JPH0843294A - Analyzing method for total blood and analyzing element - Google Patents

Abstract

PURPOSE:To extract ingredient to be detected of a total blood sample at a high extraction ratio regardless of the type of the ingredient to be detected by extracting the ingredient by using a dry analyzing element containing no hydrophilic polymer. CONSTITUTION:The dry analyzing element to be used does not contain a measuring reagent for causing chemical change due to direct reaction of a hydrophilic polymer such as gelatin and ingredient substance, and a plasma receiving layer utilizes the developed layer of a non-fibrous porous layer, etc. The sample is supplied to the blood cell separate layer of the element, and waited until the plasma ingredient permeates the receiving layer. Then, the separate layer is separated from the receiving layer, and plasma ingredient stored in the receiving layer is dried, and stabilized. The drying is enough if moisture is removed without change of properties of the substance to be detected by heating, etc. The predetermined area of the receiving layer is dipped in an extract solvent such as buffer solution, etc., after drying, to extract the ingredient to be detected, and the extract is analyzed as specimen. Then, the element from which the hydrophilic polymer is excluded, is used to efficiency extract protein ingredient.

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY In the field of clinical analysis, the present invention stably holds a small amount of whole blood sample collected from a patient or subject at home and transfers the sample to a medical institution or a laboratory. The present invention relates to a method of analyzing a whole blood sample that can be analyzed by using

[0002]

2. Description of the Related Art A dry analytical element contains all reagents necessary for detection of 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 change. Or by measuring electrical changes,
The specific component can be detected quantitatively or semi-quantitatively.

For the purpose of quantitative analysis, a liquid sample is spotted on an analytical element, and under certain conditions (eg, 37 ° C. for 5 minutes).
In general, after the reaction by incubating (warming), an optical measurement or the like is immediately performed to calculate the concentration of the specific component in the sample. Recently, a measuring instrument that automatically performs operations after incubation has been marketed, and satisfactory reproducibility and accuracy have been obtained.

However, this quantitative analysis is carried out in a specialized inspection organization or a large-scale medical institution, because the measuring instruments are expensive and the measurement operation requires proficiency.

On the other hand, it has been recently pointed out that there is a need for a technique whereby a patient or subject can collect and analyze blood or the like at home.

[0006] In particular, home care has been advocated as a response to the rapid increase in medical costs due to the rapid transition to an aging society and the development of advanced medical treatment, which will be one of the cores of the future medical system. As a specific method of implementation is 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, the most important thing is to maintain a stable physiological condition unless there is a sudden change in the disease state, to continue a certain treatment, and to continuously monitor the effect of the treatment. It will be.

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

[0009] If blood test information is continuously obtained, the daily management of the doctor will be facilitated, and the treatment will be quicker and more appropriate, so that the benefit is immeasurable. In addition, it is easy for the patient to easily feel that he / she feels 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.

As one of the feasible means, after a liquid sample collected by a patient in daily life at home etc. is spotted on a dry analytical element, the reaction is stopped and fixed by incubating at a constant temperature for a certain period of time. It is conceivable that the dry analytical element may be brought to a facility having an analytical device such as a hospital or sent by mail or the like for measurement and analysis.

However, the liquid sample and the dry analytical element are changed due to the external conditions between the reaction fixing and the measurement / analysis, for example, the fluctuation of humidity, so that the analysis accuracy is remarkably lowered and the patient's condition is accurately measured. However, there was a problem that it could not be expected.

As a means for solving the problem, the present inventor has used a dry analysis element on which a liquid sample has already been spotted until the analysis by an analyzer after stopping and fixing the reaction of the spotted liquid sample. A method for storing a dry analytical element has been proposed, which is characterized in that it is stored in a state in which moisture and air are substantially cut off (Japanese Patent Laid-Open No. 3-289543).

On the other hand, the present inventor pointed out the difficulty of long-term storage of analytical elements as a problem of conventional dry analytical elements in which all reagents are incorporated into analytical elements, and as a means for solving them,
A liquid sample is supplied, at least a hydrophilic polymer layer on a water-impermeable support, a developing layer is laminated, an analytical element not containing a measuring reagent, a step of supplying a measuring reagent solution to cause a reaction, And the analytical element that caused the reaction,
There has already been proposed a measuring method using a dry analytical element, which comprises a step of measuring using optical means (Japanese Patent Laid-Open No. 5-26865). He also pointed out that this method is also effective for home inspection.

[0014]

In the above-mentioned storage method, it is necessary to further improve the reliability of the analytical value because the reaction proceeds immediately after the blood sample is spotted. On the other hand, when an analytical element containing no measurement reagent was used and applied to the above-mentioned storage method, there was no problem in analytical accuracy.

The inventor of the present invention supplies a whole blood sample to the analysis element which does not contain the measuring reagent, separates the plasma component, then stabilizes it by drying it, and extracts the test component from it to analyze the extract. As a result of studying the method, it was found that the extraction rate of the test components varies, and in particular, there is a problem in the extraction rate of protein-based test components such as enzymes and antibodies.

An object of the present invention is to provide a method for analyzing a liquid extract by supplying a whole blood sample to an analytical element not containing a measuring reagent, separating a plasma component, stabilizing the plasma component, extracting a test component therefrom. It is an object of the present invention to provide a method for analyzing a whole blood sample that can be extracted with a high extraction rate regardless of the type of test component.

[0017]

As a result of intensive studies to solve the above problems, the present inventors have found that hydrophilic polymers such as gelatin contained in dry analytical elements interact with protein-based components such as enzymes and antibodies. The present invention is completed by finding that it acts to prevent extraction, and that by using a dry analytical element that excludes a hydrophilic polymer, protein-based components can also be efficiently extracted from the plasma receptor layer. Came to.

That is, according to the present invention, a whole blood sample is supplied to a dry analytical element which has at least a blood cell separation layer and a plasma receiving layer and does not contain a measurement reagent and a hydrophilic polymer, and the plasma component of the whole blood sample is plasma. After diffusion and permeation into the receptive layer, the blood cell separation layer is separated from the plasma receptive layer, the plasma component accumulated in the plasma receptive layer is dried and stabilized, and the plasma receptive layer is immersed in an extraction solvent to extract a test component. The present invention relates to a method of analyzing a whole blood sample, which is characterized in that the extract is used as a sample for analysis.

As the blood cell separation layer, there is disclosed in Japanese Unexamined Patent Publication No. 138756/1987.
No. 8, JP-A-2-105043, JP-A No. 3-16651, etc. The fibrous porous layer and the non-fibrous porous layer are bonded with an adhesive that is partially arranged (partial bonding). ) An integrated product, a fluorine-containing polymer having a hydrophilic surface, a microporous layer having a blood cell separating ability such as polysulfone, and the like can be used. Particularly preferred is a blood cell separation layer in which a fibrous porous layer is arranged on the blood spotting side and a non-fibrous porous layer is arranged on the plasma receiving layer side, and both are integrated by partial adhesion described later.

As a non-fiber porous layer in a blood cell separation layer in which a fibrous porous layer and a non-fibrous porous layer are integrally bonded (partially bonded) with an adhesive that is partially arranged, a Japanese Patent Publication No.
A layer of a brush polymer composed of cellulose esters described in 21677, U.S. Pat. No. 1,421,341, and the like, for example, lulose acetate, cellulose acetate / butyrate, and cellulose nitrate is preferable. 6-nylon, 6,
A microporous membrane such as polyamide such as 6-nylon, polyethylene, polypropylene or the like may be used. Others
A porous layer having continuous voids in which small polymer particles, glass particles, diatomaceous earth, etc. are bonded with a hydrophilic or non-water-absorbing polymer, as described in JP-A-677, JP-A-55-90859 and the like, can also be used.

The effective pore size of the non-fibrous porous layer is 0.8-30 μm.
m, particularly preferably 0.5 to 5 μm. In the present invention, the effective pore diameter of the non-fibrous porous layer is indicated by the pore diameter measured by the limiting bubble pressure method (bubble point method) according to ASTM F316-70. When the non-fibrous porous layer is a membrane filter made of a so-called brush polymer made by the phase separation method, the liquid passage path in the thickness direction is the most on the free surface side (that is, the glossy surface) in manufacturing the membrane. It is usually narrow, and the hole diameter when the cross section of the liquid passage is approximated to a circle is the smallest near the free surface. The minimum pore size in the thickness direction in the passage path of the volume further has a distribution in the plane direction of the filter, and the maximum value thereof determines the filtering performance for particles. Usually it is measured by the limiting bubble pressure method.

As described above, in the membrane filter made of the so-called brush polymer produced by the phase separation method, the liquid passage in the thickness direction is on the free surface side (that is, glossy surface) in the production of the membrane. The narrowest. When using this type of membrane as the non-fibrous porous layer of the analytical element of the present invention, it is preferred to direct the glossy side of the membrane filter towards the side closer to the support, ie the side facing the plasma-receiving layer.

As a material for forming the fibrous porous layer,
Filter paper, non-woven fabric, woven fabric (for example, plain woven fabric), knitted fabric (for example, tricot knitting), glass fiber filter paper and the like can be used. Of these, woven fabrics and knitted fabrics are preferable.
The woven fabric or the like may be subjected to glow discharge treatment as described in JP-A-57-66359.

Since the fibrous porous layer is used as a developing layer for a liquid sample, it is preferably a layer having a liquid metering action. Liquid metering action is the action of spreading the liquid sample spot-supplied on the surface in the direction of the surface at a constant rate per unit area without causing the components contained therein to be substantially unevenly distributed. Is. In the development layer, in order to adjust the development area, the development speed, etc., JP-A-60-222770,
The hydrophilic polymer or the surfactant as described in JP-A-63-219397, 63-112999 and 62-182652 may be contained.

The void volume (per unit area; hereinafter the same) of the fibrous porous layer may be the same as that of the non-fibrous porous layer,
May be different. In order to adjust the relationship between the void volume of the both, the void ratio or the thickness of the both may be changed, or both the thickness and the void ratio may be changed.

The microporous layer whose surface is hydrophilized and has the ability to separate blood cells specifically separates blood cells and plasma from whole blood without substantially hemolyzing so as to affect the analysis value. .

Although the blood cell / plasma separation mechanism is not clear, this microporous layer does not trap blood cells only on its surface, and the thickness of the microporous layer made of a fluorine-containing polymer and the porous development layer are combined. As it penetrates in the direction
It is believed that this is due to the so-called volumetric filtration action, in which the large blood cell component is initially formed, and then the small blood cell component is gradually formed into a void structure to retain and remove the blood cell over the entire length in the thickness direction.

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.
A microporous matrix layer (microporous layer) consisting of G
ore-Tex (WLGore and Associates), Zi
tex (manufactured by Norton), Poreflon (manufactured by Sumitomo Electric Industries, Ltd.) and the like. In addition, US 3368872 (Examples 3 and 4), US 3260413 (Examples 3 and 4), JP-A-53-92
195 (US 4201548) and the like, and polytetrafluoroethylene microporous membranes, and polyvinylidene fluoride microporous membranes and the like described in US 3649505.

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 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 membrane 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 described in JP-A-57.
-66359 (US 4783315) 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 be adjacent. The adhesive force of the adhesive used for partial adhesion with the microporous layer can be enhanced.

Even if the microporous membrane of 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 first means, as a means for imparting hydrophilicity to the microporous membrane of a fluorine-containing polymer to enhance the hydrophilicity, the outer surface and the surface of voids inside the microporous membrane of a fluorine-containing polymer are used. 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 substantially hydrophilize the.

In order to impart sufficient hydrophilicity to a fluorine-containing polymer microporous membrane so that the aqueous liquid sample can be diffused, permeated and transferred to the surface or inside of the membrane without being repelled,
Approximately 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 microporous membrane of the fluorine-containing polymer is added to the microporous membrane. The surface needs to be coated. For example, the thickness is 50
In the case of microporous membranes of μm fluorine-containing polymer, the amount of surfactant impregnated is generally from 0.05 g / m ² to 2.5 g / m ^2.
It is preferably in the range of m ² . As a method of impregnating a microporous membrane of a fluorine-containing polymer with a surfactant, an organic solvent (eg, alcohol, ester, ketone) having a low boiling point of the surfactant (boiling point is preferably in the range of about 50 ° C to about 120 ° C) is used. ) Immersing the microporous membrane of the fluorine-containing polymer in the solution and allowing the solution to substantially fully penetrate the internal voids of the microporous membrane, then gently pulling the microporous membrane out of the solution Is preferred) and dried.
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), 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 analyzing whole blood. 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), There are higher fatty acid alkanolamides.

Specific examples of the nonionic surfactant are as follows. Examples of the alkylphenoxypolyethoxyethanol include isooctylphenoxypolyethoxyethanol: (Triton X-100: oxyethylene unit average 9 to
(Containing 10) (Triton X-45: containing 5 oxyethylene units on average) nonylphenoxypolyethoxyethanol: (IGEPAL CO-630: containing 9 units of oxyethylene units on average)
Contained) (IGEPAL CO-710: Oxyethylene unit average 10
(Containing 11 to 11) (LENEX 698: containing 9 oxyethylene units on average) As the alkyl polyether alcohol, a higher alcohol polyoxyethylene ether: (Triton X-67: CA Registry No. 59)
030-15-8)

The fluorine-containing polymer microporous film may be hydrophilized by providing one or more water-insoluble water-soluble polymers in the porous space. As examples of water-soluble polymers, polyvinyl alcohol, polyethylene oxide, polyethylene glycol, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose for hydrocarbons containing oxygen, polyacrylamide, polyvinyl pyrrolidone, polyvinyl amine for those containing nitrogen, Polyethyleneimine and those having a negative charge include polyacrylic acid, polymethacrylic acid, polystyrene sulfonic acid and the like. The insolubilization may be performed by heat treatment, acetalization treatment, esterification treatment, chemical reaction with potassium dichromate, crosslinking reaction with ionizing radiation, or the like. For details, see JP-B-56-2094 and JP-B-56-1618.
No. 7 publication.

The polysulfone microporous membrane is prepared by dissolving polysulfone in dioxane, tetrahydrofuran, dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone, a mixed solvent thereof or the like to prepare a stock solution for film formation and supporting it. It can be produced by casting on the body or directly in a coagulating liquid, washing and drying. Details are disclosed in JP-A-62-27006.
A microporous membrane of polysulfone is also disclosed in JP-A-56-1264.
No. 0, JP-A-56-86941, JP-A-56-154051 and the like are also disclosed, and these can also be used. The microporous membrane of polysulfone can also be hydrophilized by containing a surfactant like the fluorine-containing polymer or by providing a water-insoluble water-soluble polymer.

The plasma receiving layer is a layer that receives the plasma portion that has exuded from the blood cell separating layer, and a known spreading layer can be used for this, for example.

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, 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 represented by a continuous void-containing three-dimensional lattice granular structure layer formed by adhering micro beads in a point-contact manner with a water-swellable adhesive, JP-A-55-1643.
56, 57-66359 and the like porous layers made of woven fabric, 60-222769 and the like knitted fabric, various filter paper, hydrophilic paper and the like layers can be mentioned. However, the present invention is not limited to these.

It is not necessary to limit the spreading layer to only one layer, and it is possible to use the developing layers of JP-A-61-4959, 62-138756, 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 when 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.

In order to control the developability,
A development control agent such as a hydrophilic polymer can be included.

Further, 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 10 to 500 μm, preferably 20 to 300 μm, more preferably 30 to 200 μm.

Between the spreading layer and the blood cell separation element, it is sufficient that the plasma portion diffuses and permeates, and may or may not be adhered. When adhering, the so-called partial adhesion method is used so as not to hinder the diffusion and penetration of plasma. Partial adhesion refers to JP 61-4959 (EP0166365A) and JP 62-1.
38756 to 138758 (EP 0226465A), which is a mode of adhesion between two adjacent porous layers or between adjacent porous layers and a non-porous layer, "a part between the interfaces of two adjacent layers" Is adhered and integrated by an adhesive that is disposed intermittently (or intermittently), and is configured such that uniform passage of liquid is not substantially obstructed between the adjacent two surfaces and between them. It's adhesive.

A method of partially disposing the adhesive on the blood cell separating element or the porous layer is disclosed in JP-A-64-1959 and JP-A-62-13.
8756 and various methods described in JP-A-64-23160 (DE 3721236A) and the like. Of these methods, the printing method is preferred. Among the printing methods, a method of transferring an adhesive to a porous layer or a detection functional layer using a printing plate (a gravure printing plate or an intaglio is preferable) roller to attach the adhesive and a method of bonding two adjacent layers are, for example, "Printing Engineering Handbook" edited by The Printing Society of Japan (Gihodo Publishing)
Co., Ltd., 1983) 839 to 853 pages, etc.

An adhesive used is disclosed in Japanese Patent Laid-Open No. 62-1387.
The various 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 aqueous pastes such as starch paste; aqueous solutions of dextrin, carboxymethyl cellulose and the like; vinyl acetate-butyl acrylate copolymer emulsion. 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.

Among these, silicone, acrylic, and phenol resin pressure sensitive adhesives are particularly useful in the present invention.

The analytical element used in the analytical method of the present invention may be composed of only the blood cell separation layer and the plasma receiving layer, but is preferably laminated on the water-impermeable support through the hydrophobic adhesive layer. preferable.

The hydrophobic adhesive layer is a layer made of a hydrophobic adhesive such as heat-sensitive, pressure-sensitive, thermosetting and photocurable,
This adhesive does not contain components that dissolve in the extraction solvent, water, that affect the analysis and quantification of the test substance, or contain only low concentrations that do not substantially affect the measured values. . As a specific example, the pressure sensitive adhesive is styrene-
Butadiene copolymers, vinyl chloride / vinyl acetate copolymers, etc., and heat-sensitive adhesives include polymethylmethacrylate, polystyrene, styrene-maleic anhydride copolymer polyvinyl chloride, etc.

The thickness of the hydrophobic adhesive layer is about 1 μm to about 100 μm.
μm is suitable.

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
Can be used. The support may be light-transmissive, colored, or light-impermeable. The support may be a conventional one having a good flatness and a soft one which is easily deformed.

The analytical element used in the present invention is characterized in that it is substantially free of a hydrophilic polymer. This hydrophilic polymer is various well-known water-soluble, swelling, and hydrophilic polymers that have been used in dry chemistry analysis elements, and the swelling rate at water absorption is about 150% at 30 ° C.
To about 2000%, preferably about 250% to about 1500% natural or synthetic hydrophilic polymer. Specifically, gelatin (eg, acid-treated gelatin, deionized gelatin, etc.) and gelatin derivatives (eg, phthalated gelatin, hydroxyacrylate-grafted gelatin, etc.) disclosed in JP-A-59-171864 and JP-A-60-108753. , Agarose, pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone and the like.

The analytical element used in the present invention does not contain a measuring reagent. 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; when the test substance is an antigen (antibody), it is an antibody (antigen); and when the test substance is a lipid, sugar, or metabolite, An enzyme is a compound that produces a detectable change. 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. Hereinafter, a specific example will be described.

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

The above-mentioned blood cell separation layer and / or plasma receiving layer interacts with a specific component in blood, particularly a test component,
It is possible to include a substance which imparts selectivity to the transfer property to the plasma receiving layer or the extraction characteristics of the extraction solvent. Examples of these are organic and inorganic compound salts for adjusting pH and ionic strength, ionic and nonionic surfactants, polysaccharides, and other compounds.

In the method of the present invention, plasma in which blood cells have been separated in the blood cell separation layer diffuses and permeates in the plasma receiving layer so as to contain a constant amount of plasma per unit area.

In order to confirm this, a dry analytical element as shown in FIG. 1 was prepared. This analysis element has a width of 7 mm,
60mm long strip-shaped plasma receiving element with a width of 7mm at one end
A 10 mm blood cell separating element is laminated by a partial adhesion method. The plasma receiving element is formed by coating a polyethylene terephthalate film (water-impermeable support) 1 having a thickness of 185 μm with a hydrophobic adhesive layer (“Cranbetter”, Kurabo) 2 having a thickness of 50 μm, and hydrophilizing it. It is a laminate of the plasma-receptive layer 3 of the treated knitted fabric. Further, the blood cell separation element is one in which the fibrous porous layer 5 and the non-fibrous porous layer 4 are bonded by the partial bonding method.

When the whole blood sample is spotted on the porous layer 5, blood cells are trapped in the porous layers 5 and 4, and the plasma receiving layer 3 is shown in FIG.
As shown in, only plasma diffuses. The amount of separated plasma depends on the amount of the sample liquid. The amount of plasma sample contained per unit area of the plasma receiving layer 3 is constant, and when the amount of separated plasma is large, the amount of plasma diffused in the plasma receiving layer 3 is also large. That is, the diffused length of the sample in the plasma receiving layer 3 is directly proportional to the amount of plasma.

According to the practice of this method, usually, in order to collect a fixed amount of plasma, the analytical element containing plasma may be cut into a fixed length.

The relationship between the spotted whole blood volume and the permeation length of plasma is shown in Table 1.

The amount of separated plasma is proportional to the length of the plasma-receptive layer to the permeation tip, and the position of the permeation tip can be visually determined. Therefore, the amount of liquid to be the basis of analysis can be determined from the permeation length.

[0070]

[Table 1]

As described above, it is desirable that the dry analytical element has a larger plasma receiving layer than the blood cell separating layer. The shape of both is not limited to the strip shape, and the point is that a certain amount of plasma can be easily collected by punching out the plasma receiving layer. For example, as shown in Figure 3, the plasma receiving element may be the same size as the blood cell separating element. On the other hand, the blood cell separation layer has the ability to retain plasma. Therefore, in the case of a whole blood sample with the highest hematocrit value, it is set just so that plasma can be supplied to the entire surface of the plasma receiving layer, that is, the area of the plasma receiving layer is set to match the permeation area of plasma at the minimum plasma volume. Therefore, when a fixed amount of whole blood sample is supplied, it is possible for the blood cell separation layer to retain the excess plasma of the whole blood sample having a smaller hematocrit value and to supply a fixed amount of plasma to the plasma receiving layer. is there.

In the analysis method of the present invention, first, a whole blood sample is supplied to the blood cell separation layer of the dry analysis element. The amount of the whole blood sample can be varied within a considerable range when the dry analytical element is, for example, a strip shape, but is constant when the area of the plasma receptive layer matches the permeation area of the whole blood sample having the maximum hematocrit value. The amount. The minimum amount of plasma required for analysis is about 1-10 μl.

After the whole blood sample is supplied, it waits until the plasma component permeates the plasma receiving layer. This time is usually about 10 to 60 seconds.

Next, the plasma separating layer is separated from the plasma receiving layer, and the plasma component accumulated in the plasma receiving layer is dried and stabilized.

The drying may be carried out by removing water without deteriorating the plasma component, particularly the test substance, and any means such as heating, depressurization, air blowing and use of a desiccant can be used.

This drying is preferably carried out under substantially constant conditions. Specifically, Japanese Patent Application No. 2-90562
(JP-A-3-289543), page 25, line 9 to page 28, line 6, especially page 27, line 13 to page 28, line 6 can be used. For example, there is a method of incubating with the plasma receiving 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 preferably 30 to 45 ° C.

Temperature fluctuation during incubation is ± 5
C., preferably ± 3 ° C., more preferably ± 1 ° 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 in which the analysis element from which the blood cell separation element has been removed is placed in the storage part of the element and kept at a constant heating temperature by the heating means. In this publication, a cover which is capable of sealing the element storage portion above the storage portion of the analysis element and is detachable is provided, and when the element storage portion is sealed by the cover, the inside of the element storage portion is closed. It is essential that the volume of the created space is an incubator designed to substantially match the volume of the analytical element, but in carrying out the method of the present invention, it is not necessary to seal with a cover, and the upper portion is always open. It doesn't matter.

Even if a dry air of 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.

As a result of further studies on the drying conditions, the present inventor has found that the plasma receiving element is preferably dried at 50 ° C. or lower in a closed container containing a desiccant. Specifically, the plasma receiving element is sealed with a desiccant in a plastic bag with a fastener or a plastic container with a lid, and the temperature is kept at 50 ° C or lower. It is preferable to keep the temperature at about 25 ° C. or lower for about 1 hour after spotting. You can put it in a refrigerator or freezer. The desiccant may be appropriately selected and used from among known hygroscopic agents that does not substantially deteriorate the test substance, but from the viewpoint of safety and dehydration ability, zeolite,
Silica gel is preferred, and zeolite is more preferred. 4 or more plasma receiving elements per 1 g of zeolite or silica gel
About 10 sheets can be dried. As a form, granular particles of about 1 to 3 mm are put in a bag having good moisture permeability, for example, a bag made of Japanese paper or polyester non-woven fabric, a bag made of nylon mesh, or the like.

The drying time varies depending on the amount and type of the liquid sample spotted, the desiccant and the type, etc.
90% of the moisture content of analytical element in about 1 to 3 hours
It is preferable that the above conditions be removed and dehydrated by a desiccant. This drying time is also affected by temperature. The higher the storage temperature, that is, the higher the vapor pressure, the shorter the drying time may be. Alternatively, it may be left in a refrigerator or a freezer to be kept at a low temperature and then dried at 1 ° C. or higher. This low-temperature drying method is particularly useful because when the test substance is an enzyme, its denaturing deterioration can be prevented.

Here, the term "dry" means that the reaction does not substantially proceed in the plasma receiving layer or the deterioration of the test substance does not proceed. Therefore, depending on the analysis target, for example, when the target is an enzyme, the water content in the hydrophilic polymer may be 20% or less, preferably 10% or less, more preferably 5% or less.

Here, the% of water is the ratio when the amount of water when the aqueous solution containing the test substance is spotted on the analytical element is 100.

It is not necessary to perform extraction immediately after drying,
The dry state can be maintained, for example, by mailing to an institution for analysis.

The extraction is carried out by extracting a fixed amount of plasma in a dry state, specifically by immersing a certain area of the plasma receiving layer in an extraction solvent to extract the test components. In the case of a plasma receiving element in which a hydrophobic adhesive layer, a water-impermeable support and the like are laminated on the plasma receiving layer, it can also be added to the extraction solvent.

Extraction solvents include water, buffer solutions, alcohols such as methyl alcohol and ethyl alcohol, acetone,
A wide range can be selected depending on the characteristics of the test substance such as ketones such as methyl ethyl ketone, esters such as ethyl acetate, and ether and chloroform.

The extraction solvent may contain a substance that specifically reacts with a specific component in plasma to insolubilize it. Examples of such substances include trichloroacetic acid, acetone, alcohol known as protein flocculants, activated carbon known as adsorbents, alumina, silica, zeolite, Ti.
There are fine particles such as O ₂ , ion exchange resins, antibodies, enzymes, enzyme-bonded compounds, particles, fine fibers, polymer emulsions and suspensions. In addition, there are low-molecular and high-molecular compounds in which an inorganic / organic salt for adjusting pH or ionic strength or a specific bonding group is incorporated in the molecule.

The extraction solvent may contain a substance that promotes the extraction of the test component. Examples of such substances include ionic and nonionic surfactants, organic / inorganic salts, polyethylene glycol, glycerin, ethylene glycol, sugars, and the like. Further, ether, chloroform, plasticizer and the like may be added as an emulsion.

The amount of extraction solvent may be about 10 to 500 times the amount of plasma liquid. Extraction may be performed at room temperature for about 1 to 10 minutes,
During that time, shaking, stirring and the like are appropriately performed.

After the extraction, the plasma receiving element is removed if necessary, and the extract is used as a sample for analysis. This analysis may be performed according to the well-known method of wet chemistry,
As the measurement reagent solution, those known in wet chemistry can be used. It is also possible to analyze using a conventional dry analytical element containing all the necessary measuring reagents.

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. Preferred test substances are CRP (C-reactive protein), HbA ₁ C (glycated hemoglobin A ₁ C), HBc (hepatitis B virus) antibody, and other immunoassay items, DNA (deoxyribonucleic acid),
White blood cells, lymphocytes and the like, and HbA ₁ C, 1.5AG (1.5 albumin glucosylate), CRP, HDL (high density lipoprotein cholesterol fraction) and the like are particularly preferable.

[0093]

【Example】

Example 1 1) Preparation of Multilayer Analytical Element 1-1: Preparation of Plasma Receptor Element A film-like heat-sensitive adhesive “Cranbetter”, Kurabo Industries was formed on a 185 μm thick polyethylene terephthalate (PET) colorless transparent smooth sheet. It was applied so as to have a thickness of 50 μm to form a hydrophobic adhesive layer.

Next, on the hydrophobic adhesive layer, a broad cloth fabric made of PET (thickness: about 150 μm, void volume: 9.8 μL / m ² ) was adhered as a plasma receiving layer by lightly pressing.

Next, 100 parts of an aqueous solution having the following composition was applied to this cloth.
The plasma receiving element was completed by applying it almost uniformly at a rate of mL / m ² and drying. Octylphenoxypolyethoxyethanol 27g (containing 10 oxyethylene units on average) Water 964.3g

1-2: Preparation of Blood Cell Separation Element A tricot knitted fabric (thickness: about 250 μm) obtained by knitting 36-gauge PET spun yarn corresponding to 50 denier was impregnated with an aqueous solution having the following composition and dried. Polyethylene glycol (average molecular weight 50,000) 2.0g Sodium tetraborate 2.0g Water 96g

Next, the above-mentioned impregnated tricot knitted fabric 80
The hot-melt adhesive, which was heated to ℃ and heated to 130 ℃ and melted on its surface, was applied in dots by transfer from a gravure roller by a gravure printing method. The dot pattern of the gravure roller is a circle with a dot diameter of 0.3 mm,
The distance between the centers of the dots was 0.6 mm, and the dot area ratio was about 20%. The amount of adhesive deposited was about 2 g / m ² . Then, pass the non-glossy surface of the cellulose acetate membrane filter with an effective pore diameter of 3.0 μm, a thickness of 140 μm and a porosity of about 80% on the surface of the hot fabric immediately after the adhesive is transferred, and pass it between the laminating rollers. , And both were laminated and adhered integrally (partially adhered) to produce a blood cell separation element.

A hot-melt type adhesive was similarly attached to the glossy side of the cellulose acetate membrane filter of this blood cell separation element using a gravure roller, and the plasma receiving element was attached by lightly pressing it. However, the areas of the blood cell separating element and the plasma receiving element were the same, and both were cut into 12 mm × 12 mm squares so as to completely overlap each other. A large number of multilayer analytical elements of the same size were prepared.

2) Preparation of whole blood sample High concentrations of BuN, GOT, and CRP (C-reactive protein) were added to whole blood, and the values in plasma were BuN: 412 mg / d, respectively.
Samples adjusted to have l, GOT: 238 u / L, and CRP: 17.5 mg / dl were prepared. TP (total protein) of this sample
Was 7.8 g / dl.

3) Plasma Separation 20 μl of the whole blood sample prepared in 2) was spotted on the blood cell separation element of the multi-layer analytical element prepared in 1), allowed to stand in the room for 30 seconds, and then the blood cell separation membrane and the plasma receptor were received using tweezers. The membrane was separated.

The same operation was repeated to obtain a plasma receiving element containing separated plasma for a total of 9 sheets. Further, a sample obtained by doubling the whole blood sample prepared in 2) with physiological saline was treated in the same manner to obtain a plasma receptor element containing plasma having different concentrations.

4) Dehydration and Drying 3 pieces of the analysis element of 3) were enclosed with 2 g of granular zeolite (drying agent) in a polyethylene bag with a width of 50 mm and a length of 75 mm, which could be hermetically sealed, and left to dry at room temperature overnight. did.

5) Extraction of Plasma A large number of plastic sample test tubes each having an inner diameter of 15 mm and a volume of 6 ml were prepared, and 500 μl of distilled water was dispensed into each of them.

The dried plasma receiving element prepared in 4) was put into one test tube three times each and capped, and then slowly stirred and extracted with a rotary shaker.

6) Sampling and measurement Five minutes after the stirring was started, the extract was transferred to a sample cup, and a Hitachi 7150 automatic analyzer was used for BuN, TP, GOT,
The component concentrations of CRP were measured. I made a cup.

7) Measurement Results Table 2 shows the average of the results obtained by measuring three sheets under the same conditions. The extraction rate was calculated at home when 30% of the plasma in whole blood was transferred to the plasma receptive membrane. The extraction rates were all 88% or more, indicating that this method enables quantitative measurement.

[0107]

[Table 2]

Example 2 In the same experiment as in Example 1, a plasma receiving element without a blood cell separating element was prepared in order to accurately examine the extraction efficiency. For the sample, further B to the control serum QPH for Fuji Dry Chem.
uN, GOT, and CRP were added and used.

After 7 μl of the sample was spotted on the plasma receiving element, the sample was enclosed with a desiccant (2 g of granular zeolite) in a sealable polyethylene pouch and left to dry at room temperature for a whole day and night.
Extraction and measurement were carried out in the same manner as in Example 1.

Comparative Example 1 In the construction of the multilayer analytical element in Example 2, only the hydrophobic adhesive layer was changed to gelatin having a film thickness of 20 μm to prepare a multilayer analytical element having a hydrophilic polymer layer, and an example was prepared. Extraction experiments were performed according to the same procedure as in 2.

Comparison of Results Example 2 (the method of the present invention) and Comparative Example were carried out simultaneously in parallel. Table 3 shows the results. With respect to BuN, which is a low molecular weight compound having high water solubility, high extraction efficiency was obtained in both the method of the present invention and the comparative example. On the other hand, there are few
And GOT and CR which have strong interaction with hydrophilic polymer layer
In P, the extraction method of the present invention showed a high extraction rate, and after 5 minutes, 90%.
In contrast to the above, in the comparative example, the extraction rate was 50% or less even after 5 minutes.

From the above, it was found that the method of the present invention is a particularly advantageous measurement method for trace components such as oxygen, antigens and antibodies, which consist of proteins having a strong interaction with hydrophilic polymers.

[0113]

[Table 3]

[0114]

EFFECT OF THE INVENTION By the analysis method of the present invention, it is possible to extract and analyze a whole blood sample with a high extraction rate regardless of the type of test component.

[Brief description of drawings]

FIG. 1 is a cross-sectional view of an example of a dry analytical element used in the method of the present invention.

FIG. 2 is a plan view showing a state of diffusion and permeation of plasma in the analysis element.

FIG. 3 is a cross-sectional view of another example of the dry analytical element used in the method of the present invention.

[Explanation of symbols]

 1 ... Support 2 ... Hydrophobic adhesive layer 3 ... Plasma receiving layer 4 ... Blood cell separation layer (non-fibrous porous layer) 5 ... Blood cell separation layer (fibrous porous layer)

Claims (3)

[Claims] 1. A whole blood sample is supplied to a dry analytical element which has at least a blood cell separation layer and a plasma receiving layer and does not contain a measuring reagent and a hydrophilic polymer, and a plasma component of the whole blood sample diffuses into the plasma receiving layer. After permeation, the blood cell separation layer is separated from the plasma receiving layer, the plasma component accumulated in the plasma receiving layer is stabilized by drying, and the plasma receiving layer is immersed in an extraction solvent to extract the test component, and this extract Method for analyzing whole blood sample characterized in that analysis is carried out by using 2. A dry analysis method which does not contain a measuring reagent and a hydrophilic polymer, in which a plasma-receptive layer is laminated on a water-impermeable support through a hydrophobic adhesive layer, and a blood cell separation layer is laminated thereon. element 3. The dry analytical element according to claim 2, wherein the plasma receiving layer has a rectangular shape, and the blood cell separating layer is laminated on one end portion in the longitudinal direction thereof.