JPS63109799A - Dry cholesterol-analyzing element - Google Patents

Abstract

PURPOSE:To obtain a dry cholesterol-analyzing element contg. a cholesterol ester-hydrolyzing enzyme in its porous liquid-developing layer and also a specific reagent in the porous layer or its water-permeating layer and having a high colorimetric analysis sensitivity to total cholesterols. CONSTITUTION:A dry cholesterol-analyzing element has a porous liquid- developing layer and at least one water-permeating layer in a state contactable with the liquid where the porous liquid-developing layer contains a cholesterol ester-hydrolyzing enzyme and also where the porous liquid-developing layer or at least one water-permeating layer contains an alkylphenoxypolyglycidol.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a dry analytical element suitable for analyzing cholesterol in body fluids such as blood, particularly total cholesterol including protein-bound cholesterol.

[Prior Art] Methods using enzymes such as cholesterol esterase to hydrolyze cholesterol esters are well known. However, it is well known that this method requires a means other than enzymes to break the protein-cholestyrol ester bond and dissociate the cholesterol ester.

It is known from US Pat. No. 3,869,349 to use proteases together with lipases. Also, U.S. Patent No. 39
No. 25164, it is known to use a surfactant such as polyethylene glycol alkyl ether together with cholesterol esterase. Further, US Pat. No. 4,275,151 and US Pat. No. 4,274,152 describe that polyethylene glycol alkylphenyl ether (alkylphenoxypolyethoxyethanol) having less than 20 ethylene glycol units is useful.

However, when a polyethylene glycol alkyl phenyl ether containing less than 20 ethylene glycol units is contained in a dry analytical element having a porous liquid spreading layer (hereinafter referred to as a porous liquid spreading layer or spreading layer) together with an enzyme, this Surfactants may cause the liquid development area within a certain period of time to be too large, reducing the sensitivity of colorimetric analysis, or the capacity of liquid development (proportionality between the amount of supplied liquid and the development area) being insufficient, or the coating liquid The problem was that the wetting properties for the spreading layer were insufficient. Furthermore, when this surfactant is contained in a layer consisting of a hydrophilic binder such as a water absorption layer, a reagent layer, a light shielding layer, or an adhesive layer between the spreading layer and the support,
Insufficient wetting of the coating solution between one layer and the next layer,
Application was often uneven.

[Problems to be Solved by the Invention] The purpose of the present invention is to ensure that the liquid development area within a certain period of time is appropriate;
An object of the present invention is to provide a dry cholesterol analysis element that has high sensitivity in colorimetric analysis of total cholesterol including protein-bound cholesterol.

Another object of the present invention is to provide a dry cholesterol analysis element that has sufficient quantitative property (proportionality between the amount of supplied liquid and the area of development) of the liquid in the developing layer and allows uniform coating of the coating liquid.

[Structure of the Invention] The above-mentioned object of the present invention is to provide a dry analytical element having at least one water-permeable layer and a porous liquid spreading layer in a state where they can come into liquid contact with each other, the developing layer having cholesterol ester hydrolysis activity ( This was achieved by means of a dry analytical element containing an enzyme with cholesterol ester hydrolase activity) and containing an alkylphenoxy polyglycidol in said rWi layer or said at least one water-permeable layer.

[Detailed Description of the Structure of the Invention] The enzyme having cholesterol ester hydrolysis activity used in the present invention may be either lipase or cholesterol esterase. For example, Chromobacterium may originate from the pancreas) or from microorganisms (e.g. Candidalgosa), especially Chromobacterium.
Those obtained from Rium viseosus are useful.

Cholesterol esterase may be derived from animal organs or microorganisms, but those originating from microorganisms are preferred 1 For example, U.S. Pat.
What is described in No. 25164 can be used (
Candidalgosa, Actinomyces sp., Streptomyces sp., Penicillium sp., etc.).

The alkylphenoxy polyglycidol used in the present invention preferably has an alkyl group having 4 to 20 carbon atoms.
Two or more alkyl groups may be substituted on the benzene ring (
(dialkyl phenoxy polyglycidol, etc.), the benzene ring may have a substituent other than an alkyl group, such as an alkoxy group, a halogen atom, etc. The number of glycidol units per molecule is preferably in the range of 7 to 20, but the coating properties ,
The number of glycidol units is selected depending on the purpose such as expansion characteristics.

It is preferable that the alkyl phenoxy polyglycidol is contained in the porous developing layer together with an enzyme having cholesterol ester hydrolyzing activity.

In order to detect and quantify cholesterol, a detection reagent composition for a known reaction can be used. There are two types of known detection reactions:

(2) Cholesterol is oxidized by the enzymatic activity of cholesprol oxidase, and the generated hydrogen peroxide or cholestenone is detected and quantified by a known coloring reaction or by the fluorescence of cholestenone.

■Cholestenone produced by the dehydrogenation reaction by the enzyme activity of cholesterol dehydrogenase can be detected and quantified by a known reaction, or it can be conjugated to the dehydrogenation reaction to form NAD or NA.
NADH or N A D P produced by reduction of DP
tl is directly detected and quantified optically, or a colored substance produced through a chemical reaction is further detected and quantified optically.

■The coloring reagent composition for detecting hydrogen peroxide produced by cholesterol oxidase [^] contains a chromogen and a coupler, and the chromogen and coupler are oxidatively coupled by hydrogen peroxide in the presence of peroxidase to form a quinone imine dye. A composition that forms and develops color, or [1'! ] Leuco pigments or autooxidative color-forming chromogens are compounds that are oxidized by hydrogen peroxide in the presence of peroxidase to become pigments and develop color.

As a chromogen, ^11. (:lil, Biocb
4-aminoanti and phosphorus (also known as 4-aminophenazine, i.e. 1
-phenyl-2,3-dimethyl-4-amino-3-pyrazolin-5-one), 1-(2,4,6-)lichlorophenyl)-2,3- described in JP-A-59-54962, etc.
Dimethyl-4-amino-3-pyrazolin-5-one.

Trii! such as 1-(3,5-dichlorophenyl)-2,3-dimethyl-4-amino-3-pyrazolin-5-one!
ftA-4-amino-3-pyrazolin-5-one, 1-phenyl-2,3- as described in Japanese Patent Publication No. 55-25840, etc.
Dimethyl-4-dimethylamino-3-pyrazoline-5-
4-aminoantipyrine analogs such as ion can be used. Among these compounds, 4-aminoantipyrine, 1-(2,4,6-)dichlorophenyl)-2
, 3-dimethyl-4-amino-3-pyrazolin-5-one, 1-(3,5-dichlorophenyl)-2,3-dimethyl-4-amino-3-pyrazolin-5-one, and the like are preferred.

Couplers include ^nn, Cl1n, Bioch
em, 6.24-2ba 1969), Special Publication 1984-25
840, Special Publication No. 58-45599, Special Publication No. 58-186
28, JP-A-55-164356, JP-A-56=124
Phenol, 2-hydroxy-1-benzenesulfonic acid, 4-hydroxy-1-benzenesulfonic acid, 3,5-dichloro-2-hydroxy-1-benzenesulfonic acid, as described in No. 398, JP-A-56-155852, etc. Phenolsulfonic acids such as 2-hydroxy-3-methoxy-1-benzenesulfonic acid (including alkali metal salts and alkaline earth metal salts); 1-naphthol, 2-naphthol; 1,
Dihydroxynaphthalene such as 7-dihydroxynaphthalene; 1-hydroxy-2-naphthalenesulfonic acid, 1-
(including alkali metal salts and alkaline earth metal salts of naphtholsulfonic acid such as hydroxy-4-naphthalenesulfonic acid); and other phenol or naphthalene derivatives. Among these compounds, 1゜7-dihydroxynaphthalene, i-hydroxy-2-naphthalenesulfonic acid (including Na salt, Ni salt, and Li salt), 3,5-dichloro-2-hydroxy-1-benzenesulfone ? ! ! (Na
salt, ni salt, L; including salt) is preferred.

As a leuco dye, 2-
(4-hydroxy-3,5-dimethoxyphenyl)-4
, 5-bis[4-(dimethylamino)phenylcoimidazole and other triarylimidazole leuco dyes, 2-(3,5-dimethoxy-4-hydroxyphenyl)-4-[4 described in JP-A-59-193352 Diarylimidazole leuco dyes such as -(dimethylamino)phenyl]-5-phenethylimidazole, JP-A-61-49
2-(2-phenyl-3-indolyl)- as described in 60
Diarylindolylimidazole leuco dyes such as 4,5-di[4-(dimethylamino)phenylcoimidazole, 2-(4-hydroxy-3,5-dimethoxyphenyl)-3- described in JP-A-61-229868; triarylmonoacylimidazole leuco dyes such as acetyl-4,5-bis[4-(diethylamino)phenylcoimidazole, 2-(4-hydroxy-3,5-dimethoxyphenyl)-3-methyl-4,5-bis , t[4-(diethylamino)phenylcoimidazole and other triarylmonoalkylimidazole leuco dyes.

■As a method for quantifying NAD 1-1 or NADPH, a colorimetric analysis method in which a formazan dye having an absorption spectrum in the visible region is formed from an electron-accepting dye precursor in the presence of an electron transfer agent has been disclosed in Japanese Patent Publication No. It is known from No. 11395, 1973, etc. Using this reaction system, NAD H or NADPH can be quantified by spectrophotometry in the visible range.

As the electron transfer agent, diaphorase (EC1,6,4°3), N-methylphenazonium methosulfate, etc. can be used.

As a formazan dye precursor, INT or 2-
(p-iodoph,enyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride, BT i.e. 3,3'-(3,3°-dimethoxy-4,4'-biphenylene)bis[2, Although nitrotetrazolium blue (
NTB or Nl3T) or 3,3°-(3,3'-
dimethoxy-4,4'-biphenylene〉bis[2-(
p-, nitrophenyl)-5-phenyltetrazolium chloride] is preferred.

In the present invention, deoxycholic acid or a salt of deoxycholic acid may be contained in a layer containing an enzyme having cholesterol ester hydrolase activity or in a layer that can be in liquid contact with the layer (l[i!i contact layer, etc.). can. Salts of deoxycholic acid include sodium salt, potassium salt,
Lithium salts and the like can be used. Instead of deoxycholic acid, any one or two of cholic acid, lithocholic acid, chenodeoxycholic acid, taurocholic acid, glycolic acid, taurodeoxycholic acid, glycodeoxycholic acid, and their sodium salts, potassium salts, lithium salts, etc. A combination of the following can be used.

The layers contained in the element (coating It per element 1z2) of each component of the reagent composition used in the element of the present invention are as follows.

Cholesterol esterase: about 5000 to about 50,000 I
υ, preferably about 100,010 to about 30,000 IU rehabilitation:
Approximately 50 QOTtl to approximately 300,000 TIJ, preferably 10,000 I
U ~ 200,000 IU Alkylphenoxypolyglycidol: Approximately 200 mg ~
About 10g, preferably about 500611? ~About 5.0g Cholesterol oxidase: Approximately 10,001U ~ Approximately 30,000I
IJ, preferably about 15,001 U to about 20,000 IU Peroxidase: about 10,001 U to about 100,000, preferably about 2
000 rU to about 60,000 IU chromogen or leuco dye: about 0.
5 mmol to about 10 mmol, preferably about 1 mmol to about 1 mmol
About 5 mmol coupler: about 2.5 mmol to about 50 mmol, preferably about 5 mmol to about 25 mmol deoxycholic acid or salt thereof: about 200 mH to about log
, preferably about 500,611? ~ Approximately 5.0 g (same for acids or salts used in place of deoxycholic acid)
) Cholesterol dehydrogenase: Approximately 200 ■U to approximately 30,000 ■U, good*L<li approximately 300IU
~About Z million TIJNAD or NADP: Approximately 10++g~
About 50 g, preferably about 30 wag to about 20 g Electron transfer agent: about IIIg to about 1000 mg, preferably about 5 g to about 500 aig Diaphorase: about 5001 U to about 20,000 IU, preferably about 7001 U to about 10,000 rU Formazan Dye precursor: about 5 IIg to about tog, preferably about 25 mg to about 5 g The present invention can be applied to various known dry analytical elements. In particular, it can be applied to an element containing a solid carrier through which both the detection reagent system and the test liquid can pass. The elements are on a light-transparent and water-impermeable support, in addition to a porous liquid spreading layer, an undercoat layer, a water absorption layer, an adhesive layer, a coloring (or coloring) reagent layer, a reactive reagent layer, a light shielding layer, and a filtration layer. Such an analytical element, which may be a multilayer structure having layers and other layers known in the art, is disclosed in US (II Patent No. 3992158,
There are those described in various specifications such as No. 5-184356 and JP-A No. 60-222769.

When a support is used, a practical configuration of the dry analytical element of the present invention is (+) having a porous liquid spreading layer on the support.

It is preferable to have a water-absorbing layer between the support and the spreading layer.

(2) It may have a reagent layer and a developing layer in this order on a support, or it may have a water absorption layer between the support and the reagent layer.

(3) A device having a second reagent layer, a first reagent layer, and a developing layer in this order on a support. A water absorption layer may be provided between the support and the second reagent layer.

(4) It may have a reagent layer, a light shielding layer, and a developing layer in this order on a support, or it may have a water-absorbing layer between the support and the reagent layer.

(5) a second reagent layer, a light shielding layer, a first reagent layer on the support,
It may have the developing layers in this order, or it may have a water-absorbing layer between the support and the second reagent layer.

In (1) to 5) above, between the water absorption layer and the reagent layer or the spreading layer, between the reagent layer and the liquid spreading layer, between the second reagent layer and the first reagent layer, or between the light shielding layer and the reagent layer or the spreading layer. A filtration layer may be provided between the layers.

The multilayer analytical element of the present invention may include a water-absorbing layer on the light-transparent water-impermeable support (possibly via another layer such as a subbing layer). The water-absorbing layer provided in the element is preferably a layer made of a hydrophilic binder, that is, a layer whose layer-forming component is a hydrophilic polymer that swells upon absorption of water.

Hydrophilic polymers that can be used in the water-absorbing layer are natural or synthetic hydrophilic polymers having a swelling ratio upon absorption of water ranging from about 1.5 to about 20, preferably from about 2.5 to about 15 at 30°C. . Examples of such hydrophilic polymers include:
Examples include gelatin (eg, alkali-treated gelatin, acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (eg, phthalated gelatin, etc.), agarose, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, and the like.

The dry thickness of the water absorption layer ranges from about 1 μl to about 100 μl, preferably from about 3 μl to about 30 μl.

Furthermore, the water-absorbing layer can contain a surfactant (cationic, amphoteric, or nonionic) and a pH buffer, if necessary.

When the water absorption layer also serves as a detection layer, the water absorption layer has light shielding properties (
Reflective or light-absorbing) fine particles may be included as necessary.

An adhesive layer may be provided on the water absorption layer, light shielding layer, V overlayer, reagent layer, etc. for adhering and laminating the spreading layer. When the adhesive layer is wet with water, or It is preferable to be made of a hydrophilic polymer that can bond the spreading layer when swollen with water. Examples include hydrophilic polymers. Among these, gelatin, gelatin derivatives, polyacrylamide, etc. are preferred, and the dry film thickness of the adhesive layer is generally in the range of about 0.5 μl to about 20 μl, preferably about 1 μm to about 1071 μm. In addition to the adhesive layer between the reagent layer and the developing layer,
Desired 211 to improve adhesion between other eyebrows! The adhesive layer that may be provided between the reagent layer and the like can be provided by applying an aqueous solution containing a hydrophilic polymer and a surfactant added as necessary on the reagent layer, etc., using a known method.

The light shielding layer is preferably a water-permeable layer in which light-reflecting fine particles are dispersed using a hydrophilic polymer as a binder.The light-reflecting fine particles are detectable particles generated in reagent R (or water absorption N) When measuring changes in color (color change, color development, etc.) from the light-transmitting support side, the color of the aqueous liquid dotted onto the developing layer, especially the red color of hemoglobin when the sample is whole blood, etc. It also functions as a light reflective layer or background layer. As examples of the light-reflecting fine particles, titanium dioxide fine particles and barium sulfate fine particles are preferable.If necessary, the above-mentioned light-shielding fine particles may be included in the developing layer.

It is preferable that the porous liquid spreading layer is a spreading layer that has a liquid metering function. Having a liquid metering function means that the porous liquid spreading layer can substantially absorb the components contained therein when the liquid sample is dotted onto its surface. It has the function of supplying the liquid sample to the lower layer by spreading it at a rate of a substantially constant amount per unit area in the lateral direction (direction along the plane of the layer) without causing it to be unevenly distributed.

The materials constituting the matrix of the development layer include 1 paper,
Non-woven fabrics, woven fabrics (e.g. plain weave such as broad, boblin, etc.), knitted fabrics (e.g. tricot knit, double tricot knit, Milanese knit, etc.), glass fiber P paper, membrane filter (plush polymer layer), or polymer micro A three-dimensional lattice structure made of beads or the like can be used. Among these, it is preferable to use fibrous layers represented by woven fabrics and knitted fabrics.For details, see JP-A-55-164356, JP-A-57-66359, and JP-A-60-222769. Please refer to the issue.

The woven fabric or knitted fabric that can be used in the dry analysis element of the present invention is preferably subjected to degreasing treatment such as washing with water to substantially remove at least the oils and fats attached during the production of yarn, m-type fabric, or knitted fabric. .

When a liquid sample is applied to the developing layer, coloring reagent layer, reaction reagent layer, or water absorption layer and an analytical operation is performed, the pH within the element is adjusted to an appropriate value within the range of about 4.5 to about 9.0. The agent may be appropriately selected from known agents capable of maintaining pH 1118 and may be included. As a vI street agent that can be used, Nippon Kagaku complete edition "Chemistry Handbook Basic Edition" (Tokyo, Marukubi: +1. Published in 1966 > pages 1312-1320, R, M, C, Dawson
Edited by eL al DaLa for Bioches
ical Re5earct1” 2nd edition (Oxford)
at the C1arendon Press, 19
Published in 1969) pp. 476-508, '[1iocl+em
istry” Vol. 5, pp. 467-477 (1966),
“^na-IyLical Bioche+*1str
y, Vol. 104, pp. 300-310 (1980).

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

The multilayer analytical element of the present invention has a side of about 15 mm to about 30 mm.
Cut into small pieces such as squares or circles of approximately the same size,
Special Publication No. 57-28331. Utsukai Showa 56-142454.
Japanese Patent Application Publication No. 57-63452, Utility Application Publication No. 58-32350
It is preferable to use it as a chemical analysis slide by placing it in the slide frame described in Japanese Patent Application Publication No. 58-501144, etc., from various viewpoints such as manufacturing, packaging, transportation, storage, and measurement operations. (
-j! Depending on the purpose.

It can be used in the form of a long tape stored in a cassette or magazine, or in small pieces attached or stored in a card with an opening.

The multilayer analysis element of the present invention can perform quantitative analysis of cholesterol, which is an analyte, in a liquid sample by the operations described in the above-mentioned patent specifications. That is, about 5. Approximately 3 from L
A sample drop of an aqueous liquid such as whole blood, plasma, serum, urine, etc. in the range of 0 uL, preferably 8 uL to 15 LIL is placed on the spreading layer, and the sample is heated in the range of about 20°C to about 40°C for 1 to 10 minutes. Incubation is carried out at a substantially constant temperature, preferably around 37° C., to induce color development or discoloration within the element by exposing it to light at or near the maximum absorption wavelength of visible or ultraviolet light. The total cholesprol content in a liquid sample can be determined by performing reflection photometry from the light-transmitting support side using a colorimetric method using a previously prepared calibration curve. Alternatively, the total cholesterol content in the liquid sample can be determined by photometrically measuring the intensity of fluorescence within the element and using a calibration curve prepared in advance. By doing so, quantitative analysis of analytes can be performed with high precision. Measurement operation was performed using Japanese Patent Application Laid-open No. 60-125543 and Japanese Patent Laid-open No. 60-2208.
82, JP 61-294367, JP 58-161
The chemical analyzer described in 867 and the like allows highly accurate quantitative analysis to be performed with extremely easy operation.

[Left below] [Example 1] A colorless transparent smooth polyethylene terephthalate (PET) film (support) having a thickness of 180 μm and provided with a gelatin undercoat is coated with an aqueous solution for forming a coloring layer having the following composition and dried to a film thickness of 180 μm. It was coated to a thickness of 15 μm and dried to form a coloring layer.

Components of aqueous solution for forming coloring layer Alkali-treated gelatin 12 g Nonylphenoxy polyglycidol (contains an average of 10 glycidol units) 0.3 g NAD
o, sg diaphorase
28001ONTI3(*)
0.2g water
95ml* 3.3'-(3,3'-dimethoxy-4,4'-biphenylene)bis[2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride] Next, on the coloring layer, An aqueous solution for forming a light-shielding layer having the composition shown below was coated to give a dry film thickness of 7 μm, and was dried to form a light-shielding layer.

Aqueous alkali-treated gelatin for forming a light shielding layer 14g Rutile titanium oxide 70 Bisui 100 + Kai 1 On the light shielding layer, apply an aqueous gelatin solution with the following composition to a dry film thickness of 2 μm, and dry to form an adhesive layer. has been established.

Aqueous alkali-treated gelatin for adhesive layer formation 4g Nonylphenoxybolyethoxyethanol (contains 10 oxyethylene units on average) 0.1° water
Approximately 30H on top of 90g adhesive layer
Water was supplied almost uniformly to the entire surface at a rate of 7 m2 to moisten it, and then a tricot tA fabric made of PET spun yarn was made hydrophilic by glow discharge (thickness of the thread was equivalent to 50 denier, and the average thickness of the cloth was 250 μm). ) were laminated with light pressure, dried and adhered to form a fabric rA open layer.

Cholesterol analysis sample I having the following composition was placed on the cloth spreading layer.
An aqueous suspension of the composition was applied almost uniformly at a ratio of 200 ml/+n' and dried to prepare an integrated multilayer analytical element for cholesterol quantification.

Ethanol 5001 polyvinylpyrrolidone (average molecule i 360,000) (10% ethanol solution) 18g 2-amino-2-ethyl-1,3-propanediol 1
g Nonylphenoxypolyethoxyethanol (contains 40 oxyethylene units on average) fzr nonylphenoxyboriglycidol (contains IO on average in glycidol units) 3 g Sodium deoxycholate 7 g The following enzyme solution was added to the above ethanol solution and suspended.

Lipase (*) 15000 Ill
Cholesterol Dehydrogenase 5000 Ill Water 51*
An integrated multilayer analytical element for cholesterol quantification produced from Chromobacterium viscosum was cut into squares of 15 mm x 15 m5 and placed in a plastic mount to complete an analytical slide for cholesterol quantification.

Distribute 10 μl of human plasma with different cholesterol concentrations.

It was spotted on the developing layer of an analysis slide, and after incubation at 37° C. for 6 minutes, the reflected optical density was measured from the PET support side using visible light with a center wavelength of 600 nm. The results are shown in Table 1.

Table 1 [Example 2] On the same PET film as in Example 1, an aqueous solution having the following composition was coated so that the dry film thickness was 15μ steel, and dried to provide a coloring layer.

Alkali-treated gelatin 12 g nonylphenoxy polyglycidol (glycidol unit average 10 units) 0.1.

Diaphorase 3000 UNTB
O, 35゜bis(vinylsulfonylmethylcarbonylamino)methane
0.12 g water
105 g Next, an aqueous suspension having the following composition was coated to a dry film thickness of 7 μm, and dried to provide a light shielding layer.

Alkali-treated gelatin 7g Rutile titanium oxide 35g Water
501 Next, an adhesive layer containing cholesterol dehydrogenase was applied using the following formulation to a dry film thickness of 4 μm and dried.

Cholesterol dehydrogenase 39000 U NAD 0.9g Alkaline processed gelatin 20.

Nonylphenoxy polyglycidol (contains an average of 10 glycidol units) 0.2 g water
After dampening the 220 g adhesive layer by supplying dampening water at a rate of 30 g/m2, the same PETvJ yarn knitted fabric as in Example 1 was laminated and adhered while being lightly pressed to form a cloth spreading layer. Ta.

Next, on this cloth spread layer, an aqueous suspension of a reagent composition for cholesterol analysis having the following composition is almost uniformly applied at a rate of 20 On + I/m2, and dried to prepare an integrated multilayer analytical element for cholesterol quantification. did.

Ethanol 500 increments 1 polyvinylpyrrolidone (average molecular weight 360,000) (10% ethanol solution) 18 g 2-amino-2-methyl-1,3-propanediol 1
g Sodium deoxycholate 7 g Nonylphenoxybolyethoxyethanol (contains an average of 40 oxyethylene units) 6 g Nonylphenoxy polyglycidol (contains an average of 10 glycidol units) 3 g The following enzyme solution was added to the above ethanol solution.

Lipase C* > 15000 [1 water 3g*
Chromobacterium viscosu＋＊
A chemical analysis slide for cholesterol standardization was completed using the elements produced in the same manner as in Example 1. Next, a calibration curve was prepared in the same manner as in Example 1, and the values shown in Table 2 were obtained.

Table 2 [Example 3] The same results as in Example 2 were obtained in the same manner as in Example 2 except that cholesterol esterase 120001U was used instead of lipase 1500010.

Claims (1)

[Claims] A dry analytical element having at least one water-permeable layer and a porous liquid spreading layer in a state where they can come into liquid contact with each other, wherein the spreading layer contains an enzyme having cholesterol ester hydrolysis activity; Dry analytical element containing alkyl phenoxy polyglycidol in one water permeable layer.