JPH06217793A - Method of measuring enzyme activity - Google Patents

Abstract

PURPOSE:To obtain a method of measurement capable of surely preserving a very small amount of an enzyme-containing liquid specimen, especially analyzing factors of humor specimen, optionally transporting and then measuring enzyme activity in sufficient accuracy. CONSTITUTION:A specimen containing an enzyme to be examined is supplied to an element for receiving the enzyme to be examined, not containing a measuring reagent to be directly reacted with the enzyme to be examined and the specimen and a desiccant are put in a container substantially not to permeate water vapor. >=90% water in the specimen is removed at <=50 deg.C, the measuring reagent to cause detection reaction is deposited on the element for receiving the enzyme to be examined and activity of the enzyme to be examined is measured. Or, the specimen is extracted from the element for receiving the enzyme to be examined, the measuring reagent to cause detection reaction is added to the extracted solution and activity of the enzyme to be examined is measured to give the method of measuring enzyme activity.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring an enzyme activity, which is effective in the field of clinical analysis and the like when a small amount of a sample containing an enzyme is collected and then analyzed.

[0002]

2. Description of the Related Art A method for diagnosing a human disease using blood, urine or the like as a sample is widely used. 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. Especially when the number of samples to be measured is large, dissolve an appropriate amount of reagent,
Since it can be prepared, the reagent cost per measurement can be reduced, which is economically advantageous. It is complicated and troublesome to combine a large amount of solution with the amount and time of addition, but since the development of clinical testing equipment has a long history and social demands were high, it is already large, medium, and large.
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. Photo film
Co., Ltd.), Ektachem (Eastman Kodak Company, USA), Dry Lab (Konica Corporation), Spotchem (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 the sample, but the measurable items are gradually increasing, and at least Analytical elements have been developed for more than 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]

The method of measuring enzyme activity using the above-mentioned wet chemistry analysis method or dry chemistry analysis element is widely known. However, a common drawback of both methods is the problem of sample preparation and supply. There is.

The amount of enzyme present is measured by its activity value. However, the enzyme is unstable and easily denatured, its activity is easily lowered by temperature and pH changes, and the enzyme solution is inactivated in the order of several hours at room temperature. The most reliable method of storing the enzyme stably for a long time is to dehydrate and dry it and store it at low temperature.

The enzyme in human plasma / serum is also unstable, and its activity decreases with the storage time. However, a freeze-dried preparation of human serum is widely commercialized as a control serum. These are shipped in a state of being dehydrated under reduced pressure and sealed in a glass bottle, and if they are refrigerated and stored, the storage period is extremely long, from one to several years. However, it is almost impossible to process and store samples such as whole blood collected from individual patients by such a method.

Further, since the wet chemistry method is premised on the permeation measurement of a transparent solution, the whole blood sample cannot be directly used as the 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.

[0015] 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, but for a person who has a constitutionally thin blood vessel and has difficulty in collecting blood. 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 serum is sedimented and separated as it is or in a state of being refrigerated and then subjected to 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 the centrifugation be performed immediately after the 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, but this can be measured in a test center that requires time to transfer the sample even if it is possible in a hospital test. If you do, it is totally different, 1
The reality is that they are often separated after more than a day.

On the other hand, since the dry chemistry analysis element contains all reagents, the reaction proceeds immediately after spotting the sample. It is well suited for immediate measurement at the blood sampling site, such as in an emergency test, but when it takes time from sample collection to analysis due to the difference between the sample collection location and the analysis location, the liquid should be maintained without degeneration. It means that it must be stored as it is, and the same problem as in the solution method described above occurs.

Further, in this method, although the measuring operation is simple, all the reagents necessary for the reaction must be incorporated into the element, and the characteristics of the reagents used differ one by one depending on the item to be analyzed. There is a big problem that it takes a lot of labor, time and equipment to optimize development and manufacturing conditions.

The object of the present invention is to be able to reliably store the analytical element after the supply of a small amount of a liquid sample containing an enzyme, particularly a body fluid sample, and to transfer it as necessary.
An object of the present invention is to provide a measuring method capable of measuring the enzyme activity with sufficient accuracy thereafter.

[0022]

The above object of the present invention is to supply a test sample containing a test enzyme to a test enzyme receiving element which does not contain a measurement reagent which directly reacts with the test enzyme, and which is substantially combined with a desiccant. Place in a container that is impermeable to water vapor, remove 90% or more of the water in the sample at a temperature of 50 ° C or lower, and then deposit a measurement reagent that causes a detection reaction on the enzyme-receiving element to be tested. A method for measuring enzyme activity, which comprises measuring the activity of the enzyme, or supplying a sample containing the test enzyme to a test enzyme receiving element that does not contain a measurement reagent that directly reacts with the test enzyme, and then supplies it with a desiccant. Put it in a container that is substantially impermeable to water vapor, remove 90% or more of the water content in the sample at a temperature of 50 ° C. or lower, and then extract the sample from the enzyme-receiving element to be tested, The activity of the test enzyme can be measured by adding a measurement reagent that causes a detection reaction. And a method for measuring enzyme activity, which comprises:

After the filter paper is soaked with body fluid, it is dried once,
A measurement method for detecting occult blood and metabolites such as amino acids in body fluids after detection is widely used. For example, as a screening detection for colorectal cancer or the like, a method in which a part of feces is soaked in filter paper, dried, and then transferred to a laboratory to measure hemoglobin (Hb) contained in the feces, a screening test for inborn errors of metabolism in newborns As a method, urine and blood of newborn baby are soaked in filter paper, left to stand and dried, then transferred to a laboratory, where a certain area is punched out and immersed in an extract to extract and measure the target component. , And the like.

These measurement targets are cancer markers such as α-fetoprotein (AFP), or metabolites such as phenylketone and vanylmandelic acid, which are indicators of inborn errors of metabolism, and their abundance is very small. , Itself is stable and does not denature or lose activity due to standing drying or extraction operation. Instead of extraction, a method is also known in which a coloring reagent solution is soaked in a dry filter paper and the degree of coloring is examined, but these direct methods have poor reproducibility of analysis, reliability, and qualitative or semi-quantitative level. The reality is that it remains.

On the other hand, although the enzyme is unstable as described above, the present invention spots a sample containing the enzyme,
The method enables quantitative measurement even after several days to several weeks, and is a method that has not been considered in the past. The present invention will be described in detail below.

First of the enzyme-accepting elements to be tested used in the present invention
The embodiment 1 is a microporous sheet made of a material having a function of expanding a component contained in an aqueous sample in a planar manner without substantially unevenly distributing the component.

As a concrete material, it is possible to appropriately select and use from the well-known non-fibrous and fibrous microporous materials which have been used in the spreading layer of the dry chemistry analysis element. 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. A non-fibrous microporous 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-57-66359. Etc., a microporous layer made of a woven fabric, a layer made of a knitted fabric disclosed in the same 60-222769, various filter papers, a microporous membrane of a fluorine-containing polymer whose surface is hydrophilized as described below, etc. However, the present invention is not limited to these.

The thickness of the microporous sheet varies depending on the use mode, but is about 20 μm to about 500 μm when dried, preferably about 50 μm to about 400 μm, particularly preferably about 80 μm to about 300 μm.
It is preferably μm and has a substantially uniform structure. The microporous sheet may contain various reagents or a part of the reagents for accelerating a desired reaction or preventing or reducing an interference or interfering reaction.

The second aspect is a sheet in which a hydrophilic polymer layer is provided on a water-impermeable support. For the hydrophilic polymer layer, various known water-soluble, swellable and hydrophilic polymers that have been used in dry chemistry analysis elements can be used. Swelling rate when absorbing water is about 30 ℃
A natural or synthetic hydrophilic polymer in the range of 150% to about 2000%, preferably about 250% to about 1500% can be used. Specifically, it is described in JP-A-59-171864, JP-A-60-108753 and the like. Disclosed gelatin (eg, acid-treated gelatin, deionized gelatin, etc.), gelatin derivative (eg, phthalated gelatin, hydroxyacrylate graft gelatin, etc.), agarose, pullulan, pullulan derivative, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, poly Examples thereof include, but are not limited to, hydroxyethyl 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 or opaque 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.

When whole blood is used as a sample, as a third mode, a mode in which a blood cell separating element is provided on a test enzyme receiving element composed of a microporous sheet or a hydrophilic polymer layer (hereinafter referred to as sample receiving Element) is preferred. Examples of the blood cell separating element include fibrous microporous layers and non-fibrous microporous layers described in JP-A-62-138756-8, JP-A-2-105043 and JP-A-3-16651. It is possible to use a layer in which the layers are adhered (partially adhered) together 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.
Of these, particularly preferred are blood cell separations in which a fibrous microporous layer is placed on the blood spotting side and a non-fibrous microporous layer is placed on the plasma receiving layer side, and both are integrated by partial adhesion. Is an element.

The non-fibrous microporous layer in the blood cell separation element in which the fibrous microporous layer and the non-fibrous microporous layer are integrally bonded (partially adhered) with an adhesive that is partially disposed (Japanese Patent Publication) A layer of a brush polymer composed of cellulose esters described in 53-21677, U.S. Pat. No. 1,421,341 and the like, for example, cellulose acetate, cellulose acetate / butyrate, cellulose nitrate is preferable. It may be a fine microporous membrane having a hydrophilic surface such as polyamide such as 6-nylon or 6,6-nylon, polyethylene, polypropylene, or Teflon. In addition, small polymer particles, glass particles, diatomaceous earth, etc. described in JP-B No. 53-21677, JP-A No. 55-90859 and the like having continuous voids bonded with a hydrophilic or non-water-absorbing polymer. Porous layers can also be used.

The effective pore size of the non-fiber microporous layer is 0.8 to 30 μm.
m, particularly preferably 0.5 to 5 μm. In the present invention, the effective pore diameter of the non-fiber microporous layer is shown by the pore diameter measured by the limiting bubble pressure method (bubble point method) according to ASTM F316-70. When the non-fiber microporous layer is a membrane filter made of so-called brush polymer made by the phase separation method, the liquid passage path in the thickness direction is on the free surface side (that is, glossy surface) during the production of the membrane. It is usually the narrowest, and the diameter hole when the cross section of the liquid passage path is approximated to a circle is the smallest near the free surface. The minimum pore size in the thickness direction in the passage path of the 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 microporous layer of the analytical element of the present invention, it is preferred to orient 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 microporous layer, filter paper, non-woven fabric, woven fabric (for example, plain woven fabric), knitted fabric (for example, tricot fabric), 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 microporous 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 microporous layer may be the same as or different from that of the non-fibrous microporous layer. In order to adjust the relationship between the void volume of the both, the void ratio or the thickness of the both may be changed, or both the thickness and the void ratio may be changed.

The microporous layer made of a fluorine-containing polymer, polysulfone, etc., whose surface has been hydrophilized, does not hemolyze 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 alone as a material for the blood cell separating element without combining with the fibrous microporous membrane.

Although the blood cell / plasma separation mechanism is not clear, this microporous layer does not trap blood cells only on its surface, and a microporous layer made of a fluorine-containing polymer and a microporous spreading layer are combined. It seems to be due to the so-called volumetric filtration action, in which as it penetrates in the thickness direction, it initially becomes a large blood cell component, then gradually becomes a small blood cell component and becomes a void structure, and retains and removes blood cells 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 described 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.

Examples of the microporous layer of these fluorine-containing polymers include polytetrafluoroethylene fibrils (fine fibers) described in JP-A-63-501594 (WO 87/02267).
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.

Among these microporous membranes of fluorine-containing polymer, those particularly suitable for the microporous layer constituting the blood cell separating element are those having 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 an analytical element, the aqueous liquid sample is repelled and does not diffuse or permeate on the surface or inside of the membrane. This is a well-known fact.

As means for imparting hydrophilicity to the microporous film of a fluorine-containing polymer to enhance hydrophilicity, an amount sufficient to substantially hydrophilize the outer surface and the surface of internal voids of the microporous film is used. Known methods include a method of impregnating a microporous film of a fluorine-containing polymer with a surfactant and a method of chemically introducing a carbonyl group or a hydroxyl group into the structure of the fluororesin film.

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 add 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 generally 0.05 g / m ² from 2.5 g / m ²
It is preferably in the range of. As a method of impregnating a microporous membrane of a fluorine-containing polymer with a surfactant, an organic solvent (eg, alcohol, ester, ketone) having a low boiling point of the surfactant (boiling point is preferably in the range of about 50 ° C to about 120 ° C) is used. )
After soaking the microporous membrane of the fluorine-containing polymer in the solution and allowing the solution to penetrate the internal voids of the microporous membrane substantially sufficiently, the microporous membrane is gently pulled up from the solution and blown (warm air The preferred method is to feed (dry) and dry. A surfactant may be contained in the microporous membrane of the fluorine-containing polymer together with the components such as the pretreatment reagent contained in the microporous layer constituting the blood cell filtration layer.

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

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

The following are specific examples of the nonionic surfactant. 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 membrane may be hydrophilized by providing one or more water-insoluble water-soluble polymers in the microporous 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 layer.

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, but by combining the above-mentioned fibrous microporous layer, blood cell separation can be achieved. Noh can be further enhanced.

Since the first to third aspects have a simple structure, they are advantageous for semi-quantitative or qualitative colorimetric measurement, assay methods for extracting a sample from an enzyme-receiving element to be tested, and the like. A fourth preferred embodiment is a test enzyme-receiving element in which at least a hydrophilic polymer layer and a microporous spreading layer are laminated on a water-impermeable support. The analyte receiving element of the fifth aspect in which a blood cell separation element is provided on the microporous spreading layer is also preferable.

Among these embodiments, at least a hydrophilic polymer layer, a microporous spreading layer is laminated on a water-impermeable support, or a microporous spreading layer is further laminated. The fifth aspect in which the blood cell separation elements are laminated is preferable.

The water-impermeable support, the hydrophilic polymer layer, the microporous spreading layer, and the blood cell separating element are respectively the above-mentioned water-impermeable support, hydrophilic polymer layer, microporous sheet, and blood cell separating element. Can be used. However, the microporous spreading layer does not have to be limited to one layer, and is disclosed in
As disclosed in -4959, 62-138756, 62-135757, 62-138758, etc., 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. 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.

In the fifth embodiment, the microporous spreading layer may be simply laminated on the water-impermeable support depending on the analysis item or the like. However, between the microporous spreading layer and the support. By providing a hydrophilic polymer layer selected from the materials described above, the universality of analysis items and the like can be dramatically improved.

Here, the partial adhesion (porous adhesion) used for the above-mentioned blood cell separating element and the like will be described. Partial adhesion refers to JP 61-4959 (EP0166365A) and JP 62-138.
756 to 138758 (EP0226465A) and the like, which is a mode of adhesion between two adjacent microporous layers or between adjacent microporous layers and a non-porous layer, wherein "between the interfaces of two adjacent layers" Configured so as to be substantially adhered and integrated by an adhesive that is partially (or intermittently) arranged on the above-mentioned two surfaces, and that the uniform passage of liquid is not substantially obstructed between the adjacent two surfaces and between them. Adhesion is done.

In the analytical element used in the present invention, between the fibrous microporous layer and the non-fibrous microporous layer in the blood cell separating element, a microporous layer having a blood cell separating ability and a fibrous microporous layer are provided. It is preferable that both the space and the blood cell separation element and the microporous spreading layer are partially bonded (porous bonded). For example, in the case of bonding between the microporous spreading layer and the blood cell separating element, an adhesive is partially arranged on the microporous spreading layer, and then the blood cell separating element is bonded while applying light pressure evenly. Is the general method. On the contrary, it is also possible to partially dispose the adhesive on the blood cell separating element and then attach the microporous spreading layer while evenly applying light pressure. Further, an adhesive is partially arranged on the microporous sheet material to be the microporous spreading layer, and the blood cell separating element is bonded to the blood cell separating element while applying light pressure evenly, or vice versa. A microporous sheet in which the agent is partially arranged, and then the microporous sheet-like material to be used as the microporous spreading layer is bonded to the hydrophilic polymer layer while applying light pressure uniformly, and then used as the spreading layer in the hydrophilic polymer layer. It is also possible to bond the shaped objects uniformly.

The method of partially disposing the adhesive on the blood cell separating element or the microporous spreading layer is disclosed in JP-A-64-1959 and JP-A-62-1959.
138756 and 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 microporous spreading layer or the blood cell separating element by using a printing plate (gravure printing plate or intaglio plate is preferable) roller and the method of bonding two adjacent layers are For example, it can be carried out by a known device and method described in “Printing Engineering Handbook” edited by Japan Printing Association (Gihodo Publishing Co., Ltd., 1983), pages 839 to 853, and the like.

An adhesive used is disclosed in Japanese Patent Laid-Open No. 62-1387.
The various adhesives described in 56 and other known adhesives described in the above-mentioned "Printing Engineering Handbook", pages 839 to 853, etc. can be used. As the adhesive, a water solvent type adhesive, an organic solvent type adhesive, or a heat-adhesive (or heat-sensitive) adhesive can be used. Examples of water-solvent type adhesives include 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.

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 need to be adhered to each other so that the diffusion and permeation of the sample can be quantitatively promoted. It may be laminated, but partial adhesion is preferable to ensure uniform diffusion.

In the measuring method of the present invention, first, a sample is spotted on a test enzyme-receiving element containing no measuring reagent to sufficiently diffuse and spread the sample, and when there is a blood cell separating element, it is preferably peeled off. Dry the enzyme-receiving element under test.

The drying is carried out by removing 90% or more of the water content of the sample supplied to the enzyme-receiving element to be tested at a temperature of 50 ° C. or less in a container in which a desiccant is present and which is substantially impermeable to water vapor. Is preferred. That is, when the sample spotted on the test enzyme receiving element is plasma, the solid content is about 8%. Plasma 10
There is about 9.2 mg of water in μl, but about 8.3 mg of this is removed by drying. As a specific drying method, the test enzyme-receiving element is enclosed with a desiccant in a plastic bag with a fastener, a plastic container with a lid, or the like. Preferred plastics are polypropylene, polycarbonate, polyethylene terephthalate, polystyrene, polyvinylidene chloride and the like, which have low moisture permeability. However, even those having relatively high moisture permeability such as polyethylene can be used if the thickness is increased. Other examples include laminates of these with aluminum foil, metal cans, glass bottles, and the like.

The desiccant may be appropriately selected from among known hygroscopic agents without substantially altering the enzyme to be tested and used, but zeolite, silica gel,
It is preferable to use activated alumina, activated carbon, calcium chloride, etc., alone or with the addition of a molding aid, to mold into powder, granules, granules or sheets. A synthetic paper, a non-woven fabric, or a moisture-permeable film is used as a packaging material, and a fixed amount of about 0.5 to 5 g is used in a packaged form. The water absorption capacity of the desiccant is about 30% of the weight of ordinary granular silica gel, about 20% of zeolite, and 60% or more of calcium chloride.
When the plasma sample is 10 μl, the amount is about 9.2 mg as described above, and when zeolite is used, it is about 1 slide per slide.
The lower limit is 50 mg. However, considering the drying speed, it is preferable to encapsulate 3 to 30 times, preferably 7 to 30 times as much as this. The upper limit is not so important. Also,
In addition to dehydration, it is also effective to coexist with enzymes and reagents that contribute to the stabilization of the sample itself, such as oxygen scavengers and preservatives.

In the method of the present invention, the temperature until 90% of the water content of the enzyme-receiving element to be tested is removed can be appropriately selected within the range where the enzyme activity does not decrease. However, at a slightly high temperature such as 60 ° C., the vapor pressure of water is high, so this value is reached in a short time, but during this time, a significant decrease in enzyme activity occurs,
Also, there is a tendency for variations to increase, so it is usually about 50 ° C.
The following is selected, preferably 35 ° C or lower, and more preferably 25 ° C or lower. However, even if the initial value decreases during this drying,
Since storage stability improves when stored at low temperature, it is possible to correct by the calibration curve by keeping the processing conditions always constant and creating the calibration curve in that state, and it is possible to dry at higher temperature. Become.

Further, at a low temperature such as 4 ° C., the drying time becomes long. Therefore, it is preferable to appropriately select the nature and amount of the desiccant used according to the drying temperature. That is, a desiccant having a high water absorption rate at a high temperature such as silica gel and a desiccant having a high water absorption rate at a low temperature such as zeolite are selected. The required amount cannot be uniquely determined because it varies depending on the spotted amount of the sample, but the type and amount can be determined by those skilled in the art using a catalog of commercially available products. Further, even at a temperature of 0 ° C. or lower such as in a freezer, dehydration drying is possible by using a desiccant having a large drying ability. However, the lower the temperature, the lower the water vapor pressure, and the longer the time required for drying.

The stability of a sample from which 90% or more of water has been removed increases, but the storage temperature of the enzyme-accepting element to be tested affects the stability of the enzyme. When a sample containing an enzyme is stored at a high temperature of 60 ° C or higher for a long time, the unstable activity of the enzyme is markedly reduced.
Stable for over a week. Of course, there is no problem in raising the temperature above 60 ° C for a short time. In the case of GGT, which has a high stability among enzymes, it can be stored at a higher temperature. However, measurement of unstable enzymes such as GPT and GOT is very important in the field of clinical testing, and a method that enables these measurements is desirable.

From this, it is preferable that the temperature at the time of drying is in the above range, and as the desiccant, it is preferable to use zeolite having a high water absorption rate under the condition of low water vapor pressure. Zeolites are crystalline hydrous aluminosilicates of alkali metals or alkaline earth metals, and are MeO.Al ₂ O ₃
A compound represented by mSiO ₂ .nH ₂ O (Me: metal ion, m.n is a positive integer). For example, 1 to 4 test enzyme-receiving elements were placed 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.
A method of putting it in a closed container together with 1 to 2 g of 4 to 20 mesh zeolite can be mentioned.

Next, the enzyme-receiving element to be tested is taken out from the closed container, and a measurement reagent solution known in the wet chemistry field corresponding to the item to be analyzed is supplied. These react with the component to be analyzed (test enzyme) and can be detected mainly by optical measurement methods, for example, color change, color development (coloring), fluorescence, luminescence, change in absorption wavelength in the ultraviolet region, generation of turbidity, etc. Therefore, the activity of the enzyme contained in the sample is quantified by a method known in the field of dry chemistry (measurement of transmission or reflection absorbance, color change, fluorescence measurement, luminescence measurement, etc.).

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 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. Furthermore, it is also possible to cut out a certain area of the enzyme-receiving element to be tested and measure it by a known extraction method.

It is also possible to measure a plurality of items using one sheet of the enzyme-receiving element to be tested. In this case, the stabilized test enzyme-receiving element is taken out of the closed container and fractionated into a plurality of partial elements. 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 performed by appropriately selecting the following method. 1. At least the microporous spreading 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 is to cause thermal fusion to destroy the porous structure and prevent the measurement reagent from spreading beyond the fusing groove in at least the microporous spreading layer. Therefore, this thermal fusing groove divides one test enzyme-accepting element into a plurality of compartments, and generally has substantially the same shape.
To about 16 equal parts, preferably about 2 to 9 equal parts, and particularly preferably about 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. Attach to an ultrasonic oscillator to raise the temperature of the thermoplastic resin by vibrating energy,
Etc. 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, the measurement reagent spreads through the hydrophilic polymer layer only by thermal fusing of the microporous spreading layer,
Measurement accuracy may be reduced. At this time, by raising the temperature to the softening point of the microporous spreading layer and the hydrophilic polymer or higher,
A fusing groove that extends from the microporous spreading layer to the support is provided.

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, paraffin 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. . What is important at this time is that these hydrophobic compounds do not merely hydrophobize only the surface of the spreading layer, but a sufficient amount penetrates in the thickness direction and the width direction. select. Width is 0.1
˜2 mm, preferably 0.5 to 1 mm.

Fractionation using a blade may be performed by a normal 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 include a fluorine-containing compound or the like as a microporous spreading layer.
It is especially useful when using materials that are not easily heat-fused.

Although the type of the test enzyme is not limited, glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), lactate dehydrogenase (LDH), alkaline phosphatase (AlP), acid phosphatase (ACP), gamma -Glutamyl transpeptidase (GGT), cholinesterase (Che), leucine aminopeptidase (LAP), creatine phosphokinase (CP)
K), lipase, creatine phosphokinase isozyme, amylase (AMy), ribonuclease (RN)
ase), esterase, aldolase (ALD), pyruvate kinase (PK), lysozyme, malate dehydrogenase, amylase isozyme, GOT isozyme,
Fibrinogen and the like can be mentioned.

In the present invention, the measuring reagent refers to a reagent that directly reacts with a target enzyme to be analyzed to cause a chemical change, that is, a substrate of the enzyme. For example, in the case of GOT, its substrates aspartic acid and α-ketoglutaric acid, high-molecular-weight starch or low-molecular-weight oligosaccharide for amylase, and L-γ for GGT.
-Glutamyl para-nitroanilide, para-nitrophenyl phosphate for ALP.

Various reagents are used for the purpose of adjusting pH and ionic strength, promoting diffusion and permeation into the material constituting the analytical element, and the like in order to proceed the reaction stably and with good reproducibility. Reagents 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.

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.

[0090]

【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 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 enzyme-receiving element to be tested. 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: Completion of multi-layer analysis slide A completed enzyme-receiving element was cut into a square chip having a side length of 15 mm, placed in an organic polymer slide frame described in JP-A-57-63452, and Fuji Drychem 5500. We have completed a multi-layer analysis slide with a shape that can be measured by an analyzer (Fuji Photo Film Co., Ltd.).

2. Measurement 2-1: Preparation of Sample A Fuji Dry Chem enzyme calibrator L (manufactured by Fuji Photo Film Co., Ltd.) was used.

2-2: Spotting and Drying of Specimen 10 μl of each of the above-mentioned specimens was spotted on the above-mentioned multilayer analysis slide, and immediately, a desiccant (2 g of granular zeolite of 12 to 20 mesh, non-woven fabric and polyethylene film) was applied. Encapsulated in a laminated pouch, Zeolum A manufactured by Shinkoshi Kasei Co., Ltd.
It was enclosed together with -4) in a moisture-proof bag (aluminum foil laminated on polypropylene), and left for a certain time in a 25 ° C. constant temperature air bath.

2-3: Measurement of enzyme activity The following measurement reagents were prepared, and GOT and GPT activities were measured at predetermined intervals using a Fuji Drychem 5500 analyzer (manufactured by Fuji Photo Film Co., Ltd.). In parallel with this, the water content in the plasma receptive element was measured by gas chromatography. The results are shown in Table 1. Here, the water content is a value when the water content without standing is 100.

GOT Tris (hydroxymethyl) aminomethane 84 mg Phosphate / 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 Oxaloacetate 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 6.6 ml

GPT Tris (hydroxymethyl) aminomethane 87 mg Phosphoric acid • 1 potassium • 2 hydrogen 103 mg L-alanine 620 mg α-ketoglutaric acid • 2Na 93 mg 20% MgCl ₂ 273 μl POD 343 IU thiamin pyrophosphate 21 mg flavin adenine dinucleotide 5 mg pyruvate oxidase 3088u Triton X-100 100 mg 2- (3,5-dimethoxy-4-hydroxyphenyl) -4- [4- (dimethylamino) phenyl] -5-phenethylimidazole 54 mg 1N NaOH 3.4 ml distilled water 6.6 ml (pH = 7.5)

[0097]

[Table 1]

From the above results, it can be seen that when about 90% of water is removed and then stored, the enzyme activity is maintained and a highly accurate measurement result can be obtained.

Example 2 / Comparative Example 1 Venous blood collected from a healthy subject was centrifuged to obtain a plasma sample. 10 μl of this sample was spotted on a required number of multi-layered analysis slides containing no reagent prepared in the same manner as in Example 1, and immediately put in a small bag containing 2 g of granular zeolite (Shin-Koshikasei Co., Ltd.).
It was placed in a moisture-proof bag laminated with polyethylene and aluminum foil of 5 × 8 cm, which can be hermetically sealed with a fastener at the entrance, and left at room temperature (20 ± 2 ° C.) for 3 hours. After 3 hours, put each moisture-proof bag in the refrigerator (4 ℃),
It was stored in an air thermostat at 25 ° C and 45 ° C. The moisture-proof bag was taken out at regular intervals, and the GOT and GPT activity values were measured in the same manner as in Example 1 using the inner slide. The measurement was performed three times under the same conditions, and the average value was used as the measurement value. As a comparative example, after the specimen was spotted on the slide, the slide was left as it was for 3 hours at room temperature (20 ± 2 ° C.), and then the same measurement was carried out for a sample enclosed in a moisture-proof bag containing a desiccant. The results are shown in Table 2.

[0100]

[Table 2]

Further, when the above sample was measured using an automatic clinical chemistry analyzer, Hitachi 7150 (manufactured by Hitachi Ltd.), GO
T = 19u / L and GPT = 16u / L. From these results, it is understood that accurate measurement is possible even after 3 days when dried with a desiccant.

Example 3 Another plasma sample prepared in the same manner as in Example 2 was spotted on the slides of the same number as in Example 2 in an amount of 10 μl each. These slides were sealed in a bag in the same manner as in Example 2 and left at the predetermined temperature shown in Table 3 for 3 hours to dry the slides. Then, it was left to stand at each temperature shown in Table 3 for 24 hours. After 24 hours, the bag was taken out, and the activity of GOT and GPT was measured in the same manner as in Example 2 using the inner slide. The results are shown in Table 3.

[0103]

[Table 3]

From this result, 24
It turns out that measurement is possible after time.

Example 4 The same experiment as in Example 2 was repeatedly measured for 10 days using control sera having different concentrations, and the daily variation was examined. The results are shown in Table 4.

[0106]

[Table 4]

From the above results, it can be seen that the repeatability of the method of the present invention is high.

Example 5 A GGT was measured using a slide treated in the same manner as in Example 3 (however, the standing temperature was also examined at a higher temperature) and the measuring reagent having the following formulation. The results (activity value of GGT: u / L) are shown in Table 5.

Formulation of GGT measuring reagent L-α-glutamyl-3-carboxy-paranitroaniline 58.5 mg glycylglycine 156 mg 2N Tris-hydrochloride buffer (pH 8.1) 10 ml

[0110]

[Table 5]

From these results, it can be seen that in the case of a highly stable enzyme such as GGT, highly accurate measurement results can be obtained regardless of the drying temperature and the standing temperature.

Example 6 A blood cell separating element having the following constitution was provided on the plasma receiving element having the constitution of Example 1. 36 PET spun yarn equivalent to 50 denier
Gauge knitted tricot knitted fabric (thickness about 250 μm)
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 adhesive (H950 manufactured by Nitta Gelatin Co., Ltd.) heated to 130 ° C. and melted was attached 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 hot fabric immediately after the adhesive was transferred, the effective pore diameter was 3.0 μm, the thickness was 140 μm, and the porosity was about 80.
% Cellulose acetate membrane filter with the non-glossy sides facing each other and passing between the laminating rollers,
Both were laminated and bonded together (partially bonded) to prepare a blood cell separating element. This blood cell separating element was adhered to and integrated with the main plasma element by the partial adhesion method by the same gravure printing method as described above. Completed multi-layer analysis element 15 mm on a side
The sample was cut into square chips and placed in an organic polymer slide frame described in JP-A-57-63452 to complete a multilayer analysis slide.

50 μl of venous blood collected from a healthy person was spotted on the above-mentioned multilayer analysis slide, and 30 seconds later, the blood cell separation element was peeled off using tweezers. Using the test enzyme-accepting element thus obtained, the same measurement as in Example 2 was carried out.
The same stable measurement result as in Example 2 was obtained.

Example 7 A multi-layer analytical slide prepared in the same manner as in Example 1 was spotted with Fuji Dry Chem Calibrator M as a sample.
After performing the same drying treatment as in Example 2, it was aged at a predetermined temperature. After a predetermined time, the test enzyme receiving element was taken out of the plastic frame and cut into a piece having a width of about 5 mm. This cut piece was put into a plastic sample tube (made by Nipro Co., Ltd.) with a volume of 2 ml, 300 μl of distilled water was added, and a rolling mixer (made by Thermal Science Industry Co., Ltd.) was used for 10 minutes at room temperature. The soluble components were extracted by stirring. A measurement reagent was added to the extract, and measurement was performed using a clinical chemistry automatic analyzer Hitachi 7150. Since the dilution ratio with the extract was 30 times, the enzyme activity value in the sample was obtained by multiplying the measured value by 30 times. The results are shown in Table 6.

[0115]

[Table 6]

The values obtained by directly diluting the same sample by 30 times were GOT = 380 u / L and GPT = 432 u / L, and were extracted in the same manner as when the test reagent was directly spotted on the enzyme-receiving element. It was confirmed that the method can also perform highly quantitative measurement.

Example 8 A central portion of a test enzyme-accepting element that had been aged under the same conditions as in Example 7 was punched out into a 10 mm disc, which was then extracted from the disc in the same manner as in Example 7, using Hitachi 7150. 1 measurement daily
As a result of examining the daily variations, the results were good as shown in Table 7.

[0118]

[Table 7]

[0119]

INDUSTRIAL APPLICABILITY According to the analysis method of the present invention, a trace amount of a liquid sample, particularly a body fluid sample containing an enzyme can be used to stably store a multi-layered analytical slide supplied with the sample for a long period of time. The enzyme activity can be measured with high analysis accuracy.

Claims (2)

[Claims] 1. A sample containing a test enzyme is supplied to a test enzyme receiving element that does not contain a measurement reagent that directly reacts with the test enzyme,
Put it in a container that is substantially impermeable to water vapor together with a desiccant, remove 90% or more of the water content in the sample at a temperature of 50 ° C. or lower, and then use a measurement reagent that causes a detection reaction in the enzyme-receiving element to be tested. A method for measuring enzyme activity, which comprises spotting and measuring the activity of a test enzyme. 2. A sample containing a test enzyme is supplied to a test enzyme receiving element that does not contain a measurement reagent that directly reacts with the test enzyme,
It is placed in a container that is substantially impermeable to water vapor together with a desiccant, and at a temperature of 50 ° C. or lower, after removing 90% or more of the water content in the sample, the sample is extracted from the enzyme-receiving element to be tested, A method for measuring enzyme activity, which comprises adding a measurement reagent that causes a detection reaction to an extract and measuring the activity of a test enzyme.