JPH05273207A - Whole blood analyzing element and measuring method using the same - Google Patents

Abstract

PURPOSE:To easily and accurately perform the examination of an examinee at home by excluding the analytical error based on the non-uniform diffusion of a whole blood sample by almost uniformly allowing the plasma part of the whole blood sample to permeate and diffuse throughout the developing layer of a multilayered analyzing element using a whole blood analyzing element and accurately performing analysis even after the elapse of a considerable time by drying the developing layer to which the plasma part is transferred. CONSTITUTION:In the measurement of a blood component, a multilayered analyzing element wherein at least one hydrophilic polymer layer and a developing layer are laminated on a water impermeable support in this order and a blood corpuscle separating element having a blood corpuscle separating layer are superposed one upon another so as to provide space between both elements and, in this state, a whole blood sample is dripped on the blood corpuscle separating element. After the sample is developed throughout the blood corpuscle separating layer, the sample dripped part is pressed to transfer the plasma part in the sample to the developing layer and the blood corpuscle separating element is removed and the substance to be examined transferred to the multilayered analyzing element is measured.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a whole blood analysis element capable of accurately analyzing a measurement target component even when a subject himself / herself spots his / her blood and a method for measuring the measurement target component using the same. Is.

[0002]

2. Description of the Related Art A dry analytical element generally contains all reagents necessary for detecting a specific component in a dry state, and a liquid sample is brought into contact therewith to cause a reaction with the reagents, resulting in an optical reaction. The specific component can be quantitatively or semi-quantitatively detected by measuring a static change or an electrical change.

For the purpose of quantitative analysis, a liquid sample is spotted on an analytical element with a microvolumetric constant volume pipette or the like, and reacted by incubating (warming) under constant conditions (for example, 5 minutes at 37 ° C.). After that, it was general to immediately perform optical measurement or the like to calculate the concentration of the specific component in the sample. Recently, measuring instruments that automatically perform the operations after incubating have been put on the market, and satisfactory reproducibility and accuracy have been obtained.

However, many of these measuring instruments require a power supply. It has a certain size. Complex, precise, and expensive. For this reason, it cannot be installed anywhere.

Further, in order to always obtain highly reliable data, it is necessary to operate under the control of at least some degree of measurement operation and technical technicians, doctors, nurses and the like. Therefore, the installation of such a device is usually limited to medical institutions such as hospitals, clinics, and inspection centers.

[0006]

If, for example, a diabetic hospital outpatient measures blood glucose several times in his or her daily life,
If the result can be brought to the hospital, the treating doctor can grasp the condition of the patient more accurately. However, there are some devices that can be placed in the home as long as they are semi-quantitative, but the accuracy is low, so they do not meet the above purpose.

[0007] In recent years, home care has been advocated as a response to the rapid increase in medical expenses due to the rapid transition to an aging society and the development of advanced treatment, and it will be one of the core of the future medical system. Specific methods of implementation are being considered.

The basis of home care is that the patient is always under proper supervision, guidance, and management by a doctor while staying in each home, and that appropriate treatment can be carried out as necessary. For example, in the case of chronic patients and the elderly, it is in a stable physiological state if there is no sudden change in pathology, and it is most important to continue certain treatment and continuously monitor the therapeutic effect. It will be.

Such continuous monitoring is easy for inpatients, but it is extremely difficult in practice when the patients are dispersed in each home. Usually, the temperature and weight are measured, and other symptoms are observed by the nurse. It is limited to subjective information such as inspection records and complaints about the patient's pain.

[0010] If blood test information is continuously obtained, the daily management of the doctor will be easy, and the treatment will be quicker and more appropriate, so the benefit is immeasurable. In addition, it is easy for the patient to easily understand that his / her daily medical condition is reported to the doctor and taken care of by him / her, and that it provides mental support for recovery.

If a doctor or a nurse can carry out a blood test for a patient at home without following the instructions of the patient to the patient, more rapid and appropriate treatment can be performed. This means going to all households considering the time required for home visits, but especially in areas where transportation is inconvenient,
Great advances in healthcare in remote areas, remote islands, and undeveloped areas,
It brings benefits.

Therefore, after the patient collects blood in his / her daily life at home or the like and spots it on the spotted dry analytical element,
It is conceivable to bring this to a facility with an analyzer such as a hospital or send it by mail or the like for measurement and analysis. In this case, the spotted whole blood sample permeates into the dry analytical element during transport and reacts with the reagent contained therein, but there is a problem that this affects the analytical result due to changes in external conditions. However, this can be eliminated by removing some or all of the reagents contained in the dry analytical element.

However, in addition to the problem of the reaction with the above-mentioned reagent, the whole blood sample spotted on the dry analytical element has a blood clot at the center and the plasma is diffused around, so that the plasma concentration becomes ring-shaped. There was a problem that it became unevenly diffused, which caused an analysis error. Patients, such as patients, are generally unfamiliar with the operation of spotting blood, unlike laboratory technicians.Therefore, there are large variations in spotting amount and position, which is accompanied by the diffusion of plasma in a ring shape. This was one of the causes of the large analysis error.

The object of the present invention is to allow even a person, such as a subject, unfamiliar with the spotting operation to permeate and diffuse plasma almost uniformly on the spreading layer of the multilayer analytical element, and thereby It is to provide a whole blood analysis element capable of eliminating an analysis error based on uniform diffusion.

Another object of the present invention is to provide a whole blood analysis element capable of analyzing a whole blood sample with high analysis accuracy by solving the problem of non-uniform diffusion of a plasma portion even after a lapse of time after spotting. Especially.

[0016]

The present invention has been made to achieve the above-mentioned object, and a blood cell separating element is provided on a multi-layer analytical element, that is, on the developing layer side with a space, and the blood cell separating element is provided. After the whole blood sample spotted on the blood cell spreads in the blood cell separation layer, the plasma spot in the sample is transferred from the blood cell separation layer to the development layer by pressing the sample spotting part to spread the plasma portion almost uniformly. It is possible to solve the problem of non-uniform diffusion by analyzing the plasma part transferred to the spreading layer, and to solve the problem of non-uniform diffusion due to the lapse of time after spotting. Could be achieved.

That is, according to the present invention, a blood cell separation element having a multilayer analysis element and a blood cell separation layer in which at least one hydrophilic polymer layer and a development layer are laminated in this order on a water-impermeable support has a space. A whole blood analysis element characterized by being placed in place, and a whole blood sample is spotted on the blood cell separation element of the element, and the sample spotting portion is pressed after the sample is spread in the blood cell separation layer. The present invention relates to a method for measuring a blood component, which comprises transferring a plasma portion in a sample to a spreading layer, removing a blood cell separation element, and measuring a test substance transferred to a multilayer analysis element.

The blood cell separating element has one or more blood cell separating layers. As the blood cell separation layer, both a non-fibrous microporous membrane and a fibrous microporous membrane can be used.
As the non-fibrous microporous membrane, Japanese Patent Publication No. 53-21677, cellulose esters described in US Pat. No. 1,421,341, for example, cellulose acetate, cellulose acetate / butyrate, a brush polymer composed of cellulose nitrate, 6-nylon, Microporous membranes of polyamide such as 6,6-nylon, polyethylene, polypropylene, etc.
-A microporous membrane made of polysulfone described in No. 27006, a microporous membrane made of nitrocellulose, JP-B-53-
For example, porous layers having continuous voids in which small polymer particles, glass particles, diatomaceous earth and the like described in 21677 and 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 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 layer thickness is about 10 μm to about 200 μm, preferably about 30 μm to about 150 μm, considering the handling property such as wrinkling during the manufacturing process, It is preferably in the range of about 50 μm to about 120 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 is formed into a microporous membrane having a large porosity and a short filtration length. 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.
-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 the fluorine-containing polymer has a low surface tension as it is and is used as a blood cell filtration layer of a dry analytical element, 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 fluorine-containing polymer microporous membrane 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 analysis 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.

Specific examples of the nonionic surfactant are as follows. 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 blood cell separation element can be provided with two or more blood cell separation layers. 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.

The multilayer analytical element is one in which at least one hydrophilic polymer layer and a developing 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 substantially unevenly distributing the components and supplying the hydrophilic polymer layer at 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 of 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 multilayer 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.

The hydrophilic polymer layer is soluble in 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 a desired reaction or for preventing or reducing an interference or interfering reaction.

As the water-impermeable support, a known water-impermeable support conventionally used in 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
Transparent film 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.

The whole blood analysis element of the present invention is characterized in that the blood cell separation element and the multilayer analysis element are arranged with a space. This space layer is to prevent the whole blood sample spotted on the blood cell separation element from diffusing into the multilayer analysis element. On the other hand, the thickness of this space layer is determined by pressing the sample spotting portion of the blood cell separating element after the whole blood sample has spread to some extent in the blood cell separating element, so that both elements come into contact and the plasma portion in the blood cell separating element is It should be possible to move to the development layer of the multi-layer analytical element. Therefore, although the thickness of this space layer varies depending on the material and thickness of both elements, the diameter of the space, etc., it is usually about 0.1 to 5 mm, and particularly about 0.1 to 1 mm is suitable in many cases. Space layer diameter is usually 3-10 mm
The degree is appropriate. A spacer is generally used as a means for forming the space layer. The shape of the spacer is
For example, in addition to a frame body having a circular window, a square window, etc., strip-shaped spacers may be arranged at both ends between both elements. 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 the analysis element of the present invention, since the blood cell separation element is removed after the plasma portion of the whole blood sample is transferred to the spreading layer, when both elements are adhered to the spacer, at least both elements can be peeled off. Keep the structure. As a means for this, at least one of them may be bonded with an adhesive having a weak adhesive force and capable of peeling, or the spacer itself may be broken to separate the two elements. It is preferable to provide a tab or the like on one of the elements, for example, the blood cell separation element, in order to facilitate separation.

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 to 4,
A configuration example is shown.

1: Analytical element containing a water-impermeable support / hydrophilic polymer layer / developing layer / spacer / hemocyte separation layer and containing no reagent which causes a chemical change by directly reacting with a test substance. Ca, GOT (glutamate oxaloacetate transaminase), GPT (glutamate pyruvate transaminase), γ-GTP (γ-glutamyl transpeptidase), glucose, LDH (lactate dehydrogenase), CPK (creatine phosphokinase), TP
It is effective for analysis of (total protein), Alb (albumin), TCHO (total cholesterol), UA (uric acid), and neutral fat.

2: A water-impermeable support / hydrophilic polymer layer / developing layer / spacer / blood cell separating layer, which contains a chromogen in the hydrophilic polymer layer and / or the developing layer. An analytical element that does not contain a reagent that reacts directly with it to cause a chemical change. As a chromogen, Ann. Clin. Biochem.,
6, 24-27 (1969), 4-aminoantipyrine (alias)
4-aminophenazone, that is, 1-phenyl-2,3-dimethyl-4-amino-3-pyrazolin-5-one), JP-A-59-54
1- (2,4,6-trichlorophenyl) -2,3-dimethyl-4-amino-3-pyrazolin-5-one described in 962 etc., 1- (3,5-dichlorophenyl) -2,3- Dimethyl-4-amino-3-pyrazoline-
Tri-substituted-4-amino-3-pyrazolin-5-one such as 5-one,
1-Phenyl-2,3-dimethyl-4 described in JP-B-55-25840
4-Aminoantipyrine analogs such as -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-one and the like are preferable.

3: Water-impermeable support / hydrophilic polymer layer / developing layer / spacer / blood cell separating layer, wherein a chromogen and other reagents (described later) are contained in the hydrophilic polymer layer and / or developing layer. Analysis elements (excluding measurement reagents). Other reagents include POD (peroxidase), N
AD (nicotinamide adenine dinucleotide), NA
Examples thereof include DP (nicotinamide adenine dinucleotide phosphate) and DIP (diaphorase).

In the configurations 2 and 3, the chromogen or other reagents can be supplied after the liquid sample is supplied and stabilized, but since most of the chromogens are water-insoluble, they are separated from the measurement reagent. It is necessary to supply the chromogen and other reagents, and it is advantageous to reproducibly produce the chromogen and other reagents by including them in the layer from the beginning.

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.

The details of these mordant polymers are described in JP-B-2-30466, JP-A-51-40191 and JP-A-54-2970.
0, 53-131089, etc. For example, as the anion mordant polymer, Japanese Patent Publication No. 30466/1990
~ Alkaline hydrolyzate of methyl vinyl ether-maleic anhydride copolymer described in column 14, polystyrene-p-sulfonic acid alkali metal salt or alkaline earth metal salt, styrene-p-sulfonic acid and hydrophilic Examples thereof include alkali metal salts or alkaline earth metal salts of copolymers with vinyl monomers. Further, regarding layers and the like that can contain these polymers, the same as described in 15 to 16 of the publication.
There is detailed description in the column.

5: 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) and barium sulfate fine particles. , Aluminum fine particles or micro flakes, and examples of light shielding fine particles include carbon black, gas black, carbon micro beads, etc., among which titanium dioxide fine particles,
Barium sulfate fine particles are preferred.

Examples of the hydrophilic polymer binder having a film-forming ability include weakly hydrophilic regenerated cellulose and cellulose acetate in addition to the above-mentioned hydrophilic polymers. Of these, gelatin, gelatin derivatives, polyvinyl alcohol, polyacrylamide, Maleic acid copolymers 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, wherein 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 indicator is referred to.

When the reaction reagents incorporated in the above reaction system are unstable like some enzymes, these are also preferably included in the measurement reagents. That is, the distribution of the reagent to be contained in the measurement reagent solution and the reagent to be contained in the analytical element can be variously changed with the analytical performance and the storage stability as indexes. 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 a material constituting the analytical element are improved, and an enzyme contained therein is contained. 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 reagents 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. Furthermore, in whole blood measurement, NaN ₃ which is effective as an inhibitor of hemoglobin catalase activity
Etc. can also 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, it may be in the form of a so-called stick like the urine test paper.

As a method of using the whole blood analysis element of the present invention, first, a 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 for 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 100 seconds is suitable.

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 rounded. The pressing surface may have a diameter of about 5 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 20 seconds. After transferring the plasma portion, the blood cell separation element is peeled off.

When the measurement is carried out immediately, the measurement reagent solution may be spotted on the spreading layer and incubated, and the measurement may be carried out by a conventional method. However, the analytical element of the present invention exerts its power in applications such as home care, and when it takes time from the plasma portion to the development layer to the measurement as described above, the analytical element may be used for a certain period of time. It is preferable to dry under constant conditions. The preferable drying method and conditions are described in detail in JP-A-3-289543.
A particularly preferred method is heating with the dry analytical element placed in a covered enclosure. As a result, a constant dry state is achieved without being affected by the ambient temperature and humidity.

The temperature range is 10 to 60 ° C., preferably 20 to 50
C., more preferably 30 to 45.degree. The temperature fluctuation during the incubation is ± 10 ° C, preferably ± 5 ° C, more preferably ± 3 ° C.

An incubator suitable for carrying out such incubation under constant conditions is described in Japanese Utility Model Publication No. 3-126499. That is, it is an incubator that keeps a constant temperature after heating by the heating means in a state where the analytical element is installed in the accommodating portion of the element, and the element accommodating portion can be sealed above the accommodating portion of the analytical element In addition, a removable cover is provided, and when the element storage part is sealed with the cover, the volume of the space created inside the element storage part is an incubator designed to substantially match the volume of the analysis element. ..

Even if a dry air having a constant temperature is blown under a constant condition, a result with good reproducibility can be obtained similarly, but it has a drawback that it is more expensive than the incubator.

Here, "drying" means that the reaction does not substantially progress in the hydrophilic polymer or the deterioration of the test substance does not progress. Therefore, depending on the analysis target, for example, when the target is an enzyme, the water content in the hydrophilic polymer may be 50%, preferably 20% or less, more preferably 10% or less.

When it takes a long time to supply the measurement reagent after stabilization, for example, when the dry analytical element is mailed to a hospital, etc., it is necessary to store it in a state in which moisture and air are substantially cut off. There is.

Details of this storage condition are also described in Japanese Patent Application Laid-Open No. 3-289543. For example, there is a method of sealing in a metal box provided with a means for removing water or a bag made of a film or sheet of an organic polymer or metal that does not allow water to permeate.

As the water removing means, any known hygroscopic agent that does not substantially change the quality of the sample can be appropriately selected and enclosed. After bagging the element, you may suffice it by letting it air out.

Using these multilayer analysis elements, analysis is carried out by the following method. A measurement reagent solution corresponding to the item to be analyzed is supplied to the analysis element taken out from the closed container to cause a reaction. This reaction is measured by a method known in the field of dry chemistry (reflection density photometry, color change, fluorescence measurement, luminescence measurement, etc.) to quantify the components contained in the sample.

As the measuring reagent solution corresponding to the item to be analyzed, a known reagent solution in wet chemistry can be used. These react with the analyte and
A change that can be detected mainly by an optical measurement method, for example, a color change, color development (coloring), fluorescence, light emission, a change in absorption wavelength in the ultraviolet region, a change in turbidity, or the like occurs.

As a method for measuring dry chemistry, a reflective optical system is usually used. Also in the method of the present invention,
The method of photometry through the water-impermeable support of the analytical element is the most applicable, but when the sample is not whole blood or after the sample is supplied, the microporous layer made of the fluorine-containing polymer and containing the surfactant is removed. In the case of performing the measurement by using a transmission photometry method, the measurement can be performed. When the water-impermeable support is opaque, it can be measured from the opposite side of the support.

The whole blood analysis element of the present invention is classified into the above-mentioned classification 1
It can be effectively used in any of the fields corresponding to category 3, and is also effective in clinical medical examinations for home care.

When blood is used as the sample, the dry analytical element generally requires only a very small amount of blood.
Blood can be collected using an appropriate device such as a capillary pipette. The multi-layer analysis element can be transferred by mail, courier, etc., and it can be sufficiently applied to in-home inspection.

FIG. 3 shows a schematic diagram of the measuring method according to the present invention.

[0080]

In the whole blood sample spotted on the blood cell separating element, the plasma portion first penetrates and diffuses in the blood cell separating layer and reaches the back surface of the blood cell separating element. Therefore, when the sample spotting portion of the blood cell separation element is pressed, only the plasma portion in the sample is transferred to the development layer of the multi-layer analytical element at a substantially uniform concentration. Then, the blood cell separating element is peeled and removed so that the measurement reagent can be supplied and the test substance transferred to the multilayer analysis element can be measured.

[0081]

【Example】

Example 1 1) Preparation of multi-layered analytical slide 10% by dry weight was placed on a support made of a photographic polyester film base having a thickness of 180 μm that had been subjected to a hydrophilic treatment.
Gelatin containing polyoxyethylene nonyl phenyl ether was coated at a coating amount of 15 g / m ² . On this hydrophilic polymer layer, a development layer made of a knitted fabric having a thickness of about 200 μm, which was made of a cotton spun yarn count and made of 100S polyester twisted yarn and whose surface was hydrophilized by glow discharge, was laminated. Further, an aqueous solution containing 10% of polyoxyethylene nonylphenyl ether and 15% of hydroxyethyl cellulose was applied from above so that the dry weight of hydroxyethyl cellulose would be 5 g / m ² . Multilayer analysis element made in this way is 15 mm × 15 mm
Cut to the size of and have a hole with a diameter of 10 mm in the center.
A multilayer analysis slide was completed by incorporating it into a styrene plastic frame consisting of two upper and lower 5 mm pressing plates and a rectangular frame for accommodating the above multilayer analysis element.

2) Preparation of blood cell separation element Water-resistant, vertical 28 mm, horizontal with 6 mm diameter holes
24 mm paper, 8 mm diameter glass fiber filter paper (GA-100 from Advantech), and 12 mm diameter polytetrafluoroethylene microporous membrane with hydrophilic surface (Sumitomo Electric Industries Ltd.
Pore Freon WPW-100-100) was laminated in this order under the laminated member with the double-sided tape.

3) Preparation of analytical element for whole blood A multilayer analysis slide was laminated on the plastic frame so that the microporous membrane of the blood cell separation element was on the bottom. By using the double-sided tape, the slide frame and the blood cell separating element are laminated with their respective functional membranes overlapping on a concentric circle,
fixed. However, a gap of about 0.2 mm was set between the porous membrane and the surface of the spreading layer of the multilayer analytical film in the slide frame.

4) Preparation of whole blood sample 10 ml of venous blood of a healthy person was collected, and heparin, which is an anticoagulant, was added to this to a concentration of 15 u / ml. Furthermore, this sample was dispensed into four test tubes. Separately from this, two calibrators L, M, H for quality control of Fuji Dry Chem are used.
A solution dissolved in ml of physiological saline was prepared. Test tube No. 1 was stored as it was without adding anything. For No.2 to No.4, gently centrifuge and absorb 0.8 ml of fine spirit with a dropper, and instead use No.2.
For calibrator L, No. 3 for calibrator M, N
To o.4, 0.8 ml of calibrator H was added and mixed to prepare a whole blood sample whose component concentrations were adjusted. Approximately 1 ml of the No. 1 to No. 4 specimens are separated and centrifuged to collect plasma.
Glucose, GPT, and amylase were measured with an automated clinical chemistry analyzer.

5) Measurement of whole blood sample 40 μl of the No. 1 whole blood sample was aspirated with a micropipette,
The slides prepared in 3) were supplied. Whole blood was drawn into the glass fiber filter paper in about 1 second. Therefore, the diameter of the circular shape is 15 mm
The paper label was used to cover the spotting holes. However, the multilayer analysis film was left for 10 seconds while being kept separated from the development layer. Then, a columnar plastic rod having a cylindrical sponge having a diameter of 8 mm and a length of 10 mm attached to the tip thereof was pressed from above the label to press-bond the blood cell separating element and the development layer of the multilayer analysis film. The weight at that time was adjusted to 600 g / cm ² by adjusting the length and hardness of the sponge. After contacting for 10 seconds, the blood cell separation element was peeled off from the multilayer analysis film. Only plasma was transferred to the analysis slide, and it was visually confirmed that it had a disc shape with a diameter of about 10 mm. Apart from whole blood, plasma samples (No.5 to No.8) obtained from No.1 to No.4 were processed in the same manner.

6) Drying of slides The slides in which the plasma was soaked in a disk shape were placed at 37 ° C (± 1
(° C), and placed in a heat block type incubator in which 12 holes each having a width of 25 mm, a length of 30 mm, and a depth of 10 mm are formed in two rows, and dehydrated and dried for 5 minutes. After the dehydration treatment, the slides were stored in a small bag of 50 mm x 100 mm, which was made of a small polyethylene and could be hermetically sealed at the entrance.

7) Measurement of Plasma Components Using a Fuji Drychem multi-item measuring device DC-5500, the concentrations of chemical components such as glucose, GPT and amylase and the activity values of enzyme components were measured. In the measurement, prepare a measuring reagent solution having the following composition in the dispenser of the measuring machine,
10 μl was set to be automatically spotted on each slide. Measurement reagent formulation: Glucose GLU GOD 1300U POD 5000U Dihydroxynaphthalene 83mg 4-Aminoantipyrine 280mg Polyhydroxyethylnonylfel 400mg ――――――――――――――――――――――― MES (2 -(N-Morphlino) etanesulfonic acid) 0.1M (pH ＝
7.3) 10ml AMYL α-glucosidase 6000U PNP-G5 540mg ―――――――――――――――― HEPES 0.1M (pH = 7.3) 10ml GPT Tris 87mg Phosphate ・ 1 potassium ・ dihydrogen 103mg L -Alanine 620 mg α-KG · 2Na 93 mg 20% MgC ₁₂ 275 μl POD 3416U TPP (cocarboxylase) 21 mg FAD (flavin adenine dinucleotide) 5 mg POPG (bilbate oxidase) 3088U 1N NaOH pH = 7.5 2- (3,5- Dimethoxy-4-hydroxyphenyl) -4- [4- (dimethylamino) phenyl] -5-phenethylimidazole 50 mg Triton X-100 (registered trademark) 200 mg ―――――――――――――――――― ―――――――――――――――――――― Distilled water 10ml Amylase α-glucosidase 6000U Paranitrophenol-G-5 540mg 100mM HEPE (pH7.3) 10ml

8) Preparation of Calibration Curve Using the Fuji Dry Chem 5500, after the No. 5 to No. 8 plasma samples were spotted on the slide, the reflection optical density from 0 to 6 minutes was measured about every 12 seconds. .. The wavelengths used for photometry were glucose: 505 mm, GPT: 640 mm and amylase: 400 nm. Regarding the change with time of the reflection optical density thus obtained, glucose was calculated as a 6-minute value-1 minute value, GPT was a 4-minute value-2 minute value, and amylase was a 5-minute value-1 minute value. did. For glucose, n = 5, GPT, AMY
L was measured at n = 10. On the other hand, a calibration curve was prepared using the values measured for plasma using Hitachi 7050 as the horizontal axis and the average value of the above measurements as the vertical axis. The results are shown in FIGS.

9) Measurement of whole blood sample The whole blood samples of No. 2 to No. 4 were also measured at n = 3 in the same manner as plasma, using the calibration curve previously prepared using plasma. The concentration and activity value were measured. The results are shown in Table 1.

[0090]

[Table 1]

10) Measurement of Simultaneous Reproducibility of Whole Blood Specimens Using the specimen of No. 1, the operations 5) to 8) were repeated. However, the same specimen was measured 10 times each, and the simultaneous reproducibility was calculated. The results are shown in Table 2.

[0092]

[Table 2]

All of them gave the results showing that whole blood samples can be measured and quantitatively measured by the method of the present invention.

[0094]

EFFECTS OF THE INVENTION By using the whole blood analysis element of the present invention, the plasma portion in a whole blood sample can be almost uniformly permeated and diffused in the spreading layer of the multilayer analysis element, which is based on the non-uniform diffusion of the sample. Analysis error can be eliminated. By transferring the plasma portion to the development layer and then drying it, the analysis can be accurately performed even after a considerable time has passed, and the subject can be easily and accurately tested at home.

[Brief description of drawings]

FIG. 1 is a schematic cross-sectional view showing the basic configuration of a whole blood analysis element of the present invention.

FIG. 2 is a schematic cross-sectional view showing another example of the configuration of the whole blood analysis element of the present invention.

FIG. 3 is a schematic cross-sectional view showing another example of the configuration of the whole blood analysis element of the present invention.

FIG. 4 is a schematic cross-sectional view showing another example of the configuration of the whole blood analysis element of the present invention.

FIG. 5 is a graph showing a glucose calibration curve obtained in an example of the present invention.

FIG. 6 is a graph showing a GPT calibration curve obtained in an example of the present invention.

FIG. 7 is a graph showing a calibration curve for amylase obtained in Examples of the present invention.

Claims (6)

[Claims] 1. A multi-layer analytical element in which at least one hydrophilic polymer layer and a developing layer are laminated in this order on a water-impermeable support, and a blood cell separating element having a blood cell separating layer are arranged with a space. Whole blood analysis element characterized by 2. The analytical element according to claim 1, which contains a reagent that directly reacts with a test substance to cause a chemical change. 3. The analysis element according to claim 1, which does not contain a reagent that causes a chemical change by directly reacting with a test substance. 4. The analytical element according to claim 1, 2 or 3, wherein the blood cell separation layer is a microporous layer made of a fluorine-containing polymer and containing a surfactant. 5. A multi-layer analytical element in which at least one hydrophilic polymer layer and a spreading layer are laminated in this order on a water-impermeable support, and a blood cell separating element having a blood cell separating layer are superposed with a space therebetween. Whole blood sample is spotted on the blood cell separation element in the state of being placed, and after the sample is spread in the blood cell separation layer, the sample spot is pressed to move the plasma part in the sample to the spreading layer, Is removed, and the test substance that has transferred to the multilayer analysis element is measured, and the method for measuring blood components is characterized. 6. At least one on a water-impermeable support.
The whole blood sample is spotted on the blood cell separation element in a state in which the multilayered analysis element in which the hydrophilic polymer layer and the development layer of the layer are laminated in this order and the blood cell separation element having the blood cell separation layer are overlapped with a space, After the sample has spread in the blood cell separation layer, press the sample spotting part to transfer the plasma part in the sample to the development layer, remove the blood cell separation element, and transfer the test substance transferred to the multilayer analysis element from the outside. Measuring method of blood component characterized by measuring by supplying a measuring reagent solution