JPH0381666A - Integral type multilayered analyzing element - Google Patents

Abstract

PURPOSE:To separate blood cell components and plasma components in the analyzing element and to allow the quantitative determination of an analyte by specifying the average value of the hole diameter on the surface of a lower microporous layer in contact with an upper microporous layer and the average value of the inter-surface distance between the boundaries. CONSTITUTION:This element is constituted of the fibrous microporous layer 11 to be spotted with a liquid sample, the upper nonfibrous porous layer 12, the lower nonfibrous porous layer 13, a nonporous reagent layer 14 into which a reagent compsn. is incorporated, and a transparent base 19. The element is so formed that the average value of the hole diameter of the surface of the porous layer 13 in contact with the porous layer 12 is larger than the average value of the hole diameter on the surface of the porous layer 12 in contact with the porous layer 13 and the average value of the inter-surface distance between the two layers at the adhesive boundary P between the porous layers 12 and 13 is larger than the average value of the hole diameter of the surface of the porous layer 12 in contact with the porous layer 13.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a dry integrated multilayer analytical element used mainly for quantifying components in biological body fluids such as blood or urine for clinical tests, especially whole blood. Specifically, it is an integrated multilayer analytical element in which multiple (two or more) microporous layers with different pore diameters are laminated with specific partial adhesion (partial adhesion with larger pore diameters on the downstream side). be.

[Conventional technology and its problems] A sheet (flat plate) for quickly and easily quantifying the target analyte (test component) in body fluids such as blood (whole blood, plasma, serum) and urine using a dry method. Many analytical test pieces, non-integral multilayer analytical elements, and integrated multilayer analytical elements have been proposed and are already in practical use. A monolayer analytical test piece in which filter paper is used as a constituent material of the layer impregnated with or supported by a reagent composition (Monoku JP Publication No. 36-4198).

U.S. patent that uses a membrane filter (hereinafter referred to as U.S. patent)
to! ! a*) 3607093) etc. o Arrange multiple layers.

An example of a laminated non-body multilayer analysis element having a structure in which the layers are not bonded is one having a glass filter layer (Japanese Patent Laid-Open No. 49-11395.US 3663374).

Those with two layers of filter paper or wi (net) (Japanese Unexamined Patent Publication No. 47
-37993, US 3802842). Arrange multiple layers.

This is an example of an integrated multilayer analysis element with a structure in which each layer is closely adhered with virtually no gaps.

US 3526480. IJS 3901657 (-Unexamined Japanese Patent Publication No. 50-147790).

US 3992158 (Japanese Patent Publication No. 49-53888),
JP 55-164356 (US 4292272),
Japanese Patent Publication No. 57-66359 (T's 4783315),
JP 6O-222769 (EP 0162302A)
, US 4042335 (Unexamined Japanese Patent Publication No. 51-40191)
, rclinical Chen+1stryJ 24
Vol., pp. 335-1350 (1978), etc.

Single-layer test pieces and non-integrated multilayer analytical elements have sufficient performance for semi-quantitative use, but generally have insufficient accuracy for quantitative use. These integrated multi-layer analysis elements (hereinafter referred to as
In a multilayer analytical element (sometimes simply referred to as a multilayer analytical element), the top layer (outermost layer) is a liquid sample that is supplied at 9 points, and a nearly constant amount per unit area is transferred to the lower functional layer (e.g., reagent layer). A microporous spreading layer (spreading layer) is provided which functions to supply a substantially constant amount of liquid sample per unit area. As a structural material for building each layer.

For the uppermost layer (floor 1), a membrane filter, a three-dimensional lattice-like fine particle structure layer, and a microporous sheet-like material typified by a tanned fabric and a knitted fabric are used. The lower functional layer is typically comprised of a hydrophilic polymer such as gelatin, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, or agar.

These integrated multilayer analytical elements prevent the analyte from penetrating or passing through the functional layer when the analyte is a lipid, polymeric substance (e.g., enzyme, antibody, immune complex); There are disadvantages that quantitative analysis may be inaccurate or impossible, and that the same degree of quantitative analysis accuracy cannot always be obtained for whole blood samples with a wide range of hematocrit values as liquid samples.

In order to solve this problem, an integrated multilayer analysis element including a structure in which each of a plurality of microporous layers is partially bonded has been proposed (for example, Japanese Patent Laid-Open No. 61-4959, EP
0166365A). The integrated multilayer analysis element has a top layer and one or two layers below it! i is a microporous layer, and the layers are partially bonded (by adhesive placed between two adjoining surfaces of the microporous layer at regular or random minute intervals). It refers to an adhesion that is bonded and integrated with minute intervals and allows liquid to pass between adjacent 2N layers; also called porous adhesion.The integrated multilayer analytical element N of
As one example of a configuration, from top to bottom, a textile fabric layer (mainly functioning as a spreading layer), an upper membrane filter layer containing titanium dioxide microparticles (functioning as a light shielding layer), and a lower side containing a reagent composition. consisting of a membrane filter layer (microporous first reagent layer), a hydrophilic polymer layer containing the reagent composition (non-porous second reagent II), a transparent support, a textile fabric layer and two membrane filters. Some layers are partially bonded between each other. When whole blood is applied to this integrated multilayer analytical element to perform quantitative analysis of analytes, red blood cells are bonded to the upper layer containing titanium dioxide microparticles. It has been found that there are cases in which the light passes through the membrane filter layer (light shielding layer) and the microporous first reagent layer and reaches the surface of the second reagent layer made of a hydrophilic polymer.

When the red blood cells reach the surface of the second reagent layer made of a hydrophilic polymer, the red color of the red blood cells cannot be blocked by the light shielding layer when measuring the optical density of the color developed in the element from the transparent support side. .

This is not preferable because it not only interferes with photometry, but also interferes with the coloring reaction caused by hemoglobin.

One proposal for solving the problems of the previous gJ is an integrated multilayer analytical element described in Japanese Patent Application Laid-Open No. 138757/1983 (EP 0226465A). In this multi-layer analysis element.

The structure includes, in order from the top, a terry cloth layer, an upper membrane filter layer, and a lower membrane filter layer (a water absorption layer containing a hydrophilic polymer or a reagent layer may be provided below). The average value of the minimum pore diameter of the membrane filter (nominal pore diameter) is selected to be larger than the average value of the minimum pore diameter of the lower membrane filter. When partially adhering multiple microporous layers in a multilayer analysis element for whole blood, the average pore diameter of the voids in the microporous layer becomes smaller sequentially from the upper side through which the whole blood, which is the sample, flows. It was designed to have a microporous layer that allows whole blood to flow quantitatively and uniformly. Blood cell components such as red blood cells remain in the voids of the microporous layer while the whole blood flows down.
The idea was that essentially only plasma would flow downward. However, even with this multilayer analytical element, it has been found that red blood cells can still pass through the upper microporous layer and the lower microporous layer and reach the surface below the lower microporous layer. Served.

By the way, it is known that when whole blood flows vigorously, blood cell components therein pass through voids or pores that are considerably smaller than the size of the blood cell components in their natural state. The phenomenon in which red blood cells reach the underside of the microporous layer in the integrated multilayer analytical element having a structure in which multiple microporous layers are partially bonded together as proposed in the above two published patents is due to this phenomenon of blood cell components. It is presumed that it is based on the nature.

In addition, Japanese Patent Application Laid-Open No. 1-138757 (DE 3721237
A) has an average base area of 0.03-5.0mm” and a thickness of 0.
．． 02'-1.0 mm dots (granules) of melt adhesive (hot melt adhesive) are placed on the polymer sheet so as to cover IO ~ 70% of the bonding surface, and then paper, fleece, etc. are placed on the polymer sheet.
Diagnostic test carriers have been described in which a microporous sheet, such as a woven fabric or two knitted fabrics, are bonded together. According to the example, this test carrier consists of two microporous sheets placed side by side on top of a polymer sheet with a slight spacing between them.
The polymer sheet and the microporous sheet are each adhesively fixed to a dot-shaped liquid sample using a molten adhesive so as to maintain a micro gap (capillary transport area) between the polymer sheet and the polymer sheet. Then, the liquid sample spotted on the first microporous sheet flows along the surface of the sheet through the capillary transport area, and flows to the laterally adjacent second porous sheet (impregnated with the coloring reagent composition). The amount of analyte is measured colorimetrically by photometrically measuring the color developed there. However, this published patent specification describes a structure in which two microporous sheets are bonded and fixed with a dot (granular material) of molten adhesive while maintaining microgaps (capillary conveyance area), and There is no description of the relationship between the pore size of the sheet and the gap in the capillary transport area, and furthermore, this test carrier is said to be capable of using blood as a liquid sample;
There is also no description of using whole blood.

[Technical Problems to be Solved] The present invention provides an integrated multilayer analytical element that can use whole blood as a liquid sample, in which blood cell components and plasma components are separated in two analytical elements, and furthermore, blood cell components and plasma components are separated in two analytical elements. The technical challenge is to provide a dry-operable integrated multilayer analytical element capable of quantitatively analyzing a wide variety of analytes, including high-molecular-weight analytes (e.g., enzymes, antibodies, immune complexes).

Another technical problem of the present invention is to provide a method for manufacturing the integrated multilayer analytical element.

[Technical rIB solution] The above problem is.

A plurality of microporous layers including a microporous layer on which a liquid sample is deposited are laminated together by partial adhesion, and at least one of the microporous layers contains a reagent composition. of an integrated multilayer analytical element, or

A plurality of microporous layers, including a microporous layer on which a liquid sample is deposited, are laminated together by partial adhesion, and a reagent composition is placed under a hydrophilic layer under the bottom layer of the microporous layers. An integrated multilayer analytical element having at least one detection function layer contained in a polymer binder laminated thereon.

At at least one of the interfaces of adjacent microporous layers that are partially bonded.

The average value of the pore diameters on the surface of the lower microporous layer facing the upper microporous layer is greater than the average value of the pore diameters on the surface of the upper microporous layer facing the lower microporous layer. and an integrated multilayer analytical element characterized in that the average value of the distance between the surfaces of the interface is larger than the average value of the pore diameters of the surface of the upper microporous layer facing the lower microporous layer. Resolved.

[Detailed Description of Specific Structure of the Invention 1 In this specification, the side on which a liquid sample is applied is referred to as the upper side of a plurality of laminated microporous layers.

The part where the liquid sample permeates is called the lower part. Alternatively, focusing on the flow of the liquid sample between the eyebrows, the side where the liquid sample is deposited is sometimes called the upstream side, and the side where the liquid sample flows into the interior and reaches it is called the downstream side. Therefore.

Upper side and upstream side have the same meaning, and lower side and downstream side have the same meaning.

In the integrated multilayer analytical element of the present invention, when adjacent microporous layers are integrated by partial adhesion, the average pore diameter on the upper surface of the downstream microporous layer (the surface facing the upper microporous layer) Both the value and the average value of the surface-to-surface distance of two adjacent microporous layers to be bonded in 9 parts are the voids on the lower surface of the upstream microporous layer (the surface facing the lower microporous layer). It is characterized by a larger than average pore diameter. In this specification, this partial adhesion is referred to as "partial adhesion with large downstream hole diameter."

The maximum value of the average value of the distance between the surfaces of two adjacent microporous layers that are partially bonded with each other with large pores on the downstream side is about 10 μm. It has been observed that when this average distance exceeds about 10 μm, plasma cannot migrate to the downstream layer. The minimum average value of the pore diameter on the bottom surface of the upstream microporous layer is approximately 1.2 μm.
If the average value of the pore diameter is smaller than this value, hemolysis may occur in the upstream microporous layer. The maximum average value of the pore diameter on the upper surface of the microporous layer on the downstream side is approximately 2
It is from 0 μm to about 200 μm. If the surface has the property of being easily wetted by plasma, plasma transfer will occur even with a large average pore diameter of up to about 200 μm. It is preferable that the (absolute) pore diameter value of each pore on the downstream side is larger than the average value of the pore diameters on the upstream side. However, this condition is not an essential condition in the present invention. The surface of the microporous layer on the downstream side is a rough surface (for example, 7W4 fabric or knitted fabric) and 9
This is because there is no problem even if some portions are in direct contact with the upstream microporous layer.

Examples of the materials constituting the microporous layers of the integrated multilayer analytical element of the present invention include the following: Non-fibrous porous layer: Patent fJ 1977-53888. US
For example, a flash ◆ polymer layer (membrane filter) consisting of cellulose esters 9 described in No. 1421341, etc., cellulose acetate, cellulose acetate butyrate, and cellulose nitrate is preferred. In addition, 6-
Microporous membranes made of polyamides such as nylon, 6,6-nylon, polyethylene, polypropylene, etc., JP-A-62-2
A microporous membrane (membrane filter) made of polysulfone described in No. 7006 can also be used.

Japanese Patent Publication No. 49-53888. Japanese Patent Publication No. 55-90859 (U
A microporous layer (three-dimensional lattice-like fine particle structure) with continuous voids in which small polymer particles, glass particles, diatoms, etc. are bonded in a dot-like manner with a hydrophilic or non-water-absorbing polymer, as described in physical layer) can also be used.

Fiber a microporous I! ! : Filter papers made of various materials, non-woven fabrics, woven fabrics, 2-knitted fabrics, fine mesh nets; glass filters, etc.

Other microporous N: composites of the above-mentioned layered materials (sheet-like materials), etc.

Among the microporous layers mentioned above, the material that causes hemolysis is:

This is not preferred because it destroys red blood cells in sample type iα and allows hemoglobin to pass through the reagent layer.

The preferred layer configuration of the integrated multi-II analytical element is, in order from the side on which the liquid sample is deposited: *fibrous porous layer, upper non-fibrous porous layer, lower non-fibrous porous layer (and , at least one detection functional layer comprising a reagent composition contained in a hydrophilic polymer binder).

It is not limited to this layer configuration.

Made by phase separation method. In membrane filters made of so-called plush polymers, the pore size distribution in the internal thickness direction is not uniform. Here, the surface of one membrane filter having a coarse pore size distribution is called 1 side, and the surface having a dense pore size distribution is called ``face''. The liquid passage path in the thickness direction of the membrane filter is narrowest on the free surface side (shiny surface) during membrane manufacturing (the surface with a dense pore size distribution;
surface>, and the surface that was in contact with the temporary support (separated surface) is wider (surface with rough pore size distribution; ■ surface; non-glossy surface).

In the integrated multilayer analytical element of the present invention, if there are two membrane filter layers glued together at 9 parts, the two layers are both on the upstream side! It is preferable to face the person. When a woven fabric or knitted fabric layer is placed above two membrane filter layers, the average pore size of the m-woven fabric or knitted fabric layer is generally coarser than the pore size distribution on one side of the upper membrane filter. .

Therefore, from the woven or knitted fabric layer to the joint surface between the upper membrane filter layer and the lower membrane filter layer, the average pore diameter of the pores becomes smaller as it goes downstream. The average distance between the surfaces of the two partially bonded membrane filter layers at the adhesive interface is kept larger than the average value of the pore diameters on one side (non-glossy side) of the upper membrane filter, The average value of the pore diameters on the upstream surface of the lower membrane filter is preferably selected to be larger than the average value of the pore diameters on the n-side (glossy surface) of the upper membrane filter. In practical terms.

For two membrane filter layers that are partially bonded, the average value of the pore diameters on the downstream surface of the upper membrane filter <the average value of the distance between the surfaces of both layers at the adhesive interface and the average value of the pore diameters on the upstream surface of the lower membrane filter The average value of the pore diameters may be increased in the order of the average value.

When partially adhering the membrane filter and the fabric, the two can be integrated by applying adhesive to the thread piles present on the fabric surface and bonding the two together.

At this time, even if the pile of threads without adhesive may come into direct contact with the surface of the membrane filter, the average value of the distance between the two surfaces will be If the pore size is larger than the average value of the pore diameter (the surface facing the microporous layer), the intended purpose will be achieved.

A method of disposing an adhesive in a microporous layer at regular or random minute intervals such as a dotted, intersecting line, parallel line or grain pattern is disclosed in Japanese Patent Application Laid-Open No. 61-4959 (E).
P O166365A), JP-A-62-138757 (
EPo 226465A), JP-A-64-23160 (
DE 3721236A).

JP 1-145567 (DE 3721237A),
Gravure printing method using a liquid adhesive as described in Japanese Patent Application No. 63-258571.

There is a method of partially adhering it to the surface of a microporous layer using the same method as intaglio printing or screen printing. Among various methods, a preferred method is to print and arrange a hydrophobic, heat-sensitive hot melt adhesive on the surface of the microporous layer in a halftone pattern using a printing method such as a gravure printing plate method or an intaglio method. be.

As an adhesive, JP-A-62-138756. Liquid adhesives, heat-sensitive hot melt adhesives, and the like described in F Printing Engineering Handbook J (edited by Japan Society of Printing, Gihodo Publishing, published in 183), pages 839 to 853, etc. can be used.

Liquid adhesives include, for example, aqueous starch glue; aqueous solutions of dextrin, carboxymethyl cellulose, polyvinyl alcohol, etc.; and vinyl acetate-butyl acrylate copolymer emulsions. Organic solvent type adhesives may also be used, but those with slow evaporation of the solvent are suitable. Heat adhesives are particularly useful. In the case of liquid adhesives, those with a viscosity of about 1000 cps or more and low stringiness are preferred.

In the case of a liquid adhesive, it is preferable that the adhesive be localized on the surface of the microporous layer and less likely to penetrate into the layer in the depth direction.

As a heat-sensitive (thermal adhesive) hot melt adhesive,
A hot melt adhesive described in Industrial Materials J Vol. 26 (No. 11), pages 4 to 5, etc. can be used. As an example,
Ethylene copolymers such as ethylene-vinyl acetate copolymer, ethylene-ethyl acrylate copolymer, and ethylene-acrylic acid copolymer, polyolefins such as low molecular weight polyethylene and atactic polypropylene, polyamides such as nylon, and polyesters. Examples include thermoplastic rubbers such as styrene block copolymers, styrene block copolymers such as SBS, styrene-butadiene rubber, butyl rubber, urethane rubber, rosin, petroleum resins, terpene resins, paraffin, and synthetic waxes. In the case of heat-sensitive hot melt adhesives, those in the form of fine threads, particles, or powder are preferred. The heat-sensitive hot-melt adhesive can be heated to a liquid state and used in the same manner as the liquid adhesive described above.

The integrated multilayer analysis element of the present invention can have various layer configurations. For example, US Pat.
-53888), JP-A-55-164356 (US 4
272292), JP-A-6O-222769 (EP O
162302A), JP-A-62-138757 (EP
Please refer to the descriptions such as O166365A).

For example, in practical terms.

(1) Place the lower microporous layer, the upper microporous layer, and the microporous layer to which the liquid sample is dotted onto the support in this order (
The reagent composition is contained in at least one of the microporous layers).

(2) On the support, place the detection layer (or water absorption I'm), the lower microporous layer, the upper microporous layer, and the microporous layer to which the liquid sample is dispensed in this order (reagent The composition is contained in at least one of the microporous layers).

(3) On the support, a detection N (or water absorption layer), a hydrophilic polymer-containing non-porous reagent layer, a lower microporous layer, an upper microporous layer, and microporous holes into which a liquid sample is dispensed. The sexual layer.

In this order.

0>IN aqueous polymer-containing non-porous reagent layer on top of the support.

The lower microporous layer, the upper microporous layer, and the microporous layer to which the i&body material is dispensed in this order.

(5) Si aqueous polymer-containing non-porous reagent layer on top of the support.

a lower microporous layer, and an upper microporous layer into which the liquid sample is dotted, in this order.

What each has is useful. In the above layer structure, the microporous layers are partially bonded to each other, and at least one of the two partially bonded interfaces has a large downstream pore diameter. Hydrophilic polymer-containing non-porous reagent layer and bottom f
The interface of the microporous layer is selected to be partially bonded or generally entirely bonded depending on the purpose.

The microporous layer to which the liquid sample is dispensed may be either fibrous or non-fibrous, but fibrous knitted or woven fabric is generally preferred. The support may be provided with an undercoat layer on its surface. The detection layer is generally

A layer in which dyes generated in the presence of an analyte diffuse and permeate into the layer and are optically detected through a transparent support, and can be composed of a hydrophilic polymer. The detection layer may contain a mordant 2, for example a cationic polymer for anionic dyes. The water-absorbing layer generally refers to a layer in which the dye generated in the presence of an analyte does not substantially diffuse into the layer, and can be composed of a hydrophilic polymer that easily swells with water.

A preferred material for the support is light-transparent, water-impermeable (transparent) polyethylene terephthalate. Cellulose esters such as cellulose triacetate may also be used. In order to firmly adhere the hydrophilic layer, an undercoat layer is provided on the surface of the support, and it is preferable to perform a hydrophilic treatment.

A reagent composition is a composition capable of producing an optically detectable substance (e.g., a dye) in the presence of an analyte, either by reacting directly with the analyte or by reacting with the analyte and other substances. A color-forming composition (indicator) that can produce an optically detectable substance (pigment or dye) as a result of reaction with an intermediate produced by reaction with a reagent. It is generally preferred that the reagent composition include an enzyme.

When whole blood is directly applied as a liquid sample, by using an enzyme that uses the analyte as a substrate, it becomes possible to specifically introduce only the analyte into the coloring reaction. As an example of a reagent composition containing an enzyme,
2-138757 (Page 348, upper left column to upper right column; EP O
226465A, page 5, lines 49 to 65), etc. can be used.

Examples of color-forming Ni compositions include: Compositions that produce dyes by oxidation of leuco dyes (e.g., U.S.
triarylimidazole leuco dyes described in JP-A No. 4089747 (JP-A-53-'26-88), and diarylimidazole leuco dyes described in JP-A No. 59-193352 (EP 0122641A), etc.).

Diazonium salt.

Compositions containing compounds that, when oxidized, produce dyes by coupling with other compounds (e.g. 4-aminoantipyrines and phenols or naphthol gl (e.g. l
, 7-dihydroxynaphthalene)).

A compound that is capable of producing a pigment in the presence of a reduced coenzyme and an electron transfer agent.

Self-developing substrates that can liberate colored substances (for quantifying enzyme activity).

Various known immune reaction reagent compositions (for quantitative analysis of antibodies, antigens, or immune complexes).

The reagent composition includes an activator and a buffer as necessary.

A hardening agent, a surfactant, etc. can be contained.

Examples of buffers that can be contained in the reagent layer of the analytical element of the present invention include carbonate, borate, phosphate and rBioc.
heIIlistryJ volume 5, pages 467-477 (19
Examples include Good's buffer described in 1966). These buffers are described in rBasic Experimental Methods for Proteins and Enzymes (Takeshi Horio et al., Nankodo, 1981), rBio
Chemistry J Vol. 5, pp. 467-477 (1966
You can make a selection by referring to literature such as 2010).

Functional layers that can be incorporated into the integrated multilayer analysis element of the present invention include a spreading layer and a blood cell needle! , 1. filtration layer.

A layer for removing specific endogenous components (pretreatment layer).

trap layer, etc.), light shielding layer, light reflection layer (background layer), diffusion prevention layer (migration prevention layer), barrier layer, semi-transparent membrane layer (dialysis membrane layer), or two or more of the above functional layers. There is a single layer with etc.

In the integrated multilayer analytical element of the present invention, the composite layer consisting of a plurality of microporous layers integrated by two-part adhesion can be used as a whole as a functional layer for developing a liquid sample or as a representative of blood cell components in whole blood. as a solid component separation functional layer that separates fine particles in a liquid sample from the liquid sample medium and supplies substantially the liquid portion (plasma in the case of whole blood) to the reagent layer, or as a solid component separation functional layer that It serves as a layer with multiple separation functions.

The multilayer analytical element of the present invention can perform quantitative analysis of analytes in liquid samples, especially whole blood, by the operations described in the above-mentioned patent specifications. A drop of liquid sample was placed on the uppermost microporous layer for a period of 1 to 10 minutes.
at a substantially constant temperature in the range of about 20°C to about 40°C;
Incubation is carried out at a substantially constant temperature, preferably around 37° C., and the optical density within the element is determined using light at or near the maximum wavelength of color, fluorescence, turbidity or ultraviolet absorption within the element from the transparent support side. The analyte content in the liquid sample can be determined by the end point method or the reaction rate method using the principle of colorimetric measurement by performing reflection photometry and using a calibration curve prepared in advance. By keeping the amount of liquid sample spotted, incubation time, and temperature constant, quantitative analysis of analytes can be performed with high precision. This measurement operation was performed in Japanese Patent Application Laid-Open No. 60-12
5543. Japanese Patent Publication No. 60-220862. JP-A-61-2
94367, JP 58-161867 (t's 44
24191) etc., highly accurate iI determination can be performed with extremely easy operation.

[Example B Scraping] The present invention will be specifically explained in detail based on an example of Example A that clearly represents the characteristics of the present invention.

The integrated multilayer analysis element shown in FIG. Geological strata)11. Upper non-fibrous porous layer, cellulose acetate membrane filter layer 12. The lower non-fibrous porous layer is cellulose acetate membrane filter F! 13. IFi substantially non-porous reagent layer 14. where the reagent composition is contained in gelatin. It is composed of a polyethylene terephthalate transparent support 19.

In the multilayer analytical element of this embodiment, membrane filter layers 12 and 13 both face upstream. The average pore size (corresponding to the average pore diameter) of the woven or knitted fabric layer 11 is generally coarser than the pore size distribution on one side of the upper membrane filter. Thus from the woven or knitted fabric layer 11 to the membrane filter layers 12 and 13.
The average pore diameter of the pores decreases toward the downstream side up to the interface with the pores. The average distance between the surfaces of the membrane filter layers [2 and 13], which are partially bonded together, is kept larger than the average pore diameter of the pores on one side of the membrane filter 12, and The average pore diameter of the pores on the upstream surface of the filter 13 is selected to be larger than the average pore diameter of the pores on one surface of the membrane filter 12.

When whole blood is spotted on the woven fabric or knitted fabric layer of the integrated multilayer analysis element having such a structure, the whole blood flow temporarily stagnates when it reaches the bonding surface between the membrane filter layers 12 and 13. However, when the liquid starts to flow down again, the blood cell components are retained within the upper membrane filter layer 12,
Does not move to lower membrane filter FI+3. The liquid transferred to the lower membrane filter layer 13 is plasma from which blood cell components have been substantially removed. The pore size and thickness of the microporous layer of each layer are determined so that the time it takes for plasma to reach the lower membrane filter layer 13 is about 1 minute to about 5 minutes. (gap distance) is preferably selected. Upper membrane filter N12: Containing a blood cell aggregation promoter helps retain blood cell components.
It is effective.

Figure 2 shows a microporous spreading layer 2 on which a liquid sample is deposited.
1. Microporous light shielding layer or light reflecting layer 22. An integrated multilayer analytical element in which a substantially non-porous reagent layer 23 containing a hydrophilic polymer binder is integrally constructed on a transparent support 29, and the spreading layer and the light shielding layer are composed of microporous layers; These two layers are integrated by partial adhesion with large pores on the downstream side. The light-shielding layer contains a color-shielding substance, which functions to shield the color of the liquid sample. Dyes and pigments are generally used as color-blocking substances. As a specific example, vl
There is a method of dyeing the porous layer with dyes or pigments. As the dye, a dispersible dye such as Seritone is preferred.

It goes without saying that the dyes are not limited to these dyes. When using a membrane filter as a microporous layer, fine particles of titanium dioxide and fine particles of barium sulfate having light shielding ability are added to a solution in which a polymer is dissolved when preparing the membrane filter.

A microporous layer can be used by mixing and dispersing a pigment of a suitable color such as carbon black, and forming a film under conditions that make fine particles or a pigment-containing polymer solution porous.

FIG. 3 shows, in order from the top, the microporous development layer 31. Microporous light shielding layer or light reflection N32. Microporous first reagent layer 3
3. a substantially non-porous second material comprising a hydrophilic polymeric binder;
It is an integrated multilayer analytical element in which the reagent layer 34 is integrally constructed on the transparent support 39, and the developing layer, the light shielding layer, and the first reagent layer are made of microporous layers, and between these three layers there is a downstream It is integrated by partially adhering the side hole with a large diameter.

This integrated multilayer analysis element is suitable for quantitative analysis of high molecular weight analytes such as various enzyme activity values, plasma proteins, antibodies, and antigens contained in whole blood by spotting whole blood as it is. It is something that When the analyte is an enzyme, the sensitivity of quantitative analysis can be increased by containing a substrate for the enzyme to be measured in the second reagent layer. When the analyte is blood Mi protein, the plasma protein can be efficiently quantitatively analyzed by containing a dye that changes color or a dye precursor that develops color depending on the protein to be measured in the second reagent layer.

When the analyte is an antibody, antigen, or immune complex, the antibody, antigen, or immune complex can be effectively quantitatively analyzed by making the microporous layer function as a site for immune reaction.

a Direct feeding [Example 1] ■ Creation of reagent layer An aqueous dispersion of an acrylic ester copolymer having the following composition was placed on a polyethylene terephthalate support with a thickness of 180 μm so that the coating amount after drying was as follows: It was applied and dried to form an undercoat layer.

Undercoat layer I? /Coating amount per Hydroxyethyl acrylate 30mg Butyl acrylate 130mg Styrene
60mg methyl methacrylate
60mg acrylic rll
A 20 mg solution of the reagent composition in a mixed solvent of methanol and water (volume ratio 6:4) was coated on top of this undercoat layer so that the coating amount after drying was as follows. A chemical reagent layer was provided.

Coating amount per 1 trr of reagent layer Polyvinyl alcohol (#A degree 9% Bromocresol green 360mg Malic acid 60mg 0mg Nonylphenoxypolyethoxyethanolone Ethylene unit average 1)
(Contains O) 80mg■ Creation of lower non-fibrous microporous layer Reagent layer of nonylphenoxypolyethoxyethanol<4
To. The n-side (glossy side) of a cellulose acetate membrane filter with a thickness of about 140 μm and an average minimum pore size of 5.0 μm was wetted almost uniformly with water containing 2% (containing 10 units on average). were laminated by applying light and uniform pressure toward the reagent layer, bonded together, and dried. This lower non-fibrous microporous layer (membrane filter N) also serves as a light shielding layer.

■ Creation of a spreading layer and hemocyte filtration layer Place a dot on one side (non-glossy side) of a cellulose acetate membrane filter (upper non-alpha fibrous microporous layer) with a thickness of about 140 μm and an average minimum pore size of 3.0 μm. Approximately 0.3m in diameter
Starch paste was applied at a solid content of 3ff1g/ff12 by screen printing through a screen with a dot spacing of about 0.6mm and an H point area ratio of about 20%. On this glue side.

Polyethylene terephthalate spinning fan-shaped tricot knitted fabric (fiber Wt microporous layer) of approximately 250 μm thickness equivalent to 100S is laminated by applying light and uniform pressure, bonded together, dried, and integrated to form a spreading layer and blood cell filtration layer. It was created.

(2) - Integrated multilayer analysis element completion step Starch glue was adhered to one side (non-glossy side) of the membrane filter (lower non-wA & microporous layer) prepared in (2) in the same manner as in step (2). The n-side (glossy side) of the membrane filter, which is the spreading layer and hemocyte filtration layer created in step ①, is laminated onto this glue surface by applying light and uniform pressure, and is dried and integrated into a single layer for quantifying albumin in whole blood. Completed body shape multi-layer analysis elements. In this multilayer analytical element, the adhesion interface between the three microporous layers is a partial adhesion with large pore diameters on the downstream side.

■Creating a slide for quantifying albumin in whole blood The multilayer analytical element obtained in step (■) was cut into a square chip of 15 mm x 15 mm, and placed in a polymer mount described in JP-A-57-63452 for quantifying albumin in whole blood. I created slides for this purpose.

[Measurement of spacing between partial adhesive surfaces] Quickly freeze the integrated multilayer analytical element and cut it with a sharp knife blade almost perpendicular to the plane of the analytical element.

The cut surface was photographed with a microscope, showing the upstream surface of the membrane filter layer, which is the lower non-fibrous microporous layer, and the downstream surface of the membrane filter, which is the upper non-wA fibrous microporous layer, of the spreading layer and hemocyte filtration layer. When the distance between the side surfaces was measured, it was found to be 3.8 μm on average.

[Evaluation of the performance of an integrated multilayer analytical element for quantifying Tnbumin in whole blood]■ Human whole blood 15% was added to the knitted fabric layer of the obtained multilayer analytical element.
When microliters were spotted and incubated at 37°C for 6 minutes, the partially adhered interface was immediately peeled off and the upstream surface of the membrane filter, which is the lower non-fibrous porous layer, was observed, no red blood cells were found. It wasn't done. In this experiment, red blood cells in whole blood spotted on the top layer (the upper knitted fabric layer of the spreading layer and hemofiltration layer) were transferred to the lower microporous layer (the upper non-fibrous microporous layer) of the spreading layer and hemofiltration layer. It became clear that the particles were completely filtered and separated up to the membrane filter layer (the membrane filter layer).

■ Whole blood samples adjusted to the albumin content listed in Table 1 were prepared. The albumin content of whole blood samples was assayed using the bromcresol green standard method.

・ Transfer 15 mL of each sample to a sample for quantifying fulbumin in whole blood.
8. Place the spot on the knitted fabric layer and heat the ink for 4 minutes at 37°C. Second, the color optical density value in the analytical element was measured by reflection photometry using visible light with a center wavelength of 640 nm from the transparent support side. Table 1 shows the correspondence between the albumin content assay value of the whole blood sample and the measured reflection optical density value. When a calibration curve was created from this correspondence, the calibration curve shown in FIG. 4 was obtained. From the calibration curve in FIG. 4, it is clear that the integrated multilayer analytical element for quantifying fulbumin in whole blood of the present invention can accurately quantitatively analyze the albumin content of whole blood.

Table 1 Example 2 ■ Formation of coloring reagent layer Thickness 1 with smooth surface and gelatin undercoat layer
A dilute aqueous NaOH solution was added to adjust the pH to 6.5 on an 80 μm polyethylene terephthalate colorless transparent sheet (support) so that the coating amount after drying was as follows. A substantially non-porous coloring reagent layer was provided by coating from an aqueous coating solution and drying.

Deionized gelatin 20gp-nonylphenoxypolyglycitol 5urfac manufactured by Orin Co., Ltd.
tant l0G) 850mg peroxidase 16000 units F A D
25mg T P P
110mg pyruvate oxidase 16000 units 2-(3,5-dimethoxy-4-hydroxyphenyl)・4-phenethyl-5-
[p-(dimethylamino)phenylcoimidazole (leuco dye) 320 mg ■ Formation of adhesive layer On the coloring reagent layer formed in the above step, coat the dilute Na a An adhesive layer was formed by coating from an aqueous solution adjusted to pH 7.0 by adding Otl aqueous solution and drying.

Coating amount per 1 trl' of adhesive layer Deionized gelatin 4gp-nonylphenoxy polyglycidol (Olin Co., Ltd. 5urfact
ant tOG) 160ff1g rare Na0)
Add an aqueous solution to adjust the pH to 7.0 ■ Lamination and treatment of the lower non-fibrous porous layer Spread water at about 25°C on the adhesive layer at a rate of about 30 g/ra2 almost uniformly over the entire surface. Supply and moisten cellulose acetate membrane filter (average minimum pore size 5.
0 μm, thickness 140 it m) side (glossy side) was laminated by applying light pressure to the adhesive layer, dried and bonded to form a lower non-fibrous porous layer.

Next, on top of this membrane filter, an aqueous dispersion adjusted to pH 7.5 by adding a dilute NaOH aqueous solution is coated on top of the membrane filter so that the amount of component coating after drying is as shown below, and dried to form a reagent layer. A lower non-fibrous porous layer (microporous reagent N) was also provided.

Component coverage per 1 trl' of the microscopic test layer Tris (
hydroxymethyl) aminomethane potassium dihydrogen phosphate α-sodium ketoglutarate and alanine hydroxypropyl methylcellulose (methoxy groups 28-30%, hydroxypropoxy groups 7
Contains ~12%; Viscosity of 2% aqueous solution at 20°C: 50 cps;
Metrose 90S) I 100) manufactured by Shin-Etsu Chemical Co., Ltd.
870mg4.4'・Monomethine-bis-[l・p-
sulfophenyl)-3-methyl-5-pyrazolone]oxonol (dye) 70II + 8ρ-octylphenoxypolyethoxyethanol 220 inug 50m8 00mg 2750 width g (oxyethylene unit average 10 content) 2701
)ag titanium dioxide fine particles (rutile type)
7.0g magnesium chloride 24
0mg ■ Impregnation treatment of the spreading layer (fibrous porous layer) Tricot knitted fabric (about 250 μm thick) made by knitting 36 gauge polyethylene terephthalate spun yarn equivalent to 50S is immersed in an aqueous solution with the following composition and dried to extract the solid components. Impregnated with.

Aqueous solution for impregnating glabellar layer (fibrous porous layer) Composition of boric acid 2.0 g Sodium tetraborate 1.0 g 25% aqueous solution of tertiary amino group-containing polymer (CH2= CH-(CH2)
Polymer of 3-N-((jla)a; viscosity 320 cp
s) 200mg purified water
96.80g ■Lamination of spread layer and upper non-fibrous porous layer The impregnated tricot knitted fabric mentioned above is heated to a temperature of 80°C, and a hot-melt adhesive layer heated and melted to a temperature of 130°C is gravure applied to its surface. A halftone dot pattern was applied using a gravure printing method in which the film was transferred from a plate roller. The gravure plate roller used had a circular dot diameter of 0.
．． The diameter of the dots was 3 mm, the distance between dot centers was 0.611 m, and the dot area ratio was about 20%. The amount of solid components deposited was approximately 3 g/m2. Next, one side (non-glossy) of a cellulose acetate membrane filter (average minimum pore size of 3.0 μm, thickness of approximately 140 μm, porosity of approximately 80%) was placed on the surface of the hot fabric immediately after the adhesive was transferred. The two were laminated (bonded together) using a partial adhesive method, with their sides facing each other and passed between laminating rollers.

■Completion of an integrated multilayer analytical element The above laminate (laminate of the spread layer and the upper non-fibrous M microporous layer) was heated to 80°C, and the downstream surface (■ side, glossy side) of the membrane filter was heated to 130°C. A hot-melt adhesive layer heated to 0.degree. C. was deposited in a halftone dot pattern using a gravure printing method in which the hot-melt adhesive layer was transferred from a gravure plate roller. The gravure plate roller used had a circular dot diameter of 0.3 mm, and the distance between the centers of the dots was i! A dot with a diameter of 0.6 mm and a dot area ratio of about 20% was used. The amount of solid components deposited was approximately 3 g/I12. Next, the downstream surface of the high-temperature membrane filter immediately after the adhesive has been transferred is transferred to the upstream surface of the membrane filter, which is the lower non-fibrous porous layer on top of the coloring reagent layer (■ side, non-glossy side). ), and the two were laminated (bonded together) through a laminating roller to complete an integrated multilayer analytical element.

Completed ALT (alanine aminotransferase)
The integrated multilayer analytical element for activity measurement has a structure in which a transparent support, a coloring reagent layer, a lower non-1A fibrous porous layer, an upper non-WA fibrous porous layer, and a porous development layer are laminated in this order. be.

The spreading layer and the upper non-fibrous porous layer act together as a spreading layer and a blood cell filtration layer. The lower non-fibrous porous layer is AL
It acts as a reaction layer that produces pyruvate in the presence of T. The coloring reagent layer acts as a layer that converts pyruvic acid produced in the lower non-fibrous porous layer into a dye via hydrogen peroxide in the presence of ALT. The produced dye is optically detected through a transparent support.

■Production of analysis slide The obtained multilayer analysis element for ALT activity measurement is 15ma+X
Cut into 15mm square chips, published in JP-A-57-634.
A biochemical analysis slide for measuring ALT activity was completed by placing it in the polymer slide frame described in 52.

[Performance evaluation of multilayer analysis element for ALT activity measurement - 1 item A
Preparation of LT-added whole blood sample Human whole blood was centrifuged to separate plasma, and a portion of the 9-separated plasma was replaced with a 7% HSA (human serum albumin) solution prepared by adding an appropriate amount of ALT. . By adding an appropriate amount of this ALT-added plasma to an appropriate amount of the separated blood cell portion and placing it on a table.

Whole blood samples with ALT activity value of 800 units/L and hematocrit value of 25%; 40%; 55% were prepared respectively.

■Performance evaluation of multilayer analysis element 20 μL of each of the above whole blood samples was spotted on the fibrous porous layer of a biochemical analysis slide for measuring ALT activity, and each analysis slide was kept at 37°C in the incubator of the analyzer for 2 hours. The reflected optical density values from 2 minutes 30 seconds to 4 minutes after spotting were measured at the center wavelength 6.
Measurement was performed using visible light of 40 nm, and the change in reflected optical density value per second was determined. The measurement results are shown in Table 2.

Table 2 From the data in Table 2, it can be seen that the integrated multilayer analytical element for measuring ALT (alanine aminotransferase) activity of the present invention has a hematocrit value of 0% (from blood W! to 55%, which is almost constant regardless of the hematocrit value). It is clear that this is a change in optical density.

That is, the multilayer analytical element for measuring ALT activity of the present invention is
It is clear that equivalent quantitative values can be obtained for both whole blood and plasma as liquid samples.

[Performance evaluation of multilayer analytical element for measuring ALT activity-2] AL
The correlation between the color-developed reflective optical density value of the integrated multilayer analytical element for measuring T activity and the ALT activity value was evaluated as follows.

(2) Preparation of whole blood samples with different ALT activity values A water-soluble amount of ALT was added to a 7% HSA aqueous solution to prepare ALT-containing H9A aqueous solutions with different activity values.

Human whole blood was centrifuged to separate plasma, and a portion of the plasma separated nine times was replaced with the aforementioned ALT-containing 7% ISA aqueous solution having different activity values. Whole blood samples having a hematocrit value of 40% and an ALT activity value shown in Table 3 were prepared by adding an appropriate amount of this ALT-added plasma to an appropriate amount of the separated blood cell portion and mixing them.

In addition, whole blood (hematocrit value: 47%) before centrifugation was also used as a liquid sample.

■Performance evaluation of multilayer analysis element 20 μL of the above whole blood sample base was spotted on the mat porous layer of a biochemical analysis slide for measuring ALT activity, and each analysis slide was kept at 37°C in the incubator of the analyzer. Reflection optical density values from 2 minutes 30 seconds to 4 minutes after spotting were measured using visible light with a center wavelength of 640 nm, and changes in reflection optical density values per second were determined. The measurement results are shown in Table 3 and Figure 5 (AL
T activity value (horizontal axis) and change in reflected optical density value per second (
(vertical axis).

The values in the bottom row of Table 3 are the measurement results for whole blood before centrifugation.

From the data in Table 3 and the graph in FIG. 5, 9 AL of the present invention
In the integrated multilayer analytical element for measuring T (alanine aminotransferase) activity, it is clear that the change in the reflected optical density value of the color is in a linear relationship with the ALT activity value in the specimen (whole blood sample). That is, it is clear that the multilayer analytical element for measuring ALT activity of the present invention can accurately measure the ALT activity value by the reaction rate method (rate assay) using whole blood as a liquid sample.

No. table 11 selections

[Brief explanation of drawings]

1 to 3 are schematic cross-sectional views showing the layer structure of an embodiment of the integrated multilayer analysis element of the present invention. FIG. 4 shows the test line of the body type multilayer analysis element for quantifying albumin in whole blood of Example 1. FIG. 5 is a graph showing the relationship between the ALT activity value (horizontal axis) and the change in reflected optical density value per second (vertical axis) of the integrated multilayer analytical element for measuring ALT activity value in whole blood of Example 2. It is. 1 2 3 4 9 1 fibrous microporous layer (1 m fabric or knitted fabric) on which the liquid sample is deposited Upper membrane filter layer Lower membrane filter layer Substantially non-porous reagent containing a hydrophilic polymer binder Layers Transparent support Microporous spreading layer Microporous light blocking layer (or light reflective N) Substantially non-porous reagent layer containing a hydrophilic polymer binder Transparent support Microporous spreading layer Microporous light Shielding layer (or light reflecting N) Microporous first reagent layer Substantially non-porous second reagent layer comprising a hydrophilic polymer binder Transparent support

Claims (2)

[Claims] (1) A plurality of microporous layers including a microporous layer on which a liquid sample is deposited are laminated together by partial adhesion, and at least one of the microporous layers is coated with a reagent composition. The lower microporous layer faces the upper microporous layer at at least one of the interfaces of adjacent partially bonded microporous layers of the integrated multilayer analytical element contained therein. The average value of the pore diameter of the surface is larger than the average value of the pore diameter of the surface facing the lower microporous layer of the upper microporous layer, and the average value of the distance between the surfaces of the interface is An integrated multilayer analytical element characterized in that the pore diameter of the surface facing the microporous layer below the porous layer is larger than the average value. (2) A plurality of microporous layers including a microporous layer on which a liquid sample is deposited are laminated and integrated by partial adhesion, and a reagent composition is further disposed under the bottom layer of the microporous layers. is contained in a hydrophilic polymer binder, at least one of the interfaces of adjacent microporous layers that are partially bonded in an integrated multilayer analytical element on which at least one detection functional layer is laminated. , the average value of the pore diameters on the surface of the lower microporous layer facing the upper microporous layer is the average value of the pore diameters on the surface of the upper microporous layer facing the lower microporous layer. an integrated multilayer analytical element, wherein the average value of the distance between the surfaces of the interface is larger than the average value of the pore diameters of the surface of the upper microporous layer facing the lower microporous layer. .