JPH06510426A - Glucose measurement reagent - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Reagent mixture for glucose measurement This invention relates to reagent mixtures, and in particular to improving the persistence of color over time. The present invention relates to measuring reagent mixtures in carrier gels.

Background of the invention Blood and sample held in the test stick or test nozzle The drug reacts and develops a color, and by observing this color, the operator can detect the blood. Fluid samples are often used to determine the amount of glucose or other components in them. It will be done. The reagent pad may contain, for example, glucose oxidase and peroxidase. An enzyme, glucose oxidase, converts glucose in blood samples into Contains 0-tolidine as a chromogen that turns blue when oxidized to phosphoric acid and hydrogen peroxide. You may. Hydrogen peroxide reacts with 0-tolidine in the presence of peroxidase. The intensity of the resulting color is determined by the amount of hydrogen peroxide released. It is therefore determined by the amount of glucose in the blood. This reagent is usually used for example f f Natural gels such as gelatin or synthetic polymers such as polyvinylpyrrolidone It is integrated with a gel matrix such as

Similar to the amount of blood that comes into soft contact with the liquid, the amount of blood that comes into contact with the reagent is A problem arises in that the color produced is affected by the time it is held. So Therefore, it is customary for such tests to be performed within a strictly defined examination time schedule. Due to the inaccuracy of keeping time schedules, the value of the result is often It's questionable.

Surprisingly, we found that the mixing ratio of the reagents in the matrix was similar to that of blood. There is no possibility that it will affect the duration of color caused by interaction with drugs. I found it. If the mixing ratio in the matrix is within a certain range, the time The colors will stand the test of time to the extent that the need for strict schedules is alleviated. It is established.

Summary of the invention Therefore, the present invention provides the enzymes glucose oxidase and peroxidase and Compatible with hydrogen peroxide resulting from the oxidation of blood glucose by glucose oxidase. Provides a blood test reagent mixture composition consisting of interacting chromogens, Glucose with oxidase and peroxidase of 300-700 international units (IU) oxidase and at least 20 IU peroxidase. The chromogen is present in 12 to 500,000 IU of glucose oxidase. It is characterized in that it is present in an amount defined by 20 g of active chromogen. gluco -s oxidase is 400-5 per 27.5-32.51 U of peroxidase. 501U is present, and the chromogen is o-tolidine and glucose oxidizer Ze500. Preferably 12 to 15 g per OOOIU are present.

Preferably, the reagent mixture is integrated with the gel matrix, especially in this case. The gel matrix contains glucose oxidase 500. per OOOIU, dry It is preferable that the weight is defined as 200 to 400 g.

The reagent mixture/gel matrix is preferably absorbed into the microporous membrane carrier. More preferable.

The enzyme reagents used herein may be present in any suitable form, e.g. Active enzyme dry powder, precursor, or active enzyme in the reagent mixture during test conditions. It may be an addition product thereof that produces an enzyme. In this way, enzyme reagents can be It can be an active enzyme such as lucose oxidase or peroxidase, or For example, their stable forms such as acetate or other salts or adducts thereof may also be used. This releases the active enzyme when the reagent mixture is wetted.

For convenience, the invention hereinafter refers to described in terms of a mixture of glucose oxidase and peroxidase. It will be done.

Similarly, from this point on, the term ``chromogenic substance'' refers to the enzyme reagent or the blood being measured. something that develops properties by interacting with one or more products produced when it reacts with a liquid It is used here to indicate a substance. Such phrases are or other substances that exhibit detectable or non-visible properties. However However, chromogenic substances that express colors within the visible spectrum are desirable. For example, dianisidine or. -Tolidine is used to prevent glucose in the blood from mixing with the reagent. Reacts with hydrogen peroxide released when reacting with glucose oxidase in the compound . Chromogenic substances can be used in the form of active substances, precursors, or adducts thereof. The adducts may be used, especially their inorganic acid salts such as hydrochloride or sulfate. The active ingredient is released during testing.

For convenience, this invention will hereinafter be referred to as a chromogenic material. - From the perspective of tolidine I can be seen.

Enzyme reagents and chromogenic substances are effectively associated with each other; can interact with each other. Typically, the reagent and the chromogenic substance are physically mixed with each other. or mix the enzyme reagent with the glucose in the blood sample immediately before use. The reaction products due to the interaction diffuse into the region containing the chromogenic substance, and the enzymes interact. Reagents and chromogenic substances are kept separate from the region in such a way that color develops there. This is also within the scope of the present invention. For example, enzyme reagents may be concentrated on one end or side of a reagent pad. However, the chromogen may be concentrated at its opposite end or face.

Conveniently, in this invention, from this point on, the enzyme reagent is distributed substantially uniformly throughout the pad. It is described in terms of a reagent mixture band containing a drug and a chromogenic substance.

The reagent mixture is conventional, e.g. phosphate buffer reagents, preservatives, anticoagulants, or Other materials such as surfactants may also be included. Such other substances are commonly the substance being tested, other components in the reagent mixture, and any reactions or interactions that may occur during the test process. It is inert to the products of use. Such other components are included in the reagent mixture. May be present in amounts commonly used.

As mentioned above, we consider that if the enzyme reagent and chromogenic material are present in the reagent mixture composition, between the substance being measured and the various components in the mixture if they were present within the ratio of The colors produced by the interaction of the two are surprisingly stable and last longer than ever before. We have discovered that this makes it possible to observe intercolours. Such enzyme reagents are usually mixed. Glucose oxidase in the compound is 400-6001U, especially 45-o-550U. IU of peroxidase, at least 201 U, usually 27.5 It is present in a proportion of ~351U. The chromogenic substance is usually glucose oxidase 5° It is present in an amount of 12 to 17 g, especially 13 to 16 g per O,0001 U. this The optimum ratio within the range can be determined by a simple trial and error test depending on the conditions and carrier. You can decide.

As mentioned above, preferably the reagent mixture is integrated with the matrix carrier medium. so that the substance being measured can penetrate the enzyme reagent or chromogenic substance. . This matrix can be, for example, gelatin, agar, meat jelly, silica gel, etc. Natural gum, jelly, gel, cellulose gel, polyvinyl chloride resin gel provided by synthetic polymers such as For convenience, in this invention, the gel It will be described in terms of using gelatin gel as a matrix.

The gel matrix has enzymatic reagents and chromogenic substances dispersed almost uniformly throughout it. can be retained. This includes enzymes in a premix of gel material and chromogen material. This is accomplished by mixing reagents and allowing the mixture to solidify into the desired shape. is convenient. Alternatively, the enzymatic reagent and chromogenic material may be kept together during mixing. Mixed with a thixotropic gel carrier that is kept in a fluid state by stirring or other operations. This may then be hardened for storage and transport prior to use. A gel layer is formed at a high concentration on the upper layer of Trix, and the lower layer containing a large amount of reagent is where it acts as a protective or covering layer, or where the enzyme reagent contains a chromogenic substance. selectively if located in a region of the matrix separated from the matrix may contain enzyme reagents and chromogenic materials heterogeneously dispersed therein.

In this case, the product that results from the interaction of the substance being tested with one or more enzyme reagents diffuses from the area of the enzyme to the area containing the chromogenic substance, and in that area separate Colors are expressed as individual stages.

For convenience, the invention hereinafter refers to reagents uniformly dispersed throughout the gel matrix. Stated in terms of mixtures.

The matrix holding the reagent mixture can be in a number of physical forms, e.g. may be integral with the disk, and the pads of the matrix are attached to the test piece or the disk. Interaction of the substance held on one side and measured with the reagents and substances in the matrix or against a white background of supporting strips or discs or another related background. observed visually. Another method is to use Matri Tsuku Z consecutively. The reagent mixture may be diffused through this area to be measured. The interaction between the substance and the enzyme occurs in the first region, and the product of this interaction is the color Diffusion to the second region where the reaction occurs. Yet another method is to use a reagent mixture matrix. The lix is absorbed or impregnated into the pores of the porous carrier and The substance to be measured is dropped onto one side, and the color developed within the Matri Noras is opposite. observed from the surface.

For convenience, the invention hereinafter refers to the nozzle or nozzle of a microporous membrane infiltrated with a reagent mixture. It will be described from the point of view of the use of the disc.

The gel matrix in conventional blood reagent mixtures is approximately 4% by dry weight. High molecular weight gelatin was present. However, the reagent mixture is absorbed into the microporous membrane. We prefer low molecular weight gelatin, usually 20.000 to 50.0 Or choose to use -9 minute -B gelatin. Gelatin like this is now Even when present in the amounts used up to We have found that cannot be held. On the other hand, the reagent mixture If the gel component in the composition exceeds about 20% by dry weight, the gel will pass through the membrane. This suppresses the diffusion of the reaction products of We found that reflecting the range. Therefore, we have glucose oxidase An amount of 250 to 325 g dry weight per 500.0 OOIU is present in the reagent mixture. More preferably, a gel matrix of low molecular weight gelatin is provided.

The present invention will now be illustrated by the following examples and unless otherwise stated. , parts and percentages are all given by weight.

The first solution was 300ml deionized water (111) 2°C of 0.5M phosphate buffer in H7. Om I, 100 ml of 2o% w/v solution of surfactant Gantrez (trade name), and a molecular weight of 25,000 to 40. Dry gelatin powder ranging from ’000 to 30 It was prepared by stirring 0 g at room temperature.

The second solution was 300 ml of deionized water, 300 ml of methoxyethanol. and 0- Prepared by stirring 15 g of tolidine hydrochloride or dianisidine hydrochloride at 60°C for 1 hour. It was.

Add the second solution dropwise into the first solution with stirring and mix at 60"C for 1 hour. I left it still.

The third solution contained glucose oxidase at 500°C in O, 1M phosphate buffer. It was prepared by mixing 001U of peroxidase with 30.0OOIU of peroxidase. This melt The liquid is mixed into another mixed solution with stirring, and 0.　1μm pore size filter filtered by.

The obtained solution was prepared using a polysulfone resin sheet (thickness: 0.2~0, 4 mm, 5 average pores). 3 liters per minute per cm under 110 gauge pressure with a diameter of 0.2 μm. 51 U of glucose oxidase per cm” of membrane.

Defined by 31 U of peroxodase, 0.2 mg of o-tolidine, and 4 mg of gelatin. determined.

For comparison, the same membrane was prepared using conventional levels of enzyme and chromogen per ICm'. impregnated with a conventional reagent mixture.

Blood samples were dropped onto the surface of multiple 6 mm diameter discs cut from each membrane. It was given down. With the reagent composition of the present invention, a blue color appeared after only 10 seconds. that color The color hue and intensity will stabilize after approximately 30 to 40 seconds, and will remain stable for another 30 to 40 seconds. Because of this situation, I had a lot of free time to observe colors. For comparison, With the current ingredient composition, the color and intensity will change within 10 to 30 seconds after dropping the blood sample. A color appeared, but after another 15 seconds the color produced became weaker and the exact color was observed. There was little or no freedom of time.

In another aspect, the present invention provides that the components of a mixture are mixed together to form a substantially homogeneous composition. A method of making a test reagent mixture of the present invention is provided which provides a mixture.

The invention further provides a method for making a test reagent mixture carried in a microporous carrier medium. The fluid reagent mixture of the present invention is contained in a carrier medium. The mixture is an example impregnated with the medium, e.g. by dipping a sheet of carrier into a solution of the mixture. It is desirable to introduce the mixture into the gel within the pores of the carrier.

Preferably, the pores of the carrier are occluded by the gel mixture, thereby Blood and cell rupture due to capillary activity through the pores is reduced. have an opening in it or attached to a test stick with an opening as the end wall of the sample receiving section. In order to Copy and translation of written amendment) Submission (Article 184-8 of the Patent Law) December 20, 1993

Claims (11)

[Claims] 1. Glucose oxidase and peroxidase are 300-700 international units (I U) of glucose oxidase and at least 20 IU of peroxidase. glucose oxidase 500,000 I Characterized by being present in an amount defined by 12-20 g of active chromogen per U The enzymes glucose oxidase and peroxidase and glucose oxidase Interacts with hydrogen peroxide resulting from the oxidation of blood glucose by oxidases A blood test reagent mixture composition comprising a chromogen. 2. The glucose oxidase is peroxidase 27.5 to 32.5 IU The chromogen is O-tolidine, and the chromogen is O-tolidine. The body is present in an amount of 12-17 g per 500,000 IU of glucose oxidase. Test reagent mixture according to claim 1, characterized in that: 3. Claim characterized in that said test reagent mixture is integrated with a gel matrix. 3. The test reagent mixture according to claim 1 or 2. 4. the gel matrix is a gelatin matrix; per 500,000 IU of glucose oxidase, 200 Test sample according to claim 3, characterized in that it is defined by ~400 g of gelatin. drug mixture. 5. The method of claim 1, characterized in that the reagent mixture is retained by a microporous membrane. A test reagent mixture as described in any of the above. 6. The molecular weight of the gelatin is 20,000 to 50,000, and glucose 250-325 g dry weight per 500,000 IU of oxidase A test reagent mixture according to claim 4 or 5, characterized in that: 7. A test reagent mixture according to claim 1, which is substantially as in the embodiments detailed heretofore. Compound. 8. that the components of a mixture are mixed with each other to provide a mixture of substantially uniform composition A method for producing a test reagent mixture according to claim 1. 9. The fluid reagent mixture according to any of claims 1 to 4 or 6 is microporous. The test according to claim 5, characterized in that the test is impregnated into the pores of a carrier membrane. Method of manufacturing reagent mixture. 10. Developed by dropping blood onto the test reagent mixture according to claim 1. A blood sample testing method characterized by observing color. 11. The reagent mixture is retained by a microporous carrier membrane, and the blood is deposited on one side of the membrane. Claim 10, wherein the color is observed from the opposite side of the film. How to put it on.