JPH08166384A - Dry analysis element for total-blood-sample analysis - Google Patents

Abstract

PURPOSE: To make it possible to measure the total blood speciment in high accuracy regardless of a hematocrit value by adding reagent component for the hemolysis of the total blood in an expansion layer. CONSTITUTION: An expansion layer has the function, which supplies the component contained in aqueous specimen into a hydrophilic polymer layer at the rate of the constant amount per unit area. For the expansion layer, porous material of fiber and non-fiber material is used. In this expansion layer, the reagent component, which performs the hemolysis of the total blood (the cytoplasmic membrane of the component having the cytoplasmic membranes of erythrocytes and leukocytes is broken or fused to have the contents discharged to plasma), is contained. As the hemolysis reagent, two kinds of surfactant having hemolytic ability and the combination of hemolysin and complement are typical examples. As the surfactant, nonion surfactant is preferable. For example, the contents of the hemolysis reagent contained in the expansion layer is about 1-50g/m<2> , preferably, about 2-20g/m<2> . Thus, the total blood specimen can be simply measured in high accuracy regardless of hematocrit.

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 capable of analyzing a whole blood sample.

[0002]

2. Description of the Related Art Recently, a biochemical examination of blood has been frequently performed, and a dry analytical element has been used for this biochemical examination.

[0003] Conventionally, in both the wet method and the dry chemical analysis, the analysis has often been carried out using serum or plasma from which red blood cells have been removed as a sample. However, the operation of separating erythrocytes from other components of blood involves a lot of labor and equipment cost, and therefore it is desirable to be able to analyze undiluted whole blood.

For performing dry chemical analysis using whole blood as a sample, conventionally, a blood cell separation layer or a blood cell separation element is provided in a multi-layer analysis element to separate blood cells (red blood cells and white blood cells) and other formed components of whole blood. Was. It is disclosed in JP-A-57-53661 and JP-A-61-96466 that glass fibers, membrane filters, cotton wool fiber products and the like can be used for the blood cell separation layer or the blood cell separation element. . It is also known to combine (laminate) two or more layers, as disclosed in JP-A-62-138756 and JP-A-3.
No. 16651 discloses a combination of a fibrous porous layer and a non-fibrous porous layer.

[0005]

[Problems to be Solved by the Invention] The whole blood sample has a large individual difference and varies in a very wide range, such as 10% for a person with a low hematocrit value and 70% for a person with a high hematocrit value. This is
This means that even if the amount of the whole blood sample supplied to the analysis element is always constant, the amount of plasma contained therein varies from 80% to 30%. As a result, there is a problem in that the amount of plasma supplied to the layer that receives plasma also fluctuates greatly, resulting in an error in the analysis result. In the dry analytical element, the development layer is usually laminated on the uppermost side. This spreading layer has the ability to spread the components contained in the spotted aqueous sample in a plane without being substantially unevenly distributed and to supply it to the layer below it at a substantially constant rate per unit area. Therefore, the above-mentioned fluctuation in plasma volume is a problem particularly in the case of whole blood samples having a high hematocrit value.

It is an object of the present invention to provide a dry analysis element for analyzing a whole blood sample which can easily measure a whole blood sample with high accuracy regardless of the hematocrit value.

[0007]

DISCLOSURE OF THE INVENTION The present invention provides a dry analytical element for analyzing a whole blood sample, which achieves the above object, wherein the analytical element is a spreading layer for spreading whole blood, and at least one reagent layer. And a dry analytical element in which a water-impermeable support is laminated in this order, characterized in that the spreading layer contains a reagent component for hemolyzing whole blood.

The spreading layer is a layer having a function of spreading the components contained in the aqueous sample in a plane without being substantially unevenly distributed and supplying the hydrophilic polymer layer at a substantially constant amount per unit area. Is. As a development layer, conventionally, it is used as a development layer of a dry analytical element (also referred to as a multilayer analytical element, a dry analytical element, a dry analytical film, etc.),
Known non-fibrous and fibrous porous materials can be used. Specifically, woven fabric spreading layers (eg, plain woven fabrics such as broad and poplin) described in JP-A-55-164356 and JP-A-57-66359, JP-A-60-222769. The knitted fabric development layer described in the official gazette (eg, tricot knitted fabric, double tricot knitted fabric,
(Milanese knitted fabric, etc.), a spreading layer comprising a woven fabric or a knitted fabric which has been subjected to an etching treatment with the alkaline etching solution described in JP-A-1-172753, JP-A-57-14.
No. 8250, a developing layer made of a papermaking paper containing an organic polymer fiber pulp; JP-B-53-21677;
Membrane filters (brush polymer layers) described in US Pat. No. 3,992,158, polymer microbeads, glass microbeads, non-fibers such as continuous microvoid-containing porous layers in which diatomaceous earth is held by a hydrophilic polymer binder, etc. Directional porous spreading layer, continuous microvoid-containing porous layer (three-dimensional lattice shape) in which polymer microbeads described in JP-A-55-90859 are adhered in point contact with a polymer adhesive that does not swell with water. It is possible to use a non-fiber isotropic porous development layer composed of a granular structure layer).

It is not necessary to limit the spreading layer to only one layer, but two or more layers are disclosed as disclosed in JP-A-61-4959, 62-138756, 62-135757 and 62-138758. Laminates can be used in which the porous layer is adhered by means of a partially arranged adhesive.

The spreading layer may contain a spreading control agent such as a hydrophilic polymer for the purpose of controlling the spreading property.

Further, various reagents for accelerating the intended detection reaction, or for reducing or preventing interference or interference reactions, or a part of the reagents can be included.

The thickness of the spreading layer is 20 to 200 μm, preferably 50 to 170 μm, more preferably 80 to 150 μm.
μm.

The woven fabric, knitted fabric or papermaking paper used for the porous spreading layer is formed by applying a physical activation treatment typified by glow discharge treatment or corona discharge treatment described in JP-A-57-66359 to fabric cloth or paper. It is applied to at least one side, or is disclosed in JP-A-55-164356 and JP-A-57-6635.
9 or the like to make the cloth or paper hydrophilic by performing a water-washing degreasing treatment, a surfactant-impregnating agent or a hydrophilic polymer-impregnating agent, or a combination of these treatment steps in an appropriate manner to make the fabric or paper hydrophilic. It is possible to increase the adhesive force with the layer on the side closer to the body).

The analysis element of the present invention is characterized in that whole blood is hemolyzed in the spreading layer, that is, the cell membrane of components having cell membranes such as red blood cells and white blood cells (also called formed components) is broken or dissolved. It is characterized in that it contains a reagent component that releases a substance into plasma. Typical examples of this hemolytic reagent are a surfactant having hemolytic ability, a combination of hemolysin (which is an immune component) and complement (eg, a combination of rabbit anti-sheep blood cell antibody and guinea pig complement). Is. Surfactants that can be used include nonionic surfactants; cationic surfactants; anionic surfactants, and preferred surfactants are nonionic surfactants.

Regarding the content of the surfactant, 5 to 11
A dry analytical element is known in which a developing layer contains a nonionic surfactant of g / m ² (JP-A-61-85200). However, this analytical element is intended to eliminate the influence of triglycerides in a dry analytical element serum sample for the determination of total cholesterol, and has not been shown to be applied to a whole blood sample to cause hemolysis.

Preferred nonionic surfactants are:
p- (1,1,3,3-tetramethylbutyl) phenoxypolyethoxyethanol (oxyethylene unit average 9 to 1
0-containing Triton X-100: Oxyethylene unit average 16-containing Triton X-165: Oxyethylene unit average 40-containing Triton X-405, all Chemical Abstract Regi
story No. 9002-93-1) alkylphenol polyethylene oxide condensate: p-nonylphenoxypolyglycidol (glycidol unit average 1
0-containing) alkylphenol polyglycidol condensate: lauryl alcohol polyethylene oxide condensate (eg Brij 35, Chemical Abst
ractRegistry No.9002-92-
0): Cetyl alcohol polyethylene oxide condensate (eg Brij 58, Chemical Abst
ractRegistry No. 9004-95-
9) Polyethylene oxide condensates of higher aliphatic alcohols such as: stearates polyethylene glycol condensates (eg Myrj 52, Myrj 59,
All are Chemical Abstract Regi
story No. 9004-99-3), a higher fatty acid ester condensate of polyethylene glycol: a polyethylene glycol condensate of a sorbitan monolaurate (eg, Tween 20, Chemical A.
btract Registry No. 9005-
There are polyethylene glycol condensates of higher fatty acid sorbitan esters such as 64-5).

The spreading layer may contain a nonionic, anionic, cationic or amphoteric surfactant in order to promote the spreading of the sample.

The surfactant contained in the spreading layer as a hemolytic reagent has a function of making the spread of the whole blood sample uniform and promoting the spread.

The content of the spreading layer of the hemolytic reagent is 1 to 5
0 g / m ² approximately, and preferably from about 2 to 20 g / m ^2.

As a method of containing and impregnating the hemolytic reagent, it may be dissolved or dispersed in a polymer solution for spreading control and then applied from above the spreading layer. Water, an organic solvent, a water-organic solvent mixed solvent or the like can be used as a solvent of the polymer solution,
Alcohol (eg, methanol, ethanol, isopropyl alcohol) or the like can be used as the organic solvent.

In the reagent layer, at least a part of the reagent composition described below, which reacts with a test component in the aqueous liquid to produce an optically detectable change, is substantially uniformly dispersed in the hydrophilic polymer binder. Is a water-absorbing, water-permeable layer. The reagent layer also includes an indicator layer, a coloring layer and the like.

The hydrophilic polymer that can be used as the binder of the reagent layer generally has a swelling ratio of 3 when absorbing water.
About 150% to about 2000% at 0 ° C, preferably about 25%
Natural or synthetic hydrophilic polymer ranging from 0% to about 1500%. Examples of such hydrophilic polymers include JP-A-58-171864 and JP-A-60.
Gelatin (eg, acid-treated gelatin, deionized gelatin, etc.), gelatin derivative (eg, phthalated gelatin, hydroxyacrylate graft gelatin, etc.), agarose, pullulan, pullulan derivative, polyacrylamide, etc. Examples thereof include polyvinyl alcohol and polyvinylpyrrolidone.

The reagent layer can be a layer which is appropriately crosslinked and cured using a crosslinking agent. Examples of the cross-linking agent include known vinyl sulfone-based cross-linking agents such as 1,2-bis (vinylsulfonylacetamido) ethane and bis (vinylsulfonylmethyl) ether for gelatin, aldehydes, aldehydes for methallyl alcohol copolymer, and two There are glycidyl group-containing epoxy compounds and the like.

The dry thickness of the reagent layer is from about 1 μm to about 1
It is preferably in the range of 00 μm, more preferably in the range of about 3 μm to about 30 μm. The reagent layer is preferably substantially transparent.

As the support, a light-transmissive and water-impermeable support that has been used in conventionally known dry analytical elements can be used. Examples of the light-transmitting / water-impermeable support are polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, cellulose ester (eg, cellulose diacetate, sucrose triacetate, cellulose acetate propionate, etc.)
Examples of the transparent support include a film or sheet having a thickness of about 50 μm to about 1 mm, and preferably about 80 μm to about 300 μm.

If necessary, an undercoat layer may be provided on the surface of the support to strengthen the adhesion between the support and the reagent layer provided on the support. Further, instead of the undercoat layer, the surface of the support may be subjected to a physical or chemical activation treatment to improve the adhesive strength.

A light-shielding layer can be provided on the reagent layer, if necessary. The light-shielding layer is water-permeable or water-permeable, in which fine particles having light-absorbing properties or light-reflecting properties (collectively referred to as light-shielding properties) are dispersed and held in a small amount of a hydrophilic polymer binder capable of forming a film. It is a sex layer. The light-shielding layer is the color of the aqueous liquid that is spot-supplied to the developing layer when the detectable changes (color change, color development, etc.) that occur in the reagent layer are reflected and measured from the side of the support having optical transparency. It also functions as a light-reflecting layer or a background layer as well as shielding red color of hemoglobin particularly when the sample is whole blood.

Examples of the light-reflecting fine particles include titanium dioxide fine particles (such as rutile-type, anatase-type or brookite-type fine crystal particles having a diameter of about 0.1 μm to about 1.2 μm), barium sulfate fine particles, Examples thereof include aluminum fine particles or fine flakes, and examples of the light absorbing fine particles include carbon black, gas black, carbon microbeads, etc. Among these, titanium dioxide fine particles and barium sulfate fine particles are preferable. . Particularly preferred are anatase type titanium dioxide fine particles.

Examples of hydrophilic polymer binders having a film-forming ability include hydrophilic polymers similar to the hydrophilic polymers used in the production of the reagent layer described above, as well as weakly hydrophilic regenerated cellulose and cellulose acetate. Among these, gelatin, gelatin derivatives, polyacrylamide and the like are preferable. In addition, gelatin and a gelatin derivative can be used by mixing a known curing agent (crosslinking agent).

The light-shielding layer can be provided by coating an aqueous dispersion of light-shielding fine particles and a hydrophilic polymer on the reagent layer by a known method and drying. Further, instead of providing the light shielding layer, the above-mentioned spreading layer may contain light shielding fine particles.

An adhesive layer may be provided on the reagent layer for adhering and laminating the above-mentioned spreading layer via a layer such as a light shielding layer in some cases.

The adhesive layer can be composed of a hydrophilic polymer which can adhere the spreading layer when it is wet with water or when it is swollen with water, thereby allowing each layer to be integrated. preferable. Examples of hydrophilic polymers that can be used for producing the adhesive layer include the same hydrophilic polymers as those used for producing the reagent layer. Of these, gelatin, gelatin derivatives, polyacrylamide and the like are preferable. The dry film thickness of the adhesive layer is generally in the range of about 0.5 μm to about 20 μm, preferably about 1 μm to about 10 μm.

The adhesive layer may be provided not only on the reagent layer but also on a desired layer in order to improve the adhesive force between other layers. The adhesive layer can be provided by a known method, such as a method of coating an aqueous solution containing a hydrophilic polymer and a surfactant and the like, which is added, on a support, a reagent layer, or the like.

In the dry analytical element of the present invention, a water absorbing layer can be provided between the support and the reagent layer. The water-absorbing layer is a layer containing a hydrophilic polymer that absorbs and swells water as a main component, and is a layer that can absorb water of an aqueous liquid sample that has reached or penetrated the interface of the water-absorbing layer. It has the effect of promoting the permeation of plasma, which is an aqueous liquid component, into the reagent layer. The hydrophilic polymer used in the water absorbing layer can be selected from those used in the reagent layer described above. Generally, gelatin or a gelatin derivative, polyacrylamide, polyvinyl alcohol, especially the above-mentioned gelatin or deionized gelatin is preferable for the water absorbing layer, and the same gelatin as the reagent layer is most preferable. The water-absorbent layer has a dry thickness in the range of about 3 μm to about 100 μm, preferably about 5 μm to about 30 μm, and the coating amount is about 3 g / m ² to about 1.
00 g / m ² , preferably about 5 g / m ² to about 30 g / m
It is in the range of ² . The water-absorbent layer may contain a pH buffer, which will be described later, a known basic polymer, etc., to adjust the pH during use (at the time of performing an analysis operation). Further, the water absorbing layer may contain a known mordant, a polymer mordant or the like.

When analyzing an ammonia-producing substrate such as ammonia or creatinine, a liquid blocking layer is provided on the first reagent layer (indicator layer). This liquid barrier layer is
When manufacturing the multilayer analysis element and / or during the analysis operation,
Micropores with through air holes that are substantially impermeable to liquids such as coating liquids and sample liquids, and interfering components (eg, alkaline components) dissolved and contained in these liquids and to which gaseous ammonia can permeate It is composed of volatile substances. This microporous material is made of a hydrophobic material that does not substantially cause capillary action due to liquid, especially water, or a material that has been hydrophobized (water repellent treatment) to the extent that capillary action does not substantially occur. Is preferably provided.

For the water repellent treatment of the microporous material, a material generally known as a hydrophobizing agent or a water repellent agent typified by silicone resin, silicone oil, fluororesin, and fluorine oil is used as it is or required. With a solvent (for example, hexane, cyclohexane, petroleum ether, etc.), the content of the hydrophobizing agent or water repellent agent is about 0.1 to about 5
It can be carried out by diluting so as to be in the range of wt% and applying it to at least one surface of the microporous material having continuous voids and its vicinity by a method such as impregnation, coating or spraying. .

Examples of the microporous substance include a membrane filter; a microporous material in which fibrous materials are intertwined with each other, or bonded or bonded (for example, paper, filter paper, felt, nonwoven fabric, etc.). ); And microporous materials consisting of woven, knitted or reticulated materials.

For example, a membrane filter made of a fluorine-containing polymer such as cellulose acetate, cellulose nitrate, regenerated cellulose, polyamide, polycarbonate of bisphenol A, polyethylene, polypropylene, polytetrafluoroethylene or the like is required. The water-repellent treated product is exemplified. When used in the integrated multilayer analysis element of the present invention,
The thickness of the membrane filter is about 30 μm to about 3 μm.
In the range of 00 μm, preferably about 70 μm to about 200 μm
It is within the range of m. The porosity (porosity) of the membrane filter is about 25% to about 90%, preferably about 60 to about 90%. The average pore size of the membrane filter is about 0.01 to about 20 μm, preferably about 0.1 to about 1.
It is within the range of 0 μm. Various types of these membrane filters are already commercially available from many manufacturers, and it is possible to select and use from among these commercially available products as necessary.

These microporous materials adhere to the above-mentioned indicator layer with practical strength. For the adhesion, the surface of the indicator layer may be put in a wet state and then stuck and dried. Here, the wet state means that the solvent in which the binder is dissolved remains, or the dried film is wetted with the soluble solvent and the binder is in a swollen state, a dispersed state, or a solution state.

A second reagent layer containing a reagent that reacts with an ammonia-producing substrate to produce ammonia is provided on the liquid barrier layer.

On the second reagent layer, it has a function of substantially preventing the ammonia generated in this reagent layer from diffusing to the endogenous ammonia trapping layer described below, and it can be used for both ammonia trapping and ammonia forming reactions. It is preferable to provide an ammonia diffusion preventing layer which is not substantially used. The ammonia diffusion preventing layer may be replaced with a layer having other functions as long as it is a layer in which the ammonia trapping and the ammonia forming reaction are not substantially performed. Examples of the layer having other functions include a cured hydrophilic polymer layer, a light shielding layer, an adhesive layer and the like.

If the analytical element is for the determination of ammonia-producing substances such as creatinine, it is already present in the aqueous liquid sample either directly on the ammonia diffusion-preventing layer or via the light-shielding layer or other intermediate layers mentioned above. An endogenous ammonia scavenging layer (hereinafter simply referred to as an endogenous ammonia scavenging layer) containing a reagent that acts on ammonia (endogenous ammonia) to change the endogenous ammonia to a state where it cannot substantially reach the reaction layer; It is preferable to provide. The endogenous ammonia-trapping layer removes coexisting endogenous ammonia before an ammonia-producing substance such as creatinine or urea nitrogen, which is an analyte (test component), reaches the reaction layer and ammonia is produced. It is a layer having a function of capturing.

As the endogenous ammonia-trapping reagent, it is preferable to use a reagent composition containing an enzyme having a catalytic ability to convert ammonia into another substance using the substrate as a substrate. Specific examples of the endogenous ammonia scavenging reagent include NADH (nicotinamide adenine dinucleotide reduced type) and / or NADPH (nicotinamide adenine dinucleotide phosphate reduced type), glutamate dehydrogenase (EC1.4.1). .3; hereinafter referred to as GLDH) and α-ketoglutaric acid (or a disodium salt thereof: hereinafter referred to as α-KG). In addition, aspartase (EC4.3.1.
A reagent composition containing 1) and fumaric acid or a fumarate may be used. In the dry analytical element of the present invention, NAD
It is preferable to use a reagent composition containing H, GLDH and α-KG as an endogenous ammonia scavenging reagent. When using a reagent composition containing GLDH or a reagent composition containing aspartase, the pH value of the endogenous ammonia-trapping layer is usually 10.0 or less, preferably 7.0.
It is preferable to use a suitable buffer so that it can be maintained within the range of 9.5.

When the analytical element is for ammonia determination,
It goes without saying that the endogenous ammonia trapping layer is not provided.

The test substance targeted by the analytical element of the present invention is not particularly limited, but it is preferable that the content in blood cells and the content in plasma are not so different from each other in that the content in plasma is known. Examples of such test substances include creatinine, glucose, urea nitrogen, inorganic phosphorus, bicarbonate and the like. The beginning of the present invention was found that the content of creatinine and the like does not change much between blood cells and plasma, and that the measured value of whole blood obtained using the analytical element of the present invention and the measured value of a plasma sample are almost the same. Because I found out that.

The dry analytical element of the present invention contains a reagent depending on the kind of the substance to be tested.

For example, when analyzing ammonia,
As a color-developing ammonia indicator, a leuco cyanine dye,
Leuco dyes such as nitro-substituted leuco dyes and leucophthalein dyes (described in US Reissue Patent No. 30267 or JP-B-58-19062): bromphenol blue, bromcresol green, bromthymol blue, quinoline blue and PH indicator such as rosolic acid (Kyoritsu Shuppan Co., Ltd., Kagaku Daiji, Vol. 10, 6
3 to 65): Triarylmethane dye precursors:
Leucobenzylidene dyes (described in JP-A-55-379 and JP-A-56-145273): diazonium salts and azo dye couplers: base bleachable dyes and the like can be used. The blending amount of the color-forming ammonia indicator with respect to the weight of the binder is preferably within the range of about 1 to 20% by weight.

The reagent for reacting with the ammonia-generating substance to generate ammonia is preferably an enzyme or a reagent containing the enzyme. An enzyme suitable for analysis is selected according to the type of the ammonia-generating substance which is an analyte. Can be used. When an enzyme is used as the above reagent,
The specificity of the enzyme determines the combination of the ammonia producing substance and the reagent. Specific examples of the combination of the ammonia-generating substance and the enzyme as a reagent include urea: urease,
Creatinine: Creatinine deiminase, amino acids:
Amino acid dehydrogenase, amino acid: amino acid oxidase, amino acid: ammonia lyase, amine: amine oxidase, diamine: amine oxidase, glucose and phosphoamidate: phosphoamidate hexose phosphotransferase, ADP: carbamate kinase and phosphate carbamol, acid amide: amide Hydrolase, nucleobase: nucleobase deaminase, nucleoside / nucleoside deaminase, nucleotide: nucleotide deaminase, guanine: guanase and the like can be mentioned. The alkaline buffer that can be used in the reagent layer when analyzing ammonia has a pH of 7.0 to 12.0, preferably 7.5 to 1
Buffering agents in the range of 1.5 can be used.

In the reagent layer for analyzing ammonia,
In addition to a reagent that reacts with an ammonia-generating substance to generate ammonia, an alkaline buffer, and a hydrophilic polymer binder having film-forming ability, a wetting agent, a binder cross-linking agent (curing agent), a stabilizer, a heavy metal ion trap, if necessary. Agents (complexing agents) and the like can be included. The heavy metal ion trapping agent is used for masking heavy metal ions that inhibit enzyme activity. Specific examples of the heavy metal ion trapping agent include EDT
A ・ 2Na, EDTA ・ 4Na, nitrilotriacetic acid (N
TA), a complexane such as diethylenetriaminepentaacetic acid.

Reagent layer, water absorption layer, ammonia diffusion prevention layer,
Endogenous ammonia trapping layer, light reflection layer, adhesive layer, spreading layer,
A surfactant may be contained in the intermediate layer, the developing layer containing the ammonia scavenging reagent composition, and the like. An example thereof is a nonionic surfactant. Specific examples of the nonionic surfactant include p-octylphenoxypolyethoxyethanol, p-nonylphenoxypolyethoxyethanol, polyoxyethylene oleyl ether, polyoxyethylenesorbitan monolaurate, p-nonylphenoxypolyglycidol, octylglucoside, and the like. There is. By including a nonionic surfactant in the developing layer, the developing action (metering action) of the aqueous liquid sample becomes better. By containing a nonionic surfactant in the reagent layer or the water absorbing layer, water in the aqueous liquid sample is easily absorbed into the reagent layer or the water absorbing layer substantially uniformly during the analysis operation, and the liquid with the developing layer is easily absorbed. Contact is rapid and substantially uniform.

Examples of glucose measuring reagent compositions include:
U.S. Pat. No. 3,992,158; JP-A-54-2679
3; JP-A-59-20853; JP-A-59-4685
4. There is an improved Trinder reagent composition containing glucose oxidase, peroxidase, 4-aminoantipyrine or a derivative thereof, and 1,7-dihydroxynaphthalene described in JP-A-59-54962.

The dry analytical element of the present invention can be prepared by a known method. The hemolytic reagent may be added in advance to the reagent aqueous solution to be applied or impregnated. As another method, an aqueous solution containing a surfactant, a hydrophilic polymer or the like for controlling the spreading area, an organic solvent (ethanol, methanol, etc.) solution, or a water-organic solvent mixed solution solution is applied on the spreading layer. It can also be impregnated. The analysis of the analyte using this can also be performed according to a known method. Incubation temperature, time, etc. may be the same as conventional ones.

The embodiments of the present invention will be described below.

[Embodiment 1] In a dry analysis element in which a spreading layer for spreading whole blood, at least one reagent layer and a water-impermeable support are laminated in this order, whole blood is hemolyzed in the spreading layer. A dry analytical element, which comprises a reagent component for

[Embodiment 2] The dry analytical element according to Embodiment 1, wherein the reagent component for hemolyzing the whole blood is a surfactant.

[Embodiment 3] The dry analytical element according to Embodiment 2, wherein the surfactant is a nonionic surfactant.

[Embodiment 4] The dry analytical element according to Embodiment 3, wherein the nonionic surfactant is alkylphenoxypolyethoxyethanol.

[0058] [Embodiment claim 5] The surfactant dry analytical element according coating amount to embodiment claim 2 is in the range of 1g / m ² ~50g / m ² in the spreading layer.

[Embodiment 6] The dry analytical element according to Embodiment 2, wherein the coating amount of the surfactant in the spreading layer is in the range of ² g / m ^{2 to} 20 g / m ² .

[Embodiment 7] The dry analytical element according to Embodiment 1, wherein the reagent composition contained in the dry analytical element is a reagent composition for measuring creatinine, urea nitrogen, inorganic phosphorus or bicarbonate. .

[Embodiment 8] The dry analytical element according to Embodiment 1, wherein the reagent composition contained in the dry analytical element is a reagent composition for measuring creatinine or urea nitrogen.

[0062]

【Example】

Example 1 On a colorless transparent polyethylene terephthalate (PET) smooth film sheet (support) having a thickness of 180 μm, ethanol was applied as a solvent to a coating amount shown in Table 1 and dried to form an indicator layer. .

[0063]

[Table 1]

From the top, the average pore diameter is 0.2 μm and the porosity is 7
A 5% polyethylene membrane filter having a thickness of 100 μm was uniformly pressed to provide a liquid barrier layer.

On this liquid barrier layer, water was used as a solvent so that the coating amount shown in Table 2 was applied, and the coating liquid was dried to provide an ammonia-forming reaction reagent layer.

[0066]

[Table 2]

On the reaction reagent layer, water was used as a solvent so that the coating amount shown in Table 3 was applied, and the coating was dried to provide an ammonia diffusion preventing layer.

[0068]

[Table 3]

On the ammonia diffusion preventing layer, water was used as a solvent so that the coating amount shown in Table 4 was applied, and the coating was dried to form an endogenous ammonia trapping layer.

[0070]

[Table 4]

*:

Embedded image In the formula, R is H or —CH ₂ CH ₂ COOH —COOH to —NH— (or —NH ₂ ) ratio is 2: 6.

Then, water was supplied onto the endogenous ammonia-trapping layer at a rate of 30 g / m ² to wet it,
Tricot knitted fabric made of pure polyethylene terephthalate with a thickness of 300 μm (made of spun yarn equivalent to 100S and having a thickness of about 25
(0 μm) was uniformly lightly pressed and laminated. Further, this knitted fabric was impregnated with ethanol as a solvent so that the coating amount shown in Table 5 was obtained, coated and dried to provide a spreading layer.

[0073]

[Table 5]

Thus, an integrated multi-layer analytical element for creatinine measurement was produced.

Comparative Example 1 As a comparative control, an analytical element for measuring creatinine was produced in the same manner as in Example 1 except that Triton X100 was omitted from the composition of the spreading layer of Example 1 (Table 5).

Measurement Example 1 In order to examine the hematocrit dependence of the analytical elements of Example 1 of the present invention and Comparative Example 1, the following test was carried out.

From human whole blood assayed to have a creatinine concentration of 7.5 mg / dL, plasma (corresponding to 0% hematocrit) and hematocrit values of 10%, 20%, 30%,
Whole blood adjusted to 40% and 50% was prepared, and 10 μl of each was spotted on this analytical element.

After each analytical element was incubated at 37 ° C. for 6 minutes, the reflection density from the support side was measured at a wavelength of 600 nm. The creatinine concentration converted from this reflection density is shown in [Table 6] and [Fig. 1]. In the figure, the solid line indicates the result obtained using the example product, and the dotted line indicates the result obtained using the comparative example product.

[0079]

[Table 6]

In the analysis element according to the present invention, the creatinine concentration in the range from plasma (corresponding to 0% hematocrit) to a whole blood sample having a hematocrit value of 50% is accurate to a test value of 7.5 mg / dL regardless of the hematocrit value. It is clear that it can be measured as any value. On the other hand, in the analysis element of the comparative example, the creatinine concentration in the range from plasma to a whole blood sample having a hematocrit value of 40% has a test value of 7.5 m.
It was found that only very inaccurate measurements were obtained, which differed from g / dL by about 20% to about 70%.

Example 2 An aqueous solution of a glucose measuring reagent composition was coated on a colorless transparent polyethylene terephthalate sheet (support) having a thickness of 180 μm and a smooth surface so that the coating amount was as shown in Table 7, and dried. Thus, a glucose measuring reagent layer was provided.

[0082]

[Table 7]

Then, water was supplied onto the reagent layer at a rate of 30 g / m ² to moisten the surface of the reagent layer almost uniformly, and then a tricot knitted fabric made of pure polyethylene terephthalate (made of spun yarn equivalent to 100S). (About 250 μm in thickness) was lightly pressed and uniformly laminated to form a spreading layer.

An ethanol solution containing a hemolytic reagent component and a hydrophilic polymer for spreading control was applied onto the spread layer of the knitted fabric so as to have the coverage shown in Table 8, impregnated and dried to form an integrated type for glucose determination. A multilayer dry analytical element was prepared.

[0085]

[Table 8]

Comparative Example 2 An integrated multi-layer dry analysis for glucose determination, which belongs to the prior art, is carried out in the same manner as in Example 2 except that the composition for coating and impregnating the knitted fabric spread layer of Example 2 does not contain a hemolytic reagent component. A device was created.

Measurement Example 2 In the dry analytical elements of Example 2 and Comparative Example 2 of the present invention, the following evaluation test was carried out in order to investigate the influence (error) of the hematocrit value of the whole blood sample on the measurement result.

From human whole blood assayed to have a glucose concentration of 380 mg / dL, plasma (corresponding to 0% hematocrit),
And hematocrit value 10%, 20%, 30%, 40
% And 50% of the whole blood was prepared, and 10 μL of the whole blood was spotted on each of the analytical elements. After incubating each analytical element at 37 ° C. for 6 minutes, visible light having a central wavelength of 550 nm was irradiated from the support side to measure the reflection optical density. Table 9 and FIG. 2 show the results of obtaining glucose concentration values from the obtained reflection optical density values using a calibration curve prepared in advance.

[0089]

[Table 9]

In the analysis element according to the present invention, the glucose concentration in the range from plasma (corresponding to 0% hematocrit) to a whole blood sample having a hematocrit value of 50%, regardless of the hematocrit value, is a precise glucose value close to the assay value of 380 mg / dL. It is clear that can be measured as On the other hand, in the analytical element of the comparative example, the glucose concentration was 380 mg / g for the whole blood sample having a hematocrit value of 20% to 30%.
Although accurate measurement values within the range of dL to about 3.7% or less were obtained, the glucose concentration ranged from about 9% to about 15% from the assay value in the range from plasma to a whole blood sample having a hematocrit value of 10%. In the range of whole blood samples with values of 40% to 50%, glucose concentrations were about 8% to about 17% of the assay value.
It turned out that only very inaccurate readings were obtained at%.

[0091]

The dry analysis element of the present invention can analyze a whole blood sample easily and accurately regardless of the hematrit value.

[Brief description of drawings]

FIG. 1 is a diagram showing a creatinine concentration assay value of 7.5 mg / using the analytical element of Example 1 of the present invention and the analytical element of Comparative Example 1.
It is a guffula which shows the test result of the measurement example which measured the whole blood sample whose hematocrit value differs from the plasma sample of dL. The horizontal axis of the graph represents the hematocrit value, and the vertical axis represents the measured creatinine concentration value.

FIG. 2 shows a glucose concentration test value of 380 mg / d using the analytical element of Example 2 of the present invention and the analytical element of Comparative Example 2.
It is a graph which shows the test result of the measurement example which measured the whole blood sample whose hematocrit value differs from the plasma sample of L. The horizontal axis of the graph represents the hematocrit value, and the vertical axis represents the measured glucose concentration value.

Claims (1)

[Claims] 1. A dry analytical element in which a spreading layer for spreading whole blood, at least one reagent layer and a water-impermeable support are laminated in this order, wherein a reagent component for hemolyzing whole blood is present in the spreading layer. A dry analysis element characterized by being included.