JPH03131758A - Dry type analyzing element for analyzing whole blood sample - Google Patents

Abstract

PURPOSE:To facilitate processing at the time of production (molding) and to allow rapid measurement as well as to enhance the reproducibility of an analyte measured value by laminating a fluffed fabric consisting of the fluffed fibers having a prescribed fiber average diameter, fiber density and average fiber length and a detecting function layer. CONSTITUTION:The fluffed fabric consisting of the fluffed fibers having 1 to 30mum fiber average size, 7.0X10<3> to 8.2X10<5> pieces/cm<3> fiber density and 0.5 to 5.0mm average fiber length and the detecting function layer are laminated or a porous layer as a development layer is held in place therebetween to form the dry analyzing element for the whole blood sample analysis. The examples of the fluffed fabric include velvet, circular knitted velour, etc. The porous layer is the blank material different from the fluffed fabric and may be formed of any of fibrous and nonfibrous porous layers. Filter paper, non-woven fabric, woven fabric, knitted fabric, etc., are usable. The analyzing element is constitut ed by successively laminating a reagent layer, light shielding layer and fluffed fabric on a base.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a dry integrated multilayer analytical element used for quantifying components in whole blood as a sample. It concerns multi-layer analysis elements.

(Technical background) (2) (1) Various components in body fluids that are important in clinical medicine, such as glucose, bilirubin, BUN (urea nitrogen), uric acid, cholesterol, and various enzymes (creatine kinase, GOT
(Glutamate oxaloacetate transaminase)
, GPT (glutamate pyruvate transaminase) and the like can be quantitatively analyzed using a wet method or a dry chemical analysis. Dry chemical analysis is an analytical method that uses analytical elements such as test pieces, analytical slides, and analytical tapes containing dry analytical reagent systems, and is easier to operate and easier to analyze than wet methods that use solution reagents. Excellent in terms of speed.

By further advancing this dry method, a dry integrated multilayer analytical element has been developed as an analytical means that can quickly perform high-precision chemical analysis on a minute amount of liquid sample. This includes, for example, Special Publication No. 5 x 103-21677 (US3, 992.
158), JP 55-164356 (US 4,29
2,272), JP-A-6O-222769 (EP 01
There are analytical elements described in 62302Al and others.

An integrated multilayer analytical element (also referred to as a multilayer analytical element) is composed of, for example, a transparent support, a reagent layer, a light-reflecting layer (light-shielding layer), and a developing layer. The reagent layer coated on the transparent support (for example, a polymer sheet) contains a reagent that reacts with the test component contained in the liquid sample and develops or changes color to an optical density depending on the amount of the component. The spreading layer spreads the deposited liquid sample uniformly (at a substantially constant rate per unit area in the surface direction without substantially unevenly distributing the components contained therein). This action is called liquid metering.
g) Action is also called spreading or spreading action.

When a fixed amount of a liquid sample, for example, a minute amount (e.g., about 10μ!!) of blood, is spotted on the surface of the developing layer of such a dry analytical element, the blood developed in the developing layer will pass through the light-reflecting layer. It passes through and reaches the reagent layer, where it reacts with the reagent and develops or changes color. After incubating the multilayer analytical element at a certain temperature for a certain period of time, the amount of color development or color change is measured reflectively from the transparent support side, and quantitative analysis is performed based on a calibration curve. The light reflecting layer has the role of eliminating the influence of the color of the liquid sample in the developing layer and forming a white background during optical density measurement.

However, even in such dry chemical analysis methods, serum or plasma from which red blood cells have been removed from whole blood is often used as a sample, as in the wet method. The operation of separating red blood cells from blood requires a lot of effort, time, and equipment.

Therefore, it is particularly desired that the dry method, which is characterized by speed and convenience, be able to directly analyze whole blood as a sample.

(Prior art) As a dry multilayer analysis element for whole blood samples,
×103~21677 (US 3,992.158),
Japanese Patent Publication No. 6O-111960 (US 4,637.978
) and Japanese Unexamined Patent Publication No. 60-27980, etc., have proposed a device provided with a filtration layer for separating blood cells and high molecular weight components in blood.

These are porous polymer membranes such as cellulose acetate membranes (
A membrane filter) or a porous layer having continuous voids formed of microparticles is used as a filtration layer to trap and remove blood cell components at or near the surface of the filtration layer. Therefore, in order to sufficiently remove blood cells, it is necessary to use a material with a sufficiently small average pore diameter. However, when the average pore size is made small, it takes time for plasma to pass through the filter layer, which significantly impairs the speed of measurement. In addition, if the pore size is small, the packed ground due to hemoglobin pigment will increase due to hemolysis, and the leakage of blood cell white components will adversely affect the coloring reaction in the detection layer, resulting in variations in measured values.
There was also the inconvenience of poor reproducibility.

On the other hand, glass fiber filter paper is also known to have a high blood cell/plasma separation function that separates blood cells and plasma from whole blood, and analytical elements using this glass fiber filter paper were also developed in Japanese Patent Application Laid-Open No. 57-53.
661 (EP O045476A), JP-A-6L-96
466 (EP O159727A), etc.

These are excellent in that they cause less hemolysis, but because they use highly brittle glass fibers, they require careful processing during the production of analytical elements, resulting in poor manufacturing suitability. Further, since the glass fiber filter paper (or glass fiber layer) is simply placed in close contact with the detection reagent layer or the development layer, its adhesive strength is low and it is easily peeled off. When peeling occurs, the amount of plasma transferred to the detection reagent layer varies, causing a problem of poor reproducibility of measured values.

(Problem to be solved by the invention) The present invention was made in view of the above circumstances, and
The object of the present invention is to provide a dry analytical element for whole blood sample analysis for quantitative analysis that is easy to process during molding, can be measured quickly, and has high reproducibility of analyte measurement values.

(Means for Solving the Problems) The above object is such that the napped fibers have an average fiber diameter of 1-3o.
μm, fiber density 7. Ox 10×103~8.2x 1
This is achieved by a dry analytical element for whole blood sample analysis, which is formed by laminating a napping cloth with 0' fibers/cm2 and an average fiber length of 0.5 to 5.0 mm and a detection function layer.

In addition, for the above purpose, the raised fibers have a fiber average diameter of 1
1-30u, fiber density 7. OX 10×103~8.2
X to 6 fibers/cm2 and average fiber length 0.5 to 5.0 m
This can also be achieved by a dry analytical element for analyzing whole blood samples, which is formed by laminating a raised blanket (m), a porous layer, and a detection function layer in this order.

(Function) The present invention was made based on the findings of the present inventors who found that a certain type of napping blanket specifically separates blood cells and plasma without causing hemolysis. It is constructed using plasma separation effect.

Although the mechanism of blood cell separation is not clear, the standing blanket does not trap blood cells only on its surface, but as it penetrates through its thickness, it gradually forms a fibrous structure, first large blood components, then smaller blood components. This seems to be due to the so-called volumetric filtration effect, which removes blood cells over the entire length in the thickness direction.

Although the porous layer is intended to function as a spreading layer, the napping cloth itself can also exhibit a certain degree of spreading action, so when the napping cloth alone is sufficient, it can have both blood cell separation and spreading effects. If the porous layer is provided, the porous layer can be omitted.

(Detailed explanation of means for solving the problem) The basic structure of the dry analytical element of the present invention is a laminated layer of a raised cloth and a detection function layer, or a porous layer as a developing layer is sandwiched between them. The smallest unit is something. However, the analytical elements of the present invention may be formed on a transparent support. Further, the detection function layer includes at least one or more reagent layers, and may be transferred to another detection layer for detection in order to facilitate the detection of the dye produced as a result of the color reaction. The multiple components will be explained below.

The fiber material of the L throne blanket is not particularly limited. This is because the blood cell/plasma separation function of the napkin is due to the chemical properties of the fibers.
Rather, it is thought that this is due to the fabric structure of the raised blanket. Therefore, the present invention can be achieved by selecting fiber diameter, fiber density, and average fiber length that allow sufficient separation of blood cells and plasma. - Strategically, the average fiber diameter is 1~
30 μm, fiber density 7. OX 103~8.2x 10
6 fibers/cm2 and an average fiber length in the range of 0.5 to 5.0 mm are desirable.

The thickness (average diameter) of the fibers of the cloth used ranges from about 1 μm to about 30 μm, preferably from about 2 μm to about 10 μm. The density of the cloth is about 0.02g/cm3 to about 1.0g/c
m3. Preferably about 0.05g/cm3 to about 0.7g/cm3
cm3 range, cloth thickness is about 100μm to about 2000μm
m, preferably in the range of about 150 μm to about 11000 LL, the porosity of the fabric is about 45% to about 70%, preferably about 50
% to about 65%.

Examples of raised blankets include velvet, circular knit velor, double raschel velor, and tricot velour.

Note that the preferable range of the amount of blood spotting is based on the average fiber length (L: μ
m) and the blood spotting amount (v:μI2), 5≦L/V
The range is ≦200.

Moxibustion test 1 The porous layer is a spreading layer that uniformly spreads blood plasma separated from blood cells with a raised blanket, and supplies the plasma to the detection function layer below. Although the material is different from that of the raised blanket, it may be formed of either a fibrous porous layer or a non-fibrous porous layer.

In the case of forming a fibrous porous layer, filter paper, nonwoven fabric, woven fabric (for example, hand-woven fabric), knitted fabric (for example, tricot knitted fabric), etc. can be used. Among these, woven fabrics and knitted fabrics are preferred. Woven fabrics, knitted fabrics, etc. are disclosed in Japanese Patent Application Publication No. 57-66359 (US 4.783,315).
A glow discharge treatment as described in . Or Tokuko Sho 59-11709, Tokuko Sho 59
Woven fabrics, knitted fabrics, etc. that have been subjected to alkali etching (alkali weight loss) treatment as described in No. 24233 can also be used.

In the case of a non-fibrous porous layer,
Preferred are flash polymer layers (membrane filters) of cellulose esters such as cellulose acetate, cellulose acetate butyrate, cellulose nitrate, as described in US Pat. In addition, polyamides such as 6-nylon and 6.6-nylon; microporous membranes such as polyethylene and polypropylene; JP-A-62-27
A microporous membrane made of polysulfone described in No. 006 can also be used. Others, Special public Showa 5×103-2167
7, Japanese Patent Publication No. 55-90859 (US 4,258.0
A porous layer having continuous voids in which polymer microparticles, glass microparticles, diatomaceous earth, etc. described in No. 011 etc. are bonded in a dot-like manner with a hydrophilic or non-water-absorbing polymer can also be used.

In the case where the raised cloth exhibits a spreading action, the multilayer analytical element of the present invention may be constructed by omitting this porous layer. However, when the raised blanket and the porous layer are laminated by partial adhesion (porous layer@) described later, these two layers are harmoniously integrated to perform blood cell separation, plasma expansion (liquid measurement), and plasma measurement. It is a preferred embodiment of the present invention to have the function of supplying to the lower detection function layer.

It is desirable that this porous layer be impregnated with/contained with a wood-philic polymer or surfactant as described below to make its spreading action more uniform.

■Wood-friendly polymer JP-A-6O-222770 (EP 0162301A)
Hydrophilic cellulose derivatives described in JP-A-6×103-21533 (EP O254202)
Methylcellulose, ethylcellulose; polyvinyl alcohol; polyvinylpyrrolidone; polyacrylic acid, etc. described in A): JP-A-62-182652 (DE 3717913A)
Hydrophilic polymers such as homopolymers or copolymers of acrylamide, N-substituted or N,N-disubstituted acrylamide, etc., as described in
58), Japanese Patent Publication No. 55164356 (US 4,292.
Nonionic surfactants such as nonylphenoxypolyethoxyethanol described in JP-A No. 60-222770, nonionic surfactants with an HLB value of 10 or more described in JP-A No. 60-222770, JP-A No. 6-103-21533 (EP 0254202)
Polyhydric alcohol ester ethylene oxide adducts described in A); polyethylene glycol fatty acid esters; higher alcohol ethylene oxide adducts: alkylphenol ethylene oxide adducts; nonionic surfactants such as higher fatty acid alkanolamide; JP-A-62-6167 (Chemical Abst
racts 107°171961w), JP-A-6×1
These hydrophilic polymers and surfactants, such as the fluorine-containing surfactants described in EP 03-196849 (EP 0278496A), can also be used in combination.

In addition, when the porous layer is omitted, or when the raised blanket also has a spreading effect even when used together with the porous layer, it is recommended to include these hydrophilic polymers and surfactants in the raised blanket. desirable.

Adequate History Plate 1 The detection function layer is a layer that receives plasma supplied from the porous layer (or the direct nap layer) and has the function of detecting the test component. It consists of at least one reagent layer, but it is also possible to allow only a color development (or color change) reaction to occur in the reagent layer, and the resulting dye to be transferred to a detection layer, in which the amount of dye is detected. .

The reagent layer is a layer containing a composition (reagent composition) that produces optically detected color development or color change, or a change in absorption wavelength in the ultraviolet region in the presence of a test component, and is a layer containing a hydrophilic composition. In some cases, a reagent composition is contained in a water-permeable layer or a porous layer made of a transparent polymer binder.

■ When the reagent layer is composed of a water-permeable layer made of a hydrophilic polymer binder, examples of the hydrophilic polymer binder include gelatin and its derivatives (e.g., phthalated gelatin); cellulose derivatives (e.g., hydroxyethyl cellulose); Agarose;
Polyacrylamide, polymethacrylamide, copolymers of acrylamide or methacrylamide and various vinyl monomers; polyvinyl alcohol; polyvinylpyrrolidone, etc. can be used. Within such a hydrophilic polymer binder, all or some of the components of the reagent composition (
In this case, the remaining components are contained in other layers) substantially uniformly.

The reagent layer using a hydrophilic polymer as a binder is
03-21677 (US 3,992.158), JP 55-164356 (US 4.292.272),
JP 54-101398 (US 4.132.528)
) JP-A-61-2961-292063 (Che Ab
structs 106゜210567y1, etc., by applying an aqueous solution or aqueous dispersion containing a reagent composition and a hydrophilic polymer onto a support or other layer such as a detection layer and drying it. Can be done. The dry thickness of the reagent layer using a wood-philic polymer as a binder is approximately 3.
The coating ranges from about 3 g/m2 to about 50 g/m'', preferably from about 5 g/m2 to about 30 g/m2.

■When the reagent layer is composed of a porous layer containing a reagent composition (
Porous reagent layer), in this case, the porous reagent layer may be a porous layer similar to the above-mentioned porous layer, or a porous layer as described in JP-A-59-120957 (EP 01
14403A) etc., a porous structure layer containing solid fine particles and a polymer as a binder can be used.

The reagent composition is contained in the porous reagent layer by impregnation or coating with a solution (or dispersion) of the reagent composition. The porous reagent layer containing the reagent composition can be used in conjunction with other water-permeable layers, such as a second reagent layer, a water-absorbing layer,
Laminated on top of the detection layer or adhesive layer. Note that this lamination is
JP 55-164356 (US 4,292.272
This can be done by adhering a porous reagent layer onto a lower layer that has been almost uniformly wetted with water as described in (1999).

Alternatively, after adhering a porous layer on the water absorption layer, detection layer, or adhesive layer by the method described in JP-A-55-164356, the solution or dispersion of the reagent composition is applied to the porous reagent layer. It may be applied to

A known method can be used to impregnate or apply a reagent composition solution to the porous reagent layer. For example, dip coating,
Doctor coating, hopper coating, curtain coating, etc. can be selected and used as appropriate.

■ Reagent composition The reagent composition may be any composition that can produce an optically detectable substance (for example, a pigment (dye)) in the presence of the test component, or that can directly react with the test component. Reagent compositions containing test components and other reagents (e.g. enzymes, even those that develop or change color, JP-A-62-138756 (Page 333, lower right @ - 334)
(upper right column of page) (EP 0226465A, page 5 line 48 ~
A composition (indicator) that produces an optically detectable substance (pigment or dye) as a result of the reaction with an intermediate produced by the reaction with a reagent composition (such as the reagent composition described on page 6, line 40)
But that's fine. For example, i) compositions that produce dyes by oxidation of leuco dyes (e.g., triarylimidazole leuco dyes described in US Pat. (imidazole leuco dyes): ii) diazonium salts; 1ii1 Compositions containing compounds that, when oxidized, produce dyes by coupling with other compounds (e.g. 4-aminoantipyrines and phenols or naphthols)
: iv) A compound consisting of a compound capable of producing a pigment in the presence of a reduced coenzyme and an electron transfer agent can be used.

Furthermore, in the case of an analytical element for measuring enzyme activity, a self-developing substrate that liberates a colored substance such as p-nitrophenol can be included in the reagent layer or porous layer.

The reagent composition may contain an activator, a buffer, a hardening agent, a surfactant, etc., if necessary. Examples of buffers that can be contained in the reagent layer of the analytical element of the present invention include carbonates, borates, phosphates, and
Examples include Good's buffer described in TryJ, Vol. 5, pp. 467-477 (1966). These buffers are used in "Basic Experimental Methods for Proteins and Enzymes"
(Takeshi Horio - Baka, Nankodo, 1981), rBio
ChemistryJ Vol. 5, pp. 467-477 (19
You can make a selection by referring to literature such as 1966).

If the coloring reaction proceeds in multiple stages, the reagent layer may be divided into two or more parts, and each layer may contain a reagent composition corresponding to each reaction stage.

1. The detection layer is a layer in which dyes generated in the presence of the test component in the reagent layer diffuse to this layer and are optically detected through the transparent support, and is composed of a wood-philic polymer. Can be done.

If necessary, the detection layer may include a mordant, such as a cationic polymer for anionic dyes, to facilitate the detection of colored dyes.

A water absorption layer may be provided below the detection layer.

The water absorption layer generates a kind of diffusion negative pressure by absorbing water, which promotes the movement, diffusion, and penetration of plasma into the porous layer and reagent layer, and the movement of dye into the detection layer. A layer in which the dye produced in a layer does not substantially diffuse into the layer. This water-absorbing layer can be made of a wood-philic polymer that easily swells with water.

Further, a light shielding layer may be provided above the detection layer and at least below the raised blanket or porous layer. This light shielding layer shields the red color of hemoglobin of red blood cells in blood when measuring detectable changes (color change, color development, etc.) occurring in the detection layer, reagent layer, etc. from the transparent support side. A layer that functions as a light reflective layer or background layer, containing titanium dioxide,
It is a layer made of a hydrophilic binder containing light-reflecting fine particles such as barium sulfate, or a porous layer containing light-reflecting fine particles.

A preferred material for the support is polyethylene terephthalate. Cellulose esters such as cellulose triacetate, polystyrene, polycarbonate of bisphenol A, etc. can also be used. A support that is light-transparent (transparent) and water-impermeable is generally used, but water-permeable, water-permeable, or light-impermeable (opaque) supports can also be used. The support is usually provided with an undercoat layer or subjected to a hydrophilic treatment in order to firmly adhere the hydrophilic layer laminated thereon.

However, if the fine fiber nonwoven fabric layer has a physical strength that can maintain the structure of the element, the support may be omitted.

blue gold The analytical elements of the invention can have various configurations.

For example, Japanese Patent Publication No. 49-53888 (US 3,992.
158) Japanese Patent Publication No. 55-164356 (US 4.292)
, 2721, JP-A-6O-222769 (EP O1
62302A), JP-A-62-138756, JP-A-62-138757, JP-A-62-138758 (
The layer structure of a multilayer analysis element described in EP 0226465A) etc. can be referred to. Note that in one aspect of the present invention,
As mentioned above, the porous layer adjacent to the raised blanket may be omitted.

Practically speaking, for example, (1) a reagent layer containing a hydrophilic polymer binder and a layer of a raised cloth are placed on the support in this order; (2) a reagent layer containing a hydrophilic polymer binder and a porous layer are placed on the support. (3) on the support; a water absorbing layer, a reagent layer containing a hydrophilic polymer binder, a porous layer, and a layer of the napped cloth in this order; (4) on the support; A detection layer, a reagent layer containing a hydrophilic polymer binder, a porous layer, and a layer of a raised blanket in this order (5) On a support, a water absorption layer, a porous layer containing a reagent, and a layer of a raised blanket in this order (6) It is useful to have a detection layer, a reagent-containing porous layer, and a raised layer on a support in this order (the support may also include an undercoat layer).

. Connection L/Main Connection Next, the partial adhesion (or porous adhesion) between the raised fabric and the porous layer or the detection function layer, which is a feature of the multilayer analysis element of the present invention, will be explained.

Partial adhesion is described in Japanese Patent Application Laid-Open No. 61-4959 (EP O16).
6365Al, JP-A-62-138756-8 (E
A mode of adhesion between two adjacent porous layers or between an adjacent porous layer and a non-porous layer described in P 0226465A) etc. "adhesive that is substantially tightly bonded and integrated by an adhesive disposed between the two adjacent surfaces, and is configured such that uniform passage of liquid between the two adjacent surfaces is substantially unobstructed." be. In the multilayer analysis element of the present invention, the interface between the adjacent raised cloth and the porous layer or the interface between the adjacent raised cloth and the detection function layer is partially bonded.

- Strategically, the adhesive is placed partially on the raised fabric and then the porous layer or the detection functional layer is bonded to the raised fabric while applying light pressure uniformly. Conversely, it is also possible to place the adhesive partially on the porous layer or the detection function layer, and then attach the raised cloth to the porous layer or the detection function layer while uniformly applying light pressure. Furthermore, adhesive is partially placed on the raised blanket to create a porous layer, and the raised blanket is adhered to a porous sheet while applying light pressure uniformly, or conversely, a porous sheet is created to create a porous layer. Place the adhesive partially on the shaped object,
Then, after uniformly pasting the standing blanket onto the porous sheet while applying pressure, the porous sheet can be uniformly pasted onto the detection function layer.

A method of partially disposing an adhesive on a raised blanket, a porous layer, or a detection function layer is described in JP-A-61-4959 and JP-A-62.
-138756, JP-A-64-23160 (DE
Various methods described in 3721236A1 and the like can be used. Among these methods, the printing method is preferred. Among the printing methods, a method in which an adhesive is transferred and adhered to a porous layer or a detection function layer using a printing plate (preferably a Clavier printing plate or an intaglio) roller, and an adjacent two
The method of pasting the layers together is described, for example, in "Printing Engineering Handbook J" (edited by the Printing Society of Japan) (Gihodo Publishing (Kimu, 1983) 838-
This can be carried out using the known apparatus and method described on page 853 and the like.

Adhesives that can be used include various adhesives described in JP-A-62-138756, as well as those described in the above-mentioned "Printing Engineering Handbook,"
Known adhesives such as those described in Jl, pages 839 to 853 can be used. Adhesives include water-based adhesives,
An organic solvent type adhesive or a thermal adhesive (hot melt type or heat sensitive) adhesive can be used.

Examples of water-based adhesives include water-based glues such as starch glue; aqueous solutions such as dextrin, carboxymethyl cellulose, and polyvinyl alcohol; and vinyl acetate-butyl acrylate copolymer emulsions. As the organic solvent type adhesive, one whose solvent evaporates slowly is suitable. Thermal adhesion (
Hot melt or heat sensitive) adhesives are particularly useful.

As the heat-adhesive hot-melt adhesive, the hot-melt adhesive described in "Industrial Materials" Vol. 26 (No. 11), pages 4-5, etc. can be used. Examples include ethylene copolymers such as ethylene-vinyl acetate copolymer, ethylene-ethyl acrylate copolymer, and ethylene-acnolic acid copolymer; polyolefins such as low molecular weight polyethylene and acid polypropylene; and nylon. Thermoplastic rubbers such as styrene block copolymers such as SBS; styrene-butadiene rubber, butyl rubber, urethane rubber; rosin, petroleum resins, terpene resins; and synthetic waxes.

(The following are blank spaces) (Example 1 and Comparative Example 1) For Glucose Determination - Mold Analysis An analytical element A was prepared in which a reagent layer, a light shielding layer, and a raised blanket were laminated in this order on an elementary support.

First, water droplets of the reagent composition were coated on the surface of a colorless transparent polyethylene terephthalate (PET) sheet (support) having a thickness of 180 μm so as to have the following coating amount, and dried to form a reagent layer.

Two drug layers? 1m2 Deionized gelatin 26g Glucose oxidase 2000U Peroxidase 3300U4- [4-(
Dimethylamino)phenyl]-5-phenethylimiguzole (leuco dye) acetate 600mg 1,7-hydroxynaphthalene 660mg
Nonylphenoxypolyethoxyethanol (containing average 10 oxyethylene units) 270mg On this reagent layer, apply an aqueous dispersion of the composition below to form a light shielding layer (dry thickness approximately 7 μm) with the following coverage: did.

Amount of layer (1 m2) Deionized gelatin 2.9 g Rutile type titanium dioxide fine particles 13 g Nonylphenoxypolyethoxyethanol (contains 10 oxyethylene units on average) 600 mg Uniformly swell the surface of this light shielding layer with water. Then, on top of that, rayon velvet (fiber average diameter of napped part 15 μm, fiber density 5.5 x 1
0'/cm2 and average fiber length of 1.3 mm) were laminated on the ground surface while applying light pressure in a -like manner, and dried and integrated in close contact to obtain an integrated multilayer analytical element (Element A).

As a comparative example, the nominal pore diameter (
Average value of effective pore diameter or minimum pore diameter) 1.2L1. An integrated multilayer analysis element (Element B) was prepared in the same manner as Element A except that a cellulose acetate membrane filter having a thickness of approximately 140 am and a porosity of approximately 80% was used.

Elements A and B each contain normal human whole blood (hemacrit value 42%, plasma glucose concentration 122 mg/mj2)2
A temperature of 0 μ°C was applied. The element was incubated at 37°C for 6 minutes and immediately exposed to a central wavelength of 540 nm and 600 nm from the support side.
Reflection absorbance within the element was measured with visible light at 40 nm. This measurement was repeated 10 times using 10 elements each, and the average value and standard deviation (SD) were determined. The results are shown in Table 1.

The reflected absorbance at 540 nm, which corresponds to the absorption of surface red blood cell hemoglobin, was not observed at all in Element A using a raised blanket, whereas Element B, a comparative example, showed a large value of 0.432. This is because the separation of blood cells is insufficient or bloodletting occurs in element B, whereas element A using a napping blanket
This shows that red blood cells were completely removed and there was almost no hemolysis.

In addition, the 640 nm reflection absorbance, which corresponds to the glucose level in plasma, was high at 1.117 for element A (and the variation was small (SD 0.003). - Blade element B was 0.
．． 761, and the variation was large (SDO,
032). This is considered to be because in element B, hemolysis occurred within the analytical element, inhibiting the pigment production reaction.

Thus, element A of the present invention exhibits excellent blood cell separation properties and non-hemolytic properties. Furthermore, the measurement sensitivity itself was excellent, and the reproducibility was also excellent.

(Example 2 and Comparative Example 2) Total Bilirubin Quantification Mold Layer An analytical element C was prepared in which a first water-absorbing layer, a second water-absorbing layer, a reagent layer, and a nonwoven fabric were laminated in this order on a required support.

First, on the surface of a colorless transparent polyethylene terephthalate (PET) sheet (support) with a thickness of 180 μm, a first water absorbing layer (dry thickness of approximately 15 μm) and a second water absorbing layer (dry thickness of approximately IOμm) were coated from an aqueous solution in this order and dried.

Amount of 1st layer: 1m" Lipolyvinyl alcohol (degree of saponification 88%, viscosity of 4% aqueous solution at 20°C 5 cps) 14g 4g nonylphenoxy polyglycidol (contains 10 glycidol units) 350mg
Amount of second absorption layer: Polyvinyl alcohol (saponification degree 99%, viscosity of 4% aqueous solution at 20°C 5 cps) 9.4 g nonylphenoxy polyglycidol (contains average 10 glycidol units) 300 mg per 1 m2
The second water-absorbing layer was uniformly moistened with water, and then subjected to a hydrophilic treatment with an aqueous solution of nonylphenoxy polyglycidol (containing an average of 10 glycidol units) and dried. Broad woven fabrics made of 100 count twine cotton yarn were laminated with uniform light pressure and dried to form the matrix of the porous reagent layer.

Next, the first reagent composition coating solution was applied onto the textile fabric in the following coating amount, and the fabric was impregnated and dried.

Further, a second reagent composition coating solution was applied onto the textile fabric in the following coating amount, impregnated, and dried to form a light-shielding layer on the upper edge of the textile fabric (porous reagent layer).

Amount of 1st sample 7-(2,×103~dihydroxypropyl)theophylline per 1 m2 [CA Registry No. 479-18
-5] 25g sulfosalicylic acid
5g Hydroxypropyl methacrylate-N
-(α-Sulfomethyl-α-methylethyl)acrylamide (6:4) copolymer 2.5g2.4-Dichlorobenzenediazonium sulfosalicylate
320mg2 reagent (per 1m2 of acid) Titanium dioxide fine particles 11g7-
(2,×103~dihydroxypropyl)theophylline 0 g Nonylphenoxypolyglycidol (containing average IO glycidol units) Ig On top of this woven fabric (porous reagent layer), fiber average diameter 10
Nylon tricot velour, made by knitting tricot pile of nylon yarn of 100 μm and shirring to a fiber length of 800 μm (napped fiber density 3.2 x 10'/cm)
2) was placed in close contact to obtain an integrated multilayer analytical element (element C) for quantifying total bilirubin.

As a comparative example, a nominal pore size of 1.
A cellulose acetate membrane filter with a diameter of 2 μm, a thickness of approximately 140 μm, and a porosity of approximately 80% was used, and the rest were element C.
An integrated multilayer analysis element (element D) was created in the same manner as above.

For elements C and D, normal human whole blood (hematocrit value 42%, plasma median bilirubin concentration 0.98 mg/dR)
) 20μρ was spotted. The element was incubated at 37°C for 6 minutes and immediately exposed to a central wavelength of 600 nm from the support side.
The reflected absorbance within the element was measured with visible light at 540 nm.

This measurement was repeated 10 times using 10 elements each, and the average value and standard deviation (SD) were determined.

The reflection absorbance at 60 Gnm, which corresponds to the amount of surface-forming pigment, was as high as 0.617 for element C (and the variation was small (SD = 0).
．． 0021° On the other hand, the value was small at 0.379, and the variation was large (SD=0.029). This is thought to be because hemolysis occurs in the element and the pigment production reaction is inhibited.

The reflected absorbance at 540 nm is the sum of the reflected absorbance of both the produced pigment and the red blood cells. Element C had a large value of 1.007, compared to 0.895. As described above, although the amount of dye production (absorbance at 600 nm) was smaller than that of Element C, the absorbance at 540 nm showed a high value. This indicates that blood cell separation is insufficient with elemental separation. On the other hand, Element C of the present invention exhibits excellent blood cell separation properties and non-hemolytic properties, and also has excellent measurement sensitivity and reproducibility.

(Example 3 and Comparative Example 3) For quantification of total cholesterol - Sculpture layer analysis A first reagent layer (coloring reagent layer), a second reagent layer (porous reagent layer), and a nonwoven fabric were laminated in this order on a bare support. Analysis element A was created.

First, a coloring reagent layer coating solution was applied to the surface of a colorless transparent PET sheet (support) with a thickness of 180 μm in the following coating amount, and dried to form a coloring reagent layer (dry thickness of approximately 20 μm).
μm).

1 m2 of color-developing drug layer [leuco dye oily organic solvent liquid co-2-(4-hydroxy-3,5-dimethoxyphenyl)]
-4-(4-(dimethylamino)phenyl 1-5-phenethylimidazole (leuco dye), acetate 15 mg Same as above leuco dye, hydrochloride 60 mgN,N
-Diethyl laurylamide 7.2g [gelatin aqueous solution] Deionized gelatin 22g bis[(
vinylsulfonylmethylcarbonyl)amino1methane
220mg of the above gelatin aqueous solution (
While stirring the gelatin content (15.8% by weight) using an emulsifier, the above leuco dye oily organic solvent solution was added, and the gelatin content was 15.8% by weight.
Stirring was continued for a minute to prepare a dispersion emulsion (coloring reagent composition aqueous dispersion), which was applied to a support and dried to form a coloring reagent layer.

Next, the surface of the coloring reagent layer is uniformly wetted with water,
On top of that, the nominal pore size (effective pore size or average value of minimum pore size) 1
．． Cellulose acetate membrane filters of 2 μm, thickness of about 140 LLm, and porosity of about 80% were laminated while applying light pressure uniformly and dried to closely integrate the coloring reagent layer and the membrane filter.

Next, an aqueous dispersion of a cholesterol measuring reagent composition having the following composition was applied onto the membrane filter in the following coating amount, impregnated, and dried to form a second reagent layer (porous reagent layer).

Cholesterol measuring reagent Acid amount In2 Cholesterol esterase? , 7kU cholesterol oxidase 3.6kU novoprotein lipase 5.3kU peroxyguse 83kU potassium ferrocyanide 490mg condensate of nonylphenoxypolyethoxyethanol and formaldehyde
400 mg Next, place this laminate on a membrane filter with an average fiber diameter of 5.3 μm and a fiber density of 6.
．． An integral multilayer analytical element (Element E) for quantifying total cholesterol was prepared by partially adhering the back side of a polyester circular knitted velour, having 6 x 104 fibers/cm3 and an average fiber length of 2.5 mm, by gravure printing.

For partial adhesion, hot melt adhesive Nikite H635 is used.
2-2A (manufactured by Nitta Gelatin ■; styrene-butadiene copolymer; softening point 76°C, viscosity at 140°C 410
0cps) was melted at 135°C, and a gravure version of the pattern shown in Figure 1 (a pattern with perfectly circular dots arranged at each vertex of a square: each dot had a depth of 50 μm and a diameter of 0) was prepared.
．． 4+nm, shallow U-shaped cross section, vertical and horizontal dot center spacing (
The film was transferred onto a membrane filter using a pitch of 0.8 mm), and the membrane filter was immediately crimped and laminated onto a napping cloth.

As a comparative example, instead of polyester circular knit velor, the nominal pore diameter is 1.2 μm, the thickness is approximately 140 μm, and the porosity is approximately 80%.
Using a cellulose acetate membrane filter,
An integrated multilayer analysis element (element F) that was otherwise the same as element E.
It was created.

Elements E and F include normal human whole blood (hematocrit value 48%, plasma median cholesterol concentration 151 mg/d).
12) 20u 12 was spotted. element is 37°C
Incubate for 6 minutes at
The reflected absorbance within the element was measured using visible light.

This measurement was repeated 10 times using 10 elements each, and the average value and standard deviation (SD) were determined.

The reflected absorbance at 540 nm, which is absorption by superficial red blood cells, was as large as 0.495 in Element F, indicating that blood cell separation was insufficient. In contrast, the reflected absorbance of Element E using the napping blanket was at the background level, indicating that red blood cells were almost completely removed and hemolysis hardly occurred.

Furthermore, the 6400 m reflection absorbance corresponding to the total cholesterol value is as high as 1.003 for element E, and the variation is small, fsD=0.005+. On the other hand, for element F, 0.351
It is small and the variation is large (SD 0.028+. This is thought to be because element F is hemolyzed within the analysis element and the pigment production reaction is inhibited.

As described above, Element E of the present invention exhibits excellent blood cell separation properties and non-hemolytic properties, and also has excellent measurement sensitivity and reproducibility.

(Effects of the Invention) As described above, the integrated multilayer analytical element of the present invention is configured to remove and separate blood cells using a standing blanket, and exhibits excellent blood cell separation properties and non-hemolytic properties. Therefore, the measured values do not vary and are highly reproducible. In addition, since a raised cloth with low brittleness is used, it is easy to manufacture and process analytical elements.

[Brief explanation of the drawing]

Figure 1 shows the pattern of the gravure printing plate (square shape Pattern with perfect circular dots placed at each vertex, depth of each dot 50μm, diameter 0.4mm, shallow U
Character-shaped cross section, vertical and horizontal dot center spacing (pitch) 0.8m
m).

Claims (2)

[Claims] (1) The raised fibers have an average fiber diameter of 1 to 30 μm and a fiber density of 7.0 x 10^3 to 8.2 x 10^6 fibers/cm^
2 and a raised blanket having an average fiber length of 0.5 to 5.0 mm;
A dry analytical element for whole blood sample analysis consisting of a stacked detection function layer. (2) The raised fibers have an average fiber diameter of 1 to 30 μm and a fiber density of 7.0 x 10^3 to 8.2 x 10^6 fibers/cm^
2 and a raised blanket having an average fiber length of 0.5 to 5.0 mm;
A dry analytical element for whole blood sample analysis comprising a porous layer and a detection function layer laminated in this order.