JP3792885B2 - Method for measuring peroxidase activity - Google Patents

Description

で表すことができる。
【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１〜１４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６〜１４のものが好ましく、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、更に、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、各々、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、炭素数が、例えば、１〜２０、特に、１〜１０のものが好ましい。
【００１５】
前記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’ブ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などが挙げられるが、勿論これらに限定されるものではない。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。
【００１６】
また、一般式（Ｉ）において、Ｘ^n-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（ＮＯ₃ ^-）、ハロゲン化物イオン（例えば、塩化物イオン、フッ化物イオン、臭化物イオン等）、（メタ）リン酸イオン（ＰＯ₃ ^-）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。
【００１７】
本発明のペルオキシダーゼ活性の測定方法において用いられる化学発光試薬の製造に供されるＮ，Ｎ−ジ置換カルボン酸アミド化合物としては、例えば、次の一般式（２）
【００１８】
【化３】

で表される化合物を挙げることができる。一般式（２）において、Ｒ_１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、炭素数６〜２０のアリール基であり、アリール基はアルキル基、ハロゲン原子、ニトロ基、水酸基、アミノ基等で置換されていてもよい。Ｒ_２は、メチル基又はエチル基であり、Ｒ_３は、炭素数１〜１０のアルキル基、炭素数２〜１０のアルケニル基、炭素数６〜２０のアリール基であり、アリール基はアルキル基、ハロゲン原子、ニトロ基、水酸基、アミノ基等で置換されていてもよい。また、Ｒ_１及びＲ_３は互いに結合して、各々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。Ｎ，Ｎ−ジ置換カルボン酸化合物の具体例を例示すると、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−ピロリドン及びＮ−エチル−２−ピロリドン等が非限定的に挙げられる。
【００１９】
Ｎ，Ｎ−ジ置換カルボン酸アミド化合物は、本発明の化学発光試薬の製造には必須であり、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類と複合体を形成した後、アクリジニウム塩構造の還元反応に伴い、より発光収率の高い化合物へと変化するものと考えられ、本発明の化学発光試薬の製造反応に反応成分として関与し、反応生成物の水溶性の付与等に寄与しているものと考えられる。従って、その使用量は、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類に対して１〜１０００当量、好ましくは１．５〜５００当量、特に好ましくは２〜３０００当量の割合でよい。
【００２０】
本発明のペルオキシダーゼ活性の測定方法に用いられる化学発光試薬の製造において、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類を、Ｎ，Ｎ−ジ置換カルボン酸アミド化合物の存在下において還元剤と接触処理させる際に、更に、蟻酸を添加することができる。蟻酸は、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類に対して０〜５０００当量、好ましくは、０．５〜１０００当量、特に好ましくは１〜５００当量の割合で用いられ、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類の還元反応を促進し、化学発光試薬の反応収率を著しく高めることができる。
【００２１】
本発明の測定系における化学発光試薬の製造に用いられる還元剤としては、水素化リチウムアルミニウム、水素化リチウムボロン、水素化ナトリウムボロン等が挙げられるが、水素化リチウムアルミニウムが特に好ましく用いられる。
【００２２】
前記の接触処理においては、反応溶媒、特に、有機溶媒を用いることが好ましい。有機溶媒としては、反応試薬に対して不活性であり、かつ、反応試薬に対する溶解性を有するものであれば、特に限定されるものではないが、テトラヒドロフラン、ジオキサン類等の環状エーテル類等が好ましく用いられる。
【００２３】
本発明の化学発光試薬の製造に用いられる紫外線の光源としては、高圧水銀灯、低圧水銀灯、殺菌灯等を非限定的に用いることができるが、高圧水銀灯が好ましく用いられる。この紫外線照射により得られる化学発光試薬は、紫外線を照射せずに製造した化学発光試薬に較べて同濃度で使用した場合に、非常に高い発光強度を示すことが認められることから、化学発光性化合物の形成反応を促進する作用を有するものと推定される。
【００２４】
反応温度は用いられる還元剤及び反応溶媒の種類によって異なるが、一般的に、−１０〜＋１５０℃、好ましくは、０〜１２０℃、特に好ましくは、２０〜９０℃の範囲の温度である。反応時間は１分〜一昼夜、好ましくは、１０分〜１２時間、特に好ましくは、３０分〜５時間の範囲で採用することができる。
【００２５】
本発明のペルオキシダーゼ活性の測定方法に用いられる化学発光試薬は、ｐＨ７．５〜１３の塩基性条件下において過剰の過酸化水素及びペルオキシダーゼの存在下で反応してペルオキシダーゼの濃度に依存した量で発光する。この反応は過酸化水素の検出及びペルオキシダーゼの定量等に利用することが可能であり、ペルオキシダーゼを標識物質として用いることにより、更に、種々の物質の定量に用いることが可能になる。
【００２６】
また、本発明のペルオキシダーゼ活性の測定方法に用いられる化学発光試薬による発光は、フェノール性化合物等の発光増強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、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。
【００２７】
本発明のペルオキシダーゼ活性の測定方法に用いられる化学発光試薬の濃度は、１０^-8〜１Ｍ、好ましくは、１０^-6〜１０^-2Ｍの範囲の濃度であり、その使用量は１０〜５００μｌ、特に、５０〜３００μｌの範囲で用いることが好ましい。また、発光増強剤の使用量は、化学発光試薬の量の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、好ましくは、１０^-4〜１０^-2Ｍの範囲である。
【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はないが、有機過酸化物、無機過酸化物等が任意に用いられ、過酸化水素が好ましく用いられる。この水素受容体の使用量は化学発光性物質に対して充分に過剰な量で用いることが必要であり、その使用量は化学発光性物質に対して３〜１万倍モル、特に、１０〜１０００倍モルの範囲で用いることが好ましい。
【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等に限定されるものではない。
【００３２】
［実施例１］
ルシゲニン１ｍｇを試験管に採り、これにＮ，Ｎ−ジメチルアセトアミド１５０μｌを加えて溶解させた後、攪拌下に１，４−ジオキサン１ｍｌ中へ加えてルシゲニンを１，４−ジオキサン中に微分散させた。次に、これに蟻酸２０μｌを加えてよく攪拌してから、水素化リチウムアルミニウム粉末５ｍｇを添加して、１ＫＷの高圧水銀灯の照射下に２５℃で３０分間攪拌して還元反応させた後、反応混合液を氷冷下に脱イオン水１ｍｌ中へ少量ずつ注入して過剰量の水素化リチウムアルミニウムを分解させた。次に、生成した水酸化アルミニウム等の沈殿を濾別してから、濾液のｐＨを１Ｎ塩酸で７．０に調整することにより新規化学発光試薬を得た。
［ペルオキシダーゼ活性の測定］
複数の化学発光測定用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０．１Ｍトリス塩酸緩衝液（pH8.4)溶液２００μｌ及びｐ−ヨードフェノール１０ｍＭトリス塩酸緩衝液（pH8.4)溶液２０μｌを加えた後、これに１０００倍に希釈した前記化学発光試薬を２０μｌ及び０．００３４重量％過酸化水素水溶液５０μｌを順次加え、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った。
【００３３】
【表１】

【００３４】
［実施例２］
ルシゲニン１ｍｇを試験管に採り、これにＮ，Ｎ−ジメチルホルムアミド１５０μｌを加えて溶解させた後、攪拌下に１，４−ジオキサン１ｍｌ中へ加えてルシゲニンを１，４−ジオキサン中に微分散させた。次に、これに蟻酸２０μｌを加えてよく攪拌してから、水素化リチウムアルミニウム粉末５ｍｇを添加して、１ＫＷの高圧水銀灯の照射下に２５℃で３０分間攪拌して還元反応させた後、反応混合液を氷冷下に脱イオン水１ｍｌ中へ少量ずつ注入して過剰量の水素化リチウムアルミニウムを分解させた。次に、生成した水酸化アルミニウム等の沈殿を濾別してから、濾液のｐＨを１Ｎ塩酸で７．０に調整することにより新規化学発光試薬を得た。
［ペルオキシダーゼ活性の測定］
複数の化学発光測定用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０．１Ｍトリス塩酸緩衝液(pH 8.4)溶液２００μｌ及びｐ−ヨードフェノール１０ｍＭトリス塩酸緩衝液(pH 8.4)溶液２０μｌを加えた後、これに１０００倍に希釈した上記化学発光試薬を２０μｌ及び０．００３４重量％過酸化水素水溶液５０μｌを順次加え、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った。
【００３５】
【表２】

【００３６】
［実施例３］
ルシゲニン１ｍｇを試験管に採り、これにＮ−メチル−２−ピロリドン１５０μｌを加えて溶解させた後、攪拌下に１ｍｌ中へ加えてルシゲニンを１，４−ジオキサン中に微分散させた。次に、これに蟻酸２０μｌを加えてよく攪拌してから、水素化リチウムアルミニウム粉末５ｍｇを添加して、１ＫＷの高圧水銀灯の照射下に２５℃で３０分間攪拌して還元反応させた後、反応混合液を氷冷下に脱イオン水１ｍｌ中へ少量ずつ注入して過剰量の水素化リチウムアルミニウムを分解させた。次に、生成した水酸化アルミニウム等の沈殿を濾別してから、濾液のｐＨを１Ｎ塩酸で７．０に調整することにより新規化学発光試薬を得た。
［ペルオキシダーゼ活性の測定］
複数の化学発光測定用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０．１Ｍトリス塩酸緩衝液(pH 8.4)溶液２００μｌ及びｐ−ヨードフェノール１０ｍＭトリス塩酸緩衝液(pH 8.4)溶液２０μｌを加えた後、これに１０００倍に希釈した上記化学発光試薬を２０μｌ及び０．００３４重量％過酸化水素水溶液５０μｌを順次加え、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った。
【００３７】
【表３】

【００３８】
［比較例１］
複数の化学発光測定用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０．１Ｍトリス塩酸緩衝液(pH 8.4)溶液２００μｌ及びｐ−ヨードフェノール１０ｍＭトリス塩酸緩衝液(pH 8.4)溶液２０μｌを加えた後、これにルミノールを５．６×１０^-5Ｍの濃度で含有する０．１Ｍトリス塩酸緩衝液(pH 8.4)１００μｌ及び０．００３４重量％過酸化水素水溶液５０μｌを順次加え、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に示すような発光強度が得られ、ＨＲＰを１×１０^-17 mol/assay まで測定できることが可能であった。
【００３９】
【表４】

【００４０】
【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になり、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及び抗体類を、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, peroxidase activity in the presence of a hydrogen acceptor by using a chemiluminescence method using a specific chemiluminescence reagent. It is related with the measuring method.
[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. 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.
[0003]
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 a colorimetric method is used for measuring peroxidase activity, there is a problem that the quantitative property in the low concentration region of peroxidase is lacking, and a fluorescence method has been developed to solve this problem. It is not yet sufficient for quantitativeness in the area. 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 chemiluminescent system has been developed that uses luminol as a chemiluminescent substance, hydrogen peroxide as a hydrogen acceptor, and p-iodophenol as a luminescence enhancer, and can quantify peroxidase activity with high sensitivity. It is possible.
[0004]
However, when applying peroxidase enzyme activity measurement to enzyme immunoassay, there are many cases where it is necessary to further enhance the quantitativeness of the enzyme in a low concentration region, and the peroxidase enzyme activity measurement is more sensitive. It was hoped that
[0005]
[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 method capable of measuring peroxidase activity with higher sensitivity.
[0006]
[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 comprising a reaction product obtained by contact treatment with a reducing agent under ultraviolet irradiation in the presence of a disubstituted carboxylic acid amide compound, hydrogen peroxide as a hydrogen acceptor, and further luminescence We found that chemiluminescent systems that use specific phenolic compounds as enhancers can quantitate peroxidase activity with higher sensitivity compared to systems that use luminol as a chemiluminescent substance. Based on this, the present invention has been achieved.
[0007]
Therefore, the first of the present invention is
Reaction product obtained by contacting N, N′-disubstituted-9,9′-bisacridinium salts with a reducing agent in the presence of an N, N-disubstituted carboxylic acid amide compound under ultraviolet irradiation. The present invention relates to a method for measuring peroxidase activity by a chemiluminescence method, wherein a peroxidase enzyme activity is measured in the presence of a hydrogen acceptor using a chemiluminescent reagent composed of a product.
[0008]
The second of the present invention is
Reaction mixture obtained by contacting N, N′-disubstituted-9,9′-bisacridinium salts with a reducing agent in the presence of an N, N-disubstituted carboxylic acid amide compound under ultraviolet irradiation. When measuring peroxidase enzyme activity in the presence of a hydrogen acceptor using a chemiluminescent reagent comprising a phenolic compound as a luminescence enhancer, the method relates to a method for measuring peroxidase activity by a chemiluminescence method. is there.
[0009]
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 reduced as chemiluminescent substances in the presence of N, N-disubstituted carboxylic acid amide compounds. A peroxidase enzyme activity is measured in the presence of a hydrogen acceptor, more preferably in the presence of a luminescence enhancer, using a chemiluminescent reagent comprising a reaction product obtained by contact treatment with an agent. . The measurement operation is arbitrary, but generally, a measurement sample containing a chemiluminescent substance, a luminescence enhancer and a peroxidase enzyme is mixed, and a hydrogen acceptor solution is added in a specific basic pH region to cause a chemiluminescent reaction. A method of measuring the chemiluminescence amount with a luminescence measuring apparatus is performed.
[0010]
The chemiluminescent reagent used in the method for measuring peroxidase activity of the present invention is an N, N′-disubstituted-9,9′-bisacridinium salt in the presence of an N, N-disubstituted carboxylic acid amide compound. It is a reaction product obtained by contact treatment with a reducing agent under ultraviolet irradiation.
[0011]
N, N′-disubstituted-9,9′-bisacridinium salts are represented by the following general formula (1):
[0012]
[Chemical 2]

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. An alkyl group, an aryl group, and a halogenated aryl group are those having 1 to 14 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. The aryl group preferably has 6 to 14 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, and are the same as each other Or it may be different. These hydrocarbon groups preferably have 1 to 20 carbon atoms, particularly 1 to 10 carbon atoms.
[0015]
Specific examples of the N, N′-disubstituted-9,9′-bisacridinium salts include N, N′-dimethyl-9,9′-bisacridinium salts, N, N′-diethyl- 9,9 ′ busacridinium salt, N, N′-diphenyl-9,9′-bisacridinium salt, N, N′-di-m-chlorophenyl-9,9′bisacridinium salt, etc. Of course, it is not limited to these. In particular, N, N′-dimethyl-9,9′-bisacridinium dinitrate (lucigenin) is preferable.
[0016]
In the general formula (I), X ⁿ⁻ is an n-valent anion, and n is 1 or 2. The anion is not particularly limited, and nitrate ion (NO ₃ ⁻ ), halide ion (eg, chloride ion, fluoride ion, bromide ion, etc.), (meth) phosphate ion (PO ₃ ⁻ And the like. Of these anions, nitrate ions are particularly preferred.
[0017]
Examples of the N, N-disubstituted carboxylic acid amide compound used for the production of the chemiluminescent reagent used in the method for measuring peroxidase activity of the present invention include the following general formula (2):
[0018]
[Chemical 3]

The compound represented by these can be mentioned. In the general formula (2), R ₁ is a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an aryl group having 6 to 20 carbon atoms. It may be substituted with an atom, nitro group, hydroxyl group, amino group or the like. R ₂ is a methyl group or an ethyl group, R ₃ is an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, or an aryl group having 6 to 20 carbon atoms, and the aryl group is an alkyl group , A halogen atom, a nitro group, a hydroxyl group, an amino group and the like may be substituted. 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. Specific examples of N, N-disubstituted carboxylic acid compounds include N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylacrylamide, N, N-dimethylpropionamide, N, N-dimethyl. Nonlimiting examples include benzamide, N-methyl-2-pyrrolidone, and N-ethyl-2-pyrrolidone .
[0019]
The N, N-disubstituted carboxylic acid amide compound is essential for the production of the chemiluminescent reagent of the present invention, and formed a complex with N, N′-disubstituted-9,9′-bisacridinium salts. Later, with the reduction reaction of the acridinium salt structure, the compound is considered to change to a compound with a higher luminescence yield, involved in the reaction for producing the chemiluminescent reagent of the present invention as a reaction component, and the water solubility of the reaction product. It is thought that it contributes to grant. Therefore, the amount used is 1 to 1000 equivalents, preferably 1.5 to 500 equivalents, particularly preferably 2 to 3000 equivalents, relative to the N, N′-disubstituted-9,9′-bisacridinium salts. Percentage is acceptable.
[0020]
In the production of the chemiluminescent reagent used in the method for measuring peroxidase activity of the present invention, N, N′-disubstituted-9,9′-bisacridinium salts are substituted with N, N-disubstituted carboxylic acid amide compounds. In addition, formic acid can be added in the contact treatment with the reducing agent. Formic acid is used in a proportion of 0 to 5000 equivalents, preferably 0.5 to 1000 equivalents, particularly preferably 1 to 500 equivalents, based on N, N′-disubstituted-9,9′-bisacridinium salts. In addition, the reduction reaction of N, N′-disubstituted-9,9′-bisacridinium salts can be promoted, and the reaction yield of the chemiluminescent reagent can be significantly increased.
[0021]
Examples of the reducing agent used for producing the chemiluminescent reagent in the measurement system of the present invention include lithium aluminum hydride, lithium boron hydride, sodium boron hydride and the like, and lithium aluminum hydride is particularly preferably used.
[0022]
In the contact treatment, it is preferable to use a reaction solvent, particularly an organic solvent. The organic solvent is not particularly limited as long as it is inert to the reaction reagent and has solubility in the reaction reagent, but cyclic ethers such as tetrahydrofuran and dioxane are preferable. Used.
[0023]
As the ultraviolet light source used in the production of the chemiluminescent reagent of the present invention, a high pressure mercury lamp, a low pressure mercury lamp, a germicidal lamp and the like can be used without limitation, but a high pressure mercury lamp is preferably used. The chemiluminescent reagent obtained by this UV irradiation is shown to exhibit a very high luminescence intensity when used at the same concentration as the chemiluminescent reagent produced without UV irradiation. It is presumed to have an action of promoting the compound formation reaction.
[0024]
While the reaction temperature varies depending on the type of reducing agent and reaction solvent used, it is generally -10 to + 150 ° C, preferably 0 to 120 ° C, particularly preferably 20 to 90 ° C. The reaction time is 1 minute to 1 day, preferably 10 minutes to 12 hours, particularly preferably 30 minutes to 5 hours.
[0025]
The chemiluminescent reagent used in the method for measuring peroxidase activity of the present invention reacts in the presence of excess hydrogen peroxide and peroxidase under basic conditions of pH 7.5 to 13, and emits light in an amount depending on the concentration of peroxidase. To do. This reaction can be used for detection of hydrogen peroxide and quantification of peroxidase. By using peroxidase as a labeling substance, it can be further used for quantification of various substances.
[0026]
In addition, it is recognized that light emission by the chemiluminescent reagent used in the method for measuring peroxidase activity of the present invention is enhanced by a light emission enhancer 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, 6-hydroxybenzothiazole, 2-naphthol, firefly luciferin and the like.
[0027]
The concentration of the chemiluminescent reagent used in the method for measuring peroxidase activity of the present invention is 10 ^{−8 to 1} M, preferably 10 ⁻⁶ to 10 ⁻² M. The amount used is 10 to 500 μl, In particular, it is preferably used in the range of 50 to 300 μl. The amount of the luminescence enhancer used is 0.01 to 100 times mol, preferably 0.1 to 10 times mol of the amount of the chemiluminescent reagent, and its concentration is 10 ^{−6 to 1} M, preferably It is in the range of 10 ⁻⁴ to 10 ⁻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, but organic peroxides, inorganic peroxides, etc. are arbitrarily used. Hydrogen oxide is preferably used. It is necessary to use the hydrogen acceptor in an amount that is sufficiently excessive with respect to the chemiluminescent substance, and the use amount thereof is 3 to 10,000 times mol, particularly 10 to 10 times that of the chemiluminescent substance. It is preferable to use in the range of 1000 times mole.
[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 etc.
[0032]
[Example 1]
Take 1 mg of lucigenin in a test tube, add 150 μl of N, N-dimethylacetamide to dissolve it, add to 1 ml of 1,4-dioxane with stirring, and finely disperse lucigenin in 1,4-dioxane. It was. Next, 20 μl of formic acid was added and stirred well, 5 mg of lithium aluminum hydride powder was added, and the mixture was stirred at 25 ° C. for 30 minutes under irradiation of a 1 KW high-pressure mercury lamp, followed by a reduction reaction. The mixed solution was poured little by little into 1 ml of deionized water under ice cooling to decompose the excess amount of lithium aluminum hydride. Next, the produced precipitate such as aluminum hydroxide was filtered off, and the pH of the filtrate was adjusted to 7.0 with 1N hydrochloric acid to obtain a novel chemiluminescent reagent.
[Measurement of peroxidase activity]
200 μl of 0.1 M Tris-HCl buffer solution (pH 8.4) with various concentrations of horseradish peroxidase (HRP) and p-iodophenol 10 mM Tris-HCl buffer solution (pH 8.4) in various chemiluminescence assay wells After 20 μl was added, 20 μl of the chemiluminescent reagent diluted 1000 times and 50 μl of 0.0034 wt% aqueous hydrogen peroxide were sequentially added thereto, and 0-5 with a luminometer (Diatron Luminous CT-9000D). As a result of integrating the luminescence amount per second, the luminescence intensity as shown in Table 1 was obtained, and it was possible to measure HRP up to 1 × 10 ⁻¹⁹ mol / assay.
[0033]
[Table 1]

[0034]
[Example 2]
Take 1 mg of lucigenin in a test tube, add 150 μl of N, N-dimethylformamide to dissolve it, add to 1 ml of 1,4-dioxane with stirring, and finely disperse lucigenin in 1,4-dioxane. It was. Next, 20 μl of formic acid was added and stirred well, 5 mg of lithium aluminum hydride powder was added, and the mixture was stirred at 25 ° C. for 30 minutes under irradiation of a 1 KW high-pressure mercury lamp, followed by a reduction reaction. The mixed solution was poured little by little into 1 ml of deionized water under ice cooling to decompose the excess amount of lithium aluminum hydride. Next, the produced precipitate such as aluminum hydroxide was filtered off, and the pH of the filtrate was adjusted to 7.0 with 1N hydrochloric acid to obtain a novel chemiluminescent reagent.
[Measurement of peroxidase activity]
200 μl of 0.1 M Tris-HCl buffer solution (pH 8.4) at various concentrations of horseradish peroxidase (HRP) and 20 μl of p-iodophenol 10 mM Tris-HCl buffer solution (pH 8.4) were added to a plurality of chemiluminescence wells. After the addition, 20 μl of the chemiluminescent reagent diluted 1000 times and 50 μl of 0.0034 wt% aqueous hydrogen peroxide solution were sequentially added thereto, and luminescence was emitted for 0 to 5 seconds with a luminometer (Luminous CT-9000D manufactured by Diatron). As a result of integrating the amounts, emission intensity as shown in Table 2 was obtained, and HRP could be measured up to 1 × 10 ⁻¹⁹ mol / assay.
[0035]
[Table 2]

[0036]
[Example 3]
1 mg of lucigenin was taken in a test tube, and 150 μl of N-methyl-2-pyrrolidone was added thereto and dissolved, and then added to 1 ml with stirring to finely disperse lucigenin in 1,4-dioxane. Next, 20 μl of formic acid was added and stirred well, 5 mg of lithium aluminum hydride powder was added, and the mixture was stirred at 25 ° C. for 30 minutes under irradiation of a 1 KW high-pressure mercury lamp, followed by a reduction reaction. The mixed solution was poured little by little into 1 ml of deionized water under ice cooling to decompose the excess amount of lithium aluminum hydride. Next, the produced precipitate such as aluminum hydroxide was filtered off, and the pH of the filtrate was adjusted to 7.0 with 1N hydrochloric acid to obtain a novel chemiluminescent reagent.
[Measurement of peroxidase activity]
200 μl of 0.1 M Tris-HCl buffer solution (pH 8.4) at various concentrations of horseradish peroxidase (HRP) and 20 μl of p-iodophenol 10 mM Tris-HCl buffer solution (pH 8.4) were added to a plurality of chemiluminescence wells. After the addition, 20 μl of the chemiluminescent reagent diluted 1000 times and 50 μl of 0.0034% by weight aqueous hydrogen peroxide solution were sequentially added thereto, and light was emitted for 0 to 5 seconds with a luminometer (Luminous CT-9000D manufactured by Diatron). As a result of integrating the amounts, emission intensity as shown in Table 3 was obtained, and HRP could be measured up to 1 × 10 ⁻¹⁹ mol / assay.
[0037]
[Table 3]

[0038]
[Comparative Example 1]
200 μl of 0.1 M Tris-HCl buffer solution (pH 8.4) at various concentrations of horseradish peroxidase (HRP) and 20 μl of p-iodophenol 10 mM Tris-HCl buffer solution (pH 8.4) were added to a plurality of chemiluminescence wells. After the addition, 100 μl of 0.1 M Tris-HCl buffer (pH 8.4) containing luminol at a concentration of 5.6 × 10 ⁻⁵ M and 50 μl of 0.0034 wt% aqueous hydrogen peroxide solution were sequentially added to the luminometer. As a result of integrating the amount of luminescence for 0 to 5 seconds with (Diatron Luminous CT-9000D), the luminescence intensity shown in Table 4 is obtained, and HRP can be measured up to 1 × 10 ^-17 mol / assay. there were.
[0039]
[Table 4]

[0040]
【The invention's effect】
The method for measuring peroxidase activity of the present invention makes it 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 and antibodies, and nucleic acids can be detected or quantified with higher sensitivity, respectively, by hybridization assays.

Claims (9)

The following general formula (1)

(In the above general formula (1), R ¹ and R ² are a methyl group, an ethyl group or a propyl group, and may be the same or different from each other, and R ³ , R ⁴ , R ⁵ and R ⁶ are Is a hydrogen atom, X is a nitrate ion or a halide ion, and n is 1.)
N, N′-disubstituted-9,9′-bisacridinium salts represented by
The following general formula (2)

(In the general formula (2), R ₁ is a hydrogen atom, a methyl group, an ethyl group or a propyl group, R ₂ is a methyl group or an ethyl group, and R ₃ is a methyl group, an ethyl group or a propyl group. R ₁ and R ₃ may be bonded to each other to form a ring together with the carbon atom and the nitrogen atom of the amide group to which each is bonded.
A chemiluminescence reagent containing a reaction product obtained by irradiating with ultraviolet rays when contacted with a reducing agent in the presence of an N, N-disubstituted carboxylic acid amide compound represented by A method for measuring peroxidase activity by chemiluminescence, which comprises measuring peroxidase enzyme activity in the presence of The following general formula (1)

(In the above general formula (1), R ¹ and R ² are a methyl group, an ethyl group or a propyl group, and may be the same or different from each other, and R ³ , R ⁴ , R ⁵ and R ⁶ are Is a hydrogen atom, X is a nitrate ion or a halide ion, and n is 1.)
N, N′-disubstituted-9,9′-bisacridinium salts represented by
The following general formula (2)

(In the general formula (2), R ₁ is a hydrogen atom, a methyl group, an ethyl group or a propyl group, R ₂ is a methyl group or an ethyl group, and R ₃ is a methyl group, an ethyl group or a propyl group. R ₁ and R ₃ may be bonded to each other to form a ring together with the carbon atom and the nitrogen atom of the amide group to which each is bonded.
A chemiluminescence reagent containing a reaction product obtained by irradiating with ultraviolet rays when contacted with a reducing agent in the presence of an N, N-disubstituted carboxylic acid amide compound represented by 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.           The N, N'-disubstituted-9,9'-bisacridinium salt is N, N'-dimethyl-9,9'-bisacridinium dinitrate. The method for measuring peroxidase activity according to claim 1.           The N, N-disubstituted carboxylic acid amide compound is N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylacrylamide, N, N-dimethylpropylamide, N, N-dimethylbenzamide, N The method for measuring peroxidase activity according to any one of claims 1 to 3, which is at least one compound selected from the group consisting of -methyl-2-pyrrolidone and N-ethyl-2-pyrrolidone.           The N, N-disubstituted carboxylic acid amide compound is N, N-dimethylformamide, N, N-dimethylacetamide or N-methyl-2-pyrrolidone, according to any one of claims 1 to 3. Method for measuring peroxidase activity. The N, N′-disubstituted-9,9′-bisacridinium salts,
The measurement of peroxidase activity according to any one of claims 1 to 5, wherein formic acid is further added when contacting with the reducing agent in the presence of the N, N-disubstituted carboxylic acid amide compound. Method.           The method for measuring peroxidase activity according to any one of claims 1 to 6, wherein the reducing agent is lithium aluminum hydride.           The method for measuring peroxidase activity according to any one of claims 1 to 7, wherein the hydrogen acceptor is hydrogen peroxide.           The said luminescence enhancer is at least 1 sort (s) of phenolic compound selected from the group which consists of p-iodophenol, p-phenylphenol, and 6-hydroxybenzothiazole, The claim in any one of Claims 2-8. Method for measuring peroxidase activity of