JPS5988097A - Analytical element - Google Patents

Abstract

PURPOSE:An analytical element having improved measuring accuracy, stability, etc., obtained by providing a substrate with a receiving layer and a developing layer containing a dyestuff-forming precursor which is reacted with a specific component to be analyzed and colored, an electron transferring agent, an oxidizing type coenzyme, a specific reagent, etc. CONSTITUTION:A substrate having light transmittance and impermeability to liquid is provided with a receiving layer and a developing layer. The different layers or the same layers are provided with an electron transferring agent, a dyestuff-forming precursor, an oxidizing type coenzyme, and a reagent capable of converting the oxidizing type coenzyme into a reducing type coenzyme through a specific component to be analyzed in a liquid specimen, to give the desired analytical element. The liquid specimen such as blood, etc. is dropped on the analytical element, the specific component to be analyzed in the specimen is treated with the reagent to form the reducing type coenzyme, the dyestuff- forming precursor is reduced with the reducing type coenzyme through the electron transferring agent, the prepared dyestuff is measured, so that the specific component in the specimen is analyzed.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an analytical element, particularly an analytical element for analyzing a specific component in a fluid, and more particularly for analyzing a specific component in a biological fluid sample via a reduced coenzyme. Regarding dry analytical elements.

In the past, many methods have been developed to analyze specific components in fluid samples, but these can be roughly divided into two types: reaction systems in which the reaction takes place in a solution, and reaction systems in which the reaction takes place in a homogeneous carrier. It can be divided into Analytical reactions in solution systems (hereinafter referred to as "wet chemistry") range widely from non-mechanical methods to automated analytical instruments. This is so remarkable that in recent years, Yuzu's automatic footstep analysis equipment for clinical testing has been introduced into clinical laboratories of hospitals.

There is.

However, the above-mentioned method has various drawbacks because the reaction is basically carried out in the form of an aqueous solution. That is, the analysis process requires a large amount of water, especially purified water or distilled water, resulting in increased energy consumption. In addition, Tanuki's automatic analysis equipment is itself extremely expensive, requires a great deal of skill to operate, is not a scale that requires a huge amount of time and effort, and its waste fluid inevitably causes environmental pollution. It has the following drawbacks.

In contrast, the analytical reaction in the same phase yarn (hereinafter referred to as dry
Analytical methods using chemistry are also widely used, but these are carried out using filter paper or the like impregnated with reagents.

The above-mentioned P paper is produced by impregnating a water-absorbing fibrous carrier such as a filter paper with a reagent bath solution, as described in, for example, U.S. Pat. No. 3,050,373 or U.S. Pat. It is made by drying.

These are commonly referred to as analytical test strips or simply test strips, and can be determined visually by placing a drop of a fluid sample on the test strip, or by immersing the test strip into a fluid sample and visually determining the change in color or concentration of the test strip. It is measured by a reflexometer to determine the concentration level of a particular component in a fluid sample.

These test pieces are useful because they are easy to handle and provide immediate results, but due to their composition, they remain in the area of cattle identification or qualitative analysis.

On the other hand, multilayer analytical elements are known that use dry chemistry, which is easier to operate than the conventional analytical methods described above, and which also have high quantitative performance.

For example, Japanese Patent Publication No. 53-21677, Japanese Patent Publication No. 55-16
No. 4356, No. 57-125847, Patent Application No. 1982-6
Multilayer analysis elements are described in No. 5446 and No. 56-189784.

According to the devices described in these documents, all reagents used in analytical reactions are contained in a single analytical device, and a certain volume of serum or whole blood is dropped onto the above device at a certain temperature for a certain period of time. After keeping warm, it is reflected from the support side! The above method, which makes it possible to determine the concentration of a substance from the reflected density after a single measurement, has far greater analytical accuracy than the conventional test strip type method, and requires the preparation of reagents in advance. It has performance equivalent to or better than wet chemistry without any

However, analytical elements using such dry chemistry are mainly used for analyzing low molecular weight compounds;
The main method used is the reaction end point measurement method (end point assay), and the initial rate method (rate assay) is still being used.
No assay has been developed to measure the activity of high molecular weight substances, especially enzymes.

In particular, a method for measuring components in a fluid sample by increasing or decreasing reduced coenzyme, i.e., reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate, is a widely applicable and useful method. It is known.

These reduced coenzymes are applied in wet chemistry, where specific components in a sample are brought into the desired reaction path and then led to the reduction reaction of the reduced coenzymes mentioned above. Detection is done by measuring the ultraviolet region using the initial velocity method, or by transmitting changes in the reduced coenzyme to a pigment-forming precursor via an electron transfer agent to form a pigment and increase the concentration of this pigment. A method of quantifying by colorimetry is known.

The former method is a method commonly used in wet φ chemistry, but there are some fatal problems when it is introduced into the analytical element of the above-mentioned dry bite mistry. Changes in the reduced coenzyme, which is the object of measurement, require measurement of absorption in the ultraviolet region of 340 nm, and it is known that its spectral absorption coefficient is extremely small. Therefore, it is necessary to measure minute changes in absorbance in the ultraviolet region, and due to the structure of the analytical element,
Because reflection photometry must be carried out, extensive and expensive measuring equipment must be used. Furthermore, in order to measure ultraviolet light, all materials must be used that do not have absorption in the vicinity of 340 nm, which is a disadvantage.

The latter method is not only much more advantageous than the former method because it forms a pigment via an electron transfer agent and can be detected in the visible region using a colorimetric method. It is very advantageous to be able to use both the reaction end point method.

Despite having such advantages, the latter method has not been widely used in wet Sake chemistry. In other words, the aforementioned electron transfer agents and dye-forming precursors have the serious disadvantage that their stability is greatly reduced when they coexist in a solution system, which may lead to the formation of undesired dyes. However, there is a decrease in accuracy when mixing the two,
Pigments derived from pigment-forming precursors are often insoluble in water, causing precipitation in the water bath, and problems such as decreased accuracy and reproducibility due to this precipitation arise.

Therefore, if the latter method is simply applied to a multilayer analytical element, the multilayer analytical element itself will not be able to demonstrate its usefulness, and furthermore, it will not be fully satisfactory in terms of stability and measurement accuracy. It is clear that this is not something to pray for.

Therefore, it has been strongly desired to develop an analytical element applying the latter method while maintaining the usefulness of the multilayer analytical element. An object of the present invention is to provide a dry analytical element for easily analyzing a specific component in a biological fluid sample via a reduced coenzyme. Analytical element for analyzing specific components in a fluid sample, which is provided on an impermeable support with a receptive layer (in both INI!I) and a developing layer (in both cases) as essential layers in sequence. at least one electron transfer agent, at least one pigment-forming precursor, at least one oxidized coenzyme, and the oxidized enzyme can be converted to a reduced coenzyme via the specific component. It is characterized in that it contains at least one type of reagent (hereinafter referred to as the reagent according to the present invention).

The electron transfer agent according to the present invention can be used to reduce the amount of reduced coenzyme produced by the reaction of one of the reagents according to the present invention with an oxidized coenzyme through the intervention of a specific component in a biological fluid sample. The electron transfer agent that is reduced in the presence of the specific component in the fluid sample that can be measured in the present invention will reduce the pigment-forming precursor and form a pigment that has an absorption in the visible region. Examples include lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), amylase <AMY), and creatine phosphokinase (CP).
K'), and triglycerides (Tω, etc.).

The reagent according to the present invention mainly refers to a substrate or an enzyme, and in some cases also includes a coenzyme.

These reagents are appropriately selected depending on the specific components in the biological fluid sample to be measured, such as LII (lactic acid for t-measurement, aspartic acid for α■, α-ketoglutaric acid, and glutamic acid dehydrogenation). enzyme (GIDH),
Alanine, α-ketoglutarate and glutamate dehydrogenase (GIDH) for GPT, maltopentose, orthophosphate, β-phosphoglucomutase (β-P(EM)), glucose oxidase (for AMY)
COD) and maltose phosphorylase (MP)
, in the case of CPK, creatine, adenosine triphosphate (
ATP), hexokinase (HK) and glucose-6-phosphate dehydrogenase (G-6-PDH), TG
In the case of lipoprotein liver 7 (LPL), glycerokinase (a()), glycerophosphate dehydration cumulative fermentation (GP)
DH) and adenosine triphosphate (ATP).

The oxidized coenzymes according to the present invention include oxidized nicotinamide adenine dinucleotide (NAD+) and oxidized nicotinamide adenine dinucleotide phosphate (NAD+).
P+) etc. The term “reduced coenzyme” refers to a reduced form of the oxidized coenzyme. NAIVE) The reduced form is NADH, N
The reduced form of ADP+ is NADPH.

The following is a reaction in which a reduced coenzyme is produced by the reaction of one or more reagents of the present invention with an oxidized coenzyme through the intervention of a specific component in a biological fluid sample according to the present invention. The formula is shown below.

OT acid 10NADH PT +NADH D-glucose-1-phosphe-1, 1-PGM β-D-glucose-1-honupeto〉phosphogluconate 10NA DH PK DP -phosphorus 1 6-phosphogluconate + NAJ) PH+ADP xiacentone phosphate+NADH Examples of electron transfer agents used in the present invention include N-methylphenazine methosulfates (for example, N-methylphenazine methosulfate, 1-medoxy N-methylphenazine methosulfate, etc.), Troubles, methylene blue, diaphorase, etc. can be used, and preferred electron transfer agents include N-methylphenazine methosulfates.

On the other hand, tetrazolium salts are usually used as the dye-forming precursor according to the present invention. Most of the above-mentioned tetrazolium salts used in the present invention become poorly soluble or insoluble in water after pigment formation, and are usually wet or insoluble.
Although chemical methods are difficult to use, the dyes formed are diffusion-resistant, preventing undesired linking, and
Examples of the above-mentioned tetrazolium salts useful in the present invention, which can be preferably used in terms of improving the quantitative properties of measurements, include 3.3'-(3,3'-dimethoxy-4
,4'-biphenylene)-bis[2-(p-nitrophenyl)-5-phenylte) Laso! j') Muchloride] (NBT), 3.3'-(3,3'-dimethoxy-4゜13
r: Su^4'-biphenylene)-bis[2,5-
diphenyl-tetrazolium chloride] (BT), 3-
(4157-dimethyltriazolyl-2) -2,4
-diphenyltetrazolium bromide (MTT), 2
-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-tetrazolium chloride (INT)
, 2.2', 5.5'-tetra-(p-nitrophenyl)-3,3'-(3-dimethoxy-4-diphenylene)-ditetrazolium chloride (TNBT), 2,3
゜5-Triphenyltetrazolium chloride (Tr)
and 3.3'-(4,4'-biphenylene)-bis[2°5-diphenyltetrazolium chloride] (NT
) etc.

The reagent of the present invention and the oxidized coenzyme contained in the analytical element of the present invention may be contained in a small amount (both one receiving layer and at least one developing layer).

The electron transfer agent and the dye-forming precursor may also be contained in any of the at least one receiving layer and at least one spreading layer, respectively. These electron transfer agents and dye-forming precursors must be contained in a state in which they do not react with each other to form undesired dyes. is preferably contained in different layers of at least one receptive layer and at least one developing layer. Even when the electron transfer agent and the pigment-forming precursor are contained in the same particle, it is preferable that the electron transfer agent and the pigment-forming precursor are contained as separate particles.

The electron transfer agent and the dye-forming precursor are contained in different layers, and the electron transfer agent is contained in one or more layers of at least one receiving layer and at least one developing layer. In an analytical element that contains a dye-forming precursor in one or more layers of one or more layers, the electron transfer agent is contained in one of the developing layers, and the dye-forming precursor is contained in the receiving layer. (Although it may be contained in the opposite state, the former state is preferable. In this case,
More preferably, a layer not containing an electron transfer agent and a dye-forming precursor is interposed between a developing layer containing an electron transfer agent and a receiving layer containing a dye-forming precursor.

When the electron transfer agent and the dye-forming precursor are contained in separate layers, the method for providing these containing layers is to dissolve the electron transfer agent or the dye-forming precursor in an aqueous binder solution, and to An example of this method is to dissolve the binder in water, then add this solution to a binder dissolved in water, and apply the dissolved solution.

When the electron transfer agent and the dye-forming precursor are contained as separate particles in the same layer of the receiving layer and the developing layer, the electron transfer agent and the dye forming precursor are contained as separate particles. Each pigment-forming precursor has a particle size of 0.
It is preferable that the thickness is 1 μ or more.

Furthermore, even when the electron transfer agent and the dye-forming precursor are contained in separate layers, the electron transfer agent and/or the dye-forming precursor can also be in particulate form.

As a method for providing a layer containing an electron transfer agent and a pigment-forming precursor as particles, the electron transfer agent and the pigment-forming precursor are each suspended in an organic solvent and then dissolved in the same organic solvent. The resulting suspension is added to a binder and then uniformly dispersed, and then applied, or the electron transfer agent and the pigment-forming precursor are suspended in an organic solvent and finely pulverized. , a method in which the electron transfer agent and the pigment-forming precursor are pulverized in an organic solvent containing the binder, and then the electron transfer agent and the pigment-forming precursor are pulverized in an organic solvent containing the binder. Examples include a method of applying a uniform coating.

In addition, when the developing layer is composed of a fibrous porous material such as F paper, the electron transfer agent and the pigment-forming precursor are added to an organic solvent containing a binder, and then the porous material is added and dispersed uniformly. There is a method of applying it.

The amount of the electron transfer agent according to the present invention contained in the analytical element of the present invention varies depending on the award of the specific component, but is usually 1 mg/in'-1 g/m": Yf
In addition, when the analytical element of the present invention contains the dye-forming precursor according to the present invention, the amount of the dye-forming precursor according to the present invention is usually 10 mg/
m'~10 g/in', preferably 50 mg/r
+12 to 3 g/m2. Historically, the amount of the oxidized coenzyme according to the present invention contained in the analytical element of the present invention is usually 1.
0mg/r'112-501m2, preferably 50mg
/in2 to 10 g/m", while the total amount of the reagent according to the present invention cannot be unconditionally determined depending on the type of the reagent, but is usually in the range of 10 mg/r'n2 to 100 g/m2. It is contained in the analytical element of the present invention.

The binder used to form the layer containing the electron transfer agent and the layer containing the dye-forming precursor is not particularly limited, but the binder of the layer containing the dye-forming precursor is a gelatin derivative. Or preferable. The method of forming the layer is not particularly limited. Also, when the electron transfer agent and the dye-forming precursor are present as separate particles in the same layer, the binder in the layer containing them is preferably a hydrophobic binder because it does not dissolve them.

The analytical element of the present invention constructed in this way has dramatically improved storage stability of the fabricated element, including the fog density of the analytical element, making it possible to obtain high discrimination ability. .

The spreading layer according to the present invention has the function of tl) uniformly distributing a constant volume of fluid sample per unit area to the receiving layer. Moreover, in addition,
No. 77, i.e. (2) the ability to remove substances or factors that inhibit the analytical reaction in the fluid sample, and/or (3) the ability to transmit measurement light through the support when performing spectrophotometric analysis. It is preferable that it has the function of performing a background effect of reflecting light. Therefore, the expansion layer according to the present invention is a layer having only the function (1) above, (
In addition to 1), it can be a layer having the functions of (2) and/or (3), or (1)
) It is also possible to separate a plurality of functions including 19- as appropriate and use a separate layer for each function. Furthermore, (1), (2) and (3
), it is also possible to use a combination of a layer with two functions and a layer with the remaining one function. An expanded layer of non-fibrous porous media, called plush polymer, consisting of cellulose acetate.
Examples include the fibrous structure spreading layer described in JP-A-56-24576, JP-A-57-125847, and % Application No. 56-65446. In particular, the fiber structure spreading layer is particularly useful as a material that can quickly transport blood cell parts, and is further useful for spreading and transporting macromolecules such as enzymes, which is one of the objects of the present invention. . The film thickness of the spreading layer in the analytical element of the present invention should be determined by its porosity, but is preferably about 100 to about 5.
00 microns, more preferably about 150-350 microns. Further, the porosity is preferably about 2 to about 85%.

The receptor layer according to the present invention is capable of absorbing a pigment-forming precursor when a specific substance in a fluid sample is finally transformed into a dye through a selected reaction via an electron transfer agent. The receptor layer is provided for the purpose of receiving a small amount of the dye that changes from the dye-forming precursor and detecting it as a change in the spectroscopically observable quantity. It is possible to contain various reagents and various additional additives as long as they do not contradict this purpose.

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

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

As usable surfactants, both ionic (anionic or cationic) and nonionic surfactants can be used, but nonionic surfactants are effective.

Examples of nonionic surfactants include 2,5-di-t-butylphenoxypolyethylene glycol, p-
Octylpheno 21, r
,,.

Examples include polyalkylene glycol derivatives of alkyl-substituted phenols such as xypolyethylene glycol and p-isononylphenoxypolyethylene glycol, and polyalkylene glycol esters of higher fatty acids. These surfactants regulate the rate of penetration of the fluid sample into the receptive layer and at the same time prevent undesirable "chromatographic phenomena".
It has the effect of suppressing the occurrence.

The above-mentioned surfactant can be used in a wide range of amounts, but it can be used in a range of 10% by weight to 0005% by weight, preferably from 6% to (i,05% by weight) based on the weight of the coating solution. be able to.

The above-mentioned liquid-impermeable, light-transparent support (hereinafter referred to as the support according to the present invention) related to the analytical element of the present invention is liquid-impermeable and light-transparent, so long as the type thereof is Any polymeric material, such as cellulose acetate, polyethylene terephthalate, polycarbonate, or polystyrene, is suitable for this purpose. In addition to the above-mentioned polymeric 22-mer materials, inorganic materials such as glass can be used as well. The thickness of the support according to the present invention is arbitrary, but is preferably about 50 to about 250 microns. Further, one side surface of the observation side of the support according to the present invention can be arbitrarily processed depending on the purpose. Furthermore, a receptive layer is laminated)f! A light-transmissive subbing layer may optionally be used on the opposite side of the support to improve the adhesion between the receptor layer and the support.

The analytical element of the present invention may be coated with a reflective layer, an undercoat, etc. as described in US Pat. No. 3,992,158, if necessary. #, a radiation blocking layer as described in U.S. Pat. No. 4,042,335, a barrier layer as described in U.S. Pat. No. 4,066,403, a microscopic layer as described in U.S. Pat. No. 4,166,093, a radiation blocking layer as described in U.S. Pat. Scavenger layer 1 described in No. 55-90859 and U.S. Pat. No. 4,110,079
The destructible body-like members described in the above can be arbitrarily combined to form an arbitrary structure suitable for the purpose of the present invention.

The various layers of these analytical elements are sequentially laminated on the support according to the present invention using a slide hopper coating method, an extrusion coating method, a dip coating method, etc., which are conventionally known in the photographic industry, and are appropriately selected according to the desired configuration. By doing so, a layer of arbitrary thickness can be applied.

Using the analytical element of the invention, the amount of a specific component in a fluid sample can be determined from the support side according to the invention by reflection spectral bow odometry according to the initial velocity method or the reaction termination method. The amount of the specific component can be determined by applying the measured value thus obtained to a calibration curve prepared in advance.

The amount of fluid sample applied to the analytical element of the present invention can be determined arbitrarily, but is preferably about 51 Ll to about 50 Ll.
μm, more preferably from 5 μJ to 20 μm. It is usually preferred to apply a fluid sample of about 10 μm.

The analytical element of the present invention can be used for the analysis of whole blood, serum, and plasma without any disadvantage. Urine in history,
Other body fluids such as lymph, spinal fluid, etc. may also be used without disadvantage.

If whole blood is used, a radiation blocking layer or other reflective layer as described above may be provided, if necessary, to prevent radiation for detection or protection from blood cells.

The analytical reaction used in the analytical element of the present invention can be arbitrarily determined depending on the purpose, but is useful, for example, in the field of clinical chemistry, and is particularly useful for analyzing components in biological fluid samples, ie, blood or urine. used.

As detailed above, according to the analytical element of the present invention, the electron transfer agent and the dye-forming precursor can be contained in the constituent layers in a state where they cannot react with each other until a fluid sample is applied. This not only improves the storage and stability of reagents, reduces fog density, and provides excellent color development, but also eliminates the occurrence of non-uniform concentrations and chromatographic phenomena, and eliminates the use of visible light. A conventional spectrophotometer can be used simply and quickly for high-precision dry quantitative analysis of components in fluid samples, especially biological fluid samples, and is extremely advantageous in practical terms.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the embodiments of the present invention are not limited thereby.

Margin Example 1 (1) Production A of analytical element-1 of the present invention Preparation of coating solution (,) 25 g of gelatin, 3.8 g of oxidized nicotinamide atenine dinucleotide (NAD+), Trito
nX-100 (product name, manufactured by Rohm & Hass)
0. ! M, lithium lactate 5g, trishydroxymethylaminemethane 3.9g, trishydroxymethylaminomethane hydrochloride 2.3f, 1. 0.2 parts of 2-bis(vinylsulfonyl)ethane was dissolved in 150 mJ of distilled water to prepare a receptor layer coating solution.

(b) 1-Methoxy-N-methylphenazine methosulfate (Meo-pMs) 6 in 100 m/xylene
Add 8M9 and 100ml of AP xylene! to NBT82O
Add N and prepare separate solutions and add 70 m of each to these.
After adding the glass beads of No. 1 and dispersing each of MeO.PN2 and NBT in fine particles in xylene using a sand grinder, the glass beads were separated from each other to prepare respective xylene dispersions. Furthermore, 300 m of xylene
/ to styrene-glycidyl methacrylate 27--copolymer [copolymerization ratio 9:1 (weight ratio)] 13f, T
Add 9g of riton
91 Da) was added and thoroughly stirred to obtain a coating solution for a developing layer.

B Analytical Element Production Layers having the following composition were sequentially coated on a transparent, undercoated polyethylene terephthalate support with a film thickness of about 180 microns using the coating solutions prepared in (a) and (b) above for analysis of the present invention. 0 (a) Receiving layer gelatin 25g/lr? Lithium lactate 5, g/fiNAD+
3.8g/Silent Triton X 1
0 0 0-5 97 m' Trishydroxymenalaminomethane 3.9f/d 28- Trishydroxymethylaminomethane hydrochloride
2.317 1.2-bis(vinylsulfonyl)ethane
A layer containing 0.2y/lri.

(b) Spreading layer powder F paper C91fl/n? Styrene-glycidyl methacrylate (9;1) copolymer 139/dMeO・PN2
68m? /rr? NBT
820 we/rr? Triton
-10091/n? A layer containing.

(2) Manufacture A of analytical element-2 of the present invention Preparation of coating liquid (-) MeO・PN2 681 in 50 ml of xylene
1F added liquid, xylene 50m/solution with NBT820 cape added, xylene 100m/lithium lactate 10f
, NAD + 3.8 f, tris hydroxy 29-dimethylaminomethane 3.9 F, and 2.3 g of tris hydroxy methyl amino methane hydrochloride were separately prepared, and 70 ml of glass beads was added to these solutions Loom/. In addition, each reagent was dispersed into fine particles in xylene using a sand grinder, and then the glass beads were separated in a furnace to obtain respective xylene dispersions.

Furthermore, styrene-methacrylate copolymer [copolymerization ratio 9:1 (weight ratio)] 1 op.
Triton
Glycidyl methacrylate copolymer [copolymerization ratio 9:1 (
Weight ratio)] 13y, Triton X 100
After adding 9 g to make a xylene solution, powdered p paper C91FK was added to this solution and thoroughly stirred to obtain a developing coating solution.

B Analytical Element Production 30 - The analytical element of the present invention was prepared by sequentially coating layers of the following composition on a transparent polyethylene terephthalate support with a subbing coating of about 180 microns using the coating solution prepared in step aXb above. -2 was produced.

(a) Acceptance j-Styrene-glycidyl methacrylate (9;1) copolymer 1 (1/m'MeO・PM
8 68”F/yn'NB'r
820■/d Lithium lactate
10f/dNAD+ a, s97'
m'97' Hydroxymethylaminomethane 3.9tt/rrl Trishydroxymethylaminomethane hydrochloride
2.3f/rdTrl
Contains ton X 100 0.5 fl/m'! -〇(b) Spreading layer powder door paper c 91g/m” styrene-
Glycidyl methacrylate (9;1) copolymer 139/n? Triton
X 100 997'tr? Contains no.

(3) Production A of analytical element-3 of the present invention Preparation of coating liquid (al) Gelatin 25fX'NAD+3.89, NBT
820m9, lithium lactate 1ON, trishydroxymethylaminomethane 3.9g, trishydroxymethylaminomethane hydrochloride 2.3f, Tr 0nX-1000,
5 g, and 1,2-bis(vinylsulfonyl)ethane Q, 4 f were dissolved in 150 ml of distilled water, and the first
A receptive layer coating solution was prepared.

(a,) Add MeOePM872N to 50ml of toluene and make 35ml! Glass peas were added thereto and dispersed into fine particles in toluene using a sand grinder, and then the glass peas were separated to obtain a toluene dispersion. Furthermore, add polystyrene to 100 mJ of toluene and 5. Dissolve Of;
The toluene dispersion liquid was mixed with this solution to obtain a second receptor layer coating liquid.

(b) Styrene-glycidyl methacrylate copolymer in 300 m/xylene [copolymerization ratio 9:1 [weight ratio]]
13F, add Triton X-10099 to make a xylene solution, add powdered paper C911 to this solution,
The mixture was thoroughly stirred to obtain a developing layer coating solution.

B Analytical element production The above (aυ, (a2) and (
Using the coating solution prepared in b), layers having the following compositions were sequentially applied to produce analytical element-3 of the present invention.

(aυ first receptor layer gelatin 259/dN B T
820 “f/m”NAD+
3.8tt/lithium lactate
10111/m' trishydroxymethylaminomethane 3.91/r1? Trishydroxymethylaminomethane hydrochloride
2.3 g/zeTrit
on X-1000,5II/1ri1.2-bis(vinylsulfonyl)ethane
0.411/rr? A layer containing.

(a2) Second receptor layer MeO@PMS 72 cape/-polystyrene
5.011/rr? (b) Spreading layer powder p paper C91fl/lri Styrene-glycidyl methacrylate (9;1) copolymer 13g/door Trilon
X 100 9111rr? A layer containing.

(4) Production A of analytical element-4 of the present invention Production of coating liquid (aυ gelatin 25g, N81820w9, N4D+3
．． 89X Triton
A receptive layer coating solution was prepared.

(-2) Add 35 m/g of glass beads to a solution of 50 ml of n-butanol, 3.9 g of trishydroxymethylaminemethane, and 2.3 g of trishydroxymethylaminomethane hydrochloride, and disperse into fine particles using a sand grinder. After this, the glass beads were separated in a furnace, and this dispersion was added to and mixed with a solution of polyvinylpyrrolidone 5'l f n-butanol (50 m/m) to prepare a second receptor layer coating solution.

(b) Add 52 mL of N-methylphenazine methosulfate (pMs) to 50 m of xylene, add 3.5 ml of glass beads, disperse in the xylene into fine particles using a sand grinder, and then place the glass beads in a furnace. This was separated to obtain a xylene dispersion. Furthermore, 313 g of styrene-glycidyl methacrylate copolymer [copolymerization ratio 9:1 (weight ratio), Triton
(Z+ was added to prepare a xylene solution, which was mixed with the above xylene dispersion. Powder p paper C91f was added to the mixed solution and stirred thoroughly to obtain a developing layer coating solution.

B. Manufacture of analytical element. On a transparent undercoated polyethylene terephthalate support with a film thickness of about 180 microns, layers with the following composition are sequentially applied using the coating solutions prepared in (a), (m), and (b) above. The analytical element-4 of the present invention was prepared by coating.

(11) First receptor layer gelatin 259/lr? NAD+
3.8y/rdNBT
820*/m' lithium lactate l
0IJ/7F1'Triton X 100 0
．． 59 /1r11.2-bis(vinylsulfonyl)
Ethane 0.2f/rr? A layer containing.

(a2) Second receptor layer polyvinylpyrrolidone 5f/-trishydroxymethylaminomethane 3.9tt/lt? Trishydroxymethylaminomethane hydrochloride
A layer containing 2.3 f/m'.

(b) Spreading layer powder filter paper C, 91 fl/rr? , styrene-glycidyl methacrylate (9;1) copolymer 131/rr? PM 8
52 */ m'Triton X-100
9f/W? A layer containing.

(5) Manufacturer of analytical element-5 of the present invention Coating solution preparation Gelatin/styrene-glycidyl methacrylate (weight ratio 95; 5) copolymer = 7/93 (weight ratio) (% Gansho 5
Polymer particles described in No. 6-155788) 150 g per 60 g
Add ml of distilled water and add NBT820 Misaki, NA
D+3.8N,) lishydroxymethylaminomethane 3
．． 9f1 Trishydroxymethylaminomethane hydrochloride2.
3g, lithium lactate 10g, T "White on X-100"
0.2 g of 1,591,1,2-bis(vinylsulfonyl)ethane was added to prepare a receiving J-coating solution.

The developing layer coating liquid used was the same as that used for Analytical Element-4 of the present invention.

B. Manufacture of analytical element Using the coating solution prepared above on a transparent undercoated polyethylene terephthalate support with a thickness of about 180 microns,
Analytical element-5 of the present invention was prepared by sequentially applying layers having the following compositions.

(8) Receptive layer gelatin/styrene-glycidyl methacrylate (95
;5) Copolymer 60y/n? NBT 820p/1NAD+
3.8y/n? Trishydroxymethylaminemethane 3.9/- Trishydroxymethylaminomethane hydrochloride
2.39/d lithium lactate 10fl/lt? Triton
X -1001,5g/-1,2-bis(vinylsulfonyl)ethane 0.2117t
A layer containing d.

(b) Spreading layer powder F paper c 91f/m'Styrene-glycidyl methacrylate (9;1) copolymer x3g/m'PM8
52 Misaki/rdTriton X-1
Layer containing 009fl/rd (6) Manufacturer of comparative analysis element-1 Coating liquid preparation gelatin 25 f, MeO・PN268”9, NBT
Cape 820, Lithium lactate 5g, NAD+3.811 Trishydroxymethylaminomethane 3.9p1 Trishydroxymethylaminomethane hydrochloride 3.99, Trishydroxymethylaminomethane hydrochloride 2.39.1.2-Bis(vinylsulfonyl)ethane 0. 2g, and Tri
ton-X O', 5f was dissolved in 150 m/distilled water to prepare a receptor layer coating solution.

The same coating liquid as that used in Analytical Element-2 of the present invention was used as the spreading layer coating liquid.

B. Manufacture of Analytical Element A comparative analytical element-1 was prepared by sequentially coating layers having the following composition using the above coating liquid on a transparent undercoated polyethylene terephthalate support having a film thickness of about 180 microns.

(-) Receptive layer gelatin 25f/dMeO・P
M S 68 “i/n?NBT
820■/door lithium lactate
5fi/lr? NAD+3
．． El/m' Trishydroxymethylaminomethane 3.9f/m' Trishydroxymethylaminomethane hydrochloride
2.3fl/rr?
Triton X 100 0.5 f/r
r11.2-bis(vinylsulfonyl)ethane
0.2tt/lr? A layer containing.

(+)) No expansion powder tear paper C911/rr? Styrene-glycidyl methacrylate (weight ratio 9:1) copolymer 13y/n? Triton
X 100 99/lt? A layer containing.

(7) Manufacture of Comparative Analytical Element-2 A comparative analytical element that is the same as Comparative Analytical Element-1 except that 15 g of polyvinylpyrrolidone was used instead of gelatin and PM852 town was used instead of MeO@PMS when preparing the coating solution.
(8) Manufacturer of Comparative Analysis Element-3 Coating liquid preparation Gelatin/styrene-glycidyl methacrylate (weight ratio 95i5) copolymer = 7/93 (weight ratio) (%) 150FM/distilled water was added to 60g of the polymer particles described in the publication, and MaO-PN2
72"7. NBT820q, NAD+3.8g, trishydroxymethylaminomethane 3.99, trishydroxymethylaminomethane hydrochloride 2.3g, lithium lactate 10 fXTritonX 1001.59, and 1.2-bis(vinylsulfonyl)ethane 0. 2f was added to prepare a receptor layer coating solution.

The same coating liquid used for the analytical element-2 of the present invention was used as the spreading layer coating liquid.

B. Manufacture of Analytical Element A comparative analytical element-3 was prepared by sequentially coating layers 1r of the following composition on a transparent undercoated polyethylene terephthalate support with a film thickness of about 180 microns using the above coating solution.

(-) Receptive layer Zeyf/styrene-glycidyl methacrylate copolymer 60f/n1MeO-PMi9
71v/m”NBT
8201"P/m'NAD+ 3.
8g/m' trishydroxymethylaminomethane 3.91/rr? Trishydroxymethylaminomethane hydrochloride
2.3f/door lithium lactate 109/rr? Triton
100 1.5 fl/rr? 1.2-bis(vinylsulfonyl)ethane 0.
A layer containing 2f/m'.

(b) Spreading layer powder r paper C91g/m' Styrene-glycidyl methacrylate (weight ratio 9:1) copolymer 10g/W? Triton
X 100 9 f/rr? A layer containing.

The reflection density (fog) of the thus obtained analytical elements 1 to 5 of the present invention and comparative analytical elements 1 to 3 immediately after manufacture and after storage at 35° C. for 3 days was determined at 2 m a x 580 n
Measurement was made from the support side using a filter of The results are shown in Table 1.

Table 1 As is clear from Table 1, the analytical element of the present invention has an extremely low capri concentration even when stored on the same day and after storage for 3 days, and the stability of the element is the same as that of the comparative analytical element. I understand.

Example 2 The compositions shown in Tables 2 and 3 were appropriately selected from the acceptance amount (1 to 12) and the spreading layer (1 to 12) of the composition (coating amount in the coating layer) as shown in Table 4. The analytical element of the present invention (6 to 1
6) and comparative analysis elements (4 to 8) were produced. Next, the reflection density of each analytical element obtained was measured, and the results were shown in Table 4. Dispersed directly.

Other coating liquids were applied as aqueous solutions. Gelatin/styrene-glycidyl methacrylate (weight ratio 95;
5) Copolymer - 2/98 polymer particles.

Rice 2 Manufactured by Toyo P Paper @ 300 mesh or less Margin As is clear from Table 4, compared to the comparative analytical elements, the analytical element of the present invention has an extremely high degree of fog even on the same day and after storage for 3 days. It can be seen that the stability of the device is improved.

Example 3 Immediately after each element of analytical element 1.4.5.8.12.14 of the present invention and comparative analytical element 1.2.3.8 manufactured in Example 1 and Example 2 was prepared and at 35°C. For each element after storage for 3 days, unikiretoLD
LDH activity 126 confirmed using H (manufactured by Chugai Pharmaceutical ■)
0 Breschi 1 unit, 2530 Breschi 1 unit and 50
10 μl of each standard serum containing 1 unit of 60 Brzeski was dropped onto the developing layer, and after incubation at 37°C for 10 minutes, λm
Reflection density was measured from the support side using a filter with ax of 580 nm. The results are shown in Table 5. As is clear from the results in Table 5, LB
While the change in color density is very low with respect to the change in H activity, the analytical element of the present invention shows a change in color density with a good start-up, and even after storage at 35°C for 3 days, LDH It was found that the color density changes well with changes in activity.

Example 4 In analytical elements 1 to 16 of the present invention and comparative analytical elements 1 to 8, α-ketoglutaric acid 4 was used instead of lithium lactate.
67M9/rr? , aspartic acid 21.3f/ld,
1200 units/nI of glutamate dehydrogenase was added, and 5.9 f/m" dipotassium monohydrogen phosphate and 1" potassium dihydrogen phosphate 2. ”
2 fl/d was added, and the analytical element for GOT according to the present invention (1
7 to 32) and comparative analytical elements for GOT (9 to 16) were produced.

For each analytical element thus obtained, the color i and capri concentration were measured in the same manner as in Example 3 and Example 1. As a result, the comparative analysis element had a high capri and a significantly small change in color density.
After storage at 5°C for 3 days, the capri of the comparative analytical element was high;
It was difficult to even distinguish whether the color concentration was due to serum dripping or not. On the other hand, the analytical element of the present invention had a low capri concentration even after storage, and showed good coloration corresponding to GOT activity.0 Example 5 In analytical elements 1 to 16 of the present invention and comparative analytical elements 1 to 8, α-ketoglutaric acid 4 was used instead of lithium lactate.
67 q/-Alanine 28.5g/W? 1200 units of glutamate dehydrogenase/hydrogenase and the same buffer as in Example 4 were added to prepare the GPT analytical element according to the present invention (
33-48) and a comparative analysis element (IY-24) were produced.

For each of the analytical elements thus obtained, the color development and capri density were measured in the same manner as in Example 3 and Example 1. As a result, comparative analysis element 9 had high fog and a significantly small change in color density. Also, 35℃,
After storage for 3 days, the capri of the comparative analytical element was high, and it was difficult to even distinguish whether the color density was due to the serum drop. On the other hand, the analytical element of Honshu4mei had a low capri concentration even after storage, and showed good coloration corresponding to GPT activity.

Example 6 Analytical elements (1 to 16) of the present invention and comparative analytical elements (1
~8), creadenphosphate disodium salt 15.0f/i, adenosine-5' instead of lithium lactate
-2 phosphate sodium salt 2.3 p/-, glucose 4.
1y/11? , magnesium sulfate hydrate 201
1/rr? , N A D P + 1.511/d, hexokinase 100 Tq/m', glucose-6-phosphate dehydrogenase 20 η/-, and trishydroxymethylaminomethane 2.71/d and its hydrochloride 3. 5f/rr? In addition to f, CP according to the present invention
Analytical elements for K (49 to 64) and comparative analytical elements (25
-32) The color development and capri density of each analytical element thus obtained were measured by the same operations as in Example 3 and Example 1. As a result, the comparative analysis element had a high capri and a significantly small change in color density.
After storage for 3 days, the capri of the comparative analytical element was high, and it was difficult to even distinguish whether the coloring concentration was due to the serum drop.On the other hand, the analytical element of the present invention had a low capri concentration even after storage, and the CPK activity was It showed good coloration corresponding to .

Patent applicant: Konishiroku Photo Industry Co., Ltd. Procedural amendment (voluntary) 2. Name of the invention analysis element 3. Person making the amendment Relationship to the case Patent applicant address: 26-2 Nishi-Shinjuku 1-chome, Shinjuku-ku, Tokyo
Name (127) Roku Konishi Photo Industry Co., Ltd. 4, Agent 6, Contents of amendment As shown in the attached document, please refer to the entire book of swords P% (no changes to the content) \-111fll# Procedural amendment (voluntary) Commissioner of the Japan Patent Office Wakasugi Kazuo-dono 1, Indication of the case Relationship with Patent Application No. 198446 Case Patent Applicant Address 1-26-2 Nishi-Shinjuku, Shinjuku-ku, Tokyo Name (Name) (127) Konishiroku Photo Industry Co., Ltd. Company 6
, The number of inventions increased by the amendment tx L8, Contents of the amendment The detailed explanation of the invention in the engraving specification t- (hereinafter simply referred to as the engraving specification) submitted on November 16, 1980 regarding the patent application in question is as follows. Correct it accordingly.

that's all

Claims (4)

[Claims] (1) On a light-transparent and liquid-impermeable support,
In an analytical element for analyzing a specific component in a fluid sample, in which a receptive layer (at least one layer) and a developing layer (at least one layer) are provided as essential layers in order,
an electron transfer agent, at least one pigment-forming precursor, at least one oxidized coenzyme, and at least one type capable of converting the oxidized coenzyme into a reduced coenzyme via the specific component. An analytical element for analyzing a specific component in a fluid sample, characterized in that it contains a reagent. (2) less (both one kind of electron transfer agent) and less (both one kind of electron transfer agent)
Analytical element according to paragraph (1), wherein the different dye-forming precursors are contained in different layers. (3) The analytical element according to claim (2), wherein the receptor layer contains a dye-forming precursor. (4) The analytical element according to claim (1), wherein the electron transfer agent and the dye-forming @carrier material are contained in the same layer as separate particles.