JPS63119697A - Element for analysis of cholesterol - Google Patents

Abstract

PURPOSE:To obtain the titled dry-type liquid analysis element composed of a liquid-permeable and liquid-retaining layer and having excellent storage stability, by including cholesterol esterase or lipase and deoxycholic acid in said layer. CONSTITUTION:The objective dry-type liquid analysis element is composed of at least one liquid-permeable and liquid-retaining layer. At least one of the layer contains cholesterol esterase or lipase, deoxycholic acid or its salt and cholesterol dehydrogenase and at least one of the layer is a porous liquid- developing layer. An excellent analysis element for the quantitative determination of cholesterol can be produced by including cholesterol esterase, a lipase originated from Chromobacterium viscosum, deoxycholic acid or its salt, or cholesterol dehydrogenase in said porous liquid-developing layer.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application] The present invention relates to an analytical element for quantifying cholesterol contained in biological fluids such as blood and other liquid samples.

[Technical Problems to be Solved] As a useful method for quantitatively analyzing cholesterol, for example, a method for quantifying hydrogen peroxide or cholestenone produced by oxidizing cholesterol in the presence of cholesterol oxidase is disclosed in Japanese Patent Publication No. 8318/1983 (applicant). :National Research and Development Cooperation)
It is known for.

It is also known from U.S. Pat. No. 3,925,164 (Boehringer Mannheim) to combine this method with the release of cholesterol by cholesterol esterase for application in the determination of total cholesterol, including bound cholesterol. . The above method is for quantifying either cholestenone produced from cholesterol or 1-1202 using a known method. Although the Trinder reagent is useful for quantifying H202, the coexisting bilirubin causes errors in the analytical results. As a method of preventing interference between this and rilubin, it is proposed to coexist with ferrocyanate, as disclosed in US Pat. No. 4,291,121 (Miles et al.).

Laboratories, Inc.); however, when a ferrocyanate is included in a dry analytical element containing cholesterol esterase, cholesterol oxidase, peroxidase, and Trinder reagent (or similar hydrogen peroxide detection reagent system), storage stability of the analytical element can be improved. It was found that sex was significantly impaired.

An object of the present invention is to provide a cholesterol analysis element that has practically no inferior cholesterol analysis strength compared to dry analysis elements containing cholesterol esterase, cholesterol oxidase, peroxidase, and Trinder reagent, and has good storage stability. That's true.

The above object of the present invention is to provide a dry liquid analytical element comprising at least one liquid-permeable and liquid-retaining layer, wherein cholesterol esterase or lipase, deoxycholic acid or a salt thereof, and cholesterol dehydrogenase are respectively contained in the layer. an analytical element for quantifying cholesterol in a liquid (a layer containing cholesterol esterase or lipase, a layer containing deoxycholic acid or its salt), at least one of which is a porous liquid development layer; layer, the layer containing cholesterol dehydrogenase may be the same layer or different from each other).

The object of the present invention is also characterized in that the porous liquid spreading layer contains at least one of lipase, deoxycholic acid or its salt, and cholesterol dehydrogenase (at least one of liquid permeability and liquid retention property). This was more preferably achieved by an analytical element for cholesterol quantification (containing lipase, deoxycholic acid or a salt thereof, and cholesterol dehydrogenase in the layer).

Cholesterol esterase used in the present invention can be of various origins.

That is, it may be obtained from animal organs or from microorganisms (for example, Candidalgosa, Actinomyces sp., Streptomyces sp., Penicillium sp., etc. described in U.S. Pat. No. 3,925,164). .

The lipase used in the present invention can be of various origins, but in particular Cbromobacterium
Those obtained from Rium viscosum are useful.

In the present invention, deoxycholic acid or a salt of deoxycholic acid is used. As the salt of deoxycholic acid, sodium salt, potassium salt, lithium salt, etc. can be used. Instead of deoxycholic acid, cholic acid, lithocholic acid, chenodeoxycholic acid, taurocholic acid, glycolic acid, taurodeoxycholic acid, glycodeoxycholic acid, and their sodium salts, potassium salts, lithium salts, etc. can be used.

Cholesterol dehydrogenase used in the present invention can be from various sources, but Noca
Those obtained from bacteria of the genus Rdia are useful.

In the analytical element of the present invention, free cholesterol is produced under the action of cholesterol esterase or lipase and deoxycholic acid or its salt, and cholestenone is produced from cholesterol under the action of cholesterol dehydrogenase. At this time, NAD or NAD
When P coexists, NAD II or NADPH
is produced, so cholesterol can be quantified by quantifying the produced NADH or NADPH.

As a method for quantifying NAD H 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 was proposed in JP-A-49 -11395 etc. Using this reaction system, NADH or NADP H can be quantified by spectrophotometry in the visible range.

As an electron transfer agent, diaphorase, N-+5etl+
ylphenazoniu++ methosulfa
te etc. can be used.

As a formazan dye precursor, INT or 2-(p-iodopl+enyl)-3-(p-ni
trophenyl)-5-phenylteLraz
olium chloride, I3T or 3°3'-(3,3'-dimeLboxy-4,4'
-biphenylene)-bis[2°5-di
phenylteLrazolium cl+Iori
de], 3.3'-(4,4°-biphenylene
e)-bis[2,5-diphenyltetraz
oliumcbroride], etc., but nitrotetrazolium blue (NTBt or NBT
) i.e. 3.3'-(3,3'-dimet!+
oxy-4,4'-bipbenylene)-bi
s[2-(p-ni Lropbenyl)-
5-pheny I tetrazo I iumeh
loridel is preferred.

The content of each component of the reagent composition used in the element of the present invention (coating Ji per element 112) is as follows.

Cholesterol esterase; approximately 500 to 50,000
IU, preferably about 1000-30000 II
J lipase; about 5,000 to 300,000 I U, preferably 10,000 to 200,000 I U Deoxycholic acid or its salt; about 0.2 to 10 g, preferably 0.5 to 5
g (same amount in case of acid or salt used in place of deoxycholic acid) Cholesterol dehydrogenase; 200-30000
ru, preferably 300-20000 I UNAD or NADP, about 110 l11-50 g, preferably 30 cocoons g-20 g Electron transfer agent; 1-11000 II1, preferably 5-5
00 mg for diaphorase 500-20000
IU, preferably 700-10000 IU formazan dye precursor; about 5B-Log, preferably 25
5 g to 5 g The present invention can be applied to various known dry analysis elements. In particular, it is applicable to elements that include a solid support through which both the detection reagent system and the test liquid can permeate. The element is a porous liquid-extended 17F on a light-transparent/liquid-impermeable support.
In addition to IM, there are undercoat layer, water absorption layer, adhesive layer, reaction reagent layer,
It may also be a multilayer structure with a light blocking layer, a filter layer, and other layers known in the art. Such analysis elements are, for example, U.S. Patent No. 992,158 and Japanese Patent Application Laid-Open No.
-164356.

When a support is used, practical configurations of the analytical element of the present invention include (1) a structure having a liquid spreading layer on the support; A water absorbing layer may be provided between the support and the liquid spreading layer.

(2) Having a reagent layer and a liquid spreading layer in this order on a support. A water-absorbing layer may be provided between the support and the reagent layer.

In this case, cholesterol esterase or lipase is contained in the liquid spreading layer or reagent layer, preferably in the liquid spreading layer.

(3) A device having a second reagent layer, a first reagent layer, and a liquid developing layer in this order on a support. A water absorption layer may be provided between the support and the second reagent layer. In this case, cholesterol esterase or lipase is contained in the liquid spreading layer, the second reagent layer, or the first reagent layer, and is preferably contained in the first reagent layer or the liquid spreading layer.

(4) A support having a reagent layer, a light guide layer, and a liquid spreading layer in this order. A water absorption layer may be provided between the support and the reagent layer. In this case, cholesterol esterase or lipase is contained in the liquid spreading layer or reagent layer, preferably in the liquid spreading layer.

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

In this case, cholesterol esterase or lipase is contained in the liquid spreading layer, the second reagent layer, or the first reagent layer, and is preferably contained in the first reagent layer or the liquid spreading layer.

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

Light transmittance that can be used in the dry analysis element of the present invention
Examples of water-impermeable supports include polymers such as polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, cellulose esters (e.g., cellulose triacetate, cellulose acetate propionate, etc.), and have a thickness of about 50 μm to about 50 μm. 1zz, preferably a film or sheet-like transparent support ranging from about 80 μm to about 300 μm.

If necessary, an undercoat layer may be provided on the surface of these supports to strengthen the adhesion between the support and the detection layer or other layer provided on the support. Further, instead of the undercoat layer, the surface of the support may be subjected to physical (eg, glow discharge, corona discharge) or chemical activation treatment to improve the adhesive strength.

In the analytical element of the present invention, a water-absorbing layer can be provided on the light-transmitting/water-impermeable support (depending on the case, via another layer such as an undercoat layer). The water-absorbing layer is generally a layer in which the dye formed in the presence of the test component does not substantially diffuse, and is made of a hydrophilic polymer that swells by absorbing water. The hydrophilic polymer used in the water-absorbing layer is generally a natural or synthetic hydrophilic polymer having a swelling ratio at 30° C. in the range of about 1.5 to about 20, preferably about 2.5 to 15.
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-absorbing layer is usually in the range of about 1 μm to about 100 μm, preferably in the range of about 3 μm to 30 μl. In the water absorption layer, surfactants (cationic,
Anionic, amphoteric, or nonionic) or tHTr agents can also be included.

In the analytical element of the present invention, a detection layer can be provided on the light-transparent/water-impermeable support (depending on the case, via another layer such as an undercoat layer). The detection layer is generally a layer in which a dye formed in the presence of the analyte is diffused and can be optically detected through a light-transparent support. The detection layer can be composed of the same kind of wood-philic polymer as the water-absorbing layer. The detection layer may contain a hardening agent, a surfactant (cationic, anionic, amphoteric, or nonionic), a cherry-blossom agent, and light-shielding (reflective or absorptive) fine particles as necessary. I can do it.

An adhesive layer for adhering and laminating the spreading layer may be provided on the layers such as the water absorption layer, light shielding layer, filtration layer, and reagent layer. Preferably, the adhesive layer comprises a hydrophilic polymer capable of adhering the spreading layer when wetted with water or swollen with water. Examples of hydrophilic polymers that can be used in the adhesive layer include hydrophilic polymers similar to those used in the water absorption layer. Among these, gelatin, gelatin derivatives, polyacrylamide, etc. are preferred. The dry film thickness of the adhesive layer generally ranges from about 0.5 µl to about 20 µl, preferably from about 1 µl to about 10 µl. The adhesive layer can be provided by applying an aqueous solution containing a hydrophilic polymer and a surfactant added as necessary onto the reagent layer, etc. by a known method.

Examples of cherry-picking agents that can be contained in the reagent layer and other layers of the analytical element of the present invention include carbonates, borates, phosphates, and [Liochemis Lry Magazine Vol. 5 (No. 2)]
, pp. 467-477 (1966), and the like.

These buffers can be selected and used with reference to known literature such as Basic Experimental Methods for r-Proteins and Enzymes 1 (written by Takeshi Horio et al., Nankodo, 1981).

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.
When measuring the detectable changes (color change, color development, etc.) that occur in a sample from the light-transmitting support side, the color of the aqueous liquid dotted onto the developing layer, especially when the sample is whole blood. In addition to blocking the red color of hemoglobin, it also functions as a light-reflecting layer or a background layer. Examples of light-reflecting fine particles include pigment fine particles, titanium dioxide fine particles (
Rutile type, anatase type or brutzite type microcrystal particles with a particle size of about 0.1 μl to about 1.2 μl),
Examples include barium sulfate fine particles. Preferred are rutile titanium dioxide fine particles.

Examples of the hydrophilic polymer binder include the hydrophilic polymers of the type used in the water absorption layer described above, as well as weakly hydrophilic regenerated cellulose. Among these, gelatin, polyacrylamide, etc. are preferred. Note that a known hardening agent can be added to gelatin or gelatin derivatives.

The light-shielding layer can be provided by applying an aqueous dispersion of light-shielding fine particles and a hydrophilic polymer onto the water-absorbing layer, reagent layer, etc. by a known method and drying it. In the analytical element of the present invention, the above-mentioned light-shielding fine particles may also be contained in the spreading layer, if necessary.

Preferably, the spreading layer is a spreading layer having a liquid metering effect. Having a liquid metering effect means that the liquid sample that is dotted onto the surface of the layer is distributed at an approximately constant rate per unit area in the plane direction of the layer, without substantially unevenly distributing the liquid sample contained therein. It has the effect of spreading the word.

Materials constituting the matrix of the spreading layer include filter paper,
Membrane filters formed from non-woven fabrics, woven fabrics (e.g. plain weave such as broadcloth), knitted fabrics (e.g. tricot knit, double tricot knit, etc.), plush polymers.
A three-dimensional lattice structure made of 1 or polymer microbeads or the like can be used. Among these,
Fibrous layers typified by woven fabrics and knitted fabrics are preferred.

For details on these, see Japanese Patent Application Laid-Open No. 55-164356,
Reference may be made to No. 57-66359 and No. 60-222769.

It is preferable that the woven fabric or knitted fabric used in the analytical element of the present invention has been subjected to a degreasing treatment such as washing with water to substantially remove oils and fats adhering to it during the production of yarn, woven fabric, or knitted fabric.

The integrated multilayer analytical element produced in accordance with the present invention is approximately -15
zv into small pieces of approximately 30J11 squares or circles of approximately the same size, and cut into small pieces such as 30J11 squares or circles of approximately the same size,
Utility Model Publication No. 55-32350 and Special Publication No. 58-5011
It is preferable from all points of view of manufacturing, packaging, transportation, storage, and measurement operations to use it as an analysis slide by placing it in a slide frame or the like as disclosed in Publication No. 44 Isosceles.

The dry analytical element manufactured according to the present invention can be prepared from approximately 5 μl to approximately 3 μl.
0μ, preferably about 8-11 to about 15μr, of an aqueous liquid sample is spot-fed onto the spreading layer, optionally at a temperature substantially in the range of about 20°C to about 45°C, preferably 35°C to 40°C. Incubate at constant temperature.

Thereafter, detectable changes such as color changes and color development within the dry analytical element are measured by reflectance photometry from one side (from the light-transmitting support side in the case of wood-shaped multilayer analytical elements) in the liquid sample using the principle of colorimetry. Analyze the component to be measured.

The present invention will be explained in more detail below using Examples.

(Margins below) [Example 1] The following composition (ao) was applied on a colorless transparent smooth film of polyethylene terephthalate with a thickness of 180 μm that was undercoated.
An aqueous solution of was applied so that the dry film thickness was 15 μm and dried (coloring layer).

(a,) Alkali-treated gelatin 12 g surfactant
0.1g (Olin 5urfacLa
nt IOG ) NAD 0
．． 8g diaphorase 28001ON
TB (*) 0.2g water
95a+l*3,3'
-(3,3'-dimcLl+oxy-4,4'-b
ipbenylene)-13is[2-(p-ni
Lropheny I)-5-pheny l-
211tetrazoliua+ cl+Ioride
] Next, an aqueous solution having the following composition ('b o ) was applied onto the above coloring layer so that the dry film thickness was 7 μm, and dried (
Light path layer) (bo) Alkali-treated gelatin 14g Rutile titanium oxide 70g Water
Apply an aqueous gelatin solution on 100 ml of the above light shielding layer so that the dry layer has a thickness of 2 μm, and wet the entire surface of the dried gelatin layer at a rate of about 30 g/vn2. After drying, a polyester tricot knitted fabric (thickness 0.5 mm) was made hydrophilic by glow discharge.
25 n1m), laminated 1- while applying light pressure.
L, dried and adhered (spreading layer).

Next, on this cloth (spreading layer), a suspension having the following composition (do) was almost uniformly applied at a rate of 200 cc/m 2 and dried to produce an integrated multilayer analytical element for cholesterol quantification. .

(do> Ethanol 500 ml Polyvinylpyrrolidone 18 g (10% ethanol solution) Polyoxyethylene nonylphenyl ether (11.40) 6 g2-
Amino-2-ethyl-1,3-propanediol 1 g Polyoxyethylene octylphenyl ether (n; 10) 3 g Sodium deoxycholate 7 g The following enzyme solution was added to the above ethanol solution and suspended.

Lipase (*) 15000 U Cholesterol dehydrogenase 5000 U Water
5ml* Chrot
The thus completed analytical film produced from Nobacterium viscosu+n was cut into 15×15 mm squares and placed in a plastic mount to complete the analytical slide for cholesterol emplacement.

10 μp of human plasma with different cholesterol concentrations was spotted on an analysis slide, kept at 37°C for 6 minutes, and then heated at a wavelength of 600°C.
Reflection optical density was measured from the support side at nm. The results are shown in Table 1.

Table 1

Claims (9)

[Claims] (1) A dry liquid analysis element comprising at least one liquid-permeable and liquid-retaining layer, wherein cholesterol esterase or lipase, deoxycholic acid or a salt thereof, and cholesterol dehydrogenase are each contained in at least one of the layers. and at least one of the layers is a porous liquid spreading layer, an analytical element for quantifying cholesterol in a liquid (layer containing cholesterol esterase or lipase, layer containing deoxycholic acid or a salt thereof, cholesterol dehydrogenase) may be the same layer or different layers). (2) The analytical element according to claim 1, wherein the porous liquid spreading layer contains cholesterol esterase. (3) The analytical element according to claim 1, wherein the porous liquid spreading layer contains lipase. (4) Chromobacterium in the porous liquid spreading layer
The analytical element according to claim 1, characterized in that it contains a lipase obtained from C. umviscosum. (5) The analytical element according to claim 1, wherein the porous liquid spreading layer contains cholesterol dehydrogenase. (6) The analytical element according to claim 1, wherein the porous liquid spreading layer contains deoxycholic acid or a salt thereof. (7) Claim 1 characterized in that the porous liquid spreading layer contains lipase and deoxycholic acid or a salt thereof.
Analysis elements described in. (8) The analytical element according to claim 1, wherein the porous liquid spreading layer contains lipase, deoxycholic acid or a salt thereof, and cholesterol dehydrogenase. (9) Chromobacterium in the porous liquid spreading layer
The analytical element according to claim 7 or 8, characterized in that it contains lipase obtained from C. umviscosum.