JP3958452B2 - Novel chemiluminescent reagent and method for producing the same - Google Patents

Description

（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、互いに同一でも異なるものでもよく、Ｒ³ 、Ｒ⁴ 、Ｒ⁵ 及びＲ⁶ は、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を示し、互いに同一でも異なるものでもよく、Ｘはｎ価の陰イオンであり、ｎは１又は２である。）
で表わされるＮ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類とＮ，Ｎ−ジ置換カルボン酸アミド化合物との複合体を含有することを特徴とする化学発光試薬に関するものである。
【０００７】
また、本発明の第二は、
下記一般式（Ｉ）
【化４】

（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、互いに同一でも異なるものでもよく、Ｒ³ 、Ｒ⁴ 、Ｒ⁵ 及びＲ⁶ は、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、互いに同一でも異なるものでもよく、Ｘはｎ価の陰イオンであり、ｎは１又は２である。）で表わされるＮ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類を溶媒の存在下または非存在下においてＮ，Ｎ−ジ置換カルボン酸アミド化合物と接触処理することを特徴とする化学発光試薬の製造方法に関するものである。
【０００８】
【発明の実施の形態】
以下、本発明につき更に詳しく説明する。
本発明の化学発光試薬は、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類とＮ，Ｎ−ジ置換カルボン酸アミド化合物との複合体を含有するものである。
【０００９】
また、本発明の化学発光試薬の製造方法は、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類を溶媒の存在下又は非存在下においてＮ，Ｎ’−ジ置換カルボン酸アミド化合物と接触処理することからなるものである。
上記Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類は、次の一般式（１）：
【００１０】
【化５】

で表され、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、各々、互いに同一でも異なるものでもよい。
【００１１】
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アルキル基又はこ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、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。
【００１２】
一般式（Ｉ）において、Ｒ³ 、Ｒ⁴ 、Ｒ⁵ 及びＲ⁶ は、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、互いに同一でもまたは異なるものでよい。炭化水素基の炭素数としては、例えば、１〜２０、好ましくは、１〜１４を挙げることができる。
また、上記一般式（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、具体的には硝酸イオン、ハロゲンイオン、リン酸イオン、硫酸イオン、スルホン酸イオン等が挙げられる。これらのなかで、特に、硝酸イオンが好ましい。
【００１３】
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を挙げることができ、対イオンとして上記の陰イオンが好ましいが、特に、対イオンが硝酸イオンである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）が好適である。
【００１４】
本発明の化学発光試薬の製造に用いられるもう一つの成分であるＮ，Ｎ−ジ置換カルボン酸アミド化合物としては、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−ピロリドン等が比限定的に挙げられるが、特に、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−メチル−２−ピロリドン等が好ましい。
【００１５】
これらのＮ，Ｎ−ジ置換カルボン酸アミド化合物のうち、常温で液体のものは溶媒を兼ねて大過剰に用いることもできるし、他の適当な溶媒を用いてもよい。また、室温で固体のものは加熱溶融させて用いてもよく、また、適当な溶媒を用いることもできる。このような溶媒としては、ジメチルスルホキサイド、ヘキサメチルホスホアミド等を挙げることができる。
【００１６】
本発明の化学発光試薬の製造においては、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類とＮ，Ｎ−ジ置換カルボン酸アミド化合物とを接触させることにより、その接触時間により発光強度が増大し、ピークを形成して減衰していく現象が観察されることから、Ｎ，Ｎ−ジ置換カルボン酸アミド化合物がＮ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類と反応して活性化状態を形成した後、経時的に変化して不活性化するものと考えられる。
【００１７】
従って、本発明の化学発光試薬の製造は、基本的にはＮ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類とＮ，Ｎ−ジ置換カルボン酸アミド化合物との接触処理によるものであり、以下に述べる反応条件下において両者を混合、保持することに行なわれる。具体的には本発明の化学発光試薬を用いて化学発光分析を行なう際に、これらの試薬の各成分を調合して所定時間反応させてから使用する方法が採用される。
【００１８】
反応条件として、反応温度は用いられる試薬及び溶媒の種類によって異なるが、一般的に−１０〜＋２００℃、好ましくは、０〜１２０℃、特に好ましくは、２０〜６０℃の範囲の温度である。反応時間は１分〜一昼夜、好ましくは、５分〜１２時間、特に好ましくは、１０分〜５時間の範囲の時間を採用することができる。また、本発明の化学発光試薬の各成分の割合は、Ｎ，Ｎ’−ジ置換−９，９’−ビスアクリジニウム塩類１モルに対してＮ，Ｎ−ジ置換カルボン酸アミド化合物当モルから大過剰の範囲でよい。
【００１９】
本発明の化学発光試薬は、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−プロモフェノール、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を非限定的に挙げることができる。
上記水素受容体としては、ペルオキシダーゼの基質になり得るものであれば特に限定されるものではなく、有機過酸化物、無機過酸化物等を任意に用いることができるが、特に、過酸化水素が好ましい。
【００２０】
本発明の化学発光試薬は、１０^-6〜１Ｍ、好ましくは、１０^-4〜１０^-2Ｍの範囲の濃度で用いられ、その使用量は１０〜５００μｌ、好ましくは、５０〜３００μｌの範囲である。発光増強剤の使用量は、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、好ましくは１０^-4〜１０^-2Ｍの範囲で用いるのが望ましい。また、化学発光反応に用いる過酸化水素等の水素受容体の量は化学発光試薬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試薬に対して３〜１万倍モル、好ましくは１０〜１０００倍モルの範囲が望ましい。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。
【００２１】
上記化学発光反応に用いられる緩衝液としては、トリス緩衝液、リン酸緩衝液、ホウ酸緩衝液、炭酸緩衝液、グリシン−水酸化ナトリウム緩衝液等を挙げることができる。これらの緩衝液の濃度は１ｍＭ〜１Ｍの範囲で用いることが好ましい。また、反応時に界面活性剤、キレート剤等の添加剤を任意に用いることもできる。
【００２２】
本発明によれば、ペルオキシダーゼ活性測定用試薬キットとして、各々、別個の容器に充填された一般式（１）で表される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ペルオキシダーゼまたはペルオキシダーゼ標識物質からなる化学発光分析用試薬キットを提供することもできる。
【００２３】
本発明の化学発光試薬は、以上説明したように、塩基性条件下に過酸化水素及びペルオキシダーゼの存在で反応して発光するが、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、さらに、種々の物質の定量に用いることが可能になる。
【００２４】
【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。もっとも、本発明は実施例等により限定されるものではない。
【００２５】
［実施例１］
ルシゲニン１ｍｇを試験管に採り、これにＮ，Ｎ−ジメチルホルムアミド２ｍｌを加えて溶解させた後、室温で９０分静置してから純水２ｍｌを加えて混合することによりルシゲニンとＮ，Ｎ−ジメチルホルムアミドとの複合体からなる化学発光試薬を得た。
【００２６】
ペルオキシダーゼ活性の測定
上記の如くして得られた化学発光試薬を直ちに２０μｌずつ複数の化学発光測定用ウェルに加え、次いで、こ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０．００３４重量％過酸化水素水溶液５０μｌを加え、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、ペルオキシダーゼ活性を濃度依存性良く測定出来ることが認められた。
【００２７】
【表１】

［実施例２］
ルシゲニン１ｍｇを試験管に採り、これにＮ，Ｎ−ジメチルアセトアミド２ｍｌを加えて溶解させた後、室温で３時間静置してから純水２ｍｌを加えて混合することによりルシゲニンとＮ，Ｎ−ジメチルアセトアミドとの複合体からなる化学発光試薬を得た。
【００２８】
ペルオキシダーゼ活性の測定
この化学発光試薬溶液を直ちに２０μｌ採り、複数の化学発光測定用ウェルに加え、次に、このウェルに各々西洋ワサビペルオキシダーゼの異なる複数の濃度水準の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０．００３４重量％過酸化水素水溶液５０μｌを加え、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、ペルオキシダーゼ活性を濃度依存性良く測定出来ることが認められた。
【００２９】
【表２】

［実施例３］
ルシゲニン１ｍｇを試験管に採り、これにＮ，Ｎ−メチル−２−ピロリドン２ｍｌを加えて溶解させた後、室温で３時間静置してから純水２ｍｌを加えて混合することによりルシゲニンとＮ−メチル−２−ピロリドンとの複合体からなる化学発光試薬を得た。
【００３０】
ペルオキシダーゼ活性の測定
この化学発光試薬溶液を直ちに２０μｌ採り、複数の化学発光測定用ウェルに加え、次に、このウェルに各々西洋ワサビペルオキシダーゼの異なる複数の濃度水準の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、更に、これに０．００３４重量％過酸化水素水溶液５０μｌを加え、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、ペルオキシダーゼ活性を濃度依存性良く測定出来ることが認められた。
【００３１】
【表３】

［比較例１］
ルシゲニン１ｍｇを純水２ｍｌに溶解し、これを２０μｌずつ複数の化学発光測定用ウェルに加え、次に、このウェルに各々西洋ワサビペルオキシダーゼ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、さらに、これに０．００３４重量％過酸化水素水溶液５０μｌを加え、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、ペルオキシダーゼ活性を正確には測定できないことが確認された。
【００３２】
【表４】

【００３３】
【発明の効果】
本発明により提供される化学発光試薬は、過酸化水素等の水素受容体とペルオキシダーゼの存在下に、ペルオキシダーゼの濃度に依存して化学発光する性質を有することを利用して、ペルオキシダーゼ酵素を高感度で検出することができる。
さらに、ペルオキシダーゼを標識物質として抗原、抗体、核酸等を標識した酵素標識物を用いて、酵素免疫測定法により抗体、抗原等をウエスタンブロット法により蛋白質を、サザーン及びノーザンブロット法によりＤＮＡ及びＲＮＡを、そして酵素標識核酸プローブを用いて核酸を各々特異的に、且つ高感度で測定することができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a chemiluminescence reagent capable of controlled chemiluminescence and used for detection and quantification of various substances by chemiluminescence analysis and a method for producing the same, and more particularly, the presence of a hydrogen acceptor. The present invention relates to a chemiluminescent reagent capable of measuring peroxidase enzyme activity and a method for producing the same.
[0002]
[Prior art]
Lucigenin (N, N′-dimethyl-9,9′-bisacridinium dinitrate), which has been used for a long time as a chemiluminescent reagent, exhibits low light in an alkaline aqueous solution, and a hydrogen acceptor, for example, It is known that when hydrogen peroxide is added, it emits intense light and is used for various microanalysis. However, since lucigenin emits light quantitatively in the presence of hydrogen peroxide and alkali, it can be used for the determination of hydrogen peroxide. However, it is a chemical that controls the light emission by this reaction and measures enzyme activity by the amount of light emitted. The luminescent enzyme immunoassay method (CLEIA) has a problem that it is difficult to use. As a means to solve this problem, glucogen oxidase is used as a labeling enzyme, and hydrogen peroxide produced by the oxidation reaction of glucose is allowed to act on lucigenin in the presence of alkali, thereby depending on the concentration of the measurement target substance. A method of emitting light is performed.
[0003]
[Problems to be solved by the invention]
However, the chemiluminescence analysis method using glucose oxidase as a labeling enzyme requires complicated preparation of the chemiluminescence reagent and the operation of the luminescence system, so that an inexpensive chemiluminescence reagent for chemiluminescence analysis using peroxidase as the labeling enzyme is used. Development was desired.
[0004]
In view of the above circumstances, an object of the present invention is to provide a novel chemiluminescent reagent capable of using a peroxidase whose substrate is a hydrogen acceptor such as hydrogen peroxide and a method for producing the same.
[0005]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the inventors of the present invention converted N, N′-disubstituted-9,9′-bisacridinium salts such as lucigenin to N, N-disubstituted carboxylic acids. A chemiluminescent reagent containing a complex obtained by contacting with an amide compound does not react with hydrogen peroxide under specific alkaline pH conditions, and depends on the amount of peroxidase in the presence of hydrogen peroxide and peroxidase Thus, chemiluminescence is found out, and the present invention has been achieved based on these findings.
[0006]
That is, the first of the present invention is
The following general formula (1)
[Chemical 3]

(In the above 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, and R ³ , R ⁴ , R ⁵ and R ⁶ represents a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group, or a halogen atom, and may be the same or different from each other, X is an n-valent anion, and n is 1 or 2. )
The present invention relates to a chemiluminescent reagent comprising a complex of an N, N′-disubstituted-9,9′-bisacridinium salt represented by formula (I) and an N, N-disubstituted carboxylic acid amide compound. is there.
[0007]
The second of the present invention is
The following general formula (I)
[Formula 4]

(In the above 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, and R ³ , R ⁴ , R ⁵ and R ⁶ is 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 may be the same or different from each other, X is an n-valent anion, and n is 1 or The N, N′-disubstituted-9,9′-bisacridinium salt represented by 2) is contacted with the N, N-disubstituted carboxylic acid amide compound in the presence or absence of a solvent. The present invention relates to a method for producing a chemiluminescent reagent.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in more detail.
The chemiluminescent reagent of the present invention contains a complex of N, N′-disubstituted-9,9′-bisacridinium salts and N, N-disubstituted carboxylic acid amide compounds.
[0009]
Further, the method for producing the chemiluminescent reagent of the present invention comprises N, N′-disubstituted-9,9′-bisacridinium salts in the presence or absence of a solvent. It comprises a contact treatment with an amide compound.
The N, N′-disubstituted-9,9′-bisacridinium salts are represented by the following general formula (1):
[0010]
[Chemical formula 5]

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.
[0011]
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 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, or branched alkyl groups thereof. . The aryl group preferably has 6 to 20 carbon atoms, and includes a phenyl group, a tolyl group, a xylyl group, and the like, and may be further 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.
[0012]
In the general formula (I), R ³ , R ⁴ , R ⁵ and R ⁶ are 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 can be different. As carbon number of a hydrocarbon group, 1-20, for example, Preferably, 1-14 can be mentioned.
In the general formula (1), X is an n-valent anion, and n is 1 or 2. Specific examples of the anion include nitrate ion, halogen ion, phosphate ion, sulfate ion, and sulfonate ion. Of these, nitrate ions are particularly preferred.
[0013]
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-diphenyl-9,9′-bisacridinium salt, N, N′-di-m-chlorophenyl-9,9′-bisacridinium salt, etc. The above anions are preferable as the counter ion, and N, N′-dimethyl-9,9′-bisacridinium dinitrate (lucigenin), in which the counter ion is a nitrate ion, is particularly preferable. .
[0014]
The N, N-disubstituted carboxylic acid amide compound which is another component used for the production of the chemiluminescent reagent of the present invention includes N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylacrylamide. , N, N-dimethylpropionamide, N, N-dimethylbenzamide, N-methyl-2-pyrrolidone and the like, but N, N-dimethylformamide, N, N-dimethylacetamide, N -Methyl-2-pyrrolidone and the like are preferred.
[0015]
Among these N, N-disubstituted carboxylic acid amide compounds, those which are liquid at room temperature can also be used in a large excess as a solvent, or other suitable solvents may be used. In addition, a solid material at room temperature may be used after being melted by heating, or an appropriate solvent may be used. Examples of such a solvent include dimethyl sulfoxide and hexamethylphosphoamide.
[0016]
In the production of the chemiluminescent reagent of the present invention, the contact time is obtained by bringing an N, N′-disubstituted-9,9′-bisacridinium salt into contact with an N, N-disubstituted carboxylic acid amide compound. As a result, it is observed that N, N-disubstituted carboxylic acid amide compound is N, N′-disubstituted-9,9′-bisacrylic acid. It is considered that after reacting with dinium salts to form an activated state, it is inactivated by changing over time.
[0017]
Accordingly, the production of the chemiluminescent reagent of the present invention is basically by contact treatment of N, N′-disubstituted-9,9′-bisacridinium salts with N, N-disubstituted carboxylic acid amide compounds. It is carried out by mixing and maintaining both under the reaction conditions described below. Specifically, when chemiluminescence analysis is performed using the chemiluminescent reagent of the present invention, a method is used in which each component of these reagents is prepared and reacted for a predetermined time.
[0018]
As reaction conditions, although reaction temperature changes with kinds of the reagent and solvent to be used, it is generally -10- + 200 degreeC, Preferably it is 0-120 degreeC, Most preferably, it is the temperature of the range of 20-60 degreeC. The reaction time may be 1 minute to 1 day, preferably 5 minutes to 12 hours, particularly preferably 10 minutes to 5 hours. In addition, the ratio of each component of the chemiluminescent reagent of the present invention is equimolar to the N, N-disubstituted carboxylic acid amide compound per mole of N, N′-disubstituted-9,9′-bisacridinium salts. To a large excess.
[0019]
The chemiluminescent reagent of the present invention 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 luminescence is enhanced by a luminescence 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-promophenol, p-chlorophenol, 6-hydroxybenzothiazole, 2-naphthol, firefly luciferin and the like can be mentioned without limitation.
The hydrogen acceptor is not particularly limited as long as it can be a substrate for peroxidase, and organic peroxides, inorganic peroxides and the like can be arbitrarily used. preferable.
[0020]
The chemiluminescent reagent of the present invention is used at a concentration in the range of 10 ^{−6 to 1} M, preferably 10 ⁻⁴ to 10 ⁻² M, and the amount used is in the range of 10 to 500 μl, preferably in the range of 50 to 300 μl. is there. The amount of the luminescence enhancer 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 1} M, preferably 10 ⁻⁴ to 10. ^It is desirable to use within the range of ^-2 M. In addition, the amount of hydrogen acceptor such as hydrogen peroxide used for the chemiluminescent reaction must be used in an excessive amount with respect to the chemiluminescent reagent. The range of 10,000 times mole, preferably 10 to 1000 times mole is desirable. 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.
[0021]
Examples of the buffer used for the chemiluminescence reaction include Tris buffer, phosphate buffer, borate buffer, carbonate buffer, glycine-sodium hydroxide buffer, and the like. The concentration of these buffer solutions is preferably used in the range of 1 mM to 1M. In addition, additives such as a surfactant and a chelating agent can be arbitrarily used during the reaction.
[0022]
According to the present invention, N, N′-disubstituted-9,9′-bisacridinium salts represented by the general formula (1) each packed in a separate container as a reagent kit for measuring peroxidase activity A reagent kit for chemiluminescence analysis comprising an N, N-disubstituted carboxylic acid amide compound and peroxidase or a peroxidase-labeled substance can also be provided.
[0023]
As described above, the chemiluminescent reagent of the present invention reacts in the presence of hydrogen peroxide and peroxidase under basic conditions, and emits light. This reaction is used for detection of hydrogen peroxide, quantification of peroxidase, and the like. In addition, by using peroxidase as a labeling substance, it can be used for quantification of various substances.
[0024]
【Example】
Hereinafter, the present invention will be specifically described with reference to Examples and Comparative Examples. However, the present invention is not limited to the examples.
[0025]
[Example 1]
Take 1 mg of lucigenin in a test tube, add 2 ml of N, N-dimethylformamide to dissolve it, let stand at room temperature for 90 minutes, and then add 2 ml of pure water and mix to mix lucigenin and N, N- A chemiluminescent reagent consisting of a complex with dimethylformamide was obtained.
[0026]
Measurement of peroxidase activity The chemiluminescence reagent obtained as described above was immediately added to each of a plurality of chemiluminescence measurement wells in an amount of 20 μl, and the wells were then charged with various concentrations of horseradish peroxidase (HRP). After adding 200 μl of 0.1 M Tris-HCl buffer solution (pH 8.4) and 20 μl of p-iodophenol 10 mM Tris-HCl buffer solution (pH 8.4), 50 μl of 0.0034 wt% aqueous hydrogen peroxide solution was added thereto. As a result of integrating the luminescence amount for 0 to 5 seconds using a luminometer (Diatron Luminous CT-9000D), the luminescence intensity shown in Table 1 is obtained, and it is recognized that the peroxidase activity can be measured with high concentration dependency. It was.
[0027]
[Table 1]

[Example 2]
Take 1 mg of lucigenin in a test tube, add 2 ml of N, N-dimethylacetamide and dissolve it, let stand at room temperature for 3 hours, add 2 ml of pure water and mix to make lucigenin and N, N- A chemiluminescent reagent composed of a complex with dimethylacetamide was obtained.
[0028]
Measurement of peroxidase activity Immediately take 20 [ mu] l of this chemiluminescent reagent solution and add it to a plurality of chemiluminescent assay wells, and then add 0.1 M Tris-HCl at different concentration levels of horseradish peroxidase to each well. After adding 200 μl of buffer solution (pH 8.4) and 20 μl of p-iodophenol 10 mM Tris-HCl buffer solution (pH 8.4), 50 μl of 0.0034% by weight aqueous hydrogen peroxide solution was added thereto, and a luminometer (diameter) was added. As a result of integrating the amount of luminescence for 0 to 5 seconds with Yatron Luminous CT-9000D), it was confirmed that the luminescence intensity as shown in Table 2 was obtained and the peroxidase activity could be measured with high concentration dependency.
[0029]
[Table 2]

[Example 3]
Take 1 mg of lucigenin in a test tube, add 2 ml of N, N-methyl-2-pyrrolidone to dissolve it, let stand at room temperature for 3 hours, add 2 ml of pure water and mix to mix lucigenin and N A chemiluminescent reagent composed of a complex with -methyl-2-pyrrolidone was obtained.
[0030]
Measurement of peroxidase activity Immediately take 20 [ mu] l of this chemiluminescent reagent solution and add it to a plurality of chemiluminescent assay wells, and then add 0.1 M Tris-HCl at different concentration levels of horseradish peroxidase to each well. Add 200 μl of buffer solution (pH 8.4) and 20 μl of p-iodophenol 10 mM Tris-HCl buffer solution (pH 8.4), and add 50 μl of 0.0034 wt% aqueous hydrogen peroxide solution to the luminometer (diameter). As a result of integrating the luminescence amount for 0 to 5 seconds with Yatron Luminous CT-9000D), it was confirmed that the luminescence intensity as shown in Table 3 was obtained and the peroxidase activity could be measured with high concentration dependency.
[0031]
[Table 3]

[Comparative Example 1]
1 mg of lucigenin is dissolved in 2 ml of pure water, 20 μl of this is added to a plurality of chemiluminescence wells, and then 0.1 M Tris-HCl buffer (pH 8. 4) Add 200 μl of solution and 20 μl of p-iodophenol 10 mM Tris-HCl buffer (pH 8.4) solution, and add 50 μl of 0.0034% by weight aqueous hydrogen peroxide solution, and add a luminometer (Luminus 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 as shown in Table 4 was obtained, and it was confirmed that the peroxidase activity could not be measured accurately.
[0032]
[Table 4]

[0033]
【The invention's effect】
The chemiluminescent reagent provided by the present invention has high sensitivity to peroxidase enzyme by utilizing the property of chemiluminescence depending on the concentration of peroxidase in the presence of hydrogen acceptor such as hydrogen peroxide and peroxidase. Can be detected.
Furthermore, using enzyme-labeled products labeled with antigens, antibodies, nucleic acids, etc. with peroxidase as a labeling substance, antibodies, antigens, etc. are analyzed by enzyme immunoassay, proteins are detected by Western blot, DNA and RNA are detected by Southern and Northern blots. In addition, each of the nucleic acids can be measured specifically and with high sensitivity using an enzyme-labeled nucleic acid probe.

Claims (7)

The following general formula (1)

(In the above 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, and R ³ , R ⁴ , R ⁵ and R ⁶ is 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 may be the same or different from each other, X is an n-valent anion, and n is 1 or And a complex of an N, N′-disubstituted-9,9′-bisacridinium salt represented by (2)) and an N, N-disubstituted carboxylic acid amide compound. Luminescent reagent. The chemiluminescence according to claim 1, wherein the N, N'-disubstituted-9,9'-bisacridinium salt is N, N'-dimethyl-9,9'-bisacridinium dinitrate. reagent. The N, N-disubstituted carboxylic acid amide compound is a compound selected from the group consisting of N, N-dimethylformamide, N, N-dimethylacetamide and N-methyl-2-pyrrolidone. The chemiluminescent reagent as described. The following general formula (1)

(In the above 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, and R ³ , R ⁴ , R ⁵ and R ⁶ is 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 may be the same or different from each other, X is an n-valent anion, and n is 1 or N, N′-disubstituted-9,9′-bisacridinium salts represented by formula (2) are contacted with an N, N-disubstituted carboxylic acid amide compound in the presence or absence of a solvent. A method for producing a chemiluminescent reagent. The chemiluminescence according to claim 4, wherein the N, N'-disubstituted-9,9'-bisacridinium salt is N, N'-dimethyl-9,9'-bisacridinium dinitrate. A method for producing a reagent. 6. The compound according to claim 4 or 5, wherein the N, N-disubstituted carboxylic acid amide compound is a compound selected from the group consisting of N, N-dimethylformamide, N, N-dimethylacetamide and N-methyl-2-pyrrolidone. The manufacturing method of the chemiluminescent reagent of description. 7. The mixing ratio of the N, N′-disubstituted carboxylic acid amide compound is an equimolar amount or more with respect to 1 mol of the N, N′-disubstituted-9,9′-bisacridinium salt. The method for producing a chemiluminescent reagent according to any one of the above.