JPH06242107A - Dry analysis element for multi-item measurement - Google Patents

Abstract

PURPOSE:To supply, preserve and transfer a body fluid sample to plural analysis elements so as to make possible analysis with satisfactory accuracy by providing a hydraulic polymer layer and each porous developing layer stacked on a water impermeable support in such a manner as to be separable by a blood corpuscle separation element. CONSTITUTION:A dry analysis element for multi-item measurement comprises a partial analysis element 3 where a hydraulic polymer layer 5 and a porous developing layer 2 are stacked on a water impermeable support 1, and a blood separation element 4 where non-fibrous and fibrous porous layers 7, 6 are partially made adhere to each other to be united in one body and partially made adhere to the developing layer 2. A transparent film made of polyethylene terephthalate or the like is used for the support 1, and a gelatin derivative or the like is used for the polymer layer 5. The separation element 4 is formed in such a manner that porous layers 6, 7 are respectively partially made adhere to the blood dropped side and the blood plasma acceptor side. woven fabric and knit fabric are suitable for the porous layer 6, and a brush polymer layer is preferable for the porous layer 7. For example, the whole blood tested body 9 is dropped, and the element 4 is removed after blood plasma 10 is developed on an element 3. The remaining element 3 is dried and then a measurement reagent is dropped to cause reaction and analyzed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an analytical element capable of measuring many items with a small amount of sample in the field of clinical analysis, etc., and an analytical method effective when it takes a long time to analyze a liquid sample. Is.

[0002]

2. Description of the Related Art A method for diagnosing human diseases using blood, urine or the like as a sample has long been practiced. As one of the methods,
There is a wet chemistry analysis method. This is a method using a so-called solution reagent and has a long history, and detection reagents have been developed for many items, and there are various measuring machines, from simple small machines to large full-automatic machines. The samples used for wet chemistry are plasma, serum, urine, etc. Usually, whole blood is not used as it is as a sample.

In wet chemistry, it is possible to divide into several groups in consideration of the stability of reagents during storage and mix them at the time of dissolution and preparation, or to divide the procedure of reagent addition into several steps. It is also possible.

Further, since an appropriate amount of reagent can be dissolved and prepared according to the number of samples to be measured, the reagent cost per measurement can be reduced. It is complicated and troublesome to combine and handle a large number of solutions automatically, but the development of clinical testing equipment has a long history and social demands were high, so it is necessary to have large, medium, and small processing capacity. Also in the field, the efficient automatic equipment has been developed and put into practical use.

On the other hand, a large number of so-called dry chemistry analysis elements, in which all reagents necessary for qualitative / quantitative analysis are incorporated into analysis elements such as reagent paper and multilayer analysis film, have been developed and commercialized. Photographic film
Co., Ltd.), Ektachem (Eastman Kodak Company, USA), Dry Lab (Konica Corporation), Spot Chem (Kyoto Daiichi Kagaku Co., Ltd.), Reflotron (Germany, Boehringer Mannheim), It is commercially available under the names such as Ceralyzer (made by Miles Laboratory, USA).

These dry chemistry analysis elements have the following features. (1) All reagents necessary for analysis are incorporated in the analysis element. (2) Simply depositing a sample (usually serum, plasma, urine, and whole blood for some items) causes the reaction required for analysis.

Dry chemistry analysis elements are roughly classified into three types according to their fields of use. Classification 1: A purpose for screening at a medical practitioner, home, etc., and a qualitative (+/-) or semi-quantitative (about 5 levels) result can be discriminated by visual inspection. Class 2: A measurement place that can be selected relatively freely by combining it with a measuring machine that features compact and simple operation. Used in emergency laboratories, pediatric wards, practitioners, small hospitals, etc. Class 3: A fully automated machine used for routine measurement in hospitals and laboratories. The composition and contents of analysis elements also differ according to the above classification.

The analysis elements provided for classification 1 are those represented by urine tests and blood glucose test strips, and the analysis operation is simple and requires no equipment, but a rough judgment (normal or abnormal, etc.) is made. ) Is obtained, and it is assumed that it will be re-measured by other analytical means that gives a quantitative result if necessary.

The analysis element according to this method is not premised on the operation by an expert such as a clinical technologist, a doctor or a nurse, and urine or whole blood can be directly used as a sample so that sample processing is not required. It is usually possible.

Those belonging to Class 2 are intended for quantitative analysis and are premised on quantitative measurement by an instrument.
The operation itself is relatively easy, though not as much as Class 1, and is not premised on an expert such as a clinical laboratory technician. As for the sample, an analytical element that can use any of whole blood, plasma, serum, and urine is being developed, but the number of measurable items in whole blood is still only 10 items, which is relatively limited.

The samples used for the class 3 fully automatic machines are usually limited to plasma, serum and urine, and whole blood cannot be used as the sample. However, the number of measurable items is gradually increasing, and analytical elements have been developed for at least 40 items.

Further, the present inventor uses a dry analytical element on which a liquid sample has been spotted as a method of storing a dry chemistry analytical element, after stopping and fixing the reaction of the spotted liquid sample, and then using an analyzer. A method for storing a dry analytical element has been developed, which is characterized in that it is stored in a state in which moisture and air are substantially blocked until analysis (JP-A-3-289543).

[0013]

A problem common to the above-mentioned wet chemistry analysis method and dry chemistry analysis element is the problem of sample preparation and supply.

Since the wet chemistry method is premised on the permeation measurement of a transparent solution, the whole blood sample cannot be used as it is as a measurement sample. That is, after collecting a whole blood sample, plasma or serum of the centrifuged supernatant is transferred to a sample cup, or the centrifuge tube itself is set in a measuring instrument instead of the sample cup. In addition to the complexity of performing these operations, there is the problem of securing sufficient plasma or serum for reliable sampling without the contamination of red blood cells.

To secure 200 μl of plasma after centrifugation, 1.5-2 ml of whole blood is usually required. Even if centrifugation and subsequent operations are carefully performed, the minimum required blood collection amount is estimated to be around 500 μl.

On the other hand, the amount of sample required for analysis measurement is 10 μl
Because it is about / items, even if 10 items are tested, 100
Even if 20 items were tested, it was only 200 μl.
The actual amount of blood collected at hospitals is 2 to 20 ml. That is,
50-100 times the final required plasma volume has been collected.

Although it is physically and physically painful for a healthy person to puncture a blood vessel by inserting a needle into a blood vessel using a syringe, it is difficult to collect blood in a person who has a constitutionally thin blood vessel. Especially for sick patients, the pain associated with blood sampling is more than you might imagine, and for patients with repeated blood sampling,
There is a great desire to minimize the amount of blood drawn.

In a blood collection room of a hospital, a practitioner, or a clinic, whole blood is collected in a test tube or a vacuum blood collection tube and then transferred to a central examination room or an examination center as it is or in a refrigerated state. That is, the collected blood is first centrifuged after being transferred, and separated into tangible components such as red blood cells and plasma or serum serving as an analysis sample.
During this time, coexistence with erythrocytes may lead to the progress of biochemical reactions that affect the analysis.
Only countermeasures have been taken against the variable factors known to have an extremely large effect on the analysis results.

In consideration of the above, it is desirable that centrifugation be performed immediately after blood collection, but this is not usually performed. This is because the analytical method using serum as a sample has been established historically, but it is necessary to complete the coagulation reaction by leaving it for at least 30 minutes to 1 hour to obtain serum. Even after adding and centrifuging, precipitation of fibrin, etc. may occur depending on the sample until the subsequent measurement time by the analytical instrument, which causes troubles in the delivery system such as the dispensing syringe and tube of the analytical instrument. Because it is easy to become.

For this reason, it is desirable to obtain a serum sample by centrifugation within a few hours after blood collection. However, even if this is possible in a hospital, it is necessary to transfer the sample in a test center. When it is used as a measurement target, it is quite different, and in many cases, it is often separated after one day or more has passed.

On the other hand, in the dry chemistry analysis element, all the reagents necessary for the reaction must be incorporated in the element, which is convenient, and the characteristics of the reagents used differ one by one depending on the items to be analyzed. In addition, there is a big problem that it takes a lot of labor, time and equipment to optimize the manufacturing conditions.

Since all the reagents are contained, the reaction proceeds immediately after spotting the sample. This means that if it takes time from sample collection to analysis because the sample collection location and analysis location are different, the sample must be stored as a liquid without being altered, As is the case, not only is it time-consuming, but it is often technically difficult.

Further, when a plurality of analysis items are analyzed, it is necessary to spot the sample on each analysis element by the number of analysis items, which is troublesome and requires a large amount of sample liquid. It tends to be.

An object of the present invention is to provide an analytical element which can easily supply a small amount of a liquid sample, particularly a body fluid sample, to a plurality of analytical elements, and can surely store and transfer the sample, and perform analysis with sufficient analytical accuracy. Especially.

[0025]

The above object of the present invention is to provide a measurement reagent in which a hydrophilic polymer layer and at least one porous spreading layer are laminated in this order on a water-impermeable support. This is achieved by a dry analytical element characterized in that one blood cell separating element is laid across in a peelable manner between the respective porous development layers of a plurality of partial analytical elements not included.

The basic constitution of the analytical element of the present invention is that a hydrophilic polymer layer, a porous developing layer serving as a plasma receiving layer, and a peelable laminate with this porous developing layer are provided on a water-impermeable support. It is composed of a stack of blood cell separating elements which may be composed of a plurality of layers.

As a blood cell separating element, Japanese Patent Laid-Open No. 138756/1987 has been used.
No. 8, JP-A-2-105043, JP-A-3-1665
The one in which the fibrous porous layer and the non-fibrous porous layer described in JP-A-1 are bonded (partially bonded) by an adhesive that is partially arranged, a fluorine-containing polymer whose surface is hydrophilized,
A microporous layer having a blood cell separating ability such as polysulfone can be used. Particularly preferred is a blood cell separation element 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.

As the non-fibrous porous layer in the blood cell separation element in which the fibrous porous layer and the non-fibrous porous layer are integrally bonded (partially bonded) with an adhesive that is partially arranged, there is a Japanese Patent Publication No.
A layer of a brush polymer composed of cellulose esters described in US Pat. No. 21,677, US Pat. No. 1,421,341 and the like, for example, cellulose acetate, cellulose acetate / butyrate, cellulose nitrate is preferable. 6-nylon,
A microporous membrane such as polyamide such as 6,6-nylon, polyethylene, polypropylene or the like may be used. Others
A porous layer having continuous voids in which small polymer particles, gas particles, diatomaceous earth, etc. are bonded with a hydrophilic or non-water-absorbing polymer, as described in JP-A-21677, 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 when the cross section of the liquid passage is approximated to a circle, the diameter hole is the smallest near the free surface. The minimum pore size in the thickness direction in the passage path of the unit further has a distribution in the plane direction of the filter, and its maximum value determines the filtering performance for particles. Usually it is measured by the limiting bubble pressure method.

As described above, in a membrane filter made of a so-called brush polymer produced by the phase separation method, the liquid passage path in the thickness direction is on the free surface side (that is, glossy surface) in the production of the film. 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 spreading layer, in order to control the spreading area, the spreading 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 or different from that of the non-fibrous porous layer. To adjust the relationship between the two void volumes,
The porosity or the thickness of both may be changed, or both the thickness and the porosity may be changed.

The microporous layer such as a fluorine-containing polymer and polysulfone whose surface is hydrophilized does not cause hemolysis to the extent that it substantially affects the analytical value, and specifically separates blood cells and plasma from whole blood. Therefore, it can be used as a material for a blood cell separation element.

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.

Examples of the fluorine-containing polymer include polyvinylidene fluoride and polytetrafluoroethylene, and polytetrafluoroethylene is preferable.

The micropore size of the microporous layer of fluorine-containing polymer is from about 0.2 μm to about 60 μm, preferably about
Range from 0.5 μm to about 20 μm, more preferably 0.5-5 μm
m range, porosity about 40% to about 95%, preferably about 50
% To about 80%, layer thickness is about 10 μm to about 200 μm
m, preferably about 30 .mu.m to about 150 .mu.m, and most preferably about 50 .mu.m to about 120 .mu.m in consideration of handleability such as wrinkling during the manufacturing process.

A microporous layer of a fluorine-containing polymer is disclosed 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 to the microporous spreading layer can be enhanced.

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 (W.L. Gore and Associates), Zite
x (manufactured by Norton), Poreflon (manufactured by Sumitomo Electric Industries, Ltd.) and the like.

Pore size (micropore size) from about 0.1 μm to about 50
μm, the layer thickness is about 20 μm to about 400 μm.

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.

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, a film having a pore size of 0.5 to 5 μm, a film thickness of 10 to 200 μm, and a porosity of at least 70% or more is preferable.

The fibril structure or the uniaxially stretched or biaxially stretched non-laminate type microporous membrane is stretched to form 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. .

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 microporous membrane of a fluorine-containing polymer has a low surface tension as it is, and even if it is used as a blood cell filtration layer of a dry analytical element, the aqueous liquid sample is repelled and diffuses and permeates on the surface and inside of the membrane. It is a well-known fact not to do. In the analytical element of the present invention, as a means for imparting hydrophilicity to the fluorine-containing polymer microporous film to enhance hydrophilicity, the outer surface and the surface of voids inside the fluorine-containing polymer microporous film are 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 surfactant to render the sample.

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, it is generally necessary to add a fine amount of the fluorine-containing polymer. 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 porous membrane coats the surface of the voids of the microporous membrane. It is necessary. For example, the thickness is 50μ
For microporous membranes of m fluorine-containing polymers, 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 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. ) 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 film include nonionic (nonionic), anionic (anionic), cationic (cationic) and amphoteric Any surfactant can be used.

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

Specific examples of the nonionic surfactant are as follows. Examples of the alkylphenoxypolyethoxyethanol include: isooctylphenoxypolyethoxyethanol: (Triton X-100: containing oxyethylene units on average 9 to 10) (Triton X-45: containing oxyethylene units on average 5) nonylphenoxypolyethoxyethanol: ( IGEPAL CO-630: Oxyethylene unit average 9
Contained) (IGEPAL CO-710: Oxyethylene unit average 10
(Containing 11 to 11) (LENEX 698: containing 9 of oxyethylene units on average) As the alkyl polyether alcohol, a higher alcohol polyoxyethylene ether: (Triton X-67: CA Registry No.59030-15-
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 microporous membrane of polysulfone 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.

A microporous layer such as a fluorine-containing polymer or polysulfone whose surface is hydrophilized can be used alone as a blood cell separating element. However, by combining the above-mentioned fibrous porous layer, the blood cell separating ability can be improved. Can be further increased.

The porous spreading layer serving as a receiving layer for plasma separated by the blood cell separating element spreads in a plane without substantially unevenly distributing the components contained in the aqueous sample, and is substantially constant per unit area. It is a layer that has a function of supplying the hydrophilic polymer layer in an amount ratio, and uses all known non-fibrous and fibrous porous materials as the spreading layer that has been used in the dry chemistry analysis element so far. You can 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
-164356, 57-66359 and the like porous layer made of a woven fabric, 60-222769 etc. a layer made of a knitted fabric, various filter papers and the like can be mentioned, but are not limited thereto. Not something.

Among these, the porous film made of a thermoplastic material is a cellulose derivative (DAC, TAC, NC, H).
MC (hydroxymethylcellulose), HEC (hydroxyethylcellulose) porous membrane, polyethylene,
Porous film made of ethylene polymer or copolymer such as polypropylene, polystyrene, vinyl chloride, porous film made of polyethylene terephthalate, polycarbonate, polysulfone, acrylic acid or methacrylic acid,
There are porous membranes made of vinyl polymers or copolymers of these esters. On the other hand, in the application of the present invention, a non-thermoplastic porous film is produced by binding a porous film of a condensation polymer such as nylon, polyamide, or polyurethane, glass particles, fine particles of an inorganic material such as diatomaceous earth with a small amount of polymer. Porous membranes, porous membranes made of polytetrafluoroethylene, filter papers, glass fiber filter papers and the like. In the present invention, when the analytical element is divided into the partial analytical elements by thermal fusing, it is preferable to use a porous membrane made of a thermoplastic material.

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

For a multi-layer analytical element having two or more spreading layers, it is essential that all layers are laminated and integrated at the time of spotting the 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.

For the purpose of controlling 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 20 to 200 μm, preferably 50 to 170 μm, more preferably 80 to 150 μm.

The partial analytical element on which the blood cell separating element is provided may be such that the above-mentioned porous development layer is simply laminated on the water-impermeable support depending on the analysis item or the like. By providing the hydrophilic polymer layer between the supports, the universality of analysis items and the like can be dramatically improved.

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

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

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

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

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,
A transparent film made of cellulose acetate propionate or the like) having a thickness of about 50 μm to 1 mm, preferably about 80 μm to about 300 μm can be used.

The support is usually a light-transmissive one, but it may be colored or light-impermeable when the measurement is performed from the spreading layer side.

If necessary, a publicly known undercoat layer or an adhesive layer may be provided on the surface of the support to strengthen the adhesion to the hydrophilic polymer layer.

Partial adhesion (porous adhesion), which is one of the features of the dry analytical element used in the present invention, will now be described. Partial adhesion refers to JP 61-4959 (EP 0166365).
A), JP-A-62-138756 to 138758 (EP 0226465A) and the like, which is a mode of adhesion between two adjacent porous layers or between adjacent porous layers and a non-porous layer, Are substantially adhered and integrated by an adhesive which is partially (or intermittently) arranged between the interfaces of the two layers, and the uniform passage of the liquid is substantially uniform between the two adjacent surfaces and between them. Adhesion is configured so as not to be hindered by.

In the analysis element of the present invention, between the fibrous porous layer and the non-fibrous porous layer in the blood cell separating element, between the microporous layer having a blood cell separating ability and the fibrous porous layer, and the blood cell separation. It is preferable that the element and the porous spreading layer are both partially bonded (porous bonded). For example, in the case of bonding between the porous spreading layer and the blood cell separating element, it is preferable to partially dispose the adhesive on the porous developing layer and then bond the blood cell separating element with a uniform light pressure. This is a common method. On the contrary, an adhesive may be partially disposed on the blood cell separating element, and then the porous spreading layer may be adhered while evenly applying a light pressure. Further, an adhesive is partially arranged on the microporous sheet material to be the porous development layer, and the blood cell separating element is bonded thereto while applying light pressure evenly, or vice versa. , And then the microporous sheet-like material to be the porous development layer is uniformly and lightly bonded together, and then the microporous sheet-like material to be the development layer on the hydrophilic polymer layer. Can be evenly stuck together.

A method of partially disposing the adhesive on the blood cell separating element or the porous spreading layer is disclosed in JP-A-64-1959 and JP-A-62-113.
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, the method of transferring and adhering the adhesive to the porous spreading layer or the blood cell separating element by using a printing plate (gravure printing plate or intaglio plate is preferable) and the method of laminating 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.

As an adhesive used, 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 water-based glues such as starch glue; dextrin, carboxymethyl cellulose,
An aqueous solution of polyvinyl alcohol or the like; a vinyl acetate-butyl acrylate copolymer emulsion is available. As the organic solvent type adhesive, one having a slow solvent evaporation is suitable. Thermal adhesive (or heat sensitive) adhesives are particularly useful.

As the heat-adhesive (or heat-sensitive) hot-melt type adhesive, the hot-melt type adhesive described in "Industrial Materials", Vol. 26 (No. 11), pp. 4-5 can be used.
Examples thereof include ethylene copolymers such as ethylene-vinyl acetate copolymer, ethylene-ethyl acrylate copolymer, ethylene-acrylic acid copolymer; polyolefins such as low molecular weight polyethylene and atactic polypropylene; nylon, etc. Polyamide; polyester-based copolymer; thermoplastic rubber such as styrene block copolymer such as SBS; styrene-butadiene rubber, butyl rubber, urethane rubber; rosin, petroleum resin, terpene resin; synthetic wax.

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

Furthermore, in the embodiment in which the blood cell separation element is peeled off after spotting the sample, the spreading layer and the blood cell separation element do not need to be adhered, and the diffusion and permeation of the sample can be quantitatively promoted. It may be laminated, but partial adhesion is preferable to ensure uniform diffusion.

Each of the plurality of partial analytical elements may be completely separated, or the support may not be separated and only the other layers may be separated. To make these partial analytical elements, individual small analytical elements, which may have a support, may be arranged at appropriate intervals,
Alternatively, the hydrophilic polymer layer and the porous spreading layer provided on a common support may be fused by using a fusing blade.
In the latter case, it is preferable to use a porous spreading layer made of a thermoplastic material, and to fuse these layers with a blade heated to a temperature equal to or higher than the softening point of the spreading layer material.

As a fusing method, an ultrasonic oscillator for an aluminum blade is used, in which a brass blade having a shape corresponding to the number of divisions, for example, a cross shape when divided into four, is heated using electricity or the like. And the temperature of the thermoplastic resin or the like is raised above the softening point by vibration energy.

In any case, the blade thickness is 0.4 to 2 mm,
The thickness is preferably 0.6 mm to 1.7 mm, more preferably 0.8 to 1.3 mm. If the blade is thin, the distance between the cut surfaces is not sufficient, and if it is too thick, lumps may remain on the support, which is not preferable.

The number of partial analysis elements is practically about 2 to 16, preferably 2 to 9, and particularly preferably 2 to 4.
It is an individual.

It is also possible to use a heated straight blade, a rotary blade or the like to perform fusing in a cross shape. More preferable is a method of using a double-edged blade having a V-shaped cross-section that can be heated by electricity or the like, and fusing while moving one or both of the plasma receiving element and the blade.

The blade angle is not particularly limited, but it is preferable to set the distance between the upper ends of the cut porous development layers to be in the range of about 0.1 to about 2 mm, preferably about 0.4 to about 1.2 mm. It is preferable to set the blade angle within the range of 60 ± 15 degrees. If this interval is too small, it may overflow the groove when the measurement reagent is supplied later. There is no restriction on the length of the blade, and you can select from the ease of manufacturing and handling,
For example, it may be about 0.5 to 7 cm.

At the time of fusing, it is preferable that the porous spread layer is flat or convex. In the case of a flat surface, the plasma receiving element or the blade is moved according to the length direction of the blade, but in the case of a convex surface, for example, the support is brought into close contact with the periphery of the drum, and the blade is brought into contact with the porous spreading layer. The drum can be rotated. Due to the relative movement of the blade and the plasma receiving element, the fused portion of the porous development layer moves to the rear side of the blade, sufficiently melts the melted cross section, and at the same time prevents the generation of thread-like residues, The flatness is improved by preventing the porous spreading layer from rising at the fusing end.

The temperature at the time of fusing is preferably set so that not only the porous spreading layer but also a part of the surface of the support is melted, but it cannot be uniquely determined because it changes depending on the moving speed. For example, when polyester is used as the material for the porous spreading layer and the support, the temperature is 400 to 500 ° C, and the moving speed is about 10 m.
It can be / minute. Generally, the temperature of heat melting is about 100 to 800 ° C., preferably about 200 to 600 ° C., and more preferably about 300 to 500 ° C. as a blade temperature.

The fusing groove is also preferably provided on the surface of the support. In principle, if the porous development layer is melted and the hydrophilic polymer is cut at the same time, there will be no mixing of measurement reagents, but
Considering the planarity of the plasma receptor element, it is difficult to constantly create such a state, and the depth of the support surface should be 5 to 5.
The conditions are set so as to form a fusing groove of 60 μm, preferably about 10 to 45 μm. The deeper the separation is complete,
Since the flatness is poor and the flatness tends to fall apart, the handling becomes worse in the subsequent steps.

It is also possible to form a plurality of fusing grooves simultaneously by using a plurality of blades. That is, a web-shaped plasma receiving element is conveyed using a drum, and a plurality of blades are installed on the rotating drum portion. Then, the fusing groove can be formed in a grid pattern by placing it on a flat surface and moving the blade, for example, at a right angle to the formed fusing groove. This plasma receiving element is cut so as to include, for example, four square areas (partial analysis elements) of equal area, and a blood cell separation element is laid over it.

In any of the above cases, the blood cell separation element may be arranged so that the sample can be supplied to each partial analysis element substantially evenly, and the shape thereof does not matter.

An embodiment of the analytical element of the present invention is shown in FIGS. The analysis element of FIG. 1 corresponds to the basic mode of the analysis element of the present invention, and blood cells are formed on the porous development layer 2 of the partial analysis element 3 in which the porous development layer 2 is laminated on the water-impermeable support 1. The separating element 4 is partially bonded. The dotted line 11 indicates that the support may or may not be cut.
However, when the support is cut, another support may be used although not shown in the figure.

The analytical element of FIG. 2 is a standard analytical element of the present invention. In the partial analytical element, a hydrophilic polymer layer 5 and a porous spreading layer 2 are laminated in this order on a water-impermeable support 1, a fibrous porous layer 6 and a non-fibrous porous layer 7 Blood cell separating element 4 in which the cells are integrated by partial adhesion
The non-fibrous porous layer 7 side is partially bonded onto the porous spreading layer 2.

Next, the test substance will be described. 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, drugs, hormones, etc.
As long as an analysis method such as a tumor marker, DNA, RNA, etc. is established, it can be an analysis target.

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 and 2
Shows a basic configuration example.

1: Analytical element composed of water-impermeable support / hydrophilic polymer layer / developing layer / blood cell separation element. Ca, GOT (glutamate oxaloacetate transaminase), GPT (glutamate pyruvate transaminase), γ-GTP (γ-glutamyl transpeptidase), glucose, LDH (lactate dehydrogenase),
It is effective for the analysis of CPK (creatine phosphokinase), TP (total protein), Alb (albumin), TCHO (total cholesterol), UA (uric acid), neutral fat and the like.

2: An analysis element having a structure of a water-impermeable support / hydrophilic polymer layer / developing layer / blood cell separating element and containing a chromogen in the hydrophilic polymer layer and / or the developing layer. As a chromogen, Ann. Clin. Biochem., 6, 24-27
4-aminoantipyrine described in (1969) (also known as 4-aminophenazone, that is, 1-phenyl-2,3-dimethyl-4-amino-3-pyrazolin-5-one), JP-A-59-54962 and the like. 1- (2,4,6-trichlorophenyl) -2,3-dimethyl-4-amino-3-pyrazolin-5-one described, 1- (3,5-dichlorophenyl)-
2,3-Dimethyl-4-amino-3-pyrazolin-5-
Tri-substituted-4-amino-3-pyrazolin-5-one such as one
On, 1-phenyl-2,3 described in JP-B-55-25840
-Dimethyl-4-dimethylamino-3-pyrazoline-5
4-Aminoantipyrine analogs such as -one can be used. Among these compounds, 4-aminoantipyrine, 1- (2,4,6-trichlorophenyl)-
2,3-Dimethyl-4-amino-3-pyrazolin-5-
On, 1- (3,5-dichlorophenyl) -2,3-dimethyl-4-amino-3-pyrazolin-5-one and the like are preferable.

3: A water impermeable support / hydrophilic polymer layer / developing layer / blood cell separating element, wherein a chromogen and other reagents (to be described later) are contained in the hydrophilic polymer layer and / or the developing layer. Analytical elements including (excluding measurement reagents). Other reagents include POD (peroxidase),
NAD (nicotinamide adenine dinucleotide), N
ADP (nicotinamide adenine dinucleotide phosphate), DIP (diaphorase) and the like can be mentioned.

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 layer by including it 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 dye forms a cationic dye, a hydrophilic polymer layer containing a polymer containing an anion atom or an atomic group bonded to a polymer chain is used as a mordant layer, and a coloring reagent. When forming an anionic 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, JP-A-54-29700 and JP-A-5-29700.
3-131089 etc. For example, as the anion mordant polymer, Japanese Patent Publication No. 2-30466, Nos. 13-14
Column, alkaline hydrolysis product of methyl vinyl ether-maleic anhydride copolymer, polystyrene-p
-Alkali metal salts or alkaline earth metal salts of sulfonic acids, alkali metal salts or alkaline earth metal salts of copolymers of styrene-p-sulfonic acid and hydrophilic vinyl monomers, and the like.

Further, layers and the like capable of containing these polymers are also described in detail in columns 15 to 16 of the publication.

5: An analytical element in which the light shielding layer is provided between the hydrophilic polymer layer and the spreading layer in the constitutions 1 to 4 above.
Whole blood can be used as a sample without removing the blood cell separation element. The light-shielding layer is a water-permeable material in which fine particles or fine powders having light-shielding properties or both light-shielding properties and light-reflecting properties (hereinafter simply referred to as “fine particles”) 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 fine flakes, and examples of light-shielding fine particles include carbon black, gas black, carbon microbeads, etc. Among these, titanium dioxide fine particles,
Barium sulfate fine particles are preferred.

Examples of hydrophilic polymer binders having film-forming ability include weakly hydrophilic regenerated cellulose and cellulose acetate in addition to the above 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.

In the slide used in the present invention, it is also possible to select a preferable constitution corresponding to a predetermined test substance and use them in combination as a partial analytical element.

In the present invention, the measuring reagent refers to a reagent which 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. A specific example will be described below.

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 para-nitrophenyl phosphate for ALP. is there.

Also, 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 and an indicator or the like directly react with each other to cause a detectable change, the indicator is referred to.

One of the objects of the present invention is to prevent the deterioration of the detection reagent which occurs during storage of the analytical element, which is a drawback of the conventional dry chemistry, and therefore the reaction reagents incorporated in the above reaction system. When is unstable like an enzyme, it is preferable that these are also contained in the measurement reagent.

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

Among the measuring reagents, in order to proceed the reaction stably and with good reproducibility, pH and ionic strength are adjusted, diffusion and permeation into the material constituting the analytical element are improved, and an enzyme contained therein is included. Various reagents may be included for the purpose of improving instability.

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

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

An example of the measuring method using the analytical element of the present invention is shown in FIG. First, the whole blood sample 9 is spotted as shown on the left side of FIG. It is appropriate that the spotted amount of this whole blood sample is about 10 to 100 μl in the case of four analysis items. After the spotting, as shown in the center of FIG. 3, the plasma 10 is allowed to develop into the partial analysis element, and the blood cell separation element 4 is removed. The remaining partial analytical element is dried unless further analysis is to be performed. As shown in the right side of FIG. 3, the dried partial analytical element is spotted with a measuring reagent to cause a reaction and analysis is performed.

In the present invention, when the interval between the supply of the liquid sample and the supply of the measurement reagent is long, the partial analytical element is preferably dried for a certain period of time under substantially constant conditions.

One preferable drying method and conditions are described in Japanese Patent Application No. 2-90562 (JP-A-3-28954), page 25, line 9 to page 28, line 6, especially page 27. Line 13-28
Details are given on page 6, line 6.

For preferred incubation methods and conditions, see Japanese Patent Application No. 2-90562 (JP-A-3-28954).
Page 25, line 9 to page 28, line 6, especially page 27,
It is described in detail from line 13 to page 28, line 6.

A preferred method is heating while the partial analytical element is placed in an enclosure in which the periphery of the partial analytical element is covered. 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.

Temperature fluctuation during incubation is ± 5
C., preferably ± 3 ° C., more preferably ± 1 ° C.

An incubator suitable for performing such incubation under constant conditions is described in Japanese Utility Model Publication No. 3-126449. In other words, 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.

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 or the like, it is necessary to store it in a state in which moisture and air are substantially cut off. There is.

Details of the storage conditions are also described in Japanese Patent Application No. 2-90562 (JP-A-2-289543), page 28, line 12 to page 30, line 11. . 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 pass therethrough.

As means for removing water, any known hygroscopic agent that does not substantially alter the quality of the sample can be selected and enclosed. After bagging the element, it may be aired out and squeezed out.

In addition, water containing a desiccant, 40 ° C. or lower in a water vapor impermeable closed container, and 25 ° C. or lower when the test substance is easily denatured, an unstable compound such as an enzyme is detected. In this case, a method of drying at 10 ° C or lower, preferably 0 ° C or lower is more preferable. Specifically, the analysis element is enclosed in a plastic bag with a fastener, a plastic container with a lid, and the like together with a desiccant, and the temperature is kept below the above temperature. The desiccant may be appropriately selected from known hygroscopic agents that do not substantially deteriorate the test substance and used. Zeolites and silica gel are preferable from the viewpoint of safety and dehydration ability, and zeolite is more preferable. As a form, particles having a diameter of about 1 to 3 mm are put into a bag made of Japanese paper having good moisture permeability, and put into a nylon mesh,
Etc. It is possible to dry 4 to 10 multilayer analytical elements per gram of zeolite or silica gel.

The drying time varies depending on the amount and type of the spotted liquid sample, the type and shape of the desiccant, the size of the container, the positional relationship with the analytical element, and the like. Generally, it is practically preferable to set the conditions such that 90% or more of the water content of the analytical element is removed and dehydrated by a desiccant in about 1 to 10 hours. This drying time is also greatly 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. When dried by this method,
You can mail it to a hospital etc. in its original form.

Here, the term "dry" means that the reaction does not substantially proceed in the hydrophilic polymer 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 a ratio when the amount of water when the aqueous solution containing the test substance is spotted on the analytical element is 100.

Using these partial analysis elements, analysis is performed by the following method. The 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 carried out 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 of 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 has the widest range of application, but when the sample is not whole blood or when the blood cell separation element is removed after the sample is supplied and the measurement is performed, the transmission photometric method is used. Can be measured. When the water-impermeable support is opaque, it can be measured from the opposite side of the support.

An example of the measuring method using the analytical element of the present invention will be described. An analysis element in which a blood cell separation element is laid over four partial analysis elements is housed in a plastic mount having fixing pieces of the analysis element at four corners. The subject or the like collects blood and spots it on the blood cell separation element, and waits for the plasma to sufficiently spread on the porous spreading layer of each partial analysis element. Then, the blood cell separating element is removed, dried in a closed container containing a desiccant at 10 ° C. or lower, and mailed to a laboratory. The inspection organization takes out the analytical element, installs it in the spotting station of the analyzer, and spots the measurement reagent on each partial analytical element. For example, glucose, urea nitrogen, cholesterol and uric acid measuring reagents are spotted on the four partial analytical elements. When the spotting is completed, the table on which the analytical element is installed is rotated 1/4 turn and reacted in the incubator. Meanwhile, the following analytical elements are installed in the spotting station to deposit the reagents. The analytical element that had been incubated in the incubator was 1/4 the size of the table.
Rotate and move to the next standby part, then rotate 1/4 turn and at the photometry station the colors of the four sections are measured at the same time,
The analytical element for which photometry has been completed is discharged from the table.

The measurement may be carried out sequentially for each partial analytical element, but it is also possible to deposit a measuring reagent on a plurality of, preferably all, partial analytical elements at the same time, incubate them, and then prepare them for photometry. .

The dry analysis element of the present invention is classified into the above-mentioned classification 1
~ It can be effectively used in any field corresponding to classification 3. When using blood as a sample,
Since dry analytical elements require very little blood,
Blood can be collected using an appropriate device such as a capillary pipette. Furthermore, the dry analytical element is compatible with whole blood, and whole blood as collected can be used as a liquid sample for measurement.
It can be transported by mail, courier, etc., if necessary, which is particularly effective for in-home inspection.

Bodily fluids other than blood, such as urine and saliva, can be directly used as specimens simply by spotting appropriate amounts thereof.

[0136]

【Example】

Example 1 1. Preparation of multi-layered analytical slide 1-1: Preparation of plasma receiving element A water absorbing layer composed of the following components was dried on a 180 μm thick polyethylene terephthalate (PET) colorless transparent smooth sheet undercoated with gelatin to have a thickness of 15 μm after drying. And was dried. Deionized gelatin 20 g p-Nonylphenoxypolyglycidol 1.5 g (containing 10 glycidol units on average) Bis [(vinylsulfonylmethylcarbonyl) amino] methane 220 mg

Next, an adhesive layer composed of the following components was applied onto this layer from an aqueous solution so that the thickness after drying was 1 μm, and dried to form an adhesive layer. Deionized gelatin 4.0 g p-nonylphenoxypolyglycidol 430 mg (containing 10 glycidol units on average)

Next, water was supplied onto the adhesive layer at a rate of about 30 g / m ² to wet the entire surface almost uniformly, and a broad cloth made of PET (thickness: about 150 μm, void volume: 9.8 μL / m ² ) Was lightly pressed, laminated, adhered and dried.

Next, 100 m of an aqueous solution having the following composition was applied to this cloth.
The plasma receiving element was completed by applying it evenly at a rate of L / m ² and drying. Hydroxypropyl methylcellulose 8.7g (contains 28-30% methoxy groups and 7-12% hydroxypropyl groups. Solution viscosity at 20 ° C in 2% aqueous solution is 50cps) Octylphenoxypolyethoxyethanol 27g (containing 10 oxyethylene units on average) Water 964.3g

1-2: Fusing with a heating blade A brass block is processed to form a thermal fusing head having a 15 mm long and 15 mm wide cross-shaped tip and a 1 mm thick blade, which is attached to the tip of a soldering iron. I installed it. The heating temperature of the thermal fusing head can be adjusted by controlling the power supply voltage with a slidac.

The plasma receiving element prepared in 1-1 was 15 × 15 mm
Then, the whole of the development layer and the water absorption layer and a part of the PET support were melt-fused by cutting with a fusing head and heating at 260 ° C. for 2 seconds, and into four equal sections (No. 1 to 4). divided.

1-3: Preparation of blood cell separating element A tricot knitted fabric (thickness: about 250 μm) in which PET spun yarn corresponding to 50 denier was knitted with 36 gauge 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, immediately after the adhesive was transferred, 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% was faced to the surface of the high temperature cloth, and was passed between the laminating rollers. , And both were laminated and adhered integrally (partially adhered) to prepare a blood cell separation element.

1-4: Preparation of analytical slide While heating the plasma receiving element divided into four sections prepared in 1-2 on a heat plate set at 60 ° C.,
The multi-layered analytical slide was completed by placing it on the blood cell separation element prepared in 1-3 and placing a weight of 50 g / cm ^{2 with} a smooth bottom surface and leaving it for 2 minutes.

2. Measurement 2-1: Preparation of sample 20 ml of heparin-containing normal whole blood was centrifuged to separate into blood cell component and plasma component. A part of the plasma component was replaced with control serum (Fuji Dry Chem Control LH) of known component concentration. Whole blood of which the component concentration was adjusted was reconstituted by mixing the blood cell component and the plasma substituted with the control serum.

2-2: Specimen spotting Whole blood as collected on the above-mentioned multilayer analysis slide was
30 μl of whole blood prepared in 2-1 for preparing a calibration curve was spotted on each of the other slides, left at room temperature for 30 seconds, and then the blood cell separating element was peeled off from the plasma receiving element with tweezers.

2-3: Dehydration and Drying The slide obtained in 2-2 was placed in a moisture-permeable bag made of Japanese paper and 5 cmX with 2 g of granular zeolite having a particle size of about 1 mm.
It was put in a 7 cm moisture-proof polyethylene pouch, sealed, and stored at room temperature for 3 hours.

2-4: Spotting and measurement of detection reagent Glucose (GLU), urea nitrogen (B
UN), cholesterol (TCHO) and uric acid (UA)
Each measurement reagent solution of was prepared.

Measurement reagent for GLU 2- (N-morpholino) ethanesulfonic acid 213 mg p-nonylphenoxypolyglycidol 800 mg (containing 10 glycidol units on average) dihydroxynaphthalene 110 mg 4-aminoantipyrine 140 mg glucose oxidase 1300U peroxidase 5000U distilled water 10.0 ml

Measurement reagent for BUN Triton-X 100 (manufactured by Rohm Antohace) 2 g o-phthalaldehyde 2 g N-1-naphthyl-N′-diethylethylenediamine oxalic acid 820 mg distilled water 10 ml

Measurement reagent for TCHO Cholesterol esterase 987U Cholesterol oxidase 600U Peroxidase 6614U Triton-X 100 (manufactured by Rohm Antohase) 0.5 g 2- (3,5-dimethoxy-4-hydroxyphenyl) -4- [4- (dimethyl Amino) phenyl] -5-phenethylimidazole 30 mg Buffer solution of the following formulation 10 ml Liquid solution of 90.9 mg of dipotassium phosphate in 100 ml of distilled water 5
To 0 ml, add 680.5 mg of phosphoric acid, 1 potassium, and 2 hydrogen to 100 distilled water.
A solution adjusted to pH 7.5 by adding a solution dissolved in ml.

Measurement reagent for UA Uricase 145U Peroxidase 6794U Triton-X100 (manufactured by Rohm Antohase) 0.5 g 2- (3,5-dimethoxy-4-hydroxyphenyl) -4- [4- (dimethylamino) phenyl] -5-phenethyl imidazole 30mg boric acid 0.15M 10ml

GLU, BUN, and T were respectively provided in sections No. 1, 2, 3, and 4 of the 4-divided slide after drying described in 2-3.
5 μl of the detection reagent solution of CHO and UA was spotted. At this time, the cross section of the thread forming the spreading layer of the slide was completely welded, and the reagent solution did not seep out. In addition, the liquid did not seep out from the water absorbing layer.

The slides that had undergone the color development reaction were incubated at 37 ° C. for 6 minutes, and a Fuji Drychem 5500 analyzer whose beam diameter was adjusted to 4 mm by adjusting the photometric beam head was used to reflect light at wavelengths corresponding to each color development. The concentration was measured. The concentration of each component was calculated by using the calibration curve based on the relationship between the concentration of each component and the reflection optical density.
= 93 mg / dL, BUN = 19 mg / dL, TCHO = 148 mg / d
L, UA = 4.4 mg / dL.

A part of the same untreated whole blood was centrifuged and the concentration of each component was measured using Hitachi 7150.
GLU = 97mg / dL, BUN = 22mg / dL, TCHO = 156m
Since g / dL and UA = 4.7 mg / dL, it was confirmed that the values obtained by the method of the present invention had the accuracy for practical use.

2-5: Repeatability The same operation as above was repeated 10 times to examine the repeatability. CV (%) is GLU = 4.6, BUN =
6.2, TCHO = 4.1, UA = 2.9, which proved to be a sufficiently reliable measurement method.

Example 2 1. Preparation of Multilayer Analytical Slide The same plasma receiving element as in Example 1 was cut using a cutter to prepare four pieces of about 7 × 7 mm. This has a width of 15 mm
Part of the same blood cell separation element as in Example 1 was adhered in a substantially cross shape so that the support of the element and the adhesive layer were in contact with each other and the corners were separated by about 1 mm on the transparent adhesive tape. Then, a multilayer analytical slide having partial analytical elements No. 1 to 4 was prepared.

2. Measurement 2-1: Preparation of Specimen In the same manner as 2-1 of Example 1, a plasma component and a plasma component whose component concentration was adjusted were obtained.

2-2: Spotting of sample The plasma component obtained by centrifugation on the multilayer analysis slide described in 1 and the plasma component prepared in 2-1 on the other slides described above 30 μl of each was spotted.

2-3: Dehydration and Drying After the blood cell separating element was peeled from the analytical slide obtained in 2-2, dehydration was carried out under the conditions described in the above publication using the constant temperature dryer described in Japanese Utility Model Publication No. 3-126499. Dried.

2-4: Preparation of Measurement Reagent Solution A measurement reagent solution having the following formulation was prepared. TP Copper sulfate pentahydrate 3.5g Tartaric acid 2.3g Lithium hydroxide 3.8g Cetylmethylammonium bromide 100mg Water 10ml

Alb Bromocresol Green 85 mg Triton-X 100 (manufactured by Rohm Antohase) 100 mg Citric acid 0.2M aqueous solution (pH 3.5) 10 ml

GGT L-α-glutamyl-3-carboxy-paranitroaniline 58.5 mg Glycylglycine 156 mg 2N Tris-HCl buffer (pH 8.1) 10 ml

GOT Trishydroxyethylaminomethane 84 mg Phosphoric acid • 1 potassium • 2 hydrogen 104 mg L-aspartic acid 431 mg α-ketoglutaric acid 93 mg 20% MgCl ₂ 273 μl POD 343IU TPP (cocarboxylase) 21 mg FAD (flavin adenine dinucleotide) 5 mg Oxalo Acetate dehydrase 24U POPG (pyruvate oxidase) 3088U 2- (3,5-dimethoxy-4-hydroxyphenyl) -4- [4- (dimethylamino) phenyl] -5-phenethylimidazole 54mg 1N NaOH 3.4ml distilled water 6.6ml

2-5: Spotting of measurement reagent and measurement TP and Al were added to No. 1 to No. 4 of the analytical element obtained in 2-3.
After spotting 5 μl each of the measurement reagent solutions for b, GOT, and GGT, incubation was performed and photometry was performed in the same manner as in the method described in 2-5 of Example 1.

Table 1 shows the values measured by the method of the present invention for the plasma obtained by separating the collected whole blood, and the values obtained by measuring the same plasma sample with the Hitachi 7150 analyzer.

[0167]

[Table 1]

From this result, it was found that the value obtained by the method of the present invention had the accuracy for practical use.

2-7: Repeatability Repeatability The same operation as above was repeated 10 times to examine repeatability. The results are shown in Table 2, and it was found that the measurement method had sufficiently high precision.

[0170]

[Table 2]

Example 3 A plasma receiving element similar to that produced in Example 1 was prepared,
The fusing groove was formed under the following conditions.

A V-shaped tool having a length of about 5 cm and a blade angle of about 60 degrees and capable of electrically heating and controlling the temperature (made of cemented carbide mainly composed of tungsten carbide) was produced. The above bite heated to about 400 ° C. was placed on a plasma receiving element having a width of about 30 cm, which was conveyed by a suction drum having a diameter of 300 mm at a speed of 10 m / min, from the surface of a PET support having a thickness of 180 μm to about 30 μm
The conditions were set so that the fusing groove was formed until the contact was made. Repeat the above operation, at intervals of about 8 mm, width of about 0.5 m
10 fusing grooves were formed. About 30 cm was cut from this web, placed on a flat suction plate, and the cutting tool was moved under the same conditions, but this time, to form a fusing groove orthogonal to the fusing groove. Molten PE on the V-shaped inner surface
A T film was formed. A part of the plasma receiving element is cut into a size of 15 × 15 mm with the fusing groove in the center, and the same blood cell separating element is provided thereon in the same manner as in Example 1, and the same method as described below. Completed a multi-layer analysis slide.

When the same measurement as in Example 1 was carried out using this multilayer analysis slide, similar excellent results were obtained for the four items.

[0174]

By using the analysis element of the present invention,
A small amount of liquid sample, particularly a body fluid sample, can be easily supplied to a plurality of analytical elements, and can be reliably stored and transferred, and analysis can be performed with sufficient analytical accuracy.

[Brief description of drawings]

FIG. 1 is a cross-sectional view of an analytical element according to a basic aspect of the present invention.

FIG. 2 is a cross-sectional view of an analytical element of a standard aspect of the invention.

FIG. 3 is a process chart showing a method of using the analysis element of FIG.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Water-impermeable support 2 ... Porous spreading layer 3 ... Partial analytical element 4 ... Blood cell separation element 5 ... Hydrophilic polymer layer 6 ... Fibrous porous layer 7 ... Non-fibrous porous layer 8 ... Water repellent 9 ... Specimen 10 ... Plasma 11 ... Division of support

Front page continued (72) Inventor Masao Kitajima 3-1146 Izumisui, Asaka-shi, Saitama Prefecture Fujisha Shin Film Co., Ltd. Within Film Co., Ltd.

Claims (2)

[Claims] 1. A porosity of a plurality of partial analytical elements containing no measurement reagent, wherein a hydrophilic polymer layer and at least one porous spreading layer are laminated in this order on a water-impermeable support. A dry analysis element, wherein one blood cell separation element is laid across in a spreadable manner in a peelable manner. 2. The dry method according to claim 1, wherein the porous spreading layer is made of a thermoplastic material, and is divided into partial analytical elements by thermal fusing to reach the support from the porous spreading layer. Analysis element.