JP2006087356A - Dry analytical element - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a dry analytical element having excellent storage stability. <P>SOLUTION: In the dry analytical element in which an analyte or a substance derived from an analyte in an organism sample is treated with a conjugated enzyme to form hydrogen peroxide, a color-developing substance is colored by using the hydrogen peroxide to qualitatively or quantitatively analyze the analyte, a coloring layer containing the color-developing substance and a reagent layer containing the conjugated enzyme are adjacently arranged on a substrate and a mediator participating in color development of the color-developing substance is not contained in the dry analytical element. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a dry analytical element for qualitatively or quantitatively analyzing an analyte in a biological sample.

  In clinical tests, it is known to use a dry analytical element to quantify an analyte (analysis target component) contained in a biological sample such as blood. For example, Patent Document 1 discloses that a layer containing a color reagent composition capable of quantifying an analyte in an aqueous liquid sample by quantifying hydrogen peroxide generated in the presence of an oxidase is light-transmissive and water-impermeable. A dry multilayer analytical element having an adhesive support on top is disclosed. The dry multi-layer analytical element of Patent Document 1 includes an oxidase, a hydrogen peroxide-degrading enzyme, an alkali ferrocyanide, and a chromogen that act specifically on an analyte or a substance derived from the analyte to generate hydrogen peroxide. A color reagent composition as an essential component is held in a dry state on a layer on one side of the support.

FIG. 3 shows a cross-sectional view of the dry multilayer analytical element of Patent Document 1. The dry multilayer analytical element 500 shown in FIG. 3 is for quantifying cholesterol. The chromogen-containing layer 200 is formed on the light-transmissive water-impermeable support 100, and the ferrocyanide alkali and the oxidation are formed thereon. A light reflecting layer 400 containing fine titanium particles, and a reagent spreading layer 520 containing cholesterol esterase, hydrogen peroxide decomposing enzyme (peroxidase) and cholesterol oxidase are provided thereon.
In order to analyze, for example, high-density lipoprotein (HDL) -cholesterol in blood using this dry multilayer analysis element 500, first, a fractionation reagent is added to plasma or serum and centrifuged. Collect HDL fraction from Qing. Then, this specimen is spotted and supplied to the reagent developing layer 520, and the color density of the colored chromogen is detected by reflection photometry. The measurement principle is as follows. That is, serum containing cholesterol penetrates into the reagent spreading layer 520 with a spread approximately proportional to its volume. In the reagent spreading layer 520, cholesterol ester contained in lipoproteins in serum is hydrolyzed into cholesterol and fatty acids by cholesterose esterase. The produced cholesterol is oxidized by cholesterol oxidase present therein to produce hydrogen peroxide. When this hydrogen peroxide is converted into water by peroxidase, ferrocyan ion as a mediator is oxidized to produce ferricyan ion. The ferricyan ions reach the chromogen layer 200, oxidize the chromogen to a colored dye to form a dye, and the amount of the dye produced correlates with HDL-cholesterol in the serum.

JP-A 63-48457

Since the dry analytical element as described above is often stored for a long period of time, further improvement in storage stability is required. However, when HDL-cholesterol was measured using a conventional dry analytical element, it became clear by the present inventors that the measured value becomes lower than the actual value after long-term storage.
Accordingly, an object of the present invention is to provide a dry analytical element having excellent storage stability.

The present invention is as follows.
1. Qualitative or quantitative analysis of analytes in biological samples by causing a conjugate enzyme to act on analytes or substances derived from analytes to generate hydrogen peroxide and coloring the chromogenic substance using the hydrogen peroxide In the dry analytical element to be analyzed, on the support, a color developing layer containing the chromogenic substance and a reagent layer containing the conjugated enzyme are laminated adjacently in this order, and a mediator involved in the color development of the chromogenic substance Dry analytical element characterized by not containing.
2. 2. The dry analytical element according to 1 above, wherein the reagent layer contains at least peroxidase and cholesterol oxidase.
3. 3. The dry analytical element according to 1 or 2 above, wherein the analyte is HDL-cholesterol.
4). 4. The dry analytical element as described in any one of 1 to 3 above, wherein the mediator is ferrocyan ion.
5. 5. The dry analytical element as described in any one of 1 to 4 above, wherein the chromogenic substance is a leuco dye.

  According to the present invention, a dry process in which storage stability is greatly improved by removing a mediator typified by ferrocyanide from a dry analytical element and adjoining a layer containing a chromogenic substance and a layer containing a conjugate enzyme. Analytical elements are obtained.

In the dry analytical element of the present invention, a conjugate enzyme is allowed to act on an analyte in a biological sample or a substance derived from the analyte to generate hydrogen peroxide, and the chromogenic substance is colored by using hydrogen peroxide. As long as the reaction system can qualitatively or quantitatively analyze the analyte, the analyte, the conjugate enzyme, and the chromogenic substance are not particularly limited. In the present specification, the term “chromogenic substance” means a dye precursor that can absorb visible light by transferring electrons.
In the following, as a preferred embodiment of the present invention, cholesterol (particularly HDL-cholesterol (high density lipoprotein cholesterol)) is used as an analyte, cholesterol oxidase is used as a conjugate enzyme that generates hydrogen peroxide, and a leuco dye is used as a chromogenic substance. The form which was done is demonstrated.
FIG. 1 shows an example of a dry analysis element according to this embodiment. In the dry analytical element 10 shown in FIG. 1, a coloring layer 12 containing a leuco dye is provided on a support 11. Further, a reagent layer 13 containing peroxidase, cholesterol esterase, and cholesterol oxidase is provided adjacent to the coloring layer 12.

  FIG. 2 shows the principle of cholesterol measurement by the dry analytical element 10 according to the above embodiment. As shown in FIG. 2, lipoprotein containing HDL-cholesterol is separated into cholesterol + cholesterol ester + protein by a surfactant (reaction 2). Next, cholesterol esterase (CE) is allowed to act on the resulting cholesterol ester to separate it into fatty acid and free cholesterol (reaction 3). Free cholesterol produced by these treatments is brought into contact with cholesterol oxidase (CO) to produce hydrogen peroxide (oxidizing substance) (reaction 4). A peroxidase is allowed to act on the hydrogen peroxide, and at the same time, a chromogen such as a leuco dye is oxidized to a colored dye to form a dye (reaction 5). This coloring dye is qualitatively analyzed or quantified by a colorimetric method.

As described above, the dry analytical element 10 according to the present embodiment has the color forming layer 12 and the reagent layer 13 containing a conjugate enzyme arranged adjacent to each other, and does not use a mediator represented by ferrocyan ion. That is, when hydrogen peroxide is reduced by peroxidase and converted to water, the leuco dye is directly oxidized. Here, the “mediator” in this specification refers to a substance that plays a role as an electron carrier between hydrogen peroxide and the chromophoric substance, involved in the coloring of a chromophoric substance such as a leuco dye.
Thus, in the present invention, by using no mediator, a dry analytical element with significantly improved storage stability can be obtained. In the dry analytical element 10 according to this embodiment, the coloring layer 12 and the reagent layer 13 are laminated adjacent to each other in this order on the support 11, thereby preventing oxidation of the coloring substance and storage stability. Will improve.

Hereinafter, each layer constituting the dry analysis element 10 will be described.
The coloring layer 12 is preferably a porous layer made of a porous medium or a layer made of a hydrophilic polymer binder in order to increase water permeability, and a layer made of a hydrophilic polymer binder. More preferred.
When a porous layer is used as the color forming layer 12, the porous medium may be fibrous or non-fibrous. As the fibrous material, for example, filter paper, non-woven fabric, woven fabric (for example, plain woven fabric), knitted fabric (for example, tricot knitted fabric), glass fiber filter paper, and the like can be used. Examples of non-fibrous materials include membrane filters made of cellulose acetate and the like described in JP-A-49-53888, JP-A-49-53888, JP-A-55-90859, JP-A-58- Any of the continuous void-containing granular structure layers composed of inorganic or organic fine particles described in Japanese Patent No. 70163 may be used. Partial adhesion described in JP-A-61-4959, JP-A-62-116258, JP-A-62-138756, JP-A-62-138757, JP-A-62-138758, etc. A laminate of a plurality of porous layers made is also suitable.

  When the color developing layer 12 is formed of a porous layer, the thickness of the color developing layer 12 is not particularly limited, but when it is provided as a coating layer, it is preferably about 1 μm to 50 μm, and more preferably 2 μm to 30 μm. In the case of a method other than coating, such as lamination by lamination, the thickness can vary greatly in the range of several tens to several hundreds of μm.

  Examples of the hydrophilic polymer that can be used when the color forming layer 12 is formed of a water-permeable layer made of a hydrophilic polymer binder include the following. Gelatin and derivatives thereof (for example, phthalated gelatin), cellulose derivatives (for example, hydroxyethyl cellulose), agarose, sodium alginate, acrylamide copolymer, methacrylamide copolymer, acrylamide or copolymers of methacrylamide and various vinyl monomers Polyhydroxyethyl methacrylate, polyvinyl alcohol, polyvinylpyrrolidone, sodium polyacrylate, copolymers of acrylic acid and various vinyl monomers, and the like.

The layer composed of the hydrophilic polymer binder is formed by the method described in JP-B-53-21677, JP-A-55-164356, JP-A-54-101398, JP-A-61-292063, etc. Thus, an aqueous solution or aqueous dispersion containing a hydrophilic polymer can be applied on a support or other layer and dried. When the coloring layer 12 is formed from a layer having a hydrophilic polymer as a binder, the thickness of the coloring layer 12 when dried is preferably in the range of about 2 μm to about 50 μm, more preferably in the range of about 4 μm to about 30 μm. Is preferably in the range of about 2 g / m ² to about 50 g / m ² , more preferably in the range of about 4 g / m ² to about 30 g / m ² .

The leuco dye contained in the color forming layer 12 is, for example, a triarylimidazole leuco dye described in a composition (eg, US Pat. No. 4,089,747) that produces a dye by oxidation of a leuco dye, No. 59-193352 and the like, and the like. Alternatively, it is also possible to use a composition (for example, 4-aminoantipyrines and phenols or naphthols) containing a compound that forms a dye upon coupling with another compound when oxidized.
The blending amount of the leuco dye in the coloring layer 12 is not particularly limited, and can be appropriately set in consideration of the molar amount of the analyte to be detected.

  In order to contain the leuco dye in the coloring layer 12 (FIG. 1), when the coloring layer 12 is composed of a porous layer, a solution of a coloring material may be applied to the porous layer. Further, when the color developing layer 12 is formed of a layer made of a hydrophilic polymer binder, a coating solution in which a color forming substance is emulsified or dispersed in a solution in which a hydrophilic polymer is dissolved or dispersed is supported. The coloring layer 12 can be formed by applying and drying on the body 11. In the case of emulsifying the chromogenic substance, a chromogenic substance, a surfactant, oil or the like can be added to the hydrophilic polymer and emulsified by a known emulsification method. On the other hand, when the color developing substance is dispersed, the color developing substance and the surfactant can be added to the hydrophilic polymer and dispersed by a known dispersion method.

  Here, since chromogenic substances such as leuco dyes are generally insoluble in water and soluble in organic solvents, it is necessary to dissolve or disperse the chromogenic substance and the conjugate enzyme in an organic solvent and apply them on the support. The use of solvents raises the problem of increasing the environmental burden. On the other hand, in a preferred embodiment of the dry analytical element according to the present invention, as described above, the coloring layer is composed of a coloring material, an oil component (for example, N, N-dimethyllauramide, stearyl alcohol, etc.), an interface, and the like. The activator and water are mixed, and the coloring layer is applied in an aqueous state in the form of an emulsion. By applying the coloring layer in water, it is not necessary to use an organic solvent, thereby reducing the burden on the environment and improving workability.

The coloring layer 12 has an enzyme activator, coenzyme, surfactant, pH buffer composition, fine powder, oxidation, for the purpose of improving various properties such as coating characteristics, diffusibility, reactivity, and storage stability. Various additives comprising an organic substance or an inorganic substance can be added, such as an inhibitor, a crosslinking agent, and the like. Examples of buffering agents that can be incorporated into the color-forming layer 12 include “Chemical Handbook Basics” edited by the Chemical Society of Japan (Tokyo, Maruzen Co., Ltd., 1966), pages 1312-1320, RMCDawson et al, “Data for Biochemical Research "Second Edition (Oxford at the Clarendon Press, 1969), pages 476-508," Biochemistry "pages 5,467-477 (1966)," Analytical Biochemistry "pages 104, 300-310 (1980) There is a pH buffer system. Buffers containing borate as specific examples of pH buffers; buffers containing citrate or citrate; buffers containing glycine; buffers containing Bicine; buffers containing HEPES; buffers containing MES Good buffering agents such as agents.
The color forming layer 12 can be a layer that has been appropriately crosslinked and cured using a crosslinking agent. Examples of cross-linking agents include known vinyl sulfone cross-linking agents such as 1,2-bis (vinylsulfonylacetamido) ethane and bis (vinylsulfonylmethyl) ether for gelatin, aldehydes such as aldehydes for methallyl alcohol copolymers, Examples include glycidyl group-containing epoxy compounds.

The reagent layer 13 is preferably a porous layer made of a porous medium or a layer made of a hydrophilic polymer binder, and more preferably a porous layer. When the reagent layer 13 is a porous layer, it is preferably a porous layer having a function of a spreading layer. The spreading layer is a layer having a so-called metering action that spreads the liquid in an area substantially proportional to the amount of the supplied liquid.
Examples of the spreading layer that can be applied to the reagent layer 13 include a textile spreading layer described in, for example, JP-A Nos. 55-164356 and 57-66359 (for example, plain fabrics such as broad and poplin), A knitted fabric development layer (for example, tricot knitting, double tricot knitting, Miranese knitting, etc.) described in JP-A-60-222769, etc., and an organic polymer fiber pulp-containing papermaking paper described in JP-A-57-148250. Expanded layer, membrane filter (brush polymer layer), polymer microbeads, glass microbeads, and diatomaceous earth described in Japanese Patent Publication No. 53-21677, US Pat. No. 3,992,158, etc. are retained in the hydrophilic polymer binder. A non-fiber isotropic porous spreading layer such as a continuous microvoided porous layer, described in JP-A-55-90859 A non-fibrous isotropic porous development layer composed of a continuous microvoided porous layer (three-dimensional lattice-like granular structure layer) in which polymer microbeads are bonded in a point contact manner with a polymer adhesive that does not swell with water Can be used.
The thickness of the reagent layer 13 is not particularly limited, and the thickness can vary greatly within a range of several tens of μm to several hundreds of μm.

As the cholesterol oxidase (COD, EC 1.1.3.6) contained in the reagent layer 13, a commercially available product can be used. Examples of commercially available cholesterol oxidase include COO-311 from Toyobo Co., Ltd., CON from Asahi Kasei Co., Ltd., Cholesterol oxidase from F. Hoffmann-La Roche Ltd., and Amano Enzyme. CHO-1 of Co., Ltd. etc. are mentioned.
The amount of cholesterol oxidase used can be appropriately changed depending on the estimated amount of cholesterol contained in the biological sample, etc. For example, the coating amount in the layer containing the enzyme is 500 to 32000 U / m ² , preferably 2000 to 13000 U / m. ² . The unit (U) referred to here was defined as 1 U in an amount necessary to oxidize 1 μmol of cholesterol per minute at 25 ° C.

A commercially available peroxidase (POD, EC 1.11.1. 7) contained in the reagent layer 13 can be used. Examples of commercially available peroxidases include PEO-301 from Toyobo Co., Ltd.
Peroxidase, can be appropriately changed by the estimation amount of the cholesterol contained in a biological sample, for example, as the coating amount of the layer that contains the enzyme, 10000~100000U / ^{m 2,} preferably, 30000~60000U / ^{m 2,} Note, In peroxidase, 13.5 times the international unit using o-dianisidine at 25 ° C. was defined as 1U.

In order for the reagent layer 13 to contain an enzyme such as cholesterol oxidase or peroxidase, an aqueous solution containing the enzyme may be applied to the development layer mentioned above, for example. The aqueous solution containing the enzyme preferably contains a surfactant that decomposes lipoprotein, cholesterol esterase that separates the resulting cholesterol ester into fatty acid and free cholesterol, and the like.
A commercially available cholesterol esterase (CE, EC 3.1.1.13) can be used. Examples of commercially available cholesterol esterase include CHE-2 from Amano Enzyme Co., Ltd. Moreover, as a surfactant that decomposes lipoprotein, a known one can be used, and examples thereof include Triton X-100.
The reagent layer 13 may contain ascorbate oxidase (ASO). A commercially available ascorbic acid oxidase can be used, and for example, it can be obtained from Roche and Toyobo Co., Ltd.

  The optimum pH of the reagent layer 13 is preferably in the range of 7 to 9, and the most preferable pH range is in the range of 7.5 to 8.5. For the adjustment of the optimum pH, a reagent such as a buffer can be used as necessary.

  As the support 11, it is preferable to use a support having a high light transmittance and impermeable to water. As the light-transmitting and water-impermeable support, known materials used for conventional dry analytical elements can be used. Specific examples thereof include polyethylene terephthalate, bisphenol A polycarbonate, polystyrene, cellulose ester (for example, cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.), and a thickness of about 50 μm to about 1 mm. Using a smooth planar support that transmits electromagnetic radiation, preferably in the range of about 80 μm to about 300 μm, for example, at least partially in the wavelength range of about 200 nm to about 900 nm. Can do. A known undercoat layer or adhesive layer can be provided on the surface of the support.

  In the dry analytical element of the present invention, it is only necessary that the color-forming layer containing the color-forming substance and the reagent layer containing the conjugated enzyme are arranged adjacent to each other, and other layers can be laminated as necessary. Examples of the other layers include a development layer, a detection layer, a light reflection layer, an adhesive layer, a water absorption layer, and an undercoat layer. Examples of such dry analytical elements are those disclosed in, for example, JP-A-49-53888, JP-A-51-40191, JP-A-55-164356, JP-A-61-4959 Can be applied to the present invention.

Further, in the dry analytical element of the present invention, the reagent layer 13 can be provided with the function of a reflective layer by adding fine particles having light reflectivity or light absorbability to the reagent layer 13. The reflective layer is a water-permeable or water-permeable layer in which light-reflecting or light-absorbing fine particles or fine particles are dispersed and held in a small amount of a hydrophilic polymer binder having a film-forming ability. When the reflective layer is photometrically reflected from the support 11 (FIG. 1) side for detectable changes (color change, color development, etc.) of the chromogenic substance, the color of the supplied aqueous liquid, particularly when the sample is whole blood It also has a function of shielding the red color of hemoglobin.
Examples of the light-reflecting fine particles include titanium dioxide fine particles (rutile type, anatase type or brookite type fine crystal particles having a particle diameter of about 0.1 μm to about 1.2 μm, etc.), barium sulfate fine particles, aluminum fine particles or Examples of the light-absorbing fine particles include carbon black, gas black, and carbon micro beads. Among these, titanium dioxide fine particles and barium sulfate fine particles are preferable. Particularly preferred are anatase-type titanium dioxide fine particles.

  The dry analytical element of the present invention can be prepared by known methods described in the aforementioned patent publications. The dry analytical element of the present invention is cut into small pieces such as a square having a side of about 10 mm to about 30 mm or a circle of approximately the same size, and is disclosed in JP-B-57-28331, JP-A-56-142454, JP-A-57-63452, It is preferable to use it as a chemical analysis slide by placing it in a slide frame described in Japanese Patent Publication Nos. 58-32350 and 58-501144, etc. from the viewpoint of manufacturing, packaging, transportation, storage, measurement operation and the like. Depending on the purpose of use, the tape can be used in a long tape form in a cassette or magazine, or a small piece can be attached to or stored in a card with an opening, or a cut piece can be used as it is.

The dry analytical element of the present invention can perform qualitative or quantitative analysis of an analyte by the same operations as those described in the aforementioned patent publications. For example, an aqueous liquid sample solution such as plasma, serum, or urine in the range of about 2 μL to about 30 μL, preferably 4 to 15 μL is spotted on the reagent layer 13. The spotted dry analytical element is incubated at a constant temperature in the range of about 20 ° C to about 45 ° C, preferably at a constant temperature in the range of about 30 ° C to about 40 ° C for 1 to 10 minutes. The color development or discoloration in the element is reflected and photometrically measured from the support side, and the amount of the analyte in the specimen can be determined based on the principle of the colorimetric measurement method using a calibration curve prepared in advance. Quantitative analysis can be performed with high accuracy by keeping the amount of liquid sample to be spotted, incubation time and temperature constant.
In the case of measuring blood HDL-cholesterol, it is desirable to prepare a HDL-cholesterol fraction from plasma or serum in advance using a fractionation reagent before spotting on the dry analytical element. Specifically, as shown in reaction (1) of FIG. 2, after adding a reagent for fractionation to plasma or serum and mixing, the mixture is centrifuged, and the supernatant is obtained as an HDL fraction. be able to. After centrifugation, low density lipoprotein (LDL) and very low density lipoprotein (VLDL) are obtained as precipitates.
Examples of the reagent for fractionation include phosphotungstic acid-Mg, heparin-Mn, dextran sulfate-Mg, heparin-Ca, and polyethylene glycol.
Centrifugation is preferably performed at 2000 to 10000 rpm for 5 to 20 minutes.

The measurement operation can be carried out with high accuracy and quantitative analysis by an extremely easy operation using a chemical analyzer described in JP-A-60-125543, JP-A-60-220862, JP-A-61-294367, and JP-A-58-161867. Depending on the purpose and required accuracy, the degree of color development may be determined visually to perform qualitative or semi-quantitative measurement.
The biological sample that can be analyzed by the present invention is preferably a liquid that can be collected from a living body, and examples thereof include blood, urine, and sputum.

Hereinafter, the present invention will be further described by examples.
[Example 1]
A colored layer aqueous solution-a having the following composition was coated on a 180 μm polyethylene terephthalate colorless transparent smooth film coated with gelatin and dried to form a colored layer. The thickness of the coloring layer after drying was 30 μm.
(Aqueous solution for coloring layer-a)
Gelatin 25.0 g / m ²
Surfactant 0.8g / m ²
Leuco dye 0.5 g / m ²
N, N-dimethyllauramide 7.5 g / m ²
Stearyl alcohol 1.1 g / m ²

  Here, the surfactant is sodium α-olefin sulfonate (OS-14, manufactured by Nikko Chemical), and the leuco dye is 2- (3,5-dimethoxy-4-hydroxyphenyl) -4- (4- Dimethylaminophenyl) -5-phenethylimidazole acetate was used.

Next, a tricot knitted fabric knitted with 36-gauge polyethylene terephthalate spun yarn equivalent to 50 denier is lightly applied to the color-developing layer after being wetted by supplying water over the entire surface at a supply amount of about 30 g / m ^2. Layered and dried. On the dried fabric, the reagent layer aqueous solution-a having the following composition was applied and dried to form a reagent layer to prepare a dry analytical element. In addition, the thickness of the reagent layer after drying was 10 μm.
(Aqueous solution for reagent layer-a)
Phosphoric acid 1K 0.3 g / m ²
Phosphoric acid 2K 5.1 g / m ²
Surfactant 1 0.5 g / m ²
Surfactant 2 1.1 g / m ²
Deoxycholic acid 3.7 g / m ²
Sucrose 4.7 g / m ²
Metroose 90SH100 0.3g / m ²
Peroxidase 46000 U / m ²
Cholesterol oxidase 4300 U / m ²
Cholesterol esterase 2500U / m ²
ASO 14000U / m ²

  Surfactant 1 was polyoxy (2-hydroxy) propylene nonylphenyl ether (Surfactant 10G, manufactured by Aurin), and surfactant 2 was polyoxyethylene octylphenyl ether (HS240, manufactured by NOF Corporation). ASO means ascorbate oxidase, and Roche's ascorbate oxidase was used here. For peroxidase, Toyobo Co., Ltd. PEO-301 was used, for cholesterol oxidase, Roche's Cholesterol oxidase was used, and for cholesterol esterase, Amano Enzyme Co., Ltd. CHE-2.

  The dry analytical element was cut into 12 mm × 13 mm square chips and placed in a slide frame (described in JP-A-57-63452) to produce a dry slide for measuring cholesterol according to the present invention.

[Comparative Example 1]
A colored layer aqueous solution-b having the following composition was applied to a colorless and transparent smooth film of 180 μm polyethylene terephthalate coated with gelatin and dried to form a colored layer. Note that the thickness of the color developing layer after drying was set to 30 μm.
(Coloring layer aqueous solution-b)
Gelatin 25.0 g / m ²
Surfactant 0.8g / m ²
Leuco dye 0.5 g / m ²
N, N-dimethyllauramide 7.5 g / m ²
Stearyl alcohol 1.1 g / m ²

  Here, the surfactant is sodium α-olefin sulfonate (OS-14, manufactured by Nikko Chemical), and the leuco dye is 2- (3,5-dimethoxy-4-hydroxyphenyl) -4- (4- Dimethylaminophenyl) -5-phenethylimidazole acetate was used.

Next, an aqueous solution having the following composition was applied on the color developing layer and dried to form a layer containing potassium ferrocyanide as a mediator. The thickness of this layer after drying was 5 μm.
Gelatin 3.3 g / m ²
Titanium dioxide 0.9g / m ²
Surfactant 0.3 g / m ²
Potassium ferrocyanide 0.7 g / m ²

Here, polyoxy (2-hydroxy) propylene nonylphenyl ether (Surfactant 10G, manufactured by Aurin) was used as the surfactant.
Next, a tricot knitted fabric knitted with 36 gauge polyethylene terephthalate spun yarn equivalent to 50 denier was applied onto the above layer with water at a supply rate of about 30 g / m ² and moistened. Layered and dried. On the dried fabric, the reagent layer aqueous solution-b having the following composition was applied and dried to form a reagent layer to prepare a dry analytical element. In addition, the thickness of the reagent layer after drying was 10 μm.
(Aqueous solution for reagent layer-b)
Phosphoric acid 1K 0.3 g / m ²
Phosphoric acid 2K 5.1 g / m ²
Surfactant 1 0.5 g / m ²
Surfactant 2 1.1 g / m ²
Deoxycholic acid 3.7 g / m ²
Sucrose 4.7 g / m ²
Metroose 90SH100 0.3g / m ²
Peroxidase 46000 U / m ²
Cholesterol oxidase 4300 U / m ²
Cholesterol esterase 2500U / m ²
ASO 14000U / m ²

  Surfactant 1 was polyoxy (2-hydroxy) propylene nonylphenyl ether (Surfactant 10G, manufactured by Aurin), and surfactant 2 was polyoxyethylene octylphenyl ether (HS240, manufactured by NOF Corporation). Here, the same peroxidase as in Example 1 was used.

  The dry analytical element was cut into a 12 mm × 13 mm square chip and placed in a slide frame (described in JP-A-57-64552) to produce a dry slide for measuring cholesterol.

[Measurement Example 1]
In order to compare the storage stability of the two, the slides prepared in Example 1 and Comparative Example 1 were stored at 45 ° C. for 0, 1, 4, and 7 days, respectively. On the other hand, human serum with HDL-cholesterol concentrations of 39, 68, 102 mg / dL and a reagent for fractionation (phosphotungstic acid / magnesium chloride) were mixed at a volume ratio of 1: 1, and allowed to stand for 10 minutes, and then 3000 rpm × 10 minutes Centrifugation gave an HDL fraction. 10 μL of this fraction was spotted on the slide, incubated at 37 ° C. for 6 minutes, and the reflection density (ODr) at 600 nm was measured with Fuji Dry Chem 5000 (Fuji Photo Film Co., Ltd.). The measured reflection density was converted to HDL-cholesterol concentration from a calibration curve (relationship between reflection density and cholesterol concentration) prepared in advance.
The results of Example 1 are shown in Table 1, and the results of Comparative Example 1 are shown in Table 2. In the table, HDL-C means HDL-cholesterol, and the unit of HDL-C concentration is mg / dL. The rate of change means the rate of change of the measured value when stored at 45 ° C. for 7 days with respect to 0 days.

  As shown in the above results, it is clear that the dry analytical element of the present invention can prevent the HDL-cholesterol level from being lowered and is excellent in storage stability.

It is sectional drawing which shows one Embodiment of the dry analysis element of this invention. It is a figure for demonstrating the measurement principle of HDL-cholesterol by the dry analytical element of this invention. It is sectional drawing which shows the dry-type multilayer analysis element of patent document 1. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Dry analytical element 11 Support body 12 Coloring layer 13 Reagent layer

Claims (5)

  Qualitative or quantitative analysis of analytes in biological samples by causing a conjugate enzyme to act on analytes or substances derived from analytes to generate hydrogen peroxide and coloring the chromogenic substance using the hydrogen peroxide In the dry analytical element to be analyzed, on the support, a color developing layer containing the chromogenic substance and a reagent layer containing the conjugated enzyme are laminated adjacently in this order, and a mediator involved in the color development of the chromogenic substance Dry analytical element characterized by not containing.   The dry analytical element according to claim 1, wherein the reagent layer contains at least peroxidase and cholesterol oxidase.   The dry analytical element according to claim 1 or 2, wherein the analyte is HDL-cholesterol.   The dry analytical element according to claim 1, wherein the mediator is a ferrocyan ion.   The dry analytical element according to claim 1, wherein the chromogenic substance is a leuco dye.

Images