JPH0799996A - Reagent for assaying peroxidase activity and assay using the same - Google Patents

Abstract

PURPOSE:To obtain the subject highly sensitive reagent capable of determining the activity of extremely minute amount of peroxidase, containing specific activity-amplifying substances together with luminol and hydrogen peroxide as substrates. CONSTITUTION:This reagent containing luminol and hydrogen peroxide as substrates contains 10-5000 (pref. 100-1000)muM, of a pyridine compound of formula I (R<1> and R<2> are each H, a lower alkyl; R<3> is H, a halogen, a lower alkyl, hydroxyl or amino) plus a phenothiazine compound of formula II (X is H, an alkali metal or an alkaline earth metal), as activity-amplifying substances.

Description

Detailed Description of the Invention

[0001]

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

[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.

[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]

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]

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]

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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]

The present invention will be described in more detail with reference to Reference Examples and Examples.

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.

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.

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]

[Table 1]

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.

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.

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.

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]

[Table 2]

[0025]

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.

[Brief description of drawings]

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.

FIG. 2 is a diagram in which luminescence was measured using 4-dimethylaminopyridine and sodium 3- (10-phenothiazinyl) propanesulfonate as an amplifying substance.

FIG. 3 is a calibration curve of peroxidase when conventional p-iodophenol is used as an amplifying substance.

FIG. 4 is a calibration curve of peroxidase when 4-dimethylaminopyridine and sodium 3- (10-phenothiazinyl) propanesulfonate are used as amplification substances.

Claims (2)

[Claims] 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. 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. ).