JPH0484898A - Testing tool - Google Patents

Abstract

PURPOSE:To obtain the title tool enabling simple, quick detection ant quantification by providing on a substrate a reagent layer with a reagent containing an oxalic acid oxidase, peroxidase and color-developing agent carried thereon. CONSTITUTION:Firstly, a substrate 2 is prepared by making e.g. a glass into a thin plate 20-500mum thick. Second, a carrier to form a reagent layer 3 0.1-2mm thick is prepared by selection from nonfibrous porous materials such as membrane filters. Third, a reagent solution is prepared by formulating e.g. gelatin with a reagent comprising (A) 0.1-3.0U per 1cm<2> of the reagent layer of an oxalic acid oxidase derived from e.g. moss, (B) 0.5-15U per 1cm<2> of the reagent layer of a peroxidase derived from e.g. leukocytes, (C) a color-developing agent such as otolidine, (D) 0.5-10mg/ml of a reaction promoter such as riboflavin, and (E) 1.0-20mg/ml of a sensitizer such as quinoline. Fourth, said carrier is immersed in this solution and then dried to obtain the reagent layer 3. Finally, the reagent layer 3 is applied on the substrate 2, thus obtaining the objective testing tool 1. When needed, using the present testing tool 1, the oxalic acid concentration in urine is determined to make a preventive diagnosis for e.g. lithangiuria.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a test device for detecting the pressure of oxalic acid or its salts in a specimen.

<Conventional technology> Approximately 80% of upper urinary tract stones and 50% of lower urinary tract stones in urolithiasis are calcium-containing stones, of which calcium oxalate stones are said to account for the majority. (Hyakude, “Epidemiology of Urolithiasis in Japan,” Journal of the Japan Urology Society, 70.975 (1970)).

The relationship between the onset of calcium oxalate stones and urinary calcium concentration or urinary oxalate concentration has long been attracting attention. Calcium has been given importance.

However, in recent years, the urinary oxalate concentration of stone patients has increased.
It has come to be recognized that there is a slight increase in urinary oxalate concentration compared to that in normal people, and urinary oxalate concentration has come to be regarded as an important risk factor for the development of urinary stones.

Conventionally, the method for detecting oxalic acid in urine is permanganese M? i! The i-method, fluorescence method, colorimetric method, etc. are known, but all of these methods require steps such as precipitation, solvent extraction, and permeation through ion-exchange resins in order to eliminate the effects of other components in urine. A pretreatment step to separate the acid was essential.

In addition, as separation analysis methods using equipment, gas chromatography (GC method), high performance liquid chromatography (HPL method)
C method) and ion chromatography method (IC method) are known, but GC method and HPLC method require derivatization for the purpose of improved separation and high sensitivity detection, and IC method requires only one sample. It was necessary to wash the column every time.

As described above, all of the conventional methods for detecting urinary oxalic acid have the drawbacks that they require many operational steps, are complicated, require skill, and require long measurement and analysis times.

<Problems to be Solved by the Invention> The present invention has been made in view of the above-mentioned shortcomings of the prior art, and its purpose is to provide a test device that can quickly detect and quantify oxalic acid in a sample with simple operations. Our goal is to provide the following.

Means for Solving the Problems> Such objects are achieved by the following inventions (1) to (4).

(1) A test device having a reagent layer supporting a reagent on a support, wherein the reagent contains oxalate oxidase, hydrogen peroxide decomposing enzyme, and a coloring agent.

(2) The test device according to (1) above, wherein the reagent layer contains riboflavin as a reaction accelerator.

(3) The test device according to (1) or (2) above, wherein the reagent layer contains quinoline or a derivative thereof as a sensitizer.

(4) The test device according to any one of (1) to (3) above, which is used for diagnosing stones.

It decomposes into oxygen atoms, and the chromogen develops color due to the action of these oxygen atoms.

<Function> In the present invention, oxalic acid in a sample can be detected by simply immersing the test device in the sample or dropping the sample onto the test device. That is, oxalic acid in the sample reacts with the reagent supported on the reagent layer, which causes the reagent layer to develop a color, and the degree of coloration can be visually determined or optically measured to determine whether or not the oxalic acid in the sample is present in the sample. oxalic acid can be detected and quantified.

In this case, the typical coloring principle of the reagent layer is as follows:
As shown in the formula below, oxalic acid in the sample and oxygen taken in from the outside air are decomposed into carbon dioxide and hydrogen peroxide by oxalate oxidase, and this hydrogen peroxide is converted to peroxidase (hydrogen peroxide decomposing enzyme). Example: The test device of the present invention will be described in detail below based on a preferred example shown in the accompanying drawings.

FIG. 1 is a cross-sectional front view showing an example of the configuration of the test device of the present invention. As shown in the figure, the test device 1 has a reagent layer 3 disposed on one side of a support 2.

The support 2 has a plate shape, for example, with a thickness of about 20 to 500 mm, and is made of a material that is inert to the specimen.

Specific constituent materials of the support 2 include polyethylene terephthalate, cellulose ester (cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.), polycarbonate of bisphenol A, polymethyl methacrylate, polyvinyl chloride, polypropylene, polystyrene. , various resins such as polyvinyl alcohol, or glass. Further, the support 2 may be a stack of sheets made of two or more of the above materials.

The reagent layer 3 is composed of a carrier supporting a reagent to be described later.

The carrier is preferably made of a non-fibrous or fibrous porous material.

Typical examples of non-fibrous porous materials include filter paper and membrane filters, as well as dispersions of porous materials such as diatomaceous earth and microcrystalline materials dispersed in a binder, and microspherical beads of glass or resin that are bonded to each other. Examples include porous aggregates bonded at points.

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 thickness of the carrier, that is, the thickness of the reagent layer 3 is not particularly limited, but is preferably about 0.1 to 2 mm, particularly about 0.5 to 1 mm.

The main drugs that make up the reagent are oxalate oxidase,
Examples include hydrogen peroxide-degrading enzymes and coloring agents (chromogens).

Examples of oxalate oxidase include those derived from barley seedlings and moss (Mnium menziesii).

Mnium insigne, Mnium oval
e, Hylocomium splendens, E
urohynchium 5tokesii.

Rhytidiadelphns triquetru
s) Examples include white rice.

The amount of oxalate oxidase supported on the reagent layer 3 is not particularly limited, but is 90.1 to 3 per 1°ff12 area of the reagent layer.
．． Ou (unit) degree, especially 0.3 to 1. It is preferable to set it to about Ou.

Examples of hydrogen peroxide decomposing enzymes include peroxidase and caffrase.

As the peroxidase, peroxidases derived from various animals and plants, such as those derived from horseradish, figs, white blood cells, and milk, can be used.

The amount of hydrogen peroxide decomposing enzyme supported on the reagent layer 3 is not particularly limited, but is 0.5 to 1 cII12 of the area of the reagent layer.
About 15u (unit), especially 1.5 to 5. It is preferable to set it to about Ou.

As a coloring agent, benzidine or its compound such as o-tolidine, m-)-lysine, benzidine, tetramethylbenzidine, 0-methylbenzidine, 0-dianisidine, 2,7-diamitfluorene, or 4-aminoantipyrine ( 4-AAP) and a coupling agent that causes coupling with the 4-AAP, 3-methyl-
Benzothiazolinone hydrazone (MBTH) and MBT
Examples include a combination of H and a coupling agent that causes coupling.

As the coupling agent, p-chlorophenol,
Phenol derivatives such as 2.4-dichlorophenol, 2.4-dibromophenol, 2,4.6-trichlorophenol, naphthalene derivatives such as 4-chloro-1-naphthalene, 1,7-sihydronaphthalene, or N, N-
Dimethylaniline, N,N-diethyl-m-toluidine, N-ethyl-N-sulfopropyl-m-toluidine,
N-ethyl-N-(2-hydroxy-3-sulfopropyl) m-toluidine (TOOS) 5,6,7°8-
Tetrahydro-1-naphthylamine, N-ethyl-N-
Examples include aniline derivatives such as (2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline.

In addition, the reagent layer 3 may optionally contain a pH adjuster, a surfactant (wetting agent), a light reflective substance, a reaction accelerator, a stabilizer,
A sensitizer, oxidizing agent, thickening agent, etc. may be added.

Examples of the pH adjuster include phosphoric acid (phosphate buffer), citrate buffer, borate buffer, Tris buffer, and Gud buffer.

The surfactant (wetting agent) is used to uniformly wet (penetrate) the sample liquid when the reagent layer 3 and the sample come into contact, and includes polyvinylpyrrolidone, sodium cholate, sodium lauryl sulfate, and sodium dodecyl sulfate. , alkyl sulfates such as sodium tetradecyl sulfate, alkyl benzene sulfonates such as sodium dodecylbenzenesulfonate, sodium dioctyl sulfosuccinate, sodium dibutyl sulfosuccinate, sodium di-2-ethylhexyl sulfosuccinate (Aerosol-OT)
Examples include dialkyl sulfosuccinic salts such as.

The light-reflecting substance is used to eliminate the influence of red blood cells on color density measurement when the sample is whole blood, and includes fine particles such as titanium oxide, barium sulfate, aluminum, and various ceramics.

The reaction accelerator is for promoting the reaction of oxalate decomposition by oxalate oxidase to further shorten the detection time, and includes, for example, riboflavin.

The amount of this reaction accelerator added is not particularly limited, but the concentration in the immersion liquid is about 0.5 to 10 mg/mj, particularly 1.
It is preferably about 0 to 5.0 mg/+nj.

Among the stabilizers, those for improving color stability after coloring include a copolymer of methyl vinyl ether and maleic anhydride, or a half ethyl ester thereof.

In addition, as stabilizers for preventing changes over time during storage of test devices, Japanese Patent Publication No. 56-43238, Japanese Patent Application No. 01
-238148 (especially 2-mercaptobenzimidazole) and the like.

The sensitizer enhances the activity of the coloring reaction of the chromogen by hydrogen peroxide-degrading enzyme, and includes quinoline and its derivatives, such as quinine, cinchonine, 6-medoxyquinoline, quinaldine, 8-amino- Examples include 6-medoxyquinoline, 2-quinolitool, isoquinoline, benzo(f)quinoline, and 3-aminoquinoline. The presence of such a sensitizer usually accelerates the oxidation reaction and increases the color intensity of the oxidized chromogen, increasing the sensitivity of the test device.

The amount of the sensitizer added is not particularly limited, but the concentration in the immersion liquid is about 1,0 to 20 mg/mj, and especially about 10 to 1.
It is preferable to set it to about 5 mg/ml.

The thickener is used to prevent the drug from flowing out of the carrier in the reagent layer 3, and includes polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, acrylate,
Examples include polymers such as polyacrylamide, poly(hydroxyethyl methacrylate), poly(hydroxyethyl acrylate), carboxymethyl cellulose, gelatin, gum arabic, and the like.

The above reagent (including additives) may be supported on a carrier by impregnating a liquid carrier containing the reagent (for example, by immersing the carrier in an immersion liquid), and then drying it.

When there are two or more types of liquids, impregnation and drying are repeated.

Note that such a reagent layer 3 can be formed by applying, for example, double-sided adhesive tape (not shown), adhesive, or other holding device (for example, the holding member described in Japanese Patent Application No. 63-334198) to the surface of the support 2. Fixed by

Further, the reagent layer 3 may be obtained by applying a liquid containing a reagent or the like to the surface of the support 2 and drying it.

In this case, the reagent layer 3 contains a predetermined reagent etc. in a hydrophilic binder (binder) such as gelatin, polyvinyl alcohol, polyvinylpyrrolidone, agarose, polyvinylbenzene sodium sulfonate, polyurethane, polyvinylbrobionate, etc. (dispersed) composition.

In addition to the various additives described above, a hardening agent (crosslinking agent) may be added to such a coating type reagent layer 3.

As a hardening agent, it crosslinks gelatin etc.
For example, N,N'-hexamethylene-1,6-bis(
1-aziridinecarboxamide) (HDLI), trimethylpropane-triβ-aziridinylpropionate (TAZM), tetramethylolmethanetri-β
- Aziridine rust conductors such as aziridinyl propionate (TAZO).

The thickness (dry film thickness) of such a coating type reagent layer is 1, but not particularly limited, about 1 to 100 mm, especially 5 to 30 mm.
It is preferable to set it to about 7J11.

In the illustrated example, there is only one reagent layer 3, but a plurality of reagent layers each containing reagent components of different compositions may be laminated. In particular, a layer in which a pre-light distribution reflective material is dispersed (light reflective layer) may be separately provided on the surface of the reagent layer 3.

Although the test device of the present invention has been described above with reference to the configuration example shown in the drawings, the present invention is not limited thereto. In particular, the layer structure of the test device can be arbitrary, and may include a developing layer, a light reflecting layer, an absorbing layer (sample amount adjustment layer), a protective layer, an oxidizing agent-containing layer, and other layers provided for various purposes. There may be layers.

The test device of the present invention can be used for specimens (e.g., whole blood, plasma,
It is used to detect oxalic acid in body fluids such as serum, urine, feces, saliva, lymph fluid, cerebrospinal fluid, or other test fluids).
Among these, it is particularly preferable to use it for detecting oxalic acid in urine.

In other words, since urinary oxalate concentration is closely related to the development of urinary tract stones and kidney stones, measuring urinary oxalate concentration with the test device of the present invention can be used to detect urinary tract stones, etc. Prevention and diagnosis can be performed easily and quickly. In this sense, the test device of the present invention is preferably used for testing and diagnosing stones, particularly urinary stones and kidney stones.

In this case, the test device contains 0.1 to 50 mg per 1 dj of urine.
In particular, it is preferable that 0.5 to 10 mg of oxalic acid can be quantitatively determined.

The test device of the present invention is suitable for medical use, but is not limited to this, and can also be applied to other fields such as analysis of food and environmental samples.

In particular, since oxalic acid is toxic, the Food Sanitation Act stipulates the permissible amount in food, and it can be used to determine whether the amount of oxalic acid in food is within the permissible range. Furthermore, in beer and the like, oxalic acid derived from raw materials may precipitate and reduce its commercial value, so it can also be used to control the quality of beer.

<Experiment example> Experimental examples of the present invention will be described below.

(Experiment Example 1) Prepare solutions 1 and 2 with the compositions shown below, and make a solution of approximately 1.
First, add solution 1 to a mm filter paper (Toyosha No. 1650).
was impregnated and dried (40'C, 160 minutes), and then impregnated with Solution 2 and dried (40'C, 110 minutes).

After that, cut this into a size of 511IIl x 5■,
A test device for detecting urinary oxalic acid was obtained by attaching the sample to a polystyrene sheet support having a thickness of 0.3 mm using double-sided adhesive tape.

In addition, this test device has a concentration of l, o to 50 + ag per 1 dj of urine.
of oxalic acid can be quantified.

5000 u Peroxidase (derived from horseradish) 24000 u Riboflavin (reaction accelerator) 50 mg 0.2M citrate buffer 10 mJO, 1% sodium alginate Dissolved in 10 mJ 1 cap Utetramethylbenzidine (color developer) Standard urine with a known oxalic acid concentration was prepared as a sample in the dissolution method in 500+mg 3-aminoquinoline (sensitizer) 250mg benzene.

Standard urine is obtained by collecting human urine from a normal person, and measuring the oxalate concentration in the human urine using an oxalate measurement kit (manufactured by Sigma).
An aqueous oxalic acid solution was added to give a concentration of 20 and 50111 g/dj to prepare standard urine. In addition, the human urine was diluted with distilled water, and the oxalic acid concentration was 1 and 2, respectively.
Standard urine of ff1g/dj was prepared.

The test device was immersed in each standard urine for about 1 second, and after 3 minutes had passed, the color intensity of the test device was measured using a reflection spectrophotometer (MCPD-200, manufactured by Tenba Denshi Co., Ltd.).

The results are shown in FIG.

This figure 2 shows the maximum absorption wavelength 670 of the reflected light spectrum.
The logarithmically converted value of the relative reflected light intensity in nm is plotted for each urinary oxalate concentration.

(Experimental Example 2) The coloring agent in Solution 2 in Experimental Example 1 was 4-AAP:30.
The urinary oxalate concentration was measured in the same manner as in Experimental Example 1 except that 0mg and TOO5:350mg were used.
Calibration curve similar to Figure 2 (maximum absorption wavelength of spectrum 56
5 nm) cannot be obtained.

(Experimental Example 3) The coloring agent in Solution 2 in Experimental Example 1 was o-tolidine: 5
When the urinary oxalic acid concentration was measured in the same manner as in Experimental Example 1 except that the amount was changed to 00 mg, a calibration curve similar to that shown in FIG. 2 (maximum absorption wavelength of the spectrum: 630 nap) was obtained.

<Effects of the Invention> As described above, according to the present invention, oxalic acid in a sample such as urine can be rapidly detected or quantified with a simple operation, and the detection accuracy is also high.

Explanation of symbols 1...Test tool 2...Support 3...Reagent layer applicant representative person same Terumo Corporation

Claims (1)

[Scope of Claims] (1) A test device having a reagent layer carrying a reagent on a support, the reagent containing oxalate oxidase, hydrogen peroxide decomposing enzyme, and a coloring agent. Characteristic test equipment. (2) The test device according to claim 1, wherein the reagent layer contains riboflavin as a reaction accelerator. (3) The test device according to claim 1 or 2, wherein the reagent layer contains quinoline or a derivative thereof as a sensitizer. (4) The test device according to any one of claims 1 to 3, which is used for diagnosing stones.