JPH02210265A - Analysis element - Google Patents

Abstract

PURPOSE:To rapidly and conveniently perform the quantitative analysis of the specified component under HDL-fractionation by providing a layer contg. an agent for fractionating the high-specific-gravity lipoprotein in a liq. sample contained in the element above the layer in which the measurement reaction of the specified component is carried out. CONSTITUTION:At least one reagent layer is placed on a support, and a porous development layer is set thereon. The specified component is analyzed in the fractionation of the high-specific-gravity lipoprotein in the liq. sample. At this time, a reagent for fractionating the lipoprotein is incorporated in the element layer contg. the reagent for detecting the specified component, the lipoprotein fraction not to be measured is settled as an insoluble precipitate, and only the specified component under HDL fractionation is measured. In this case, the layer contg. the fractionating agent on the liq. dripping surface side is provided above the layer contg. the reagent used in the measurement reaction of the specified component, and the IDL and VLDL contained in the liq. dripped in the layer are precipitated. The precipitate is collected by the matrix constituting the layer, a liq. contg. only the HDL fraction is supplied to the lower layer, and the pretreatment for fractionation is dispensed with.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an analytical element, particularly an analytical element for quantifying a specific component in a liquid. The present invention relates to an analytical element used for quantitative analysis of specific components, particularly lipid components, contained in fractions (abbreviated as HDL).

[Conventional technology]

In the past, many methods have been developed for analyzing specific components in liquid samples, but these can be roughly divided into two types: reaction systems in which the reaction takes place in a solution, and reaction systems in which the reaction takes place in a solid phase carrier.
It can be divided into 8 categories. Analytical reactions in solution systems (
Wet chemistry (hereinafter referred to as wet chemistry) is widely known, from manual methods that do not use any machines at all to automatic analysis equipment. Particularly, progress has been remarkable in the field of clinical chemistry, and in recent years various automatic quantitative analysis instruments for clinical tests have been introduced into clinical laboratories of hospitals.

However, the above-mentioned method basically requires 1. It has the following drawbacks. That is, the analysis process requires a large amount of water, especially purified pure water or distilled water, resulting in increased energy consumption.

In addition, various automatic analysis devices are themselves extremely expensive, require a great deal of skill to operate, and not only require a great deal of time and effort, but their waste fluids inevitably pollute the environment. It has the disadvantage of causing

In contrast, analytical reactions in solid phase systems (hereinafter referred to as dry reactions)
Analytical methods using chemistries (chemical methods) are also widely used, but these are carried out using 2F paper or the like impregnated with reagents.

The f paper mentioned above is, for example, US Patent No. 3,050,375 or US Patent No. 3. [No. 161,523] As described in each specification, etc., it is made by impregnating a water-absorbing fibrous carrier such as f paper with a reagent solution and drying it. These are generally called analytical test strips or simply test pieces.
The concentration of specific components in the liquid sample is determined by dropping a liquid sample onto the test piece or by immersing the test piece in the liquid sample and measuring the change in color or concentration of the test piece with the naked eye or with a reflection densitometer. This determines the level.

Although these test pieces are useful because they are easy to handle, easy to use, and provide immediate results, their composition limits them to semi-quantitative or qualitative analysis.

On the other hand, multilayer analytical elements are known that use dry chemistry, which is easier to operate than the conventional analytical methods described above, and that also have high quantitative performance. For example,
No. 3-21677, Jikai @ No. 55-164556, No. 5
Multilayer analytical elements are described in Japanese Patent Application No. 7-125847, Japanese Patent Application No. 56-65446, and Japanese Patent Application No. 56-189784.

According to the devices described in these documents, all reagents used in analytical reactions are contained in a single analytical device, and a certain volume of serum or whole blood is dropped onto the above device at a certain temperature for a certain period of time. After keeping it warm, the reflection density is measured from the support side, and it is possible to determine the substance concentration from this reflection density.

The above method has a much higher analytical accuracy than the conventional test strip type method, and has performance equivalent to or better than wet chemistry without having to mix the reagent in advance.

In recent years, it has become indispensable in clinical medicine to quantitatively analyze specific components in blood, especially specific components in HDL fractions. Conventionally, when measuring a specific component in an HDL-fraction, there are two steps: an operation for fractionating lipoproteins, and an operation for measuring a specific component in the fractionated HDL.

To fractionate HDL, ultracentrifugation, gel P passing method, polyanion precipitation method, etc. are used. A dry analytical element using the above-mentioned dry chemistry for measuring cholesterol is described in JP-A-61-177997 and other publications.

[Problem to be solved by the invention]

In order to quantitatively measure the content of a specific component in the HDL-fraction in biological body fluids, especially blood, it is necessary to go through the two steps of VC') button back fractionation operation as described above, followed by analysis of the specific component. It has nine parts that require complicated and time-consuming operations.

An object of the present invention is to provide an analytical element that eliminates these disadvantages.

[Means to solve the problem]

To summarize the present invention, the present invention relates to an analytical element, which has at least one reagent layer on a support and a porous spreading layer above the reagent layer, and which is used for high-density Bottonbac fractionation of a liquid sample. In an analytical element for analyzing a specific component, the element has a layer t-containing a fractionating agent for fractionating high specific gravity botanicals in a liquid sample, and the core layer is used for measuring the specific component. It is characterized by being provided above the layer where the reaction takes place.

As a result of intensive studies, the present inventors have succeeded in obtaining an analytical element that can easily, quickly, and accurately measure a specific component in an HDL-fraction by converting a two-step operation into one step.

The present invention contains a reagent used for lipoprotein fractionation in the layer of an analytical element that requires a detection reagent for a specific component, converts the lipoprotein fraction that is not subjected to measurement into an insoluble precipitate, and
This method was completed by making it possible to measure only specific components in the fraction.

As the reagent used in the above-mentioned Noribotanbaku fraction, reagents commonly used in polyanion precipitation methods can be used. For example, any combination of a polyanion such as heparin, dextran sulfate, or sodium phosphotungstate and a divalent metal ion such as magnesium, calcium, or manganese can be used.

These reagents insoluble precipitate the low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) fractions, but do not precipitate the HDL fraction.

In conventional methods, these reagents are added to a test substance such as serum to precipitate LDL and VLDL, followed by centrifugation. It requires a two-step operation to measure the following.

The analytical element according to the present invention has a layer containing the above-mentioned fractionating agent above the layer containing the reagent system used in the reaction for measuring a specific component, that is, on the side on which the liquid is dropped. The layer containing the fractionating agent is preferably the uppermost layer, that is, the layer with which the liquid comes into contact for the first time, and the LDL and VLDL contained in the dropped liquid precipitate in this sea. In this case, advantageously, the matrix of substances constituting the layer traps the resulting precipitate, so that the lower layer is supplied with a liquid containing only the HDL fraction. Therefore, pretreatment for fractionation was no longer necessary.

Furthermore, whole blood can be used as a sample by providing this pigeon with a blood cell filtration function as described in JP-A-61-96466. In this case, plasma separation can be performed at the same time, which is more efficient.

This layer containing the fractionating agent can also serve as a spreading layer that supplies a constant volume t of liquid per unit area to the lower layer.

The spreading layer according to the present invention has the function of (1) uniformly distributing a fixed volume of liquid sample per unit area to the reagent layer. Moreover, in addition,
The performance described in Publication No. 7, that is, (2) the ability to remove substances or factors that inhibit analytical reactions in a liquid sample, and/or (3) the ability to transmit through the support when performing spectrophotometric analysis. It is preferable that it has the function of performing a background effect of reflecting measurement light. Therefore, the development layer according to the present invention is a layer having only the function +1) above;
In addition to (1), it may be a layer having functions (2) and/or (3), or (11)
It is also possible to separate a plurality of functions including + as appropriate and use a separate layer for each function. Furthermore +11. +2
A layer having two functions among the functions 1 and (3);
The remaining single functional layers can also be used in combination.

In the present invention, expansion/i1 includes a first expansion layer having functions (1) and (2), and a first expansion layer having functions (2) and/or (3) in addition to (1). Preferably, it consists of two development layers.

For the first spreading layer, for example, a material used as a filtering material having the function of removing blood cells from whole blood can be used.

Commonly available commercially available glass fiber filter membrane filter-1 cotton wool fiber products may be mentioned. Although any of these can be used in the analytical element of the present invention, glass fiber filters are particularly preferred. Examples of glass fiber filters include those described in JP-A No. 57-53661, C) A-100 (thickness: approximately 450 μm), and GA-200 (commercially available from Toyo Roshi Co., Ltd.).
Thickness approx. 750 μffi] and GB-10OR (thickness approx.
00 μm). In order to contain the fractionating agent in this layer, the particle size is 5 to 10% in a solvent that does not dissolve the fractionating agent.
It is obtained by dispersing it into fine particles of about μW1, immersing it in the filter medium, and drying it. Since the fractionating agent is in the form of fine particles, it can be trapped in the matrix of the filter medium, so it can be contained efficiently. The filter material containing the fractionating agent thus obtained is placed on the second spreading layer.

For installation, conventional methods can be used. For example, physical pressure bonding, adhesion using various adhesives, etc. can be used.

By using this stiff filter material, it is possible to measure specific components using whole blood as a sample.

The first spreading layer may also be a skin having a particle combination structure as described in JP-A-58-9mm67. It is composed of a particle assembly that is a non-swellable three-dimensional lattice with interconnecting voids with a porosity of 25 to 80 cm, is non-swellable, impermeable to the sample solution, and has reactive groups. It is characterized by consisting of heat-stable organic polymer particles with a size of 1 to 10 μm. When coating this layer, it is desirable to disperse a fractionation reagent in the coating solution so that the fractionation reagent is physically adsorbed onto the particles after coating and drying, thereby forming a fractionation filtration layer.

In addition to the fractionation reagent, surfactants, buffers, etc. can be added to these first spreading layers as desired. In this fractionation filtration layer, when a sample solution is dropped, lipoprotein components that are not subjected to measurement by the fractionation reagent form a precipitate and are captured within the constituent layer. The measured HDL component does not form a precipitate and permeates into the lower layers. However, in this case, in order to prevent the precipitate that was not captured within the layer from penetrating into the lower layer together with the sample solution, the layer below this bottle, that is, the layer located on the opposite side from the surface where the liquid is supplied, Preferably, the second spreading layer has a filtering function. At the interface between the first developing layer and the second developing layer, the precipitate is completely captured, and a liquid containing only the HDL fraction that does not precipitate below the second developing layer is supplied. This second development layer preferably has functions (2) and/or (3) in addition to (1) above, in order to supplement the functions of the first development layer. for example,
The second spreading layer is a spreading layer of a non-fibrous porous medium called a plush polymer consisting of titanium dioxide and cellulose diacetate, which is described in Japanese Patent Publication No. 53-21.677. Spreading layer of hydrophilized fabric described in Japanese Patent Application Laid-open No. 164356, JP-A-57-94658
No. 57-125847, No. 57-197466, and No. 58-70161, etc., the fiber structure development layer described in each publication, JP-A No. 58-90167, the particle combination structure development layer described in each publication Examples include layers.

The thickness of the second spreading layer in the analytical element of the present invention should be determined by its porosity, and is preferably about 100 to about 500 μm, more preferably about 150 to 35 μm.
It is 0 μm. Further, the porosity is preferably about 20 to about 8
It is 5 yen.

Furthermore, various other additives such as preservatives, buffers, surfactants, etc. can be added as desired.

In particular, surfactants can be effectively used to adjust the permeation rate when a liquid sample is applied to the element of the present invention.

As usable surfactants, it is possible to use both ionic (anionic or cationic) and nonionic surfactants, but nonionic surfactants are effective.Nonionic surfactants Examples of agents include 2,5-di-
Examples include polyalkylene glycol derivatives of alkyl-substituted phenols such as t-butylphenoxypolyethylene glycol, p-octylphenoxypolyethylene glycol, and p-innonylphenoxypolyethylene glycol, and polyalkylene glycol esters of higher fatty acids. These surfactants have the effect of regulating the rate of penetration of the liquid sample into the receiving layer and at the same time suppressing the occurrence of undesirable "chromatographic phenomena."

The above-mentioned surfactant can have a t selected from a wide range, but it should be 10% by weight or more based on the weight of the coating solution (LO
It is possible to use i% by weight of O5, preferably 6% by weight to α05% by weight.

The analytical element according to the present invention can be used to measure many components by containing measurement reagents suitable for each target substance to be measured. For example, by containing commonly used reagents such as cholesterol, triglycerides, and phospholipids,
Quantitative analysis of each component contained in the HDL fraction can be performed.

Quantitative analysis of HDL-cholesterol will be described below as an example, but the present invention is not limited to quantitative analysis of HDL-cholesterol.

It is known that cholesterol exists as a mixture of cholesterol nysteltate and free cholesterol in HDL fractionated by a fractionating agent. These are detected by the reactions described below.

Cholesterol + Fatty Acid Cholesterol Oxidase (2) Cholesterol Cholestemono 10 HIO + 31 H2O2 Detection Reaction (1) and (2) It is desirable to contain it in a layer below the spreading layer, preferably in the second spreading layer.

At this time, in order to make the enzyme be incorporated into the core layer, it is desirable that the core layer be coated with water or a similar system. However, when this method is used, migration of water-soluble reagents from the reagent layer is induced, making it impossible to achieve sufficient analytical performance. To solve this problem, it is recommended to coat the layer with a non-aqueous solvent, but if the enzyme is directly dispersed in the layer, the enzyme will be denatured and deactivated by the organic solvent, resulting in a significant decrease in sensitivity. cause

In order to solve the above problems, in the present invention, it is preferable to use protein and/or polypeptide compounds that do not contain interfering substances, as described in JP-A-61-177997.

As used in the present invention, "a substance that substantially interferes with the analysis and the reaction" refers to a specific component desired for analysis and a
# means a protein and/or polypeptide compound that is substantially free of elementary activity and does not contain substances that substantially interfere with the reaction that induces the specific component into a detectable substance.

Preferred examples of the protein and/or polypeptide compounds defined above include albumin (bovine serum albumin, ovalbumin, etc.), globulin, gelatin, and gelatin decomposition products.

Although the ratio of the enzyme to the protein and/or polypeptide compound that does not contain interfering substances can be selected from a wide range, the amount of each protein is preferably 1 to 1 to 5000 by weight; In particular, it is particularly suitable to use the ratio in the range of 1:1 to 1:200.

In the present invention, examples of organic solvents for forming a porous layer containing a mixture of an enzyme and a protein and/or a polypeptide compound include xylene, benzene, toluene, methanol, ethanol, butanol, acetone, chloroform, Examples include non-aqueous solvents such as tetrahydro-7rane, but preferred among these are water-immiscible organic solvents such as xylene, benzene, toluene, butanol, and chloroform.

When peroxidase is used, a hydrogen donor and a coupler are used in combination according to conventional methods.

Examples of hydrogen donors include 4-substituted antipyrine disclosed in JP-A No. 48-52158 and JP-A No. 55-2.
2-hydrazinopendithiazoline disclosed in Publication No. 0471, p-halogenophenol disclosed in Publication No. 55-148100 (hereinafter referred to as
-174099), and also JP-A-157-157
No. 192, No. 57-94655, and No. 57-174
It is disclosed in the No. 099 Specification Publication. - or p-phenylenediamine compounds.

Preferred among these are 4-substituted antipyrine and p-phenylenediamine compounds, especially 4-substituted antipyrine and p-phenylenediamine compounds.
Preferred are aminoantipyrine and p-7 unilenediamine compounds listed as specific examples in JP-A No. 57-94655.

The hydrogen donor can be used in a widely selected amount, but in the range αo1 to too6 mmol/m2, preferably α05 to 50 mmol/m2.

Examples of couplers include acylacetamide compounds disclosed in JP-A-57-94654;
Pyrazolone compounds disclosed in No. 94656 and No. 57-174099, phenol compounds disclosed in No. 57-94655 and No. 57-174099, No. 57-94655 and No. 57-
Examples thereof include naphthol compounds disclosed in Japanese Patent No. 174099 and N,N-disubstituted aniline compounds disclosed in Japanese Patent No. 57174099. Preferred among these are pyrazolone compounds, phenol compounds, and naphthol compounds, especially those disclosed in Japanese Patent Application Laid-open No. 5
No. 7-94656, No. 57-94655, No. 57-9
Compounds listed as specific examples in No. 4655 and No. 57-174099, and 7-chloro-5-(
2-(2-hexyldecylsulfonylene ethyl)-6-
Methyl-pyrazolo-(3,2-a)-s-)lyazole is preferred, the coupler can be used in widely selected amounts, but α, 1 to 100 mmol/m”, preferably α
It can be used in a range of 5 to 50 mmol/mz.

The analytical element of the present invention includes, on a support, at least one reagent layer comprising a hydrophilic colloid and containing at least one reagent that reacts with a component in a liquid sample, and above the reagent layer, a reagent layer containing at least one reagent that reacts with a component in a liquid sample. Development that permeates or passes into the reagent layer#
It is made by laminating layers in sequence.

The support according to the present invention may be of any conventionally known type, and suitable ones include those that are liquid-impermeable and light-transparent, such as cellulose acetate, polyethylene terephthalate, polycarbonate, or polystyrene. A variety of polymeric materials are suitable for this purpose. Furthermore, in addition to the above polymer materials, inorganic materials such as glass can be used as well. Although the thickness of the support according to the present invention is arbitrary, it is preferably about 50 to about 250 μm. Further, one side surface of the observation side of the support according to the present invention can be arbitrarily processed depending on the purpose. Furthermore, it is possible to improve the adhesion between the reagent layer and the support by using a light-transmitting undercoat layer on the side of the support on which the reagent layer is laminated, depending on the case.

The reagent layer according to the present invention is composed of a hydrophilic binder, and can be separated into two or more reagent layers.

As the binder, gelatin, gelatin derivatives such as pentatalated gelatin, water-soluble cellulose derivatives such as hydroxyethylcellulose and carboxymethylcellulose sodium salt, polyvinyl alcohol, polyacrylamide, polymethacrylamide, poly(mono- or dialkyl-substituted) acrylic mid, Poly(mono or dialkyl substituted)
Examples include methacrylamide and water-soluble copolymers thereof.

The analytical element of the present invention may include, for example, the reflective layer described in U.S. Pat. No. 5,992,158, the nine undercoat layers,
Radiation blocking skin as described in U.S. Pat. No. 4,042,555, U.S. Patent No. 4.066.403 #1
-Barrier layer described in iF, U.S. Pat. No. 4,166,09
Migration prevention layer described in specification No. 5, JP-A-55
The scavenger layer described in Japanese Patent No. 4,110,079 and the breakable bond-like member described in US Pat.

The various layers of these analytical elements are sequentially formed on the support according to the present invention by appropriately selecting and using a slide hopper coating method, an extruded metal cloth method, a dip coating method, etc., which are conventionally known in the photographic industry, according to the desired configuration. By laminating, layers of arbitrary thickness can be applied.

f of a specific component in a liquid sample using the analytical element of the present invention.
t-1 It can be measured by reflection spectrophotometry from the support side according to the present invention according to the initial opening method or the reaction termination method. The amount of the specific component can be determined by applying the measured values obtained by K in this manner to a friend calibration curve prepared in advance.

The amount of liquid sample applied to the analytical element of the present invention can be determined arbitrarily, but is preferably about 5 μt to about 50 μt.
Lt, more preferably 5 tit.

It is 20μt. Preferably, a typical 10 ttl liquid sample is applied.

The analytical element of the present invention can be used for the analysis of whole blood, serum, and plasma without any disadvantage. Furthermore, other body fluids such as urine, lymph, spinal fluid, etc. may be used without any disadvantage. If whole blood is used, the aforementioned radiation blocking layer or other reflective layer can be provided, if necessary, to avoid interference of the radiation for detection by blood cells.

The analytical reaction used in the analytical element of the present invention can be arbitrarily determined depending on the purpose, but is useful in the field of clinical chemistry, in particular for analyzing components in biological liquid samples, such as blood or urine. used.

〔Example〕

Hereinafter, the unexploded BAf will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 A reagent layer having the following composition was coated on a transparent subbed polyethylene terephthalate support having a film thickness of 180 mm.

Gelatin 15097m” Peroxidase 13000
U/;+s” ascorbic acid oxidase
9500 U/m” Sodium triisopropylnaphthalene sulfonate
α56121.2-bis(vinylsulfonylethane)
0.2097m” dibutyl phthalate
1.0297m'' Sodium azide α169/reagent After coating the above reagent layer and drying, coat an adhesive layer with the following composition.

After coating and drying the above adhesive layer, a second spreading layer having the following composition was coated.

2F Fiber for raw material for paper (manufactured by Toyo Roshi Co., Ltd.)? t
o 97m"Polyoxyethylene/lauryl ether 1AQf/m" Hydrochloric acid 4-N,N diethylamino-2-(2'-methanesulfonamidoethyl)aniline a 1897m" After coating and drying the above second developing layer, the following composition A first spreading layer was applied.

Poly(styrene-glycidyl methacrylate)
90/10] (average particle size approximately 5 Am) ethylenediamine 15α0wa4-α1f7-dextran [sodium acid α029
7m” Magnesicum Chloride [
L4697m” Sodium Chloride
Analytical element (1) of the present invention was prepared as described above.

The analytical element of the present invention (
Analytical element +11 of the present invention was evaluated in comparison with a conventional analytical element similar to 1).

;Various human serum 5' with different Restylol contents before fractionation operation! II and diluted each half with physiological saline to prepare an analytical element of the present invention.

Then, 10 μty4 was applied to a conventional analytical element, and the reflected optical density (Dr) at a wavelength of 546 nm was measured after incubation at 37° C. for 7 minutes.

Table 1 shows the results.

Table 1 Table 2 Fractionation was performed using the fractionation reagent attached to the above five types of human serum "f," HDL-Cholesterol N (manufactured by Chugai Pharmaceutical Co., Ltd., Uniquila). amount of fractionation reagent were mixed, left to stand for 10 minutes, and centrifuged at 3000 rpm for 10 minutes. 9. Transfer each supernatant to the analytical element (1) of the present invention and the conventional analytical element (#4) by 10 μt. The reflected optical density (Dr) at a wavelength of 546 nm Vc was measured after incubation at 57° C. for 7 minutes.The results are shown in the second section.

From the results in Tables 1 and 2, it is clear that with the analytical element of the present invention (No. 11), good color development corresponding to the HDL-cholesterol concentration can be obtained even when serum that has not undergone fractionation is used; In the device, if you do not use serum that has been subjected to fractionation,
It can be seen that color development corresponding to the HDL-cholesterol concentration cannot be obtained.

Example 2 After applying and drying the reagent N of Example 1, approximately 50 f/n!
After applying and moistening water, a tricot knitted fabric made of polyethylene terephthalate spun yarn (66 gauge, 50D) was pressed and dried.

Thereafter, an aqueous solution having the following composition was applied so as to be 1- to form a second developed layer.

Polyoxyethylene lauryl ether 1502 Cholesterol oxidase 1
80 oυ cholesterol ester 5-ase
1800U ascorbic acid oxidase
7500U water 00f After coating and drying the second spreading layer, the first spreading layer prepared as described below was adhered using an α-cyanoacrylate adhesive.

Teifactant IOG 50 f/B” Dextran Sulfate Sodium
102V-Magnesium Chloride
146V-Sodium Chloride
110V - The above reagent was pulverized in xylene using a sand grinder until the average particle size was about 5 μm, and a glass fiber filter C manufactured by Toyo Roshi Co., Ltd.
) B100R (A disc-shaped piece with a diameter of 7 cm was impregnated with the above content and dried.

In this manner, the analytical element (2) of the present invention was manufactured.

I (various types of human whole blood asit with different DL-cholesterol levels) were prepared, and 20
Reflection optical density (Dr) at wavelength 546 nm after dropping in μt increments and incubating for 7 minutes at 37°C
was measured. Table 6 shows the results.

Table 3 From the results shown in Table 3, it can be seen that the analytical element (2) of the present invention can produce good color development in accordance with the HDL-cholesterol content of the whole blood sample.

[Effects of the Invention] As explained in detail above, the analytical element of the present invention enables highly accurate quantitative analysis of specific components in HDL fractions in an extremely quick and easy manner without the need for complicated fractionation operations. Therefore, it is very effective in practice.

Claims (1)

[Claims] 1. An analytical element for analyzing a specific component in a high-density lipoprotein fraction of a liquid sample, which has at least one reagent layer on a support and a porous development layer above it,
The element has a layer containing a fractionating agent that fractionates high-density lipoproteins in a liquid sample, and the layer is provided above the layer that performs the measurement reaction of the specific component. Characteristic analytical elements.