JPH06213886A - Multiple-item analysis method - Google Patents

Abstract

PURPOSE:To analyze multiple items with a sufficient accuracy simply by supplying a small amount of liquid sample, especially a humor sample, to an analysis element once, and further surely store and transfer analysis elements after supplying the sample. CONSTITUTION:At least hydrophilic polymer layer and porous development layer are laminated in this order on a water-unpermeable support. A specimen is spotted at a multilayer analysis element which does not contain a measurement reagent, the porous development layer is fractionated after drying, and then a measurement reagent causing each different detection reaction to occur is spotted to each fractionated part, and then measurement is made.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an analytical method which can be used for measuring many items with a small amount of sample in the field of clinical analysis, and when it takes a long time to analyze a liquid sample. .

[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 and the like, and normally 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. Furthermore, since an appropriate amount of reagent can be dissolved and prepared according to the number of measurement samples, the reagent cost per measurement can be reduced. Although it is complicated and troublesome to combine and automate a large number of solutions, clinical test equipment has a long history and social demands have been high.
Efficient automatic equipment has been developed and put into practical use in fields requiring small processing capacity.

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 application fields. 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. Category 2: The measurement location can be selected relatively freely by combining it with a measuring machine characterized by small size 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 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 by this method is not premised on the operation by specialists such as clinical technologists, doctors and nurses, and it is possible to directly sample urine or whole blood so that sample processing is not required. It is normal.

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.

[0008] 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 a 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 method for storing a dry chemistry analytical element after stopping and fixing the reaction of the deposited liquid sample on the analytical element on which the liquid sample has been spotted.
We have already developed a method for storing analytical elements, which is characterized in that it is stored in a state in which moisture and air are substantially blocked until analysis by an analyzer (Japanese Patent Laid-Open No. 3-289543).

[0010]

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 measurement of permeation of a transparent solution, the whole blood sample cannot be directly used 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 analytical measurement is about 10 μl / item, so even if 10 items are tested, 100 μl, and if 20 items are tested, 200
Only μl. The actual amount of blood collected at a hospital is 2 to 20
ml. That is, 50 to 100 times the blood volume required finally is collected.

[0012] 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 the serum is sedimented and separated as it is or in a state of being refrigerated to perform a central examination. Transfer to room or inspection center.
Alternatively, the collected blood is first centrifuged after the transfer, and separated into tangible components such as red blood cells and plasma or serum serving as an analysis sample. During this period, coexistence with erythrocytes may lead to the progress of biochemical reactions that affect the analysis, but against variable factors known to have extremely large effects on the analysis results, such as glycolysis inhibition and anticoagulation. Only measures are taken.

In consideration of such matters, it is desirable that centrifugation be performed immediately after blood collection, but this is often not 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 if you add and centrifuge
This is because fibrin may be deposited depending on the sample before the subsequent measurement time by the analytical instrument, which easily causes troubles in the delivery system such as the dispensing syringe and the tube of the analytical instrument.

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 test, it takes time 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 analysis target item. 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. Furthermore, when analyzing a plurality of analysis items, it is necessary to spot a sample on each analysis element as many as the number of analysis items, which is not only time-consuming but also tends to increase the required sample liquid volume. is there.

The object of the present invention is to perform a multi-item analysis with sufficient accuracy by only supplying a small amount of a liquid sample, particularly a body fluid sample, to the analysis element once, and further to reliably store the analysis element after the sample is supplied. -To provide a multi-item analysis method that can be transferred.

[0019]

The above object of the present invention is to provide at least a hydrophilic polymer layer on a water-impermeable support,
A porous spreading layer is laminated in this order, a sample is spotted on a multi-layer analytical element that does not contain a measuring reagent, and after drying, at least the porous spreading layer is cut, and then cut into each fraction. This was achieved by a multi-item analysis method characterized by spotting measurement reagents that cause different detection reactions and performing the measurement.

The basic constitution of the multilayer analytical element used in the present invention is composed of a water-impermeable support, a hydrophilic polymer layer, and a porous developing layer which serves as a plasma receiving layer. The porous spreading layer may be composed of a plurality of layers, and may have a blood cell separating element which may be composed of a plurality of layers on the layer.

When whole blood is used as the sample, it is preferable to provide a blood cell separating element on the porous spreading layer. As the blood cell separating element, the fibrous porous layer and the non-fibrous porous layer described in JP-A-62-138756-8, JP-A-2-105043, JP-A-3-16651 and the like can be used. One that is bonded (partially bonded) with an adhesive that is partially arranged,
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. Of these, a particularly preferable one 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. is there.

As a non-fiber porous layer in a blood cell separation element in which a fibrous porous layer and a 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 having a hydrophilic surface such as polyamide such as 6,6-nylon, polyethylene, polypropylene, or Teflon may be used. In addition, Japanese Patent Publication No. 53-21677 and Japanese Patent Publication No. 55-908
It is also possible to use a porous layer having continuous voids in which small polymer particles, glass particles, diatomaceous earth, etc. described in No. 59 and the like are bonded with a hydrophilic or non-water-absorbing polymer.

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 the membrane filter made of the 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 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 or knitted fabric 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 made of a fluorine-containing polymer, polysulfone, etc., whose surface is hydrophilized, specifically separates blood cells and plasma from whole blood without causing hemolysis to the extent that it substantially affects the analytical value. Therefore, it can be used alone as a material for the blood cell separation element without being combined with the fibrous porous membrane. Although the blood cell / plasma separation mechanism is not clear, this microporous layer does not trap blood cells only on its surface, but in the thickness direction that combines the microporous layer made of a fluorine-containing polymer and the porous development layer. As it penetrates,
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 pore size of the microporous layer made of a fluorine-containing polymer is about 0.2.
μm to about 60 μm, preferably about 0.5 μm to about 20 μm
, More preferably 0.5 to 5 μm, the porosity is about 40% to about 95%, preferably about 50% to about 80%, and the layer thickness is about 10 μm to about 200 μm, preferably about 3
0 μm to about 150 μm, most preferably about 50 μm to about 12 considering the handling property such as wrinkles in the manufacturing process.
It is in the range of 0 μm.

A microporous layer of a fluorine-containing polymer is disclosed in JP-A-57.
-66359 (US4783315) physical activation treatment (preferably glow discharge treatment or corona discharge treatment) is applied to at least one side of the microporous layer to make the surface of the microporous layer hydrophilic, and to adjoin the adjacent fine particles. The adhesive strength of the adhesive used for partial adhesion with the porous 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. Other,
US3368872, US3260413, JP-A-53-92195 (US4201
548) and the like, and the microporous membrane of polyvinylidene fluoride described in US3649505.

Of these microporous membranes of fluorine-containing polymer, those particularly suitable for the microporous layer constituting the blood cell separation element are those having a pore size that is small enough 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-laminated 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 an analytical element, the aqueous liquid sample is repelled and does not diffuse or permeate into the membrane surface or inside. This is a well-known fact. As a means for imparting hydrophilicity to the microporous film of a fluorine-containing polymer to enhance the hydrophilicity, a sufficient amount of a surfactant is used to substantially hydrophilize the outer surface and the surface of voids inside the microporous film. Known methods such as a method of impregnating a microporous film of a fluorine-containing polymer with a fluorine-containing polymer and a method of chemically introducing a carbonyl group or a hydroxyl group into the structure of the fluorine resin film can be used.

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

Surfactants used for hydrophilizing a microporous film of a fluorine-containing polymer include nonionic (nonionic), anionic (anionic), cationic (cationic), 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.

The following are specific examples of the nonionic surfactant. Examples of the alkylphenoxy polyethoxy ethanol include isooctyl phenoxy polyethoxy ethanol: (T
riton X-100: oxyethylene unit average 9 to 10 contained) (Triton X-45: oxyethylene unit average 5 contained) Nonylphenoxypolyethoxyethanol: (IGEP
AL 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: (Tri
ton X-67: CA Registry No.59030-15-8)

The fluorine-containing polymer microporous membrane may be hydrophilized by providing one or more water-insoluble water-soluble polymers in its 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 membrane-forming stock solution 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 having a hydrophilic surface 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 having a function of supplying the hydrophilic polymer layer in an amount ratio. The material constituting the porous spreading layer has a softening point of about 50 to 300 ° C, preferably 60 to 2
A thermoplastic resin of about 50 ° C. is preferable. Preferred thermoplastic materials include cellulose esters such as cellulose acetate, cellulose acetate / butyrate, and cellulose nitrate, polyolefins such as polyethylene, polypropylene, polystyrene, polyamides such as various nylons, polyesters such as polyethylene terephthalate, and the like. Etc. can be mentioned. In particular,
It is possible to appropriately select and use from among non-fibrous and fibrous porous materials known as a spreading layer used in dry chemistry analysis elements. For example, a non-fibrous isotropic microporous medium layer represented by a membrane filter (brushed polymer) disclosed in JP-A-49-53888, and polymer microbeads disclosed in JP-A-55-90859. Is a non-fibrous porous layer typified by a continuous void-containing three-dimensional lattice granular structure layer formed by adhering in a point contact manner with a water-swellable adhesive, JP-A-55-164356 and JP-A-57-356356.
A porous layer comprising the woven fabric disclosed in 66359, etc.
Examples thereof include, but are not limited to, a layer made of a knitted fabric disclosed in -222769 and the like, various filter papers, and the like. The porous spreading layer can contain a material which does not have thermoplasticity in a range which can be melt-cut as described later, for example, titanium oxide, barium sulfate or the like.

The above-mentioned microporous film of a fluorine-containing polymer whose surface has been hydrophilized can also be used.

The porous spreading layer does not have to be limited to one layer, and it is not limited to the above-mentioned ones.
7, two or more layers can be used in a stacked manner as disclosed in JP-A-62-138758 and the like. For multi-layer analytical elements in which two or more spreading layers are stacked, it is essential that all layers are laminated and integrated when spotting a sample, but they need not be integrated in the subsequent process. . If necessary, it can be used in a state where the first developing layer and the second developing layer are separated from each other.

The spreading layer may contain a nonionic, anionic, cationic or amphoteric surfactant in order to accelerate the spreading of the sample. Further, a development control agent such as a hydrophilic polymer may be included for the purpose of controlling the development property. 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 1
The thickness is 70 μm, more preferably 80 to 150 μm.

The analytical element used in the present invention may be such that the above-mentioned porous spreading layer is simply laminated on the water-impermeable support depending on the analysis item or the like, but it may be disposed between the porous spreading layer and the support. By providing the hydrophilic polymer layer, universality of analysis items and the like can be dramatically improved.

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

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 μm to about 100 μm when dried.
m, preferably about 3 μm to about 50 μm, particularly preferably about 5 μm to about 30 μm, and a substantially uniform structure is preferred. The hydrophilic polymer layer may contain various reagents or a part of the reagents for promoting the intended reaction or for preventing or reducing the interference or interfering reaction.

As the water-impermeable support, a known water-impermeable support conventionally used 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 when it is measured from the spreading layer side, it may be colored or light-impermeable. If necessary, a publicly known undercoat layer or an adhesive layer may be provided on the surface of the support to strengthen the adhesion with the hydrophilic polymer layer.

Partial adhesion (porous adhesion), which is one of the features of the analytical element used in the present invention, will be described. Partial adhesion refers to JP 61-4959 (EP0166365).
A), JP-A-62-138756 to 138758 (EP0226465A) and the like, which are modes of adhesion between two adjacent porous layers or between adjacent porous layers and a non-porous layer. It is substantially adhered and integrated by an adhesive partly (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 that is structured so as not to be hindered.

In the analytical element used in 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 It is preferable that the blood cell separation 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.

The method of partially disposing the adhesive on the blood cell separating element or the porous spreading layer is described in JP-A-64-1959 and JP-A-62-13.
8756, various methods described in JP-A-64-23160 (DE3721236A) 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 the adhesive used, there is disclosed in JP-A-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 adhesive, the hot-melt 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.

Further, 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 have to be adhered to each other so that the diffusion / permeation of the sample progresses quantitatively. It may be laminated, but partial adhesion is preferable to ensure uniform diffusion.

In the analysis method of the present invention, first, a sample is spotted on a multilayer analysis element containing no measurement reagent to sufficiently diffuse and develop the sample, and when a blood cell separation element is present, it is preferably peeled off to perform multilayer analysis. Before the element is dried and the reagent is spotted, at least the porous development layer is cut, and the measurement reagent that causes different detection reaction is spotted on each of the parts fractionated by the cutting, and the measurement is performed. It has a feature.

The sample is whole blood, plasma, serum or the like, and the spotted amount may be about 15 to 50 μl. After spotting, leave the sample for 30 seconds to 1 minute to spread the sample.

Drying is carried out in a closed container in which a desiccant is present.
It is preferable to dry at 25 ° C or lower when the test substance is easily denatured at 25 ° C or lower, and at 10 ° C or lower, preferably 0 ° C or lower in the case of an unstable compound such as an enzyme. As the closed container, a plastic bag impermeable to water, steam, etc. can be used. 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. It is preferable to keep the temperature at 25 ° C. or lower for at least 1 hour after spotting. 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. It is possible to dry at least about 4 multilayer analysis elements (up to about 10 sheets) per 1 g of zeolite or silica gel. As a form, particles having a diameter of about 1 to 3 mm may be put in a bag having good moisture permeability, for example, a bag made of polyester nonwoven fabric, a bag made of nylon mesh, or the like.
The drying time varies depending on the amount and type of the deposited 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 the desiccant in about 1 to 10 hours, particularly in about 1 to 3 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.

As another preferable drying method, Japanese Patent Laid-Open No. 3-212058 can be used.
-28954, page 25, line 9 to page 28, line 6, especially line 2
Incubation can be mentioned in detail on page 7, line 13 to page 28, line 6.

In this method, the analytical element on which the specimen has been spotted is heated, preferably in a state in which the analytical element is placed in a surrounding enclosure. As a result, a constant dry state is achieved without being affected by the ambient temperature and humidity. Temperature range is 10
~ 60 ℃, preferably 20 ~ 50 ℃, more preferably 30 ~ 45 ℃
Is. The temperature fluctuation during the 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. 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 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.

In the incubation method, when it takes a long time to supply the measurement reagent after stabilization,
For example, when mailing an analysis element to a hospital or the like, it is necessary to store the analysis element in a state in which moisture and air are substantially cut off.

Details of the storage conditions are also described in 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 the water removing means, any known hygroscopic agent that does not substantially change the quality of the sample can be appropriately selected and enclosed. After bagging the analytical element, the air may be sufficed and squeezed out.

When dried in the above-mentioned closed container containing a desiccant, it can be stored and transferred as it is.

Here, "drying" 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.

Next, the dried and stabilized analytical element on which the sample is spotted is taken out from the closed container, and if there is a blood cell separating element, it is peeled off and fractionated. The purpose of the fractionation is to prevent a decrease in measurement accuracy due to the mixing of a plurality of measurement reagents that are subsequently spotted.

Fractionation is carried out by appropriately selecting the following method. 1. At least the porous development layer is heated to a temperature equal to or higher than the softening point and thermally fused. 2. A water repellent compound is used. 3. Cut off with a normal blade. Each method will be described below.

The thermal fusing groove in the method of the present invention causes thermal fusion of the porous structure during fusing to break the porous structure, and prevents the measurement reagent from spreading at least in the porous spreading layer beyond the fusing groove. . Therefore, this heat-fusing groove divides one multi-layered analytical element into a plurality of sections, and the shape is usually approximately the same, and is divided into 2 to 16 equal parts, preferably 2 pieces.
˜9 equal parts, particularly preferably 2 to 4 equal parts.
As a method of forming the heat-fusing groove, an aluminum blade having a shape according to the number of fractions to be cut, for example, a cross-shaped brass blade to be heated when using 4 fractions, is heated using electricity or the like. For example, the temperature of the thermoplastic resin may be increased by attaching it to an ultrasonic oscillator and using vibration energy. It is also possible to use a heated straight blade, a rotary blade or the like to perform fusing in a cross shape. In any case, the blade thickness is 0.4 to 2 mm, preferably 0.6 mm to 1.7 mm, and 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, a lump of thermoplastic resin may remain, which is not preferable.

In some cases, only by thermal fusing of the porous spreading layer, the measuring reagent may spread through the hydrophilic polymer layer and the measuring accuracy may be lowered. At this time, the fusing groove which reaches the support from the porous development layer is provided by raising the temperature to the softening point of the porous development layer or the hydrophilic polymer or higher.

The following method can be mentioned as the fractionation using a water-repellent compound. As the water-repellent compound, generally known non-volatile organic compounds can be used in addition to silicone, fluorine compounds and the like. These compounds may be dispersed or dissolved in water or an organic solvent, and the development layer is treated by a method such as gravure printing, silk screen printing, painting with a brush, or extruding from the tip of a thin tube. . At this time, it is important that these hydrophobic compounds not only make only the surface of the spreading layer hydrophobic, but that a sufficient amount penetrates in the thickness direction and the width direction,
Select the viscosity of the compound or solution. The width is 0.1 to 2 mm, preferably 0.5 to 1 mm.

Fractionation using a blade may be performed by a usual cutting method. In this case, it is possible to cut even the water-impermeable support.

Fractionation using a water-repellent compound and fractionation using a blade are particularly useful when a material that is not easily heat-fused, such as a fluorine-containing compound, is used as the porous spreading layer.

Using these fractionated analytical elements, analysis is carried out by the following method. In each of the divided compartments of the analytical element, the measurement reagent solution (2-10
supply about 1 μl) to cause the reaction. This reaction
The components contained in the sample are quantified by measurement by a method known in the field of dry chemistry (reflection density photometry, color change, fluorescence measurement, luminescence measurement, etc.).

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 components to be analyzed, and cause changes that can be detected mainly by optical measurement methods, such as color changes, color development (coloring), fluorescence, luminescence, changes in absorption wavelength in the ultraviolet region, and changes in turbidity. .

As a method of measuring dry chemistry, a reflection optical system is usually used. Also in the method of the present invention,
The method of photometry through the water-impermeable support of the analytical element is the most applicable, but when the sample is not whole blood or 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.

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 analytical element used in the present invention can have various configurations described below depending on the item or sample to be measured.

1: Analytical element having a structure of water-impermeable support / hydrophilic polymer layer / developing layer. The target is a sample solution that does not contain a large amount of strongly colored particle components such as plasma, serum, urine, and red blood cells. Alternatively, it is effective when erythrocytes or hemoglobin itself are to be analyzed.

2: Analytical element composed of water-impermeable support / hydrophilic polymer layer / developing layer / blood cell separation element. An analysis element having a blood cell separation element is effective in the case of an analysis using a body fluid containing colored particles as a sample, such as whole blood.
GOT (glutamate oxaloacetate transaminase), GPT (glutamate pyruvate transaminase), γ-GTP (γ-glutamyl transpeptidase, abbreviation GGT), glucose, LDH (lactate dehydrogenase), CPK (creatine phosphokinase), TP
(Total protein), Alb (albumin), TCHO (total cholesterol), UA (uric acid), BUN (urea nitrogen), neutral fat, inorganic ions such as Na, K, Ca, Cl, P, Mg, etc. It is effective for analysis.

3: Analytical element comprising 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.

4: A water-impermeable support / hydrophilic polymer layer / developing layer / blood cell separating element, and a chromogen and other reagents (described later) in the hydrophilic polymer layer and / or 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 3 and 4, the chromogen or other reagents can be supplied after the liquid sample is supplied / stabilized, but most of the chromogens are water-insoluble and 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.

5: Analytical element containing a 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.

6: An analytical element in which the light shielding layer is provided between the hydrophilic polymer layer and the spreading layer in the above constitutions 1 to 5.
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 the hydrophilic polymer binder having a film-forming ability include weakly hydrophilic regenerated cellulose and cellulose acetate in addition to the above-mentioned hydrophilic polymers, and among them, 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.

7: An analytical element in which the water-impermeable and gas-permeable layer (hereinafter referred to as a barrier layer) is provided between the hydrophilic polymer layer and the spreading layer in the above constitutions 1 to 5. 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 method of the present invention, a sample is spotted on one analytical element and then fractionated into a plurality of compartments. It cannot be mixed and used. Therefore, different reaction systems may be used even if the items to be analyzed are the same. For example, even when measuring the same BUN,
When the analytical element having the configuration 2 is used and when the analytical element having the configuration 7 is used, measurement reagents having different formulations are used.

In the present invention, the measuring 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. 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.

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 the substance to be analyzed 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.

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

An example of the measuring method using the analytical element of the present invention is shown in FIG. As shown in the figure, as this analytical element, one having a blood cell separating element on a porous developed layer is used, and the analytical element is housed in a plastic mount having fixing pieces of the analytical element at four corners. ing. The subject or the like collects blood, spots it on the blood cell separation element of the analysis element, and waits for the plasma to sufficiently spread in the porous spreading layer. Then, the blood cell separating element is peeled off, dried at a predetermined temperature in a closed container containing a desiccant, and transferred to a place where a measuring instrument is installed. There, the analysis element is taken out, and first, a heat-fusing groove is formed by the method described above to divide it into four. This analytical element is installed in the spotting station of the analyzer, and the measurement reagent is spotted in each section. In the drawing, glucose, urea nitrogen, cholesterol and uric acid measuring reagents are spotted on four compartments. 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 incubated in the incubator rotates the table 1/4 turn and moves to the next standby part, then 1
/ 4 rotation is performed, and the color development of the four sections is measured at the same time by the photometry station, and the analytical element for which the photometry is completed is discharged from the table.

The measurement may be carried out sequentially for each compartment, but a plurality of, preferably all, compartments should be coated with the measurement reagent at the same time, incubated, and then photometrically performed at the wavelength corresponding to each measurement item. You can also leave it.

The analysis method of the present invention can be effectively used in any of the fields corresponding to the above-mentioned categories 1 to 3. When using blood as a sample, the analysis element requires only a very small amount of blood, so it is possible to collect blood using an appropriate instrument such as a capillary pipette. It can be a liquid sample for measurement. Furthermore, it is possible to transfer the dried analysis element by mail, courier, etc., if necessary.
It is also effective in clinical medical examinations for home care.

[0106]

【Example】

Example 1 1. Preparation of Multilayer Analytical Slide 1-1: Preparation of Plasma Receptor Element On a 180 μm thick transparent polyethylene terephthalate (PET) colorless transparent undercoated sheet, a water absorbing layer consisting of the following components was dried to a thickness of 15 μm. 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 consisting of the following components was formed on this layer after drying. Of 1 μm in thickness from the aqueous solution 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 to the adhesive layer at a rate of about 30 g / m ² to wet the entire surface almost evenly and to be made of PET. Broad woven fabric (thickness about 150 μm, void volume 9.8 μL / m ² ) was lightly pressed to laminate, adhere, and dried. Next, an aqueous solution having the following composition was applied to this cloth at a rate of 100 mL / m ² almost uniformly and dried to complete the plasma receiving element. 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: Preparation of blood cell separation 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) 2.0 g Sodium tetraborate 2.0 g Water 96 g Next, the impregnated tricot knitted fabric is heated to 80 ° C.,
A hot-melt type adhesive (H950, manufactured by Nitta Gelatin Inc.), which was heated and melted at 130 ° C., was adhered to the surface 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 dots is 0.6 mm, and the dot area ratio is about 20%. The amount of adhesive deposited was about 2 g / m ² . Then, on the surface of the high-temperature fabric immediately after the adhesive was transferred, the effective pore size was 3.0 μm, the thickness was 140 μm, and the porosity was about 80%.
The non-glossy surface of the cellulose acetate membrane filter of No. 1 was faced to each other and passed through a laminating roller, and both were laminated to be bonded and integrated (partially bonded) to form a blood cell separating element.

1-3: Preparation of Multilayer Analytical Element This blood cell separating element was adhered to the base of the analytical element prepared in the step 1-1 by the partial adhesion method by the gravure printing method similar to the step 1-2, and integrated. Made into That is, in the same manner as in the step 1-2, after the hot-melt adhesive (H950 manufactured by Nitta Gelatin Inc.) that has been heated and melted on the surface of the membrane filter of the blood cell separation element is attached in a dot shape by the gravure printing method. Immediately, the broad woven cloth as the base of the plasma receiving element prepared in the step 1-1 was opposed to the ground side, and both were passed through a laminating roller and laminated to be bonded and integrated.

1-4: Completion of multi-layer analysis slide The completed multi-layer analysis element was cut into a square chip having a side length of 15 mm and placed in an organic polymer slide frame described in JP-A-57-63452, and a Fuji Drychem 5500 analyzer ( Fuji Photo Film Co., Ltd.) completed a multi-layer analysis slide with a measurable shape.

2. Measurement 2-1: Preparation of sample 20 ml of heparin-containing whole blood of a healthy person was collected, and a part thereof was taken and centrifuged to separate into a blood cell component and a plasma component. A part of the plasma component was replaced with a control serum (Fuji Dry Chem Control LH) having a known component concentration to adjust the component concentration. Whole blood with the adjusted component concentration was reconstituted by remixing the blood cell component and the plasma replaced with the control serum.

2-2: Spotting of Specimen 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 analytical slide obtained in 2-2 was put in a moisture-permeable bag made of polyester nonwoven fabric, and a granular zeolite having a particle size of about 1 mm (Shin-Koshikasei Co., Ltd.) It was put together with 2 g in a polyethylene pouch laminated with 5 cm × 7 cm moisture-proof aluminum foil, sealed, and stored at room temperature for 3 hours.

2-4: Fusing with a heating blade A brass block is processed to prepare 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. By applying a fusing head to the dried slide prepared in 2-3 and heating at 260 ° C. for 2 seconds, all of the development layer and the water absorption layer and a part of the PET support were fused, and four sections (No. .1 to 4).

2-5: Preparation of Measurement Reagent Solution 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

BUN measurement reagent Triton-X 100 (produced by Rohm and Haas) 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 and Haas) 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 Phosphoric acid / dipotassium 870.9 mg dissolved in distilled water 100 ml Into 50 ml of liquid, phosphoric acid / 1 potassium / hydrogen 680.5 mg was dissolved in distilled water 100 ml Liquid adjusted to pH 7.5 by adding liquid.

Measurement reagent for UA Uricase 145U Peroxidase 6794U Triton X-100 (manufactured by Rohm and Haas) 0.5 g 2- (3,5-dimethoxy-4-hydroxyphenyl) -4- [4- (dimethylamino) phenyl] -5-Phenethylimidazole 30mg Boric acid 0.15M 10ml

2-6: Spotting and measurement of measuring reagent Section No. 1, 2 of the 4-fractionation slide prepared in 2-4,
3 and 4, GLU, BUN, adjusted in 2-5,
5 μl of the measurement reagent solution of TCHO 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. Incubate the slides that have developed the color reaction for 6 minutes at 37 ° C, and measure the reflection optical density at the wavelength corresponding to each color development using the Fuji Drychem 5500 Analyzer with the beam diameter adjusted to 4 mm by adjusting the photometric beam head. did. The concentration of each component was calculated using the relationship between the concentration of each component and the reflection optical density as a calibration curve. GLU = 103 mg / dL, BUN
= 21 mg / dL, TCHO = 157 mg / dL, UA = 4.6 mg / dL. Centrifuge a portion of the same untreated whole blood to obtain a Hitachi 71
When the concentration of each component was measured using 50, the result is GL
U = 101 mg / dL, BUN = 20 mg / dL, TCHO = 154 mg /
dL, UA = 4.3 mg / dL, and it was confirmed that the values obtained by the method of the present invention had the accuracy for practical use.

2-7: Repeatability The same operation as above was repeated 10 times to examine the repeatability. The coefficient of variation CV (%) is GLU = 4.
2, BUN = 5.1, TCHO = 3.7, UA = 2.8, which proved to be a sufficiently precise measurement method.

Example 2 The same method as in Example 1 was repeated except that the slide was blown by the following method using ultrasonic waves, and the same good result was obtained. Fusing blade Aluminum head with the same shape as in Example 1 Ultrasonic oscillator device BRANSON Ultrasonic oscillator device 8700 WELD TIME Set to 0.125 seconds Hold TIME set to 0.2 seconds

Example 3 1. Preparation of Multilayer Analysis Slide 1-1: Preparation of Multilayer Film A water absorbing layer having the following formulation was provided on the same support as in Example 1. (Dry weight) Polyvinyl alcohol PVA KL506 23.8 g / m ² (manufactured by Kuraray Co., Ltd.) Epoxy-based cross-linking agent Araldite DY022 0.8 g / m ² (manufactured by Ciba-Geigy) Surfactant Surfactant 10G 2.0 g / m ² (manufactured by Ohrin) ) Next, 2% of a surfactant (Surfactant 10G manufactured by Ohrin Co., Ltd.) was uniformly applied so as to have a coating amount of 20 ml / m ² , and the surface of the water absorbing layer was swollen to make the surface hydrophilic. A 150 μm thick polyester plain weave (manufactured by Kuraray Co., Ltd.) was uniformly laminated and adhered, and dried. Furthermore,
24g hydroxypropyl cellulose (manufactured by Shin-Etsu Chemical) and 4g nonionic surfactant (HS24 manufactured by NOF CORPORATION)
1600 g of an aqueous solution containing 0) was prepared and uniformly coated on a plain fabric with a coating amount of 250 g / m ² and dried to form a multilayer film.
completed.

1-2: Preparation of Multi-layer Analytical Slide The multi-layer film produced in 1-1 was incorporated into a plastic frame in the same manner as in 1-4 of Example 1 to complete an analytical slide.

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: Specimen spotting The plasma component obtained by centrifugation on the multilayer analysis slide described in 1-2, and 2-1 on the other slides described above.
20 .mu.l of each of the plasma components prepared in 1. was spotted.

2-3: Dehydration and Drying The analytical slide obtained in 2-2 was dehydrated and dried using the constant temperature dryer described in Japanese Utility Model Laid-Open No. 3-126499 under the conditions described therein.

2-4: Fusing by heating blade In the same manner as 2-4 of Example 1, four sections (No. 1 to No. 1) were used.
It was divided into 4).

2-5: 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 (Rohm and Haas) 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

2-6: Spotting and measurement of measuring reagent No. 1 to No. 4 of the four-fractionated analytical elements obtained in 2-4
Then, 5 μl of each of the measurement reagent solutions for TP, Alb, GOT, and GGT was spotted, and then incubated and photometrically 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.

[0132]

[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.

[0135]

[Table 2]

Example 4 Using an analytical slide similar to that made in Example 3,
The following experiments and evaluations were conducted. 10 μl of plasma was spotted on the plasma receiving element, immediately sealed with a desiccant (2 g of zeolite) in the same manner as in Example 1, and left at room temperature (about 20 ° C.). After standing for a certain period of time, the amount of water in the plasma receiving element was measured using gas chromatography, and at the same time, the Glu concentration and GOT activity were measured in the same manner as in Examples 1 and 3. The results are shown in Table 3.

[0137]

[Table 3]

In any of the cases, it was found that the analysis result with excellent stability was obtained after 1 hour of standing and for 7 days.

[0139]

According to the analysis method of the present invention, many items can be easily measured using a very small amount of a liquid sample, especially a body fluid sample. In addition, the analytical element on which these samples are spotted can be reliably stored and transferred, and it is possible to measure many items with sufficient analytical accuracy.

[Brief description of drawings]

FIG. 1 is a diagram illustrating an embodiment of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Masashi Ogawa 3-11-46 Izumisui, Asaka-shi, Saitama Fuji Shashin Film Co., Ltd.

Claims (2)

[Claims] 1. A sample is spotted on a multi-layer analytical element containing no measurement reagent, in which at least a hydrophilic polymer layer and a porous development layer are laminated in this order on a water-impermeable support,
After drying, at least the porous development layer is fractionated, and then each of the fractionated portions is spotted with a measurement reagent that causes a different detection reaction, and the measurement is performed. 2. The multi-item analysis method according to claim 1, wherein the porous spreading layer is made of a thermoplastic resin, and the fraction is heated to a temperature equal to or higher than the softening point of the thermoplastic resin and melted.