JP2000035427A - Manufacture of dry type analysis element and dry type analysis element - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve conservative performance of an alalysis element by supplying water solution containing at least enzyme and disaccharide to the developed layer of a dry type analysis element formed on a transparent supporting body, and exposing it to the hot air to dry it. SOLUTION: Water solution containing enzyme such as cholesterol esterase, cholesterol oxidase, or peroxidase and disaccharide such as tolehalose, sucrase, maltose, or lactose is spread on the developed layer of an analysis element formed on a light transmitting and liquid impermeable supporting body. After spreading, it is exposed to the hot air of about 70 deg.C, for example, at temperature of 30-70 deg.C, and adjusted at dew point of 0-10 deg.C to be dried. This drying temperature is changed according to a formed analysis element, for example, for an analysis element used for determination of total cholesterol, about 55-70 deg.C is most favorable.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a dry analytical element for quantifying components contained in biological fluids such as blood and other liquid samples, and a method for producing the same.

[0002]

2. Description of the Related Art Dry analytical elements for quantifying components in blood are widely known, for example, commercially available under the names of Fuji Dry Chem (Fuji Photo Film Co., Ltd.), Vitros (Johnson & Johnson) and the like. I have. In the case of Fuji Dry Chem, dry analytical elements for quantifying general chemical substances such as total cholesterol, neutral fat, glucose, total bilirubin, total protein, uric acid, calcium, and inorganic phosphorus in blood, and creatine kinase in blood Dry analytical elements for measuring the activity of enzymes such as ze, amylase, transaminase, alkaline phosphatase, lactate dehydrogenase, and transpeptidase.

[0003] As a useful method for quantitative analysis of cholesterol, which is one of the components in blood, for example, a method for quantifying hydrogen peroxide or cholestenone produced by oxidizing cholesterol in the presence of cholesterol oxidase is disclosed in Japanese Patent Publication No. 54-8318. No. (applicant: National Research and Development Corporation).

It has also been known from US Pat. No. 3,925,164 (Boehringer Mannheim) to combine this method with the release of cholesterol by cholesterol esterase for application to the determination of total cholesterol, including bound cholesterol. Have been. The method described above comprises cholesterone and H20 derived from cholesterol.
2 is quantified by a known method. H2
To quantify 02, the use of a grinder reagent is useful,
Coexisting bilirubin causes errors in the analysis results. As a method for preventing this bilirubin interference, coexistence of a ferrocyanide salt is known from US Pat. No. 4,291,121 (Miles Laboratories). It has been found that the addition of a ferrocyanide salt to a dry analytical element containing the same or a similar hydrogen peroxide detection reagent system) significantly impairs the storage stability of the analytical element.

[0005] As a useful method for quantitative analysis of CPK (creatine kinase), which is another component in blood, creatine phosphate (hereinafter abbreviated as CP) and ADP as substrates, and the ATP produced is glucose and hexokinase ( Hereinafter, HK is abbreviated as glucose-6-phosphate (G6).
P) and further converted to NADH or NADPH by NAD + or NADP + and glucose-6-phosphate dehydrogenase (hereinafter abbreviated as G6PDH),
A method of measuring the CPK enzyme activity by measuring the increase in H or NADPH at a wavelength of 340 nm is disclosed in
No. 9988, which is widely used as the SSCC method and the like.

[0006] However, this method has a drawback that the photometry in the ultraviolet region is required, so that the equipment becomes expensive, and that there are interferences from many compounds having absorption in the ultraviolet region.

The NADH produced by the enzymatic reaction is converted from the electron-accepting dye precursor and the electron transfer agent by a colorimetric analysis method for forming a formazan dye having an absorption spectrum in the visible region by a spectrophotometry in the visible region. Can be determined. Attempts have been made to introduce a reaction system used for the above-mentioned CPK activity measurement into a dry analytical element which can perform quick and simple analysis without the need for complicated solution preparation work as compared with the solution method. No. 88097, JP-A-61-2541
No. 99.

[0008] These known CPK activity measuring elements have a porous carrier, and creatine phosphate, ADP, CPK are added to the porous carrier or a water-permeable layer in liquid contact with the porous carrier.
Activators thiol compounds, glucose, HK, N
Contains AD +, G6PDH, electron transfer agent, and formazan dye-forming tetrazolium salt and gelatin. More preferably, a porous layer, a water-permeable layer and a water-impermeable / light-permeable support which are in liquid contact with the porous layer are integrally laminated in this order, and the porous layer or the liquid contact therewith. Creatine phosphate, ADP, CP in related water permeable layer
Thiol compounds that are K activators, glucose, HK,
A dry multilayer for measuring CPK enzyme activity, comprising NAD +, G6PDH, and an electron transfer agent, wherein at least one of said water-permeable layers (in liquid contact with the porous layer) is made of gelatin and contains a formazan dye-forming tetrazolium salt. It is an analysis factor. However, it was difficult to say that these analytical elements had sufficient storage performance.

Several techniques for stabilizing macromolecules such as enzymes and proteins using saccharides are known. For example, JP-A-8-187095 (Toyobo) discloses a method for stabilizing cholesterol oxidase by freeze-drying using a saccharide and a reagent kit for measuring cholesterol.
Japanese Patent Publication No. 7-79694 (Quadrant) discloses a method for protecting a protein, which is carried out by drying at a temperature above freezing point in the presence of trehalose, and impregnation with trehalose used for the protein. A disclosed porous substrate is disclosed. However, what is disclosed in these publications is a technique for preventing inactivation of macromolecule activity during drying, which is immobilized in a matrix structure by drying with hot air at about 70 ° C. from around room temperature. In a dry analytical element having an enzyme, no technique has been suggested that the storage stability of the analytical element is improved by adding a disaccharide.

[0010]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing an enzyme-containing dry analytical element for quantifying components contained in a biological fluid such as blood and other liquid samples, and a method for producing a dry analytical element containing the enzyme. Another object of the present invention is to provide a dry analytical element having improved storage stability.

[0011]

An object of the present invention is to provide a method for producing a dry analytical element having at least a color-forming reagent layer and a developing layer on a transparent support, wherein an aqueous solution containing at least an enzyme and a disaccharide in the developing layer is provided. At about 30-70 ° C
And a dry analysis element manufactured by the method.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention can be applied to various known dry analysis elements containing enzymes. In particular, the present invention can be applied to an element containing a solid carrier through which both the detection reagent system and the test liquid can pass. The element consists of an undercoat layer, a porous liquid spreading layer,
It may have a multilayer structure having a water absorbing layer, an adhesive layer, a reaction reagent layer, a light shielding layer, a filtration layer, and other known layers.
Such an analysis element is disclosed in, for example, JP-A-63-11969.
7, No. 63-158000, and JP-A-63-3249.
No. 9, No. 63-283600 and the like.

The disaccharides that can be used in the present invention include trehalose, sucrose, maltose, lactose, etc. Among them, trehalose and sucrose are preferred. These disaccharides are 0.5-4
0 g / m ^2, preferably present in an amount of 4~20g / m ^2.

Hereinafter, a dry analytical element for quantifying cholesterol in blood will be described as one of the examples to which the present invention can be applied. Such a dry analytical element is described in, for example, JP-A-63-119697. However, when a support is used, a practically usable configuration of the dry analytical element is as follows.

(1) One having a liquid developing layer on a support. A water absorbing layer may be provided between the support and the liquid developing layer.

(2) One having a reagent layer and a liquid developing layer on a support in this order. A water absorbing layer may be provided between the support and the reagent layer. In this case, it is preferable that cholesterol esterase, cholesterol oxidase, peroxidase, and disaccharide are contained in the liquid developing layer.

Cholesterol esterase (E.C.
3.1.1.13) is an enzyme that converts cholesterol ester contained in the sample into cholesterol, and cholesterol oxidase (EC 1.1.3.6) is used.
Is an enzyme that oxidizes cholesterol to produce hydrogen peroxide. Peroxidase (EC 1.11.1.
7) is an enzyme involved in a reaction for oxidatively coloring a coloring reagent composition using hydrogen peroxide. Examples of cholesterol esterase include E. coli. C. Not only the enzymes classified in 3.1.1.13 can be used, but also other single enzymes having cholesterol esterase activity (eg,
Lipase MR and lipase 3000 having cholesterol esterase activity described in JP-B-56-45599.
R), and combinations of two or more enzymes (eg,
Lipase having cholesterol esterase activity described in JP-A-45599 (eg, Lipase MR, Lipase 30)
00R), and a protease (eg, chymotrypsin (E.
C. 3.4.21.1), papain (EC 3.4.
21.2), promelain (EC 3.4.22.
4), a combination with pronase R) can be used.

Cholesterol oxidase (E.C.
1.1.3.6) is an enzyme that oxidizes cholesterol to generate hydrogen peroxide. Cholesterol oxidase can be appropriately selected from commercially available enzymes and used. If necessary, it can be used together with coenzyme FDA (flavin adenine dinucleotide).

As a peroxidase, Japanese Patent Publication No. Sho 56-4
No. 5599, Japanese Patent Publication No. 57-5520, and other peroxidases of plant and animal origin (EC 1.1).
1.1.7), microorganism-derived peroxidases described in JP-B-58-5034 (EC 1.11.1.
7) can be used. Of these, non-specific peroxidases of plant or microbial origin are preferred. Horseradish, as an example of a preferred peroxidase,
Peroxidase extracted from radish, Cochliob
peroxidase extracted from microorganisms of the genus olus or Curvularia.

The encapsulating reagent layer contains all or a part of the coloring reagent composition and is formed of a hydrophilic polymer. The hydrophilic polymer used in the reagent layer is generally a natural or synthetic hydrophilic polymer having a swelling ratio upon absorption of water at 30 ° C. in the range of about 1.5 to about 20, preferably about 2.5 to 15. Examples include gelatin (eg, alkali-treated gelatin, acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (eg, phthalated gelatin, etc.) agarose, polyacrylamide, polyvinyl alcohol, polyvinyl pyrrolidone, and the like. The dry thickness of the reagent layer is usually in the range of about 1 μm to about 100 μm, preferably in the range of about 3 μm to 30 μm. The reagent layer may contain a surfactant (cationic, anionic, amphoteric, or nonionic) or a buffer, if necessary.

The chromogenic reagent composition contained in the chromogenic reagent layer contains a chromogen and a coupler. The chromogen and the coupler are oxidatively coupled with hydrogen peroxide in the presence of peroxidase to form a quinone imine dye to form a color. Or a leuco dye that is oxidized by hydrogen peroxide in the presence of peroxidase to become a dye and develop color.

As the chromogen, Ann. Clin. Bi
ochem. , 6, 24-27 (1969).
-Aminoantipyrine (also known as 4-aminophenazone, i.e., 1-phenyl-2,3-dimethyl-4-amino-3-pyrazolin-5-one), JP-A-59-54962.
No. 1- (2,4,6-trichlorophenyl)
-2,3-dimethyl-4-amino-3-pyrazolin-5
-One, 1- (3,5-dichlorophenyl) -2,3-
Tri-substituted-4-amino-3-pyrazolin-5-ones such as dimethyl-4-amino-3-pyrazolin-5-one; 1-phenyl-2,
3-dimethyl-4-dimethylamino-3-pyrazolin-
4-Aminoantipyrine analogs such as 5-one can be used. Among these compounds, 4-aminoantipyrine, 1- (2,4,6-trichlorophenyl)
-2,3-Dimethyl-4-amino-3-pyrazolin-5
-One, 1- (3,5-dichlorophenyl) -2,3-
Dimethyl-4-amino-3-pyrazolin-5-one and the like are preferred.

As the coupler, Ann. Clin. B
iochem. 6, 24-27 (1969);
5-25840, JP-B-58-45599, JP-B-58-18628, JP-A-55-164356, JP-A-56-124398, and JP-A-56-155852.
2-hydroxy-1-benzenesulfonic acid, 4-hydroxy-1-benzenesulfonic acid, 3,5-dichloro-2-hydroxy-1-benzenesulfonic acid, 2-hydroxy-3-methoxy Phenolsulfonic acids such as -1-benzenesulfonic acid (including alkali metal salts and alkaline earth metal salts); 1-naphthol,
2-naphthol; dihydroxynaphthalene such as 1,7-dihydroxynaphthalene; naphtholsulfonic acid such as 1-hydroxy-2-naphthalenesulfonic acid and 1-hydroxy-4-naphthalenesulfonic acid (such as alkali metal salts and alkaline earth metal salts). Other phenol or naphthol derivatives. Of these compounds,
1,7-dihydroxynaphthalene, 1-hydroxy-2
-Naphthalenesulfonic acid (including Na salt, K salt and Li salt) and 3,5-dichloro-2-hydroxy-1-benzenesulfonic acid (including Na salt, K salt and Li salt) are preferred.

As the leuco dye, there are JP-B-57-551.
2- (4-hydroxy-3,5-dimethoxyphenyl) -4,5-bis [4- (dimethylamino) described in No. 9
Triphenylimidazole leuco dyes such as [phenyl] imidazole; and 2-arylimidazole described in JP-A-59-193352.
(3,5-dimethoxy-4-hydroxyphenyl) -4
A diarylimidazole leuco dye such as-[4- (dimethylamino) phenylto5-phenethylimidazole;
Diarylindolimidazole leuco dyes such as 2- (2-phenyl-3-indolyl) -4,5-di "4- (dimethylamino) phenylimidazole" described in JP-A-61-4960; Triarylmonoacylimidazole leuco dyes such as 2- (4-hydroxy-3,5-dimethoxyphenyl) -3-acetyl-4,5-bis [4- (diethylamino) phenyl] imidazole described in 229868; -(4-hydroxy-3,5-dimethoxyphenyl) -3-methyl-4,5
And triarylmonoalkylimidazole leuco dyes such as -bis [4- (diethylamino) phenyl] imidazole.

Examples of the light-permeable and water-impermeable support that can be used in the dry analytical element include polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, and cellulose esters (eg, cellulose triacetate, cellulose acetate protease). Thickness of about 50 μm to about 1 m consisting of a polymer such as pionate
m, preferably in the range from about 80 μm to about 300 μm. If necessary, an undercoat layer may be provided on the surface of the support to enhance the adhesion between the support and the detection layer or other layers provided on the support. Further, instead of the undercoat layer, the surface of the support may be subjected to physical (for example, glow discharge or corona discharge) or chemical activation treatment to improve the adhesive strength.

The analytical element may be provided with a water-absorbing layer on a light-permeable / water-impermeable support (in some cases, via another layer such as an undercoat layer). The water-absorbing layer is generally a layer in which the dye formed in the presence of the test component is not substantially diffused, and is made of a hydrophilic polymer that absorbs water and swells. The hydrophilic polymer used in the water-absorbing layer is generally a natural or synthetic hydrophilic polymer having a swelling ratio upon absorption of water in the range of about 1.5 to about 20, preferably about 2.5 to 15, at 30 ° C. Examples include gelatin (eg, alkali-treated gelatin, acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (eg, phthalated gelatin, etc.) agarose, polyacrylamide, polyvinyl alcohol, polyvinyl pyrrolidone, and the like. The dry thickness of the water-absorbing layer is usually in the range of about 1 μm to about 100 μm, preferably in the range of about 3 μm to 30 μm. The water-absorbing layer may contain a surfactant (cationic, anionic, amphoteric, or nonionic) or a buffering agent, if necessary.

The analytical element may be provided with a detection layer on a light-permeable / water-impermeable support (in some cases via another layer such as an undercoat layer). The detection layer is generally a layer in which the dye formed in the presence of the test component diffuses and can be optically detected through the light-transmitting support. The detection layer is
It can be composed of the same kind of hydrophilic polymer as the water absorbing layer. Hardening agents, surfactants (cationic,
Anionic, amphoteric, or nonionic), buffer, and light shielding (reflective or absorptive) fine particles can be contained as necessary.

On the water absorbing layer, the light shielding layer, the filtering layer, the reagent layer, etc., an adhesive layer for bonding and laminating the spreading layer may be provided. The adhesive layer is preferably made of a hydrophilic polymer that can adhere the spread layer when wet with water or when swollen with water. Examples of the hydrophilic polymer that can be used for the adhesive layer include the same hydrophilic polymers as those used for the water absorbing layer. Of these, gelatin, gelatin derivatives, polyacrylamide and the like are preferred. The dry thickness of the adhesive layer is generally about 0.5μ.
m to about 20 μm, preferably about 1 μm to about 10 μm
Range. The adhesive layer can be provided by a method in which an aqueous solution containing a hydrophilic polymer and a surfactant or the like added as necessary is applied onto a reagent layer or the like by a known method.

Examples of buffers that can be contained in the reagent layer and other layers of the analytical element include carbonates, borates, phosphates and Biochemistry, Vol. 5, No. 2, p. Page (1966)
Good's buffer and the like. These buffers are used in "Basic Experimental Methods for Proteins and Enzymes"
(Takeichi Horio et al., Minamido, 1981).

The light-shielding layer is preferably a water-permeable layer in which light-reflective fine particles are dispersed using a hydrophilic polymer as a binder. The light-reflective fine particles were spotted and supplied to the developing layer when the detectable change (color change, color development, etc.) generated in the reagent layer (or the water-absorbing layer) was reflected and measured from the light-transmitting support side. It blocks the color of the aqueous liquid, particularly the red color of hemoglobin when the sample is whole blood, and also functions as a light reflection layer or background layer. Examples of the light-reflecting fine particles include pigment fine particles such as titanium dioxide fine particles (rutile, anatase or brookite fine crystal particles having a particle diameter of about 0.1 μm to about 1.2 μm, etc.), and barium sulfate fine particles. And the like. Preferred are rutile-type titanium dioxide fine particles. Examples of the hydrophilic polymer binder include, in addition to the above-mentioned hydrophilic poly of the kind used in the water absorbing layer, weakly hydrophilic regenerated cellulose and the like. Among them, gelatin, polyacrylamide and the like are preferable. Known hardeners can be added to gelatin and gelatin derivatives. The light-shielding layer can be provided by applying an aqueous dispersion of the light-shielding fine particles and the hydrophilic polymer onto the water-absorbing layer, the reagent layer, and the like by a known method, and drying.

The analysis element may contain the light-shielding fine particles as described above in the spreading layer, if necessary. The spreading layer is preferably a spreading layer having a liquid measuring action. Having a liquid metering action means that a liquid sample supplied by spot application on the surface thereof has a substantially constant ratio per unit area in the plane direction of the layer without substantially unevenly distributing the portion contained therein. Has the effect of spreading.

Materials constituting the matrix of the spreading layer include paper, non-woven fabric, woven fabric (eg, plain weave such as broad), knitted fabric (eg, tricot knit, double tricot knit, etc.), and a membrane formed from a brush polymer. A filter or a three-dimensional lattice-like structure composed of polymer microbeads or the like can be used. Among these, a fibrous layer represented by a woven fabric and a knitted fabric is preferable. These are described in detail in JP-A-55-1.
Nos. 64356, 57-66359 and 60-2
Reference may be made to U.S. Pat. It is preferable that these woven fabrics and knitted fabrics have been substantially removed of oils and fats attached at the time of production of yarns, woven fabrics or knitted fabrics by a degreasing treatment such as washing with water.

These layers can be laminated by applying an aqueous solution containing a water-soluble polymer such as gelatin, or by laminating a nonwoven fabric or the like.

It is preferable that the enzyme and the disaccharide are contained in the same layer. In the slide for total cholesterol and the slide for CPK, it is particularly preferable to include them in the spreading layer. In this case, it can be produced by applying a solution containing the enzyme and the disaccharide on the spreading layer and drying.

When the solution containing the enzyme and the disaccharide is dried after being applied, the drying conditions are important, and the drying is generally performed by blowing hot air at around room temperature to 70 ° C. For example, warm air adjusted to a temperature of 30 to 70 ° C and a dew point of 0 to 10 ° C can be used.

The drying temperature differs depending on the dry analytical element to be manufactured. For example, in an analytical element used for quantification of total cholesterol, about 30 to 70 ° C., preferably about
0-70 ° C, more preferably about 55-70 ° C. On the other hand, in the analysis element used for quantification of CPK, about 30 to
70 ° C., preferably in the range of about 30-45 ° C.

The integrated multilayer analytical element manufactured by the present invention is cut into small pieces such as a square having a side of about 15 mm to about 30 mm or a circle having substantially the same size.
No., JP-A-54-156079, and JK-56-125
No. 424, No. Sho 55-32350 and Tokuyo Sho 58
It can be used as an analysis slide in a slide frame or the like disclosed in JP-A-501144.

Further, JP-A-6-50959 and JP-A-6-2
The invention can also be applied to an analysis element having no slide frame disclosed in, for example, Japanese Patent No. 73424.

The dry analytical element produced in the present invention has a capacity of about 5
μl to about 30 μl, preferably about 8 μl to about 15 μl
One aqueous liquid sample is spotted onto the spreading layer and optionally incubated at a substantially constant temperature in the range of about 20 ° C to about 45 ° C, preferably 35 ° C to 40 ° C.
Then, from one side (from the side of the light-transmitting support in the case of the integrated multilayer analytical element), a detectable change such as color change or color development in the dry analytical element is measured by reflection. Analyze the components to be measured.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[0041]

EXAMPLES Example 1 [Preparation of Analytical Element for Total Cholesterol Measurement] 180 μm thick colorless and transparent PET coated with gelatin
(Polyethylene terephthalate) on a smooth film,
A leuco dye solution having the following composition was applied (190 cc /
m ² ) and dried to form a color reagent layer having a dry thickness of about 20 μm.

Gelatin 25 g / m ² Leuco dye 474 mg / m ² shown below

[0043]

Embedded image

An aqueous solution having the following composition was applied (50 cc / m ² ) on the color reagent layer and dried to form a reflective layer having a dry thickness of about 3 μm.

Gelatin 3 g / m ² Titanium oxide 930 mg / m ² Potassium ferrocyanide 712 mg / m ²

Water was supplied to the entire surface of the reflective layer at a rate of about 30 mg / m ² to wet it.
Tricot knitted fabric (thickness of about 250 μm) in which ET spun yarn is knitted with 36 gauge is overlaid with light pressure.
The fabric was dried to form a knitted fabric spreading layer.

The thus-developed spreading layer contains an enzyme-containing aqueous solution containing a reagent as an aqueous solution so as to have the following composition, and the aqueous solution has a content per m ^{2 of} 0.75, 1.5, 4.
An aqueous solution to which trehalose was added so as to be 5, 9.0 or 15.0 g was almost uniformly penetrated (150 cc).
/ M2), dried with hot air adjusted to a temperature of 60 ° C and a dew point of 10 ° C to prepare an analytical element for measuring total cholesterol.

Peroxidase 43500 U / m ² Cholesterol esterase 2300 U / m ² Cholesterol oxidase 4600 U / m ² Ascorbate oxidase 13000 U / m ² Potassium phosphate 5 g / m ²

[Evaluation of slide storage performance] The above-mentioned analytical element was sealed and packaged in an aluminum laminate bag under an environment of 10% humidity, stored at 45 ° C for 7 days, and then 10 µl of a liquid having a total cholesterol amount of 200 mg / dl. Reflection optical density (OD) after 6 minutes when spotted and kept at 37 ° C. in a closed container
r (6)) was measured at a wavelength of 505 nm. In FIG.
FIG. 4 shows the relationship between the ratio of ODr (6) on slides after storage at 45 ° C. for 7 days to ODr (6) on slides before storage at 5 ° C. for 7 days and trehalose content.

From FIG. 1, it is clear that the total cholesterol analysis element according to the present invention has better storage performance over time than the conventional analysis element.

FIG. 2 shows the relationship between the trehalose content and ODr (6) for the analysis elements before storage at 45 ° C. for 7 days. From now on, in the analysis element before storage,
It turns out that sufficient performance is shown without adding trehalose.

Example 2 A color developing reagent layer and a reflective layer were formed on a PET film in the same manner as in Example 1, and a knitted fabric spreading layer was formed thereon in the same manner as in Example 1. Then preparing an enzyme-containing aqueous solution having the same composition as prepared in Example 1, glucose to the solution and the solution, mannitol, sucrose, maltose, trehalose, as content per m ² is each 4.5g The added aqueous solution is allowed to penetrate almost uniformly (15
0 cc / m ² ) and dried under the same conditions as in Example 1 to prepare various analytical elements for measuring total cholesterol.

Each analysis element created as described above is
After storage at 5 ° C. for 7 days, the storage performance was evaluated in the same manner as in Example 1. The results are shown in FIG.

FIG. 3 shows that the addition of a disaccharide to the total cholesterol analysis element improves the storage performance, and in particular, the effect of tresulose is great.

Example 3 [Preparation of Analytical Element for Creatine Kinase Measurement] 180 μm thick colorless and transparent PE coated with gelatin
A formazan dye precursor solution having the following composition was applied (163 cc / m ² ) on the T-smooth film and dried to form a color reagent layer having a dry thickness of about 20 μm.

Gelatin 20 g / m ² Formazan dye precursor 800 mg / m ² shown below

[0057]

Embedded image

After water was supplied to the entire surface of the coloring reagent layer at a rate of about 30 mg / m ² to wet it, a tricot knitted cloth (thickness about 250 μm) obtained by knitting a PET spun yarn equivalent to 50 denier by 36 gauge was used. The layers were overlapped under light pressure and dried to form a knitted fabric spreading layer.

An enzyme-containing aqueous solution containing a reagent as an aqueous solution so as to have the following composition, and a disaccharide so that the content per m 2 becomes 1.3 and 6.3 g, respectively, are added to the thus formed spreading layer. The obtained aqueous solution was substantially uniformly permeated (126 cc / m ² ) and dried by blowing hot air adjusted to a temperature of 35 ° C. and a dew point of 0 ° C. to prepare an analytical element for creatine kinase measurement.

Hexokinase 9762 U / m ² Glucose-6-phosphate dehydrogenase 13938 U / m ² Diaphorase 1132 U / m ² Ascorbate oxidase 4993 U / m ² Creatine phosphate 1.85 g / m ²

Each of the analysis elements created as described above is
Creatine kinase was stored at 74 ° C for 74 days.
The absorbance at 540 nm when the solution containing 0 U / l was spotted at 10 μl each and kept at 37 ° C. in a closed vessel was measured at 2.5 minutes and 4.0 minutes, and the fluctuation was calculated. FIG. 4 shows the results.

FIG. 5 shows changes in the residual activity of the enzyme G6PDH contained in the analysis element using sucrose as a disaccharide.

From the above results, it is clear that the creatine kinase analysis element according to the present invention has better storage performance than the conventional creatine kinase analysis element.

[0064]

According to the present invention, an enzyme-containing dry analytical element having excellent storage performance can be obtained.

[Brief description of the drawings]

FIG. 1 shows the amount of trehalose contained in the total cholesterol analysis element and the color density (ODr) before and after storage of the analysis element.
(6)) The ratio relationship is shown.

FIG. 2 shows the relationship between the amount of trehalose and the color development density (ODr (6)) for the analysis element before storage over time.

FIG. 3 shows the storage performance of a total cholesterol analysis element containing various disaccharides.

FIG. 4 shows the effect of adding a disaccharide on the preservation of creatine kinase assay elements.

FIG. 5 shows the effect of sucrose addition on the reduction of enzyme activity during storage in a creatine kinase assay.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/48 C12Q 1/48 Z G01N 33/92 G01N 33/92 A // G01N 33/70 33/70 F term (reference) 2G045 AA13 AA16 BB21 BB49 CA25 CB03 DA20 DA69 FB01 FB11 FB16 GC12 HA14 4B063 QA01 QQ03 QQ27 QQ76 QR02 QR03 QR04 QR07 QR12 QR41 QR43 QR66 QR84 QS39 QX01

Claims (7)

[Claims] In a method for producing a dry analytical element having at least a color-forming reagent layer and a developing layer on a transparent support, an aqueous solution containing at least an enzyme and a disaccharide is supplied to the developing layer. A method for producing a dry analytical element, comprising drying by applying hot air. 2. The method according to claim 1, wherein said enzyme is peroxidase, cholesterol esterase and cholesterol oxidase, and said disaccharide is at least one selected from trehalose, sucrose and maltose. Element manufacturing method. 3. The method for producing a dry analytical element according to claim 1, wherein said hot air is at a temperature of about 40 to 70 ° C. 4. The method for producing a dry analytical element according to claim 1, wherein said hot air is at a temperature of about 55 to 70 ° C. 5. The method for producing a dry analytical element according to claim 1, wherein the enzymes are hexokinase, glucose-6-phosphate dehydrogenase and diaphorase, and the disaccharide is sucrose. 6. The method for producing a dry analytical element according to claim 1, wherein the hot air is at about 30 to 45 ° C. 7. A dry analytical element produced by the method according to claim 1, 2, 3, 4, 5, or 6.