JPH05264539A - Whole blood analysis element - Google Patents

Abstract

PURPOSE:To reduce analysis errors by laminating blood cell separation layers comprising a finely porous film having a hydrophilic surface with a specified pore size to prevent unevenness of permeation and diffusion when blood cells are separated by a simple means. CONSTITUTION:More than one blood cell separation layers which comprise a finely porous film having a hydrophilic surface with a pore size of below 10mum are laminated to form an analysis element. The blood cell separation layer can utilize any of a non-fibrous finely porous film, for example, made of cellulose acetate or cellulose acetate/butylate and a fibrous finely porous film such as filter paper or unwoven cloth. The blood cell separation film preferable is a finely porous film comprising a polymer containing fluorine such as polyvinylidene fluoride. Among finely porous films of the polymer containing fluorines, those suitable for composing a blood cell filter layer are so small in pore size as not to let blood cells pass in substance and high in void with a less film thickness. To be more specific. the pore size is in a range of 0.2-60mum, preferably 1-10mum.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a dry multi-layer analytical element capable of analyzing a whole blood sample.

[0002]

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

[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.

In order to carry out a dry chemical analysis using whole blood as a sample, blood cells (red blood cells and white blood cells) and other high molecular weight components of whole blood must be separated by some means in the analytical element. One of the means is to provide a blood cell separation layer in the multilayer analysis element. This blood cell separation layer has glass fiber,
It is disclosed in JP-A-57-53661 and JP-A-61-96466 that a membrane filter, a cotton wool fiber product and the like can be used. It is also known to combine two or more layers, and it is disclosed in JP-A-62-138756 and JP-A-3-
Japanese Patent No. 16651 and the like disclose combining a fibrous porous layer and a non-fibrous porous layer. However,
The use of two or more layers of the same blood cell separating layer is not disclosed in any document.

[0005]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The blood cell portion in a whole blood sample has a large hematocrit value and is as large as 70%.
Therefore, the blood cell separation layer is likely to be clogged, and the permeation / diffusion of the plasma portion is likely to be uneven. As a result, there is a problem in that the analysis result varies depending on the measurement site of the analysis element, resulting in an error.

An object of the present invention is to provide a whole blood analysis element which prevents non-uniformity of permeation / diffusion which occurs when separating blood cells by a simple means and reduces analysis error based on the non-uniformity.

[0007]

The present invention has been made to solve the above problems, and achieves the object by a whole blood analysis element characterized in that two or more blood cell separation layers are laminated. It was done.

As the blood cell separation layer, either a non-fibrous microporous membrane or a fibrous microporous membrane can be used. As the non-fibrous microporous membrane, Japanese Patent Publication No. 53-21677, U.S. Pat.
Cellulose esters described in 1,341, etc., for example, cellulose acetate, cellulose acetate / butyrate, a brush polymer composed of cellulose nitrate, 6-
Microporous membranes made of nylon, polyamide such as 6,6-nylon, polyethylene, polypropylene, etc., JP-A-62-27006
, A microporous membrane made of polysulfone, a microporous membrane made of nitrocellulose, Japanese Patent Publication No. 53-21677.
A porous layer having continuous voids in which small polymer particles, glass particles, diatomaceous earth and the like described in JP-A-55-90859 are bonded with a hydrophilic or non-water-absorbing polymer can be used.
As the fibrous microporous membrane, filter paper, non-woven fabric, woven fabric (for example, plain woven fabric), knitted fabric (for example, tricot stitch), glass fiber filter paper and the like can be used.

The preferred blood cell separation membrane is a microporous membrane composed of a fluorine-containing polymer. Examples of the fluorine-containing polymer include polyvinylidene fluoride and polytetrafluoroethylene, and polytetrafluoroethylene is preferable.

As the microporous layer of these fluorine-containing polymers, fibrils (fine fibers) of polytetrafluoroethylene described in JP-A-63-501594 (WO 87/02267) can be used.
Microporous matrix layer consisting of (microporous layer), Go
re-Tex (WL Gore and Associates), Zitex (Norton
Manufactured by Sumitomo Electric Co., Ltd., etc. In addition, US3368872 (Examples 3 and 4), US3260413
(Examples 3 and 4), JP-A-53-92195 (US4201548)
A microporous membrane of polytetrafluoroethylene as described in
There is a microporous membrane of polyvinylidene fluoride described in US3649505.

Of these microporous membranes of fluorine-containing polymer, those which are particularly suitable for the microporous layer constituting the blood cell filtration layer have a pore size that is so small as to substantially prevent the passage of red blood cells and a membrane thickness. It is thin and has a high porosity. Specifically, it is in the range of about 0.2 μm to about 60 μm, preferably about 1 μm to about 20 μm, more preferably 1 to 10 μm, and the porosity is about 40% to about 95%, preferably about 50% to about 95%.
%, More preferably about 70% to about 95%, the thickness of the layer is about 10 μm to about 200 μm, preferably about 30 μm to about 150 μm, and the most preferable in consideration of handling such as wrinkling in the manufacturing process. Is in the range of about 30 μm to about 80 μm.

In forming the microporous film of these fluorine-containing polymers, one kind or two or more kinds of fluorine-containing polymers may be mixed, or one kind or two or more kinds of polymers or fibers containing no fluorine may be mixed. It may be mixed with and formed into a film.

The structure is not stretched, uniaxially stretched, biaxially stretched, one-layer non-laminated type, two-layer laminated type, for example, laminated to another membrane structure such as fiber. There are membranes.

The fibril structure or the uniaxially-stretched or biaxially-stretched non-laminate type microporous membrane produces a microporous membrane having a large porosity and a short filtration length by stretching. Microporous membranes with short filtration length are characterized by high accuracy of quantitative analysis because they are less likely to be clogged by formed components (mainly red blood cells) in blood and the time required for separating blood cells and plasma is short. .

A microporous layer of a fluorine-containing polymer is disclosed in JP-A-57 / 57
-66359 (US4783315) physical activation treatment (preferably glow discharge treatment or corona discharge treatment) is applied to at least one side of the microporous layer to make the surface of the microporous layer hydrophilic, and to adjoin adjacent fine particles. The adhesive strength of the adhesive used for partial adhesion with the porous layer can be enhanced.

Even if the microporous membrane of a fluorine-containing polymer has a low surface tension and is used as a blood cell filtration layer of a dry analytical element as it is, the aqueous liquid sample is repelled and diffuses on the surface or inside of the membrane. It is a well-known fact that it does not penetrate. In the analysis element of the present invention, as a means for imparting hydrophilicity to the fluorine-containing polymer microporous film to enhance the hydrophilicity, the surface of the voids inside and outside of the fluorine-containing polymer microporous film is substantially hydrophilic. The problem of repelling the aqueous liquid sample was solved by impregnating the microporous membrane of the fluorine-containing polymer with a sufficient amount of a surfactant to render it soluble.

In order to impart sufficient hydrophilicity to a microporous film of a fluorine-containing polymer so that the aqueous liquid sample can be diffused, permeated and transferred to the surface or inside of the film without being repelled,
About 0.01% to about 10%, preferably about 0.1% to about 5.0%, more preferably 0.1% to 1%, of the void volume of the fluorine-containing polymer microporous membrane, of the pore volume of the microporous membrane. The surface needs to be coated. For example, the thickness is 50
When the microporous membrane of fluorine-containing polymer [mu] m, the amount of surfactant to be impregnated is preferably generally in the range of 0.05 g / m ² of 2.5 g / m ^2. As a method of impregnating a microporous film of a fluorine-containing polymer with a surfactant, an organic solvent (eg, alcohol, ester, ketone) having a low boiling point of the surfactant (preferably having a boiling point of about 50 ° C to about 120 ° C) is used. )
After soaking the microporous membrane of the fluorine-containing polymer in the solution and allowing the solution to penetrate the internal voids of the microporous membrane substantially sufficiently, the microporous membrane is gently pulled up from the solution and blown (warm air The preferred method is to feed (dry) and dry. A surfactant may be contained in the microporous membrane of the fluorine-containing polymer together with the components such as the pretreatment reagent contained in the microporous layer constituting the blood cell filtration layer.

Surfactants used for hydrophilizing a microporous film of a fluorine-containing polymer include nonionic (nonionic), anionic (anionic), cationic (cationic) and amphoteric. Any surfactant can be used.

Among these surfactants, the nonionic surfactant has a relatively low effect of hemolyzing erythrocytes, and is therefore advantageous in a multilayer analytical element for taking whole blood as a sample. As the nonionic surfactant, alkylphenoxy polyethoxy ethanol, alkyl polyether alcohol, polyethylene glycol monoester, polyethylene glycol diester, higher alcohol ethylene oxide adduct (condensate), polyhydric alcohol ester ethylene oxide adduct (condensate), Higher fatty acids such as alkanolamides.

The following are specific examples of the nonionic surfactant. Examples of the alkylphenoxypolyethoxyethanol include: isooctylphenoxypolyethoxyethanol: (Triton X-100: containing oxyethylene unit average 9 to 10) (Triton X-45: containing oxyethylene unit average 5) nonylphenoxypolyethoxyethanol: ( IGEPAL CO-630: containing 9 units of oxyethylene units on average) (IGEPAL CO-710: containing 10 to 11 units of oxyethylene units on average) (LENEX 698: containing 9 units of oxyethylene units on average) As an alkyl polyether alcohol, higher alcohols such as polyoxyethylene Ether: (Triton X-67: CA Registry No.59030-15-8)

The whole blood analysis element of the present invention comprises two blood cell separation layers.
It is characterized by using more than one layer. It is preferable that the respective layers are laminated by changing the web direction of the microporous membrane used as the blood cell separation layer or by inverting them. The number of layers is usually two or three. Since a plurality of layers are stacked and used, each layer can be thinner than before. For example, in the case of two layers, one having a thickness of 1/2 may be used, and in the case of three layers, one having a thickness of 1/3 may be used.

The blood cell separation layers need only be laminated so that plasma can diffuse and permeate through the layers.
It may or may not be adhered. When gluing,
Adhesion is performed by the so-called partial adhesion method so as not to hinder the diffusion and penetration of plasma. Partial adhesion refers to JP 61-4959 (E
P0166365A), JP-A-62-138756-138758 (EP02264).
65A) or the like, which is a mode of adhesion between two adjacent porous layers or between adjacent porous layers and a non-porous layer, which is "partially (or intermittently between the interfaces of two adjacent layers. ) Is substantially adhered to and integrated with the adhesive disposed in (1), and is configured so that the uniform passage of the liquid is not substantially hindered between the adjacent two surfaces. ..

The method of partially disposing the adhesive on the blood cell separation layer is described in JP-A-64-1959, JP-A-62-138756, and JP-A-64-23.
Various methods described in 160 (DE3721236A) and the like can be used. Of these methods, the printing method is preferred. Among the printing methods, a method of transferring and adhering the adhesive to the porous layer or the detection function layer using a printing plate (a gravure printing plate or an intaglio plate is preferable) and a method of bonding two adjacent layers are, for example, 839-853, "Printing Engineering Handbook" edited by Japan Society of Printing Science (Gihodo Publishing Co., Ltd., 1983)
It can be carried out by a known device and method described in pages and the like.

The adhesive used is disclosed in Japanese Patent Laid-Open No. 62-1387.
Various kinds of adhesives described in 56 and other known adhesives described in the above-mentioned "Printing Engineering Handbook", pages 839 to 853, etc. can be used. As the adhesive, a water solvent type adhesive, an organic solvent type adhesive, or a heat adhesive (or heat sensitive) adhesive can be used. Examples of water-solvent type adhesives include water-based glues such as starch glue; dextrin, carboxymethyl cellulose,
An aqueous solution of polyvinyl alcohol or the like; a vinyl acetate-butyl acrylate copolymer emulsion is available. As the organic solvent type adhesive, one having a slow solvent evaporation is suitable. Thermal adhesive (or heat sensitive) adhesives are particularly useful.

As the heat-adhesive (or heat-sensitive) hot-melt type adhesive, the hot-melt type adhesive described in "Industrial Materials", Vol. 26 (No. 11), pp. 4-5 can be used. Examples thereof include ethylene copolymers such as ethylene-vinyl acetate copolymer, ethylene-ethyl acrylate copolymer, ethylene-acrylic acid copolymer; polyolefins such as low molecular weight polyethylene and atactic polypropylene; nylon, etc. 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; synthetic wax.

Of these, silicone-based, acrylic-based, and phenolic-based pressure-sensitive adhesives are particularly useful in the present invention.

In the whole blood analysis element of the present invention, it is sufficient that two or more layers of the same blood cell separation layer are laminated, and a different kind of receptor layer or blood cell separation layer can be further incorporated on this layer.
In that case, a combination of a fibrous porous layer and a non-fibrous porous layer as disclosed in JP-A-3-16651 can be used.

Other layers are commonly used for whole blood analysis elements,
At least one hydrophilic polymer layer and a spreading layer are laminated in this order on a water-impermeable support.

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 with a substantially constant amount per unit area. As the spreading layer that has been used in the dry chemistry analysis element so far, all known non-fibrous and fibrous porous materials can be used.
Specifically, a non-fibrous isotropic microporous medium layer represented by a membrane filter (brushed polymer) disclosed in JP-A-49-53888 and a polymer disclosed in JP-A-55-90859. A non-fibrous porous layer typified by a continuous void-containing three-dimensional lattice granular structure layer formed by adhering micro beads in a point contact manner with a water non-swelling adhesive, JP-A-55-16
4356, 57-66359 and the like, a porous layer made of a woven fabric, a layer made of a knitted fabric, and the like disclosed in 60-222769, etc. can be mentioned, but not limited thereto.

It is not necessary to limit the development layer to only one layer, and it is possible to use the development layers disclosed in JP-A-61-4959, 62-138756, 62-135757 and 62-135757.
As disclosed in 62-138758 and the like, two or more layers can be stacked and used.

For a multi-layer analytical element having two or more spreading layers, it is essential that all layers are laminated and integrated at the time of spotting a sample, but they are integrated in the subsequent process. No need. If necessary, it can be used in a state where the first developing layer and the second developing layer are separated from each other.

The spreading layer may contain a nonionic, anionic, cationic or amphoteric surfactant in order to accelerate the spreading of the sample. Further, a development control agent such as a hydrophilic polymer may be included for the purpose of controlling the development property. Furthermore, various reagents for accelerating the target 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.

Between the spreading layer and the blood cell separating layer, it is sufficient that the plasma portion diffuses and permeates, and it may or may not be adhered. However, in the case of adhesion, it is preferable to use the above-mentioned partial adhesion.

The hydrophilic polymer layer is soluble in the known water used in dry chemistry analysis elements,
Various swelling and hydrophilic polymers can be used.
A natural or synthetic hydrophilic polymer having a swelling ratio upon absorption of water at 30 ° C. of about 150% to about 2000%, preferably about 250% to about 1500% can be used. Akira
59-171864, 60-108753 and the like (eg, acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (eg, phthalated gelatin, hydroxyacrylate graft gelatin, etc.), agarose, pullulan, pullulan derivatives, Examples thereof include polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, etc., but are not limited thereto.

Instead of the hydrophilic polymer layer, paper having a hydrophilic surface or a polymer porous membrane can be used.

The thickness of the hydrophilic polymer layer is about 1 when dried.
μm to about 100 μm, preferably about 3 μm to about 50 μm, particularly preferably about 5 μm to about 30 μm, and preferably substantially transparent.

The hydrophilic polymer layer may contain various reagents or a part of reagents for promoting the intended reaction or for preventing or reducing the interference or interfering reaction.

As the water-impermeable support, a known water-impermeable support conventionally used for dry chemistry analysis elements can be used. Specifically, polyethylene terephthalate, bisphenol A polycarbonate, polystyrene, cellulose ester (for example,
Cellulose diacetate, cellulose triacetate,
Cellulose acetate propionate, etc.),
Thickness about 50 μm to 1 mm, preferably about 80 μm to about 300 μm
Can be used. The support is usually a light-transmissive one, but it may be colored or light-impermeable when the measurement is performed from the spreading layer side. If necessary, a publicly known undercoat layer or an adhesive layer may be provided on the surface of the support to strengthen the adhesion with the hydrophilic polymer layer.

In the whole blood analysis element of the present invention, the blood cell separation layer and the development layer can be arranged with a space. This space layer is for preventing the whole blood sample spotted on the blood cell separation layer from diffusing into the spreading layer.
On the other hand, the thickness of this space layer is
By pressing the sample spotting portion of the blood cell separation layer after the development to some extent, both layers should come into contact with each other and the plasma portion in the blood cell separation layer should be able to transfer to the development layer. Therefore, although the thickness of this space layer varies depending on the material and thickness of both layers, the diameter of the space, etc., it is usually about 0.1 to 5 mm, especially 0.1 to 1 mm.
Often the appropriate degree. The diameter of the space layer is usually 3 to
10mm is suitable. A spacer is generally used as a means for forming the space layer. The shape of the spacer may be, for example, a frame body having a circular window, a square window, or the like, and strip-shaped spacers may be arranged at both ends between both layers. The material of the spacer may have a certain degree of physical strength and can be selected from a wide variety of materials. For example, it may be a water-impermeable material such as that used for a support or a water-permeable material such as used for a spreading layer or a blood cell separation layer. The spacer may be glued to both elements or simply sandwiched and held. As the means, various known means can be used. In order to remove the blood cell separation layer after transferring the plasma part of the whole blood sample, when both layers are adhered to the spacer, at least both layers should be peeled off. Is preferred. The means is
An adhesive having a weak adhesive force and capable of peeling may be used for bonding at least one of them, or the spacer itself may be destroyed to separate the two layers. It is preferable to provide a tab or the like on one element, for example, the blood cell separation layer, to facilitate the peeling.

In the present invention, the target test substance is not particularly limited. In addition to enzymes, lipids, inorganic ions, metabolites, proteins, etc., which are usually measured in the field of clinical testing, various globulin, immune antigens, biologically derived components such as immune antibodies,
If analysis methods such as drugs, hormones, tumor markers, etc. are established, they can be analyzed.

The dry analytical element used in the present invention is
Various configurations described below can be adopted depending on the item or sample to be measured. 1: A water-impermeable support / hydrophilic polymer layer / developing layer / spacer (yes / no) / blood cell separation layer / blood cell separation layer, which includes a reagent that directly reacts with a test substance to cause a chemical change Not an analysis element. Ca, GOT (glutamate oxaloacetate transaminase), GPT (glutamate pyruvate transaminase), γ-GTP (γ-glutamyl transpeptidase), glucose, LDH
(Lactate dehydrogenase), CPK (creatine phosphokinase), TP (total protein), Alb (albumin), TCH
It is effective for analysis of O (total cholesterol), UA (uric acid), and neutral fat.

2: Water-impermeable support / hydrophilic polymer layer / developing layer / spacer (yes / no) / blood cell separation layer / blood cell separation layer, wherein the hydrophilic polymer layer and / or the development layer are colored. An analytical element that contains a drug substance but does not contain a reagent that reacts directly with a test substance to cause a chemical change. As the chromogen, 4-type described in Ann. Clin. Biochem., 6, 24-27 (1969).
Aminoantipyrine (also known as 4-aminophenazone, namely 1-phenyl-2,3-dimethyl-4-amino-3-pyrazoline
-5-one), 1- (2,4,6-trichlorophenyl) -2,3-dimethyl-4-amino-3-pyrazolin-5-one described in JP-A-59-54962, 1- ( 3,5-dichlorophenyl) -2,3-dimethyl
4-Substituted-4-amino such as 4-amino-3-pyrazolin-5-one
-3-pyrazolin-5-one, 1-described in JP-B-55-25840
4-Aminoantipyrine analogs such as phenyl-2,3-dimethyl-4-dimethylamino-3-pyrazolin-5-one can be used. Among these compounds, 4-aminoantipyrine, 1- (2,4,6-trichlorophenyl) -2,3-dimethyl-4-amino-3-pyrazolin-5-one, 1- (3,5- Dichlorophenyl) -2,3-dimethyl-4-amino-3-pyrazolin-5-
ON and the like are preferable.

3: Water-impermeable support / hydrophilic polymer layer / developing layer / spacer (yes / no) / blood cell separation layer / blood cell separation layer, wherein the hydrophilic polymer layer and / or the development layer contain Analytical elements including chromogens and other reagents (excluding measurement reagents described below). Other reagents include POD
(Peroxidase), NAD (nicotinamide adenine dinucleotide), NADP (nicotinamide adenine dinucleotide phosphate), DIP (diaphorase) and the like. In the configurations 2 and 3, the chromogen or other reagent supplies a liquid sample.
It can be supplied after stabilization, but many chromogens are insoluble in water and must be supplied separately from the measurement reagents. Produced by including these chromogens and other reagents in the layer from the beginning. There are advantages such as better reproducibility.

4: Analytical element containing mordant layer. When the coloring reagent forms an ionic dye, a mordant layer can be provided between the water-impermeable support and the reagent layer. The efficiency of optical detection can be improved by transferring and trapping the dye generated in proportion to the amount of the test substance in the sample to the mordant layer. For example, when the coloring pigment forms a cationic dye, the mordant layer is a hydrophilic polymer layer containing a polymer containing an anion atom or an atomic group bonded to a polymer chain, and the coloring reagent is an anion. In the case of forming a volatile dye, a hydrophilic polymer layer containing a polymer containing a cation atom or an atomic group bonded to a polymer chain can be used as the mordant layer.
For details of these mordant polymers, see Japanese Patent Publication No. 2-
30466, JP-A-51-40191, JP-A-54-29700, JP-A-53-131089
Etc. For example, as the anion mordant polymer, an alkaline hydrolyzate of methyl vinyl ether-maleic anhydride copolymer described in JP-B No. 2-30466, column 13 to 14, a polystyrene-p-sulfonic acid Examples thereof include an alkali metal salt or an alkaline earth metal salt, an alkali metal salt or an alkaline earth metal salt of a copolymer of styrene-p-sulfonic acid and a hydrophilic vinyl monomer, and the like. Further, layers and the like capable of containing these polymers are also described in detail in columns 15 to 16 of the publication.

5: An analytical element in which the light shielding layer is provided between the hydrophilic polymer layer and the spreading layer in the constitutions 1 to 4 above.
The light-shielding layer is a water-permeable material in which fine particles or fine powder (hereinafter, simply referred to as fine particles) having both light-shielding properties and light-reflective properties are dispersed and held in a hydrophilic polymer binder having a small film-forming ability. Or a water permeable layer. The light shielding layer shields the color of the supplied aqueous liquid sample, especially the red color of hemoglobin contained in the whole blood sample, when the detectable change (color change, color development, etc.) is reflected and measured from the light-transmissive support side. In addition, it also functions as a light reflection layer or a background layer. Examples of fine particles having both light-shielding property and light-reflecting property are titanium dioxide fine particles (such as rutile-type, anatase-type or brookite-type fine crystal particles having a particle size of about 0.1 μm to about 1.2 μm), barium sulfate fine particles, and aluminum fine particles. Alternatively, there are fine flakes and the like, and examples of the light-shielding fine particles include carbon black, gas black, carbon microbeads, etc. Among these, titanium dioxide fine particles and barium sulfate fine particles are preferable. As the hydrophilic polymer binder having a film-forming ability, there are weakly hydrophilic regenerated cellulose, cellulose acetate and the like in addition to the above-mentioned hydrophilic polymers, and among them, gelatin, gelatin derivatives, polyvinyl alcohol, polyacrylamide, maleic acid Polymers and the like are preferred. A known hardening agent (crosslinking agent) can be mixed and used for gelatin and a gelatin derivative.

6: An analytical element in the above constitutions 1 to 4, in which a water-impermeable and gas-permeable layer (hereinafter referred to as a barrier layer) is provided between the hydrophilic polymer layer and the spreading layer. It is effective for analysis of BUN (urea nitrogen), CRE (creatinine), CO _{2 and the} like which generate ammonia gas by the reaction. Either whole blood or plasma can be used as the sample. As the barrier layer, the uniform polymer coating layer disclosed in JP-A-52-3488, the membrane filter disclosed in JP-A-58-77661 and the like can be used.

Here, the measurement reagent refers to a reagent that directly reacts with a test substance to be analyzed to cause a chemical change.
That is, when the enzyme is a test substance, its substrate is, and when the test substance is an antigen (antibody), it is an antibody (antigen),
When the test substance is a lipid, sugar, or metabolite that produces a change detectable by an enzyme, the enzyme is the enzyme. In addition, when these reactions are caused by a general chemical reaction by a chemical reagent other than an enzyme, it refers to a corresponding chemical substance. A specific example will be described below.

When the test substance is GOT, which is an enzyme,
Its substrates aspartic acid and α-ketoglutaric acid, high-molecular-weight starch or low-molecular-weight oligosaccharides for amylase, L-γ-glutamylparanitroanilide for GGT, paranitrophenylphosphate for ALP, etc. Is.

Further, glucose is glucose oxidase, uric acid is uricase, cholesterol is cholesterol esterase or cholesterol oxidase, neutral fat is lipase or esterase, and urea is urease.

When an analyte is a protein, albumin, Ca, inorganic phosphorus or the like, and a test substance directly reacts with an indicator or the like to cause a detectable change, the term "indicator" is used.

When the reaction reagents incorporated in the above reaction system are unstable like some enzymes, it is preferable that these are also contained in the measurement reagent.

That is, regarding the distribution of the reagent to be contained in the measurement reagent solution and the reagent to be contained in the analytical element,
It can be variously changed by using the analytical performance and storage stability as an index. Of course, even if there is only one analysis target, the above distribution differs depending on the assembly of the detection reaction system.

Among the measuring reagents, in order to proceed the reaction stably and with good reproducibility, pH and ionic strength are adjusted, diffusion and permeation into the material constituting the analytical element are improved, and an enzyme contained therein is included. Various reagents can be included for the purpose of improving instability. It is also possible to include a reagent for inhibiting a reaction that competes with the detection reaction.

Examples of such a reagent include bilirubin oxidase and ascorbate oxidase. Further, for isozyme detection, a compound that inhibits an enzyme derived from a specific organism, such as an inhibitor of P-type amylase, can be included.

Further, in the measurement of whole blood, NaN ₃ or the like which is effective as an inhibitor of the catalase activity of hemoglobin can be added.

The whole blood analysis element used in the present invention is cut into a small piece such as a square having a side of about 5 mm to about 30 mm or a circle of about the same size, and is disclosed in JP-A-57-63452, JP-A-54-156079, and JP-A-54-156079. 56-142454, 58-32350, 58-501144
It is preferable to use it as an analytical slide by accommodating it in the slide frame described in the above etc. from the viewpoint of production, packaging, transportation, storage, measurement operation and the like.

However, a so-called stick form similar to the urine test paper may be used.

The method of using the whole blood analysis element of the present invention may be the same as other known whole blood analysis elements. When a space layer is provided between the blood cell separation layer and the spreading layer, first, the whole blood sample is spotted near the center of the blood separation element, and in this state the whole blood sample is sufficiently spread in the blood cell separation layer. Wait to do. The waiting time is at the shortest until the blood cell separation element is pressed so that the minimum amount of plasma required for analysis permeates and diffuses to the extent that it can migrate to the developing layer, and the longest time is to evaporate the water content in the plasma and spread it to the developing layer. The time before the transition to becomes difficult. Usually, about 10 to 200 seconds is appropriate.

Next, the sample spotting part is pressed to transfer the plasma part in the sample to the spreading layer of the multilayer analytical element. A stick or the like may be used as the pressing means. The shape of the rod may be a square rod or the like, but is usually a round rod. The tip may be flat or may be round, and may be a hollow cylinder having no pressure in the center. The diameter of the pressing surface may be about 2 to 10 mm. It is suitable that the pressing force is lightly pressed, and the pressing time may be such that the transition is performed, for example, about 1 to 60 seconds.

After transferring the plasma portion, the blood cell separating element is peeled off and measured.

[0062]

The blood cell separation layer has a direction which is inevitably generated during production, and the pore distribution is uneven. Therefore, by stacking two or more blood cell separation layers, it is possible to prevent uneven diffusion / permeation due to structural unevenness of the blood cell separation layers.

[0063]

【Example】

Example 1 (1) Preparation of Analytical Element Polyvinyl containing 0.2% of the nonionic surfactant p-nonylphenoxypolyglycidol (containing an average of 10 glycidol units) on a 180 μm thick transparent film base of polyethylene terephthalate with a subbing. An alcohol (KL506 manufactured by Kuraray Co., Ltd.) to which an epoxy compound (Araldite DY022 manufactured by Ciba-Geigy Co., Ltd.) was added as a cross-linking agent was applied and dried so that the dry film thickness was about 15 μm. Polyester knitted fabric
Laminated according to the method described in 62-224299. Furthermore, an ethanol solution containing 1.5% hydroxyethyl cellulose (HPC manufactured by Shin-Etsu Chemical Co., Ltd.) and 0.25% nonionic surfactant (HS240 manufactured by NOF CORPORATION) was applied at a flow rate of 250 g / m ² for the purpose of controlling development. And dried. (2) Preparation of blood cell separation element A membrane in which the surface of the microporous membrane of polytetrafluoroethylene and the inner surface of the pores are made hydrophilic by a hydrophilic polymer (Sumitomo Electric Co., Ltd., Poreflon wpw-100-100), Two and three sheets were stacked and cut into a disk shape with a diameter of 12 mm. Separately, a glass fiber filter paper (CA-55, Advantech Co.) with a diameter of 8 m is used.
Cut out to a disk of m. A plastic single-sided adhesive tape is punched out to a diameter of 15 mm having a hole of 6 mm in diameter, and the disc of the microporous membrane and the disc of the glass fiber filter paper are further cut on the side where the glass fiber filter paper is closer to the adhesive tape It was glued so that it would be located in. For comparison, a slide was prepared using the same Poreflon membrane and one sheet. (3) Assembly of whole blood analysis element The coated film prepared in (1) above is cut out into a square of 15 mm × 15 mm, and a plastic frame made of high-impact polystyrene having a hole with a diameter of 10 mm in the center (length: 28 mm,
Width: 24 mm, thickness: 1.5 mm)
The same form of analytical element as described in 3452 was made. Center above the hole on the top of this analytical element and follow (2) above.
The blood cell separation element prepared in 1. was adhered and fixed at a position where the development layer of the analysis element and the microporous membrane of the blood cell separation element were in close contact with each other to complete the whole blood analysis element. (4) Preparation of total protein measuring reagent solution A reagent having the following composition was dissolved in 20 ml of water to prepare a measuring reagent solution utilizing the Biuret reaction. Copper sulphate (pentahydrate) 3.5 g Tartaric acid 2.3 g Lithium hydroxide 3.8 g Cetylmethylammonium bromide 0.1 g (5) Preparation of samples 2 to 1 ml of whole blood samples from heparin collected from healthy subjects
Divided into a third three test tubes. A small amount of bovine serum albumin was added to the second and third test tubes to prepare whole blood samples having different total protein concentrations. For these, a part of them was centrifuged, and the total protein concentration of the plasma sample was determined using a Hitachi 7050 analyzer. (6) Spotting of sample 30 μl of the whole blood sample prepared in (5) was spotted on the whole blood analysis element prepared in (3) above. Initially, whole blood formed a drop on the glass fiber filter paper and rose, but gradually penetrated into the lower layer. After the whole blood was spotted and allowed to stand for 1 minute, one end of the adhesive tape was pinched with tweezers, and the blood cell separation element was peeled and removed from the plastic slide frame. On the coated film piece fixed in the plastic slide frame, it was possible to visually confirm that only the plasma was transferred into a circle with a diameter of about 10 mm. In the experiment using one POREFLON membrane, a filtration pattern of plasma corresponding to the unevenness of the membrane quality of the POREFLON membrane itself was obtained, but when two or more layers were superposed, the filtration pattern was uniform. (7) Dehydration of separated plasma The analytical element that received the plasma obtained in (6) was placed in an aluminum heat block set at 37 ° C according to the method described in Japanese Patent Application No. 2-90562 and left for 5 minutes. It was then dehydrated and dried. (8) Measurement of total protein amount Fuji Dry Chem 5500 Analyzer (Fuji Photo Film
(Made by Co., Ltd.), set the analytical element obtained in (7), and set it in the dispenser (4).
10 μl of the measurement reagent solution prepared in 1. was aspirated, spotted according to a usual measurement operation, incubated for 6 minutes, and then the optical reflection density at 540 nm was measured. (9) Results Using the same sample, the measurement was repeated 10 times for slides having the same structure. When one piece of Poreflon was used, the observed variation in measured values was obviously small when two and three sheets were stacked. Number of Poreflon 1 2 3 3 Average (mg / dl) 8.7 8.5 8.4 S D (mg / dl) 0.5 0.25 0.27 C V (%) 5.7 2.9 3.2

[0064]

EFFECTS OF THE INVENTION The analysis element of the present invention can separate the plasma portion even in a whole blood sample and diffuse / permeate it uniformly, and can eliminate the variation in the analysis value due to non-uniform diffusion.

Claims (1)

[Claims] 1. A whole blood analysis element, characterized in that two or more blood cell separation layers each comprising a microporous membrane having a pore diameter of 10 μm or less and at least the surface of which is hydrophilic are laminated.