JPH07308200A - Composition for detecting and measuring activity of peroxidase-like substance and detecting and measuring oxidizing substance and measuring method therefor - Google Patents

Abstract

PURPOSE:To obtain the subject composition useful for activity of a peroxidase- like substance or detecting and measuring an oxidizing substance, as a chromogen, having excellent stability, sensitivity and reproducibility, suitable for enzyme immunoassay for medical diacrisis. CONSTITUTION:An alkoxyphenol derivative of formula I (either R1 or R2 is an alkoxy group and other is H, a halogen, COOH, CO or CHO), a phenol derivative of formula II [R3 is COOH, a group of the formula R4COOH (R4 is an alkylene or an alkenylene)], an aminobenzoic acid derivative of formula III (R5 to R8 are each H, a halogen, an alkyl, amino, an alkoxy, COOH, etc.) or an aminohydroxypyrimidine derivative of formula IV (R9 and R10 are each- be-t-h-H, an alkyl, amino, COOH or an alkoxy) as a chromogen is blended with a peroxidase-like substance or hydrogen peroxide, etc., to give the objective composition useful for medical diacrisis by activity of a peroxidase-like substance or detecting and measuring an oxidizing substance.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates to stable compositions for detecting and measuring peroxidase-like substances and oxidative substances. More specifically, this composition is intended to be used for medical diagnosis or biological evaluation.

[0002]

2. Description of the Related Art Conventionally, various methods have been used to detect and measure peroxidase-like substances or oxidizing substances. However, the most commonly used method is a method of colorimetrically quantifying the amount of dye formed by oxidizing a chromogen, which is an oxidizable color-forming substance, in the presence of a peroxide.
Although various chromogens are used, the most widely used, o-phenylenediamine (OPD), is susceptible to oxidation, and is extremely unstable especially in a solution state, and thus is oxidizable. There is a problem that the oxidation reaction proceeds non-enzymatically just by allowing the substance to coexist for several hours. Therefore, the color forming liquid must be prepared immediately before use and must be used immediately after the color forming liquid preparation. There is also a problem that the background rises during measurement.

Therefore, as a means for preventing non-enzymatic reaction of OPD, a method of adding an additive to the reaction solution is used.
(JP-A-62-294099 and JP-A-1-243998) have been used.

[0004]

The object of the present invention is that it can be prepared more easily without adding additives,
Moreover, the present invention provides a composition for detecting and measuring a peroxidase-like substance and an oxidizing substance which are excellent in storage stability and sensitivity, and provide a highly reproducible experimental system by using this composition. To do.

[0005]

Means for Solving the Problems As a result of intensive studies, the inventors have found that a chromogen represented by the following general formula (1):
The above object was achieved by a composition for detecting and measuring the activity of a peroxidase-like substance or an oxidant containing a compound represented by (2), (3) or (4) or a salt thereof.

[0006]

[Chemical 5]

In the formula, _one of R ₁ and R ₂ represents an alkoxy group, and the other represents a hydrogen atom, a halogen atom or a carboxyl group which may have a substituent, a carbonyl group or an aldehyde group. .

[0008]

[Chemical 6]

In the formula, R ₃ is a carboxyl group or --R ₄ --COO
Represents the H group. However, R ₄ represents an alkylene group or an alkenylene group.

[0010]

[Chemical 7]

In the formula, R ₅ , R ₆ , R ₇ and R ₈ each independently represent a hydrogen atom, a halogen atom, an alkyl group, an amino group, an alkoxy group or a carboxyl group. However, R
Any one of ₅ , R ₆ , R ₇ and R ₈ is a carboxyl group, but neither R ₇ nor R ₈ is a carboxyl group at the same time.

[0012]

[Chemical 8]

In the formula, R ₉ and R ₁₀ each independently represent a hydrogen atom, an alkyl group, an amino group, a carboxyl group or an alkoxy group.

In another embodiment, a composition containing a compound represented by the general formula (1), (2), (3) or (4) or a salt thereof is used as a chromogen. This is achieved by a method for quantifying oxidative substances.

When the oxidizing substance is hydrogen peroxide,
Furthermore, the effect is remarkable.

In the presence of a peroxidase-like substance,
There is also an embodiment by a method for quantifying a peroxidase-like substance, characterized in that the compound represented by the general formula (1), (2), (3) or (4) is oxidized and the coloration thereof is colorimetrically quantified. .

When the peroxidase-like substance is peroxidase and the peroxidase is horseradish peroxidase, the effect is more remarkable.

Furthermore, in another embodiment, a composition containing a compound represented by the above general formula (1), (2), (3) or (4) or a salt thereof is used as a chromogen. There is a method of enzyme immunoassay.

The present invention provides an alkoxyphenol derivative or a phenol derivative as a hydrogen donor / chromogen.
Alternatively, an aminobenzoic acid derivative or an aminohydroxypyrimidine derivative is used, which is a method for detecting and measuring a peroxidase-like substance or an oxidizing substance, and an enzyme immunoassay method using an antibody or an antigen molecule bound with peroxidase.

As the chromogen, the general formula (1),
By using the composition containing the compound represented by (2), (3) or (4) or a salt thereof, the color forming liquid containing an oxidizing substance becomes considerably stable as compared with the color forming liquid using OPD. Exists. By using the color forming liquid comprising the composition of the present invention, it is possible to considerably prevent the background from increasing during measurement, and it is possible to obtain good reproducible results.

The compound represented by the general formula (1) used in the present invention will be described. Examples of the alkoxy group represented by R ₁ or R ₂ include a methoxy group and an ethoxy group. Specifically, the compound represented by the general formula (1) includes 4-methoxyphenol, 4-ethoxyphenol, 2-ethoxyphenol, 2-hydroxy-5-methoxyacetophenone, 5-methoxysalicylic acid, 2-hydroxy-5. -Methoxybenzaldehyde, 6-hydroxy-3-anisaldehyde, methyl 2-hydroxy-5-methoxybenzoate, 4-methoxy-2-nitrophenol, 2-chloro-4-methoxyphenol, vanillin, vanillic acid and the like can be mentioned.

The use concentration of the compound represented by the general formula (1) is 0.1 to 100 mg / ml, preferably 1 to 10 mg / ml.

In the compound represented by the general formula (2) used in the present invention, examples of the alkylene group or alkenylene group represented by R ₄ include methylene group, ethylene group, propylene group and vinylene group. Specific examples thereof include p-hydroxybenzoic acid, p-hydroxycinnamic acid, p-hydroxyphenylacetic acid, m-hydroxybenzoic acid, m-hydroxyphenylacetic acid and o-hydroxyphenylacetic acid. The use concentration of the compound represented by the general formula (2) is 0.1 to 1
It is 00 mg / ml, preferably 1 to 10 mg / ml.

In the compound represented by the general formula (3) used in the present invention, R ₅ , R ₆ , R ₇ and R ₈ are each independently a hydrogen atom, a halogen atom, an alkyl group, an amino group or an alkoxy group. Represents a group or a carboxyl group, R ₅ ,
Any one of R ₆ , R ₇ and R ₈ is a carboxyl group, but neither R ₇ nor R ₈ is a carboxyl group at the same time. Specifically, 3,4-diaminobenzoic acid, 3,5-diaminobenzoic acid, o-aminobenzoic acid, m-aminobenzoic acid,
p-aminobenzoic acid, 4-amino-2-chlorobenzoic acid, 4-amino-3-methylbenzoic acid, 3,4-diaminobenzoic acid, 4-amino-3-methoxybenzoic acid, 4-aminophthalic acid, etc. Can be mentioned. The use concentration of the compound represented by the general formula (3) is
It is 0.1 to 100 mg / ml, preferably 1 to 10 mg / ml.

In the compound represented by the general formula (4) used in the present invention, R ₉ and R ₁₀ each independently represent a hydrogen atom, an alkyl group, an amino group, a carboxyl group or an alkoxy group. However, the alkyl group and the alkoxy group preferably have 3 or less carbon atoms. Specifically, 2,4-diamino-6-hydroxypyrimidine, 4,5-diamino-6-hydroxypyrimidine, 4-amino-2,6-dihydroxypyrimidine, 6-hydroxy-2,4,5-triamino Pyrimidine, 4,5
-Diamino-2,6-dihydroxypyrimidine, 4-amino-6-
Examples thereof include hydroxy-2-methylpyrimidine, 4-amino-6-hydroxypyrimidine, 4-amino-6-hydroxy-2-methoxypyrimidine and the like. The use concentration of the compound represented by the general formula (4) is 0.1 to 100 mg / ml, preferably 1 to 1
It is 0 mg / ml.

The composition described in the present invention means an alkoxyphenol derivative represented by the general formula (1), a phenol derivative represented by the general formula (2), and a general formula (3).
An aminobenzoic acid derivative represented by: or an aminohydroxypyrimidine derivative represented by the general formula (4),
Or containing those salts as a chromogen,
As long as this condition is satisfied, the form, the type and the number of constituent components are not particularly limited. That is, they consist of a part or all of the reagents constituting the color forming liquid and may be in the form of powder, solid or liquid.

In the present invention, the chromogen means a substance which is converted into a dye by reacting (causing a structural change) by itself. As described above, the composition includes a chromogen, and may be the chromogen itself, or may be composed of a chromogen and a component necessary for color development or a component for assisting color development. .

The components necessary for color development in the present invention include four components of chromogen, peroxidase-like substance, oxidizing substance and buffer substance. Examples of components that assist color development include stabilizing the chromogen. Nonionic surfactant (JP-A-62-294099), a chelating agent (JP-A-63
-199270), tripolyphosphoric acid (Japanese Patent Laid-Open No. 1-243998) and the like.

The enzyme immunoassay using the antibody or antigen molecule bound with peroxidase in the present invention is
"Enzyme Immunoassay" edited by Eiji Ishikawa et al., 19
Enzyme immunoassay as described on pages 30-49 of the first edition issued in 1982 and pages 8-19 of "Enzyme Immunoassay" by P.TIJSSEN, translated by Eiji Ishikawa, published by Tokyo Kagaku Dojin in the first edition in 1989. Any measuring method can be applied as long as it is a method of measuring the amount of peroxidase activity using a peroxidase-labeled antibody or antigen and a color-forming liquid composed of an oxidizing substance and a chromogen.

The peroxidase-like substance referred to in the present invention is specifically horseradish peroxidase, cytochrome C peroxidase, lactoperoxidase,
Examples include myeloperoxidase, glutathione peroxidase, and metals such as iron, gold and silver, and metal compounds such as hemoglobin, and horseradish peroxidase is preferable.

Further, the oxidizing substance described in the present invention means an alkoxyphenol derivative represented by the general formula (1), a phenol derivative represented by the general formula (2), and a general formula in the presence of peroxidase. It is capable of oxidizing the aminobenzoic acid derivative represented by (3) or the aminohydroxypyrimidine derivative represented by the general formula (4), and hydrogen peroxide, alkyl hydroxyperoxide, p-
Examples thereof include nitroperoxybenzoic acid, cumene hydroxyperoxide and urea peroxide, and hydrogen peroxide is particularly preferable. The concentration of oxidizing substances is 0.001 to 0.1%
Is preferred. If 0.001% or less, the sensitivity will be poor, and 0.1%
The above is not preferable in terms of reproducibility because the enzyme activity is inhibited and a good determination cannot be performed. More preferably, it is 0.005 to 0.02%. As long as they satisfy the above conditions, they are excellent in stability and background as compared with the reagents that have been conventionally used for the same purpose.

According to the present invention, detection of a peroxidase-like substance,
Alternatively, when applied to activity measurement, the method can be performed in the same manner as the conventionally performed method. For example, a chromogen described in the present invention, a coloring solution composed of hydrogen peroxide and a buffer solution (additives may be further added if necessary) and a sample containing a peroxidase-like substance are mixed, Temperature (usually near room temperature) for a specified time (usually 5 ~
After reacting for 60 minutes, a reaction terminator such as sulfuric acid, hydrochloric acid, an aqueous solution of sodium hydroxide and sodium azide is added to stop the reaction. Then, the peroxidase activity of the peroxidase-like substance can be determined by measuring the absorbance. The hydrogen peroxide concentration at this time is 0.00
1 to 0.1% is preferable. More preferably, 0.005-0.02%
Is.

When the present invention is applied to the detection or measurement of oxidizing substances, it can be carried out in the same manner as the conventional method. That is, a chromogen described in the present invention and a chromogenic solution composed of horseradish peroxidase and a buffer solution (additives may be added if necessary) and a sample containing an oxidizing substance are mixed. After reacting at a predetermined temperature (usually around room temperature) for a predetermined time (usually 5 to 60 minutes), a reaction terminator such as sulfuric acid, hydrochloric acid, sodium hydroxide aqueous solution, sodium azide is added to stop the reaction. After that, the oxidizable substance can be measured by measuring the absorbance.

When the present invention is applicable to an enzyme immunoassay using an antibody or antigen molecule to which a peroxidase is bound, the obtained peroxidase activity is used as a standard substance according to the method usually used in EIA, ELISA and the like. The concentration (or amount, unit) of the substance to be measured in the sample can be determined by comparison with the calibration curve of the peroxidase activity obtained by using.

In the present invention, one or more buffer substances may be included for pH regulation. The concentration is 0.01
~ 1.0M, preferably 0.05-0.5M. Good buffer material
Known as MES, PIPES, MOPS, HEPE
S, TES, Tris and hydrochloric acid, Tricine and Tricine Na or
Phosphates, borates, citrates, acetates, carbonates and the like can be used. The pH range is not particularly limited as long as the substance having a peroxidase-like activity exhibits a peroxidase activity of pH 2.5 to 9.0, but is preferably pH 4.5 to 8.0.

The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.

[0037]

【Example】

Example 1 Stability Measurement of Coloring Solution (1) Preparation of Coloring Solution 100 mM buffer solution (type depends on each alkoxyphenol derivative; conditions shown in Table 1) has a hydrogen peroxide concentration of 0.005% by weight. Hydrogen peroxide solution is added. Each of the various alkoxyphenol derivatives is dissolved in this buffer solution to a concentration of 10 mM. These mixed liquids (color-developing liquids) were well stirred to prepare aqueous color-developing agent solutions as shown in Table 1.

[0038]

[Table 1]

(2) Stability of Coloring Solution with Time Each of the prepared coloring solutions was allowed to stand at room temperature, and the absorbance of these solutions was measured with the wavelengths shown in Table 1 which are compatible with each other. The results are shown in Table 2.

[0040]

[Table 2]

The compound of the present invention is more stable in color forming solution over time than the comparative o-phenylenediamine.

Example 2 Measurement of stability of dye after color development (1) Coloring reaction 300 μl of each color forming solution prepared by the formulation shown in Example 1 is dispensed. Add 80 μl of 1 mU / ml peroxidase solution and stir well. After reacting for 30 minutes at room temperature, 1 ml each of the reaction stopping solutions shown in Table 3 was added to stop the reaction.

(2) Stability of dyes with time The color-developing solution in which the reaction was stopped was left at room temperature, and the absorbance of these solutions was measured over time to examine the stability of coloration. As a result, the results shown in Table 3 were obtained.

[0044]

[Table 3]

From these results, it can be seen that the color former of the present invention is stable even after color development.

Example 3 Determination of Hydrogen Peroxide A 100 mM Tris-hydrochloric acid buffer solution (pH 7.5) was used to adjust the hydrogen peroxide concentration to 0.
Hydrogen peroxide solution was added so that the concentration was about 0.02%. Vanillin was added to each of these buffer solutions to a concentration of 10 mM, and the contents were stirred well to confirm dissolution. 1mU / ml horseradish peroxidase solution was added to 300μl of each coloring solution prepared.
Add 0 μl, stir well, and leave at room temperature for 30 minutes. Then, the reaction was stopped by adding 1N sodium hydroxide aqueous solution, and
Absorbance at 0 nm was measured.

As a result, the results shown in FIG. 1 were obtained.
From this result, the amount of hydrogen peroxide and the obtained absorbance are almost linear until the concentration of hydrogen peroxide is 0.01%.
It was suggested that oxidative substances can be quantified within this range.

Example 4 Measurement of Peroxidase Activity Using a color developing solution containing 4-methoxyphenol and a color developing solution containing o-phenylenediamine left at room temperature for 3 days, color development after standing for a long time was examined. Further, as a control, the color developing solution prepared immediately before the experiment was used for each.

100 mM citrate-phosphate buffer (pH 5.0)
Then, hydrogen peroxide solution was added so that the hydrogen peroxide concentration would be 0.005%. 4-Methoxyphenol and o-phenylenediamine were added to each of the buffer solutions so as to have a concentration of 10 mM, and they were thoroughly stirred to confirm dissolution. This solution at room temperature
Leave for 3 days. In the same manner, a color developing solution containing each of these two types of compounds was prepared immediately before the start of the experiment. To 300 μl of each prepared coloring solution, 80 μl of horseradish peroxidase solution having a concentration of 0.1 to 20.0 mU / ml was added, stirred well, and left at room temperature for 30 minutes. Then, 1N H ₂ SO ₄ was added to stop the reaction, and the absorbance at 400 nm (4-methoxyphenol) and 492 nm (o-phenylenediamine) was measured. The results are shown in Fig. 2.

From FIG. 2, the color-forming liquid containing 4-methoxyphenol, which is the system of the present invention, is superior in stability over time to the color-forming liquid containing o-phenylenediamine, and has reproducibility even after standing. Good results have been obtained. Also, the relationship between horseradish peroxidase concentration and absorbance became almost linear, suggesting that peroxidase-like substances can be quantified within this range.

Example 5 Measurement of the amount of galactosyl transferase The color development after standing for a long time was examined using a color development solution containing 4-methoxyphenol and a color development solution containing o-phenylenediamine that were left at room temperature for 3 days. In addition, the color developing solution prepared immediately before the experiment was used as a control for each.

The anti-cancer-related galactosyltransferase (GAT) monoclonal antibody MAb8513 described in Japanese Patent Laid-Open No. 3-259093 was added to a 0.1M carbonate buffer containing 1M sodium chloride.
Antibody-immobilized beads were obtained by diluting to 10 μg / ml in (pH9.15) and immersing polystyrene beads (Sekisui Chemical Co., Ltd.) in this solution for 24 hours at 4 ° C. to immobilize the antibody. .

Next, a 0.02 M phosphate buffer solution (150 mM N) containing 1% bovine serum albumin (BSA) as a blocking solution.
aCl-containing pH 7.3 and hereinafter referred to as PBS) is prepared. The antibody-immobilized beads were transferred to this solution, immersed and left at 37 ° C. for 10 hours to coat (block) the antibody non-binding site on the bead surface. Through these series of operations, the antibody-immobilized beads used below were obtained.

Cancer patient pool serum containing GAT was used as a sample. 50 μl of this sample was added with 0.02 M phosphate buffer (pH 6.
200 μl of 5, 150 mM NaCl and 0.01% Tween 20) was added, and the antibody-immobilized beads prepared by the method described above were immersed therein to carry out a temporary reaction (45 ° C., 2 hours). After the reaction, washing operation was performed with PBS. HRP-labeled anti-GAT antibody MAb8628 (Japanese Patent Laid-Open No. 3-259093) labeled with horseradish peroxidase (HRP) was appropriately diluted in a PBS solution containing 1% BSA, and 250 μl thereof was added so as to correspond to the antibody immobilized on beads. Then, the secondary reaction was performed. After that, a washing operation with PBS was similarly performed. In addition, 4-methoxyphenol at a concentration of 3 mg / ml and 0.02% hydrogen peroxide or a concentration of 3 mg / ml
300 μl of a citrate buffer solution (pH 5.0) containing o-phenylenediamine and 0.02% hydrogen peroxide was added, and color reaction was performed at room temperature for 30 minutes. After the reaction, 1 ml of 1N sulfuric acid was added to stop the reaction, and the absorbance at 400 nm for 4-methoxyphenol and the absorbance at 492 nm for o-phenylenediamine were measured. The results are shown in Fig. 3.

It can be seen from FIG. 3 that the color developing solution containing 4-methoxyphenol, which is the system of the present invention, is superior in stability over time after long-term storage of the color developing solution, as compared with the color developing solution containing o-phenylenediamine. The result was good and reproducible. Also,
It was suggested that the relationship between the concentration and the absorbance was almost linear even in the calibration curve using the GAT standard substance, and that the performance was equivalent to that of the conventionally used OPD.

Example 6 Measurement of stability of color developing solution (1) Preparation of color developing solution 100 mM buffer solution (type depends on each phenol derivative; conditions are shown in Table 4) Hydrogen peroxide concentration is 0.005% by weight Hydrogen peroxide solution is added. The various phenol derivatives are dissolved in this buffer so that the concentration of each of them is 10 mM. These mixed liquids (color-developing liquids) were well stirred to prepare aqueous color-developing agent solutions as shown in Table 4.

[0057]

[Table 4]

(2) Stability of Coloring Liquid with Time Each of the prepared coloring liquids was allowed to stand at room temperature, and the absorbance of these solutions was measured with the wavelengths shown in Table 4 compatible with each other. The results are shown in Table 5.

[0059]

[Table 5]

From Table 5, it can be seen that the color forming liquid containing the compound represented by the general formula (2) of the present invention is more stable than the color forming liquid containing o-phenylenediamine.

Example 7 Determination of Hydrogen Peroxide A 100 mM Tris-hydrochloric acid buffer solution (pH 7.5) was used to adjust the hydrogen peroxide concentration to 0.
Hydrogen peroxide solution was added so that the concentration was about 0.02%. P-Hydroxybenzoic acid was added to each of these buffer solutions to a concentration of 10 mM, and the contents were stirred well to confirm dissolution. To 300 μl of each prepared coloring solution, 80 μl of 1 mU / ml horseradish peroxidase solution was added, stirred well, and allowed to stand at room temperature for 30 minutes. Thereafter, the reaction was stopped by adding a 1N sodium hydroxide aqueous solution, and the absorbance at 470 nm was measured.

As a result, the results shown in FIG. 4 were obtained.
From FIG. 4, up to 0.01% concentration of hydrogen peroxide, the amount of hydrogen peroxide and the obtained absorbance are almost linear, suggesting that the oxidizable substance can be quantified within this range.

Example 8 Measurement of Peroxidase Activity Using a color developing solution containing p-hydroxycinnamic acid and a color developing solution containing o-phenylenediamine left at room temperature for 3 days, the color development after standing for a long time was examined. Further, as a control, the color developing solution prepared immediately before the experiment was used for each.

The buffer was 100 mM for p-hydroxycinnamic acid.
Tris-hydrochloric acid buffer solution (pH 7.5) and o-phenylenediamine 100 mM citric acid-phosphate buffer solution (pH 5.0) were used, and the hydrogen peroxide concentration was adjusted to 0.005%. Hydrogen water was added. To these buffers, p-hydroxycinnamic acid and o-phenylenediamine were added so as to have a concentration of 10 mM, respectively, and well stirred to confirm dissolution. The solution was left at room temperature for 3 days. In the same manner, a color forming liquid containing each of these two types of compounds was prepared immediately before the start of the experiment.
80 μl of a horseradish peroxidase solution having a concentration of 0.1 to 20.0 mU / ml was added to 300 μl of each color-developing solution prepared, and after stirring well, the mixture was allowed to stand at room temperature for 30 minutes. Then, 1N sodium hydroxide aqueous solution was used for the color developing solution of p-hydroxycinnamic acid, and 1N H ₂ SO ₄ was used for the color developing solution of o-phenylenediamine.
Was added to stop the reaction. Then, the respective absorbances at 470 nm (p-hydroxycinnamic acid) and 492 nm (o-phenylenediamine) were measured. The results are shown in Fig. 5.

From FIG. 5, the color developing solution containing p-hydroxycinnamic acid, which is the system of the present invention, is superior in stability over time to the color developing solution containing o-phenylenediamine, and reproduced even after standing. Good results were obtained. Also, the relationship between horseradish peroxidase concentration and absorbance became almost linear, suggesting that peroxidase-like substances can be quantified within this range.

Example 9 Measurement of Galactosyltransferase Amount A color developing solution containing p-hydroxycinnamic acid and a color developing solution containing o-phenylenediamine left at room temperature for 3 days was used to examine the color development after standing for a long time. . In addition, the color developing solution prepared immediately before the experiment was used as a control for each.

Anticancer-related galactosyltransferase (GAT) monoclonal antibody MAb8513 described in JP-A-3-259093 was added to 0.1M carbonate buffer containing 1M sodium chloride.
Antibody-immobilized beads were obtained by diluting to 10 μg / ml in (pH9.15) and immersing polystyrene beads (Sekisui Chemical Co., Ltd.) in this solution for 24 hours at 4 ° C. to immobilize the antibody. .

Next, a 0.02 M phosphate buffer solution (150 mM N) containing 1% bovine serum albumin (BSA) as a blocking solution.
aCl-containing pH 7.3 and hereinafter referred to as PBS) is prepared. The antibody-immobilized beads were transferred to this solution, immersed and left at 37 ° C. for 10 hours to coat (block) the antibody non-binding site on the bead surface. Through these series of operations, the antibody-immobilized beads used below were obtained.

Cancer patient pool serum containing GAT was used as a sample. 50 μl of this sample was added with 0.02 M phosphate buffer (pH 6.
200 μl of 5, 150 mM NaCl and 0.01% Tween 20) was added, and the antibody-immobilized beads prepared by the method described above were immersed therein to carry out a temporary reaction (45 ° C., 2 hours). After the reaction, washing operation was performed with PBS. HRP-labeled anti-GAT antibody MAb8628 (Japanese Patent Laid-Open No. 3-259093) labeled with horseradish peroxidase (HRP) was appropriately diluted in a PBS solution containing 1% BSA, and 250 μl thereof was added to correspond to the antibody immobilized on beads. Then, the secondary reaction was performed. After that, a washing operation with PBS was similarly performed. Furthermore, p-hydroxycinnamic acid at a concentration of 3 mg / ml and 0.02% hydrogen peroxide or o-at a concentration of 3 mg / ml
300 μl of a citrate buffer solution (pH 5.0) containing phenylenediamine and 0.02% hydrogen peroxide was added, and color reaction was carried out at room temperature for 30 minutes. After the reaction, 1N for p-hydroxycinnamic acid
Add 1N of sodium hydroxide solution to o-phenylenediamine.
The reaction was stopped by adding 1 ml each of sulfuric acid, and the absorbance was measured at 470 nm for p-hydroxycinnamic acid and 492 nm for o-phenylenediamine. Results are shown in FIG.

From FIG. 6, it can be seen that the color developing solution containing p-hydroxycinnamic acid, which is the system of the present invention, is stable over time after being stored for a long time, as compared with the color developing solution containing o-phenylenediamine. Excellent and reproducible results were obtained. Also,
It was suggested that the relationship between the concentration and the absorbance was almost linear even in the calibration curve using the GAT standard substance, and that the performance was equivalent to that of the conventionally used OPD.

Example 10 Measurement of Stability of Coloring Solution (1) Preparation of Coloring Solution Hydrogen peroxide was added to 100 mM citric acid-phosphate buffer (pH 5.0) so that the hydrogen peroxide concentration was 0.005% by weight. Add water. The various aminobenzoic acid derivatives are dissolved in this buffer so that the concentration of each derivative is 10 mM. These mixed liquids (color-forming liquids) were well stirred to prepare a color-forming agent aqueous solution. It is currently widely used as a reference substance for color formers.
Phenylenediamine was used.

(2) Stability of Coloring Liquid with Time Each of the prepared coloring liquids was allowed to stand at room temperature, and the absorbance of these solutions was measured with wavelength at each wavelength shown in Table 6 in accordance with each time. The results are shown in Table 6.

[0073]

[Table 6]

From Table 6, it can be seen that the color forming liquid containing the compound represented by the general formula (3) of the present invention is more stable than the color forming liquid containing o-phenylenediamine.

Example 11 Determination of hydrogen peroxide Hydrogen peroxide solution was added to 100 mM citric acid-Na phosphate (pH 5.0) so that the hydrogen peroxide concentration was 0 to 0.02%. To each of these buffers, 3,4-diaminobenzoic acid was added so as to have a concentration of 10 mM, and the mixture was stirred well to confirm dissolution. To 300 ml of each color solution prepared, 80 ml of a horseradish peroxidase solution of 1 mU / ml was added, stirred well, and allowed to stand at room temperature for 30 minutes.
Leave for minutes. Then, 1N H ₂ SO ₄ was added to stop the reaction, and 492
Absorbance at nm was measured.

As a result, the results shown in FIG. 7 were obtained.
From this result, the amount of hydrogen peroxide and the obtained absorbance are almost linear until the concentration of hydrogen peroxide is 0.01%.
It was suggested that oxidative substances can be quantified within this range.

Example 12 Measurement of peroxidase activity Using a color developing solution containing 3,4-diaminobenzoic acid and o-phenylenediamine left at room temperature for 3 days, the color development after standing for a long time was examined. As a control, each color-developing solution prepared immediately before the experiment was used.

The buffer is 100 mM citrate-phosphate buffer.
(pH 5.0) was used, and hydrogen peroxide solution was added so that the concentration of each hydrogen peroxide was 0.005%. To these buffers, 3,4-diaminobenzoic acid and o-phenylenediamine were added so as to have a concentration of 10 mM, respectively, and well stirred to confirm dissolution. The solution was left at room temperature for 3 days. In the same manner, a color forming liquid containing each of these two types of compounds was prepared immediately before the start of the experiment. 0.1 to 300 μl of each prepared coloring solution
Horseradish peroxidase solution at a concentration of ~ 20.0 mU / ml
Add 80 μl, stir well, leave at room temperature for 30 minutes,
The reaction was stopped by adding 1 NH ₂ SO ₄ . Then, the absorbance at 492 nm was measured. The result is shown in FIG.

It can be seen from FIG. 8 that the color developing solution containing 3,4-diaminobenzoic acid, which is the system of the present invention, is superior in stability over time to the color developing solution containing o-phenylenediamine, and even after standing. Good results with good reproducibility were obtained. Also, the relationship between horseradish peroxidase concentration and absorbance became almost linear, suggesting that peroxidase-like substances can be quantified within this range.

Example 13 Measurement of the amount of galactosyl transferase A color developing solution containing 3,4-diaminobenzoic acid and a color developing solution containing o-phenylenediamine left at room temperature for 3 days was used to examine the color development after standing for a long time. It was In addition, the color developing solution prepared immediately before the experiment was used as a control for each.

Anti-cancer-related galactosyltransferase (GAT) monoclonal antibody MAb8513 described in Japanese Patent Laid-Open No. 3-259093 was added to 0.1M carbonate buffer containing 1M sodium chloride.
Antibody-immobilized beads were obtained by diluting to 10 μg / ml in (pH9.15) and immersing polystyrene beads (Sekisui Chemical Co., Ltd.) in this solution for 24 hours at 4 ° C. to immobilize the antibody. .

Next, as a blocking solution, a 0.02 M phosphate buffer solution (150 mM N) containing 1% bovine serum albumin (BSA) was prepared.
aCl-containing pH 7.3 and hereinafter referred to as PBS) is prepared. The antibody-immobilized beads were transferred to this solution, immersed and left at 37 ° C. for 10 hours to coat (block) the antibody non-binding site on the bead surface. Through these series of operations, the antibody-immobilized beads used below were obtained.

Cancer patient pool serum containing GAT was used as a sample. 50 μl of this sample was added with 0.02 M phosphate buffer (pH 6.
200 μl of 5, 150 mM NaCl and 0.01% Tween 20) was added, and the antibody-immobilized beads prepared by the method described above were immersed therein to carry out a temporary reaction (45 ° C., 2 hours). After the reaction, washing operation was performed with PBS. HRP-labeled anti-GAT antibody MAb8628 (Japanese Patent Laid-Open No. 3-259093) labeled with horseradish peroxidase (HRP) was appropriately diluted in a PBS solution containing 1% BSA, and 250 μl thereof was added to correspond to the antibody immobilized on beads. Then, the secondary reaction was performed. After that, a washing operation with PBS was similarly performed. Furthermore, 3 mg / ml concentration of 3,4-diaminobenzoic acid and 0.02% hydrogen peroxide or 3 mg / ml concentration of o-
300 μl of a citrate buffer solution (pH 5.0) containing phenylenediamine and 0.02% hydrogen peroxide was added, and color reaction was carried out at room temperature for 30 minutes. After the reaction, 1 ml of 1N sulfuric acid was added to stop the reaction, and the absorbance at 492 nm was measured. The results are shown in Fig. 9.

It can be seen from FIG. 9 that the color developing solution containing 3,4-diaminobenzoic acid, which is the system of the present invention, is stable over time after long-term storage of the color developing solution, as compared with the color developing solution containing o-phenylenediamine. The result was excellent and reproducible. Also,
It was suggested that the relationship between the concentration and the absorbance was almost linear even in the calibration curve using the GAT standard substance, and that the performance was equivalent to that of the conventionally used OPD.

Example 14 Stability of Coloring Solution (1) Preparation of Coloring Solution 50 mM citrate-phosphate buffer (pH 5.0) was added with hydrogen peroxide solution so that the hydrogen peroxide concentration was 0.005% by weight. Add. The various aminohydroxypyrimidine derivatives are dissolved in this buffer so that the concentration of each of them is 10 mM.
These mixed liquids (color-forming liquids) were well stirred to prepare a color-forming agent aqueous solution. As a color developing agent, o-phenylenediamine, which is widely used at present, was used.

(2) Stability of Coloring Solution with Time Each prepared coloring solution was allowed to stand at room temperature, and the absorbance of these solutions at 492 nm was measured with time. The results are shown in Table 7.

[0087]

[Table 7]

From Table 7, it can be seen that the color forming liquid containing the compound represented by the general formula (4) of the present invention is more stable than the color forming liquid containing o-phenylenediamine.

Example 15 Quantification of hydrogen peroxide Hydrogen peroxide solution was added to 100 mM citric acid-Na phosphate (pH 5.0) so that the hydrogen peroxide concentration was 0 to 0.02%. To each of these buffer solutions, 2,4-diamino-6-hydroxypyrimidine was added so as to have a concentration of 10 mM, and the mixture was stirred well to confirm dissolution. To 300 μl of each prepared coloring solution, 80 μl of a horseradish peroxidase solution of 1 mU / ml was added, stirred well, and allowed to stand at room temperature for 30 minutes. Then, 1N H ₂ SO ₄ was added to stop the reaction, and the absorbance at 492 nm was measured.

As a result, the results shown in FIG. 10 were obtained.
From this result, the amount of hydrogen peroxide and the obtained absorbance are almost linear until the concentration of hydrogen peroxide is 0.01%.
It was suggested that oxidative substances can be quantified within this range.

Example 16 Measurement of peroxidase activity Using a coloring solution containing 2,4-diamino-6-hydroxypyrimidine left at room temperature for 3 days, color development after standing for a long time was examined. As a control, each color-developing solution prepared immediately before the experiment was used.

The buffer is 100 mM citrate-phosphate buffer
(pH 5.0) was used, and hydrogen peroxide solution was added so that the concentration of each hydrogen peroxide was 0.005%. To these buffers, 2,4-diamino-6-hydroxypyrimidine and o-phenylenediamine were added so as to have a concentration of 10 mM, respectively, and well stirred to confirm dissolution. The solution was left at room temperature for 3 days. In the same manner, a color forming liquid containing each of these two types of compounds was prepared immediately before the start of the experiment. 80 μl of a horseradish peroxidase solution having a concentration of 0.1 to 20.0 mU / ml was added to 300 μl of each color-developing solution prepared, stirred well, and allowed to stand at room temperature for 30 minutes, and 1N H ₂ SO ₄ was added to stop the reaction. Then, the absorbance at 492 nm was measured. The result is shown in Figure 1.
Shown in 1.

From FIG. 11, the color developing solution containing 2,4-diamino-6-hydroxypyrimidine, which is the system of the present invention, is superior in stability over time to the color developing solution containing o-phenylenediamine. Good results with good reproducibility were obtained even after standing. Also, the relationship between horseradish peroxidase concentration and absorbance became almost linear, suggesting that peroxidase-like substances can be quantified within this range.

Example 17 Measurement of amount of galactosyl transferase About color development after left standing for a long time using a color developing solution containing 2,4-diamino-6-hydroxypyrimidine and a color developing solution containing o-phenylenediamine left at room temperature for 3 days Study was carried out.
In addition, the color developing solution prepared immediately before the experiment was used as a control for each.

Anti-cancer-related galactosyl transferase (GAT) monoclonal antibody MAb8513 described in JP-A-3-259093 was added to 0.1 M carbonate buffer containing 1 M sodium chloride.
Antibody-immobilized beads were obtained by diluting to 10 μg / ml in (pH9.15) and immersing polystyrene beads (Sekisui Chemical Co., Ltd.) in this solution for 24 hours at 4 ° C. to immobilize the antibody. .

Next, a 0.02 M phosphate buffer solution (150 mM N) containing 1% bovine serum albumin (BSA) was used as a blocking solution.
aCl-containing pH 7.3 and hereinafter referred to as PBS) is prepared. The antibody-immobilized beads were transferred to this solution, immersed and left at 37 ° C. for 10 hours to coat (block) the antibody non-binding site on the bead surface. Through these series of operations, the antibody-immobilized beads used below were obtained.

Cancer patient pooled serum containing GAT was used as a specimen. 50 μl of this sample was added with 0.02 M phosphate buffer (pH 6.
200 μl of 5, 150 mM NaCl and 0.01% Tween 20) was added, and the antibody-immobilized beads prepared by the method described above were immersed therein to carry out a temporary reaction (45 ° C., 2 hours). After the reaction, washing operation was performed with PBS. HRP-labeled anti-GAT antibody MAb8628 (Japanese Patent Laid-Open No. 3-259093) labeled with horseradish peroxidase (HRP) was appropriately diluted in a PBS solution containing 1% BSA, and 250 μl thereof was added to correspond to the antibody immobilized on beads. Then, the secondary reaction was performed. After that, a washing operation with PBS was similarly performed. Furthermore, 3,4-diamino-6 at a concentration of 3 mg / ml
-Add 300 μl of citrate buffer (pH 5.0) containing hydroxypyrimidine and 0.02% hydrogen peroxide or o-phenylenediamine at a concentration of 3 mg / ml and 0.02% hydrogen peroxide,
Color reaction was carried out at room temperature for 30 minutes. After the reaction, add 1N sulfuric acid to 1
The reaction was stopped by adding ml, and the absorbance at 492 nm was measured. The results are shown in Figure 12.

From FIG. 12, the system of the present invention, 2,4-diamino
The color developing solution containing -6-hydroxypyrimidine was superior to the color developing solution containing o-phenylenediamine in long-term stability after long-term storage, and reproducible results were obtained. In addition, the relationship between the concentration and the absorbance was almost linear in the calibration curve using the GAT standard substance, and it was suggested that the performance is equivalent to that of the OPD that has been used conventionally.

[0099]

As described above, the color developing solution using the composition of the present invention is extremely stable even in the presence of an oxidizing substance, as compared with the color developing solution using o-phenylenediamine which has been conventionally used. It can be seen that it exists and is easy to operate. Furthermore, by using the composition of the present invention, it became possible to perform an experiment with excellent background characteristics and high reproducibility.

[Brief description of drawings]

FIG. 1 is a diagram showing the relationship between hydrogen peroxide concentration and color development when vanillin is used.

FIG. 2 is a diagram showing a calibration curve in which a color developing solution containing 4-methoxyphenol and o-phenylenediamine was colored with horseradish peroxidase.

FIG. 3 is a diagram showing a calibration curve of galactosyltransferase concentration in serum by EIA method using a color developing solution containing 4-methoxyphenol and o-phenylenediamine.

FIG. 4 is a diagram showing the relationship between hydrogen peroxide concentration and color development when p-hydroxybenzoic acid is used.

FIG. 5 is a diagram showing a calibration curve in which a coloring solution containing p-hydroxybenzoic acid and o-phenylenediamine was colored with horseradish peroxidase.

FIG. 6 is a diagram for obtaining a calibration curve of galactosyltransferase concentration in serum by EIA method using a color developing solution containing p-hydroxybenzoic acid and o-phenylenediamine.

FIG. 7 is a diagram showing the relationship between hydrogen peroxide concentration and color development when 3,4-diaminobenzoic acid is used.

FIG. 8 is a diagram showing a calibration curve in which a color developing solution containing 3,4-diaminobenzoic acid and o-phenylenediamine was colored with horseradish peroxidase.

FIG. 9 is a view showing a calibration curve of galactosyltransferase concentration in serum by EIA method using a color developing solution containing 3,4-diaminobenzoic acid and o-phenylenediamine.

FIG. 10 is a graph showing the relationship between hydrogen peroxide concentration and color development when 2,4-diamino-6-hydroxypyrimidine is used.

FIG. 11 2,4-diamino-6-hydroxypyrimidine and o
-A diagram showing a calibration curve in which a color-forming liquid containing phenylenediamine was colored with horseradish peroxidase.

FIG. 12 is a diagram showing a calibration curve of galactosyltransferase concentration in serum obtained by EIA method using a color developing solution containing 2,4-diamino-6-hydroxypyrimidine and o-phenylenediamine.

[Explanation of symbols]

1 Coloring liquid containing o-phenylenediamine immediately after preparation 2 Coloring liquid containing o-phenylenediamine 3 days after preparation 3 Coloring liquid containing 4-methoxyphenol immediately after preparation 4 Coloring liquid containing 4-methoxyphenol 3 days after preparation 5 Coloring liquid containing p-hydroxybenzoic acid immediately after preparation 6 Coloring liquid containing p-hydroxybenzoic acid 3 days after preparation 7 Coloring liquid containing 3,4-diaminobenzoic acid immediately after preparation 8 3,4 after preparation 3 days -Coloring liquid containing diaminobenzoic acid 9 Coloring liquid containing 2,4-diamino-6-hydroxypyrimidine immediately after preparation 10 Coloring liquid containing 2,4-diamino-6-hydroxypyrimidine 3 days after preparation

Claims (10)

[Claims] 1. A composition for detecting and measuring an activity of a peroxidase-like substance or an oxidant containing a compound represented by the following general formula (1) or a salt thereof as a chromogen. [Chemical 1] In the formula, _one of R ₁ and R ₂ represents an alkoxy group, and the other represents a hydrogen atom, a halogen atom or a carboxyl group which may have a substituent, a carbonyl group or an aldehyde group. 2. A composition for detecting or measuring the activity or oxidizing substance of a peroxidase-like substance, which comprises a compound represented by the following general formula (2) or a salt thereof as a chromogen. [Chemical 2] In the formula, R ₃ represents a carboxyl group or a —R ₄ —COOH group.
However, R ₄ represents an alkylene group or an alkenylene group. 3. A composition for detecting and measuring the activity or oxidizing substance of a peroxidase-like substance, which comprises a compound represented by the following general formula (3) or a salt thereof as a chromogen. [Chemical 3] In the formula, R ₅ , R ₆ , R ₇ , and R ₈ each independently represent a hydrogen atom, a halogen atom, an alkyl group, an amino group, an alkoxy group, or a carboxyl group. However, R ₅ , R ₆ , R ₇ ,
Any one of R ₈ is a carboxyl group, but R ₇ , R
Both ₈ cannot be carboxyl groups at the same time. 4. A composition for detecting and measuring an activity of a peroxidase-like substance or an oxidizing substance, which comprises a compound represented by the following general formula (4) or a salt thereof as a chromogen. [Chemical 4] In the formula, R ₉ and R ₁₀ each independently represent a hydrogen atom, an alkyl group, an amino group, a carboxyl group or an alkoxy group. 5. A chromogen as defined by the general formula (1),
A method for quantifying an oxidizing substance, which comprises using a composition containing a compound represented by (2), (3) or (4) or a salt thereof. 6. The method for quantifying an oxidizing substance according to claim 5, wherein the oxidizing substance is hydrogen peroxide. 7. The compound represented by the general formula (1), (2), (3) or (4) is oxidized in the presence of a peroxidase-like substance, and its coloration is colorimetrically determined. And a method for quantifying a peroxidase-like substance. 8. The method for quantifying a peroxidase-like substance according to claim 7, wherein the peroxidase-like substance is peroxidase. 9. The method for quantifying a peroxidase-like substance according to claim 7, wherein the peroxidase is horseradish peroxidase. 10. The chromogen as defined by the general formula (1),
An enzyme immunoassay method comprising using a composition containing the compound represented by (2), (3) or (4) or a salt thereof.