JPH02243945A - Whole blood analyzing element - Google Patents

Abstract

PURPOSE:To analyze the specific components in a whole blood sample with high accuracy regardless of the hematocrit value of the blood by forming the adhesive part of a multilayered analyzing element to many discontinuous dotty parts distributing at a prescribed density. CONSTITUTION:First to third porous layers are successively and integrally laminated via or without via a water permeable layer on a water impermeable light transmittable base. A reagent compsn. which generates an optical change detectable in the presence of the component to be detected is incorporated into one of three layers of these porous layers and the water permeable layer. Three layers of the porous layers are tightly adhered and integrated by partially disposed adhesive agents between the respective adjacent surfaces, by which the very small through-parts to the extent of not hindering the uniform permea tion of liquid are formed. The adhesive parts are formed as the many discontinu ous dotty parts distributing at the density of average <200 per 1cm<2> at this time.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing a dry chemical analysis element used for quantifying a specific substance in biological fluids, such as blood.

[Technical background] Solution reagents are used for quantitative analysis of various metabolic components in body fluids that are important in clinical medicine, such as glucose, rirubin, urea nitrogen, uric acid, cholesterol, and various enzymes (creatine kinase, AST, ALT, etc.) There is a wet method and a dry chemical analysis, that is, an analysis method that uses analysis elements such as test pieces, analysis slides, and analysis tapes into which a dry analytical reagent system is introduced. Dry chemical analysis is superior to wet methods in terms of ease of operation and speed of analysis.

Dry multilayer analysis elements have been developed as an analytical means that can quickly perform highly accurate chemical analysis on minute amounts of liquid samples, such as Japanese Patent Publication No. 53-21677 and Japanese Patent Application Laid-open No. 55-16.
No. 4356 and JP-A No. 60-222769. Dry multilayer analytical elements include, for example, a transparent support, a reagent layer,
It consists of a reflective layer and a spreading layer. A reagent layer coated on a transparent support, such as a plastic film, contains a reagent that reacts with the analyte component contained in the liquid sample and develops a color with an optical density depending on the composition of the component. The spreading layer uniformly spreads the spotted liquid sample over an area approximately proportional to the amount of liquid. When a liquid sample, for example, whole blood, is dropped onto the surface of the developing layer of such a dry analytical element at a constant rate, the blood developed in the developing layer passes through the reflective layer and reaches the reagent layer, where it reacts with the reagent. After one analytical element that develops color is kept at a constant temperature for an appropriate period of time to allow the color development reaction to proceed, the reflected optical density is measured in a specific wavelength range from the transparent support side, and quantitative analysis is performed based on a calibration curve.

The reflective layer has the role of preventing the optical measurement from being affected by the liquid sample in the developing layer.

Conventionally, in both wet and dry chemical analysis, serum or plasma from which blood cell components have been removed has often been used as a sample. However, since the operation of separating blood cell components from blood involves a lot of labor and equipment cost, it is desirable to be able to analyze whole blood as it is.

In dry chemical analysis using whole blood as a sample, blood cell components and plasma must be separated by some means in the analytical element.

The dry analytical element described in Japanese Patent Publication No. 53-21677 is provided with a filtration layer in order to separate high molecular weight components of blood cells and whole blood. However, as described in JP-A-60-111960, it takes a very long time to remove blood cell components using a filtration layer provided in the analytical element.
In addition, there was a risk that some of the analyte in the plasma would be lost in the filtration layer, making the analysis inaccurate.

[Prior art and its drawbacks] A soft analytical element that separates blood cells in the analytical element and quickly diffuses the test component in the plasma into the reagent layer is
This was proposed in Japanese Patent Application Laid-Open No. 138756/1983. This analysis element has a first non-fibrous porous layer, a second non-fibrous porous layer, and a fibrous porous layer integrally laminated in this order,
The three porous layers are brought into close contact with each other by an adhesive that is partially arranged to form micro-penetrations between adjacent surfaces so that uniform permeation of liquid is substantially unobstructed. The analytical element is an integrated multilayer analytical element that is bonded together, and one of the porous layers contains a reagent composition that develops a color in the presence of a specific component.

To manufacture such a whole blood analysis element, adhesive is partially placed between the adjacent surfaces of two adjacent porous layers, and minute penetrations (continuous ) is formed. As a method for this purpose, JP-A No. 82-138758 describes a method in which a liquid adhesive is partially attached to the surface of one porous layer using the same method as gravure printing, and the layer is overlapped with the other porous layer and bonded. A method is proposed. However, when performing whole blood analysis using this multilayer analysis element, the hematocrit value (
It has been experienced that even blood with the same component content in plasma can have different analysis results depending on the volume percentage of blood cells (volume percentage) in the blood.

In Japanese Patent Application No. 62-170468, in order to reduce the above-mentioned hemadrit dependence, the pore volume of the porous layer close to the support is set to 1/2 or less of the pore volume of the porous layer above it. Although it has been proposed, its effectiveness has not yet been sufficient.

[Technical Problems to be Solved] The present invention separates and removes red blood cells in whole blood from plasma in an analytical element to avoid interference by red blood cells, and prevents the diffusion of test components in plasma into a reagent layer. The technical BH is to provide a dry analytical element that allows rapid analysis of a specific component in a whole blood sample with high accuracy regardless of the hematocrit value of the blood.

[Means for solving technical problems] The above-mentioned problem of the present invention is to form a first porous layer and a second porous layer on a water-impermeable light-transparent support with or without a water-permeable layer. A porous layer and a third porous layer are integrally laminated in this order, and a reagent composition that causes a detectable optical change in the presence of a test component is applied to the three porous layers and the water permeable layer. contained in at least one of the sexual layers, and
The porous layers of the layer are in substantially intimate contact with each other by locally disposed adhesive such that micro-penetrations are formed between adjacent surfaces to the extent that uniform liquid transmission is substantially unimpeded. A multilayer analysis element which is glued and integrated by means of a multilayer analysis element, characterized in that said glued part consists of a large number of discrete point-like parts distributed with an average density of less than 200 dots per cm'. Solved by elements.

[Details of specific configuration of the invention] One or more of the three porous layers partially bonded with the analytical element of the present invention may be non-fibrous, or one or more may be fibrous. A 0 mm quality porosity may be provided on top of the non-fibrous porous layer, or vice versa.

As the non-fibrous porous layer, Japanese Patent Publication No. 53-21877,
Layers of plush polymers consisting of cellulose esters such as cellulose acetate, cellulose acetate/butyrate, cellulose nitrate, as described in U.S. Pat. No. 1,421,341, are preferred;
6,6-Polyamides such as nylon, microporous membranes such as polyethylene, polypropylene, etc. may be used.6 JP-A-62-
It may be a microporous membrane made of polysulfone as described in No. 27006, small polymer particles, glass particles, diatomaceous earth, etc. as described in Japanese Patent Publication No. 53-21677, Japanese Patent Application Laid-open No. 55-90859, etc. Porous layers with continuous voids bound by hydrophilic or non-water-absorbing polymers are also available. However, among the above materials, materials that cause hemolysis are undesirable because they destroy red blood cells in the sample blood and allow hemoglobin to permeate into the reagent layer.

When using a membrane filter made of a so-called plush polymer made by a phase separation method as a non-fibrous porous layer, the liquid passage path in the thickness direction is formed on the free surface side (i.e. the shiny side) during membrane manufacturing. Usually, the pore diameter is the narrowest, and when the cross section of the liquid passage path is approximated as a circle, the pore diameter is the smallest near the free surface. The minimum pore diameter in the thickness direction in the unit passage meridian further has a distribution in the plane direction of the filter, and its maximum value determines the filtration performance against particles.Normally, it is measured by the critical bubble pressure method. When using this type of membrane in the porous layer of the analytical element of the present invention, it is preferable to orient the glossy side of the membrane filter to the side far from each liquid receiving surface of the porous layer. If the layer has a porous layer, a membrane filter is placed on the side far from the residue receiving layer.
It is preferable to face the glossy side.

As the material constituting the fibrous porous layer, P paper, nonwoven fabric, woven fabric (for example, plain weave fabric), knitted fabric (for example, Tricot?-m), glass fiber 1 paper, etc. can be used.

Among these, woven fabrics, knitted fabrics, etc. are preferable; woven fabrics, etc. may be subjected to glow discharge treatment as described in JP-A No. 57-66359; for the spreading layer, RF & open-face boxes, adjusting the spreading speed, etc. In order to
It may contain a hydrophilic polymer or a surfactant as described in No. 182652.

In the analytical element of the present invention, the three porous layers are partially arranged to form minute penetrations between adjacent surfaces so that uniform permeation of liquid is substantially unobstructed. They are substantially closely bonded and integrated using an adhesive. That is, the adhesive is partially placed between the adjacent surfaces of two adjacent porous layers, so that minute penetrations (which may be continuous) are formed in the areas where the adhesive is not present. do.

To do this, as described in JP-A No. 62-138756, a liquid adhesive is partially applied to the surface of one porous layer using the same method as gravure printing, and the layer is overlapped with the other porous layer. There is a way to glue them together. Instead of a gravure roller, an intaglio printing cylinder can be used to partially apply the adhesive using an intaglio printing method. The shape, size, spacing, area ratio, etc. of the adhesive-applied portion on the gravure roller or intaglio cylinder are as described in JP-A-62-138756.

Various adhesives can be used, and the description in JP-A-62-138756 can be referred to. For example, water-based glues such as starch glues are useful.

Organic solvent-based adhesives such as aqueous solutions of polyvinyl alcohol, dextrin, carboxymethyl cellulose, etc., vinyl acetate-butyl acrylate copolymer emulsions, etc. may also be used, but adhesives with slow evaporation of the solvent are suitable.

Heat adhesives are particularly useful.

E7) Melt-type adhesives, that is, thermal adhesives, are available from Industrial Materials Vol. 26, No. 11, page 4 to 5.
Hot melt materials such as those described on page 1 can be used. That is, ethylene copolymers such as ethylene-vinyl acetate copolymer, ethylene-ethyl acrylate copolymer, and ethylene-acrylic acid copolymer, polyolefins such as low molecular weight polyethylene and acid polypropylene, and nylon. Various materials such as polyamide, polyester copolymer, thermoplastic rubber such as styrene block copolymer such as SBS, styrene-butadiene rubber, butyl rubber, urethane rubber, rosin, petroleum resin, terpene resin, paraffin, synthetic wax, etc. can be used.

The thickness of the two porous layers bonded according to the method of the invention may be the same or different, typically 50 to 600 μm,
Preferably it is an 80 to 300μ pot.

In the analytical element of the present invention, the part where the porous layer is bonded between adjacent surfaces is composed of a large number of discontinuous dots distributed at an average density of less than 200 points per ctn2. If distributed at intervals, the intervals are approximately 0.
This condition is satisfied if there are 7-bacteria or more.

The ratio of the bonded portion to the total area, that is, the area ratio, is normally in the range of 10% to 30%.

The analytical elements of the invention can have various configurations.

For example, U.S. Patent No. 3,992,158, JP-A-55-1
No. 64356 (U.S. Pat. No. 4,292,272),! l
VrM No. 62-138756, 621:11875
No. 7, No. 62-138758 can be referred to.

Practically speaking, for example, (1) a non-fiber first layer is placed on a liquid-impermeable light-transmitting support;
A porous layer, a non-fibrous porous layer, and a fibrous third porous layer in this order. (2) On the support, an adhesive layer (or water absorption layer), a non-fibrous first porous layer, and a non-fibrous third porous layer (3) On the support, a detection layer, a non-fibrous first porous layer, a non-fibrous second porous layer, mM! (4) on the support, a reagent layer, a non-fibrous first porous layer,
It is useful to have a non-fibrous second porous layer and a fibrous third porous layer, respectively in this order (the support may include an undercoat layer); the detection layer generally contains the analyte component. A layer in which dyes and the like generated in the presence of a fluorophore diffuse and can be optically detected through a light-transmitting support, and can be composed of a hydrophilic polymer. The detection layer corresponds to the liquid permeable layer in the present invention. The detection layer may contain a mordant, such as a cationic polymer for anionic dyes, and the water-absorbing layer generally refers to a layer in which the dye formed in the presence of the analyte does not substantially diffuse and swells. It can be constructed from easily hydrophilic polymer. In (3) and (4) above, a water-absorbing layer may be provided between the support and the detection layer or reagent layer.

A preferred material for the light-transparent, water-impermeable support is polyethylene terephthalate. In order to firmly adhere the hydrophilic layer, which may be a cellulose ester such as cellulose triacetate, an undercoat layer is usually provided or a hydrophilic treatment is performed.

A reagent composition is a composition capable of producing an optically detectable substance, such as a dye, in the presence of a test component, and is capable of producing an optically detectable substance, such as a dye, by directly reacting with the test component or by reacting with the test component and other substances. A composition capable of producing an optically detectable substance, such as a dye, as a result of reaction with an intermediate produced by reaction with a reagent of
compositions that produce dyes by oxidation of leuco dyes (e.g., U.S. Pat. No. 4,089,747;
Compositions containing compounds that produce dyes by coupling with other compounds when oxidized (e.g., 4-aminoantipyrine) and phenols or naphthols), compounds that can form a dye in the presence of a reduced coenzyme and an electron transfer agent, etc. can be used. In addition, in the case of an analytical element for measuring enzyme activity, for example, p
- A self-developing substrate capable of liberating a colored substance such as nitrophenol can be included in the reagent layer or developing layer.

In order to incorporate a reagent composition that produces color development in the presence of the analyte component into at least one of the aforementioned non-fibrous porous layers, the porous layer may be pre-impregnated or coated with a suitable solution or dispersion of the reagent composition. It is also useful to adhere the spreading layer onto another water-permeable layer, such as a reagent layer, as described in JP-A-55-184358.

After adhering the porous layer onto other water-permeable layers (e.g., undercoat layer, adhesive layer, water absorption layer) by the method described in JP-A-55-164356, a solution or dispersion of the reagent composition is applied. The liquid may be applied to the porous layer.

Known methods can be used for impregnating or coating the porous layer. For example, dip coating, doctor coating, extrusion coating, hopper coating, curtain coating, etc. are selected and used as appropriate.

The reagent composition may be comprised entirely in the first porous layer;
The second porous layer may contain a separate porous or non-porous layer (e.g. a detection layer). The first porous layer may contain a composition (indicator) that reacts with the intermediate to give a dye or the like.

A portion of the reagent composition may be included in a substantially uniform layer (eg, a detection layer) with a hydrophilic polymer as a binder.

Examples of hydrophilic polymers include gelatin and derivatives thereof (e.g., phthalated gelatin), cellulose derivatives (e.g., hydroxyethyl cellulose), agarose, acrylamide polymers, methacrylamide polymers, acrylamide or methacrylamide, and various vinyl monomers. copolymers, etc. can be used.

After coating a uniform layer containing a reagent composition using a hydrophilic polymer as a binder, a porous layer containing no reagent composition was coated.
5-164356, the reagent composition can be substantially contained in the first porous layer.

The reagent composition may contain an activator, a buffer, a hardening agent, a surfactant, etc. as necessary. Examples of Mm agents that may be included in the reagent layer of the analytical element of the present invention include: are carbonates, borates, gJ salts and [1ioch phase 1sLry magazine Vol. 5 No. 2, page 467,
Gutsud (G) described on page 7 (1966)
Km agent by Mr. ood) can be mentioned. These buffers are described in "Basic Experimental Methods for Proteins and Enzymes J (Takeshi Horio et al., Nankodo, 1981), the above-mentioned Bioehemis.
The selection can be made by referring to literature such as volume 5 of try magazine.

The reagent composition may also contain an enzyme, for example, as disclosed in Japanese Patent Application Laid-open No. 6
2,-138756, pages 18 to 20 can be used.

The porous layer that receives the t1c liquid is often used as a spreading layer for liquid samples, and is preferably a layer that has a liquid metering effect. This is an effect of spreading the liquid sample in the direction of the surface at a rate of approximately constant amount per unit area without substantially unevenly distributing the components contained therein. As materials constituting the fibrous porous layer intended as a spreading layer, R paper, non-woven fabric, M fabric (for example, plain weave fabric), H4 fabric (for example, tricot knit), glass fiber 2 paper, etc. are used. be able to.

Among these, woven fabrics, knitted fabrics, etc. are preferable; woven fabrics, etc. may be subjected to glow discharge treatment as described in VJ Publication No. 57-66359; JP-A-60-222770, JP-A-63
-219397, 63-112999, 62-
It may contain a hydrophilic polymer or a surfactant as described in No. 182652.

An adhesive layer for adhering and laminating the porous layer may be provided on continuous layers such as a support, an undercoat layer, a water absorption layer, and a detection layer. It is preferably made of a hydrophilic polymer capable of adhesion, such as gelatin, gelatin derivatives, polyacrylamide, starch, etc.

The second porous layer is used to measure the hemoglobin of red blood cells in whole blood when measuring detectable changes (color change, color development, etc.) in the detection layer, reagent layer, etc. from the light-transmitting support side. It can block red light and function as a light reflective layer or background layer. The second porous layer may contain light reflective particles such as titanium oxide or barium sulfate dispersed with a hydrophilic polymer as a binder, and the third porous layer may also contain light reflective particles. It's okay.

[Example 1] 1-1. Vertical Giant melon soup Eight liquids of leuco dye having the following composition A were prepared.

A.

2-(4-hydroxy-3,5-dimethoxyphenyl)
4-(4-(dimethylamino)phenyl]5-phenethylimidazole acetate 5.7g2-(4-hydroxy-3,5-dimethoxyphenyl)-4-C4-
(Dimethylamino)phenyl] 5-phenethylimidazole hydrochloride 0.8 gN, N-diethyl laurylamide 1048yjushiagimeN A gelatin solution having the following composition was prepared.

B: Alkali-treated gelatin 300g water
190
0 g Bis[(vinylsulfonylmethylcarbonyl)aminocomethane 3.0g1
t1@J-Setting Solution A was added to Solution B while stirring it at about 5,700 revolutions/minute using a TK Auto Homo Mixer (emulsifier manufactured by Tokushu Kikai Kogyo Co., Ltd.), and dispersed for about 30 minutes to prepare an emulsion.

1-2 yuan area 1 tsubo @ nasal drip The above emulsion is coated with gelatin, thickness 180μ
It was applied onto a transparent polyethylene terephthalate (PET) film (support) at a rate of 150 g per lea and dried to form a coloring reagent layer.

1-3.91- - Method: Wet the surface of the above coloring reagent layer uniformly with water at about 25°C (about 30g/I2
cellulose acetate membrane filter (microfilter FM30 manufactured by Fuji Photo Film Co., Ltd.) with effective pore diameter of 1.2μ, cocoon thickness of 140μ, and porosity of approximately 80%.
0) were superimposed and dried to adhere the membrane filter to the coloring reagent layer.

Next, on top of this membrane filter, the following composition C, which had been prepared separately from TfQ, was applied at a rate of LLonl per 1 m 2 and dried to form a first porous layer.

C: Water 1223° Cholesterol esterase 1.7g Cholesterol oxidase 10. +8゜riboprotein lipase 1.94゜peroxidase
8.86Fi Ferocyan 1 Hycarium
5.6g surfactant 4.6
゜(In the following formula [I], n=p-nonyl, n-10,
mixture of m = 2 and m = 3) [1] Cellulose acetate membrane filter with an effective pore diameter of 3 μm, a thickness of 140 μm, and a porosity of approximately 80% to form the second porous ττgFJ (microfilter manufactured by Fuji Photo Film Co., Ltd.) FM300) and a tricot knitted fabric (thickness approximately 250 μR) made by knitting 36 gauge PET spun yarn equivalent to 50 denier to form the test T1 development layer, respectively. The substituent was a p-nonyl group, n=10, a mixture of m:2 and m=3) was immersed in a 2% by weight aqueous solution to fill the voids with the liquid, then taken out and dried.

1-58, 2 and then apply a hot-melt adhesive heated to 130°C to the surface of the impregnated tricot knitted fabric preheated to 80°C using a gravure printing method and apply it using a gravure roller. It was attached in the form of dots by transfer from. The gravure roller had a dot diameter of 0.45 ma, a dot center spacing of 0.91, a dot area ratio of about 20%, and an attached solid component jt (per unit area) of about 3 bi/l112.

Immediately face the non-glossy side of the impregnated membrane filter (which will become the second porous layer) to the surface of the fabric (which will become the spreading layer) to which the high-temperature adhesive has adhered, and pass both of them between laminating rollers. Laminated/(integrated adhesive).

1-6. The above-mentioned multilayered material (spreading layer and second porous layer) was adhered onto the surface of the first porous layer by the same dot adhesion method as described above, and was fixed integrally. That is, a heated hot-melt adhesive is applied to the membrane filter surface of the multilayered product in a halftone dot pattern by transfer from a gravure roller using a gravure printing method, and then the first porous layer (support The surfaces of (and (on top of the coloring reagent layer)) faced each other, and both were passed between a laminating roller and bonded together to be laminated.

The dot diameter of the gravure roller used was 0.4 mm, the distance between dot centers was 0.8 mm, and the dot surface was T? i rate is about 20
%, the attached solid component Ji (per unit surface precision) is approximately 3g/
It was a2.

The analytical film for measuring total cholesterol activity thus completed is integrally laminated with the support, the coloring reagent layer, the first porous layer, the second porous layer, and the cloth spreading layer in this order. The cloth spreading layer and the second porous layer work together as a hemofiltration layer. The first porous layer acts as a reaction layer that generates ferric ions in the presence of cholesterol.6 The coloring reagent layer acts as a reaction layer that generates ferric ions in the presence of cholesterol. The dye is detected optically through a transparent support.

The obtained analytical film for quantitative analysis of total cholesterol was cut into square chips of 151 on each side, and was published in JP-A-58-323.
A biochemical analysis slide for total cholesterol quantification was completed by fitting it into the slide frame described in No. 50.

[Comparative Example] In place of the gravure roller used in 1-6 of Example 1 to integrate the first porous layer and the heavy N material, dots with a diameter of 0.3
mm, dot center spacing 0, 6 m+*, and a gravure roller with a dot area ratio of approximately 20% was used, except for the implementation rI
A biochemical analysis slide for total cholesterol quantification was completed using the same procedure as A1.

[Comparative Example 2] 1-6 of Example 1. Instead of the gravure roller used to integrate the first porous layer and the layered material, dots with a diameter of o, 2
1. Dot center spacing 0.4mm, dot area ratio approximately 20%
A biochemical analysis slide for quantifying total cholesterol was completed in the same manner as in Example 1, except that a gravure roller was used.

[Measurement Example 1] The color development of the analysis slide due to total cholesterol in whole blood is
Evaluation was made as follows.

A human whole blood sample containing 183 mg/d1 of plasma total cholesterol and a hematocrit value of 39% and a human plasma sample collected by centrifugation were collected, and the plasma was added to the whole blood to determine the hematocrit value. 25% whole blood, then centrifuge the whole blood to remove the red blood cell portion (lower layer)
The blood was divided into a plasma portion (supernatant) and a plasma portion (supernatant), and the plasma was removed from the supernatant to obtain whole blood with a hematocrit value of 55%. That is, three types of human whole blood samples containing 183 mg/di of total cholesterol and hematocrit values of 25%, 39%, and 55%, respectively, were prepared.

Next, 10 μl of the three types of human whole blood samples were spotted on the development layer of the analysis slides prepared in Example 1, Comparative Example 1, and Comparative PA2. After vation, P
The color optical density of the analysis slide was measured by reflection photometry from the ET support side, and the value was converted to ODt (transmission optical density) (C-nicalCbemistry, Vol.
24.1335 (1978)) in the three types of human whole blood samples (mg/d).
N) was calculated. The results are shown in Table 1.

Table 1 From Table 1, it can be seen that the analysis slide of Example 1 using a gravure roller (dot surface coverage ratio of approximately 20%) with a dot diameter of 0.4+wm and a dot center spacing of 0.81 had a dot diameter of 0.4+wm. Comparative Example 1 using a gravure roller with a 3 m dot center spacing of 0.6 mm and a dot diameter of 0.2 mm,
Compared to the analysis slide of Comparative Example 2 using a gravure roller with a dot center spacing of 0.4 mm (the dot area ratio of the gravure roller is about 20% in both cases), the influence of the hemadocrit value of the sample blood on the measurement of cholesterol level. It is clear that this is difficult to accept.

[Example 2] 2-1. Water Absorption: A gelatin aqueous solution is applied onto a 180 μm thick, colorless transparent polyethylene terephthalate (PET) film (supporting material) primed with bamboo gelatin so that the film thickness after drying is 7 μm, dried and water absorbed. formed a layer.

2-2.1 Wet the surface of the above water absorption layer uniformly with water at about 25°C (approximately 3
Cellulose acetate membrane filter with 0g/m2), effective pore size of 3μ, thickness of 140μ, and porosity of approximately 80%.
(Micro filter FM30 manufactured by Fuji Photo Film Co., Ltd.)
0) were stacked on top of each other, dried, and a membrane filter was adhered to the water absorption layer.

Next, apply the following composition [1] on top of this membrane filter.
] was applied at the indicated rate and allowed to dry.

Composition [1]: Gelatin 1.2g/
la' polyolefin particles 41
g7m' (Mitsui Petrochemical Industries, Ltd. Chemipearl M2
O0, particle size approx. 5μ, rolling density 0.92) Surfactant 0.22g
/s2 (polyoxyethylene nonylphenyl ether) trishydroxymethylaminomethane 0.32g
/m2 potassium phosphate 0
．． 32, / 2α-ketogel tar acid
0.1 g/va2L-sodium aspartate 0.5 g7m2 Oxaloacetate decarboxylase 25201U/m'FAD
6 g/piece 2T P P

24 mg7m2 pyruvate oxidase 7000 II/m2 peroxidase 12801U/mouse 2
Pigment (Structure below > 0.36
g/m2 (do not apply after adjusting pi to 7.5 with dilute NaOH solution) Dye: 2-(3,5-dimeLho)
xy-4-hydroxyphenyl)4-phen
ethy 5- (4-d 1sle thy l
a+* 1nopbeny l ) 1Taidazo
Further, two of the following compositions were applied so that the coating amount after drying was as described, and dried to form a first porous layer (light reflection/shielding layer).

Composition [2] Titanium oxide microcapsules (below) 39 g7
m2 Methyl cellulose 2g/Yu2
(65SH50 manufactured by Shin-Etsu Chemical Co., Ltd.) Solvent of methylcellulose: Methanol composition! i! J [
The titanium oxide microcapsules used in 2] were prepared as follows.

12 g of monoisobutyl diphenylethane, 8 g of methacrylic acid ester/acrylic acid copolymer (1,100,000 molecules)
was melted at 80°C, and titanium oxide powder (Ishiya Sangyo) was added to it.
Co., Ltd. PF656, average particle size: 0.2μ, 15g was added and dispersed for 8 hours in an electric bowl. The following mixture was added to this dispersion and mixed well with a glass rod.

Methacrylic acid ester/acrylic acid copolymer * (molecular weight approximately 100,000 tons, 12 g) Ethyl acetate, 18.5 g, Paraffin oil (average number of carbon atoms: 12), 12 g *Fujikura Kasei Acrybase MM2002-2 Next, add the following mixture to this. , and mixed well with a glass rod to obtain liquid (I).

Ethyl acetate 14g Silicone oil (KF96 1000 manufactured by Shin-Etsu Chemical Co., Ltd.)
4g1.1.1-)S(p-incyanatomethylbenzylaminocarbonyloxymethyl)propane 28g Add diethylenetriamine 2.5% aqueous solution Log to 180g of 4% methylcellulose aqueous solution, and add it to this while stirring in a household mixer. Add the above solution (I) and disperse the 0
For dispersion, Kenko Denki Sangyo Co., Ltd.'s Shoe King Mixer M is used.
Using X915C', set the voltage to 40V with a voltage regulator,
After stirring for 20 seconds in mayonnaise mode (low speed), voltage 100
V. Dispersion was performed for 3 minutes and 30 seconds in powder grinding mode (high speed). Furthermore, 64 g of a 5% aqueous solution of diethylenetriamine was added,
The 0-reaction mixture was reacted at 0°C for 3 hours, washed with water, and dried to obtain titanium oxide microcapsules.

2-3, 2 and 1- of Example 1
Same as 4 2-4. 1- of Example 1 of 2 and
Same as 5 2-5. 1 - The multi-layered material (ryL open layer and second porous layer) of Example 1-6
It was adhered onto the surface of the first porous layer by the same dot adhesion method as described above, and was fixed integrally.

The analytical film for measuring GOT activity thus completed consists of a support, a water absorption layer, a first porous layer (which also serves as a coloring reagent layer),
The second porous layer and the fabric spreading layer are integrally laminated on the kite.

The fabric spreading layer and the second porous layer cooperate to act as a blood cell filtering layer. The first porous layer acts as a reaction and dye formation (color development) layer in the presence of the GOT. The dye is optically detected through the transparent support.

2-61 minutes analysis time (DELTA) Completed chemical analysis slides.

[Comparative Example 3] Example 2-2-5. Instead of the gravure roller used to integrate the first porous layer and the layered material, a dot with a diameter of 0.3 m was used.
A biochemical analysis slide for GOT activity measurement was completed in the same manner as in Example 2, except that a gravure roller with a dot center spacing of 0.6 I and a dot area ratio of about 20% was used.

[Comparative Example 4 Example 2-2-5. Instead of the gravure roller used to integrate the first porous layer and the layered material, a dot with a diameter of 0.2 m was used.
+s, dot center spacing 0.4+am, dot area ratio approximately 2
A biochemical analysis slide for measuring GOT activity was completed in the same manner as in Example 2, except that a 0% gravure roller was used.

[Measurement Example 2] Color development of analysis slides by GOT in whole blood was evaluated as follows.

Pig-derived G was added to a human whole blood sample with a hematocrit value of 36%.
OT (manufactured by Sigma, USA) with GOT activity of 280 units/l
Prepare a plasma sample collected by centrifuging it, first add the plasma to the whole blood to obtain a hematocrit value of 25% whole blood, then centrifuge the whole blood. The red blood cell part (lower layer) and the plasma part (
supernatant), remove the plasma from the supernatant, and reduce the hematocrit value to 5.
It was made into 5% whole blood. That is, GOT activity is 280 units/
1, and the hematocrit values are 25% and 36%, respectively.
, 55% three types of human whole blood samples were prepared.

Next, 10 μl of the three types of human whole blood samples were spotted on each of the analysis slides prepared in Example 1, Example 2, and Example 1313. , PE with visible light with a center wavelength of 640
The color optical density of the 5 analysis slides was measured by reflection photometry from the T support side, and the value was converted to 0Dt (transmission optical density) (Clinica I Chemistry, Vol. 2
4.1335 (1978)) to calculate the GOT activity value (unit/l) in the 3ai human whole blood sample. The results are shown in Table 2.

It is clear that GOT activity measurement is less affected by the hematocrit value of the sample blood than the analysis slides of Comparative Example 4 and Comparative Example 4.

Claims (11)

[Claims] (1) A first porous layer, a second porous layer, and a third porous layer are placed on a water-impermeable light-transparent support with or without a water-permeable layer. at least one of the three porous layers and the water-permeable layer includes a reagent composition that produces a detectable optical change in the presence of the analyte component; The three porous layers are brought into close contact with each other by partially placed adhesives so that minute penetrations are formed between adjacent surfaces to the extent that uniform liquid permeation is not substantially impeded. A multilayer analytical element which is glued and integrated as a single piece, characterized in that the glued part consists of a large number of discrete point-like parts distributed at an average density of less than 200 points per cm^2. analysis elements. (2) The area ratio of the bonded part is 10% to 30%
The analytical element of claim (1) which is. (3) The analytical element of claim (1) or (2), wherein all of the reagent composition is contained in at least one of the first porous layer and an optional water-permeable layer. (4) Claims in which at least a component of the reagent composition that directly contributes to color development is contained in the first porous layer (
1) or (2) analysis element. (5) The analytical element according to any one of claims (1) to (4), wherein the second porous layer is a non-fibrous porous layer. (6) The analytical element according to any one of claims (1) to (5), wherein the first porous layer is a non-fibrous porous layer. (7) The analytical element according to any one of claims (1) to (5), wherein the first porous layer is a fibrous porous layer. (8) The analytical element according to any one of claims (1) to (7), wherein the third porous layer is a fibrous porous layer. (9) The analytical element according to any one of claims (1) to (7), wherein the third porous layer is a non-fibrous porous layer. (10) The analytical element according to any one of claims (1) to (9), which has another water-permeable layer between the support and the first porous layer. (11) The analytical element according to any one of claims (1) to (10), further comprising another water-permeable layer at a position farther from the support than the third porous layer.