JPH0391497A - Integrated multi-layer analytic element containing reagent composition for determination of ammonium ion - Google Patents

Abstract

PURPOSE:To enable quantitative determination of urea nitrogen, ammonia, etc., in high accuracy by successively laminating a reagent layer having a specific composition, a developing layer and a pyruvic acid removing layer on a light-transmitting water-impermeable substrate to form a multi-layer analytic element. CONSTITUTION:An integrated multi-layer analytic element is produced by successively laminating a reagent layer, a developing layer and a pyruvic acid removing layer on a light-transmitting water-impermeable substrate. The reagent layer contains a glutamic acid synthetase, pyruvic acid kinase, pyruvic acid oxidase and a hydrogen peroxide detection reagent. The pyruvic acid removing layer contains a reagent containing an enzyme acting on pyruvic acid as a base and free from generation of hydrogen peroxide. The light-transmitting and water-impermeable substrate is e.g. a transparent snbstrate made of a polymer such as polyethylene terephthalate and having a thickness of about 50mum to 1mm.

Description

[Detailed description of the invention] - [Industrial application field] The present invention is directed to analyte components (analytes) that can generate ammonium ions such as urea nitrogen, ammonia, and creatinine.
The present invention relates to an integrated multilayer analytical element containing a reagent system for measuring .

[Conventional technology]

Urea is an intermediate and final product of protein metabolism! Elementary (B
Measurement of UN), ammonia, creatinine, etc. is necessary for diagnosing renal disease and determining the course of treatment. Conventionally,
These are analyzed by a wet method. For example, urea nitrogen is analyzed by a colorimetric method (D, J
, Jung, Cl1n, Chem, , 2L 113
6. (1975)), ammonium ions produced by the action of urease on urea were quantified using Nessler's reagent or by colorimetric fixation using the indophenol method. The indophenol method has since been improved by many researchers (for example, H,0kuda
et al, Tokushima J.

T! xpt1. Med, , 12. (1) 11-
23. (1965)). This method has the problem that the reaction is carried out under strongly acidic conditions, so the equipment is easily corroded, and there are also problems that the sensitivity is low and the measurement takes time.

Regarding the enzymatic method, a method has been developed that uses glutamate dehydrogenase to determine the amount of decrease in NADPH by measuring UV absorption (A, Mondzac et al., J.
, Lab, Cl1n, Med, , 66, 526. (19
65)), this method also had problems with sensitivity and accuracy.

A method has also been developed in which hydrogen peroxide produced by the action of glutamine synthetase in the presence of glutamate and ATP and further action of pyruvate kinase and pyruvate oxidase is colorimetrically fixed using peroxidase and 4-aminoantipyrine. (Japanese Unexamined Patent Publication No. 62-3800).

On the other hand, a dry method is also known as a method that allows rapid quantitative determination with simple operations. Examples of analytical instruments used in the dry method include:
Japanese Analytical Chemistry Full-length Analysis Library 3 “Clinical Chemistry Analysis ■Nitrogen-containing Components” 2nd edition (Tokyo Kagaku Dojin, published in 1979) 1
Pages 3-14, "Clinical Examination" Volume 5 (No. 6) 387-3
91 pages (published in 1961), US Pat. An indicator mJi (containing the pH indicator bromcresol green) was provided in this layer.
A reagent strip for UN quantitative analysis is disclosed. Japanese Patent Application Publication 1973-
Publication No. 93494 discloses a BUN quantitative analysis method in which a piece of filter paper is impregnated with pH-supporting dibromthymol blue, then further impregnated with a mixture of urease and a barrier former made of a hydrophilic polymer, and finally an ethyl cellulose semipermeable membrane is provided. Disclosed is a test piece for In addition, Japanese Patent Application Laid-open No. 52-3488 (Japanese Patent Publication No. 5B-19062) discloses a support drug layer on a water-impermeable transparent support, which allows ammonia vapor to penetrate uniformly and prevents liquid water and alkaline interference. A barrier layer consisting of a homogeneous (non-porous) thin layer of a hydrophobic polymer such as cellulose acetate butyrate that cannot be penetrated by water, a reagent layer containing urease and an alkaline agent, and a porous spreading layer are laminated together in this order. An integrated multilayer analysis element for BUN quantitative analysis has been proposed.

[Problem to be solved by the invention]

In all conventional dry methods, ammonia gas is diffused within the test piece or analysis element, and there is a problem in that this diffusion lacks quantitative properties.

An object of the present invention is to provide an analytical element that can quantify intermediates and final products of protein metabolism, such as urea nitrogen (BUN), creatinine, ammonia, etc., with high precision using a simple method.

[Means to solve the problem]

The present inventors conducted intensive studies to achieve the above objective, and found that the present inventors have developed an ammonium ion detection method that can basically be used commonly for measuring protein metabolic intermediates and final products, and is linked to color reactions. As a reaction principle, we focused on a method in which the above-mentioned glutamine synthetase is combined with pyruvate kinase and pyruvate oxidase, and the produced hydrogen peroxide is determined colorimetrically. However, when this reaction system was incorporated into a dry analytical element, a new problem arose in that pyruvic acid contained in the sample was detected at the same time, resulting in an error. Therefore, the present inventors further investigated and came up with a means to efficiently remove this pyruvic acid without affecting the above-mentioned reaction system, thereby achieving the above-mentioned object.

The present invention comprises glutamate ξ synthase, pyruvate kinase, pyruvate oxidase, and a hydrogen peroxide detection reagent composition, and includes at least a light-transparent water-impermeable support, all of the enzymes and reagent compositions described above. Alternatively, in a multilayer analysis element in which a reagent layer containing a part of the pyruvic acid and a developing layer are laminated in this order, the pyruvic acid removal layer containing an enzyme reaction reagent composition that uses pyruvic acid as a substrate but does not generate any hydrogen peroxide is added to the reagent layer. The present invention relates to an integrated multilayer analytical element containing an ammonium ion measuring reagent composition on the side opposite to the support.

The reaction system for detecting ammonium ions in the analytical element of the present invention proceeds as follows.

S Glutamic acid 10 ATP 114'';: Glutamine 10 ADP + Phosphate K ＾DP + Phosphoenolvirvic acid → 8 TP + Pyruvate Color or color change GS: Glutamine synthetase PK: Pyruvate kinase POP C Pyruvate oxidase POD = Peroxidase glutamine EC6.3.1.2] is E. coli (
Escherichia coli ATCC9637, etc.), Bacillus stearothrmophile
It can also be isolated from sheep brain, bovine brain, etc. Ba
The enzyme produced by Cillus stearothrmophilus is a thermostable enzyme that is commercially available and is particularly preferred as the analytical element of the present invention. pituvate kinase, (EC2.7.1.40) is f3acillus
ste-aroLhrmophilus, Bac
In addition to being produced equivocally, it can be obtained from the muscles of animals such as rats, rabbits, dogs, and chickens, the hearts of pigs, and the like. Pyruvate oxidase [ECI・2・3・6] is Lacto
It can be produced by various bacteria, including Bacillus delbrueckii. All of these enzymes are commercially available and can be easily purchased and used. On the other hand, for use in the analytical elements of the present invention, these enzymes do not need to be pure products; for example, it is also possible to use crude enzymes obtained from fermentation liquids of microorganisms or by means such as precipitation.

As the hydrogen peroxide detection reagent composition, a reagent composition containing peroxidase is preferred. Peroxidase is derived from plants such as wasabi, potatoes, radish, and figs, from animals such as white blood cells and milk, and from cochliobolus (Co
genus chliobolus, Curvu
Those derived from microorganisms such as the genus Laria are known, and any of these can be used, but it is convenient to use commercially available products.

Moreover, this reagent composition further includes a coloring reagent composition.
The coloring reagent composition includes a chromogen (also called a hydrogen donor) and a coupler, and the chromogen and coupler are oxidatively coupled with hydrogen peroxide in the presence of peroxidase to form a quinoneimine dye to form a color, or Leuco pigments or self-oxidizing chromogenic chromogens are metal compounds that are oxidized by hydrogen peroxide in the presence of peroxidase to become pigments and develop color.

As chromogens, Ann, Cl1n, Biochen+
, + 6.2427 (1969) (also known as 4-aminophenazine, i.e. 1
1-(2,4,6-trichlorophenyl)-2,3- as described in JP-A-59-54962, etc.
Dimethyl-4-amino-3-pyrazilin-5-one, 1
-(3,5-dichlorophenyl)-2,3-dimethyl-
Tri-substituted - such as 4-amino-3-pyrazolin-5-one
4-amino-3-pyrazolin-5-one, 1977-
4-aminoantipyrine analogs such as 1-phenyl-2,3-dimethylamino-3-pyrazolin-5-one described in 25840 and the like can be used. Among these compounds, 4-aminoantivirine, 1-(2,4,
6-)dichlorophenyl)-2,3-dimethyl-4-amino-3-pyrazolin-5-one, 1-,(3,5-dichlorophenyl)-2,3-dimethyl-4-amino-3
-pyrazolin-5-one and the like are preferred.

Couplers include Ann, Cl1n, and Biochen.
, + 6+ 24-27 (1969), Special Publication 1977-
25840, Special Publication Showa 5B-45599, Special Publication No. 58-
18628, Japanese Patent Application Publication No. 164356, Japanese Patent Application Publication No. 1987-
124398, phenol described in JP-A-56-155852, etc.; 2-hydroxy-1-benzenesulfonic acid;
4-Hydroxy-1-benzenesulfonic acid, 3゜5-dichloro-2-hydroxy-l-benzenesulfonic acid, 2
-hydroxy-3-methoxy-1benzenesulfonic acid,
Phenolsulfonic acids (including alkali metal salts and alkaline earth metal salts) such as 2-hydroxy-3-methoxy-1-benzenesulfonic acid, dihydroxynaphthalenes such as 1-naphthol, 2-naphthol, and 1゜7-dihydroxynaphthalene. ; Naphtholsulfonic acids (including alkali metal salts and alkaline earth metal salts) such as 1-hydroxy-2-naphthalenesulfonic acid and 1-hydroxy-4-naphthalenesulfonic acid, and other phenols or naphthol derivatives. Among these compounds, 1,7-dihydroxynaphthalene, 1-hydroxy-2-naphthalenesulfonic acid (including Na, K, and Li salts). 3,5-
Dichloro-2-hydroxy-1-benzenesulfonic acid (
(including Na salt, K salt, and Li salt) are preferred.

As a leuco dye, 2-
(4-hydroxy-3,5-dimethoxyphenyl)4.
Triarylimidazole leuco dyes such as 5-bis[4-(dimethylaξ])phenyl]iξdazole: 2-(3,5-dimethoxy-4-hydroxyphenyl)-4[described in JP-A-59-193352] Diaryl-dazole leuco dyes such as 4-(dimethylamino)phenyl-5-phenethyl-dazole; JP-A-61-4960
2-(2-phenyl-3-indolyl)-4, as described in
diarylindolylimidazole leuco dyes such as 5-di[4-(dimethylamino)phenylcoimidazole;
2-(4-hydroxy-3°5-dimethoxyphenyl)-3-acetyl-4 described in JP-A-61-229868
,5=bis[4-(diethylamino)phenylcoimidazole and other triarylmonoacylimidazole 04 codyes, 2-(4-hydroxy-3,5-dimethoxyphenyl)-3-methyl-4,5-bis[4 -(diethylamino)phenylcoimidazole and other triarylmonoalkyl dazole leuco dyes.

The integrated multilayer analytical element of the present invention has at least a light-transparent water-impermeable support, a reagent layer, and a developing layer laminated in this order.

As the light-transmitting and water-impermeable support, any known support used in general multilayer analytical elements may be used.

Specific examples thereof include polymers such as polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, and cellulose esters (cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.) with a thickness of about 50 nm to about 1 mm, preferably. A transparent support ranging from about 80 μm to about 300 μm can be used. If necessary, a known undercoat layer or adhesive layer may be provided on the surface of the support as described in the above-mentioned patent specifications to strengthen the adhesion with a hydrophilic layer or the like provided on the support.

The reagent layer is a layer containing all or part of the enzyme and reagent composition described above, and is a non-porous, water-absorbing layer or a microporous, water-permeable layer.

The hydrophilic polymer binder used in the substantially non-porous, water-absorbing reagent layer has a swelling rate of about 1 at 30'C upon water absorption.
Natural or synthetic hydrophilic polymers ranging from 50% to about 2000%, preferably from about 250% to about 1500%. Examples include gelatin (acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (phthalated gelatin,
(hydroxyacrylate grafted gelatin, etc.), agarose, pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, and the like. The dry thickness of the substantially non-porous reagent layer is from about 3 to about 5 On, preferably from about 5 to about 30 μm.
m range, and the coating amount is about 3g/rrf to about 50g
/n (preferably in the range of about 5 g/n to about 30 g/n). Preferably, the reagent layer is substantially transparent.

The microporous and water-permeable reagent layer is made of the same fabric as the fabric interlayer described later, a paper made from organic polymer fiber valves as described in JP-A-57-148250, and JP-A-57-125847.
A fibrous porous layer such as a porous layer formed by applying a dispersion of fibers and a hydrophilic polymer described in Japanese Patent Publication No. 53-21
677, U.S. Pat. No. 3,992,158, etc., membrane filter-N (plush polymer layer), an isotropic porous layer containing continuous microvoids, in which fine particles such as polymer microbeads are bonded in point contact with a hydrophilic polymer binder;
An isotropic porous layer containing continuous voids (three-dimensional lattice-like two-granular structure N), etc., in which polymer microbeads described in JP-A-55-90859 are brought into point contact with a polymer adhesive that does not swell with water. A layer in which a reagent composition is contained in a microporous layer such as a non-fibrous isotropic porous layer, or a layer in which a reagent composition is contained in a microporous structure composed of solid fine particles and a hydrophilic polymer binder. It is. The dry layer thickness of the microporous reagent layer is about 7 μm to about 250 μm, preferably about 10 μm.
It ranges from μm to about 250 uII+.

When a developing layer is provided on the microporous reagent layer, the gap between the two layers is as described in JP-A-61-4959 and JP-A-62-13875.
It is preferable to use porous adhesion as described in No. 6 and the like.

One embodiment of the multilayer analysis element of the present invention is a multilayer structure in which the development layer contains the above-mentioned reagent composition, and a water absorption layer or a detection layer is provided below the development layer (through an adhesive layer if necessary). There is an analytical element. In another embodiment, the polymer binder of the reagent layer is a polymer that dissolves or becomes solubilized in the solvent of the aqueous liquid sample, such as polyvinyl alcohol, polyvinylpyrrolidone, carboximityl cellulose, sodium alginate, unhardened gelatin, agar, etc. There is a multilayer analysis element constructed by using a microporous reagent layer to form a reagent layer, on which the microporous thin sheet-like member used as the microporous reagent layer is laminated and adhered as a porous detection layer.

The reagent layer can contain a known pH buffer composition, a polymer pH buffer, a basic polymer, an acidic polymer, a polymer mordant, and the like.

As the developing layer, a membrane filter (plush polymer layer) disclosed in Japanese Patent Publication No. 53-21677, etc., a continuous microporous porous layer made of polymer chlorine beads, glass porphyry beads, and diatomaceous earth held in a hydrophilic polymer binder; A porous layer containing continuous micropores (three-dimensional lattice-like granular structure) made of polymer microbeads, glass microbeads, etc., disclosed in JP-A-55-90859, bonded in point contact with a polymer adhesive that does not swell with water. Non-fibrous isotropic porous spread layer such as JP-A-55-164356
The fabric spreading layer disclosed in Japanese Patent Application No. 57-66359, the fabric spreading layer disclosed in Japanese Patent Application No. 59-79158, the organic polymer fiber valve disclosed in Japanese Patent Application No. 57-148250, etc. Examples include a fibrous porous spread layer such as a spread layer made of paper containing.

The spreading layer is laminated directly or via an adhesive layer if necessary.

In addition to the above-mentioned layers, the multilayer analysis element may include a detection layer or mordant layer disclosed in JP-A-51-40191, JP-A-53-131089, etc., and ξ disclosed in JP-A-54-29700, etc., as necessary. Glasion prevention layer, JP-A-51-40191
It is possible to incorporate an intermediate layer disclosed in et al., a reagent-containing fine particle dispersed layer disclosed in JP-A-56-8549, and the like.

The spreading layer, reagent layer, water absorption layer, detection layer, adhesive layer, etc. can contain a surfactant, preferably a nonionic surfactant. Examples of nonionic surfactants include p-octylphenoxypolyethoxyethanol, p-nonylphenoxypolyglycidol, polyoxyethylene oleyl ether, polyoxyethylene sorbitan monolaurate, oxytyl glucoside, and the like. By including a nonionic surfactant in the reagent layer, water absorption layer, or detection layer, water in an aqueous liquid sample is easily absorbed substantially uniformly into the reagent layer, water absorption layer, or detection layer during analysis operations, and Liquid contact with the spreading layer is rapid and substantially uniform. By containing a nonionic surfactant in the spreading layer, the spreading action (metering action) of the aqueous liquid sample becomes better. The content of the nonionic surfactant in the spreading layer is in the range of about 10 g to about 3.0 g, preferably about 20 g to about 2.0 g per rrf of the spreading layer.

When the analytical element of the present invention is used for urea nitrogen analysis, for example, urease is used, and when it is used for creatinine analysis, creatinine diminase, taleatinine axidohydrolase, taleatinine axidinohydrolase, and urease are used in the reagent layer, developing layer, or other layers. It may be contained in an appropriate layer.

The integrated multilayer analytical element of the present invention is an analytical element as described above, in which a pyruvate removal layer containing an enzyme reaction reagent composition that uses pyruvate as a substrate but does not produce hydrogen peroxide is opposed to a support for the reagent layer. It is characterized by being placed on the side.

Such an enzyme reaction reagent composition includes phosphoninolpyruvate synthase (EC2.7.9.2) and A
Combination of TP, pyruvate sulfate dikinase [EC2・
7.9.1] and ATP and phosphate combination, pyruvate carboxylase (EC6.4.1.1) and ATP and carbonate combination, pyruvate dehydrogenase and phosphate and electron acceptor combination, lactate dehydrogenase [EC1・
1, 1, 27] and a combination of NADH and an acid.

By using a combination of phosphooninolpyruvate synthase and ATP, pyruvate in a sample can be converted to phosphooninolpyruvate in the presence of divalent manganese ions, and pyruvate phosphate dikinase and AT
By using a combination of P and phosphate, pyruvate can be converted to phosphooninolpyruvate in the presence of magnesium and ammonium or potassium ions. Pyruvate carboxylase and ATP
Pyruvate can be converted to oxaloacetate in the presence of biotin and magnesium ions by using a combination of and carbonate, and thiamine pyrophosphate (TPP) can be converted by using a combination of pyruvate dehydrogenase and a phosphate and electron acceptor. ), magnesium ion and FAD
Pyruvate can be converted to acetyl phosphate in the presence of . Additionally, pyruvate can be converted to lactic acid in the presence of zinc ions by using a combination of lactate dehydrogenase, NADH, and acid. The reaction system for removing pyruvic acid is preferably one in which reverse reaction does not occur or is difficult to occur.

It is also preferable to further introduce a reaction system coupled to the above enzyme reaction to convert the product into another product or to return the by-product acid to the compound before the reaction. - For example, a reduced acceptor produced by pyruvate dehydrogenase, such as reduced 1-methoxy-5-methylzenadinium methyl sulfate (PMS), is reacted with a tetrazonium salt to form a diformazane dye, which is the reverse reaction. This is preferable because there is no need to take this into account. There are many conjugate reaction systems mentioned above, and one suitable for the present invention can be selected from them.

The enzyme reaction reagent composition may further contain a pH buffer, an enzyme activator, and the like.

The pyruvic acid removal layer containing the above enzymatic reaction reagent composition is provided on the opposite side of the reagent layer to the support, and is applied to the sample before enzymatically reacting the pyruvic acid generated from ammonia in the sample or from the target component. Converts the pyruvic acid contained in it into other substances. This pyruvic acid removal layer may be newly provided independently, or the enzymatic reaction reagent composition may be contained in the development layer, light shielding layer, etc., so that they can also be used.

When newly provided, for example, the hydrophilic polymer binder layer or microporous layer described in the above-mentioned reagent layer can be used. Further, the number of layers for removing pyruvic acid is not limited to one layer, and each component of the enzyme reaction reagent composition may be divided into two or more layers and contained therein.

On the other hand, some of the components of the reagent composition for detecting glutan ξ synthase, pyruvate kinase, pyruvate oxidase, and hydrogen peroxide may be contained in a layer other than the reagent layer, but even in that case, the components may be contained in the sample. It is necessary to arrange the sample so that the reaction of converting ammonia generated from the target component into pyruvic acid occurs after the pyruvic acid previously contained in the sample is removed by the pyruvic acid removal layer.

The content in the integrated multilayer analytical element of the present invention is glutaξ
200 to 30,000 IU/rr, preferably about 500 to 20,010/n-r, and 2,000 to 60,000 IU/rr of pyruvate kinase. degree, preferably 30
00 to 40,000/po, pyruvate oxidase 5oo
o~100,000 IU/about, preferably 10,000~50,000 IU/
Approximately rrf is appropriate. On the other hand, when using a combination of pyruvate dehydrogenase, phosphate, and electron acceptor in the enzyme reaction reagent composition for removing pyruvate, the amount of pyruvate dehydrogenase is about 3000 to 100,000 IU/ryf, preferably 5000 to 50,000 IU. /M.

In the case of other enzymes, the appropriate content can be determined in the same way as above or by conducting experiments if necessary.

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

The multilayer analytical element of the present invention has a side length of about 15 plates to about 3011I.
Cut into small pieces, such as +1 square or circular pieces of approximately the same size, Special Publication No. 57-28331, Utility Publication No. 56-14245
4. Japanese Patent Application Publication No. 57-63452, Utility Model Application No. 58-32350
, Japanese Patent Publication No. 58-501144, etc., and used as a chemical analysis slide is preferable from various viewpoints such as manufacturing, packaging, transportation, storage, and measurement operations. Depending on the purpose of use, it can be used in the form of a long tape and stored in a cassette or magazine, or small pieces can be attached or stored in a card with an opening.

The multilayer analytical element of the present invention can analyze analytes in liquid samples by the operations described in the aforementioned patent specifications. For example about 5 pi to about 30 ptl, preferably 8
Aqueous liquid sample droplets of whole blood, plasma, serum, urine, etc. in the range of pe to 15 μe are placed on the developing layer, and the sample is heated at a temperature of about 20°C to about 40°C for 1 to 10 minutes. Incubation is carried out at a constant temperature, preferably at a substantially constant temperature around 37° C., and color development or discoloration within the element is carried out using light at or near the absorption maximum wavelength of visible or ultraviolet light. The content of the analyte in the liquid sample can be determined by performing reflection photometry from the light-transmitting support side and using a previously prepared calibration curve based on the principle of colorimetric measurement.

Measurement operations are performed using JP-A-60-125543 and JP-A-60-2.
20862, JP-A-61-294367, JP-A-58-
161867, JP-A No. 63-313063, etc., highly accurate quantitative analysis can be carried out with extremely easy operation.

[Effect]

In the reaction reagent system for ammonia measurement employed in the analytical element of the present invention, glutamine synthetase reacts ammonium ions with glutamic acid to convert them into glutamine, and pyruvate kinase converts the ADP produced at this time into glutamine. Reacts with acid to produce pyruvic acid. Pyruvate oxidase converts this pyruvate into acetyl phosphate, producing hydrogen peroxide. This hydrogen peroxide is quantified colorimetrically using a hydrogen peroxide detection reagent composition. In such a reaction system, pyruvic acid previously contained in the sample is also detected, causing errors. In the analytical element of the present invention, before pyruvate is generated from ammonia in the sample or from the target component, errors are removed by converting this pyruvate into another substance using an enzyme reaction reagent composition for removing pyruvate. -ing For example, if phosphooninolpyruvate synthase and ATP are used in this enzyme reaction reagent composition, pyruvic acid is changed to phosphooninoleic acid to prevent it from reacting in the reaction system for ammonia measurement and analysis. There is.

〔Example〕

Example 1 185n thick colorless transparent P pillar T with gelatin undercoat layer
A reagent layer for ammonia measurement having the following composition was coated on the film support so that the layer thickness after drying was about 15 μm, and then dried.

Reagent composition for ammonia measurement (coating amount per bottle) Glutamine synthetase 7000 υpyruvate kinase 28000 UL-glutamic acid
4.1g Phosphoninolpyruvin fm
600mgATP
1.5 g pyruvate oxidase 4200
0 U peroxidase 14000
U thiamine pyrophosphate chloride (TPP) 300 mgFA
D 50 mgMg” (
Magnesium sulfate) 1570 mg2-(3,
5-dimethoxy-4-hydroxyphenyl)-4-(4
-(dimethylago)phenyl-5-phenethylimidazole 2.5g deionized gelatin
15 g nonylphenoxyboriethoxyenanol (containing an average of 10 oxyethylene units) 5 g On top of this reagent layer, 0.2 g of nonylphenoxyboriethoxyenanol (containing an average of 10 oxyethylene units), 8 g
titanium dioxide fine particles (rutile type, particle size 0.25-0
．． A light-shielding layer having a dry layer thickness of about 5 nm was provided by coating and drying an aqueous dispersion containing 1 g of deionized gelatin and 1 g of deionized gelatin.

Next, on this light shielding layer, an endogenous pyruvic acid removing layer having the following composition was applied and dried so that the layer thickness after drying was 1 on.

Reagent composition for pyruvate removal layer (coating amount per lrI′f) Pyruvate dehydrogenase (Lactobacillus
flusdelbrueckii)
5000 U phosphoric acid (pFI7.5 phosphate buffer)
40 mmolFAD
50 tagTPP
300 rngM
g ~ (magnesium sulfate) 1.0 g
-Methoxy-5-methylzenadinium methyl sulfate
7.5gNTB
2.5 g deionized gelatin
Furthermore, on this layer for removing endogenous pyruvate, a solution of 4 g of deionized gelatin and 0.2 g of nonylphenoxy polyethoxyenanol (containing an average of 10 oxyethylene units) dissolved in 1001 d of water was applied. The mixture was dried to form an adhesive layer having a dry layer thickness of about 2 nm.

On the other hand, it is a knitted fabric made of washed and degreased tricot a fabric with a thickness of about 25 On made of PET spun yarn with a thickness equivalent to about 50 D, and one side of which has been treated with glow discharge for 60 seconds (1.6 kW/rrr; oxygen concentration adjusted under reduced pressure). prepared.

Next, the adhesive layer is almost uniformly wetted with water, and the above-mentioned tricot material fabric is placed on top of it, with the electrode side surface at the time of glow discharge treatment facing the adhesive layer, and the entire wrap is passed between pressure rollers to form the adhesive layer. A knitted fabric development layer was provided by uniformly coating the fabric.

Next, an aqueous solution for impregnating the knitted fabric development layer having the following composition was applied onto the knitted fabric development layer at a rate of 150 d per thread and dried to prepare an integrated multilayer analytical element for determining ammonia concentration.

Composition of aqueous solution for fabric spreading layer impregnation treatment Methyl cellulose (2% aqueous solution, viscosity 50 cps at 20"C) 20
g Octylphenoxyethoxyethanol (average number of reduced units in OE unit 30. HLB value 17.4) 10
g water 100
0 g This analytical element was cut into square chips of 15 mm x 15 mm and placed in a plastic mount described in Japanese Patent Application Laid-Open No. 57-63452 for measurement. Healthy and normal bottom blood was collected with heparin and used as the specimen. Ammonium chloride and potassium pyruvate were each added to the plasma, and the respective ammonia and pyruvate concentrations were measured using a standard wet method to obtain the assay values. When the color optical density within the element was measured by reflection photometry from the transparent support side at a wavelength of 625 nm, the following results were obtained.

(00K is reflective optical density) The results are shown in FIG. The graph in FIG. 1 is a calibration curve, and from this calibration curve, it can be seen that the integrated multilayer analytical element of the present invention is free from interference from pyruvic acid contained in blood.
It is clear that the ammonia content in blood can be determined with high accuracy.

Example 2 An integrated multilayer analytical element for quantifying ammonia concentration was prepared in the same manner as in Example 1, except that the following composition was used as the pyruvic acid removal layer.

Pyruvic acid removal layer composition (coating amount per bottle) Lactic acid dehydrogenase (derived from pig heart) 6000UNADH65
0mg deionized gelatin 5.0g Example 1
A similar measurement was performed to give a calibration curve independent of endogenous pyruvate.

〔Effect of the invention〕

The analytical element of the present invention eliminates the influence of pyruvic acid contained in a sample, and can accurately quantify urea nitrogen, taleatinin, etc. using ammonia and its measurement reagent system.

[Brief explanation of drawings]

FIG. 1 shows the results of measuring ammonia in blood containing various concentrations of pyruvate using the analytical element of the present invention.

Claims (1)

[Claims] (1) An integrated multilayer analytical element in which a reagent layer and a developing layer are laminated in this order on a light-transparent water-impermeable support, the reagent layer containing glutamine synthetase, pyruvate kinase, pyruvate A reagent composition that contains all or some of the components of an ammonium ion measurement reagent composition including oxidase and a hydrogen peroxide detection reagent composition, and further contains an enzyme that uses pyruvate as a substrate but does not produce hydrogen peroxide. An integrated multilayer analytical element comprising a reagent composition for measuring ammonium ion, characterized in that it has a pyruvate removal layer containing a pyruvate above the reagent layer.