JP2731982B2 - Integrated multilayer analytical element - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to an improved integrated multi-layer analytical element for the analysis of analytes in an aqueous liquid sample.
More specifically, dry-operable and particularly useful for clinical analysis for the quantitative analysis of analytes in aqueous liquid samples such as biological fluids such as blood (whole blood, plasma, serum), cerebrospinal fluid, lymph, saliva, urine, etc. The integrated multi-layer analysis element.

[0002]

2. Description of the Related Art As a method for quantitatively analyzing an analyte in an aqueous liquid sample, particularly a biological fluid, a colorimetric method using a dry integrated multilayer analytical element is carried out in clinical examinations and general chemical analysis fields. It has become.

[0003] Multilayer analysis elements have come to be used for quantitative analysis of blood components in clinical analysis. It has been recognized that poor reproducibility often occurs. Variations in measured values are often caused by trace amounts of the same component substance as the analyte component in the analysis element. As an example of eliminating the dispersion of the measured values by reducing the content of the same component substance as the component to be analyzed in the analysis element, a cloth (a woven cloth or a knitted cloth) used for a developing layer in a multilayer analysis element for quantitative calcium analysis is used. Examples found to be solved by reducing the amount of calcium contained as a contaminant or a contaminant in cloth (such as cloth) (JP-A-64-25061). An example in which the problem is solved by reducing the amount of calcium contained as a contaminant or a contaminant in the binder polymer or other components used (Japanese Patent Application Laid-Open No. 2-136746).
Etc.

[0004]

However, Japanese Patent Laid-Open No. 2-13 / 1990
It has been found that even if gelatin having a reduced amount of calcium described in JP-A-6746 is used, the problems of variations in measured values and variations in background optical density values cannot be sufficiently solved.

SUMMARY OF THE INVENTION It is an object of the present invention to provide an improved integrated multi-layer analytical element capable of performing high-precision and accurate quantitative analysis of an analyte in an aqueous liquid sample by a simple dry operation.

Another object of the present invention is to provide an improved method for accurately and quantitatively analyzing the components to be analyzed in a blood sample using a blood sample (whole blood, plasma, serum) having no or low dilution ratio. The purpose of the present invention is to provide a multi-layered analysis element.

The present inventors have conducted intensive studies to achieve the above object, and as a result, by using a gelatin having a reduced ammonia content in a multilayer analytical element for ammonia quantitative analysis, the measurement of the multilayer analytical element for ammonia quantitative analysis was carried out. It has been found that the cause of the value variation is eliminated. In addition, an increase in the ammonia content in the solution over time after dissolving the reagent layer coating solution containing gelatin as a binder for dispersing and containing the indicator increases the background optical density value of the multi-layer analysis element, so that the ammonia concentration is increased. Increases the positive error. Gelatin selected in the integrated multilayer analysis element of the present invention also has a small increase in ammonia content,
The problem of increasing the positive error was solved.

[0008] Gelatin used as a binder in the indicator layer or the like also contains metal ions and anions, which may cause variations in measured values of not only calcium and ammonia as components to be analyzed but also other components to be analyzed. It is estimated that it had become. Since metal ions often have a strong inhibitory effect on an enzyme reaction, it is highly likely that the measured value of the analyte component varies in a reagent assembly containing an enzyme as a component of the detection reagent. Electrolytes in blood (Na, K,
Cl, etc.) in the determination of N contained in the binder
It is presumed that problems such as variations in measured values and variations in background optical density values have occurred due to a, K, Cl, and the like. The present invention has also solved these problems.

[0009]

SUMMARY OF THE INVENTION The above objects of the present invention are achieved by providing at least an indicator on a light-permeable, water-impermeable support, which binds to an analyte and makes an optically detectable change. An integrated multilayer analytical element having a reagent layer and a porous developing layer containing one kind in this order, wherein the binder of the reagent layer is gelatin having the following physical properties. NH ₃ content: 25 ppm or less Ca ²⁺ ion content: 5 ppm or less An aqueous solution containing gelatin and sodium tetraborate decahydrate is adjusted to pH 10 at 45 ° C. with a 5N-NaOH aqueous solution to have a gelatin content of 8 wt. %, sodium tetraborate decahydrate content 45 ° C. of an aqueous solution of gelatin is 2 wt%, NH ₃ content increases in pH10 is 70ppm or less in 2 hours.

[0010] Preferably, the integrated multilayer analytical element according to claim 1, wherein the gelatin further has the following physical properties. Cl ^- ion content: 30 ppm or less Fe ion content: 10 ppm or less Na ⁺ ion content: 5 ppm or less K ⁺ ion content: 2 ppm or less Mg ion content: 2 ppm or less Mn ion, Cu ion, Zn ion, Pb ion, Cr ions, NO ₃ ^- ions, SO ₄ ^2-ion content: was achieved by following the 1 ppm.

The gelatin used in the integrated multilayer analytical element of the present invention has the above-mentioned physical properties. More specifically, the content of NH ₃ is 25 ppm or less, preferably
It is 15 ppm or less. An aqueous solution containing gelatin and sodium tetraborate is adjusted to pH 10.0 at 45 ° C. with a 5N—NaOH aqueous solution at a temperature of 45 ° C. and pH 10 of an aqueous gelatin solution having a gelatin content of 8.0% by weight and a sodium tetraborate content of 2.0%. NH ₃ content increases 70ppm or less in 2 hours, preferably 50
It is less than ppm. The Ca ²⁻ ion content is below 5 ppm, preferably below 2 ppm, most preferably below 1 ppm. F
e ion content is 10 ppm or less, preferably 5 ppm or less,
Most preferably, it is 3 ppm or less. Cl ^- content of ions 30ppm or less, preferably 15ppm or less, and most preferably 10
It is less than ppm. Na ⁺ ion content is 5ppm or less, preferably 2ppm or less, and most preferably at 1ppm or less, K ⁺
The ion content is 2 ppm or less, preferably 1 ppm or less, most preferably 0.5 ppm or less. Mg ion content is 2ppm
Or less, preferably 1 ppm or less, most preferably 0.5 ppm or less. Mn ions, Cu ions, Zn ions, Pb ion, Cr ion, NO ₃ ^- ion, the content of SO ₄ ^2-ionic each 1ppm or less, preferably 0.2ppm or less, and most preferably 0.1ppm or less.

As a suitable gelatin for the present invention, a commercially available gelatin produced by various different processes was compared in detail, and as a result, it was found that Nitta Gelatin P.
Gelatin represented by -3408 was found to be particularly preferred.

[0013] The integrated multilayer analytical element of the present invention is a reagent comprising, on a light-permeable, water-impermeable support, at least one type of indicator that changes with an analyte by binding to an analyte. And a porous spreading layer in this order.

As the light-transmitting water-impermeable support, a light-transmitting (transparent) material used in conventionally known multilayer analytical elements can be used.
A water-impermeable support can be used. Specific examples thereof include polyethylene terephthalate, bisphenol A polycarbonate, polystyrene, and a polymer such as cellulose ester (eg, cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.) having a thickness of about 50 μm to about 1 mm, preferably About 8
Transparent from 0μm to about 300μm, i.e. wavelength of about 200n
A film-like (sheet-like) or flat-plate-like support having a smooth surface that transmits electromagnetic radiation in at least a part of the wavelength range from m to about 900 nm can be used. If necessary, a known undercoat layer or adhesive layer may be provided on the surface of the support to enhance the adhesion between the water-absorbing layer or the reagent layer provided on the support and the support. The support is preferably substantially transparent, but if necessary, titanium dioxide fine particles, barium sulfate fine particles,
Optical performance can be adjusted by dispersing and containing a small amount of carbon black or the like.

[0015] The reagent layer is a water-absorbing material in which a reagent composition comprising at least one detectable changing indicator associated with the analyte in the aqueous liquid sample is substantially uniformly dispersed in the binder. It is a water-permeable layer. The aforementioned gelatin is used as a binder used for this reagent layer. The dry thickness of the reagent layer ranges from about 5 μm to about 50 μm, preferably from about 7 μm to about 30 μm, with a coverage of about 5 g / m ² to about 50 g / m ^2.
m ² , preferably in the range from about 7 g / m ² to about 30 g / m ² . The pH at the time of analysis operation can be adjusted by incorporating a pH buffer and a known basic polymer described later in the reagent layer. Further, the reagent layer may contain a known mordant, a polymer mordant and the like. The reagent layer is preferably substantially transparent, but if necessary, a small amount of titanium dioxide fine particles, barium sulfate fine particles, carbon black or the like may be dispersed and contained in the layer to adjust the optical performance.

Examples of the porous spreading layer include a woven fabric spreading layer described in JP-A-55-164356 and JP-A-57-66359 (eg, plain woven fabric such as broad and poplin), and
No. 60-222769, a knitted fabric spreading layer (eg, a tricot knitted fabric, a double tricot knitted fabric, a Miranese knitted fabric, etc.), and a woven fabric etched with an alkali etching solution described in JP-A-1-172753. Or a developing layer comprising a knitted fabric, a developing layer comprising an organic polymer fiber pulp-containing papermaking paper described in JP-A-57-148250;
No. 53-21677, U.S. Pat. No. 3,992,158, etc., such as a membrane filter (brush polymer layer), polymer microbeads, glass microbeads, and a porous layer containing continuous microvoids in which diatomaceous earth is held by a hydrophilic polymer binder. Fiber isotropic porous spreading layer, continuous microvoid-containing porous layer (three-dimensional) in which polymer microbeads described in JP-A-55-90859 are bonded in a point contact manner with a polymer adhesive that does not swell with water. A non-fiber isotropic porous development layer composed of a lattice-like granular structure layer) can be used.

The woven fabric, knitted fabric or paper used for the porous spreading layer may be subjected to a physical activation treatment typified by a glow discharge treatment or a corona discharge treatment described in JP-A-57-66359. It is applied to at least one side, or is successively performed by appropriately performing a water-washing degreasing treatment described in JP-A-55-164356, JP-A-57-66359, or the like, or a hydrophilic treatment such as impregnation with a hydrophilic polymer, or an appropriate combination of these treatment steps. Thereby, the fabric or paper can be made hydrophilic, and the adhesive force with the lower layer (the side closer to the support) can be increased.

In the multilayered analysis element of the present invention, various layers other than the above-mentioned layers can be incorporated according to the kind of the component to be analyzed. For example, when analyzing calcium or the like, a water absorbing layer can be provided between the support and the reagent layer. The water-absorbing layer is a layer mainly composed of a hydrophilic polymer that absorbs water and swells, and is a layer that can absorb the water of the aqueous liquid sample that has reached or penetrated the interface of the water-absorbing layer. It has the effect of promoting the penetration of the plasma, which is an aqueous liquid component, into the reagent layer. The hydrophilic polymer used in the water-absorbing layer is a polymer having a swelling ratio upon absorption of water at 30 ° C. of about 150% to about 2000%, preferably about 250% to about 1500%. Specific examples of the hydrophilic polymer include the above-mentioned gelatin, gelatin described in JP-A-59-171864, JP-A-60-115859, gelatin such as deionized gelatin, phthalated gelatin, and hydroxyacrylate graft. Gelatin derivatives such as gelatin, JP-A-59-171864, JP-A
Examples include agarose, pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone described in JP-A-60-115859 and the like, and methallyl alcohol binary or ternary copolymers described in JP-A-62-32109. These hydrophilic polymers may be used alone or
More than one species can be used in combination. Generally, gelatin or a gelatin derivative, polyacrylamide, or polyvinyl alcohol, particularly the above-mentioned gelatin or deionized gelatin, is preferable for the water-absorbing layer, and the above-mentioned gelatin, which is the same as the reagent layer, is most preferable. The dry thickness of the water absorbing layer is about 3μm
From about 100 μm to about 100 μm, preferably from about 5 μm to about 30 μm,
The coating amount is about 3 g / m ² to about 100 g / m ² , preferably about 5 g / m ^2
It ranges from ² to about 30 g / m ² . The pH of the water-absorbing layer at the time of use (at the time of performing an analysis operation) can be adjusted by incorporating a pH buffer described below, a known basic polymer, and the like. Further, the water absorbing layer may contain a known mordant, a polymer mordant and the like.

Further, a hydrophilic non-porous intermediate layer that does not allow at least protein to permeate can be provided between the developing layer and the reagent layer. The hydrophilic non-porous intermediate layer is made of a hydrophilic polymer binder or a cross-linked hydrophilic polymer binder similar to that used in the water-absorbing layer, and has a thickness that does not allow passage of high molecular weight proteins, particularly albumin and globulin. It is. For the hydrophilic non-porous intermediate layer (hereinafter sometimes referred to as an intermediate layer), the above-mentioned gelatin, gelatin having a low calcium ion content, deionized gelatin or a gelatin derivative, polyacrylamide, polyvinyl alcohol and the like are generally used. It is preferable to use the above-mentioned gelatin, which is the same as the reagent layer, and most preferable. The hydrophilic polymer used for the intermediate layer can be an intermediate layer appropriately crosslinked and cured using a crosslinking agent. Examples of crosslinking agents include 1,
Known vinylsulfone-based cross-linking agents such as 2-bis (vinylsulfonylacetamide) ethane and bis (vinylsulfonylmethyl) ether; aldehydes; aldehydes to methallyl alcohol copolymers; and epoxy compounds containing two glycidyl groups. The dry thickness of the intermediate layer ranges from about 5 μm to about 50 μm, preferably from about 7 μm to about 30 μm, with a coverage of about 5 g / m ² to about 50 g / m ² , preferably about 7 g / m ²
g / m ² to about 30 g / m ² .

On the reagent layer or an optional intermediate layer, a deionized gelatin having a very small content of calcium ions, other metal cations and anions, etc. for the purpose of firmly bonding and integrating the spread layer. A known adhesive layer made of the same hydrophilic polymer as used for the water-absorbing layer can be provided. The dry thickness of the adhesive layer ranges from about 0.5 μm to about 5 μm.

When analyzing ammonia, a liquid blocking layer (hereinafter, also referred to as a barrier layer) through which gaseous ammonia can pass is provided on the reagent layer. The barrier layer is used for producing a multilayer analysis element (specifically, when a reaction layer described later is provided on the barrier layer by coating) and / or during an analysis operation, such as a liquid such as a coating solution or a sample solution. Means a layer made of a substance that does not substantially pass or pass an interfering component (for example, an alkaline component, etc.) dissolved and contained in the liquid and that can pass gaseous ammonia. The barrier layer is roughly classified into two types in structure. One embodiment is an air barrier layer (hereinafter, referred to as an air barrier layer) which is made of a porous material having continuous voids and in which an air layer substantially acts as a barrier layer.
Another embodiment is a polymer barrier layer (hereinafter, referred to as a polymer barrier layer) which is a homogeneous nonporous thin layer made of a hydrophobic (or poorly hydrophilic) polymer.

Examples of porous materials having continuous voids constituting the air barrier layer include membrane filters: porous materials in which fibrous materials are entangled with each other or bonded or bonded (eg, Paper, filter paper, felt, non-woven fabric, etc.); and porous materials composed of woven fabric, knitted fabric or reticulated material.

In the polymer barrier layer, which is a homogeneous nonporous thin layer made of a hydrophobic (or poorly hydrophilic) polymer, it is preferable to use a polymer with low hydrophobicity or low hydrophilicity as the polymer. Specific examples of polymers having poor hydrophobicity or hydrophilicity include cellulose acetate, propionate, cellulose acetate butyrate, polycarbonate of bisphenol A, polyethylene, polypropylene, ethylene-vinyl acetate copolymer, polyurethane, polystyrene, and polyvinyl chloride. , Vinyl chloride / vinyl acetate copolymer, polyamide (nylon), polymethyl methacrylate and polyvinyl butyral. These polymers can be used alone or as a mixture of these polymers.

The thickness of the polymer barrier layer is usually about 0.1 to
It is preferably in the range of 6 μm, in particular in the range of about 0.2 to 3 μm. The polymer barrier layer is JP-B-58-19
It can be provided by applying and drying an organic solvent solution of a polymer according to the methods described in JP-A No. 062 and JP-A-60-21452, respectively.

The adhesive intermediate layer that can be provided between the barrier layer and the reagent layer is made of a polymer composition that exhibits adhesiveness in the atmosphere at a humidity of 10 to 85% and a normal ambient temperature (about 0 to 40 ° C.). Layer. The adhesive intermediate layer is described in JP-A-60-21.
It can be provided according to the materials and methods described in US Pat. The polymer composition constituting the tacky intermediate layer may be a single polymer or a mixture of two or more known polymers having a glass transition point (Tg) of 0 ° C. or lower, or a known tackifier (tackifier) if necessary. ), A surfactant and the like. The thickness of the adhesive intermediate layer is usually in the range of about 0.1 to 6 μm, preferably in the range of about 1 to 4 μm. Specific examples of the polymer that can be used for the adhesive intermediate layer include vinyl acetate / butyl acrylate copolymer, poly (ethyl acrylate), and styrene / butyl acrylate.
Examples thereof include butyl acrylate / acrylic acid / N- (hydroxymethyl) acrylamide quaternary copolymer, and butyl acrylate / (ethyl acetoacetate) methacrylate / 2-acrylamido-2-methylpropanesulfonic acid ternary copolymer. When the barrier layer is a polymer barrier layer composed of a homogeneous nonporous thin layer of a hydrophobic (and poorly hydrophilic) polymer, it is preferable to provide an adhesive intermediate layer.

On the reagent layer, there is provided a function of substantially preventing the ammonia generated in the reagent layer from diffusing into the ammonia trapping layer. It is preferable to provide a prevention layer. The ammonia diffusion preventing layer can be replaced with a layer having another function as long as the layer does not substantially perform the ammonia capture and ammonia generation reaction. Examples of the layer having another function include a cured hydrophilic polymer layer, a light shielding layer, an adhesive layer, and the like.

If the analytical element is for the determination of ammonia-generating substances, the ammonia (endogenous endogenous) already present in the aqueous liquid sample may be directly on the ammonia diffusion preventing layer or via a light shielding layer or another intermediate layer described later. It is preferable to provide an endogenous ammonia-trapping layer (hereinafter simply referred to as an endogenous ammonia-trapping layer) containing a reagent that acts on the ammonia to change the endogenous ammonia to a state in which it cannot substantially reach the reaction layer. . The endogenous ammonia-trapping layer removes coexisting endogenous ammonia before an ammonia-generating substance such as creatinine or urea nitrogen, which is an analyte (test component), reaches the reaction layer to cause a reaction in which ammonia is generated. This is a layer having a function of capturing.

As the endogenous ammonia capturing reagent, it is preferable to use a reagent composition containing an enzyme having a catalytic ability to convert ammonia into another substance using ammonia as a substrate. Specific examples of the endogenous ammonia capture reagent include NADH (nicotinamide-adenine-dinucleotide reduced form), and / or
Or NADPH (nicotinamide adenine dinucleotide phosphate reduced form), glutamate dehydrogenase (EC1.4.1.3; hereinafter referred to as GlDH) and α-ketoglutarate (or its disodium salt: hereinafter referred to as α-KG) Can be mentioned. Also,
A reagent composition containing aspartase (EC 4.3.1.1) and fumaric acid or a fumarate salt may be used. In the integrated multilayer analytical element of the present invention, it is preferable to use a reagent composition containing NADH, GlDH and α-KG as an endogenous ammonia capture reagent. When a reagent composition containing GLDH or a reagent composition containing aspartase is used, the pH value of the endogenous ammonia-trapping layer is usually 10.0 or less, preferably in a range of 7.0 to 9.5. It is preferable to use a suitable buffer.

When the analytical element is for ammonia determination,
It goes without saying that the endogenous ammonia trapping layer is not provided.

The light shielding layer which can be provided between the barrier layer and the endogenous ammonia trapping layer is made of fine particles or fine powder having light shielding properties or having both light shielding properties and light reflection properties (hereinafter simply referred to as fine particles). Is a water-permeable or water-permeable layer dispersed and held in a small amount of a hydrophilic (or weakly hydrophilic) polymer binder capable of forming a film. The light-shielding layer is a color of an aqueous liquid sample that is spotted and supplied to a porous developing layer described below when a detectable change (color change, color development, etc.) generated in the indicator layer is reflected and measured from the support side. In addition to blocking red and the like due to hemoglobin in the case of whole blood, it also functions as a light reflection layer and a background layer. Examples of the fine particles having both light shielding properties and light reflecting properties include titanium dioxide fine particles, barium sulfate fine particles, aluminum fine particles, and fine flakes.

The integrated multi-layer analysis element of the present invention contains a reagent according to the type of the component to be analyzed. For example, when analyzing calcium, a known chelating indicator is used as an indicator in the reagent composition contained in the reagent layer. Specific examples of chelating indicators for calcium analysis include o-cresolphthalein complexone (3,3′-bis [[di (carboxymethyl) amino] methyl] -o-cresolphthalein [2411-89-4], The numbers in [] below are in Chem
ical Abstracts Registry Number), Eriochrome Black T (1- (1-hydroxy-2-naphthylazo) -6
-Nitro-2-hydroxynaphthalene-4-sulfonic acid monosodium salt [1787-61-7]), methylthymol blue complexone (3,3'-bis [[di (carboxymethyl) amino] methyl] thymol sulfonephthalate) Rein tetrasodium salt [1945-77-3]), thymolphthalein complexone (3,3'-bis [[di (carboxymethyl) amino] methyl thymolphthalein [1913-93-5]), 3,6 -Screw
[(4-Chloro-2-phosphonophenyl) azo] -4,5-dihydroxy-2,7-naphthalenedisulfonic acid disodium salt (chlorophosphonazo-III) [1914-99-4], 3-[( 4-chloro-2-phosphonophenyl) azo] -4,5-dihydroxy-
Described in the "Dotite Reagents General Catalog 12th Edition" (Kumamoto City, Dojindo Research Laboratories, 1980), such as 2,7-naphthalenedisulfonic acid disodium salt (chlorophosphonazo-I) [1938-82-5] There are indicators. Among these indicators, o-cresolphthalein complexone and chlorophosphonazo-III are preferable in that the most accurate quantitative analysis can be performed for both total calcium and calcium ions. If necessary, the reagent composition may be contained in two or more separate layers (for example, a reagent layer and a water-absorbing layer).

If necessary, masking of magnesium ions such as 8-hydroxyquinoline, 8-hydroxyquinoline-5-sulfonic acid, 8-hydroxyquinoline-4-sulfonic acid, and 8-hydroxyquinoline sulfate to shield magnesium ions. It is preferable to include an agent. The layer containing the magnesium ion masking agent is a reagent layer and / or
Or an intermediate layer, an adhesive layer, or a spreading layer above (a side farther from the support). The coverage of the magnesium ion masking agent ranges from about 1.5 times to about 10 times, preferably from about 2 times to about 5 times the indicator coverage.

In the case of analyzing calcium, the environmental pH value at the time of performing the analysis operation with the aqueous liquid sample spotted thereon is buffered to a desired value in the range of about 3.0 to about 12.0, preferably about 4.0 to about 11.5. It is appropriately selected from known buffering agents that can be contained. The pH buffer is contained in at least one of a reagent layer, a water-absorbing layer, an intermediate layer, and an adhesive layer described below (closer to the support) than the developing layer. Among these layers, it is preferable to include them in the reagent layer and / or the water absorbing layer.

As a buffer which can be used, "Chemical Handbook Basic Edition" edited by The Chemical Society of Japan (Maruzen, Tokyo, published in 1966), 1312-1320
RMCDawson et al, “Data for Biochemical Res”
earch "2nd edition (Oxford at the Clarendon Press, 1969
476-508; "Biochemistry" 5 , 467-477 (1966); "Analytical Biochemistry" 104, 300-3.
There is a pH buffer composition described on page 10 (1980) and the like. pH 3.
Specific examples of pH buffers ranging from 0 to 12.0 include 2- (N-morpholino) ethanesulfonic acid (MES); 3,3-dimethylglutaric acid; imidazole; tris (hydroxymethyl) aminomethane (Tris). Buffer; Buffer containing phosphate; Buffer containing borate; Buffer containing carbonate; Buffer containing glycine; N, N-bis (2-hydroxyethyl) glycine (Bicine); 4- (2 -Hydroxyethyl) -1-piperazinepropanesulfonic acid (HEPPS), Na salt or K salt, etc .; 4
-(2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), Na salt or K salt; 3-[[2-hydroxy-1,1-
Bis (hydroxymethyl) ethyl] amino] -1-propanesulfonic acid (TAPS), Na salt or K salt; β-hydroxy-4
-(2-hydroxyethyl) -1-piperazinepropanesulfonic acid (HEPPSO), Na salt or K salt; 3- (cyclohexylamino) -1-propanesulfonic acid (CAPS), Na salt or K salt; and the like There are acids, alkalis or salts optionally combined with any of the above. Specific examples of preferred buffers include MES; 3,3-dimethylglutaric acid; imidazole; Tris-sodium borate; Bicine; HEPPS; HEPPS sodium salt; HEPPSO; HEPPSO sodium salt; TAPS; TAPS sodium salt; CAPS; CAPS sodium salt. CAPS potassium salt and the like.

When analyzing ammonia, a leuco dye such as a leucocyanine dye, a nitro-substituted leuco dye and a leucophthalein dye (US Pat. No. Re. 30267 or JP-B-58-19062): pH such as bromophenol blue, bromocresol green, bromothymol blue, quinoline blue and rosolinic acid
Indicator (see Kyoritsu Shuppan Co., Ltd., Dictionary of Chemistry, Vol. 10, pp. 63-65): Triarylmethane dye precursor: Leucobenzylidene dye (JP-A-55-379 and JP-A-56-145273)
Described in each publication): diazonium salts and azo dye couplers: base bleachable dyes and the like. It is preferable that the compounding amount of the coloring ammonia indicator with respect to the weight of the binder is in the range of about 1 to 20% by weight.

The reagent which reacts with the ammonia-generating substance to produce ammonia is preferably an enzyme or a reagent containing the enzyme. An enzyme suitable for analysis is selected according to the type of the ammonia-generating substance which is an analyte. Can be used. When using an enzyme as the above reagent,
The combination of the ammonia producing substance and the reagent is determined from the specificity of the enzyme. Specific examples of combinations of ammonia producing substances / reagents include urea / urease, creatinine / creatinine deiminase, amino acid / amino acid dehydrogenase, amino acid / amino acid oxidase, amino acid / ammonia lyase, amine / amine oxidase, diamine / amine oxidase, glucose and Phosphoamidate / phosphoamidate hexose phosphotransferase, ADP / carbamate kinase and carbamate phosphate, acid amide / amide hydrolase, nucleobase / nucleobase deaminase, nucleoside / nucleoside deaminase, nucleotide / nucleotide deaminase, guanine / guanase and the like. be able to. An alkaline buffer that can be used in the reagent layer when analyzing ammonia has a pH of
Buffers ranging from 7.0 to 12.0, preferably 7.5 to 11.5 can be used.

In the reagent layer for analyzing ammonia,
In addition to a reagent that reacts with an ammonia-generating substance to generate ammonia, an alkaline buffer, and a hydrophilic polymer binder having a film-forming ability, a wetting agent, a binder cross-linking agent (curing agent), a stabilizer, and a heavy metal ion trap as required Agents (complexing agents) and the like.
Heavy metal ion trapping agents are used for masking heavy metal ions that inhibit enzyme activity. Specific examples of heavy metal ion trapping agents include EDTA
・ 2Na, EDTA ・ 4Na, nitrilotriacetic acid (NTA),
Complexes such as diethylenetriaminepentaacetic acid can be mentioned.

A reagent layer, a water absorbing layer, an ammonia diffusion preventing layer,
Endogenous ammonia trapping layer, light reflecting layer, adhesive layer, spreading layer,
A surfactant can be contained in the intermediate layer, the developing layer containing the ammonia capture reagent composition, and the like. An example is a nonionic surfactant. Specific examples of nonionic surfactants include p-octylphenoxypolyethoxyethanol, p-nonylphenoxypolyethoxyethanol, polyoxyethylene oleyl ether, polyoxyethylene sorbitan monolaurate, p-nonylphenoxypolyglycidol, octyl glucoside, and the like. There is. By including a nonionic surfactant in the spreading layer, the spreading action (metering action) of the aqueous liquid sample is further improved. By including a nonionic surfactant in the reagent layer or the water-absorbing layer, the water in the aqueous liquid sample is easily and substantially uniformly absorbed by the reagent layer or the water-absorbing layer during the analysis operation, and the The contact is rapid and substantially uniform.

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

The multilayer analytical element of the present invention has a side of about 15 mm to 30 mm.
It is cut into small pieces such as a square of mm or a circle of almost the same size, and Japanese Patent Publication No. 57-28331, Japanese Utility Model Publication No. Sho 56-142454, Japanese Patent Application Laid-Open No. Sho 57-63452, Japanese Utility Model Publication No. Sho 58-32350. ,
From the viewpoints of various kinds such as production, packaging, transportation, storage and measurement operation, it is preferable to use the slides described in JP-T-58-501144 in a slide frame and use them as chemical analysis slides. Depending on the purpose of use, it can be used in the form of a long tape in a cassette or magazine, or can be used by sticking or storing a small piece on a card having an opening.

The multilayer analytical element of the present invention can analyze an analyte in a liquid sample by the operations described in the above-mentioned various patent specifications and the like. For example, about 5 μl to 30 μl, preferably 8 μl
A droplet of an aqueous liquid sample such as whole blood, plasma, serum, or urine in the range of μl to 15 μl is spotted on the developing layer.
Incubate at a substantially constant temperature in the range of 20 ° C. to 40 ° C., preferably at a substantially constant temperature near 37 ° C., to reduce the color or discoloration in the element to the absorption maximum wavelength of visible or ultraviolet light or The content of the analyte in the liquid sample can be determined based on the principle of colorimetric measurement using a calibration curve prepared in advance by performing reflection photometry from the light-transmitting support side using light having a wavelength in the vicinity thereof. Alternatively, the intensity of the fluorescence in the element can be measured, and the analyte content in the liquid sample can be determined using a calibration curve prepared in advance. By keeping the amount of the liquid sample to be spotted, the incubation time and the temperature constant, quantitative analysis of the analyte can be performed with high accuracy. The measurement operation is extremely easy with a chemical analyzer described in JP-A-60-125543, JP-A-60-220862, JP-A-61-294367, JP-A-58-161867 and the like. Can perform high-precision quantitative analysis.

[0042]

【Example】

Example 1 The contents of various components in gelatin suitable for the integrated multilayer analytical element of the present invention and commercially available deionized gelatin were measured. Table 1
Shows the results. The NH ₃ content was determined by adding an aqueous solution containing gelatin and sodium tetraborate to a 5N NaOH aqueous solution at 45 ° C.
The NH ₃ content (ppm) when an aqueous gelatin solution having a gelatin content of 8% by weight and a sodium tetraborate decahydrate content of 2% by weight adjusted to H10.0 and left at 45 ° C. is shown.

[0043]

[Table 1]

Example 2, Comparative Examples 1, 2, 3 and 4 Colorless and transparent polyethylene terephthalate (P) having a thickness of 180 μm
ET) On a film (support), coating layers were successively applied using an aqueous solution so as to have the following component coating amounts, and were laminated by drying.

The gelatin used was as follows. Multilayer analytical element of Example 2: Gelatin G of Table 1 Multilayer analytical element of Comparative Example 1: Commercial gelatin I of Table 1 Multilayer analytical element of Comparative Example 2: Commercial gelatin II of Table 1 Multilayer analytical element of Comparative Example 3: Table 1 Commercial gelatin III Multi-layer analytical element of Comparative Example 4: Commercial gelatin I of Table 1 was prepared by the procedure described in JP-A-2-136746 to obtain a calcium content of 2.
Reduced to 0 ppm

Citric acid, polyvinylpyrrolidone, titanium dioxide fine particles, two types of nonionic surfactants, CAPS, sodium hydroxide, and 8-hydroxyquinoline-5-sulfonic acid all have a calcium content of 1.0 ppm or less. Was. Water absorption layer: Gelatin 7.7 g / m ² Nonylphenoxy polyglycidol 250 mg / m ² (containing 10 glycidol units on average) 1,2-bis (vinylsulfonylacetamide) ethane 190 mg / m ²

After the water-absorbing layer was dried, an indicator layer was applied thereon by using an aqueous solution so as to have a coating amount of the following composition, and was provided by drying. Indicator layer: Gelatin 16.8 g / m ² nonylphenoxypolyglycidol 1.1 g / m ² (glycidol unit mean 10-containing) 3- (cyclohexylamino) -1-propanesulfonic acid 2.8g / m ² (CAPS) o- cresol phthalein Complexone 150 mg / m ² 8-hydroxyquinoline-5-sulfonic acid 560 mg / m ² aqueous solution was adjusted to pH 11.0 with NaOH. Adhesive layer: was adjusted to a gelatin 1.46 g / m ² nonylphenoxypolyglycidol 100 g / m ² (glycidol unit mean 10 containing) titanium dioxide particles 850 mg / m ² aqueous solution with NaOH pH 11.0.

Next, water is supplied almost uniformly to the surface of the adhesive layer to wet it, and then a tricot knitted fabric whose calcium content is reduced by the operation described in JP-A-64-25061 is almost completely wetted. The layers were laminated by applying light pressure as described above. Then, an ethanol solution containing citric acid and polyvinylpyrrolidone was applied from above the spread layer so as to have the following coating amount, and dried to prepare an integrated multilayer analytical element for total calcium determination. Citric acid and polyvinylpyrrolidone coating composition: Polyvinylpyrrolidone 4.1 g / m ² (average molecular weight about 1.2 million) Citric acid 600 mg / m ² Nonylphenoxypolyethoxyethanol 8.6 g / m ² (containing an average of 40 oxyethylene units)

Performance Evaluation Test In order to evaluate the performance of the obtained multilayer analytical element for total calcium determination, the total calcium content was determined to be 9.80 mg / dL (measured value by the colorimetric reagent method for measuring total calcium amount). Control serum (Monitrol IX; American Dade)
The total calcium content was measured 10 times using each method, and the results shown in Table 2 were obtained.

[0050]

[Table 2]

From the results in Table 2, it is clear that the measured value of total calcium content by the multilayer analytical element of the present invention is very close to the test value by the standard method. On the other hand, the measured values of the total calcium content by the multilayer analytical elements of Comparative Examples 1, 2, 3 and 4 which are the prior art are far from the test values. From the results in Table 2, it is clear that the performance of the multilayer analytical element of the present invention is superior to that of the multilayer analytical element according to the prior art.

Example 3, Comparative Examples 5, 6, 7, and 8 Colorless and transparent polyethylene terephthalate (P) having a thickness of 180 μm
ET) Coating layers were sequentially applied on a film (support) so as to have the following component coating amounts, dried, and laminated.

The gelatin used in the reaction layer is as follows. Multilayer analytical element of Example 3: Gelatin G of Table 1 Multilayer analytical element of Comparative Example 5: Commercial gelatin I of Table 1 Multilayer analytical element of Comparative Example 6: Commercial gelatin II of Table 1 Multilayer analytical element of Comparative Example 7: Table 1 Commercial gelatin III Multilayer analytical element of Comparative Example 8: Commercial gelatin I of Table 1 was prepared by the procedure described in JP-A-2-136746 to obtain an ammonia content of 22.
Indicator layer: polyvinyl methyl ether 2.2 g / m ² (weight average molecular weight about 40,000) Bromophenol blue 140 mg / m ² NaOH 8.4 mg / m ²

Liquid permeation blocking layer Polyethylene membrane filter (average pore size 0.2 μm)
m, a porosity of about 75%, and a thickness of 100 μm) were uniformly pressed on the indicator layer.

Reaction layer: Gelatin 16 g / m ² Sodium tetraborate decahydrate 4 g / m ² p-Nonylphenoxypolyglycidol 200 mg / m ² (glycidol unit average: 10) An aqueous solution was adjusted to pH 11.0 with NaOH. 0.2% p-
Nonylphenylphenopolypolyglycidol (glycidol unit average: 10) Swell almost uniformly with an aqueous solution, and immediately knit fabric (polyethylene terephthalate, gauge number 40, N
25% weight loss treatment with aOH aqueous solution, thickness 350μm,
After passing between the pressure rollers while keeping the porosity close to 80%, they were dried and uniformly laminated. Developing layer: polyvinylpyrrolidone 7.8 g / m ² (average molecular weight about 1.2 million)

Performance Evaluation Test Ammonium sulfate was added to and dissolved in a 7% human serum albumin aqueous solution so that the concentration of ammonia nitrogen became 40, 100, and 300 μg / dL, respectively, to prepare an evaluation test solution. Using this, the ammonia nitrogen concentration was measured 10 times each, and Table 3
The results described in the above section were obtained.

[0057]

[Table 3]

From the results shown in Table 3, it is clear that the measured value of the ammonia nitrogen concentration by the multi-layer analysis element of the present invention is very close to the test value by the standard method. on the other hand,
The measured values of the ammonia nitrogen concentration by the multilayer analysis elements of Comparative Examples 5, 6, 7, and 8 which are the prior art are far from the test values. From the results in Table 3, it is clear that the performance of the multilayer analytical element of the present invention is superior to that of the conventional multilayer analytical element.

Example 4, Comparative Examples 9, 10, 11 and 12 The reaction layer aqueous solutions of Example 3, Comparative Examples 5, 6, 7 and 8
Example 4 and Comparative Examples 9, 10, and 11, respectively, except that a multilayer analysis element was prepared after being left at 2 ° C. for 2 hours.
And 12 multi-layer analysis elements were created.

A performance evaluation test was performed in exactly the same manner as in Example 3 and Comparative Examples 5, 6, 7, and 8, and the results shown in Table 4 were obtained.

[0061]

[Table 4]

From the results shown in Table 4, the measured value of the ammonia nitrogen concentration by the multilayer analysis element of the present invention was as follows.
It is clear that even after standing for 2 hours, a measurement value close to the test value by the standard method can be obtained. On the other hand, the measured values of the ammonia nitrogen concentration by the multi-layer analysis elements of Comparative Examples 9, 10, 11, and 12, which are the prior art, greatly increase from the test values. Table 4
It is clear from the results that the performance of the multilayer analytical element of the present invention is superior to that of the conventional multilayer analytical element.

[0063]

According to the present invention, it is possible to eliminate variations in measured values in the integrated multilayer analytical element. Further, in the integrated multi-layer analysis element for ammonia analysis, it is possible to suppress an increase in the background optical density value over time.

Claims (2)

(57) [Claims] 1. A reagent layer and a porous developing layer, each containing at least one kind of indicator that changes optically by binding to a component to be analyzed, on a light-permeable water-impermeable support. In the integrated multi-layered analysis element having the following sequence, the binder of the reagent layer is gelatin having the following physical properties: an integrated multi-layered analysis element NH ₃ content: 25 ppm or less Ca ²⁺ ion content: 5 ppm or less Gelatin An aqueous solution containing sodium tetraborate decahydrate and a 5N-NaOH aqueous solution adjusted to pH 10 at 45 ° C. has a gelatin content of 8% by weight and a sodium tetraborate decahydrate content of 2% by weight. Increase of NH ₃ content of gelatin aqueous solution at 45 ° C and pH10 is less than 70ppm in 2 hours 2. The integrated multilayer analytical element according to claim 1, wherein the gelatin further has the following physical properties: Cl ^- ion content: 30 ppm or less Fe ion content: 10 ppm or less Na ⁺ ion content: 5 ppm or less K ⁺ ion content: 2 ppm or less Mg ion content: 2 ppm or less Mn ion, Cu ion, Zn ion, Pb ion, Cr ion, NO ₃ ^- ion, SO ₄ ^2- ion content: 1 ppm or less for each