JPH03259077A - Analyzing element - Google Patents

Abstract

PURPOSE:To eliminate measuring error originated from coexisting substances by setting conditions to ingredients in both layers of a reagent layer and a porous developing layer on a support in a dry-type analytical element for analyzing GOT or GPT in a biological fluid sample. CONSTITUTION:The objective analyzing element providing a reagent layer and porous developing layer on a support, containing a buffer and a substrate and used for analyzing glutamic acid oxaloacetic acid transaminase (hereinafter referred to as GOT) or glutamic acid pyruvic acid transaminase (hereinafter referred to as GPT). The element contains at least one substrate for GOT or GPT in at least the reagent layer and also contains ingredients necessary for reaction after the second reaction of analytical reaction system in at least the above-mentioned porous developing layer. The ingredients necessary for the reaction after the above-mentioned second reaction include enzymes and substrates, etc., in the reaction post-second reaction.

Description

[Detailed description of the invention]

[0001]

[Industrial application field]

The present invention provides analytical elements, particularly glutamate oxaloacetate transaminase (hereinafter abbreviated as GOT) or glutamate pyruvate transaminase (GOT) in a fluid sample.
Regarding the analytical element for analyzing GPT (hereinafter abbreviated as GPT),
More specifically, GOT or GPT in a biological fluid sample
This invention relates to a dry analytical element for analyzing. [0002]

[Conventional technology]

Various configurations of dry analytical elements for analyzing GOT or GPT in biological fluid samples are known. Among them, glutamate dehydrogenase (GIDH)
An analytical element that utilizes a reaction system in which a reduced coenzyme produced under the involvement of an oxidized coenzyme and a pigment-forming precursor is transferred to a pigment-forming precursor via an electron transfer agent to form a pigment is disclosed, for example, in JP-A No. 59
-88097, 59-91896, 63-52
No. 897, No. 63-59900, No. 63-61939
No. 63-71199 and JP-A-61-262
It is described in No. 660, etc. [0003] Also, an analytical element for GPT that transfers hydrogen peroxide generated under the involvement of pyruvate oxidase to a hydrogen donor and a coupler via peroxidase to form a dye, and an analytical element for oxaloacetate decarboxylase (OAC) and pyruvate. GOT analytical elements that transfer hydrogen peroxide generated under the involvement of oxidase (POP) to a hydrogen donor and a coupler via peroxidase to form a dye are disclosed, for example, in Japanese Patent Publication No. 1-46119 and Japanese Patent Application Laid-open No. 1983-1989. 208998
No. 62-299, No. 62-79362, No. 62
-195299, 63-32498, 63-13
No. 7699 describes U). [0004]

Problem to be Solved by the Invention 1 However, these disclosed analytical elements are susceptible to the effects of coexisting substances such as glutamic acid or pyruvic acid in biological fluid samples, and therefore have poor accuracy.
It is still far from completely satisfactory, and further improvements are desired. [0005] In addition, oxaloacetic acid or GP produced by the reaction of GOT
The pyruvate produced by the reaction of T is processed by malate dehydrogenase (
With the involvement of MDH) or lactate dehydrogenase (LDH), reduced nicotinamide adenine dinucleotide (NADH)
oxidized nicotinamide adenine dinucleotide (NA
D) [0006] In these cases as well, if pyruvic acid or oxaloacetate is present as a coexisting substance in the reaction system, the accuracy of GOT or GPT analysis may be affected. Adversely affect. [0007] An object of the present invention is to provide an analytical element for detecting GOT or GPT that has improved accuracy by eliminating errors caused by coexisting substances. [0008] Means for Solving the Problems To summarize the present invention, the present invention relates to an analytical element, which has a reagent layer on a support, a porous spreading layer above the reagent layer, and a buffer layer. An analytical element for analyzing GOT or GPT in a biological fluid sample containing an agent and a substrate, wherein at least one of the substrates of GOT or at least one of the substrates of GPT is contained in at least the reagent layer; Moreover, it is characterized in that the porous development layer contains at least the components necessary for the second and subsequent reactions of the reaction system of the analysis. [0009] The reaction system for quantitatively analyzing GOT or GPT in the present invention is illustrated below. [0010]

[Chemical formula 1] (COT quantitative analysis reaction system) Reaction system (1) <First reaction> L-aspartic acid deca-ketogluta OT Ruic acid -→ Oxaloacetate L-glutamic acid Second reaction>
L-glutamic acid + NAD” 4σ-ketoglutaru Seiyu NADH + NH. Reaction system (2) 1st reaction L-aspartic acid = α-ketogluta Reaction〉oxaloacetic acid 10NADH1H” ”→malic acid + NA
D” Reaction system (3) <1st reaction> L-aspartic acid + α-)・gluta O
T Ruic acid-→Oxaloacetic acid-TL-Glutamic acid AC Second reaction Oxaloacetic acid-→Pyruvic acid Third reaction Pyruvate ±02÷Hopo3'-Diacetyl acetate 1
CO2+H202 [0011]

[Chemical formula 2] Reaction system (1st reaction) L-aspartic acid + α-ketoglutaric acid 4 oxaloacetic acid 10 L-glutamic acid (2nd reaction) oxaloacetic acid ↑ 2,4-dinylhydrazine hydrazone (GPT quantitative analysis reaction system) Reaction system (1) 1st reaction L-alanine + a-ketoglutarate 4 pyruvate 1 L-glutamic acid C, IDH 2nd reaction L-glutamic acid + NAD"-> a-ketoglutar reaction system (2) <First reaction> L-alanine semi-α-ketoglutaric acid” 1→
Pyruvate + L-glutamic acid LDI (〈Second reaction〉Pyruvate + NADH + l(”-→lactic acid +
NAD+ reaction system (3) <1st reaction> L-alanine + a-ketodaltaric acid 1 → pyruvic acid + L-glutamic acid OP <2nd reaction> pyruvic acid + 02 + HOPO3 ``-1> acetylacetic acid + GO2 10H202 [0012] Quantitative analysis is It is carried out by the following method.In the above reaction system, a reaction system in which NADH is consumed (GOT (2) or GOT
In the case of PT (2) reaction system), the decrease in NADH is directly measured. Reaction system in which NADH is produced (GOT (1)
or GPT (1) reaction system), an electron transfer agent (
For example, in the presence of diaphorase (DIA) or N-methylphenazine methosulfates, a pigment-forming precursor (eg, a tetrazolium salt) is reduced by NADH to form a pigment having absorption in the visible region. Also, H2O. The reaction system that generates (GOT (3) or GPT (3)
reaction system), in the presence of peroxidase, H
2O. By reacting a hydrogen donor with a coupler, a dye having absorption in the visible region is formed. These formed dyes are measured as reflection density using an optical densitometer. Then, the amount of pigment formed per specified time is determined as a reflection density difference, and a conversion formula (reflection density of GOT or Convert to GOT or GPT activity value using the formula for converting to GPT activity value). [0013] The reaction system in which the third reaction or further the fourth reaction is performed in the quantitative analysis reaction system described above is (GOT quantitative analysis reaction system) (
1) and (3) (GPT quantitative analysis reaction system) (1)
) and (3). The reaction is shown below. [0014]

[Chemical formula 3] (Quantitative analysis reaction system of GOT) Reaction system (1) (Third reaction) Reaction system (Quantitative analysis reaction system (Fourth reaction)) Reaction system (3) (Third reaction) [0015] The present invention The components necessary for the reactions after the second reaction are the enzymes and substrates for the reactions after the second reaction, and each reaction system will be specifically shown. [0016] (GOT quantitative analysis reaction system) (1) GIDH1 oxide coenzyme, electron transfer agent, pigment forming precursor, (2) reduced coenzyme, malate membrane hydrogenase, (3) oxaloacetate decarboxylase , phosphoric acid (or its salt) pyruvate oxidase, peroxidase, hydrogen donor, coupler (4) 2,4-dinitrophenylhydrazine, alkali, (quantitative analysis reaction system of GPT) (1) GIDH1 oxidized coenzyme, electron transfer (2) Reduced coenzyme, lactate dehydrogenase (3) Pyruvate oxidase, peroxidase, phosphoric acid (or its salt) Hydrogen donor, coupler In the present invention, GOT or GPT is analyzed. As the reaction system, both the reaction system (1) in which GIDH is used in the second reaction or the reaction system (3) are preferable. The second reaction refers to reacting the product of the first reaction (specifically, oxaloacetate or glutamic acid in the case of GOT, pyruvic acid or glutamic acid in the case of GPT) via an enzyme or forming a pigment. The third reaction is one in which the product of the second reaction is further reacted, or a reaction is caused to form a dye. The fourth reaction is a reaction in which the product of the third reaction is further reacted to form a dye. [0017] In the reaction system (1), the oxidized coenzymes used in the second reaction include oxidized nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD) and Flavin adenine dinucleotide phosphate (FADP) can also be used.In the present invention, NAD is particularly preferably used.The amount of the above-mentioned oxidized coenzyme contained in the analytical element of the present invention is usually 10 mg/m2 to 50 g. /m2 is preferably 100 mg/m2 to 20 g/m2. [0018] When measuring GOT, the substrate according to the present invention is L
- Aspartic acid or its salts (e.g. sodium salt, potassium salt, etc.) and α-ketoglutaric acid or its salts (e.g. sodium salt, potassium salt, etc.), and when measuring GPT, L-alanine and α-ketoglutaric acid. or a salt thereof (e.g. sodium salt, potassium salt, etc.) [00
19] The amount of the substrate according to the present invention contained in the analytical element of the present invention is usually 0.1 to 500 mmol/in the case of L-aspartic acid or its salt and L-alanine or its salt.
m2, preferably 1 to 200 mmol 7 m2, and in the case of α-ketoglutaric acid or its salt, usually 0.0
1~100mm○1/m2, preferably 0.1~50m
It is m01/m2. [0020] The NADH produced by the reaction in (1) reduces the electron transfer agent through the third reaction and subsequent reactions, and the reduced electron transfer agent further reduces the pigment forming precursor and produces a visible region. Forms a dye that has absorption. [0021] As the electron transfer agent, N-methylphenazine methosulfates, meltlab-methylene blue, diaphorase, etc. can be used, and preferred electron transfer agents include N-methylphenazine methosulfates and diaphorase. be able to. [0022] The amount of the electron transfer agent contained in the analytical element of the present invention is
Although it varies depending on the amount of specific components such as GOT and GPT, in the case of electron transfer agents other than diaphorase, it is usually 1 to 1.
000mg/m2, preferably 10-500mg/m2
It is. [0023] Furthermore, when diaphorase is contained in the analytical element of the present invention, the reagent layer can contain usually 100 to 1075 U/m, preferably 500 to 50,000 U/m2. When contained in the porous development layer, the porous development layer usually contains 1
0 to 100,000 U/m2, preferably 100 to 50,000 U/m2
can be contained. [0024] On the other hand, as the dye-forming precursor, tetrazolium salts are usually used. [0025] Examples of the above-mentioned tetrazolium salts useful in the present invention include those described in JP-A-63-7119 and JP-A-63-59900. 3'-dimethoxy-4,4'-biphenylene)-bis-C2-(p-nitrophenyl)-
5-tuunyltetrazolium fluoride] and 3.3'
-(4,4'-biphenylene)-bis(2,5-diphenyltetrazolium chloride), which is preferably contained in the reagent layer and porous development layer according to the present invention. [0026] The amount of the dye-forming precursor contained in the analytical element of the present invention is usually 10 mg/m 2 to 10 g/m 2 in the reagent layer,
Preferably it is 50 mg/m2 to 3 g/m2. When contained in the porous spreading layer, the porous spreading layer usually contains 5
mg/m~10g/m'', preferably 10mg/m2~
It is 3g/m2. [0027] As the buffering agent according to the present invention, Nippon Kagaku complete edition "Chemistry Handbook Basic Edition" [Tokyo, Maruzen Co., Ltd., published in 1966] pp. 1312-1320, R, M, C, Dowson (R, M
``Data for Biochemical Research'', edited by C. Dawson et al.
chemical Research) J 2nd edition (Oxford at the Clendon Press, 1969)
Published in 2013), pp. 476-508, Biochemistry (Bi
Analytical Biochemistry Volume 5, Page 467 (1966)
ical Biochemistry), Vol. 104, pp. 300-310 (1980). [0028] Specific examples of preferred buffers include, for example, a combination of Tris buffer (tris hydroxymethylaminomethane and tris hydroxymethylaminomethane hydrochloride); potassium dihydrogen phosphate-disodium hydrogen phosphate; tris-sodium borate; tris-citric acid; Citric acid-sodium dihydrogen phosphate;
Bicine: HEPPS: HEPPS sodium salt;
HEPES potassium salt: EPPS: EPPS sodium salt, TAPS: TAPS sodium salt: T
ES; TES sodium salt: TES potassium salt, etc. [0029] Although it is generally preferred that the pH buffer be present in the layer containing the enzyme, the low molecular weight pH buffer will migrate between the layers following permeation of water, which is the medium of the spotted aqueous fluid sample. pH buffering agents can be included in all layers containing enzymes and coloring reagent compositions. However, if it is present in the same layer as the dye-forming precursor, it may cause blurring, etc., so it is preferably contained in a layer separate from the dye-forming precursor. Since these are laminated without being mixed during manufacturing and sample application, it is preferable that the buffering agent is dispersed in the binder, and the adhesive layer (second layer provided between the spreading layer and the reagent layer) is used. reagent layer)
It is preferable to contain it. [00301 The amount of the buffer according to the present invention contained in the analytical element of the present invention is usually 1 mm01/m2 to 1 mol/m2, preferably 10 to 300 mm01/m2.

○031 The above-mentioned GIDH can be used in higher plants (EC1, 4, 1, 2), mammalian liver (BCl, 4.1.3), yeast or Escherichia coli (
EC1, 4, 1, 4) can be used, preferably mammalian liver (PCl, 4.1
．． 3) The origin is used. [0032] GIDH is contained in the porous spreading layer, and preferably contained in the reagent layer. The amount of GIDH contained in the analytical element of the present invention is usually 1,000,000 to 1,000,000 U/m2, preferably 500 to 500,000 U/m2 in the porous spreading layer. When it is also contained in the reagent layer, the amount in the reagent layer is usually 1 to 1 million U/m2, preferably 300 to 500,000 U/m2. [0033] Other additives include adenosine-5'-diphosphoric acid (A
DP) can also be added into the device to enhance the substrate property isomerism of GIDH to L-glutamate. [0034] When ADP is contained in the analytical element of the present invention, the amount of ADP contained in the porous spreading layer is usually 0.05 to 10.
0mmol 7m2, preferably 0.5-50mmol
It is 7m2. When it is contained in the reagent layer, it is usually 0.05 to 100 mmol/m2, preferably 0.5 to 50 mmol/7 m2. [0035] When the above-mentioned electron transfer agent, GIDH1 oxidized coenzyme, and ADP are contained in the porous spreading layer, it is preferable to use one obtained by freeze-drying these individually or in an appropriate combination together with bovine serum albumin (BSA). Furthermore, when performing freeze-drying, it is also possible to add stabilizers or activators such as enzymes and coenzymes in addition to the above-mentioned components. [0036] When the analytical element of the present invention contains the bellokindase, the content is preferably 500 to 50,000 U/m
It is 2. [0037] When the pyruvate oxidase (POP) is contained in the analytical element of the present invention, the content is preferably 50
It is 0 to 50,000 U/m2. [0038] When the oxaloacetate decarboxylase is contained in the analytical element of the present invention, the content is preferably 500 to
It is 100,000 U/m2. [0039] When the above-mentioned enzymes are contained in the spreading layer, those obtained by freeze-drying them individually or in an appropriate combination with bovine serum albumin (BSA) are preferably used. [00401 When the above-mentioned coupler is included in the analytical element of the present invention, it can be incorporated into the analytical element of the present invention according to Japanese Patent Application Laid-Open No. 57-94653 (Japanese Patent Publication No. 63-37903).
, No. 57-94654, No. 57-94655 (Special Publication No. 1-661), No. 60-47696, No. 63-1
No. 27158, No. 63-205564, JP-A-1-3
Those disclosed in No. 5369 can be used, but are not limited thereto. Further, it is preferable to use the diffusion-resistant coupler described in JP-A-60-47696 in the analytical element of the present invention. The coupler content is 1 x 10 per 5000 U of pyruvate oxidase.
~3 to 10 mol is preferred.

When the coupler is contained in the developing layer, it can be dissolved in an organic solvent and then added to the coating solution. [0042] When the above-mentioned coupler, especially a diffusion-resistant coupler, is also included in the reagent layer, it is preferable to add it by an oil protection method used in photographic technology. Specifically, a diffusible coupler is dissolved in a solvent, an anionic surfactant and/or
Alternatively, it may be mixed with an aqueous solution containing a hydrophilic colloid such as gelatin containing a nonionic surfactant, and emulsified and dispersed using a high-speed rotation mixer, a colloid mill, an ultrasonic dispersion device, or the like. [0043] The hydrogen donor used in the present invention is disclosed in JP-A-60-47696.
The hydrogen donors described in this issue are used. When it is contained in the developing layer, it can be added to the coating solution after being dissolved in methanol or the like. [0044] When the phosphoric acid (or its salts such as sodium, potassium, magnesium, calcium, cobalt, etc.) is contained in the analytical element of the present invention, the content is POPOIU (37
The activity of producing 1 μmol of hydrogen peroxide per minute at °C is defined as 1 unit (U), preferably 0.3 to
It is 5 μmol. [0045] When the analytical element of the present invention contains POP, it is preferable to contain flavin adenine dinucleotide (FAD), and the content thereof is preferably 0°3 to 30 nmol relative to POPIU. PO in the analytical element of the present invention
When containing P, thiamine pyrophosphate (TP
P) is preferably contained, and thiamine diphosphoric acid (TPP) is preferably used as TPP. Its content can be used in a ratio of 5 to 500 nmol relative to POPIU. [0046] When the analytical element of the present invention contains POP, a salt or a complex containing divalent or trivalent metal ions capable of activating POP and capable of releasing these can be added. The specific metal ion is Ca2+
CO2+ Mn2+A13+Mg2+. Specific examples of preferred metal salts include magnesium chloride (II);
Manganese(II) phosphate Manganese(II) hydrogen phosphate
Manganese(II) chloride Manganese(II) hydrogen phosphate
can be given. The divalent or trivalent metal ion can be used in a proportion of 5 to 10 nmol per IU of POP. The analytical element of the present invention can contain ascorbate oxidase in order to reduce the influence of ascorbic acid in the liquid sample. The preferred content is 5
00 to 50,000 U/m2. [0047] The binder described in JP-A-63-59900 is used as the binder constituting the reagent layer and the adhesive layer according to the present invention. [0048] The binder for forming the reagent layer according to the present invention is preferably gelatin and its derivatives. [0049] As the binder for forming the adhesive layer of the present invention, a copolymer described in JP-A-61-262660 is preferably used. [0050] Various other additives such as preservatives and surfactants can be added to the reagent layer and the adhesive layer as desired. [0051] In particular, surfactants can be effectively used to improve coating properties or promote reactions related to the present invention. [0052] As the surfactant that can be used, nonionic surfactants that can be used regardless of whether they are ionic (anionic or cationic) or nonionic are effective. Specifically, the surfactant described in JP-A-63-59900 is used. [0053] The amount of these surfactants contained in the reagent layer and the adhesive layer is usually 10 mg/m2 to 20 g/m2, preferably 100 mg/m2 to 10 g/m2. [0054] The above-mentioned support according to the analytical element of the present invention is disclosed in JP-A-63-
The one described in No. 59900 is used. The thickness of the support according to the present invention is arbitrary, but preferably 50 to 250 μm.
It is m. [0055] The porous spreading layer according to the present invention is disclosed in Japanese Patent Application Laid-open No. 63-59.
The porous spreading layer described in No. 900 is used. In particular, the fiber structure spreading layer and particle combination structure spreading layer are useful as materials capable of rapidly transporting blood cell portions. [0056] The thickness of the porous spreading layer in the analytical element of the present invention is preferably about 100 to 600 μm, more preferably about 150 to 600 μm.
4001-tm. Further, the porosity is preferably about 20 to 85%.

[0057] In addition, the porous spreading layer includes a part of the substrate and JP-A-63-71
Enzyme inhibitors described in No. 199 can also be included.

[O○58] Various other additives such as preservatives and surfactants can also be added as desired. As the surfactant, the above-mentioned surfactants that can be used in the reagent layer or the adhesive layer can be used. [0059] The surfactant modulates the rate of penetration of the fluid sample into the reagent layer;
At the same time, it has the effect of controlling the occurrence of undesirable "chromatographic phenomena." [0060] The surfactants can be used in widely selected amounts, but typically 10 [0061] The various layers of these analytical elements can be deposited using slide hopper coating methods,
An extrusion coating method, a penetration coating method, etc. can be appropriately selected and used. [0062] Using the analytical element of the present invention, the amount of a specific component in a fluid sample can be measured from the support side according to the present invention by reflection spectrophotometry according to a modified initial velocity method or a reaction endpoint method. The amount of the specific component can be determined by applying the measured value thus obtained to a calibration curve prepared in advance. [0063] The amount of fluid sample applied to the analytical element of the present invention can be determined arbitrarily, but is preferably about 5μ to about 50μ
m, more preferably from 5 μm to 20 μm. It is usually preferred to apply a 10 μm fluid sample.

The analytical element of the present invention can be used without any disadvantage in the distribution of other body fluids such as whole blood, serum and plasma, urine, lymph, spinal fluid, etc. If whole blood is used, a radiation blocking layer or other reflective layer as described above may be provided, if necessary, to avoid interference of the radiation for detection by blood cells. [0065] The analytical reaction used in the analytical element of the present invention can be arbitrarily determined depending on the purpose, but is useful, for example, in the field of clinical chemistry, and is particularly useful for the analysis of components in biological fluid samples, ie, blood or urine. used for analysis. [0066]

【Example】

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. [0067] Example 1 (Analytical element for GPT) Reagent layers R1 to R7 shown in the table below were provided on a transparent subbed polyethylene terephthalate support with a film thickness of 180 μm. [0068]

[Table 1] Book ~ Drug layer (R) [0069] On the above reagent layer, the adhesive layer a1 and the spreading layer 81 shown in the table below are further added.
- S9 were sequentially combined to create analytical elements 1 to 7 of the present invention and comparative analytical elements -(1) to (2) shown in Table 1. [00701

[Table 2] Contact arrival layer ■ *BASF product name: N-Bini le Pi B Li de hmm monoacetic acid vinyl copolymer (weight ratio 20: [0071]

[Table 3] [0072]

[Table 4] :Hi2< [0073] The adhesive layer was provided by applying a coating solution containing each component onto the reagent layer. Butanol was used as a solvent for the coating solution, and each component was added and dispersed to form a coating solution. [0074] The spreading layer was provided by applying a coating liquid containing each component onto the adhesive layer. Xylene was used as a solvent for the coating liquid, and each component was added and dispersed to form a coating liquid. [0075] GIDH, diaphorase was lyophilized with BSA and 1
It was made into a fine powder of 0.00 mesh or less and added to the coating solution. [0076] α-ketoglutaric acid, oxalic acid, 3.3'-(4,4'-
Biphenylene)-bis(2,5-diphenyltetrazolium chloride) was dissolved in methanol and added to the coating solution. [0077] Analytical elements-1 to 7 of the present invention and comparative analytical elements = (1
) to (2), 7% containing O ~ 15 mg/dl glutamic acid and having 45 kU and 92 kU GPT
After spotting a 10 μm aqueous human serum albumin (ISA) solution, incubating at 37°C, measuring the reflection density 7 minutes and 11 minutes after spotting using a 546 nyn filter, and determining the difference in reflection density. The results shown in Table 2 were obtained. [0078]

[Table 5] Table 2 [0079] As is clear from the results in Table 2, analytical element-1 of the present invention
It can be seen that the influence of glutamic acid is relatively small in samples 7 to 7 compared to analytical elements -(1) to (2). [0080] Example-2 Human serum with various GPT activities (40 samples) was spotted at 10 μm onto the analytical elements-1 to 7 of the present invention prepared in Example-1 and the comparative analytical element-(1). After that, incubate at 37°C, and the reflection density after 3.5 minutes and 7 minutes was set to 5.
Measurement was performed using a 46 nm filter, and the difference in reflection density was determined. On the other hand, automatic chemical analyzer Hitachi 7050 [
Hitachi, Ltd.] and Autocera GPT (Daiichi Chemical Co., Ltd.) were used to test the human serum (40 samples).
GPT activity was measured (2-152 kU). This GP
A calibration curve was created from the difference between T activity and the above-mentioned reflection density, and the difference in reflection density was converted into GPT activity. A correlation coefficient (a) was determined from this converted GPT activity and the GPT activity measured using the Hitachi 7050 and Autocera GPT. The results are shown in Table 3. [0081]

[Table 6] Table [0082] As is clear from the results of Table 3, analytical element-1 of the present invention
It can be seen that the correlation, that is, the accuracy of the analysis, is improved in the comparative analysis elements -(1) to (2) in the comparative analysis elements -(1) to (2). [0083] Example 3 (Analytical element for GOT) Reagent layers R8 to R14 shown in the table below were provided on a transparent subbed polyethylene terephthalate support with a film thickness of 180 μm. [0084]

[Table 71 Reagent layer (R) [0085] On the above reagent layer, adhesive layer a2 and spreading layer 5I shown in the table below are further added.
Analytical elements 8 to 14 of the present invention and comparative analytical elements -(3) to (4) shown in Table 4 were created by sequentially combining O-315. [0086] [Table 8] Adhesive layer * BASF product name: N-vinylpyrrolidone monovinyl acetate copolymer (weight ratio 20:

[Table 9] [0087] exhibition Open layer (S) [0088]

[Table 10] Table [0089] The adhesive layer was provided by applying a coating solution containing each component onto the reagent layer. Phthanol was used as a solvent for the coating liquid, and each component was added and dispersed to form a coating liquid. [00901 The spreading layer was provided by applying a coating liquid containing each component onto the adhesive layer. Xylene was used as a solvent for the coating liquid, and each component was added and dispersed to form a coating liquid. [0091] GIDH, diaphorase was lyophilized with BSA and 1
It was made into a fine powder of 0.00 mesh or less and added to the coating solution. [0092] α-ketoglutaric acid and 3,3'-(4,4'-biphenylene)-bis(2,5-diphenyltetrazolium chloride) were dissolved in methanol and then added to the coating solution. [0093] Analytical elements-8 to 14 of the above invention and comparative analytical elements-(3
) to (4), human serum with various GOT activities (4)
0 sample) was spotted at 10 μm, incubated at 37°C, and the reflection density was measured at 546 nm after 3.5 minutes and 7 minutes.
The difference in reflection density was determined using a filter. On the other hand, the GOT activity of the human serum (40 samples) was measured using an automatic chemical analyzer Hitachi 7050 [manufactured by Hitachi, Ltd.] and Autocera GOT [manufactured by Daiichi Kagaku Yakuhin Co., Ltd.]. (6-162 kU). A calibration curve was created from the difference between this GOT activity and the above-mentioned reflection density, and the difference in reflection density was converted into GOT activity. This converted GOT activity and the G measured using the above Hitachi 7050 and Autocera GOT
The correlation coefficient (a) was determined from the OT activity. The results are shown in Table 5. [0094]

[Table 11] Table b [0095] As is clear from the results in Table 5, analytical element-8 of the present invention
It can be seen that the correlation, that is, the accuracy of the analysis, is improved in the comparative analysis elements -(3) to (4) for the comparative analysis elements -(3) to (4). [0096] Example 4 (Analytical element for GOT) Reagent layers R15 to R22 shown in the table below were provided on a transparent subbed polyethylene terephthalate support having a film thickness of 180 μm. [0097]

[Table 12] Reagent layer (R) [0098] On the above reagent layer, the adhesive layer a2 shown in Example 3 was provided,
Furthermore, the analytical element of the present invention and the comparative analytical element-(5)~ shown in Table 6 are provided in combination with the developing layers S16 to S20'2 shown in the table below.
(6) was created. [0099]

[Table 13] [,0100]

[Table 14] Table [0101] The adhesive layer was provided by applying a coating solution containing each component onto the reagent layer. Butanol was used as a solvent for the coating solution, and each component was added and dispersed to form a coating solution. [0102] The spreading layer was provided by applying a coating liquid containing each component onto the adhesive layer. Xylene was used as a solvent for the coating liquid, and each component was added and dispersed to form a coating liquid. [0103] Peroxidase, pyruvate oxidase, oxaloacetate decarboxylase, and ascorbate oxidase were freeze-dried together with BSA, and added to the coating solution in the form of a fine powder of 100 mesh or less. [0104] α-Ketoglutaric acid, 5,5-dimethyl-1,3-cyclohexanedione, 4-N hydrochloric acid, N-diethylamino-2(2'-methanesulfonamidoethyl)aniline was dissolved in methanol and added to the coating solution. did. [0105] 7-chloro-3-(2-hexyldecylsulfonylethyl)-6-methyl-pyrazolo-[3,2-C,l-3
-) Riazole was dissolved in ethyl acetate and then added to the coating solution. [0106] Analytical elements-15 to 20 of the above invention and comparative analytical elements-(
5) to (6), human serum with various GOT activities (4
0 sample) was spotted at 10 μm, incubated at 37°C, and the reflection density after 3.5 minutes and 7 minutes was measured at 546 nm.
The difference in reflection density was determined using a filter of m. On the other hand, the GOT activity of the human serum (40 samples) was measured using an automatic chemical analyzer Hitachi 7050C (manufactured by Hitachi, Ltd.) and Autocella GOT (manufactured by Daiichi Chemical Co., Ltd.). (8-171 kU). A calibration curve was created from the difference between this GOT activity and the above-mentioned reflection density, and the difference in reflection density was converted into GOT activity. This converted GOT activity and the GOT measured using the above Hitachi 7050 and Autocera GOT
The correlation coefficient (a) was determined from the OT activity. The results are shown in Table 7. [0107]

[Table 15] Table 7 [0108] As is clear from the results in Table 7, analytical element-1 of the present invention
It can be seen that the correlation, that is, the accuracy of the analysis, is improved for samples No. 5 to No. 20 compared to comparative analysis elements (5) to (6). [0109] Example 5 (Analytical element for GOT) Reagent layers R23 to R30 shown in the table below were provided on a transparent subbed polyethylene terephthalate support with a film thickness of 180 μm. [01101

[Table 16] Reagent layer (R) [0111] On the above reagent layer, the adhesive layer a1 shown in Example 1 was provided,
Further, the development layers S21 to S26 shown in the table below are provided in combination, and Table 8
Analytical elements 21 to 26 of the present invention and comparative analytical elements shown in
(7) to (8) were created. [0112]

[Table 17] [0113]

[Table 18] Table [0114] The adhesive layer was provided by applying a coating solution containing each component onto the reagent layer. Butanol was used as a solvent for the coating solution, and each component was added and dispersed to form a coating solution. [0115] The spreading layer was provided by applying a coating liquid containing each component onto the adhesive layer. Xylene was used as a solvent for the coating liquid, and each component was added and dispersed to form a coating liquid. [0116] Peroxidase, pyruvate oxidase, oxaloacetate decarboxylase, and ascorbate oxidase were freeze-dried together with BSA and added to the coating solution in the form of a fine powder of 100 mesh or less. [0117] α-Ketoglutaric acid, 5,5-dimethyl-1,3-cyclohexanedione, 4-N hydrochloric acid, and N-diethylamino-2(2'-methanesulfonamidoethyl)aniline were dissolved in methanol and then added to the coating solution. did. [0118] 7-chloro-3-(2-hexyldecylsulfonylethyl)-6-methyl-pyrazolo-[3,2-C) -3-
) Riazole was dissolved in ethyl acetate and then added to the coating solution. [0119] Analytical elements-21 to 26 of the above invention and comparative analytical elements-(
7) to (8), human serum with various GPT activities (4
0 sample) was spotted at 10 μm, incubated at 37°C, and the reflection density after 3.5 minutes and 7 minutes was measured at 546 nm.
The difference in reflection density was determined using a filter of m. On the other hand, automatic chemical analyzer Hitachi 7050C manufactured by Hitachi, Ltd.] and Autocella GPT (Daiichi Chemical Co., Ltd.)
GPT of the above human serum (40 samples) using
Activity was measured. (4-158 kU). A calibration curve was created from the difference between this GPT activity and the above-mentioned reflection density, and the difference in reflection density was converted into GPT activity. A correlation coefficient (a) was determined from this converted GPT activity and the GPT activity measured using the Hitachi 7050 and Autocera GPT. Table 9 shows the results.
Shown below. [01201 [Table 19] Table [0121] As is clear from the results in Table 9, the analytical elements-21 to 26 of the present invention were compared to the comparative analytical element-(7
) to (8), it can be seen that the correlation, that is, the accuracy of the analysis is improved. [0122]

Effect of the Invention 1 The analytical element of the present invention has improved quantitative sensitivity and accuracy, and has been able to raise the level of this technical field. 【Document name】

【Filing date】 【Reference number】 【address】 [Display of incident] 【application number】

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[Procedural amendment 1]

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[Correction method] [Contents of correction] Procedural amendment March 18, 1991 639201H01

Claims (3)

[Claims] Claim 1: A method for detecting glutamate oxaloacetate transaminase or glutamate pyruvate transaminase in a biological fluid sample, the support having a reagent layer and a porous spreading layer thereabove, containing a buffer and a substrate. In the analytical element for analysis, at least one of the substrates for glutamate oxaloacetate transaminase or glutamate pyruvate transaminase is contained in at least the reagent layer, and at least the components necessary for the reactions after the second reaction of the analysis reaction system are contained in the reagent layer. An analytical element characterized by containing a porous spreading layer. Claim 2: The second
2. The analytical element according to claim 1, wherein at least one of the subsequent reactions is a reaction in which a dehydrogenase is contacted. 3. The analytical element according to claim 1, wherein at least one of the reactions after the second reaction is a reaction catalyzed by pyruvate oxidase.