JPH03103194A - Testing agent and testing tool - Google Patents

Abstract

PURPOSE:To provide the subject testing agent containing a specific oxidation color-developing indicator, an oxidizing agent and a stabilizer having a specific structure, resistant to the change with time during storage and useful for the detection of glucose in urine and blood. CONSTITUTION:The objective testing agent contains (A) an oxidation color- developing indicator developing color by the oxidation of (a) a hydroperoxide which may be formed by reaction in the presence of (b) a peroxidase or a substance having the action of peroxidase, (B) an effective amount of an oxidizing agent and (C) a stabilizer of formula (R<1> to R<4> are H, lower alkyl, halogen, amino, nitro, carboxyl or alkoxy) and is useful for the determination of the component (a) with a peroxidase by a peroxidase-like reaction. The component B is the compound of formula MXO4 (M is univalent cation; X is halogen) selected from NaIO4 and HIO4. The component A is preferably benzidine, etc., and the component C is preferably 2-mercapto benzimidazole.

Description

Detailed Description of the Invention Industrial Field of Application The present invention relates to a test agent for measuring hydroperoxides or hydrogen peroxide or other hydroperoxides produced by a pre-reaction using a peroxidase- or peroxidase-like reaction; The present invention relates to a test agent for detecting peroxidase or a peroxidase-active substance, particularly for detecting glucose in urine and blood, and for detecting blood in urine, feces, and liquid, and a test device carrying this test agent.

BACKGROUND OF THE INVENTION Recently, rapid diagnostic agents have become extremely important in medical practice and clinical trials, and rapid diagnostic agents based on absorbable carriers are important. In many cases, an absorbent carrier such as paper is impregnated with the drug necessary for the detection reaction.
When immersed in body fluids, a color reaction occurs if the substance to be detected is present. Rapid diagnostic agents that have become very important in medical diagnosis include, for example, rapid diagnostic agents for detecting glucose in urine and blood, and for detecting blood in urine, feces, and fluids.

This test is based on the following principle.

When detecting glucose in body fluids, for example, glucose is oxidized to gluconic acid by glucose oxidase (COD), while atmospheric oxygen is reduced to hydrogen peroxide. This hydrogen peroxide is produced by peroxidase (PO
D) or by oxidizing the oxidized color indicator to the corresponding dye by means of peroxidase-active substances, the concentration of the color being a measure of the amount of glucose present.

Furthermore, when detecting blood, active oxygen is generated from peroxide contained in test strips etc. in advance due to the peroxidase-like activity of hemoglobin, and oxidizes the oxidized color indicator.

Generally, the oxidation color indicator of the above-mentioned rapid diagnostic agent is
Particularly useful belonging to a particular class of compounds are the benzidines, heterocyclic azines and phenols, especially the components of guaiac resin (JP 53-126996).

By the way, recent processed foods, soft drinks, etc. contain vitamin C.
It contains a large amount of ascorbic acid, and vitamins are also taken in large quantities for health management reasons.

Therefore, a large amount of ascorbic acid is contained in the blood, and excess ascorbic acid is contained in the urine and excreted from the body. Therefore, when performing urine or blood tests using diagnostic agents such as those mentioned above, ascorbic acid has strong reducing properties, so active oxygen generated by peroxidase or peroxidase-active substances is consumed in the redox reaction with ascorbic acid. A problem has arisen in that the reaction of the oxidation indicator is inhibited and sufficient color development cannot be obtained.

Therefore, the applicant has disclosed a technique in which an effective amount of an oxidizing agent is added to a test agent to eliminate the influence of reducing substances such as ascorbic acid (Japanese Patent Laid-Open No. 60-86467
issue).

However, this test agent has the disadvantage that it is oxidized and discolored during storage prior to use of the test agent (test device) I due to the influence of oxygen and moisture in the air.

<Problems to be Solved by the Invention> The present invention has been made in view of the above-mentioned drawbacks of the prior art, and its purpose is to provide a test agent and a test device that prevent changes over time during storage. be.

Means for Solving the Problems> These objects are achieved by the present invention described in (1) to (8) below.

(1) A test agent for measuring hydroperoxide or a hydroperoxide produced by a reaction using peroxidase or a peroxidase-like reaction, or a test agent for measuring peroxidase or a peroxidase-active substance, wherein the hydroperoxide has peroxidase or peroxidase-active substances. 1. A test agent comprising an oxidized coloring indicator that develops color when oxidized during a reaction with a substance, an effective amount of an oxidizing agent, and a stabilizer represented by the following general formula (2).

General formula ■ p4 B (in the formula, R'R''R' and R4 may be the same or different, and each represents a hydrogen atom, a lower alkyl group, a halogen atom, an amino group, a ditro group, a carboxyl group, or represents an alkoxy group) (2) The test agent according to (1) above, wherein the oxidizing agent is MXO4 (M is an atom that can become a monovalent cation, and X is a halogen atom).

(3) Said MXO. is N a I O 4 or HIO
．． The test agent according to (2) above.

(4) The oxidized color indicator is benzidine, o-tolidine, 3.3',5.5'-tetra(lower alkyl)
The test agent according to any one of (1) to (3) above, which is selected from the group consisting of benzidine and 2,7-diaminofluorene.

(5) The test agent according to any one of (1) to (4) above, wherein the stabilizer is 2-mercaptobenzimidazole.

(6) The test agent according to any one of (1) to (5) above, which contains polyvinylpyrrolidone and a copolymer of methyl vinyl ether and maleic anhydride as additives.

(7) A test device comprising a carrier supporting the test agent according to any one of (1) to (6) above.

(8) The test device according to (7) above, wherein the surface of the carrier is coated with a semi-permeable membrane.

Specific constitution> The test agent of the present invention measures hydroperoxide or hydrogen peroxide or other hydroperoxide generated by a pre-reaction using peroxidase or a peroxidase-like reaction, or measures peroxidase or a peroxidase-active substance. It is something.

This test agent contains an oxidizing agent to eliminate the influence of reducing substances such as ascorbic acid. The oxidizing agent preferably includes oxyacids or their salts and metal salts.

Examples of the oxygen acid or its salt include MXO, MXO. M
XO. M. Examples include H2XOa (where M is an atom that can be a monovalent cation, such as Na, K, H, etc., and X is a halogen atom, such as I, Br%Cβ, etc.).

Specific examples include HC Shinobu 0, NaCβO, KC (20%
HBrO, NaBr'0% KBrO, HIO, NaIO
%KIO, HCf20, N a C Q O s K
C Q O 3H B f” O aN a B r
O s, K B r O s, HIO. , N a I
OsKI03, HCgO. , NaCj204, KC
j204HBrO. , N a B r O 4,
KBrO 4, HIO. N a I 0 4
K I 0 4 N a s H
* There are IOsK3Hz IOs, etc.
Among them, MXO4 is preferable, and M
IO4, most preferably N a IO < or HIO4.

Metal salts include ferric chloride, cuprous chloride, copper sulfate,
Examples include copper acetate, mercury acetate, bismuth acetate, and lead acetate.

The oxidizing agent does not oxidize the oxidized color support agent during the storage period of the test agent, but when used in the test, it is necessary to prevent the reaction of the oxidized indicator from being inhibited by reducing substances such as ascorbic acid present in the specimen. Only the amount effective to do so will be used. Specifically, it is affected by the amount of the oxidized color indicator contained in the test agent and the reducing substance present in the specimen, but it is usually 0. 3 to 100 mmol/tolerance, preferably 0. 5-50 mmol/I2
The concentration is about.

Oxidized color indicators (coloring agents) that develop color when oxidized are used as a measurement system for oxygen generated by peroxidase or bele oxidase-like reactions, such as reactions that produce oxidized condensates (phenol-based and 4-aminoantibiline, etc.), methanol, etc. It is involved in the reaction in which is oxidized to formaldehyde and produces a condensate with acetylacetone, and in the reaction in which iodide such as potassium iodide is oxidized to produce iodine.

Examples of the oxidation color indicator include benzidine compounds, heterocyclic azines, phenols, and guaiac resin components. Specifically, o-tolidine, m-tolidine, benzidine, 3,3', 5,5'-tetra(lower alkyl)benzidine, 0-methylbenzidine, 2,7-diaminofluorene or these in various ratios. A mixture containing 4,4°-diaminodiphenyl, O-phenylenediamine, m-phenylenediamine, p-phenylenediamine, 2.3-tolylenediamine, 2.4-tolylenediamine, 2.5- Tolylene diamine, various substituted phenylene diamines such as 2.6-}lylene diamine, pyrogallic acid, gallic acid, phloroglucinol, hydroquinone,
Examples include phenols such as leucoindophenol, guaiacol, pyridine derivatives, substituted azines, and leucomalachite green. Among them, 0-tolidine and 2,7-diaminofluorene are especially preferred because they lack stability and are highly effective in the present invention in which a stabilizer described below is added. These oxidized color indicators are usually about 2 to 250 mmol/°C, preferably 10 to 250 mmol/°C, relative to the immersion liquid.
50 mmol/I;! ．． It is used at a concentration of about

Hydroperoxides used in the present invention include bis[4-(α-hydroperoxyisobrobyl)benzyl]ether, 2,5-dimethylhexane-2,5-dihydroperoxide, cumene hydroperoxide, 2.5-dimethylhexane-2,5-dihydroperoxide,
-dimethylhexanone-2,5-dihydroperoxide, diisobrobylbenzene hydroperoxide, t-butyl hydroperoxide, p-menthane hydroperoxide, 4-methylphenylisobrobyl hydroperoxide, and the like.

The stabilizer added to the test agent of the present invention prevents the oxidized coloring indicator from being oxidized and developing color due to changes over time during storage, that is, due to factors such as oxygen, moisture, and light in the air.

This stabilizer is a substance represented by the general formula below.

General formula I R4 (in the formula, RI Rg R3 and R4 may be the same or different from each other, and each represents a hydrogen atom, a lower alkyl group, a halogen atom, an amino group, a nitro group, a carboxyl group, or an alkoxyl group) Examples of the lower alkyl group include those having 1 to 5 carbon atoms, such as a methyl group, an ethyl group, and a probyl group.

Examples of the halogen atom include F%C°C, Br, I, and the like.

Examples of the alkoxy group include a methoxy group, an ethoxy group, a broboxy group, a butoxy group, and the like.

Specific examples of the substance shown in the formula include 2-mercaptobenzimidazole, 2-mercaptomethylbenzimidazole, and the like. Among these, 2-mercaptobenzimidazole is particularly preferred. The reason is 2-
Mercaptobenzimidazole is the best stabilizer and is fat-soluble, so it does not have a negative effect on the reaction system of the oxidized color indicator, nor does it have a negative effect on the oxidation reaction system used to eliminate ascorbic acid. It is from.

The amount of such a stabilizer added depends on the amount of the oxidizing agent added, but is set to an amount that sufficiently functions as a stabilizer and does not reduce sensitivity. Based on this, about 0.1 to 5 mmol 72, preferably 0.1 to 5 mmol, preferably 0.1 to 5 mmol to the normal immersion liquid. It is used at a concentration of about 5 to 1.5 mmol/Q.

In addition, in the above-mentioned system that does not contain an oxidizing agent, the same effect can be obtained with a stabilizer other than the above-mentioned general formula I, but since the present invention contains an oxidizing agent, the suitable stabilizer is the above-mentioned stabilizer. Limited to those shown in General Ceremony.

The test agent of the present invention is used as a solution in which each drug constituting the test agent is dissolved in one solvent, or as a solution in which each drug is divided into several parts and each part is dissolved in a solvent with the same or different composition. Ru.

That is, the test agent consists of one type of solution or a combination of two or more types of solutions.

As the solvent, one or more solvents may be appropriately selected and used, for example, from water, benzene, toluene, xylene, methanol, ethanol, isopropanol, acetone, methyl ethyl ketone, chloroform, carbon tetrachloride, and the like.

Alternatively, the solution test agent may be dried to form a powder or granular test agent.

Alternatively, as described below, it is used as a test device in which a test agent is supported on a carrier.

If necessary, various additives as described below may be added to such a test agent.

Contact between the test device and the specimen (for example, by dipping the test device into the specimen)
pH adjustment (pH = 4-8, preferably 5-7)
Examples of buffering agents include citric acid-sodium citrate, sodium tartrate-sodium monotartrate, malic acid-borax, potassium hydrogen phthalate-dipotassium phthalate, sodium hydrogen succinate-disodium succinate, and the like.

Wetting agents used to uniformly wet (penetrate) the sample liquid when the test device and sample come into contact include alkyl sulfates such as polyvinylpyrrolidone, sodium lauryl sulfate, sodium dodecyl sulfate, and sodium tetradecyl sulfate. Salts, alkylbenzene sulfonates such as sodium dodecylbenzene sulfonate, dialkyl sulfosuccinates such as sodium dioctyl sulfosuccinate, sodium diheptyl sulfosuccinate, and the like.

Among these, polyvinylpyrrolidone in particular has the effect of promoting permeation of the specimen and has the advantage of increasing the speed of color development.

Further, in order to improve the coloring stability after coloring, a copolymer of methyl vinyl ether and maleic anhydride, or a half ethyl ester thereof, etc. may be added.

Furthermore, in the test agent, a drug that enhances peroxidase activity in hemoglobin, etc. may be added. Typical enhancers include quinoline and its derivatives, such as quinine, cinchonine, 6-methoxyquinoline, quinaldine, 8-amino-6-methoxyquinoline, 2-quinolinol, isoquinoline, benzo(f)quinoline, 3-aminoquinoline, etc. can be mentioned. The presence of these enhancers usually accelerates the oxidation reaction rate and the color intensity of the oxidized chromogen, increasing the sensitivity of the test device.

Further, when used as a test device, a thickening agent may be used to prevent the drug from flowing out from the carrier. Typical thickening agents include polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, acrylates, polyacrylamide, poly(hydroxyethyl methacrylate), poly(hydroxyethyl acrylate)
, polymers such as carboxymethylcellulose, gelatin,
Examples include gum arabic.

The carrier in the test device is preferably made of a non-fibrous or fibrous porous material.

Typical examples of non-fibrous porous materials include filter paper and Memplan filters.Other examples include dispersions of porous materials such as diatomaceous earth and microcrystalline materials dispersed in binders, and microspherical beads of glass and resin that are bonded to each other. Examples include porous aggregates that are dot-bonded.

Furthermore, examples of the fibrous porous material include woven or knitted fabrics, nonwoven fabrics, short fiber aggregates, and the like.

Examples of the fiber material include natural fibers such as cotton, silk, and wool, and resins such as glass and nylon.

The test agent may be supported by impregnating the carrier with a liquid constituting the test agent (for example, by immersing the carrier in an immersion liquid), and then drying the carrier. When there are two or more types of liquids, impregnation and drying are repeated.

Furthermore, the test device of the present invention preferably has a surface of a carrier carrying a test agent coated with a semi-permeable membrane. The presence of this semipermeable membrane allows the amount of analyte that permeates the carrier to be adjusted.

Examples of the constituent material of the semipermeable membrane include cellulose ethers such as 1-methyl cellulose and carboxymethyl cellulose, cellulose esters such as cellulose acetate, cellulose-based materials such as ion-exchanged cellulose, and polyvinylidene fluoride.

Further, the bore size of the semipermeable membrane is preferably about 0.1 to 100 mm, particularly about 1 to 10 mm.

The thickness of the semi-permeable membrane is about 0.1 to 4, especially 1 to 4.
~51 is preferred.

A method for forming such a semi-permeable membrane is, for example, by dissolving the above-mentioned cellulose material in two or more organic solvents having different boiling points (e.g., benzene/ethanol), and applying the solution to a carrier carrying a test agent. or apply to the surface of
The carrier was immersed in the solution, and then heated at about 20 to 60°C for 5 minutes.
This is done by drying for about 20 minutes.

In this case, the ratio of cellulosic material to organic solvent is O. 1 to 1 wt% is preferable.

The test agent and test device of the present invention are applied to detect substances such as glucose, uric acid, cholesterol, triglycerides (neutral fats), free fatty acids, hemoglobin, myoglobin, and white blood cells.

Example Hereinafter, specific examples of the present invention will be described.

(Example 1 of the present invention) Solutions 1, 2, and 3 having the compositions shown below were prepared, and filter paper (
Toyosha No. 5250), first impregnated with solution 1 and dried (40°C, 60 minutes), then impregnated with solution 2 and dried (4
0°C, 10 minutes), then impregnated with solution 3 and dried (40°C).
, 10 minutes) to obtain a test device for detecting glucose in urine.

In addition, this test device contains 30 to 2000 mg per 1 dj of urine.
It produces a blue color that can be used to quantify glucose.

Solution 1 Sodium metaperiodate (oxidizing agent) Glucose oxidase Peroxidase Tartrazine 10% Polyvinylpyrrolidone (K30) Aqueous solution 5% Gantrez ANl39 (Copolymer of methyl vinyl ether and maleic anhydride) Aqueous solution Citrate buffer (pH 5.5) Solution 2 0-tolidine (oxidized color indicator) 2-mercabutobenzimidal (stabilizer) Acetone solution 3 Ethylcellulose benzene-ethanol (4:1) 150mg 3.0g 100mg 150mg 30m2 70ml 50mj 1.0g 80mg 100ml 550mg 100ml (Comparative Example 1) A test device for detecting glucose in urine was obtained in the same manner as in Inventive Example 1 except that Solution 2 did not contain 2-mercabutopenzimidazole.

(Example 2 of the present invention) Solutions 4, 5, 6, and 7 having the compositions shown below were prepared, and a filter paper (No. 5250 manufactured by Toyosha Co., Ltd.) was first impregnated with solution 4.
Dry (40°C, 10 minutes), then impregnate with solution 5, dry (40°C, 60 minutes), then impregnate with solution 6, dry (4
0°C, 10 minutes), then impregnated with solution 7 and dried (40°C).
, 10 minutes) to obtain a test device for detecting occult blood in urine.

Solution 4 Bis[4-(α-hydroberoxyisobrobyl)penzyl]ether 800mg Sodium dioctyl sulfosuccinate
1.5g ethanol
100ml solution 5 10% polyvinylpyrrolidone (K30) aqueous solution 5% Gantrez AN139 aqueous solution Citric acid Sodium citrate Sodium metaperiodate (oxidizing agent) Savonin distilled aqueous solution 6 o-Tolidine (oxidized color indicator) 2-Mercaptobenzimidazole (stable) agent) Acetone solution 7 Ethyl cellulose benzene-ethanol (4:1) 100 mg 100 mg 20 ml 1.0 g 80 mg 100 mg 550 mg 100 mg (Comparative example 2) Same as inventive example 2 except that solution 6 does not contain 2-mercabutobenzimidazole A test device for detecting occult blood in urine was obtained.

(Comparative Example 3) A test device for detecting occult blood in urine was obtained in the same manner as in Inventive Example 2, except that 200 mg of penzimidazole was used instead of 2-mercaptobenzimidazole in Solution 6.

(Comparative Example 4) A test device for detecting occult blood in urine was prepared in the same manner as in Inventive Example 2, except that in Solution 6, 690 mg of dilaurylthiodibrobionate, which is a mercapto compound, was used instead of 2-mercaptobenzimidazole. Obtained.

The test devices of Invention Examples 1 and 2 and Comparative Examples 1, 2, 3, and 4 were examined for changes over time during storage. Each test device was placed in an aluminum can containing a desiccant and left in an environment with a temperature of 20 to 30° C. and a humidity of 40 to 60% RH.

The test devices of Examples 1 and 2 of the present invention showed no color development or discoloration at all even after two months had passed.

In contrast, the test devices of Comparative Examples 1 and 2 developed a blue color immediately after production.

Further, both of the test devices of Comparative Examples 3 and 4 turned brown immediately after production.

<Effects of the Invention> As described above, according to the test agent and test device of the present invention, changes over time during storage can be prevented.

Out teenager wish Reason same Terumo Corporation

Claims (8)

[Claims] (1) A test agent for measuring hydroperoxide or a hydroperoxide produced by a reaction using peroxidase or a peroxidase-like reaction, or a test agent for measuring peroxidase or a peroxidase-active substance, wherein the hydroperoxide has peroxidase or peroxidase-active substances. 1. A test agent comprising: an oxidized coloring indicator that develops color when oxidized during a reaction with a substance; an effective amount of an oxidizing agent; and a stabilizer represented by the following general formula I. General formula I ▲ Numerical formulas, chemical formulas, tables, etc. , halogen atom, amino group, nitro group, carboxyl group, or alkoxy group) (2) The test agent according to claim 1, wherein the oxidizing agent is MXO_4 (M is an atom that can become a monovalent cation, and X is a halogen atom). (3) The MXO_4 is NaIO_4 or HIO_4
The test agent according to claim 2. (4) The oxidized color indicator is selected from the group consisting of benzidine, o-tolidine, 3,3′,5,5′-tetra(lower alkyl)benzidine, and 2,7-diaminofluorene. Item 3. Test agent according to any one of Items 1 to 3. (5) The test agent according to any one of claims 1 to 4, wherein the stabilizer is 2-mercaptobenzimidazole. (6) The test agent according to any one of claims 1 to 5, which contains polyvinylpyrrolidone and a copolymer of methyl vinyl ether and maleic anhydride as additives. (7) A test device comprising the test agent according to any one of claims 1 to 6 supported on a carrier. (8) The test device according to claim 7, wherein the surface of the carrier is coated with a semi-permeable membrane.