JPS62115368A - Integral type multi-layer analyzing element for analyzing cholesterol - Google Patents

Abstract

PURPOSE:To decrease the variance of a color forming optical density value according to the difference in production lots by integrally laminating a water absorptive layer contg. a hydrophilic polymer and porous development layer on a light transmittable and water impermeable base and incorporating a specific reagent compsn. therein. CONSTITUTION:The water absorptive layer and porous development layer are integrally laminated on the light transmittable and water impermeable base. The color forming reagent compsn. consisting of cholesterol esterase, cholesterol oxidase, peroxidase, chromogen and coupler and the cholesterol analyzing reagent compsn. contg. ferrocyanide are incorporated therein. The water absorptive layer is formed of the hydrophilic polymer and swells by absorbing water to receive and accumulate the detection seeds. The fibrous development layer to which the entire components of the reagent compsn. for analysis are liable to be incorporated and held is used for the porous development layer. 1-(Halogen substd. phenyl)-2,3-dimethyl-4-amino-3-pyrazoline-5-on is used for the chromogen. The compd. selected from the group consisting of phenol, naphthol deriv. having a sulfo group and dihydroxy naphthalene is used for the coupler.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an improvement in a dry integrated multilayer analytical element for analyzing cholesterol contained in various body fluids such as blood and other aqueous samples. .

[Conventional technology]

Recently, as a form of dry analysis element, an integrated multilayer analysis is being developed, in which a transparent support is provided with a water-absorbing reagent layer containing a color reaction reagent and a hydrophilic polymer binder, and a porous development layer for developing the sample. Elements have been developed and various improvements have been made. This multilayer analytical element can analyze various samples by changing the reagents, and is also known for cholesterol (1'-CLINICAL CHE
MISTRYj2♂(j'), //j9-//12(/
9♂2)l Tokukoaki! 4 tons! ! Publication No. 99, etc.).

A conventional multilayer analysis element for cholesterol analysis has a gelatin layer and a developing layer stacked on a transparent support, and cholesterol esterase, cholesterol esterase, and a developing layer are stacked on a transparent support, as shown below.
Enzymes such as cholesterol oxidase and peroxidase, leuco dye, etc. were added. And the pH of the gelatin layer is 6°! (1'-CLI
NICALCHEMISTRYJ 2r (r),
/1tq−//≦2(/ri/, 2)).

[Problem that the invention seeks to solve]

In conventional multilayer analytical elements for cholesterol analysis,
Leuco dye develops color during storage before use in analytical procedures (Capri)
There was a problem that it was easy to clean and had poor storage stability. Since this leuco dye is more likely to develop color when exposed to air, it had to be used within a short period of time, especially after opening the package.

As a method of suppressing color development (capri) during storage of elements and after opening the package and keeping the color development rate as fast as that of leuco dyes, guaminoantipyrine (t or its derivatives) was used as a color reagent composition. (chromogen) and cohydroxy 3. ! -dichlorobenzenesulfonic acid (coupler)
Using a combination of the above, the pH during analysis was set to 7. ! From 9. θ
An integrated multilayer analytical element has been proposed that includes a cholesterol analysis reagent composition containing a pH buffer to maintain the pH within the range of . (Patent application Shoto-Irott 2) However, when using the above-mentioned integrated multilayer analytical element for cholesterol analysis and using guaminoantipyrine as the chromogen, the color optical density value may vary from production lot to production lot. found.

On the other hand, Tokukai Akira! Niichi/! ! ♂! The specification states that guamino-7enazone (4t-aminoantipyrine,
i.e. /-phenyl-2,3->methyl-4-amino-3-pyrazolin-y-one (common name -) and 3. as a coupler. ! -dichloro-hydroxybenzenesulfonic acid (t& is its alkali metal salt), and using a coloring reagent composition for cholesterol analysis in combination with ferrocyan ion such as potassium ferrocyanide or sodium ferrocyanide. It is described that the negative errors caused by this method can be eliminated, and at the same time, it is possible to quantitatively analyze cholesterol with high sensitivity. When we focused on the high sensitivity of this color reagent composition and applied it to an integrated multilayer analytical element for cholesterol analysis, it became highly sensitive, but there was still variation (variation) in color optical density values from production lot to production lot. It was found that it remained large and did not improve.

[Purpose of the invention]

An object of the present invention is to provide an integrated multilayer analytical element for cholesterol analysis that exhibits stable performance and has extremely small fluctuations (variations) in color optical density values due to differences in manufacturing lots.

Another object of the present invention is to provide an integrated multilayer analytical element for cholesterol analysis that does not develop color (capri) during storage before use in analytical operations or after opening the package and is highly sensitive.

Another object of the present invention is to provide a highly sensitive integrated multilayer analytical element for cholesterol analysis that exhibits rapid color development during analytical operations.

Another object of the present invention is to provide an integrated multilayer analytical element for cholesterol analysis that can perform accurate measurements over a wide concentration range from low to high cholesterol concentrations in a liquid sample (with a wide dynamic range). .

The present invention provides at least 7
A water-absorbing layer containing a hydrophilic polymer and a porous spreading layer are integrally laminated in this order, and contain cholesterol esterase, cholesterol oxidase, hydrogen oxidase, a coloring reagent composition consisting of a chromogen and a coupler, and ferrocyanide. In an integrated multilayer analytical element for cholesterol analysis containing a cholesterol analysis reagent composition, (a) the chromogen is /-(halogen-substituted phenyl)-1,3-dimethyl-4-amino-3-pyrazoline-! -on, medium) An integrated multilayer analytical element for cholesterol analysis, wherein the coupler is a compound selected from the group consisting of phenol, a phenol derivative having a sulfo group, naphthol, a heptol derivative having a sulfo group, and dihydroxynaphthalene. It is.

As the light-transparent water-impermeable support of the integrated multilayer analytical element of the present invention, any light-transparent (transparent) water-impermeable support used in conventionally known multilayer analytical elements can be used. Specific examples include polyethylene terephthalate,
Bisphenol A polycarbonate, polystyrene,
Made of polymers such as cellulose esters (e.g., cellulose diacetate, cellulose triacetate, cellulose acetate pyrovionate, etc.) about a thickness! Transparent, i.e., wavelength-containing crystals ranging from θ μm to about 7 mm, preferably from about lθ μm to about 300 μm, i.e., from about 9θθn
A film-like (sheet-like) or flat support having a smooth surface that transmits electromagnetic radiation in at least part of the wavelength range within the range of rn can be used. Optical performance can be adjusted by dispersing fine particles of titanium dioxide, fine particles of barium sulfate, carbon black, etc. in the support, if necessary. A known undercoat layer or adhesive layer may be provided on the surface of the support, if necessary, to strengthen the adhesion between the support and a water absorption layer, reagent layer, etc. provided on the support.

Water absorption N/-C halogen-substituted phenyl)-X.

3-dimethyl-teramino-3-pyrazoline-! -one (hereinafter sometimes referred to as 9-aminoantipyrine halogen-substituted derivative), and euhydroxy-3,! -Dichlorobenzenesulfonic acid: 6E This is a layer in which pigments produced by oxidative cupping reaction are accumulated, and is formed from a hydrophilic polymer binder, and is formed from only the hydrophilic polymer binder or from this and other components. It is.

The water-absorbing layer is a layer that absorbs water and swells, receives and accumulates the detection species, and it should function as a detection layer that absorbs water and swells and contains a mordant with a negative or positive charge that can mordantly fix the detection species. I can do it. The hydrophilic polymer binder has a swelling rate of about /! at 30°C upon absorption of water. 0% to about 200
The polymer is a natural or synthetic hydrophilic polymer with a .theta. ratio of .theta., preferably in the range of about 200% to about 1/600%. As an example,
Tokukai Akira! 9-/7//6g, θ-IC#';#
Gelatin (acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (phthalated gelatin, hydroxyacrylate grafted gelatin, etc.) disclosed in No. 3 etc., agarose,
Examples include pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol, and polyvinylpyrrolidone. The dry thickness of the water absorbing layer is approximately 7 μm to approximately 100 μm.
θ0 μm, preferably from about 3 μm to about 0 μm, particularly preferably from about 5 μm to about 30 μm. The water-absorbing layer may contain a known mordant, basic polymer, acidic polymer, etc. It is desirable that the water-absorbing layer be substantially transparent, but if necessary, the optical performance can be adjusted by incorporating a small amount of titanium dioxide fine particles, barium sulfate fine particles, carbon black, etc., dispersed in the water-absorbing layer.

As a porous development layer, JP-A-Shojj-/d4t3j6,%
Fabric development layer (e.g., plain weave such as broad, boblin, etc.) described in 1987 F7-41?19, etc., patent application Sho J9-79/J♂
Knitted fabric development layer (e.g., tricot knit, double tricot knit, Milanese knit, etc.) described in JP-A-57-4T12
A spreading layer made of paper made from organic polymer fiber pulp as described in No. 30, Tokukosho! 3-co/ni77, U.S. Patent 39ri2
7! Non-fibrous isotropic porous development such as membrane filters (single layer of plush polymer), polymer micropeas, glass microbeads, diatomaceous earth held in a woody polymer binder, and continuous microvoids-containing porous layers described in et al. Layer, Tokukai Akira! ! A non-fibrous isotropic material consisting of a continuous microvoid-containing porous layer (three-dimensional lattice-like granular structure layer) in which the polymer microbeads described in -90/rj9 are bonded in point contact with a polymer adhesive that does not swell with water. A porous spreading layer or the like can be used.

The porous spreading layer can contain all the components of the reagent composition for cholesterol analysis. Alternatively, among the reagent compositions, cholesterol esterase and cholesterol oxidase can be contained in the porous development layer, and the other components can be contained in the reagent layer.

Among these porous spreading layers, in an embodiment in which a reagent composition containing at least one enzyme is contained and held in the spreading layer, the woven fabric spreading layer and the knitted fabric spreading layer are easy to contain and hold the reagent composition in the spreading layer. A fibrous spread layer typified by Layer is preferable.

The woven fabric or knitted fabric used for the porous development layer may be subjected to a physical activation treatment such as glow discharge treatment or corona discharge treatment described in JP-A-2-6-3J on at least one side of the fabric. , or JP-A-Shojj-/641
336, Tokukai Sho! 7-64339 etc., hydrophilic treatment such as hydrophilic polymer impregnation, or a suitable combination of these treatment steps to make the cloth hydrophilic, and the lower side (the side closer to the support) It is possible to increase the adhesion force with the layer.

When the reagent layer is provided as a separate layer, the reagent layer contains all or part of a reagent for cholesterol analysis including an enzyme and a coloring reagent composition. When the reagent layer contains part of the reagent for cholesterol analysis, it is preferable that the enzymes cholesterol esterase and cholesterol oxidase be contained in the porous development layer, and the remaining reagent components be contained in the reagent layer. The reagent layer is a substantially non-porous, water-absorbing layer comprising a hydrophilic polymeric binder, or a microporous, water-permeable layer.

The hydrophilic polymeric binder used in the substantially non-porous, water-absorbing reagent layer acts as a medium to substantially uniformly dissolve or disperse the reagent, and also absorbs and absorbs water in the liquid sample. It has at least the function of allowing the test component to reach the reagent layer. The hydrophilic polymer binder can be appropriately selected from the same polymers as the water-absorbing hydrophilic polymer binder used in the hydrophilic layer. The dry thickness of the substantially non-porous reagent layer is from about 3 μm to about 10 ml, preferably about ! The reagent layer is preferably substantially transparent, but if necessary, fine particles of titanium dioxide, fine particles of barium sulfate, carbon black, etc. may be dispersed in the reagent layer to provide optical transparency. In addition to being able to adjust the performance, it can also serve as a light shielding layer.

As a microporous and water-permeable reagent layer, Japanese Patent Application No. 9-7
A microporous structure layer composed of solid fine particles and a hydrophilic polymer binder as a supporting medium as described in Japanese Patent Application No. 9/J9, etc., and a cloth as described in Japanese Patent Application No. 9/2A2. woven fabric or knitted fabric, etc.), pore size approximately 0.1
Approximately from μm! μm membrane filter (single layer of plush polymer), nonwoven fabric with continuous micropores penetrating both surfaces, hot paper, and polymer micropeas are brought into point contact with a polymer adhesive or polymer crosslinking agent that does not swell with water. A microporous sheet material such as a microporous layer containing continuous micropores (three-dimensional lattice-like granular structure layer) penetrating both sides can be used. The dry layer thickness of the microporous reagent layer is determined depending on the case of the microporous structure layer and the three-dimensional lattice-like particulate structure layer.
μm to about 20 μm1 preferably about 70 μm to about 30 μm
μm range, in the case of microporous sheets from 410 μm to about 30 μm, preferably about ! Qμm to about θθμ
m range. Optical performance is adjusted by dispersing titanium dioxide particles, barium sulfate particles, carbon black, etc. in the microporous reagent layer, as part or all of the solid particles, or in the polymer medium of a membrane filter. In addition, it can also serve as a light shielding layer.

The reagent layer can contain a known pH buffer composition, a polymer pH buffer, a basic polymer, an acidic polymer, a polymer mordant, and the like.

Tokuko Shoj on top of the reagent layer or water absorption layer? -2/477,
Tokuko Akira ♂-/♂Otsu 2! , Tokukai Akira! Goo = 9200, Tokukai Akira! A known light shielding layer described in the specifications of J-/4G 3!6 and the like can be provided.

The light-shielding layer is a water-permeable layer in which fine particles or fine powder (hereinafter simply referred to as fine particles) having light-shielding properties or both light-shielding properties and light-reflecting properties are dispersed and retained in a small amount of a hydrophilic polymer binder with film-forming ability. or a water-permeable layer. The light-shielding layer blocks the color of the aqueous liquid sample dotted onto the developing layer when measuring the color developed in the hydrophilic layer by reflection from the light-transmitting support side, especially the red color of hemoglobin contained in the whole surface sample, and also blocks the light. It also functions as a reflective or background layer. The dry layer thickness of the light shielding layer is approximately 3 μm to approximately 30 μm.
1 preferably in the range of about jμm to about 20μm.

An adhesive layer can be provided for adhering and laminating the spreading layer, light shielding layer, etc. An adhesive layer is essential when a porous spreading layer is provided on a light shielding layer.

The adhesive layer mainly consists of a hydrophilic polymer that can adhere and integrate the spreading layer when wetted with water or swollen with water. Hydrophilic polymers that can be used in the adhesive layer include the same hydrophilic polymers as those used in the hydrophilic layer, and among these, gelatin, gelatin derivatives, polyacrylamide, and the like are preferred. The dry layer thickness of the adhesive layer ranges from about 0.5 μm to about 20 μm, preferably from about 0.5 μm to about 70 μm. The adhesive layer can contain a surfactant. The surfactant is preferably a nonionic surfactant, particularly a nonionic surfactant having a chain structure in which r to /j oxyethylene groups or oxypropylene groups are connected.

The adhesive layer is formed by applying an aqueous solution containing a hydrophilic polymer and a surfactant added as desired to a reagent layer, a hydrophilic layer,
Alternatively, it can be provided by coating on a light shielding layer.

When a porous spreading layer is bonded and integrated on a microporous reagent layer, adhesion using a partially arranged adhesive (micro-through holes) as described in Japanese Patent Application No. 1997-19-1, 2+, 2 & 7 is used. It is preferable to use the adhesive bonding method.

One (or more) water absorption layer or reagent layer can be provided as required. When a reagent layer is provided, a water absorption layer can be provided between the reagent layer and the support.

Next, the cholesterol analysis reagent composition contained in the integrated multilayer analytical element for cholesterol analysis of the present invention will be explained. The reagent composition for cholesterol analysis is a coloring reagent composition consisting of cholesterol esterase, cholesterol oxidase, peroxidase, a chromogen and a coupler, and a composition whose main components are ferrocyanide.

Cholesterol esterase (E, C, 3, /, /
.

/3) is an enzyme that converts cholesterol ester contained in a sample into cholesterol. Tokukai Sho is the best cholesterol esterase! Otsu-￥! It is also possible to use the combination of lipase and protease described in the TJ99 specification. Cholesterol oxidase (E, C, /
, /, 3.4) is an enzyme that oxidizes cholesterol to produce hydrogen peroxide. Hydrogen oxidase (E, C, /, / /, 7゜2) uses this hydrogen peroxide to oxidize the halogen-substituted derivative (chromogen) of guaminoantipyrine to form cohydroxy-3,! -An enzyme that couples with dichlorobenzenesulfonic acid (coupler).

The chromogen is represented by the general formula [/] /-(halogen-substituted phenyl)-2,! -jime fk-daramino 3
-Pyrazoline-! -on is used.

In the general formula [/], the number attached to the pyrazoline nucleus indicates the position number. X represents a halogen atom (eg, chlorine atom, bromine atom, iodine atom), and n is /.

Represents ko or 3. When n is co or 3, X may be the same or different halogen atoms, and X may be bonded to the phenyl group at any position.

Me represents a methyl group.

The halogen atom of the halogen-substituted phenyl group at position 7 is preferably a chlorine atom.

Examples of the halogen-substituted phenyl group at position 7 include 3-chlorophenyl group, 2g-chlorophenyl group: g-bromophenyl group: 4t-iodophenyl group: 2°! -dichlorophenyl group: 3T4t-') chlorophenyl group: J, j-dichlorophenyl group; 3l-t-dibromophenyl group; λ,
, 6-dolychlorophenyl group preference I L<,! ,
Most preferred are the j-dichlorophenyl group and the J,4,6-trichlorophenyl)dichlorophenyl group.

7-(,-rogen-substituted phenyl)-2,! represented by the general formula [/] -Dimethyl-guamino-3-pyrazoline-! Specific examples of -on are shown in Table 1.

/-(halogen-substituted phenyl) -X, 3-dimethyl-4-amino-3-pyrazoline-! As -one, either a free amine or an acid addition salt (eg, hydrogen chloride salt) can be used. These compounds have the following properties:
t, t -11Jchlorophenyl)-1,3-dimethyl-4-amino-3-pyrazolin-y-one and/-
(3,j--,p,chlorophenyl)-2,3-dimethyl-q-amino-3-pyrazoline-! -on is preferred.

As a coupler, CHEMICAL ABSTRACTS, 7 Otsu, /377/ld (/9
7.2); Same? , 2. /4tx+7. zu (／wa♂
θ)；Tokuko Akira! ! -2 Aug θ, Tokko Akira! r-4t! j
9ri, Tokko Akira! ! -/♂Niko11 Tokukai Shojet-/64
To t3j, tokukai shoj niichi/cog 39♂, tokukai sho! ≦−／
! ! Phenols described in specifications such as rj2, US Pat. No. 3992/r♂; phenol derivatives: /-naphthol; conaphthol; /-naphthol derivatives; λ-naphthol derivatives; dihydroxynaphthalene (e.g., /, 2-dihydroxynaphthalene); Dihydroxynaphthalene derivatives can be m-. Phenol derivatives and naphthol derivatives have a sulfo group (-8O3H) or an alkali metal salt or alkaline earth metal salt thereof (-8O3M: M is a metal cation; hereinafter, including the -803M group, simply a sulfo group) on the benzene ring or naphthalene ring, respectively. Phenol derivatives and naphthol derivatives substituted with 7 to 3 groups (sometimes referred to as sulfo-substitution) are preferred. In addition to sulfo groups, phenol derivatives and naphthol derivatives contain other types of residues, such as alkyl groups (methyl, ethyl, propyl, isopropyl, etc.), halogen atoms (chlorine, bromine, etc.), alkoxy groups (methoxy It is also possible to use a derivative in which seven or more groups (such as ethoxy groups, ethoxy groups, etc.) are bonded to a benzene term or a naphthalene ring.

Specific examples of phenol derivatives having a sulfo group include co-hydroxy-/-benzenesulfonic acid; g-hydroxy-7-benzenesulfonic acid: j.

j-dichloro-hydroxy-/-benzenesulfonic acid; sulfonated 2,41-dichlorophenol; no-hydroxy-3-methoxy-/-benzenesulfonic acid, and their sodium salts, potassium salts, and lithium salts. Specific examples of naphthol derivatives having a sulfo group include /-hydroxy-j-naphthalenesulfonic acid; /-
Hydroxy-4-naphthalenesulfonic acid and its sodium, potassium, and lithium salts. The most preferred compound as a coupler is 3. j-dichloro-cohydroxy/-benzenesulfonic acid (or its sodium salt, potassium salt, lithium salt).

Ferrocyanide includes ferrocyan ion (hexacyanoferrate (II) ion; that is, CFe (CN
)6) 40) Compounds containing or capable of releasing can be used. As the ferrocyanide, alkali metal salts, alkaline earth metal salts, and mixed metal salts of hexacyanoferric (■) acid can be used.

Specific examples of ferrocyanide include sodium ferrocyanide Na4[Fe(CN)al: 7E0 potassium cyanide 4 CFe(CN)6]: lithium hexacyanoferrate(II); magnesium hexacyanoferrate(If): There is calcium hexacyanoferrate(II). Among these, potassium ferrocyanide and sodium ferrocyanide are preferred.

Examples of peroxidase include plant-derived and animal-derived peroxidases (EC/) described in Japanese Patent Publication No. 4-'463-9, Japanese Patent Publication No. 7-JJ-20, etc.

//, /, 7), Tokko Akira! ! -6036 etc. Peroxidase of microbial origin (EC/, //.

7.2) can be used. Among these, non-specific peroxidases of plant or microbial origin are preferred. Preferred examples of the oxidase include oxidase extracted from horseradish and radish, and Cochl oxidase.
Examples include oxidase extracted from microorganisms of the genus Iobolus and Curvularia.

The coating amount of each component of the cholesterol analysis reagent composition used in the element of the present invention in the element is as follows (the coating amount is element/m2).

Cholesterol esterase; about 7,000 to about! 1,000 IU, preferably about 7,000 to about 30,000 IU Cholesterol oxidase: about 7,000 to about 30,000 IU, preferably about /jθ0 to about 1,000 IU
U peroxidase: about 7,000 to about 700,000 IU, preferably about 2,000 to about 60,000 IU Guaminoantipyrine halogen-substituted derivative: about O,S
~about 70 mmol, preferably about/~about! mmol 2-hydroxy3. j-dichlorobenzenesulfonic acid: approx. 2. j ~ approx! 0 mmol, preferably about ! ~About 2! Millimorph ferrocyanide; approx. 0. /~about 70 mmol, preferably about 0°! ~about!
In addition to the above-mentioned reagent components, the integrated multilayer analytical element for cholesterol analysis of the present invention can contain other components as appropriate, depending on the type of liquid sample to be applied and the accuracy of analysis. For example, a protein dissociating agent such as a nonionic surfactant, an anti-capri agent such as dimedone, a sensitivity adjusting agent, a buffering substance for adjusting pH, etc. are appropriately added. Nonionic surfactants include polyhydric alcohol ester ethylene oxide adducts (condensates), polyethylene glycol monoesters, polyethylene glycol diesters, higher alcohol ethylene oxide adducts (condensates),
Alkylphenol ethylene oxide adduct (condensate)
etc. Specific examples of nonionic surfactants are listed below. It goes without saying that the nonionic surfactants used in the present invention are not limited to these compounds.

Chemical structure of surfactant POE (10) Sorbitan monooleate PEG (4t
00) Monostearate lauryl alcohol EO 10 mole condensate POE (10)
Octylphenyl ether POE (/, t) Octylphenyl ether POE (/, 2) Nonylphenyl ether (u) POE: Polyethylene oxide PEG: Polyethylene glycol EO: Ethylene oxide () The numbers in parentheses are the reduced number of ethylene oxide units of the present invention. The pH value when performing analysis operations using a liquid sample for the body type multilayer analysis element is approximately j,! to about 7.0, preferably from about 7.7 to about 2.j, and may be appropriately selected from known buffering agents.

Examples of buffering agents that can be used include "Chemical Handbook Basic Edition" edited by the Chemical Society of Japan (Tokyo, Maruzen ■, published in 1994.4). /
Pages 2-7320, R, M, C, Dawson et
Volume a1 “Data for BiochemicalR
esearchJ 2nd edition (Oxford at the
Published by Clarendon Press, / 9 years) 4
t7+-! θ1 page, [BiochemistryJj
, 14≦2 pages onwards (/9+g year), [Anal
yticalBiochemistryJ /θ<j,
There is a pH buffer system described on pages 300-310 (/9/θ).

As a specific example of a pH buffering agent in the range of pH 2.t to 9.0, tris(hydroxymethyl)aminomethane (Tris
); Buffers containing phosphate; Buffers containing borate; Buffers containing sulfuric acid or sulfates; Buffers containing glycylate; N,N-bis(2-hydroxyethyl); ) Bicine; N-cohydroxyethylpiperazine-N'-, 2-hydroxypronoξ-3-sulfonic acid (HEPPS) Na salt or base ring SN-, 2-hydroxyethylpiperazine-N'
-3-sulfonic acid (EPPS) Na salt or salt, etc.: N
-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS) Na salt or salt, etc.:
N-,! -Hydroxyethylpiperazine-N'-coethanesulfonic acid (HEPES) Na@ or salts, etc.;
and acids optionally combined with any of these,
There are alkalis or salts. Specific examples of preferred buffers include potassium dihydrogen phosphate-disodium hydrogen phosphate; Tr
is-sodium borate; Tris-sodium borate-E
DTA-2Na salt; Triacic acid; Triacic acid-sodium dihydrogen phosphate: Bicine; HEPPS; HE
PPS sodium salt; EPPS: EPPS sodium salt; TAPS: TAPS sodium salt, etc.

Since the pH buffer is preferably present in the enzyme-containing layer, it is generally preferable to include the pH buffer in the enzyme-containing porous development layer or reagent layer. On the other hand, the pH buffering agent in small molecules can move between layers as the water, which is the medium of the spotted aqueous liquid sample, penetrates, so it is possible that the pH buffering agent is contained in all the layers containing the enzyme and the coloring reagent composition. For example, the pH buffer may be contained only in the water absorption layer.

The multilayer analytical element of the present invention can be prepared by known methods as described in the aforementioned patent specifications.

An example of a method of incorporating a reagent composition containing an enzyme into the developing layer is disclosed in JP-A-39-7/♂4g and Japanese Patent Application JP-A-59-79/J.
2. As described in Japanese Patent Application No. 59-79/J, etc., after providing a porous development layer on the water absorption layer, an aqueous solution containing an enzyme-containing reagent composition or a solution containing an organic solvent is applied from above the development layer. There is a way to apply it.

The multilayer analytical element of the present invention has two sides of about /jmm to about 3θmm.
Cut into small pieces such as squares or circles of approximately the same size,
Tokuko Shoj7-2733/, Tokuko Shoj≦-/Gukog! Gu,
Tokukai Akira! 7-Otsu 3 Gujko, Mikiakiaki! ? -3 pieces 3! θ、Tokumyo Akira! ♂－! 0//4tq etc. can be used as a chemical analysis slide in the slide frame described in the manufacturing, packaging,
It is preferable from various viewpoints such as transportation, storage, and measurement operations. Depending on the purpose of use, it can be used in the form of a long tape and stored in a cassette or magazine, or small pieces can be attached or stored in a card with an opening.

The multilayer analysis element of the present invention can analyze cholesterol in a liquid sample by the operations described in the above-mentioned patent specifications. In other words, from about jμl to about 30μ! preferably l
From μl/! A sample tube of aqueous liquid such as whole blood, plasma, serum, etc. in the μj range was spotted on the development layer, and
Inkation is carried out at a substantially constant temperature in the range of about 2000°C to about 32°C, preferably at a substantially constant temperature around 32°C, and the water-absorbing layer or reagent-containing spreading layer is injected from the light-transparent support side. The cholesterol content in a liquid sample can be determined by reflection photometry of the optical density of the liquid sample, and by using a calibration curve prepared in advance, based on the principle of colorimetric measurement. By keeping the amount of aqueous liquid sample spotted, incubation time, and temperature constant, quantitative analysis of the test component can be performed with high precision. This measurement operation was performed by JP-A-J4-7774T4, JP-A-KAI! ? -27! Otsu 6, Tokukai Akira! Using the chemical analyzer described in R-/6/R67, etc., highly accurate measurements can be performed with extremely easy operation.

[Function] The light-transparent water-impermeable support functions as a support for the multilayer analytical element, and its light transparency makes it possible to measure color development by irradiating light from the back side of the multilayer analytical element. I have to. Since it is water-impermeable, it has the effect of stopping the permeation of the aqueous sample deposited on it. The water-absorbing layer is a layer that receives and permeates hydrophilic compounds, and particularly receives and accumulates water-soluble coloring dyes.

The water-absorbing layer also has the function of improving the developing action (metering action) of the developing layer in a short time by absorbing water from the aqueous liquid sample deposited on the porous developing layer. It has become.

The porous developing layer is a layer that develops an aqueous liquid sample spotted thereon and transfers it directly or indirectly to a water absorption layer or a reagent layer. When the developing layer contains an enzyme and a coloring reagent composition, the developing layer also functions as a layer in which the coloring reaction described below proceeds.

Cholesterol esters present in the sample are hydrolyzed by cholesterol esterase and converted to cholesterol, and cholesterol is oxidized by the action of cholesterol oxidase to produce hydrogen peroxide. This hydrogen peroxide oxidizes the teraminoantipyrine halogen-substituted derivative by the action of hydrogen oxidase, and couples the teraminoantipyrine with dichlorobenzenesulfonate to form a quinoneim7 dye, which develops color. It is presumed that during this coloring reaction, ferrocyanide ions increase the efficiency of the coloring reaction, increasing sensitivity and widening the dynamic range.

When a water-absorbing layer is provided, the water-absorbing layer also functions as a layer in which the formed dye is accumulated.

Example / 24 g/m2 of gelatin and 0.03 g polyoxyethylene nonylphenyl ether (containing ♂~/!oxyethylene units) on a polyethylene terephthalate (PET) film with a thickness of 720 μm and undercoated with gelatin.
/m2, bis(vinylsulfonylmethyl)ether 30
A gelatin layer (water absorption layer) was formed by coating and drying to a thickness of 20 μm after drying so as to have a concentration of mg/m 2 .

Next, water is supplied as dampening water to this water absorption layer at a rate of 3θg/m2, and the tricot knitted fabric is made of polyethylene terephthalate (PFI:T) spun yarn (!0 denier) that is made hydrophilic by glow discharge. Average thickness ko!Oμ
m) was pressure-bonded and laminated to provide a spread layer. .

Then, in the developing layer, make sure that the following component coverage is achieved.
An aqueous solution of a coloring reagent composition for cholesterol analysis was applied onto the knitted fabric development layer, impregnated, and dried to prepare an integrated multilayer analytical element for cholesterol quantification. The process from preparing the reagent composition to applying and drying the analytical element was repeated in the evening (J lot) to prepare the analytical element.

Component coverage of color reagent composition for cholesterol analysis (7
per m2) Methyl cellulose (methoxy group content 2 tati, viscosity in aqueous solution λo'C //2 cps) j, Og titanium dioxide fine particles (rutile type; particle size θ, ko! ~ θ, 4t 02m) 2< tg octylphenoxypolyethoxyethanol (contains 9-10 oxyethylene units) 0.7g cholesterol oxidase (EC/, /, 3.t) 3roolU cholesterol esterase (EC3, /, /, /J) 2oooIU
k oxidase (EC/, /to/, 7) 29θ0■U cohydroxy-J, J--sodium dichlorobenzenesulfonate 7. ♂g/-(2,
II, t-)lichlorophenyl)-1,3-dimethyl-4-amino-3-pyrazoline-ohone 0.61 g Potassium ferrocyanide 0.7 g Dipotassium phosphate-hydrogen 11.3 g 5N-Sodium hydroxide aqueous solution Note:! The IJium aqueous solution was added to the analytical element as a control serum Motorol IX (pH 7).
, g)/θ μl was applied, the pH value of the developed layer was 2°!
Add the amount.

Comparative example/-/ /-(z, g, za-trichlorophenyl)-1.

3-dimethyl-4-amino-3-pyrazoline-! The same procedure as in Example 1 was carried out except that guaminoantipyrine (/-phenyl-cot-3-dimethyl-4-amino-3-pyrazolin-!-one) was used at 0.3 g/7 m2 instead of -one. Integrated multilayer analytical element for cholesterol quantification (
Comparative example /-/) was prepared.

Comparative Example/-λ An analytical element (Comparative Example/-, 2) was prepared in the same manner as in Example/, except that potassium ferrocyanide was omitted from the reagent composition of Comparative Example/-/.

The completed element was cut into square chips of /jmm x /jmm, and the JP-A-7-634T! A chemical analysis slide for cholesterol quantification was completed by mounting it in the plastic mount disclosed in 2. Cholesterol concentration value taken from henolin blood on each slide/! Com g /di (C, C, A11
A drop of 70 μl of human plasma (determined by the solution analysis method according to the method of Ain et al.) was applied and incubated at 32° C. for 6 minutes.The reflected optical density of the color was measured from the PET support side using measurement light with a center wavelength of 410 nm. (Reflection optical density was measured from plasma spotting on 70 analytical slides of each lot.) In this way, variations in color-developed reflection optical density were investigated, and the results shown in Table 1 were obtained.

From the results in Table 2, it can be seen that the multilayer analytical element using the guaminoantipyrine halogen-substituted derivative and ferrocyanide of the present invention has little variation in color reflective optical density between different production lots, and produces an analytical element with uniform performance. It is clear that it can be done.

On the other hand, when guaminoantipyri/ is used, regardless of whether ferrocyanide is added, there is a large variation in color reflective optical density values between production lots, making it impossible to produce analytical elements with uniform performance. is clear.

Example 2 and Comparative Example 2 Chromogen t/-(3,!-dichlorophenyl)-co,
Instead of 3-dimethyl-4-amino-3-pyrazolin-j-one hydrogen chloride addition salt θ, jul, 0.7 jg of potassium ferrocyanide was added (Example 2), and without the addition of potassium ferrocyanide (Comparative Example, 2) A chemical analysis slide for cholesterol quantification was prepared in the same manner as in Example.

70μ of heparinized human plasma with different cholesterol concentrations was spotted on each slide, and after incubation at 32°C for 6 minutes, the center wavelength was measured. P with 4t0nm measurement light
The reflected optical density value of color development was measured from the ET support side. For this result and each human plasma, C, C, A11ain
A calibration curve was created based on the cholesterol concentration values determined by solution analysis according to the method of et al. The obtained calibration curve is shown in FIG.

From the calibration curve, it was found that the multilayer analytical element for cholesterol quantification using the halogen-substituted derivative of Nori-aminoantipyrine and ferrocyanide of the present invention has a high sensitivity because the slope of the calibration curve is large. It is clear that since the slope is large, the measurable region (dynamic range) is wide from the low concentration region to the high concentration region with highly accurate cholesterol quantification. On the other hand, it is clear that the conventional multilayer analysis element without the addition of ferrocyanide has a low sensitivity because the slope of the calibration curve is small, and the measurement accuracy is poor in the high concentration region and the dynamic range is narrow.

[Brief explanation of drawings]

FIG. 1 is a graph showing the calibration curve of the integrated multilayer analytical element for cholesterol determination of Example 2 and Comparative Example 2. In the graph, the solid line represents the analytical elements of Example 2 (combination of 4t-aminoantipyrine halogen-substituted derivative and ferrocyanide), and the broken line represents the analytical elements of Comparative Example 2 (using 4t-aminoantipyrine halogen-substituted derivative, no addition of ferrocyanide). A calibration curve is shown.

Claims (8)

[Claims] (1) A water-absorbing layer containing at least one hydrophilic polymer and a porous spreading layer are integrally laminated in this order on a light-transparent water-impermeable support, and cholesterol esterase, cholesterol oxidase, peroxidase,
In an integrated multilayer analytical element for cholesterol analysis containing a coloring reagent composition consisting of a chromogen and a coupler and a cholesterol analysis reagent composition containing ferrocyanide, (a) the chromogen is a 1-(halogen-substituted phenyl )-2,3-dimethyl-4-amino-3-pyrazoline-
(b) the coupler is a compound selected from the group consisting of phenol, a phenol derivative having a sulfo group, naphthol, a naphthol derivative having a sulfo group, and dihydroxynaphthalene. Multilayer analysis elements. (2) The halogen-substituted phenyl group of the chromogen is a 3-chlorophenyl group; 4-chlorophenyl group; 4-bromophenyl group; 4-iodophenyl group; 2,5-dichlorophenyl group; 3,4-dichlorophenyl group; Claim 1 which is one selected from the group consisting of 3,5-dichlorophenyl group; 2,4,6-trichlorophenyl group
Elements listed in Section. (3) The chromogen is 1-(2,4,6-trichlorophenyl)-2,3-dimethyl-4-amino-3-pyrazolin-5-one or 1-(3,5-dichlorophenyl)
-2,3-dimethyl-4-amino-3-pyrazoline-5
-on. (4) The element according to claim 1, wherein the coupler is a compound selected from the group consisting of phenol derivatives having a sulfo group and naphthol derivatives having a sulfo group. (5) The coupler is a compound selected from the group consisting of 3,5-dichloro-2-hydroxy-1-benzenesulfonic acid, 1-hydroxy-2-naphthalenesulfonic acid, and alkali metal salts thereof. Elements listed in Range 1. (6) The element of claim 1, wherein the coupler is 3,5-dichloro-2-hydroxy-1-benzenesulfonic acid, or its sodium or potassium salt. (7) The element according to claim 1, wherein the ferrocyanide is an alkali metal salt of hexacyanoferrate (II). (8) The element according to claim 1, wherein the ferrocyanide is potassium ferrocyanide or sodium ferrocyanide.