JP4028648B2 - Method for measuring peroxidase activity - Google Patents

Description

で表わすことができる。
【００１３】
上記一般式（１）において、Ｒ¹ 及びＲ² はアルキル基、アリール基及びハロゲン化アリール基からなる群より選択され、互いに同一でも又は異なるものでもよい。アルキル基、アリール基及びハロゲン化アリール基は、炭素数１〜２０を有するものであり、好ましいアルキル基は炭素数１〜１０のものである。例えば、メチル基、エチル基、プロピル基、ブチル基、ペンチル基、ヘキシル基、ヘプチル基、オクチル基、ノニル基及びデシル基等の直鎖状又はそれらの分岐状アルキル基を挙げることができる。また、アリール基は炭素数６〜２０のものが好ましく、例えば、フェニル基、トリール基、キシリル基等を挙げることができ、さらにアルキル基で置換されたものでもよい。アリール基としては、特に、フェニル基が好ましい。ハロゲン化アリール基としてはハロゲン化フェニル基、ハロゲン化トリル基、ハロゲン化キシリル基等を挙げることができ、特に、クロロフェニル基が好ましい。
【００１４】
一般式（１）において、Ｒ³ 、Ｒ⁴ 、Ｒ⁵ 及びＲ⁶ は、各々、水素原子、アルキル基、アリール基、アルコキシ基、アリーロキシ基及びハロゲン原子からなる群より選択され、互いに同一でも又は異なるものでもよい。これらの炭化水素基としては、炭素数１〜２０、好ましくは１〜１０のものを挙げることができる。例えば、炭素数１〜２０の直鎖状又は分岐状アルキル基、アルコキシ基、炭素数６〜２０のアリール基、アリーロキシ基を挙げることができ、アリール基、アリーロキシ基にはアルキル基が置換されたものでもよい。
【００１５】
また、一般式（１）において、Ｘはｎ価の陰イオンであり、ｎは１又は２である。陰イオンとしては、特に限定されるものではなく、硝酸イオン、ハロゲンイオン（例えば、塩素イオン、フッ素イオン、臭素イオン等）、リン酸イオン等を挙げることができる。これらの陰イオンのなかで、特に硝酸イオンが好ましい。
【００１６】
上記Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の具体例としては、Ｎ，Ｎ’−ジメチル−９，９’−ビスアクリジニウム塩、Ｎ，Ｎ’−ジエチル−９，９’−ビスアクリジニウム塩、Ｎ，Ｎ’−ジプロピル−９，９’−ビスアクリジニウム塩、Ｎ，Ｎ’−ジイソプロピル−９，９’−ビスアクリジニウム塩、Ｎ，Ｎ’−ジブチル−９，９’−ビスアクリジニウム塩、Ｎ，Ｎ’−ジイソブチル−９，９’−ビスアクリジニウム塩、Ｎ，Ｎ’−ジフェニル−９，９’−ビスアクリジニウム塩、Ｎ，Ｎ’−ジ−ｍ−クロロフェニル−９，９’−ビスアクリジニウム塩等が挙げられ、勿論これらに限定されるものではないが、特に、Ｎ，Ｎ’−ジメチル−９，９’−ビスアクリジニウムジナイトレート（ルシゲニン）が好ましい。
前記化学発光試薬の製造に必要なＮ，Ｎ−ジ置換カルボン酸アミド化合物は、下記一般式（２）
【００１７】
【化４】

で表わすことができる。
【００１８】
上記一般式（２）において、Ｒ₁ は水素原子、炭素数１〜１０のアルキル基、炭素数２〜１０のアルケニル基及び炭素数６〜２０のアリール基からなる群より選択され、該アリール基はアルキル基、ニトロ基、水酸基、アミノ基及びハロゲン原子等からなる群より選択される基で置換されていてもよい。Ｒ₂ はメチル基及びエチル基からなる群より選択され、Ｒ₃ は炭素数１〜１０のアルキル基、炭素数２〜１０のアルケニル基及び炭素数６〜２０のアリール基からなる群より選択され、アリール基は、アルキル基、ニトロ基、水酸基、アミノ基及びハロゲン原子等からなる群より選択される基で置換されていてもよい。Ｒ₁ 及びＲ₃ のアルキル基としては、例えば、メチル基、エチル基、プロピル基、ブチル基、ペンチル基、ヘキシル基、ヘプチル基、オクチル基、ノニル基、デシル基等の直鎖状又は分岐状のものを挙げることができる。また、Ｒ₁ 及びＲ₃ は互いに結合して、それぞれが結合しているカルボニル基の炭素原子及びアミド基の窒素原子と共に環を形成していてもよい。
【００１９】
上記Ｎ，Ｎ−ジ置換カルボン酸アミド化合物の具体例としては、Ｎ，Ｎ−ジメチルホルムアミド、Ｎ，Ｎ−ジメチルアセトアミド、Ｎ，Ｎ−ジメチルアクリルアミド、Ｎ，Ｎ−ジメチルプロピオンアミド、Ｎ，Ｎ−ジメチルベンズアミド、Ｎ−メチル−２−ピロリドンが非限定的に挙げられる。
【００２０】
また、化学発光試薬の製造に必要なギ酸の存在は、得られた化学発光試薬の発光反応時において発光強度を大幅に増大させる効果を有し、光照射による反応において化学発光性物質の生成量を増大させる作用を示すものと考えられる。この作用はギ酸に特異的であり、酢酸、プロピオン酸、安息香酸等の有機酸類やホルムアルデヒド、アセトアルデヒド、ベンズアルデヒド等のアルデヒド類にはその効果は全く認められない。
【００２１】
化学発光試薬の製造に用いる光照射用の光線としては、波長領域が約２９０〜８００ｎｍの範囲の紫外可視部が用いられ、特に、約４００〜８００ｎｍの範囲の可視光が望ましい。これらの光源としては、高圧水銀灯、低圧水銀灯、殺菌灯、蛍光灯及び白熱電灯等を非限定的に用いることができるが、白熱電灯が好ましく用いられる。この光照射により得られる化学発光試薬は、光源を用いずに自然光の下で製造した化学発光試薬に較べて、同じ原料ルシゲニン濃度で製造し同量で使用した場合に短時間で非常に高い発光強度を示すことと、この反応を光遮蔽下に実施した場合にはペルオキシダーゼ濃度依存性を有する化学発光試薬が得られないことから、化学発光性化合物の生成には光照射が重要な役割を担っていることが認められる。
【００２２】
上記Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類は、ｐＨ８付近では過酸化水素等の水素受容体ともペルオキシダーゼ存在下の水素受容体とも顕著な発光反応は起こさないが、これはＮ，Ｎ’−ジ置換−９，９’−ビスアクリジニウムカチオンが対イオン、特に硝酸イオンと安定な塩を形成しているためと考えられる。しかし、Ｎ，Ｎ−ジ置換カルボン酸アミド化合物等の極性の高い化合物の存在下で光照射を行なうことにより、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウムカチオンへの対アニオンからの電荷移動が促進され、イオン性の高い塩類からラジカル性を有する電荷移動錯体に変化し、この錯体がＮ，Ｎ−ジ置換カルボン酸アミド化合物の配位又は溶媒和によって安定化され、この安定化されたビラジカル性化合物が水素受容体の酵素分解により生成する活性酸素と反応して、励起状態のジオキセタン構造を経て発光するのでペルオキシダーゼ濃度に依存した発光強度が得られるものと考えられる。
【００２３】
この光反応におけるギ酸の作用については、明確な反応機構は不明であるが、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウムカチオンへの電荷移動に関与してその反応を促進する作用を有するものと考えられる。
【００２４】
これらＮ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類、Ｎ，Ｎ−ジ置換カルボン酸アミド化合物及びギ酸の使用量は、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類に対してＮ，Ｎ−ジ置換カルボン酸アミド化合物を１〜１００００倍モルの量で用い、さらに、同種及び／又は異種のＮ，Ｎ−ジ置換カルボン酸アミド化合物及び／又はその他の溶媒を反応溶媒として用いることもできる。ギ酸はＮ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類に対して１〜１００００倍モル、好ましくは１０〜１０００倍モル、更に好ましくは５０〜５００倍モルの量で用いることができる。
【００２５】
上記化学発光試薬は、ｐＨ７．５〜１３の塩基性条件下において過剰の水素受容体の存在下、ペルオキシダーゼの濃度に依存した量で発光する。この発光は、フェノール性化合物等の発光促進剤によって増強することが認められる。このようなフェノール性化合物としては、ｐ−ヒドロキシ桂皮酸、ｐ−フェニルフェノール、ｐ−（４−クロロフェニル）フェノール、ｐ−（４−ブロモフェニル）フェノール、ｐ−（４−ヨードフェニル）フェノール、ｐ−ヨードフェノール、ｐ−ブロモフェノール、ｐ−クロロフェノール、２，４−ジクロロフェノール、ｐ−クマル酸、６−ヒドロキシベンゾチアゾール、２−ナフトール、ホタルルシフェリン等が非限定的に挙げられる。
【００２６】
本発明のペルオキシダーゼ活性の測定法において、上記化学発光試薬を用いて化学発光分析を行なう場合には、１０^-8〜１Ｍ、好ましくは１０^-6〜１０^-2Ｍの範囲の濃度で用いられ、その使用量は１０〜５００μｌ、特に５０〜３００μｌの範囲が望ましい。
【００２７】
上記発光促進剤の使用量は、化学発光試薬の０．０１〜１００倍モル、好ましくは０．１〜１０倍モルの範囲であり、その濃度は１０^-6〜１Ｍ、特に１０^-4〜１０^-2Ｍの範囲で用いるのが望ましい。
【００２８】
また、化学発光反応に用いられる水素受容体としてはペルオキシダーゼ酵素の基質となり得るものであれば、特に限定されるものでなく、有機過酸化物、無機過酸化物等が任意に用いられ、特に過酸化水素が好ましい。水素受容体の使用量は化学発光試薬に対して充分に過剰な量であることが必要であり、その使用量は化学発光試薬に対して３〜１００００倍モル、好ましくは１０〜１０００倍モルの範囲が望ましい。
【００２９】
また、ペルオキシダーゼを標識物質として抗体、核酸等を標識して種々の物質を定量する場合には、特に限定されるものではないが、ペルオキシダーゼとして、西洋ワサビペルオキシダーゼ（ＨＲＰ）が好ましく用いられる。
【００３０】
化学発光反応に用いられる塩基性緩衝液としては、トリス緩衝液、リン酸緩衝液、ほう酸緩衝液、炭酸緩衝液、グリシン−水酸化ナトリウム緩衝液等を任意に用いることができる。これらの緩衝液の濃度は１ｍＭ〜１Ｍの範囲で用いるのが望ましい。また、反応時には界面活性剤、キレート剤等の添加剤を任意に用いることもできる。
【００３１】
【実施例】
以下、実施例及び比較例を示し、本発明を具体的に説明するが、本発明は下記実施例に限定されるものではない。
［実施例１］
化学発光試薬の調製
ルシゲニン１．０ｍｇを試験管に採り、これにＮ，Ｎ−ジメチルホルムアミド２ｍｌ及びギ酸２０μｌを加えて均一に溶解させた後、３０℃の温度の水浴中で２５０Ｗのコピーランプを２時間照射することにより化学発光性物質を含有する化学発光試薬を得た。
【００３２】
ペルオキシダーゼ活性の測定
この化学発光試薬を８×１０^-4Ｍのｐ−ヨードフェノール７５ｍＭトリス塩酸緩衝液(pH8.0) 溶液で５００倍に希釈して化学発光試薬溶液を調製した。また、化学発光測定用マイクロプレートの複数のウェルに西洋ワサビペルオキシダーゼ（ＨＲＰ）の種々の濃度水準の７５ｍＭトリス塩酸緩衝液（pH8.0)溶液１００μｌを充填し、これに溶液注入装置から前記化学発光試薬溶液１００μｌ及び０．００１７重量％過酸化水素の７５ｍＭトリス塩酸緩衝液(pH8.0) 溶液５０μｌを順次注入して発光反応させた後、この発光量をルミノメーター（ダイアヤトロン社製ルミナスＣＴ−９０００Ｄ）で０〜５秒間発光量を積算した結果、表１に示すような発光強度が得られ、ＨＲＰを１×１０^-19 ｍｏｌ／ａｓｓａｙまで測定することが可能であり、ギ酸を添加しないで反応させた比較例１に較べて感度及び発光強度において改善が認められた。
【００３３】
【表１】

【００３４】
［比較例１］
化学発光試薬の調製
ルシゲニン１．５ｍｇを試験管に採り、これにＮ，Ｎ−ジメチルホルムアミド１ｍｌを加えて溶解させた後、３０℃の温度の水浴中で２５０Ｗのコピーランプを５時間照射することにより化学発光性物質を含有する化学発光試薬を得た。
【００３５】
ペルオキシダーゼ活性の測定
この化学発光試薬を８×１０^-4Ｍのｐ−ヨードフェノール７５ｍＭトリス塩酸緩衝液(pH8.0) 溶液で５００倍に希釈して化学発光試薬溶液を調製した。また、化学発光測定用マイクロプレートの複数のウェルに西洋ワサビペルオキシダーゼ（ＨＲＰ）の種々の濃度水準の７５ｍＭトリス塩酸緩衝液(pH8.0) 溶液１００μｌを充填し、これに溶液注入装置から前記化学発光試薬溶液１００μｌ及び０．００１７重量％過酸化水素の７５ｍＭトリス塩酸緩衝液(pH8.0) 溶液５０μｌを順次注入して発光反応させた後、この発光量をルミノメーター（ダイアヤトロン社製ルミナスＣＴ−９０００Ｄ）で０〜５秒間発光量を積算した結果、表２に示す発光強度が得られ、ＨＲＰを５×１０^-19 ｍｏｌ／ａｓｓａｙまで測定可能なことが認められた。
【００３６】
【表２】

【００３７】
［実施例２］
化学発光試薬の調製
ルシゲニン１．５ｍｇを試験管に採り、これにＮ，Ｎ−ジメチルアセトアミド１ｍｌ及びギ酸２０μｌを加えて均一に溶解させた後、３０℃の温度の水浴中で２５０Ｗのコピーランプを３時間照射することにより化学発光性物質を含有する化学発光試薬を得た。
【００３８】
ペルオキシダーゼ活性の測定
この化学発光試薬を８×１０^-4Ｍのｐ−ヨードフェノール７５ｍＭトリス塩酸緩衝液(pH8.0) 溶液で５００倍に希釈して化学発光試薬溶液を調製した。また、化学発光測定用マイクロプレートの複数のウェルに西洋ワサビペルオキシダーゼ（ＨＲＰ）の種々の濃度水準の７５ｍＭトリス塩酸緩衝液（pH8.0)溶液１００μｌを充填し、これに溶液注入装置から前記化学発光試薬溶液１００μｌ及び０．００１７重量％過酸化水素の７５ｍＭトリス塩酸緩衝液(pH8.0) 溶液５０μｌを順次注入して発光反応させた後、この発光量をルミノメーター（ダイアヤトロン社製ルミナスＣＴ−９０００Ｄ）で０〜５秒間発光量を積算した結果、表３に示すような発光強度が得られ、ＨＲＰを１×１０^-19 ｍｏｌ／ａｓｓａｙまで測定することが可能であり、ギ酸を添加しないで反応させた比較例２に較べて感度及び発光強度において改善が認められた。
【００３９】
【表３】

【００４０】
［比較例２］
化学発光試薬の調製
ルシゲニン１．５ｍｇを試験管に採り、これにＮ，Ｎ−ジメチルアセトアミド１ｍｌを加えて溶解させた後、３０℃の温度の水浴中で２５０Ｗのコピーランプを７時間照射することにより化学発光性物質を含有する化学発光試薬を得た。
【００４１】
ペルオキシダーゼ活性の測定
この化学発光試薬を８×１０^-4Ｍのｐ−ヨードフェノール７５ｍＭトリス塩酸緩衝液(pH8.0) 溶液で５００倍に希釈して化学発光試薬溶液を調製した。また、化学発光測定用マイクロプレートの複数のウェルに西洋ワサビペルオキシダーゼ（ＨＲＰ）の種々の濃度水準の７５ｍＭトリス塩酸緩衝液(pH8.0) 溶液１００μｌを充填し、これに溶液注入装置から前記化学発光試薬溶液１００μｌ及び０．００１７重量％過酸化水素の７５ｍＭトリス塩酸緩衝液(pH8.0) 溶液５０μｌを順次注入して発光反応させた後、この発光量をルミノメーター（ダイアヤトロン社製ルミナスＣＴ−９０００Ｄ）で０〜５秒間発光量を積算した結果、表４に示す発光強度が得られ、ＨＲＰを５×１０^-19 ｍｏｌ／ａｓｓａｙまで測定可能なことが認められた。
【００４２】
【表４】

【００４３】
［実施例３］
化学発光試薬の調製
ルシゲニン１．５ｍｇを試験管に採り、これにＮ−メチル−２−ピロリドン１ｍｌ及びギ酸２０μｌを加えて均一に溶解させた後、３０℃の温度の水浴中で２５０Ｗのコピーランプを２時間照射することにより化学発光試薬を得る。
【００４４】
ペルオキシダーゼ活性の測定
この化学発光試薬を８×１０^-4Ｍのｐ−ヨードフェノール７５ｍＭトリス塩酸緩衝液(pH8.0) 溶液で５００倍に希釈して化学発光試薬溶液を調製した。また、化学発光測定用マイクロプレートの複数のウェルに西洋ワサビペルオキシダーゼ（ＨＲＰ）の種々の濃度水準の７５ｍＭトリス塩酸緩衝液（pH8.0)溶液１００μｌを充填し、これに溶液注入装置から前記化学発光試薬溶液１００μｌ及び０．００１７重量％過酸化水素の７５ｍＭトリス塩酸緩衝液(pH8.0) 溶液５０μｌを順次注入して発光反応させた後、この発光量をルミノメーター（ダイアヤトロン社製ルミナスＣＴ−９０００Ｄ）で０〜５秒間発光量を積算した結果、表５に示す発光強度が得ら、ＨＲＰを５×１０^-19 ｍｏｌ／ａｓｓａｙまで測定可能なことが認められ、光照射せずに反応を行なった比較例３に較べて感度及び発光強度において改善が見られた。
【００４５】
【表５】

【００４６】
［比較例３］
化学発光試薬の調製
ルシゲニン１．５ｍｇを試験管に採り、これにＮ−メチル−２−ピロリドン１ｍｌを加えて溶解させた後、３０℃の温度の水浴中で２５０Ｗのコピーランプを３時間照射することにより化学発光試薬を得た。
【００４７】
ペルオキシダーゼ活性の測定
この化学発光試薬を８×１０^-4Ｍのｐ−ヨードフェノール７５ｍＭトリス塩酸緩衝液(pH8.0) 溶液で５００倍に希釈して化学発光試薬溶液を調製した。また、化学発光測定用マイクロプレートの複数のウェルに西洋ワサビペルオキシダーゼ（ＨＲＰ）の種々の濃度水準の７５ｍＭトリス塩酸緩衝液(pH8.0) 溶液１００μｌを充填し、これに溶液注入装置から前記化学発光試薬溶液１００μｌ及び０．００１７重量％過酸化水素の７５ｍＭトリス塩酸緩衝液(pH8.0) 溶液５０μｌを順次注入して発光反応させた後、この発光量をルミノメーター（ダイアヤトロン社製ルミナスＣＴ−９０００Ｄ）で０〜５秒間発光量を積算した結果、表６に示す発光強度が得られ、ＨＲＰを１×１０^-18 ｍｏｌ／ａｓｓａｙまで測定可能なことが認められた。
【００４８】
【表６】

【００４９】
【発明の効果】
本発明のペルオキシダーゼ活性の測定方法により、ペルオキシダーゼ酵素活性を従来から広く使用されているルミノールを用いる測定系よりも高感度で測定することが可能であり、ペルオキシダーゼを標識物質に用いる酵素免疫測定法により抗原、抗体類を、また、ハイブリダイゼーションアッセイ法により核酸類を各々より高感度で検出又は定量することができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring peroxidase activity using a chemiluminescence method, and more specifically, high sensitivity in the presence of a hydrogen acceptor by using a chemiluminescence method using a specific novel chemiluminescence reagent. The present invention relates to a method for measuring peroxidase activity.
[0002]
[Prior art]
Peroxidase is an enzyme that catalyzes the oxidation reaction of a hydrogen donor in the presence of a hydrogen acceptor. Such catalytic activity is also observed in non-enzymatic substances such as hemoglobin, and is collectively referred to as peroxidase activity. Since the catalytic activity of peroxidase is highly sensitive and easy to detect, it is used not only for detection of peroxidase enzyme activity but also as a reagent for analysis that serves as an indicator of various substances to be detected in a wide range of fields.
[0003]
As described above, analysis methods using peroxidase as a labeling substance include immunoassays using antigen-antibody reactions, nucleic acid hybridization assays using nucleic acid complementation, other avidin-biotin, hormone-hormone receptors, sugars. -Used for analysis systems utilizing specific affinity such as lectins.
[0004]
In addition, for the measurement of such peroxidase activity, a colorimetric method or a fluorescent substance is used which oxidizes a hydrogen donor using hydrogen peroxide as a hydrogen acceptor, generates a dye, and measures the color development amount with a spectrophotometer or the like. A fluorescence method or the like in which the fluorescence is generated and measured with a fluorescence spectrophotometer is performed. However, when using a colorimetric method for measuring peroxidase activity, there is a problem that the peroxidase is not quantitative in the low concentration region, and a fluorescence method has been developed to solve this problem. The quantitative properties in the low concentration region are not yet sufficient. In order to solve this problem, a method for measuring peroxidase activity by a chemiluminescence method has been developed. The chemiluminescence method is a method for quantifying peroxidase enzyme activity by measuring the amount of luminescence emitted when the chemiluminescent substance is excited through an intermediate due to the catalytic activity of the peroxidase enzyme and returns from this state to the ground state. A chemiluminescence system has been developed that uses luminol as the chemiluminescent substance, hydrogen peroxide as the hydrogen acceptor, and p-iodophenol as the luminescence enhancer, and quantifies peroxidase activity with high sensitivity. Is possible.
[0005]
However, when applying peroxidase enzyme activity measurement to enzyme immunoassay, there are many cases where it is necessary to further improve the quantitativeness of the enzyme in a low concentration region, and further increase in sensitivity in the measurement of peroxidase enzyme activity. Was desired.
[0006]
[Problems to be solved by the invention]
Therefore, in view of the above circumstances, an object of the present invention is to provide a novel measurement method by a chemiluminescence reaction capable of measuring peroxidase activity with higher sensitivity.
[0007]
[Means for Solving the Problems]
Therefore, as a result of intensive studies to achieve the above problems, the present inventors have determined that N, N′-disubstituted-9,9′-bisacridinium salts are used as chemiluminescent reagents. Using a chemiluminescent reagent containing a chemiluminescent substance obtained by reaction under light irradiation in the presence of a disubstituted carboxylic acid amide compound and formic acid, using hydrogen peroxide as a hydrogen acceptor, and further emitting light We found that the luminescence system using a specific phenolic compound as an enhancer can quantify peroxidase activity with higher sensitivity compared to the luminescence system using luminol as a chemiluminescent substance. Based on this, the present invention has been achieved.
[0008]
Therefore, the first of the present invention is
A chemiluminescent reagent prepared by reacting N, N′-disubstituted-9,9′-bisacridinium salts with light irradiation in the presence of an N, N-disubstituted carboxylic acid amide compound and formic acid The present invention relates to a method for measuring peroxidase activity by a chemiluminescence method, characterized in that peroxidase enzyme activity is measured in the presence of a hydrogen acceptor.
[0009]
The second of the present invention is
A chemiluminescent reagent prepared by reacting N, N′-disubstituted-9,9′-bisacridinium salts with light irradiation in the presence of an N, N-disubstituted carboxylic acid amide compound and formic acid The present invention relates to a method for measuring peroxidase activity by a chemiluminescence method, wherein a phenolic compound is used as a luminescence enhancer when measuring peroxidase enzyme activity in the presence of a hydrogen acceptor.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in more detail.
In the method for measuring peroxidase activity of the present invention, N, N′-disubstituted-9,9′-bisacridinium salts are irradiated with light in the presence of an N, N-disubstituted carboxylic acid amide compound and formic acid. Using a chemiluminescent reagent containing a chemiluminescent substance obtained by reacting, in the presence of a hydrogen acceptor, more preferably, a luminescence enhancer is added to measure peroxidase enzyme activity. In general, a measurement sample containing a chemiluminescent reagent, a luminescence enhancer, and a peroxidase enzyme is mixed, and a hydrogen acceptor solution is added in a specific basic pH region to perform a chemiluminescent reaction. A method of measuring the amount with a luminescence measuring device is performed.
[0011]
The chemiluminescent reagent used in the method for measuring peroxidase activity of the present invention includes N, N′-disubstituted-9,9′-bisacridinium salts, N, N-disubstituted carboxylic acid amide compounds, and presence of formic acid. It is a reaction product obtained by making it react under light irradiation below.
Next, a chemiluminescent reagent is demonstrated.
The N, N′-disubstituted-9,9′-bisacridinium salts are represented by the following general formula (1):
[0012]
[Chemical 3]

It can be expressed as
[0013]
In the general formula (1), R ¹ and R ² are selected from the group consisting of an alkyl group, an aryl group, and a halogenated aryl group, and may be the same or different from each other. The alkyl group, aryl group and halogenated aryl group have 1 to 20 carbon atoms, and preferred alkyl groups are those having 1 to 10 carbon atoms. Examples thereof include linear or branched alkyl groups such as methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group and decyl group. In addition, the aryl group preferably has 6 to 20 carbon atoms, and examples thereof include a phenyl group, a tolyl group, and a xylyl group, and may further be substituted with an alkyl group. As the aryl group, a phenyl group is particularly preferable. Examples of the halogenated aryl group include a halogenated phenyl group, a halogenated tolyl group, a halogenated xylyl group, and the like, and a chlorophenyl group is particularly preferable.
[0014]
In the general formula (1), R ³ , R ⁴ , R ⁵ and R ⁶ are each selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group and a halogen atom, It may be different. Examples of these hydrocarbon groups include those having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms. For example, a linear or branched alkyl group having 1 to 20 carbon atoms, an alkoxy group, an aryl group having 6 to 20 carbon atoms, and an aryloxy group can be exemplified, and the aryl group and the aryloxy group are substituted with an alkyl group. It may be a thing.
[0015]
In the general formula (1), X is an n-valent anion, and n is 1 or 2. The anion is not particularly limited, and may include nitrate ion, halogen ion (for example, chlorine ion, fluorine ion, bromine ion, etc.), phosphate ion, and the like. Of these anions, nitrate ions are particularly preferred.
[0016]
Specific examples of the N, N′-disubstituted-9,9′-bisacridinium salts include N, N′-dimethyl-9,9′-bisacridinium salts, N, N′-diethyl- 9,9′-bisacridinium salt, N, N′-dipropyl-9,9′-bisacridinium salt, N, N′-diisopropyl-9,9′-bisacridinium salt, N, N '-Dibutyl-9,9'-bisacridinium salt, N, N'-diisobutyl-9,9'-bisacridinium salt, N, N'-diphenyl-9,9'-bisacridinium salt N, N′-di-m-chlorophenyl-9,9′-bisacridinium salt and the like, and of course, but not limited to, N, N′-dimethyl-9,9 '-Bisacridinium dinitrate (lucigenin) is preferred.
The N, N-disubstituted carboxylic acid amide compound necessary for the production of the chemiluminescent reagent is represented by the following general formula (2).
[0017]
[Formula 4]

It can be expressed as
[0018]
In the general formula (2), R ₁ is selected from the group consisting of a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, and an aryl group having 6 to 20 carbon atoms, and the aryl group May be substituted with a group selected from the group consisting of an alkyl group, a nitro group, a hydroxyl group, an amino group, a halogen atom, and the like. R ₂ is selected from the group consisting of a methyl group and an ethyl group, and R ₃ is selected from the group consisting of an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, and an aryl group having 6 to 20 carbon atoms. The aryl group may be substituted with a group selected from the group consisting of an alkyl group, a nitro group, a hydroxyl group, an amino group, a halogen atom and the like. Examples of the alkyl group for R ₁ and R ₃ include linear or branched groups such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, and a decyl group. Can be mentioned. R ₁ and R ₃ may be bonded to each other to form a ring together with the carbon atom of the carbonyl group and the nitrogen atom of the amide group to which they are bonded.
[0019]
Specific examples of the N, N-disubstituted carboxylic acid amide compound include N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylacrylamide, N, N-dimethylpropionamide, N, N- Nonlimiting examples include dimethylbenzamide and N-methyl-2-pyrrolidone.
[0020]
In addition, the presence of formic acid necessary for the production of the chemiluminescent reagent has the effect of greatly increasing the luminescence intensity during the luminescent reaction of the obtained chemiluminescent reagent, and the amount of chemiluminescent substance produced in the reaction by light irradiation. It is thought that it shows the effect | action which increases. This effect is specific to formic acid, and its effect is not recognized at all in organic acids such as acetic acid, propionic acid, and benzoic acid, and aldehydes such as formaldehyde, acetaldehyde, and benzaldehyde.
[0021]
As the light beam for light irradiation used for the production of the chemiluminescent reagent, an ultraviolet-visible part having a wavelength region of about 290 to 800 nm is used, and visible light of about 400 to 800 nm is particularly desirable. As these light sources, high-pressure mercury lamps, low-pressure mercury lamps, germicidal lamps, fluorescent lamps, incandescent lamps and the like can be used without limitation, but incandescent lamps are preferably used. The chemiluminescent reagent obtained by this light irradiation has a very high luminescence in a short time when manufactured with the same raw material lucigenin concentration and used in the same amount as compared with the chemiluminescent reagent manufactured under natural light without using a light source. Light irradiation plays an important role in the production of chemiluminescent compounds because it shows strength, and when this reaction is carried out under light shielding, a chemiluminescent reagent that is dependent on peroxidase concentration cannot be obtained. It is recognized that
[0022]
The N, N′-disubstituted-9,9′-bisacridinium salts do not cause a remarkable luminescence reaction at a pH of around 8 with either a hydrogen acceptor such as hydrogen peroxide or a hydrogen acceptor in the presence of peroxidase. This is presumably because the N, N′-disubstituted-9,9′-bisacridinium cation forms a stable salt with the counter ion, particularly nitrate ion. However, by irradiating with light in the presence of a highly polar compound such as an N, N-disubstituted carboxylic acid amide compound, a pair to the N, N′-disubstituted-9,9′-bisacridinium cation is obtained. Charge transfer from the anion is promoted, and the salt is changed from a highly ionic salt to a radical charge transfer complex, and this complex is stabilized by the coordination or solvation of the N, N-disubstituted carboxylic acid amide compound, This stabilized biradical compound reacts with active oxygen produced by enzymatic decomposition of the hydrogen acceptor and emits light through an excited dioxetane structure, so that it is considered that the emission intensity depending on the peroxidase concentration can be obtained.
[0023]
Regarding the action of formic acid in this photoreaction, the clear reaction mechanism is unknown, but it is involved in charge transfer to the N, N'-disubstituted-9,9'-bisacridinium cation to promote the reaction. It is thought that it has the effect | action which carries out.
[0024]
The amount of these N, N′-disubstituted-9,9′-bisacridinium salts, N, N-disubstituted carboxylic acid amide compounds and formic acid used is N, N′-disubstituted-9,9′-. The N, N-disubstituted carboxylic acid amide compound is used in an amount of 1 to 10,000 times moles relative to the bisacridinium salt, and the same and / or different N, N-disubstituted carboxylic acid amide compound and / or Other solvents can also be used as the reaction solvent. Formic acid is used in an amount of 1 to 10000 times mol, preferably 10 to 1000 times mol, more preferably 50 to 500 times mol for N, N′-disubstituted-9,9′-bisacridinium salts. Can do.
[0025]
The chemiluminescent reagent emits light in an amount depending on the concentration of peroxidase in the presence of excess hydrogen acceptor under basic conditions of pH 7.5-13. It is recognized that this light emission is enhanced by a light emission accelerator such as a phenolic compound. Such phenolic compounds include p-hydroxycinnamic acid, p-phenylphenol, p- (4-chlorophenyl) phenol, p- (4-bromophenyl) phenol, p- (4-iodophenyl) phenol, p -Iodophenol, p-bromophenol, p-chlorophenol, 2,4-dichlorophenol, p-coumaric acid, 6-hydroxybenzothiazole, 2-naphthol, firefly luciferin, and the like.
[0026]
In the method for measuring peroxidase activity of the present invention, when chemiluminescence analysis is performed using the chemiluminescent reagent, it is used at a concentration in the range of 10 ^{−8 to 1} M, preferably 10 ⁻⁶ to 10 ⁻² M. The amount used is preferably in the range of 10 to 500 μl, particularly 50 to 300 μl.
[0027]
The amount of the luminescence accelerator used is in the range of 0.01 to 100 times mol, preferably 0.1 to 10 times mol of the chemiluminescence reagent, and its concentration is 10 ^{−6 to} 1M, particularly 10 ⁻⁴ to 10 ^It is desirable to use within the range of ^-2 M.
[0028]
The hydrogen acceptor used in the chemiluminescence reaction is not particularly limited as long as it can serve as a substrate for the peroxidase enzyme, and organic peroxides, inorganic peroxides and the like are arbitrarily used. Hydrogen oxide is preferred. The usage amount of the hydrogen acceptor needs to be an excessive amount with respect to the chemiluminescent reagent, and the usage amount is 3 to 10000 times mol, preferably 10 to 1000 times mol of the chemiluminescent reagent. A range is desirable.
[0029]
Further, when various substances are quantified by labeling antibodies, nucleic acids and the like using peroxidase as a labeling substance, horseradish peroxidase (HRP) is preferably used as the peroxidase.
[0030]
As the basic buffer used for the chemiluminescence reaction, a Tris buffer, a phosphate buffer, a borate buffer, a carbonate buffer, a glycine-sodium hydroxide buffer, or the like can be arbitrarily used. The concentration of these buffers is desirably used in the range of 1 mM to 1M. Further, during the reaction, additives such as a surfactant and a chelating agent can be arbitrarily used.
[0031]
【Example】
EXAMPLES Hereinafter, although an Example and a comparative example are shown and this invention is demonstrated concretely, this invention is not limited to the following Example.
[Example 1]
Preparation of chemiluminescent reagent: Take 1.0 mg of lucigenin in a test tube, add 2 ml of N, N-dimethylformamide and 20 μl of formic acid to dissolve it uniformly, and then add 250 W in a water bath at a temperature of 30C. The chemiluminescent reagent containing a chemiluminescent substance was obtained by irradiating a copy lamp of No. 2 for 2 hours.
[0032]
Measurement of peroxidase activity This chemiluminescent reagent was diluted 500 times with 8 x ^10-4 M p-iodophenol 75 mM Tris-HCl buffer (pH 8.0) to prepare a chemiluminescent reagent solution. In addition, a plurality of wells of a microplate for chemiluminescence measurement were filled with 100 μl of 75 mM Tris-HCl buffer solution (pH 8.0) having various concentrations of horseradish peroxidase (HRP). 100 μl of a reagent solution and 50 μl of a 0.0017 wt% hydrogen peroxide 75 mM Tris-HCl buffer (pH 8.0) solution were sequentially injected to cause a luminescence reaction, and then the amount of luminescence was measured using a luminometer (Luminous CT As a result of integrating the luminescence amount for 0 to 5 seconds at 9000 D), the luminescence intensity as shown in Table 1 was obtained, and it was possible to measure HRP up to 1 × 10 ⁻¹⁹ mol / assay without adding formic acid. Improvements in sensitivity and emission intensity were observed as compared to Comparative Example 1 which was reacted.
[0033]
[Table 1]

[0034]
[Comparative Example 1]
Preparation of chemiluminescent reagent 1.5 mg of lucigenin was placed in a test tube, 1 ml of N, N-dimethylformamide was added to dissolve it, and then a 250 W copy lamp was placed in a water bath at a temperature of 30C. A chemiluminescent reagent containing a chemiluminescent substance was obtained by irradiation for a period of time.
[0035]
Measurement of peroxidase activity This chemiluminescent reagent was diluted 500 times with 8 x ^10-4 M p-iodophenol 75 mM Tris-HCl buffer (pH 8.0) to prepare a chemiluminescent reagent solution. In addition, a plurality of wells of a chemiluminescence measurement microplate are filled with 100 μl of 75 mM Tris-HCl buffer solution (pH 8.0) having various concentrations of horseradish peroxidase (HRP). 100 μl of a reagent solution and 50 μl of a 0.0017 wt% hydrogen peroxide 75 mM Tris-HCl buffer (pH 8.0) solution were sequentially injected to cause a luminescence reaction, and then the amount of luminescence was measured using a luminometer (Luminous CT As a result of integrating the light emission amount for 0 to 5 seconds at 9000 D), the light emission intensity shown in Table 2 was obtained, and it was confirmed that HRP could be measured up to 5 × 10 ⁻¹⁹ mol / assay.
[0036]
[Table 2]

[0037]
[Example 2]
Preparation of chemiluminescent reagent 1.5 mg of lucigenin was put in a test tube, 1 ml of N, N-dimethylacetamide and 20 μl of formic acid were added and dissolved uniformly, and then 250 W in a water bath at a temperature of 30C. The chemiluminescent reagent containing a chemiluminescent substance was obtained by irradiating a copy lamp of 3 hours.
[0038]
Measurement of peroxidase activity This chemiluminescent reagent was diluted 500 times with 8 x ^10-4 M p-iodophenol 75 mM Tris-HCl buffer (pH 8.0) to prepare a chemiluminescent reagent solution. In addition, a plurality of wells of a microplate for chemiluminescence measurement were filled with 100 μl of 75 mM Tris-HCl buffer solution (pH 8.0) having various concentrations of horseradish peroxidase (HRP). 100 μl of a reagent solution and 50 μl of a 0.0017 wt% hydrogen peroxide 75 mM Tris-HCl buffer (pH 8.0) solution were sequentially injected to cause a luminescence reaction, and then the amount of luminescence was measured using a luminometer (Luminous CT As a result of integrating the luminescence amount for 0 to 5 seconds at 9000 D), the luminescence intensity as shown in Table 3 was obtained, and HRP could be measured up to 1 × 10 ⁻¹⁹ mol / assay without adding formic acid. Improvements were observed in sensitivity and emission intensity as compared to Comparative Example 2 which was reacted.
[0039]
[Table 3]

[0040]
[Comparative Example 2]
Preparation of chemiluminescent reagent 1.5 mg of lucigenin was placed in a test tube, 1 ml of N, N-dimethylacetamide was added and dissolved therein, and then a 250 W copy lamp was placed in a water bath at a temperature of 30C. A chemiluminescent reagent containing a chemiluminescent substance was obtained by irradiation for a period of time.
[0041]
Measurement of peroxidase activity This chemiluminescent reagent was diluted 500 times with 8 x ^10-4 M p-iodophenol 75 mM Tris-HCl buffer (pH 8.0) to prepare a chemiluminescent reagent solution. In addition, a plurality of wells of a chemiluminescence measurement microplate are filled with 100 μl of 75 mM Tris-HCl buffer solution (pH 8.0) having various concentrations of horseradish peroxidase (HRP). 100 μl of a reagent solution and 50 μl of a 0.0017 wt% hydrogen peroxide 75 mM Tris-HCl buffer (pH 8.0) solution were sequentially injected to cause a luminescence reaction, and then the amount of luminescence was measured using a luminometer (Luminous CT As a result of integrating the light emission amount for 0 to 5 seconds at 9000 D), the light emission intensity shown in Table 4 was obtained, and it was confirmed that HRP could be measured up to 5 × 10 ⁻¹⁹ mol / assay.
[0042]
[Table 4]

[0043]
[Example 3]
Preparation of chemiluminescent reagent: Take 1.5 mg of lucigenin in a test tube, add 1 ml of N-methyl-2-pyrrolidone and 20 μl of formic acid to dissolve it uniformly, and then in a water bath at a temperature of 30C. A chemiluminescent reagent is obtained by irradiating a 250 W copy lamp for 2 hours.
[0044]
Measurement of peroxidase activity This chemiluminescent reagent was diluted 500 times with 8 x ^10-4 M p-iodophenol 75 mM Tris-HCl buffer (pH 8.0) to prepare a chemiluminescent reagent solution. In addition, a plurality of wells of a microplate for chemiluminescence measurement were filled with 100 μl of 75 mM Tris-HCl buffer solution (pH 8.0) having various concentrations of horseradish peroxidase (HRP). 100 μl of a reagent solution and 50 μl of a 0.0017 wt% hydrogen peroxide 75 mM Tris-HCl buffer (pH 8.0) solution were sequentially injected to cause a luminescence reaction, and then the luminescence was measured using a luminometer (Luminous CT-manufactured by Diatron). As a result of integrating the luminescence amount for 0 to 5 seconds at 9000 D), it was found that the luminescence intensity shown in Table 5 was obtained, and that HRP could be measured up to 5 × 10 ⁻¹⁹ mol / assay, and the reaction was performed without light irradiation. Compared to the comparative example 3 performed, improvements in sensitivity and emission intensity were observed.
[0045]
[Table 5]

[0046]
[Comparative Example 3]
Preparation of chemiluminescent reagent Take 1.5 mg of lucigenin in a test tube, add 1 ml of N-methyl-2-pyrrolidone to dissolve it, and then apply a 250 W copy lamp in a water bath at a temperature of 30C. A chemiluminescent reagent was obtained by irradiation for 3 hours.
[0047]
Measurement of peroxidase activity This chemiluminescent reagent was diluted 500 times with 8 x ^10-4 M p-iodophenol 75 mM Tris-HCl buffer (pH 8.0) to prepare a chemiluminescent reagent solution. In addition, a plurality of wells of a chemiluminescence measurement microplate are filled with 100 μl of 75 mM Tris-HCl buffer solution (pH 8.0) having various concentrations of horseradish peroxidase (HRP). 100 μl of a reagent solution and 50 μl of a 0.0017 wt% hydrogen peroxide 75 mM Tris-HCl buffer (pH 8.0) solution were sequentially injected to cause a luminescence reaction, and then the luminescence was measured using a luminometer (Luminous CT-manufactured by Diatron). As a result of integrating the light emission amount for 0 to 5 seconds at 9000 D), the light emission intensity shown in Table 6 was obtained, and it was confirmed that HRP could be measured up to 1 × 10 ⁻¹⁸ mol / assay.
[0048]
[Table 6]

[0049]
【The invention's effect】
According to the method for measuring peroxidase activity of the present invention, it is possible to measure peroxidase enzyme activity with higher sensitivity than the measurement system using luminol which has been widely used in the past, and by enzyme immunoassay using peroxidase as a labeling substance. Antigens, antibodies, and nucleic acids can be detected or quantified with higher sensitivity by hybridization assays.

Claims (6)

The following general formula (1)

(In the above general formula (1), R ¹ and R ² are selected from the group consisting of methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, phenyl group and halogenated phenyl group. or may be ^{different, R 3, R 4, R} 5 and R ⁶ are both hydrogen atom, X ^n-is nitrate ion, halogen ion or phosphate ion.)
N, N′-disubstituted-9,9′-bisacridinium salts represented by the following general formula (2)

(In the above general formula (2), R ₁ is selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group and a phenyl group, R ₂ is a methyl group or an ethyl group, and R ₃ is A methyl group, an ethyl group, a propyl group, or an isopropyl group, and R ₁ and R ₃ may be bonded to each other to form a ring together with the carbon atom of the carbonyl group and the nitrogen atom of the amide group to which each is bonded. Good.)
Peroxidase in the presence of a hydrogen acceptor using a chemiluminescent reagent containing a chemiluminescent substance obtained by reacting under light irradiation in the presence of N, N-disubstituted carboxylic acid amide compound represented by A method for measuring peroxidase activity by chemiluminescence, which comprises measuring enzyme activity.     The method for measuring peroxidase activity according to claim 1, wherein the additional component of the chemiluminescent reagent is a luminescence reinforcing agent.     The N, N'-disubstituted-9,9'-bisacridinium salt is N, N'-dimethyl-9,9'-bisacridinium dinitrate (lucigenin). Method for measuring peroxidase activity of     The method for measuring peroxidase activity according to claim 1, wherein the N, N-disubstituted carboxylic acid amide compound is N, N-dimethylformamide, N, N-dimethylacetamide or N-methyl-2-pyrrolidone.     The method for measuring peroxidase activity according to any one of claims 1 to 3, wherein the hydrogen acceptor is hydrogen peroxide or a hydrogen peroxide source.   The peroxidase activity according to any one of claims 2 to 5, wherein the luminescence enhancer is at least one phenolic compound selected from p-iodophenol, p-phenylphenol and 6-hydroxybenzothiazole. Measuring method.