JPH0799996A - Reagent for measuring peroxidase activity and measuring method using the same - Google Patents
Reagent for measuring peroxidase activity and measuring method using the sameInfo
- Publication number
- JPH0799996A JPH0799996A JP26781393A JP26781393A JPH0799996A JP H0799996 A JPH0799996 A JP H0799996A JP 26781393 A JP26781393 A JP 26781393A JP 26781393 A JP26781393 A JP 26781393A JP H0799996 A JPH0799996 A JP H0799996A
- Authority
- JP
- Japan
- Prior art keywords
- activity
- general formula
- peroxidase
- compound represented
- measuring
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102000003992 Peroxidases Human genes 0.000 title claims abstract description 29
- 108040007629 peroxidase activity proteins Proteins 0.000 title claims abstract description 29
- 230000000694 effects Effects 0.000 title claims abstract description 14
- 238000000034 method Methods 0.000 title claims abstract description 14
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 6
- -1 phenothiazine compound Chemical class 0.000 claims abstract description 29
- 239000000126 substance Substances 0.000 claims abstract description 24
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000000758 substrate Substances 0.000 claims abstract description 18
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 15
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims abstract description 15
- JUJWROOIHBZHMG-UHFFFAOYSA-N pyridine Substances C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims abstract description 15
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229950000688 phenothiazine Drugs 0.000 claims abstract description 12
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims abstract description 10
- 125000003277 amino group Chemical group 0.000 claims abstract description 9
- 229910052783 alkali metal Inorganic materials 0.000 claims abstract description 6
- 150000001340 alkali metals Chemical class 0.000 claims abstract description 6
- 229910052784 alkaline earth metal Inorganic materials 0.000 claims abstract description 6
- 150000001342 alkaline earth metals Chemical class 0.000 claims abstract description 6
- 125000005843 halogen group Chemical group 0.000 claims abstract description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 5
- 150000001875 compounds Chemical class 0.000 claims 1
- 238000005259 measurement Methods 0.000 abstract description 12
- 238000000691 measurement method Methods 0.000 abstract description 4
- 239000007853 buffer solution Substances 0.000 description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 238000004020 luminiscence type Methods 0.000 description 11
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 10
- 230000003321 amplification Effects 0.000 description 10
- 238000003199 nucleic acid amplification method Methods 0.000 description 10
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 7
- 229910052708 sodium Inorganic materials 0.000 description 7
- 239000011734 sodium Substances 0.000 description 7
- 239000000872 buffer Substances 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 4
- VSMDINRNYYEDRN-UHFFFAOYSA-N 4-iodophenol Chemical compound OC1=CC=C(I)C=C1 VSMDINRNYYEDRN-UHFFFAOYSA-N 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- BSKHPKMHTQYZBB-UHFFFAOYSA-N 2-methylpyridine Chemical compound CC1=CC=CC=N1 BSKHPKMHTQYZBB-UHFFFAOYSA-N 0.000 description 2
- ITQTTZVARXURQS-UHFFFAOYSA-N 3-methylpyridine Chemical compound CC1=CC=CN=C1 ITQTTZVARXURQS-UHFFFAOYSA-N 0.000 description 2
- FKNQCJSGGFJEIZ-UHFFFAOYSA-N 4-methylpyridine Chemical compound CC1=CC=NC=C1 FKNQCJSGGFJEIZ-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- RSAZYXZUJROYKR-UHFFFAOYSA-N indophenol Chemical class C1=CC(O)=CC=C1N=C1C=CC(=O)C=C1 RSAZYXZUJROYKR-UHFFFAOYSA-N 0.000 description 2
- 125000000587 piperidin-1-yl group Chemical group [H]C1([H])N(*)C([H])([H])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- UBQKCCHYAOITMY-UHFFFAOYSA-N pyridin-2-ol Chemical compound OC1=CC=CC=N1 UBQKCCHYAOITMY-UHFFFAOYSA-N 0.000 description 2
- 125000004214 1-pyrrolidinyl group Chemical group [H]C1([H])N(*)C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- CFJZQNZZGQDONE-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;butanedioic acid Chemical compound OCC(N)(CO)CO.OCC(N)(CO)CO.OC(=O)CCC(O)=O CFJZQNZZGQDONE-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- ONVGBCNKVCPJCI-UHFFFAOYSA-N 3-(10h-phenothiazin-10-ium-10-yl)propane-1-sulfonate Chemical compound C1=CC=C2N(CCCS(=O)(=O)O)C3=CC=CC=C3SC2=C1 ONVGBCNKVCPJCI-UHFFFAOYSA-N 0.000 description 1
- ICBUOQBBEUSCAJ-UHFFFAOYSA-N 4-(2-piperidin-1-ylethyl)pyridine Chemical compound C1CCCCN1CCC1=CC=NC=C1 ICBUOQBBEUSCAJ-UHFFFAOYSA-N 0.000 description 1
- NUKYPUAOHBNCPY-UHFFFAOYSA-N 4-aminopyridine Chemical compound NC1=CC=NC=C1 NUKYPUAOHBNCPY-UHFFFAOYSA-N 0.000 description 1
- PVMNPAUTCMBOMO-UHFFFAOYSA-N 4-chloropyridine Chemical compound ClC1=CC=NC=C1 PVMNPAUTCMBOMO-UHFFFAOYSA-N 0.000 description 1
- GCNTZFIIOFTKIY-UHFFFAOYSA-N 4-hydroxypyridine Chemical compound OC1=CC=NC=C1 GCNTZFIIOFTKIY-UHFFFAOYSA-N 0.000 description 1
- MTPBUCCXRGSDCR-UHFFFAOYSA-N 4-piperidin-1-ylpyridine Chemical compound C1CCCCN1C1=CC=NC=C1 MTPBUCCXRGSDCR-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- HOPRXXXSABQWAV-UHFFFAOYSA-N anhydrous collidine Natural products CC1=CC=NC(C)=C1C HOPRXXXSABQWAV-UHFFFAOYSA-N 0.000 description 1
- 125000002490 anilino group Chemical group [H]N(*)C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 229910052788 barium Inorganic materials 0.000 description 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- UTBIMNXEDGNJFE-UHFFFAOYSA-N collidine Natural products CC1=CC=C(C)C(C)=N1 UTBIMNXEDGNJFE-UHFFFAOYSA-N 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 125000001664 diethylamino group Chemical group [H]C([H])([H])C([H])([H])N(*)C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000004914 dipropylamino group Chemical group C(CC)N(CCC)* 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 125000000031 ethylamino group Chemical group [H]C([H])([H])C([H])([H])N([H])[*] 0.000 description 1
- 229960004979 fampridine Drugs 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 125000000250 methylamino group Chemical group [H]N(*)C([H])([H])[H] 0.000 description 1
- ODKLEQPZOCJQMT-UHFFFAOYSA-N n,n-diethylpyridin-4-amine Chemical compound CCN(CC)C1=CC=NC=C1 ODKLEQPZOCJQMT-UHFFFAOYSA-N 0.000 description 1
- LSCYTCMNCWMCQE-UHFFFAOYSA-N n-methylpyridin-4-amine Chemical compound CNC1=CC=NC=C1 LSCYTCMNCWMCQE-UHFFFAOYSA-N 0.000 description 1
- 150000002990 phenothiazines Chemical class 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000008362 succinate buffer Substances 0.000 description 1
- GFYHSKONPJXCDE-UHFFFAOYSA-N sym-collidine Natural products CC1=CN=C(C)C(C)=C1 GFYHSKONPJXCDE-UHFFFAOYSA-N 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
(57)【要約】
【構成】 （イ）ルミノールと過酸化水素とからなる基
質と、（ロ）一般式
【化１】
（式中、Ｒ1 及びＲ2 は水素原子又は低級アルキル基、
Ｒ3 は水素原子、ハロゲン原子、低級アルキル基、水酸
基又はアミノ基である。）で表されるピリジン化合物と
一般式
【化２】
（式中、Ｘは水素原子、アルカリ金属又はアルカリ土類
金属である。）で表されるフェノチアジン化合物とから
なる活性増幅物質と、からなるパーオキシダーゼ活性測
定用試薬、及びパーオキシダーゼと、ルミノールと過酸
化水素とからなる基質と、前記一般式（Ｉ）で表される
ピリジン化合物と前記一般式（II）で表されるフェノチ
アジン化合物とからなる活性増幅物質とにより発生する
化学発光を測定することからなるパーオキシダーゼ活性
の測定方法である。
【効果】 本発明の活性増幅物質を用いることにより、
今まで測定することができなかった極微量のパーオキシ
ダーゼの活性を測定することができる。さらに本発明を
ＥＩＡに用いると従来の測定法に比べ高感度な測定が可
能である。(57) [Summary] [Structure] (a) A substrate composed of luminol and hydrogen peroxide, and (b) a general formula: (In the formula, R 1 and R 2 are a hydrogen atom or a lower alkyl group,
R 3 is a hydrogen atom, a halogen atom, a lower alkyl group, a hydroxyl group or an amino group. ) And a general formula: (In the formula, X is a hydrogen atom, an alkali metal or an alkaline earth metal.) An activity-amplifying substance comprising a phenothiazine compound represented by: and a peroxidase activity-measuring reagent comprising: a peroxidase; Measuring chemiluminescence generated by a substrate composed of hydrogen peroxide, and an activity-amplifying substance composed of the pyridine compound represented by the general formula (I) and the phenothiazine compound represented by the general formula (II). Is a method for measuring peroxidase activity. [Effect] By using the activity-amplifying substance of the present invention,
It is possible to measure the activity of a very small amount of peroxidase, which could not be measured until now. Furthermore, when the present invention is applied to EIA, highly sensitive measurement is possible as compared with the conventional measurement method.
Description
【０００１】[0001]
【産業上の利用分野】本発明は、パーオキシダーゼ活性
測定用試薬及びその試薬を用いた測定方法に関する。更
に詳しくは、（イ）ルミノールと過酸化水素とからなる
基質と、（ロ）一般式BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent for measuring peroxidase activity and a measuring method using the reagent. More specifically, (a) a substrate composed of luminol and hydrogen peroxide, and (b) a general formula
【化５】 （式中、Ｒ1 及びＲ2 は水素原子又は低級アルキル基で
あり、Ｒ3 は水素原子、ハロゲン原子、低級アルキル
基、水酸基又はアミノ基である。）で表されるピリジン
化合物と一般式[Chemical 5] (In the formula, R 1 and R 2 are a hydrogen atom or a lower alkyl group, and R 3 is a hydrogen atom, a halogen atom, a lower alkyl group, a hydroxyl group or an amino group.) And a general formula.
【化６】 （式中、Ｘは水素原子、アルカリ金属又はアルカリ土類
金属である。）で表されるフェノチアジン化合物とから
なる活性増幅物質と、からなるパーオキシダーゼ活性測
定用試薬、及びパーオキシダーゼと、ルミノールと過酸
化水素とからなる基質と、前記一般式（Ｉ）で表される
ピリジン化合物と前記一般式（II）で表されるフェノチ
アジン化合物とからなる活性増幅物質とにより発生する
化学発光を測定することからなるパーオキシダーゼ活性
の測定方法である。[Chemical 6] (In the formula, X is a hydrogen atom, an alkali metal or an alkaline earth metal.) An activity-amplifying substance comprising a phenothiazine compound represented by: and a peroxidase activity-measuring reagent comprising: a peroxidase; Measuring chemiluminescence generated by a substrate composed of hydrogen peroxide, and an activity-amplifying substance composed of the pyridine compound represented by the general formula (I) and the phenothiazine compound represented by the general formula (II). Is a method for measuring peroxidase activity.
【０００２】[0002]
【従来の技術】酵素免疫測定法（ＥＩＡ）は、高感度な
測定法として技術開発が行われている。中でも化学発光
基質を用いる測定法は、高感度化の達成のために有力な
手段であり、例えばルミノールを用いた化学発光法では
比色法の１０-14 ｍｏｌに比べパーオキシダーゼの検出
感度が１０-16 ｍｏｌであるとの報告がなされている
（蛋白質核酸酵素、別冊Ｎｏ．３１，ｐ５１〜６３，１
９８７年参照）。しかし化学発光法は、発光時間が短い
などの欠点を有しているため汎用されていなかった。そ
こでこの欠点を補う試みがなされ、ｐ−ヨードフェノー
ルを活性増幅物質として用いた化学発光法（Ｈ．Ａｌｌ
ａｎ，Ｊ．Ｍｉｃｒｏｂｉｏｌｏｇｙ Ａｐｒ．，６３
０〜６３６，３５，（１９８８）参照）、あるいはイン
ドフェノール誘導体とフェノチアジン誘導体とを組み合
せて活性増幅物質として用いたパーオキシダーゼ活性の
化学発光測定法（特開平２−１７４６９４号参照）等が
見い出された。2. Description of the Related Art The enzyme immunoassay (EIA) has been developed as a highly sensitive assay. Among them, the measurement method using a chemiluminescent substrate is a powerful means for achieving high sensitivity. For example, the chemiluminescence method using luminol has a detection sensitivity of peroxidase of 10 as compared with 10 −14 mol of the colorimetric method. -16 mol has been reported (Protein Nucleic Acid Enzyme, separate volume No. 31, p51-63, 1)
987). However, the chemiluminescence method has not been widely used because it has drawbacks such as a short emission time. Therefore, attempts have been made to compensate for this drawback, and a chemiluminescence method using p-iodophenol as an activity-amplifying substance (H. All.
an, J. Microbiology Appr. , 63
0-636, 35, (1988)), or a chemiluminescence assay for peroxidase activity using an indophenol derivative and a phenothiazine derivative in combination as an activity-amplifying substance (see JP-A-2-174694). It was
【０００３】[0003]
【発明が解決しようとする課題】しかしながら、これら
の活性増幅物質を用いる化学発光法は、持続した発光が
得られ、ある程度の高感度化が達成されたものの、微量
成分の測定には更なる感度が要求されているのが現状で
ある。そして有効な測定方法の出現が久しく期待されて
いた。However, although the chemiluminescence method using these activity-amplifying substances achieves continuous luminescence and achieves a certain degree of high sensitivity, it is more sensitive to the measurement of trace components. Is currently required. And the appearance of an effective measurement method was expected for a long time.
【０００４】[0004]
【課題を解決するための手段】本発明者は、更なる高感
度を求めて鋭意研究した結果、化学発光を利用しパーオ
キシダーゼ活性を測定するためルミノールと過酸化水素
とを基質として用いる方法において、前記一般式（Ｉ）
で表されるピリジン化合物と前記一般式（II）で表され
るフェノチアジン化合物とがパーオキシダーゼの活性増
幅物質となることを見い出し、本発明を完成した。Means for Solving the Problems As a result of earnest research for further high sensitivity, the present inventor has found that in a method using luminol and hydrogen peroxide as substrates for measuring peroxidase activity by utilizing chemiluminescence. , The general formula (I)
The present invention was completed by discovering that the pyridine compound represented by and the phenothiazine compound represented by the general formula (II) serve as activity-amplifying substances for peroxidase.
【０００５】前記一般式（Ｉ）で表されるピリジン化合
物において、Ｒ1 及びＲ2 は水素原子又は低級アルキル
基であり、この低級アルキル基としては炭素数１〜４の
低級アルキル基であり、例えばメチル基、エチル基、プ
ロピル基、イソプロピル基、ブチル基、ｓｅｃ−ブチル
基、ｔ−ブチル基等を挙げることができる。In the pyridine compound represented by the general formula (I), R 1 and R 2 are a hydrogen atom or a lower alkyl group, and the lower alkyl group is a lower alkyl group having 1 to 4 carbon atoms, Examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a sec-butyl group, and a t-butyl group.
【０００６】Ｒ3 は水素原子、ハロゲン原子、低級アル
キル基、水酸基又はアミノ基である。ハロゲン原子とし
ては、例えば塩素、臭素、ヨウ素等を挙げることができ
る。低級アルキル基としては、前記Ｒ1 及びＲ2 と同じ
炭素数１〜４の低級アルキル基の他、アミノ基、ジメチ
ルアミノ基、ピペリジノ基等で置換されたアルキル基を
挙げることができる。またアミノ基としては、無置換の
アミノ基の他、メチルアミノ基、エチルアミノ基、ジメ
チルアミノ基、ジエチルアミノ基、ジプロピルアミノ
基、１−ピロリジニル基、ピペリジノ基、４−メチルピ
ペリジノ基、４−エチルピペリジノ基、フェニルアミノ
基等の置換アミノ基を挙げることができる。R 3 is a hydrogen atom, a halogen atom, a lower alkyl group, a hydroxyl group or an amino group. Examples of the halogen atom include chlorine, bromine, iodine and the like. Examples of the lower alkyl group include lower alkyl groups having 1 to 4 carbon atoms, which are the same as R 1 and R 2, and an alkyl group substituted with an amino group, a dimethylamino group, a piperidino group or the like. As the amino group, in addition to an unsubstituted amino group, a methylamino group, an ethylamino group, a dimethylamino group, a diethylamino group, a dipropylamino group, a 1-pyrrolidinyl group, a piperidino group, a 4-methylpiperidino group, and a 4-ethylpiperidino group. And substituted amino groups such as phenylamino group.
【０００７】前記一般式（Ｉ）で表されるピリジン化合
物としては、例えばピリジン、２−メチルピリジン、３
−メチルピリジン、４−メチルピリジン、４−ヒドロキ
シピリジン、２−ヒドロキシピリジン、４−クロロピリ
ジン、４−アミノピリジン、４−ジメチルアミノピリジ
ン、４−ジエチルアミノピリジン、４−ピペリジノピリ
ジン、４−（２−ピペリジノエチル）ピリジン、４−
（Ｎ−メチルアミノ）ピリジン、コリジン等を挙げるこ
とができる。Examples of the pyridine compound represented by the general formula (I) include pyridine, 2-methylpyridine, and 3
-Methylpyridine, 4-methylpyridine, 4-hydroxypyridine, 2-hydroxypyridine, 4-chloropyridine, 4-aminopyridine, 4-dimethylaminopyridine, 4-diethylaminopyridine, 4-piperidinopyridine, 4- ( 2-piperidinoethyl) pyridine, 4-
(N-methylamino) pyridine, collidine, etc. can be mentioned.
【０００８】前記一般式（II）で表されるフェノチアジ
ン化合物において、Ｘは水素原子、アルカリ金属又はア
ルカリ土類金属である。アルカリ金属としては、例えば
ナトリウム、カリウム等を、さらにアルカリ土類金属と
してはカルシウム、バリウム等を挙げることができる。In the phenothiazine compound represented by the general formula (II), X is a hydrogen atom, an alkali metal or an alkaline earth metal. Examples of the alkali metal include sodium and potassium, and examples of the alkaline earth metal include calcium and barium.
【０００９】前記一般式（II）で表されるフェノチアジ
ン化合物としては、例えば３−（１０−フェノチアジニ
ル）プロパンスルホン酸、３−（１０−フェノチアジニ
ル）プロパンスルホン酸ナトリウム、３−（１０−フェ
ノチアジニル）プロパンスルホン酸カリウム等を挙げる
ことができる。Examples of the phenothiazine compound represented by the general formula (II) include 3- (10-phenothiazinyl) propanesulfonic acid, sodium 3- (10-phenothiazinyl) propanesulfonate and 3- (10-pheno). Examples thereof include potassium thiazinyl) propane sulfonate.
【００１０】本発明の測定は、パーオキシダーゼを含む
被検液と、ルミノールと過酸化水素とからなるパーオキ
シダーゼの基質と、前記一般式（Ｉ）で表されるピリジ
ン化合物及び一般式（II）で表されるフェノチアジン化
合物とからなる活性増幅物質とにより発生する化学発光
を測定することにより行うことができる。In the measurement of the present invention, a test solution containing peroxidase, a substrate for peroxidase consisting of luminol and hydrogen peroxide, a pyridine compound represented by the general formula (I) and a general formula (II) are used. The activity can be measured by measuring the chemiluminescence generated by the phenothiazine compound represented by
【００１１】測定を実施するに当って、前記一般式
（Ｉ）で表されるピリジン化合物及び前記一般式（II）
で表されるフェノチアジン化合物は、それぞれ１０〜５
０００μＭ用いることができるが、反応を効率よく行う
ためには１００〜１０００μＭ用いることが好ましい。
測定に用いる基質は、ルミノールを１００〜１０００μ
Ｍ、好ましくは２００〜４００μＭ用い、さらに過酸化
水素を１００〜１０００μＭ、好ましくは２００〜５０
０μＭ用いることが経済的にも有利である。In carrying out the measurement, the pyridine compound represented by the general formula (I) and the general formula (II)
The phenothiazine compound represented by
Although 000 μM can be used, it is preferable to use 100 to 1000 μM for efficient reaction.
The substrate used for measurement is luminol 100-1000 μm.
M, preferably 200 to 400 μM, and further hydrogen peroxide to 100 to 1000 μM, preferably 200 to 50
It is economically advantageous to use 0 μM.
【００１２】測定は、緩衝液中で行うことが好ましく、
例えばトリシン−水酸化ナトリウム緩衝液、トリス−塩
酸緩衝液、トリス−コハク酸緩衝液、リン酸緩衝液等を
挙げることができる。緩衝液は、ｐＨが７．０〜９．０
のものを用いることができる。測定は、１０〜４０℃で
行うことができるが、効率よく行うためには２０〜３０
℃で実施することが好ましい。The measurement is preferably carried out in a buffer solution,
Examples thereof include tricine-sodium hydroxide buffer solution, Tris-hydrochloric acid buffer solution, Tris-succinate buffer solution, and phosphate buffer solution. The buffer solution has a pH of 7.0 to 9.0.
Can be used. The measurement can be performed at 10 to 40 ° C, but 20 to 30 is required for efficient measurement.
It is preferably carried out at ° C.
【００１３】一般に化学発光の測定は、パーオキシダー
ゼを含む被検液と共に前記一般式（Ｉ）で表されるピリ
ジン化合物、前記一般式（II）で表されるフェノチアジ
ン化合物および前記基質を含む試験液を入れた試験管を
フォトンカウンターの中に入れ測定を行なうことができ
る。In general, chemiluminescence is measured by a test solution containing a pyridine compound represented by the general formula (I), a phenothiazine compound represented by the general formula (II) and the substrate together with a test solution containing peroxidase. The test tube containing the can be placed in a photon counter for measurement.
【００１４】また本発明の方法をＥＩＡに用いる場合に
は、通常測定に用いられるポリスチレンビーズ、磁性粒
子、容器の壁等の固相上にサンドイッチ法、競合法等の
免疫測定法により補足されたパーオキシダーゼ標識体の
活性を上記方法に従い測定することにより達成される。When the method of the present invention is used for EIA, it is supplemented by an immunoassay method such as a sandwich method or a competitive method on a solid phase such as polystyrene beads, magnetic particles, and the wall of a container which are usually used for measurement. It is achieved by measuring the activity of the peroxidase-labeled product according to the above method.
【００１５】[0015]
【実施例】本発明を以下参考例及び実施例により更に詳
細に説明する。The present invention will be described in more detail with reference to Reference Examples and Examples.
【００１６】参考例１ １．３４ｍＭ３−（１０−フェノチアジニル）プロパン
スルホン酸ナトリウムを含むトリシン−水酸化ナトリウ
ム増幅物質緩衝液ｐＨ＝８．３（５０ｍＭ）、及び１．
０ｍＭ過酸化水素と０．７５ｍＭルミノールとを含むト
リシン−水酸化ナトリウム基質緩衝液ｐＨ＝８．３（５
０ｍＭ）をそれぞれ作成した。Reference Example 1 Tricine-sodium hydroxide amplification material buffer solution containing 1.34 mM sodium 3- (10-phenothiazinyl) propanesulfonate, pH = 8.3 (50 mM), and 1.
Tricine-sodium hydroxide substrate buffer containing 0 mM hydrogen peroxide and 0.75 mM luminol pH = 8.3 (5
0 mM) was prepared.
【００１７】参考例２ 参考例１で作成した増幅物質緩衝液２００μｌにパーオ
キシダーゼ（１ｎｇ／ｍｌ）１０μｌを加え、更に参考
例１で作成した基質緩衝液１３５μｌを加え、５秒間の
発光を添加直後（０分）から２分おきに１０分までクリ
ニルーマット（ベルトールド社製フォトンカウンター）
で測定した。その結果を図１に示す。Reference Example 2 To 200 μl of the amplification substance buffer solution prepared in Reference Example 1, 10 μl of peroxidase (1 ng / ml) was added, and further 135 μl of the substrate buffer solution prepared in Reference Example 1 was added and immediately after addition of luminescence for 5 seconds. From (0 minutes) to every 2 minutes for 10 minutes Clinil mat (Berthold photon counter)
It was measured at. The result is shown in FIG.
【００１８】実施例１ １．３４ｍＭ３−（１０−フェノチアジニル）プロパン
スルホン酸ナトリウムを含むトリシン−水酸化ナトリウ
ム増幅物質緩衝液に表１に示すピリジン化合物１．０ｍ
Ｍを加え増幅物質緩衝液を作成した。この増幅物質緩衝
液２００μｌにパーオキシダーゼ（１ｎｇ／ｍｌ）１０
μｌを加え、更に参考例１で作成した基質緩衝液１３５
μｌを加え、６分後クリニルーマットで５秒間の発光を
測定した。ピリジン化合物を添加しない増幅物質緩衝液
を用いて測定を行った結果を基準としてその相対活性を
求めた。その結果を表１に示す。Example 1 Tricine-sodium hydroxide amplifying material buffer containing 1.34 mM sodium 3- (10-phenothiazinyl) propane sulfonate 1.0 m pyridine compound shown in Table 1
M was added to prepare an amplification substance buffer. 200 μl of this amplification substance buffer was added to 10 parts of peroxidase (1 ng / ml).
μl was added, and the substrate buffer solution 135 prepared in Reference Example 1 was further added.
μl was added, and after 6 minutes, luminescence was measured for 5 seconds using a Clinylmat. The relative activity was determined on the basis of the result of the measurement using the amplification substance buffer solution to which the pyridine compound was not added. The results are shown in Table 1.
【００１９】[0019]
【表１】 [Table 1]
【００２０】実施例２ 実施例１で作成した４−ジメチルアミノピリジンを用い
た増幅物質緩衝液２００μｌにパーオキシダーゼ（１ｎ
ｇ／ｍｌ）１０μｌを加え、更に参考例１で作成した基
質緩衝液１３５μｌを加え、５秒間の発光を添加直後
（０分）から２分おきに１０分までクリニルーマットで
測定した。その結果を図２に示す。Example 2 200 μl of an amplification substance buffer solution containing 4-dimethylaminopyridine prepared in Example 1 was added to peroxidase (1 n
(g / ml), and then the substrate buffer solution (135 μl) prepared in Reference Example 1 was added, and luminescence for 5 seconds was measured immediately after addition (0 minutes) and every 2 minutes for 10 minutes with a Clinylmat. The result is shown in FIG.
【００２１】参考例３ ８．０ｍＭｐ−ヨードフェノール、１．０ｍＭルミノー
ル、１．０ｍＭ過酸化水素を含むトリシン−水酸化ナト
リウム緩衝液２００μｌを４本の試験管に入れ、パーオ
キシダーゼ（０ｎｇ／ｍｌ，１ｎｇ／ｍｌ，０．１ｎｇ
／ｍｌ，０．０１ｎｇ／ｍｌ）をそれぞれ１０μｌづつ
加え、更に参考例１で作成した基質緩衝液１３５μｌを
加え６分後の５秒間の発光をクリニルーマットで測定し
た。その結果を図３に示す。Reference Example 3 200 μl of a tricine-sodium hydroxide buffer solution containing 8.0 mM p-iodophenol, 1.0 mM luminol and 1.0 mM hydrogen peroxide was placed in four test tubes, and peroxidase (0 ng / ml, 1 ng / ml, 0.1 ng
/ Ml, 0.01 ng / ml) was added in an amount of 10 μl each, and further 135 μl of the substrate buffer solution prepared in Reference Example 1 was added, and the light emission for 5 seconds after 6 minutes was measured by a Clinylmat. The result is shown in FIG.
【００２２】実施例３ 実施例１で作成した３−（１０−フェノチアジニル）プ
ロパンスルホン酸ナトリウム及び４−ジメチルアミノピ
リジンを含む増幅物質緩衝液２００μｌを４本の試験管
に入れ、パーオキシダーゼ（０ｎｇ／ｍｌ，１ｎｇ／ｍ
ｌ，０．１ｎｇ／ｍｌ，０．０１ｎｇ／ｍｌ）各１０μ
ｌを加え、更に参考例１で作成した基質緩衝液１３５μ
ｌを加え６分後５秒間の発光をクリニルーマットで測定
した。その結果を図４に示す。Example 3 200 μl of an amplification material buffer solution containing sodium 3- (10-phenothiazinyl) propanesulfonate prepared in Example 1 and 4-dimethylaminopyridine was placed in four test tubes, and peroxidase (0 ng) was added. / Ml, 1ng / m
l, 0.1 ng / ml, 0.01 ng / ml) 10μ each
1 was added, and the substrate buffer solution (135 μm) prepared in Reference Example 1 was further added.
After 6 minutes, luminescence was measured for 5 seconds with a Clinylmat. The result is shown in FIG.
【００２３】実施例４ １．３４ｍＭ３−（１０−フェノチアジニル）プロパン
スルホン酸ナトリウム、０．０６７ｍＭフェノールイン
ドフェノールを含むトリシン−水酸化ナトリウム増幅物
質緩衝液に表２に示すピリジン化合物１．０ｍＭを加え
た増幅物質緩衝液２００μｌにパーオキシダーゼ（０．
１ｎｇ／ｍｌ）１０μｌを加え、さらに参考例１で作成
した基質緩衝液１３５μｌを加え、６分後５秒間の発光
をクリニルーマットで測定した。その結果を表２に示
す。Example 4 1.34 mM sodium 3- (10-phenothiazinyl) propane sulfonate, 0.067 mM phenol Indophenol A tricine-sodium hydroxide amplification material buffer containing 1.0 mM pyridine compound shown in Table 2 was added. 200 μl of amplification material buffer was added to peroxidase (0.
1 ng / ml) (10 μl) was further added, and the substrate buffer solution (135 μl) prepared in Reference Example 1 was further added, and after 6 minutes, luminescence for 5 seconds was measured by Clinylmat. The results are shown in Table 2.
【００２４】[0024]
【表２】 [Table 2]
【００２５】[0025]
【発明の効果】本発明の活性増幅物質は、発光反応の全
発光量又は発光信号（シグナル）／バックグラウンド比
を大きくする。そこで本発明の活性増幅物質を用いるこ
とにより、今まで測定することができなかった極微量の
パーオキシダーゼの活性を測定することができる。さら
に本発明をＥＩＡに用いると従来の測定法に比べ高感度
な測定が可能である。The activity-amplifying substance of the present invention increases the total luminescence amount or luminescence signal / background ratio of the luminescence reaction. Therefore, by using the activity-amplifying substance of the present invention, it is possible to measure the activity of a very small amount of peroxidase, which could not be measured until now. Furthermore, when the present invention is applied to EIA, highly sensitive measurement is possible as compared with the conventional measurement method.
【図１】３−（１０−フェノチアジニル）プロパンスル
ホン酸ナトリウムを増幅物質として用い、発光を測定し
た図である。BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram in which luminescence was measured using sodium 3- (10-phenothiazinyl) propanesulfonate as an amplifying substance.
【図２】４−ジメチルアミノピリジンと３−（１０−フ
ェノチアジニル）プロパンスルホン酸ナトリウムとを増
幅物質として用い、発光を測定した図である。FIG. 2 is a diagram in which luminescence was measured using 4-dimethylaminopyridine and sodium 3- (10-phenothiazinyl) propanesulfonate as an amplifying substance.
【図３】従来法のｐ−ヨードフェノールを増幅物質とし
て用いたときのパーオキシダーゼの検量線である。FIG. 3 is a calibration curve of peroxidase when conventional p-iodophenol is used as an amplifying substance.
【図４】４−ジメチルアミノピリジンと３−（１０−フ
ェノチアジニル）プロパンスルホン酸ナトリウムを増幅
物質として用いたときのパーオキシダーゼの検量線であ
る。FIG. 4 is a calibration curve of peroxidase when 4-dimethylaminopyridine and sodium 3- (10-phenothiazinyl) propanesulfonate are used as amplification substances.
Claims (2)
る基質と、 （ロ）一般式 【化１】 で表されるピリジン化合物（式中、Ｒ1 及びＲ2 は水素
原子又は低級アルキル基であり、Ｒ3 は水素原子、ハロ
ゲン原子、低級アルキル基、水酸基又はアミノ基であ
る。）と一般式 【化２】 で表されるフェノチアジン化合物（式中、Ｘは水素原
子、アルカリ金属又はアルカリ土類金属である。）とか
らなる活性増幅物質と、からなるパーオキシダーゼ活性
測定用試薬。1. A substrate consisting of (a) luminol and hydrogen peroxide, and (b) a general formula: A pyridine compound represented by the formula (wherein R 1 and R 2 are hydrogen atoms or a lower alkyl group, and R 3 is a hydrogen atom, a halogen atom, a lower alkyl group, a hydroxyl group or an amino group) and the general formula: Chemical 2] And a phenothiazine compound represented by the formula (wherein, X is a hydrogen atom, an alkali metal or an alkaline earth metal), and a reagent for measuring peroxidase activity.
化水素とからなる基質と、一般式 【化３】 で表されるピリジン化合物と一般式 【化４】 で表されるフェノチアジン化合物とからなる活性増幅物
質とにより発生する化学発光を測定することからなるパ
ーオキシダーゼ活性の測定方法（式中、Ｒ1 及びＲ2 は
水素原子又は低級アルキル基、Ｒ3 は水素原子、ハロゲ
ン原子、低級アルキル基、水酸基又はアミノ基であり、
Ｘは水素原子、アルカリ金属又はアルカリ土類金属であ
る。）。2. A peroxidase, a substrate composed of luminol and hydrogen peroxide, and a compound of the general formula: And a pyridine compound represented by the general formula: A method for measuring peroxidase activity, which comprises measuring chemiluminescence generated by an activity-amplifying substance comprising a phenothiazine compound represented by the formula (wherein R 1 and R 2 are hydrogen atoms or lower alkyl groups, and R 3 is A hydrogen atom, a halogen atom, a lower alkyl group, a hydroxyl group or an amino group,
X is a hydrogen atom, an alkali metal or an alkaline earth metal. ).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP05267813A JP3102228B2 (en) | 1993-10-01 | 1993-10-01 | Peroxidase activity measuring reagent and measuring method using the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP05267813A JP3102228B2 (en) | 1993-10-01 | 1993-10-01 | Peroxidase activity measuring reagent and measuring method using the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0799996A true JPH0799996A (en) | 1995-04-18 |
| JP3102228B2 JP3102228B2 (en) | 2000-10-23 |
Family
ID=17449967
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP05267813A Expired - Fee Related JP3102228B2 (en) | 1993-10-01 | 1993-10-01 | Peroxidase activity measuring reagent and measuring method using the same |
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| Country | Link |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7622486B2 (en) | 2004-09-23 | 2009-11-24 | Reddy Us Therapeutics, Inc. | Pyridine compounds, process for their preparation and compositions containing them |
| US7803573B2 (en) * | 2007-02-23 | 2010-09-28 | Cyanagen Srl | Method for increasing light emission from a chemiluminescent reaction |
-
1993
- 1993-10-01 JP JP05267813A patent/JP3102228B2/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7622486B2 (en) | 2004-09-23 | 2009-11-24 | Reddy Us Therapeutics, Inc. | Pyridine compounds, process for their preparation and compositions containing them |
| US8129136B2 (en) | 2007-02-20 | 2012-03-06 | Cyanagen Srl | Kit for performing an assay |
| US7803573B2 (en) * | 2007-02-23 | 2010-09-28 | Cyanagen Srl | Method for increasing light emission from a chemiluminescent reaction |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3102228B2 (en) | 2000-10-23 |
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