JPH06258319A - Test paper for detection of oxypurine in urine - Google Patents

Abstract

PURPOSE:To semiquantitatively detect the existence of oxypurine in urine by a method wherein two layers as a development layer an a reaction layer are formed on a base material and the respective layers are impregnated with inspection reagents. CONSTITUTION:In a sheet of test paper 1, at least two regions as a development- layer part 3 and a reaction-layer part 4 are formed on a sheet of paper 2 as a base material. The development-layer part 3 is impregnated with ascorbic acid oxydase, peroxidase and N-ethyl-N-m-toluidine sodium salt, and the reaction-layer part 4 is impregnated with 4-amino-antipurine and xanthineoxydase. When the parts are impregnated with these constituent components, H2O2 which is generated by the combination of oxypurine with the xanthineoxy-dase is guided to a coloring system by using the oxi4izable color reagent of the proxydase, the 4-aminoantipurine and the Methyl-N-m-tolui4 ine sodium salt. Then, when the reaction-layer part 4 is colored, the oxypurine is detected.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a test strip which can be easily used as a clinical test method and can semi-quantitatively detect oxypurine in urine.

[0002]

2. Description of the Related Art Chemical tests and quantification of body fluid components such as urine and blood in clinical tests are important test methods for the treatment and prevention of pathological conditions, and have been used in various clinical tests. ing. In particular, with the spread of medical examinations, grasping clinical test values is now indispensable for early confirmation of pathological conditions, and the accuracy and the speedup and simplification of test methods are being promoted.

At present, as a means for such speeding up and simplification, test papers found in urine or blood tests are in widespread use. This is a sheet impregnated with a test reagent required for detection and quantification of the target component in the test liquid, which is simply immersed in the test liquid or dropping the test liquid. It is capable of detecting and quantifying, and can omit complicated preparation of a reagent at the time of inspection, can make accurate judgment even if it is not an expert, and is widely used in clinical examination.

[0004]

By the way, hyperuricemia is a congenital or acquired metabolic disease which is based on hyperuricemia which is caused by metabolic disorder of purine nucleotide which is one of intracellular high energy carriers. It is said that Gout is an acute monoarthritis caused by hyperuricemia. It most often develops in the phalangeal joint of one side and is relieved even if left untreated, but joint pain is severe and it is not uncommon to be unable to walk. Moreover, since there are no symptoms until an attack occurs, it is inevitable that a clinical examination by confirming the amount of uric acid excreted in urine is necessary to grasp hyperuricemia at an early stage.

As the largest factors inducing this hyperuricemia (gout), excessive alcohol intake, skeletal muscle exercise, mental stress and their synergistic effects have been widely highlighted. The clues to these clarifications were motor hyperuricemia and the results of ethanol exercise. These results have greatly changed the etiology of hyperuricemia, and have also made great impacts on the improvement of prevention and treatment methods. To date, the cause of most hyperuricemia is thought to be these disorders of energy metabolism regulation,
It has become the most important issue to measure not only the uric acid level in blood but also the exposure of hypoxanthine and xanthine, which are precursors of uric acid, to extracellular fluid. However, although methods such as high-performance liquid chromatography and enzyme colorimetric method have been developed as the measurement methods up to now, this method is actually quite complicated and a method that can be easily applied clinically is still successful. Absent.

Therefore, it has been desired to develop a method for measuring the exposure of hypoxanthine and xanthine, which are the precursors of uric acid, to the extracellular fluid by a simpler and more accurate measurement method. The present inventors have conducted extensive studies to develop such a simple and accurate measurement method,
Oxypurines such as xanthine, which is a precursor of uric acid,
Focusing on excretion in the urine with a constant energy load, semi-quantitative detection of the presence of this oxypurine inevitably leads to the quantification of uric acid levels in the urine, and therefore hyperuricemia I found that the inspection test paper of can be made.

[0007]

SUMMARY OF THE INVENTION In order to achieve the above object, the present invention has two layers of a developing layer and a reaction layer formed on a substrate, and ascorbic acid oxidase is provided in the developing layer portion. Peroxidase, N-ethyl-N- (2-
A test strip for detecting oxypurine in urine, which is obtained by impregnating hydroxy-3sulfopropyl) -m-toluidine sodium salt and impregnating a reaction layer portion with 4-aminoantipyrine and xanthine oxidase. Furthermore, the present invention provides a test paper for detecting oxypurine in urine, which has an interfering substance removal layer formed by providing an unimpregnated layer between the spreading layer and the reaction layer in the above-mentioned constitution.

The present invention will be described in detail below with reference to the drawings. FIG. 1 shows a test paper according to a specific embodiment of the present invention, in which at least two regions of a spreading layer portion 3 and a reaction layer portion 4 are formed on a filter paper 2 which is a base material. Then, the developing layer portion 3 is impregnated with ascorbic acid oxidase, peroxidase, and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine sodium salt, and the reaction layer portion 4 is It is impregnated with 4-aminoantipyrine and xanthine oxidase.

In this embodiment, the unimpregnated layer portion 5 is provided between the spreading layer portion 3 and the reaction layer portion 4, and the unimpregnated layer portion 6 is provided below the spreading layer portion 3. However, these layers 5 and 6 do not necessarily have to be provided, and it is sufficient that the two layers of the developing layer portion and the reaction layer portion are divided on the filter paper. In the above configuration, the ascorbic acid oxidase impregnated in the spreading layer portion 3 on the filter paper is intended to decompose urinary ascorbic acid and eliminate the influence of ascorbic acid. That is, ascorbic acid is a reducing substance, and since it interferes with the inhibition of color development in the next reaction layer portion, it is impregnated to remove this influence.

Further, peroxidase acts on H ₂ O ₂ to oxidize an oxidation indicator into a corresponding dye. Furthermore, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine sodium salt was added to the reaction layer portion 4 in the presence of peroxidase as an oxidation indicator.
Is similarly impregnated with 4-aminoantipyrine as an aniline derivative that forms an oxidative condensate with H ₂ O _{2. By} impregnating these into the development layer portion 3, the test liquid is reacted with the reaction layer. Ascorbic acid, which has a strong reducing property, is removed and decomposed while being developed in the portion 4. In addition, xanthine oxidase is an enzyme that specifically acts on oxypurine in a test solution and produces H ₂ O ₂ corresponding to oxypurine which is a target component. You can use those of milk origin.

By impregnating these constituents, H ₂ O ₂ generated by the combination of oxypurine-xanthine oxidase is converted into peroxidase and 4-aminoantipurine, N-ethyl-N- (2-hydroxy-).
3-Sulfopropyl) -m-toluidine sodium salt is introduced into a coloring system using an oxidizable coloring reagent, and it is detected by the coloring of the reaction layer portion. The test paper for detecting oxypurine in urine of the present invention may be any one in which at least the above-mentioned respective components are impregnated in the development layer part and the reaction layer part which are sectioned on the filter paper. As long as the purpose is quantitative, it may be further impregnated with other components that do not interfere with the purpose. Such other ingredients include thickeners, buffers, D-mannitol,
Examples include bovine serum albumin and the like.

The thickener is used to prevent the outflow of the drug as a constituent component impregnated in the test paper of the present invention, and a typical example of such a thickener is polyvinyl. Alcohol, polyacrylamide, gelatin,
Carboxymethyl cellulose, polyvinylpyrrolidone,
Examples include sodium alginate and the like. Further, it is preferable to impregnate the developing layer portion and the reaction layer portion with a buffering agent.
It is impregnated for the purpose of keeping to ~ 8, and as such a buffering agent, sodium citrate-sodium hydroxide, veronal sodium-hydrochloric acid, potassium monophosphate-dibasic sodium phosphate, Examples include potassium monophosphate-sodium hydroxide. Further, D-mannitol and bovine serum albumin are preferably impregnated in both the reaction layer portion and the development layer portion, and are impregnated as a stabilizer for preventing the inactivation of the enzyme when the test paper is dried.

The substrate used in the present invention includes filter paper, glass fiber, non-woven fabric made of plastic material, and the like.
Any substance that does not react with the impregnating liquid or dissolves in the impregnating liquid and has absorbency may be used. Among them, filter paper generally used as clinical test paper is preferably used. In the present invention, the unimpregnated layer 5 is provided between the spreading layer 3 and the reaction layer 4 in the above-described specific structure. However, by providing the unimpregnated layer 5, the interfering substance removal layer is formed in urine. An oxypurine detection test strip is provided. The purpose of providing this interfering substance removal layer is to remove these interfering components because it may be difficult or impossible to make determination if the urine of the subject contains colored components. This is for forming a layer.

Next, a method for producing the test paper of the present invention will be described. Second
As shown in the figure, a rectangular filter paper (for example, ADVANT
EC TOYFILTER PAPER CHROM
(ATOGRAPHY 514A 20 × 400 mm) is evenly divided into 4 sections, the upper layer portion is the reaction layer portion 4, the next layer is the unimpregnated layer portion 5, and the next is the development layer portion 3, and these reaction layer portion 4 and the development layer portion. 3 is coated with the components to be impregnated respectively. Then, the filter paper 2 thus impregnated is freeze-dried, and then cut into a desired test paper to manufacture the desired test paper of the present invention.

[0015]

According to the above-mentioned constitution of the present invention, since it can be easily measured, it is possible to perform mass screening for oxypurineuria which should be called a preparative group of hyperuricemia. This will open up a completely new perspective for the prevention of the development of gout and hyperuricemia, and at the same time, it will be useful for elucidating the detailed mechanism of the onset of hyperuricemia based on these results. I can say. In particular, the test paper of the present invention can detect and quantify the target component in the test liquid by the shade of the color tone of the reaction layer portion by a quick and simple method by simply immersing it in urine as the test liquid and developing it. In addition, the preparation of complicated reagents at the time of examination can be omitted, and even an unskilled person can make accurate determination, which greatly improves the actual clinical examination.

[0016]

EXAMPLES The present invention will be described more specifically below with reference to examples. Needless to say, the following examples do not limit the present invention. A development layer reagent and a reaction layer reagent composed of the following components are prepared.

Reaction layer reagent : 2-fold concentration McIlvaine buffer solution (pH 7.4) 0.2 ml 0.4M sodium phosphate 18.17 ml 0.2M citric acid prepared from 1.83 ml 50 mM 4-aminoantipyrine 0.1 ml 20% (w / v) D-mannitol 0.125 ml 20% (w / v) bovine serum albumin 0.125 ml 250 U / ml xanthine oxidase 0.05 ml 2.5% (w / v) polyvinyl alcohol 0. 3 ml (degree of polymerization: 2000) Deionized distilled water 0.1 ml Total 1.0 ml

Developing layer reagent : 2-fold concentration McIlvaine buffer (pH 7.4) 0.2 ml 0.4M sodium phosphate 18.17 ml 0.2M citric acid prepared from 1.83 ml 100 mM N-ethyl-N- (2- Hydroxy 3-sulfopropyl) -m-toluidine sodium salt dihydrate 0.1 ml 10% (w / v) D-mannitol 0.05 ml 10 (w / v) bovine serum albumin 0.05 ml 4000 U / ml Ascorbate oxidase 0.05 ml 1000 U / ml Peroxidase 0.05 ml 2.5% (w / v) polyvinyl alcohol 0.4 ml (Polymerization degree: 2000) Deionized distilled water 0.14 ml Total 1.0 ml

Then, a rectangular filter paper of 20 × 400 mm (ADVANTEC TOYO FILTER PAP
ER CHROMATO GRAPHY 514A) is evenly divided into four sections in the lateral direction, and from the top, the first section is the reaction layer section, the second section is the unimpregnated layer section, the third section is the developed layer section, and the fourth section is unimpregnated. A layer portion is formed, and the reaction layer reagent component and the development layer reagent component adjusted as described above are applied to the reaction layer portion and the development layer portion, respectively, to complete the impregnation. Then, the filter paper thus impregnated was freeze-dried, and then the filter paper was cut in the longitudinal direction into a width of 20 × 5 ml to obtain a desired test paper for detecting oxypurine in urine.

Model test : Using the test paper for detecting oxypurine in urine produced as described above, a detection test using a model test solution was carried out. The following model test solutions were prepared. (1) 200 μM xanthine-containing physiological saline (2) Xanthine 500 μM-containing physiological saline (3) Hypoxanthine 200 μM-containing physiological saline (4) Hypoxanthine 500 μM-containing physiological saline (5) Inosine 200 μM-containing physiological saline (6) Saline containing 500 μM inosine (7) Saline (control solution) 100 μl of each test solution is dispensed into each sample cup. The test paper for detecting urinary oxypurine obtained above is placed in the liquid in each sample cup with the development layer portion facing downward, and the bottom 2 mm of the test paper is immersed. In this state, the test liquid is started to spread to the reaction layer portion above the development layer portion. 30 minutes after the development, the shade of the reddish purple color tone of the reaction layer portion is judged.

Judgment: Test liquid color tone (1) Saline containing 200 μM xanthine ++ (2) Saline containing 500 μM xanthine ++ (3) Saline containing 200 μM hypoxanthine ++ (4) Containing 500 μM hypoxanthine Saline +++ (5) Saline containing 200 μM inosine ± (6) Saline containing 500 μM inosine ± (7) Saline (control solution) −

Test with urine test solution: Human urine sample 1
00 μl was dispensed into each sample cup and tested in the same manner as above. The shade of the reddish purple color of the reaction layer portion 30 minutes after the development of each test paper was compared with the reddish purple color tone of the reaction layer portion in the above model test to determine the concentration of oxypurine in urine. . As a result, although the oxypurine concentration was negative, the higher the oxypurine concentration was, the more positive the urine sample was, and the reaction layer portion was strongly reddish purple.

[Brief description of drawings]

FIG. 1 is a test paper according to a specific example of the present invention.

FIG. 2 is a diagram showing a method for producing the test paper of the present invention.

[Explanation of symbols]

 1 Test paper 2 Filter paper 3 Development layer part 4 Reaction layer part 5,6 Unimpregnated layer part

Claims (6)

[Claims] 1. A development layer and two reaction layers are formed on a substrate, and ascorbic acid oxidase is provided in the development layer portion.
Peroxidase, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine sodium salt was impregnated, and 4-aminoantipyrine was added to the reaction layer portion.
A test paper for detecting oxypurine in urine, which is impregnated with xanthine oxidase. 2. The test paper for detecting oxypurine in urine according to claim 1, wherein the base material is a filter paper, a non-woven fabric made of a glass fiber or a plastic material. 3. The test paper for detecting oxypurine in urine according to claim 1, wherein the base is a filter paper. 4. The test paper for detecting oxypurine in urine according to claim 1, which is further impregnated with a thickener, a buffer, D-mannitol, and bovine serum albumin. 5. The thickener is selected from polyvinyl alcohol, polyacrylamide, gelatin, carboxymethylcellulose, polyvinylpyrrolidone and sodium alginate, and the buffer is sodium citrate-sodium hydroxide, veronal sodium-hydrochloric acid, monophosphoric acid. The test paper for detecting oxypurine in urine according to claim 1, which is selected from potassium-dibasic sodium phosphate and monobasic potassium phosphate-sodium hydroxide. 6. The interfering substance removing layer is formed by providing an unimpregnated layer between the spreading layer and the reaction layer.
The test paper for detecting oxypurine in urine according to [4].