[0074]
(2) Preparation of serum sample
Mix 8 volumes of a human serum sample with an AMY activity value of 64 U / l and 2 volumes of an aqueous solution containing hemoglobin-free hemoglobin or hemoglobin prepared by lysing human erythrocytes at a concentration of 2,500 or 5,000 mg / dl. Serum samples with hemoglobin concentrations of 0, 500, or 1,000 mg / dl, respectively, were prepared.
[0075]
(3) Measurement of AMY activity in serum samples
Measurements were made using a Hitachi 7150 automatic analyzer.
Add 300 μl of the AMY reagent prepared in (1) above to 5 μl of the serum sample prepared in (2) above and react at 37 ° C., then add AMY activity measuring reagent at main wavelength 405 nm and subwavelength 546 nm 2 minutes 12 seconds The AMY activity value was calculated by the following equation from the amount of change in absorbance per minute from after (11 points) to 4 minutes and 12 seconds (21 points). The K value of 3,885 in this equation is calculated assuming that the molar extinction coefficient (ε) of 2-chloro-4-nitrophenol is 15.7.
[0076]
As a control, the same measurement was performed using physiological saline instead of a serum sample, and the obtained change in absorbance was used as a reagent blind test value, which was subtracted from the change in absorbance during the measurement of the serum sample. Was used to calculate the AMY activity value.
[0077]
AMY activity value (U / l) = (Absorbance change per minute) × 3,885
[0078]
(4) Measurement results
Table 9 shows the measurement results.
The numerical values shown in Table 9 are the values of the serum samples having a hemoglobin concentration of 500 mg / dl or 1,000 mg / dl when the AMY activity measurement value of a serum sample having a hemoglobin concentration of 0 mg / dl is 100%. The measured value of AMY activity is expressed as a relative ratio (%).
[0079]
[Table 9]
As is apparent from Table 9, even in the measurement using the AMY activity measuring reagent of the present invention, when the sodium azide concentration in the measuring reagent is 0.00007% or more and 0.05% or less, errors that occur in the hemolyzed sample can be suppressed. I understand.
Therefore, it was confirmed that the enzyme activity measurement reagent and measurement method of the present invention can obtain an accurate enzyme activity value without causing an error in a hemolyzed sample regardless of the measurement item.
[0081]
【The invention's effect】
The enzyme activity measuring reagent and the enzyme activity measuring method of the present invention include an azide in the enzyme activity measuring reagent in an amount of 0.00007% to 0.05%, so that even in a hemolyzed sample, there is an error in the measured value. Generation | occurrence | production can be suppressed and the enzyme activity in a biological sample can be measured correctly.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram in which the influence of hemolysis of a sample on the measured value is examined using the γ-GT activity measuring reagent of the present invention.
[Formula 1]
Claims (3)
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| JP14216398A JP4517066B2 (en) | 1998-05-07 | 1998-05-07 | Enzyme activity measuring reagent containing a trace amount of azide and measuring method |
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| JP14216398A JP4517066B2 (en) | 1998-05-07 | 1998-05-07 | Enzyme activity measuring reagent containing a trace amount of azide and measuring method |
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| Publication Number | Publication Date |
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| JPH11313696A JPH11313696A (en) | 1999-11-16 |
| JP4517066B2 true JP4517066B2 (en) | 2010-08-04 |
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| Country | Link |
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