JPH03254699A - Analysis and reagent and analytical element used therefor - Google Patents

Abstract

PURPOSE:To carry out a sensitive quantitative analysis of a specified component by using a peroxidase-marked substance as the specified component of an analytical object, using H2O2, an aromatic primary amine and a specified aromatic secondary amine as the substrates and measuring the amount of a pigment generated in the enzymatic reaction. CONSTITUTION:A specified component as an analytical object is adsorbed on a carrier (e.g. nitrocellulose membrane) and a marked substance prepared by marking a substance (e.g. antibody) capable of binding to a specified component through a specific bonding with peroxidase is made to react therewith to bond the specified component adsorbed on the carrier thereto. An enzymatic reaction is then carried out using hydrogen peroxide, an aromatic primary amine compound and one compound selected from formula I (R is substitute; (m) is 0 or 1-4; Y is substituent having 0.3-1.5 Hammett substituent constant p; X is H or substituent eliminatable by reaction with oxidized compound of aromatic primary amine) and formula II as the substrates, and the amount of a pigment generated in the enzymatic reaction due to peroxidase is measured, thus analyzing the specified component.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for analyzing a specific component, and more particularly to a method for analyzing a specific component using a peroxidase enzymatic reaction, and a coloring reagent and an analytical element used therein.

[Conventional technology]

Various analytical methods for detecting specific components such as biological components have been developed, but these methods are highly accurate even when using intermediate amounts to detect the specific component and a substance that can specifically bind to it. (hereinafter referred to as specific binding substances), such as antigens and antibodies, certain sugar chains and lectins, biotin and avidin, protein A and IgG, hormones and receptors, enzymes and substrates, etc. A method using specific binding reactions. It has been known.

Generally, a specific component is analyzed by using a specific binding substance (hereinafter referred to as a label) attached with some type of label and detecting the signal of the label that changes depending on the specific component.

In particular, a method in which a specific component supported directly or indirectly on a support is reacted with a label, the label is immobilized as a complex of both, and a signal from the label substantially corresponding to the specific component is detected. is used as appropriate.

For example, electrophoresed protein biological components (specific components) are transferred from a gel onto a nitrocellulose membrane, labeled,
For example, a method of detecting a signal by reacting with an antibody label,
A method of detecting a signal by reacting a label with a specific component such as a lipid spread on a TLC plate, and detecting a signal between DNA and the DNA on a membrane! These methods include a method of reacting with aa complementary DNA and detecting a signal, or an immunohistochemical staining method.

By these methods, it is possible to obtain a great deal of information not only on the quantitative determination of a specific component and the reactivity of a specific component with a specific binding substance, but also on the properties, state of existence, etc. of the specific component or specific binding substance.

For example, a signal is detected on a complex made by binding a specific component of a living body such as a protein or nucleic acid transferred and supported on a membrane after electrophoresis, or a lipid component developed on TLC, and a label for the specific component. In this method, information such as the molecular weight, isoelectric point, or polarity of a specific component can be obtained from the position and mobility of the signal of the specific component.

In addition, in the immunohistochemical staining method, information such as the location and state of specific components of interest on the tissue can be obtained.

In the above-mentioned measurement of a specific component, in which a signal is detected substantially in accordance with the amount of a specific component on a complex conjugate supported directly or indirectly on a support, the specific component of interest is in a trace amount, so it is difficult to use a label. It is essential that am is detected with high sensitivity and that the am detection method has high resolution in order to obtain more information on a specific component.

Radioactive isotopes, fluorescent substances, luminescent substances, enzymes, etc. are used as specific binding substances.

Radioactive isotopes have problems such as attenuation of radioactivity, disposal, exposure to radiation, and the large expense required for equipment, and also have the disadvantage of requiring a complicated operation for detecting a signal on a label supported on a support.

Fluorescent substances or luminescent substances require special equipment and equipment.

On the other hand, when enzymes are used, the procedure is relatively simple and the produced pigment can be easily visualized and quantified. Conventionally, mW
As one enzyme, peroxidase, alkaline phosphatase, β-galactosidase, etc. have been used. In the method of producing and depositing a dye by enzymatic reaction on a complex conjugate carried on a support, peroxidase is mainly used as a labeling enzyme. -1-naphthol etc. have been used.

[Problem to be solved by the invention]

Diaminobenzidine. -Dianisidine has the drawback of being highly toxic and prone to background.

4-Chloro-1-naphthol is slightly more sensitive than others, but the sensitivity is not sufficient for measuring trace amounts of specific components or clearly obtaining more information about specific components. .

Other methods using conventionally known compounds or combinations thereof are also useful; When quantifying , glutamate pyruvate transaminase (GPT), etc. by converting them into hydrogen peroxide, the concentration of hydrogen peroxide to be quantified is very low, so the discrimination sensitivity for these quantifications is still not sufficient. .

An object of the present invention is to provide a simple, highly sensitive, high-resolution, and rapid method for analyzing specific components in the determination of hydrogen peroxide or a substance that generates hydrogen peroxide in the presence of a substance that has a peroxidizing effect. , a coloring reagent used therein, and an analytical element.

[Means to solve the problem]

To summarize the present invention, the first invention of the present invention relates to a method for analyzing a specific component. In the method for analyzing the specific component, which is measured by the amount of dye produced by the enzyme reaction, hydrogen peroxide, an aromatic primary amine compound, and the following general formula [I] or [-n): [Formula In the formula, R represents a substituent, and m represents 0 or an integer of 1 to 4. When m is an integer of 2 to 4, the plurality of R's may be the same or different.

Y is a substituent with a Hammett's substituent constant σp of 0.3 or more and 1.5 or less, and X represents a substituent that leaves by reaction with hydrogen or an oxidized product of an aromatic primary amine compound] It is characterized by using a compound that

Further, the second invention of the present invention relates to a coloring reagent, which comprises an aromatic primary amine compound and a compound represented by the general formula [I] or [II] in the first invention. Features.

Furthermore, a third invention of the present invention relates to a coloring reagent for hydrogen peroxide analysis, and is characterized by comprising a substance having a peroxidizing effect and the coloring reagent of the second invention.

Furthermore, a fourth invention of the present invention relates to another coloring reagent for hydrogen peroxide analysis, characterized in that it consists of a substance that generates hydrogen peroxide and the coloring reagent of the third invention. do.

A fifth invention of the present invention relates to an analytical element, which is used in the analytical method of the first invention, and has at least a reagent layer on a support and a spreading layer above the reagent layer. The analytical element is characterized in that each component of the coloring reagent of the fourth invention is contained in either the same or different layers.

Next, the present invention will be explained in detail.

In the present invention, the specific component may be directly supported on the support by physical adsorption, chemical bonding, etc., or may be supported indirectly via one or more specific binding substances. Alternatively, the specific component may be directly or indirectly supported on the support, and then the label may be reacted to form the composite conjugate, or the composite conjugate may be formed after forming the composite conjugate. may be directly or indirectly supported on the support. Furthermore, the label forms a complex conjugate with the specific component and is supported on the support, but the specific component and the label may be bound directly or via one or more other specific binding substances. May be combined.

Further, in the present invention, the labeled body may be one in which a specific binding substance is labeled with peroxidase and an anti-peroxidase antibody.

Analytical biochemistry using conventional hydrogen peroxide and 4-chloro-1-naphthol as substrates (
^analytical Biochemistry)
, Vol. 119, pp. 142-147 (1982), the method of the present invention shortened the color development time, produced clear spots, and increased sensitivity by more than 10 times. Moreover, as a result, the amount of peroxidase label can be reduced, which is advantageous in terms of cost. In the present invention, the target specific component is a substance or a group of substances from which a specific binding substance that specifically binds to the specific component is obtained.

For example, proteins, nucleic acids, hormones, lipids, complex carbohydrates,
Examples include glycolipids, polysaccharides, enzymes, vitamins, antigens, antibodies, and the like.

Further, the specific binding substance that can be used in the present invention is a substance that can specifically bind to a specific component or another specific binding substance, and can be appropriately used depending on the specific component. Examples include proteins, nucleic acids, hormones, lipids, complex carbohydrates, glycolipids, polysaccharides, enzymes, vitamins, antigens, antibodies, lectins, protein A1 avidin, biotin, receptors, coenzymes, enzyme substrates, toxins, complements, and Examples include complexes of these.

Supports that can be used in the present invention include membranes such as cellulose, acetate, and nitrocellulose, gel supports such as polyacrylamide, silica gel supports such as TLC plates, plate-shaped and bead-shaped plastics, glass, metals, and fibers. etc. In addition, in histochemical staining,
The tissue itself can also be used as a support.

There are no particular limitations on the substituent represented by R in general formulas [I] and [II]. Typical examples include multiple groups such as alkyl, aryl, anilino, acylamino, sulfonamide, alkylthio, arylthio, alkenyl, and cycloalkyl, but also halogen, cycloalkenyl, alkynyl, heterocycle, sulfonyl, sulfinyl, Phosphonyl, acyl, carbamoyl, sulfamoyl, cyano, alkoxy, sulfonyloxy, aryloxy, heterocyclicoxy, siloxy, acyloxy, carbamoyloxy, amino, alkylamino, imide, ureido, sulfamoylamino, alkoxycarbonylamino, aryloxycarbonyl Examples include polygroups such as amino, alkoxycarbonyl, aryloxycarbonyl, heterocyclic thio, thioureido, carboxyl, hydroxy, mercapto, nitro, and sulfonic acid, as well as spiro compound residues, bridged hydrocarbon compound residues, and the like.

Among the substituents represented by R, the alkyl group preferably has 1 to 32 carbon atoms, and may be straight or branched.

As the aryl group, a phenyl group is preferred.

Examples of the acylamino group include an alkylcarbonylamino group and an arylcarbonylamino group.

Examples of the sulfonamide group include an alkylsulfonylamine group and an arylsulfonylamino group.

Examples of the alkyl component and aryl component in the alkylthio group and arylthio group include the above-mentioned alkyl groups and aryl groups.

The alkenyl group preferably has 2 to 32 carbon atoms, and the cycloalkyl group preferably has 3 to 12 carbon atoms, particularly 5 to 7 carbon atoms, and the alkenyl group may be linear or branched.

The cycloalkenyl group has 3 to 12 carbon atoms, especially 5
-7 is preferred.

Sulfonyl groups include alkylsulfonyl groups, arylsulfonyl groups, etc.; sulfinyl groups include alkylsulfinyl groups, arylsulfinyl groups, etc.; phosphonyl groups include alkylphosphonyl groups, alkoxyphosphonyl groups, aryloxyphosphonyl groups, and arylphosphonyl groups. Acyl groups include alkylcarbonyl groups, arylcarbonyl groups, etc.; carbamoyl groups include alkylcarbamoyl groups, arylcarbamoyl groups, etc.; sulfamoyl groups include alkylsulfamoyl groups,
Arylsulfamoyl groups, etc.; Acyloxy groups include alkylcarbonyloxy groups, arylcarbonyloxy groups, etc.; carbamoyloxy groups include alkylcarbamoyloxy groups, arylcarbamoyloxy groups, etc.; ureido groups include alkylureido groups, arylureido groups, etc. ; As the sulfamoylamino group, an alkylsulfamoylamino group, an arylsulfamoylamino group, etc.; as a heterocyclic group, a 5- to 7-membered one is preferable, and specifically, a 2-furyl group, a 2-thienyl group , 2-pyrimidinyl group, 2-benzothiazolyl group, 1-pyrrolyl group, ■-tetrazolyl group, etc.; as the heterocyclic oxy group, one having a 5- to 7-membered heterocycle is preferable, for example, 3.4.5.6 tetrahydro Pyranyl-2-oxy group, 1-phenyltetrazole-5-oxy group, etc.; 5 to 7 as a heterothio group
A membered heterocyclic thio group is preferable, such as a 2-pyridylthio group, a 2-penzothiazolylthio group, a 2,4-diphenoxy-1,3,5-)lyazole-6 monothio group; as a siloxy group, trimethyl Siloxy group, triethylsiloxy group, dimethylbutylsiloxy group, etc.; imide group is succinimide group, 3-heptadecylsuccinimide group, phthalimide group, glutarimide group; ring Nispiro compound residue is spiro[3,3] heptane
1-yl, etc.; as a bridged hydrocarbon compound residue, bicycloC2,2,1
1 hebutan-1-yl, tricyclo[3,3,1,13
"7]Decan-1-yl, 7,7-dimethyl-bicyclo[2,2,1)hebutan-1-yl, and the like.

Among the above substituents, R is an alkyl group, an aryl group, a carboxyl group, an oxycarboxyl group, a cyano group, a hydroxy group, an alkoxy group, a ryoryloxy group, an amine group, an amide group, a sulfonamide group, etc. Polygroups, halogens, etc. are preferred.

m represents 0 or an integer of 1 to 4, and when m is 2 to 4,
A plurality of R's may be the same or different.

Further, a plurality of R's may be bonded to each other to form a ring, and the iron ring is preferably a saturated or unsaturated 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, etc. Examples include a pyridine ring and a quinoline ring.

The above group may further have a substituent such as a long-chain hydrocarbon group or a diffusion-resistant group such as a polymer residue.

Examples of groups that can be separated by reaction with the oxidized product of the aromatic primary amine compound represented by X include halogen (chlorine,
bromine, fluorine, etc.) and alkoxy, aryloxy, heterocyclic oxy, acyloxy, sulfonyloxy, alkoxycarbonyloxy, aryloxycarbonyl, alkyloxalyloxy, alkoxyoxalyloxy, alkylthio, arylthio, heterocyclic thio, alkyloxythiocarbonyl thio, acylamino, sulfonamide, nitrogen-containing heterocycle bonded by N atom, alkyloxycarbonylamino, ary-nioxycarbonylamino,
Examples include multiple groups such as carboxyl.

In general formulas [I] and [I[], the atomic group represented by Y has a Panot substituent constant σp of 0.3 or more and 1.5
The following substituents, typically cyano group, nitro group, sulfonyl group (e.g. octylsulfonyl group, phenylsulfonyl group, trifluoromethylsulfonyl group, pentafluorophenylsulfonyl group, etc.) β-carboxyvinyl group, sulfinyl group groups (e.g. t-butylsulfinyl group, tolylsulfinyl group, trifluoromethylsulfinyl group, pentafluorophenylsulfinyl group, etc.) β, β-dicyanovinyl group, halogenated alkyl group (e.g. trifluoromethyl group, perfluorooctyl group, ω-hydroperfluorododecyl group, etc.) formyl group, carboxyl group, carbonyl group (e.g. acetyl group, pivaloyl group, benzoyl group, trifluoroacetyl group, etc.) Alkyl and aryloxycarbonyl group (
(e.g. ethoxycarbonyl group, phenoxycarbonyl group, etc.) 1-tetrazolyl group, 5-chloro-1-tetrazolyl group, carbamoyl group (e.g. dodecylcarbamoyl group, phenylcarbamoyl group, etc.) sulfamoyl group (e.g. trifluoromethylsulfamoyl group, phenyl sulfamoyl group, ethylsulfamoyl group, etc.).

Preferred among these substituents are a cyano group, a sulfonyl group, and a sulfamoyl group.

Typical specific examples of compounds used in the present invention are shown below.

These couplers of the present invention are organic synthesis
Collective Volume 4 (Organic 5yn-the
ses Co11ective Vol+ne 4)
It can be synthesized by referring to the methods described on pages 180 and 172, etc.

Examples of aromatic primary amine compounds that can be used in the present invention include 〇- or p-aminophenol compounds, 〇- or p-phenylenediamine compounds, and salts thereof.

Preferred is a p-phenylenediamine compound represented by the following general formula [II[].

B \/ In the formula, A and B represent hydrogen or an alkyl group, and A and B
together with nitrogen, D, E, G and J are hydrogen, halogen, hydroxy group, amino group, alkoxy group, acylamido group, arylsulfonamide group,
Represents an alkylsulfonamide group or an alkyl group.

The alkyl group represented by A and B has 1 to 1 carbon atoms.
6 is preferred, and 1 to 4 are particularly preferred. Examples include methyl group, ethyl group, and butyl group. These alkyl groups may have a substituent, and examples of the substituent include a ureido group, a tetrahydrofuryl group, a carboxyl group, a methanesulfonamide group, a sulfo group, a methoxy group, an ethoxy group, a methoxyethoxy group, and a methoxyethoxy group. Examples include ethoxy group and methoxytetraethoxy group.

D, G and J are preferably hydrogen, an alkoxy group, an alkylsulfonamide group, or an arylsulfonamide group, and more preferably hydrogen. E is preferably hydrogen, an alkyl group or an acylamido group, more preferably an alkyl group having 1 to 3 carbon atoms, especially a methyl group. In addition, as salts of the compound represented by the general formula , phosphorous acid esters, organic boron compounds, salts of organic or inorganic acids such as hydrochloric acid and sulfuric acid, and p-)luenesulfonates, hydrochlorides and sulfates are particularly preferred.

Typical examples of the aromatic primary amine compound according to the present invention are shown below, but the present invention is not limited thereto.

Exemplary compound (III-1) N,N-diethyl-3-methyl-4-aminoaniline (In-2) N,N-diethyl-4-aminoaniline (III-3) N-carbamidomethyl-N-methyl- 4
-Aminoaniline (III-4)N-carbamidomethyl-N-tetrahydrofurfuryl-3-methyl-4-aminoaniline (I[[-5)N-ethyl-N-carboxymethyl3-
Methyl-4-aminoaniline (II [-6) N-carbamidomethyl-N-ethyl-3
-Methyl-4-aminoaniline (III-7) N-ethyl-N-tetrahydrofurfuryl-3-methyl-4-aminephenol (III-8)
3-acetylamino-4-aminodimethylaniline (DI-9) N-ethyl-N-β-methanesulfonamidoethyl-4-aminoaniline (III-10) N-ethyl-N-β-methanesulfonamidoethyl- 3-Methyl-4-aminoaniline (In-11) N-methyl-N-β-sulfoethyl-p
-phenylenediamine(II[-12)N-ethyl-N-hydroxyethyl-
3-methyl'''4-aminoaniline (I[l-13)N
-ethyl-N-[2-(2-methoxyethoxy)ethyl-3-methyl-4-aminoaniline (III-14)N-ethyl-N-(2-[:2(2-
methoxyethoxy)ethoxy]ethyl)-3-methyl-
4-Aminoaniline (In-15)N-ethyl-N-[2-(2[2-C2
-(2-Methoxyethoxyethoxy)ethoxy]ethoxy]ethoxy)ethyl]3-methyl-4-aminoaniline (III-16) N,N-diethyl-3-methanesulfonamidoethyl-4-aminoaniline.

Various substances can be used as the substance having a peroxidizing effect according to the present invention, and a typical example thereof includes peroxidase. Peroxidase is an enzyme that catalyzes the reaction in which hydrogen peroxide oxidizes another substance. This peroxidase is a complex protein that generally contains iron porphyrins, which can be found in horseradish, potato, fig sap, turnip (a plant peroxidase), milk (lactoperoxidase) and white blood cells (
It is also present in microorganisms and can be obtained by extraction or fermentation.

Also, ``acta, chemical power, Scandinavian power, (^cta)
Chem, 5cand,) Volume 4, Nos. 422-434
Synthetic peroxidases can also be used in the present invention, such as those disclosed in Theorell and Maehly, 1950. In addition to peroxidase, methemoglobin, oxyhemoglobin, hemoglobin, alkaline hematin,
Hemin and hemin derivatives, etc. can also be used in the present invention.

In addition to enzymes, substances exhibiting a peroxidizing effect such as iron thiocyanate, iron stannate, ferrous ferrocyanate, and nichrome salts adsorbed on silica gel (eg, potassium chromium sulfate) can also be used.

Among these, peroxidase is preferred.

In the method of the present invention, hydrogen peroxide or hydrogen peroxide produced by the action of a substance that produces hydrogen peroxide oxidizes the aromatic primary amine compound by the action of a substance having a peroxidizing action.

The resulting oxidized aromatic primary amine compound undergoes a coupling reaction with the compound represented by the general formula [I] or [II] according to the present invention, thereby resolving the dye. The produced dye has a main visible absorption in the range of 500 to 700 nm, and exhibits color development with extremely high sensitivity.

Information about the pigment produced can be read visually or using methods known in the art, such as a spectrophotometer. Alternatively, the information may be read after adding a suitable organic solvent to dissolve the dye.

The analysis method of the present invention can be carried out, for example, as follows.

Adding to the fluid sample a compound represented by the general formula CI] or [II], an aromatic primary amine compound, and a peroxidizing substance as a reaction catalyst, either simultaneously or in an appropriate order;
The mixture is heated between 4°C and 40°C, preferably between 20°C and 40°C.
temperature in the range of p) 13.0 to 9.0, preferably pH 4.5 to 8.0, for a time range of 1 minute to 60 minutes, and absorbance of the reaction mixture containing the produced dye. Measure.

The method of the invention is particularly useful for trace determination of hydrogen peroxide. The hydrogen peroxide concentration in the fluid sample is preferably between 0.01 and 200 μg/ml in the total color reaction solution. In this range of hydrogen peroxide concentration, the general formula [I
] or [II], the compound represented by 0.10 to 10
0μrrIole/-1 aromatic primary amine compound is
0.01-10 μmole/-1 peroxidizing agent, preferably peroxidase, preferably used in an amount of 0.01-100 units/- (in the total color reaction solution).

The compound represented by the general formula [I] or CI[] can be prepared using a small amount of a hydrophilic organic solvent such as methanol, ethanol, N
, N-dimethylformamide (DMF), etc. and may be added. The molar ratio of the compound represented by general formula CI] or [II] and the aromatic primary amine compound is 1:10
0 to 100 to 1 is appropriate, and 1 to 10 to 1
is preferred.

In addition, in the method of the present invention, in addition to the compound represented by general formula [I] or [II], an aromatic primary amine compound, and a peroxidizing substance, a substance that generates hydrogen peroxide is used as a coloring reagent. When combined, the substance to be finally analyzed is led to hydrogen peroxide by the action of the substance, thereby making it possible to analyze various target substances.

Substances in biological fluid samples that can be analyzed by this method include glucose, cholesterol, uric acid, talleatinine, triglycerides, lactic acid, free fatty acids,
Glutamate oxaloacetate transaminase (GOT)
) Glutamate pyruvate transaminase (G
PT) Cholinesterase, creatine phosphokinase, lactate dehydrogenase, etc.

The substances used in these analyzes include, for example, glucose oxidase, uricase, cholesterol oxidase, glycerol oxidase, glycerin-3-
Phosphate oxidase, sarcosine oxidase, pyruvate oxidase, D-aspartate oxidase,
D (or L)-amino acid oxidase, L-gulono-T
-lactone oxidase, L-turposoxidase,
L-2-hydroxy acid oxidase, 6-hydroxy-
D-nicotine oxidase, 6-hydroxy-L-nicotine oxidase, pyridoxamine phosphate oxidase, pyridoxine oxidase, hexose oxidase, ○-aminophenol oxidase, amine oxidase (containing pyridoxal or flavin), xanthine oxidase, alcohol oxidase, ethanol Various examples include amine oxidase, N'-methyl-L-lysine oxidase, choline oxidase, alcohol oxidase, and sulfite oxidase.

These substances may be of any origin and may be used in solid powder form or as a solution.

When using this substance, hydrogen peroxide may be generated in advance, but it is preferable that the hydrogen peroxide production reaction and the coloring reaction proceed simultaneously.

The timing of adding the substance to the coloring reagent solution may be arbitrary, and the amount used is arbitrarily selected depending on the type and target substrate.

Further, as another example of implementing the method of the present invention, an analytical element containing a compound represented by shell formula (I) or [II], an aromatic primary amine compound, and a peroxidizing substance may be mentioned. can.

The analytical element of the present invention is of a dry type in which reagents necessary for analysis are incorporated into the element in a dry state. These analytical elements include single-layered ones that use filter paper, membrane filters, etc. as the material for the reagent-supporting layer (Japanese Patent Publication No. 36-4198, U.S. Patent No. 3.607.093, etc.); Glass filters and membrane filters are used as non-integral multilayer analysis elements based on simple lamination or peeling (Japanese Patent Application Laid-open No. 11395/1983) 2
A piece of filter paper covered with a net (Japanese Patent Application Laid-open No. 1510-1989)
No. 96, U.S. Patent No. 3,526, 480).

Furthermore, the analytical element of the present invention is preferably an integrated multilayer analytical element (Japanese Patent Publication No. 53, 1983) having at least one reagent layer and a porous spreading layer on a liquid-impermeable, light-transmitting support.
-21677, JP-A-55-164359, JP-A-55
-90859, 57-197466, 57-1
No. 01760, No. 57-101761, No. 58-90
167 etc.).

The above-mentioned reagent layer can be provided as a layer by coating a water-soluble polymer or a hydrophilic and organic solvent-soluble polymer as an indizer on the support. Examples of water-soluble polymer binders include gelatin, gelatin derivatives such as phthalated gelatin, water-soluble cellulose derivatives such as hydroxyethylcellulose and carboxymethylcellulose sodium salt, polyvinyl alcohol, poly(N-vinylpyrrolidone), polyacrylamide, polymethacrylamide, and acrylamide. Copolymers of acrylic acid esters, poly(mono- or dialkyl-substituted) acrylamides, poly(mono- or dialkyl-substituted) methacrylamides, and water-soluble copolymers thereof, etc., preferably gelatin, polyacrylamide, and acrylamide and acrylic acid. Copolymers of esters are used. As a hydrophilic and organic solvent soluble polymer binder, poly(N
-vinylpyrrolidone) poly(N-vinylimidazole) poly(N-vinyltriazole), derivatives thereof or copolymers thereof, cellulose derivatives such as ethylcellulose and methylcellulose, and the like. These polymer binders are mainly hydrophilic polymeric substances that are soluble in alcohols, such as ethanol, proponol, butanol, and the like.

The above polymer binder can be arbitrarily selected depending on the specific component selected and its analytical reaction. In addition, if the selected analytical reaction is composed of two or more types of reagents,
The reagents may be contained mixed together in the same reagent layer, or two or more reagents may be contained in two or more separate reagent layers. These may be determined by the mechanism of action of the analytical reaction itself, and their configuration is arbitrary as long as it does not have an undesirable effect.

The thickness of the reagent layer can be arbitrarily selected as desired, but is preferably 1 to 200 μm, and more preferably 5 to 100 μm.

The porous spreading layer has the function of (1) uniformly distributing a fixed volume of fluid sample to the reagent layer per unit area; Furthermore, the performance described in Japanese Patent Publication No. 53-21677, namely (2) the ability to remove substances or factors that inhibit analytical reactions in fluid samples, and/or (3)
It is preferable that it has the function of performing a background effect of reflecting measurement light transmitted through the support when performing spectrophotometric analysis. Therefore, the porous development layer according to the present invention may be either a layer having only the function (1) above, or a layer having functions (2) and/or (3) in addition to (1). Alternatively, it is also possible to separate a plurality of functions including (1) as appropriate and use a separate layer for each function.

Furthermore, it is also possible to use a combination of a layer having two of the functions (1), (2), and (3) and a layer having the remaining one function. For example, the above-mentioned special public service
A spread layer of a non-fibrous porous medium called plush polymer consisting of titanium dioxide and cellulose diacetate described in Japanese Patent Application No. 21677, and a spread layer of a hydrophilized woven fabric described in JP-A-55-164356. , JP-A-57-94658, JP-A-57-12847, JP-A-57-
Examples include the fiber structure spreading layer described in each publication such as No. 197466 and No. 58-70161, and the particle bond structure spreading layer described in JP-A-58-90167.

In particular, the fiber structure spreading layer and particle combination structure spreading layer are particularly useful as materials capable of rapidly transporting blood cell portions. The thickness of the spreading layer in the analytical element of the present invention should be determined by its porosity, and is preferably about 100 to 600 μm, more preferably about 15 μm.
It is 0 to 400 μm. In addition, the porosity is preferably about 2
It is 0-85%.

Depending on the specific component selected and its analytical reaction, the porous spreading layer can contain a reagent that directly or indirectly interacts with the specific component in the fluid sample, as in the case of the reagent layer described above. .

Furthermore, various other additives such as preservatives and surfactants may also be added as desired.

In particular, surfactants can be effectively used to adjust the permeation rate when a fluid sample is applied to the analytical 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-
Examples include polyalkylene glycol derivatives of alkyl-substituted phenols such as t-butylphenoxypolyethylene glycol, p-octylphenoxypolyethylene glycol, and p-isononylphenoxypolyethylene glycol, and polyalkylene glycol esters of higher fatty acids.

These surfactants have the effect of regulating the rate of penetration of the fluid sample into the reagent layer and at the same time suppressing the occurrence of undesirable "chromatographic phenomena."

The surfactant can be used in an amount selected within a wide range, but ranges from 25% by weight to 0.5% by weight based on the weight of the coating solution.
0.05% by weight, preferably 15% to 0.05% by weight
Can be used.

The above-mentioned liquid-impermeable, light-transparent support (hereinafter referred to as the support according to the present invention) can be of any type as long as it is liquid-impermeable and light-transparent, but examples include cellulose acetate, Various polymeric materials such as polyethylene terephthalate, polycarbonate or polystyrene can be used, as well as inorganic materials such as glass.

Although the thickness of the support according to the present invention is arbitrary, it is preferably 5 to 250 μm. 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, on the side of the support on which the reagent layer will be laminated,
Optionally, a light-transparent subbing layer can be used to improve adhesion between the reagent and the support.

The above-mentioned integrated multilayer analytical element may include, if necessary, for example, a reflective layer as described in U.S. Pat. No. 3,992,158, an undercoat layer, a radiation blocking layer as described in U.S. Pat. Barrier layer described in U.S. Pat. No. 4.066,403, Migration prevention layer described in U.S. Pat. No. 4.166,093, JP-A-55-9085
Scavenger layer described in Publication No. 9 and US Pat. No. 4.11
The breakable bod-like members described in the specification of No. 0.079 can be arbitrarily combined to form an arbitrary structure that meets the purpose of the present invention.

The various layers of these analytical elements are formed on the support according to the present invention by appropriately selecting the slide hopper coating method, extrusion coating method, dip coating method, etc. conventionally used in the photographic industry according to the desired configuration. By sequentially laminating layers, layers of arbitrary thickness can be applied.

The method of adding the compound represented by the general formula [I] or [I[] of the present invention to the liquid for forming the analytical element of the present invention is selected as appropriate depending on the chemical structure of the compound, etc. be able to.

For example, various methods can be used, such as a method in which the material is added after being dissolved in water, an aqueous buffer solution, an organic solvent, etc., a solid dispersion method, a latex dispersion method, an oil-in-ice emulsion dispersion method, and the like.

For the oil-in-water emulsion dispersion method, a method of dispersing hydrophobic additives such as a cutter, which is conventionally known in the photographic industry, can be applied.
Usually, it is dissolved in a high boiling point solvent and/or a low boiling point solvent and mixed with an aqueous solution containing a hydrophilic colloid such as gelatin containing an anionic surfactant and/or a nonionic surfactant,
It can be used after being emulsified and dispersed using a high-speed rotation mixer, colloid mill, flow mixer, ultrasonic dispersion device, etc.

Examples of high boiling point solvents include organic acid amides, carbamates, esters, ketones, urea derivatives, etc.
Di-n-butyl phthalate, tricresyl phosphate, triphenyl phosphate, diisooctyl acetate, di-n-butyl sebacate, )'J-n-hexyl phosphate, N, N-diethyl caprylamide, N,
N-diethyl laurylamide, n-pentadecyl phenyl ether, dioctyl phthalate, n-nonylphenol, 3-pentadecyl phenylethyl ether, 2.
Examples include 5-di-5eC-amylphenylbutyl ether, monophenyl-di-O-chlorophenyl phosphate, and fluorinated paraffin. Among these, dialkyl phthalates, particularly those having an alkyl group having 1 to 6 carbon atoms, are preferred.

Examples of low-boiling solvents include methyl acetate, propyl acetate, butyl acetate, butyl propionate, cyclohexanol, diethylene glycol monoacetate, nitromethane, carbon tetrachloride, chloroform, cyclohexane, tetrahydrofuran, methyl alcohol, acetonitrile, DMF, dioxane, and methyl ethyl ketone. etc.

Examples of anionic surfactants include alkylbenzenesulfonic acids and alkylnaphthalenesulfonic acids, and examples of nonionic surfactants include sorbitan sesquioleate and sorbitan monolaurate.

The analytical element of the present invention comprises the above-mentioned substance having a peroxidizing effect,
In addition to the compound represented by general formula [I] or [II],
Depending on the type of component to be analyzed, it can contain various reagents such as a substance that generates hydrogen peroxide and other enzymes, substrates, M buffers, preservatives, surfactants, and hardening agents. .

The substances with peroxidizing action according to the invention can be used in widely selected amounts, for example in the case of peroxidases from 100 to 1,000.000 U/
m”, preferably 1.000-100.000U
/ m” range.

As mentioned above, according to the method of the present invention, the produced pigment exhibits color development with extremely high sensitivity. It also reacts sensitively to uric acid, creatinine, GOTSGPT, etc.) and is particularly useful for quantifying trace components.

In the present invention, the substance having a peroxidizing effect according to the present invention, the compound represented by the general formula [I] or [II], may be used in the reagent layer, the porous layer spreading layer, and other It can be contained in any of the layers.

When quantifying hydrogen peroxide or a substance that generates hydrogen peroxide using the analytical element of the present invention, the analytical element is immersed in a fluid sample or the fluid sample is dropped onto the analytical element.
It can be determined by reflection spectrophotometry according to the initial productivity or reaction end point method. The amount of hydrogen peroxide or a substance that generates hydrogen peroxide can be determined by applying the measured value thus obtained to a calibration curve prepared in advance.

The fluid sample applied to the analytical element of the present invention may be a biological or non-biological fluid sample as long as it contains hydrogen peroxide or a substance that generates hydrogen peroxide. For example, blood (
(including plasma and serum), lymph fluid, urine, etc.

Further, in the case of a test piece, the amount of the fluid sample to be used is arbitrary as long as it is at least the amount that allows the absorbent carrier containing the reagent to be sufficiently impregnated with the fluid sample. On the other hand, in the case of a body type multilayer analysis element, the volume is also optional, but preferably about 50 μl to about 5 μl, and more preferably about 20 μl to about 5 μm. Usually about 10
Preferably, μl of fluid sample is applied.

〔Example〕

EXAMPLES Hereinafter, the present invention will be specifically explained using Examples, but the scope of the present invention is not limited by the Examples.

Example 1 Measurement of antigen on trocellulose membrane) A phosphate buffer solution (hereinafter referred to as PBS) was added to a nitrocellulose membrane (manufactured by Bio-Rad; thickness 0.45 μm) that had been washed with pure water and air-dried.
lμl of serially diluted goat IgG was spotted.

After air drying, blocking was performed overnight at 4°C with a 1% bovine serum albumin (BSA)-PBS solution, and then a peroxidase-labeled rabbit anti-goat IgG antibody (manufactured by Kappel;
(1500 times diluted with 1% BSA-PBS solution) at 4°C for 2 hours. 0.05% Tween (Tw
een) 20 (polyoxyethylene sorbitan monolaurate; manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) - washed five times with a PBS solution and immersed in a coloring substrate reagent solution. The following three types of coloring substrate test solutions were used and compared.

(1): Dissolve 3 mg of 4-chloro-1-naphthol in 1 ml of methanol, and make 5 m7! of 0.05M
) Lis-HCl buffer (pH 7, 4,200d NaCl
(hereinafter referred to as TBS), and then 20 μl of 3% hydrogen peroxide solution.

(2) = 3■ Compound Example I-2 was dissolved in 1 rnl of methanol, 5-TBS was added, 1■ Compound Example II [
-10 (sulfate) and an additional 20 μl of 3% hydrogen peroxide.
Add.

(3): Compound example I-2 of (2), compound example ■-10 (
Compound Example l-13, Compound Example lll- instead of sulfate)
Prepared in the same manner as (2) using 1 (hydrochloride).

(4) = Compound example I-2 of (2), compound example ■-10 (
Compound Example I [-1, Compound Example m-16 instead of sulfate]
Prepared in the same manner as (2) using (p-)luenesulfonate).

After reacting for 15 minutes, it was thoroughly washed with water and air-dried. When using the coloring substrate test solution (1), the spot contains 10 ng of goat Ig.
GL could not be detected, but the coloring substrate test solution (2) ~
When (4) was used, a spot of 1 ng of goat IgG was detectable.

Example 2 (Screening for antibodies and hybridomas) 50 μg of α1-antitrypsin was injected intraperitoneally into Balb/C mice (female, 6 weeks old) together with Freund's complete adjuvant. After 3 weeks, further α1-antitrypsin 50
μg of α1-antitrypsin was injected intraperitoneally with incomplete Freund's adjuvant, and after another 2 weeks, 30 μg of α1-antitrypsin was administered.
was dissolved in PBS and injected intravenously. Final immunity 3 p.
Take out the m cells and transform them into mouse myeloma cells X according to the standard method.
Merged with 63.6.5.3. The cells were divided into five 96-well plates and cultured in HAT selection medium. Three weeks after the fusion, antibodies were measured in the wells in which hybridoma colonies had formed using the following method.

Cut nitrocellulose into four square pieces, each with α
1-Antitrypsin 500μg/mf PBS solution 1μ
Spotted l. 96-well microtiter plate
Add one plate per hole and incubate at 4℃ with 1% BSA-PBS solution.
, blocked for one night. After washing with PBS, 40 μl of hybridoma culture supernatant was added and reacted at room temperature for 2 hours. 3 in 0.05% Tween-20-PBS solution
After washing twice, peroxidase-labeled goat anti-mouse immunoglobulin antibody (manufactured by Kappel; 1% BSA-PBS)
40 μl of 1500-fold diluted solution) was added and reacted at room temperature for 2 hours. 0.05% Tween-20-
After washing three times with PBS solution, a substrate test solution for color development was added, and those in which a dye was formed on the nitrocellulose were determined to be positive for antibody activity. As a coloring substrate test solution (1): Example 1 (1)
), (2): Two types similar to (2) of Example 1 were used for comparison.

If (1) test solution was used among the 350 wells measured, 1
Although antibody activity-positive hybridomas could be detected in only 5 wells, antibody activity-positive hybridomas could be detected in 23 wells when the test solution (2) of the present invention was used.

Example 3 (Comparison of color development sensitivity) For 50 μl of a fluid sample (0.5 μl/- hydrogen peroxide solution), 50 μl of a color reagent solution prepared by the following method was added.
After mixing at a ratio of rnl and leaving at room temperature for 5 minutes, the absorbance of the produced dye at λ[OaX was measured using a hydrogen peroxide blank as a control.

Preparation of coloring reagent solution Add 0.5 μmole of the compound of general formula [■] or [II] to 1 m of DMF! , compound of general formula [III] 12.5 μm
ole and 10 units of peroxidase in PBS (pH 7).
, 4) and 5- respectively, and then both solutions are mixed to obtain a coloring reagent solution.

Coloring Reagent Absorbance Detection Wavelength (nm) It can be seen that the coloring reagent of the present invention produces coloring dyes with high sensitivity.

Example 4 Measurement of glucose (1) Preparation of coloring reagent solution 20 units of glucose oxidase, 7 units of peroxidase
, 5 j11th position, Compound Example 111-1 Hydrochloride 12, 5.
4 moles were dissolved in PBS (pH 7,4) 5-, and 0.5 μmole of compound example n-1 was dissolved in DMFlrnl, and then both solutions were mixed to obtain a coloring reagent solution.

(2) Preparation of glucose solution 10μ of glucose was added to PBS (pH 7,4) 5rn
This is used as a glucose solution.

(3) Measurement of glucose Take 40 μl of coloring reagent solution 5- and glucose solution,
After incubating for 10 minutes at 7°C, a glucose blank was used as a control. The absorbance was measured at 20 nm and the value was 1.03.

On the other hand, for comparison, measurement was performed using phenol and 4-aminoantipyrine hydrochloride instead of ll-1, II[-1, and the absorbance was 0.18. As is clear from this, the method of the present invention is a measurement method with significantly higher sensitivity than conventional methods.

Example 5 Total cholesterol analysis element film thickness 180 μm
A reagent layer, an intermediate layer, and a developing layer having the compositions shown below were sequentially provided on a transparent subbed polyethylene terephthalate support, and analytical elements 1 to 3 of the present invention and comparative analytical element - (1 )It was created.

Reagent layer (R-1) Compound example I-64, 15 g/m'' dibutyl phthalate 2.08 g/m'
Gelatin 16.5g/m' Peroxidase 12.500 U/m" Potassium phosphate buffer pH 6,8 3,25g/m" Sodium triisopropylnaphthalene sulfonate 0.93g/m'1.2-
Bis(vinylsulfonyl) ethane 0.11 g/m" Sodium azide 0.18 g/m' Used in addition to aqueous solution and ultrasonic dispersion.

Reagent layer (R-2) Compound example 11-8 4, 40 instead of compound example I-6
Prepared in the same manner as reagent layer R-1 except that 2.20 g/m' of dibutyl phthalate was used.

Reagent layer (R-3) 4.46 of compound example n-17 instead of compound example I-6
g/m', 2.23 g/m of dibutyl phthalate
Prepared in the same manner as R-2 except that 2 was used.

Reagent layer (R-4) 4-aminoantipyrine hydrochloride 1.59 g/m
'1.7-hydroxynaphthalene 1.18g/m''
Gelatin 16.5 g/m' Peroxidase 12.500U/m' Potassium phosphate buffer PH6,8 3,25 g/m' Sodium triisopropylnaphthalenesulfonate 1.2-bis(vinylsulfonyl) ethane 0.11 g/m 'Sodium azide 0.18 g/m' Intermediate layer (common to Examples and Comparative Examples) (I-1) N-vinylpyrrolidone-vinyl acetate copolymer weight ratio 2:8> 1.35 g/m ' 0.93 g/m'' Spreading layer (S-1) Fiber for opening paper raw material 91.0 g/m' [Toyo opening paper ■, 40-100 mesh] Styrene-glycidyl methacrylate copolymer (weight ratio 9:1) 23.0 g/m'
Trioxyethylene monolaurate 11.7 g/m' Compound example lll-10 (sulfate) 0.14 g/m' Dimezone 1.75 g/m' Cholesterol esterase 2500 U/m' Cholesterol oxidase 2500 U/m' Bovine serum albumin 2 .5g/m' Cholesterol esterase and cholesterol oxidase were dissolved in water together with bovine serum albumin, freeze-dried, and then pulverized.

The above development layer is applied using xylene solvent.

Developing layer (S-2) From the developing layer (S-1) of the example sample, compound example ■-1
0 Developed layer (S-1) except for sulfate and dimezone
Prepared similarly.

Table-1 Table-2 The above analytical elements of the present invention-1 to 3n and comparative analytical elements-(
For 1), 10 μl of human serum with various total cholesterol concentrations was dropped onto the developing layer, incubated at 37°C for 7 minutes, and then reflected using a 650 nm (546 nm for comparative analysis element) filter. The concentration was measured from the support side, and the results shown in Table 2 were obtained.

As is clear from the results in Table 2 above, the analytical elements 1 to 3 of the present invention show better coloring density differences with respect to the difference in total cholesterol concentration than the comparative analytical element-(1), and can be used for discrimination. It can be seen that the ability, that is, the quantitative sensitivity is high.

Example 6 Analytical element for glucose A reagent layer, an intermediate layer, and a developing layer having the compositions shown below were sequentially provided on a transparent undercoated polyethylene terephthalate support having a film thickness of 180 μm to form an analytical element 4 of the present invention shown in Table 3 below.
5 and comparative analysis element-(2) were prepared.

Reagent layer (R-5) Compound example 1-11 4.79 g/m' dibutyl phthalate 2.40 g/m'' gelatin
15.5g/rtx'' Helloxidase 33.0OOU/m' Glucose oxidase 21.000U/m' Potassium phosphate buffer (pH 6,1) 2,82 g/m' Sodium triisopropylnaphthalene sulfonate 0.96 g / m'Sodium azide 0.11g/m'1.2-
Bis(vinylsulfonyl) ethane 0.10 g/m'
Compound Eny 11 was dispersed and used in the same manner as described in Example 5.

Reagent layer (R-6) Instead of compound example ■-11, 4.86 g of n-4/
m'% dibutyl phthalate 2.43 g/m'
Prepared in the same manner as reagent layer R-4 except that .

Reagent layer (R-7) 4-aminoantipyrine hydrochloride 1.79 g/m
'1.7-hydroxynaphthalene 1.33 g /
m' gelatin 15.5 g /
m'peroxidase 33.0OOU/
m'glucose oxidase 21.0OOU/
m' Potassium phosphate buffer (pH 6,1) 2.82 g/m' Sodium triisopropylnaphthalene sulfonate 0.96 g/m' Sodium azide 0.11 g/m'1
．． 2-bis(vinylsulfonyl) ethane 0.10 g/m''
Interlayer (common to Examples and Comparative Examples) (I-2) N-vinylpyrrolidone-vinyl acetate copolymer (weight ratio 2:8) 1,40 g/m' Spreading layer (S-3) Slip raw material Fiber 91.0 g/m”
Toyo Kuchishi ■, 40-100 mesh] Styrene-glycidyl methacrylate copolymer (weight ratio 9:1) 23. Og/m'
Trito:/X -1009,1g/m'' Compound example ■-16 (p-toluenesulfonate) 0.15g/m' Dimezone 1.90g/m' Developing layer (S-4) Compound example ■- Compound example ■-10 (sulfate) in place of 16
The developing layer (S-
Prepared in the same manner as 3).

Developing layer (S-5) Developing layer (S-5) Except for removing compound example m-16 (p-toluenesulfonate) dimezone from the developing layer (S-3>
Prepared in the same manner as 3).

Table-3 Analytical elements of the present invention-4 and 5 and comparative analytical elements-(
For 2), 10μ of human serum with various glucose concentrations was added.
After dropping it onto the developing layer and incubating for 7 minutes at 37°C, the reflection density was measured from the support side using a 650 nm (546 nm for comparative analytical elements) filter, and the results are shown in Table 4. Obtained.

Table 4 As is clear from the results in Table 4 above, the analytical element 4.5 of the present invention shows a better coloring density difference with respect to the difference in glucose concentration than the comparative analytical element - (2). , it can be seen that the discrimination ability, that is, the quantitative sensitivity is high.

〔Effect of the invention〕

As described above in detail, the analytical element of the present invention has the remarkable effect of significantly improving quantitative sensitivity.

Claims (1)

[Scope of Claims] 1. A method for analyzing a specific component to be analyzed, in which the specific component to be analyzed is measured by the amount of a dye produced by an enzymatic reaction of peroxidase, using a labeled substance having peroxidase as a label. As substrates for the reaction, hydrogen peroxide, an aromatic primary amine compound, and the following general formula [
I] or [II]: ▲There are mathematical formulas, chemical formulas, tables, etc.▼...[I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼...[II] [In the formula, R represents a substituent, m represents 0 or an integer of 1-4. When m is an integer of 2 to 4, the plurality of R's may be the same or different. Y is a substituent with a Hammett's substituent constant σp of 0.3 or more and 1.5 or less, and X represents a substituent that leaves by reaction with hydrogen or an oxidized product of an aromatic primary amine compound. A method for analyzing a specific component, characterized by using a compound that is 2. A coloring reagent comprising an aromatic primary amine compound and a compound represented by the general formula [I] or [II] according to claim 1. 3. A coloring reagent for hydrogen peroxide analysis, comprising a substance having a peroxidizing effect and the coloring reagent according to claim 2. 4. A coloring reagent for hydrogen peroxide analysis, comprising a substance that generates hydrogen peroxide, and the coloring reagent according to claim 3. 5. An analytical element used in the analytical method according to claim 1, which has at least a reagent layer and a developing layer above the reagent layer on the support, wherein the analytical element contains each component of the coloring reagent according to claim 4. , in either the same or different layers.