JP2009244231A - Immunological detecting method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunological detecting method which is simple, quick, and highly sensitive. <P>SOLUTION: The immunological detection method is composed of: (a) a process for forming a composite containing an analysis object and a first protein (Y) which is capable of specifically recognizing the analysis object in an analyte which is modified with a label containing a metal activating the analyte or specimen containing the analyte by the action of the Y; (b) a process for separating the composite and the first protein Y which is free from the composite; (c) a coloring process for making an organic peroxy acid formed by the reaction of an organic peroxy acid precursor and a peroxide, and a color fixing agent act on the separated composite; and (d) a process for detecting pigment formed in the coloring process. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an immunological detection method capable of detecting a sample containing an analysis object simply, rapidly, and with high sensitivity. In particular, the present invention relates to an immunological detection method for detecting an analyte with high sensitivity using an amplified color reaction.

  There are very many physiologically active substances or environmental pollutants such as natural products, toxins, hormones, or agricultural chemicals that act in extremely small amounts. Therefore, instrumental analysis methods capable of highly sensitive analysis have been widely used for qualitative and quantitative measurement of these substances. However, the instrumental analysis method has low specificity, requires a long time for analysis including a sample pretreatment step, and is complicated in operation, and thus is inconvenient for the purpose of rapid and simple measurement that has been required in recent years. On the other hand, immunological measurement methods have high specificity and are much simpler to operate than instrumental analysis, and thus have gradually become popular in the field of measuring physiologically active substances or environmental pollutants. Among these, there are immunological measurement methods such as latex agglutination method, RIA method or EIA method (enzyme immunoassay method), but latex agglutination method does not always satisfy the quick and easy measurement or detection sensitivity. Uses a radioisotope as a labeling substance, so a special environment is required.

  Enzyme immunoassay has been developed as an immunoassay with little danger to the human body and is used in measurement systems for various substances. However, in clinical chemistry analysis, the measurement target is a biological sample (mainly serum, urine, etc.), and the measured value is often used for diagnosis or follow-up of pathological conditions, and therefore higher sensitivity and higher accuracy for that purpose. It is difficult to completely satisfy the demands of accurate measurement with the colorimetric method. In order to satisfy this requirement, a chemiluminescent enzyme immunization method has been proposed (see, for example, Patent Document 1). The chemiluminescent enzyme immunoassay measures the enzyme activity by measuring the amount of luminescence emitted when the chemiluminescent substance enters an excited state through an intermediate due to the catalytic activity of the enzyme and returns to the ground state from this state, This is a method for quantifying the amount of a substance to be measured having a correlation with the enzyme activity. In this method, since a chemiluminescent substance is caused to emit light by a chemical reaction, a light source is unnecessary, and since there is no increase in background due to the light source, it is possible to increase the sensitivity of measurement.

However, in the case of a proliferating analyte such as a virus, detection at an earlier stage, that is, with a small amount of virus is required, and a more sensitive and simple detection method has been desired.
JP 2000-146968 A

  An object of the present invention is to provide an immunological detection method capable of detecting a sample containing an analysis object simply, rapidly, and with high sensitivity.

  The above-described problems of the present invention have been solved by the following means.

<1> An immunological detection method,
a) A first protein (Y) capable of specifically recognizing an analyte in a specimen modified by a label containing a metal is allowed to act on the specimen or a sample containing the specimen, and the analyte and Y are Forming a complex comprising:
b) a step of separating the complex from the first protein (Y) not forming the complex;
c) a color developing step in which the organic peracid produced by the reaction of the organic peracid precursor and peroxide, and the color former act on the separated complex;
d) An immunological detection method comprising a step of detecting color development produced by the color development step.
<2> The immunological detection method according to <1>, wherein the organic peracid precursor is a compound having an alkanoylamino group or an alkanoyloxy group.
<3> The immunological detection method according to <2>, wherein the alkanoyl in the alkanoylamino group or alkanoyloxy group is acetyl.
<4> The immunological detection method according to any one of <1> to <3>, wherein the color development step is performed within 1 hour after the reaction between the organic peracid precursor and the peroxide.
<5> The immunological detection method according to any one of <1> to <4>, wherein the color former comprises a reducing agent and a color former.
<6> The immunological detection method according to any one of <1> to <5>, wherein the metal-containing label contains at least one of silver, gold, or platinum atoms.
<7> The immunological detection method according to <6>, wherein the metal-containing label is silver.
<8> The immunological detection method according to <6>, wherein the metal-containing label is silver halide.
<9> The immunological detection method according to any one of <5> to <8>, wherein the reducing agent is a compound represented by the following general formula (I):

(Wherein R ₁ , R ₂ , R ₃ and R ₄ each independently represents a hydrogen atom or a substituent, and R ₅ and R ₆ each independently represents an alkyl group, an aryl group, a heterocyclic group, an acyl group, Or a sulfonyl group, which may have a substituent, and R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₂ and R ₅ , or R ₄ and R ₆ are bonded to each other. And may form a 5- to 7-membered ring).
<10> The Cp is represented by the following general formulas (C-1), (C-2), (C-3), (M-1), (M-2), (M-3), (Y-1). The immunological detection method according to any one of <5> to <9>, comprising at least one compound selected from the group consisting of: (Y-2) and (Y-3):

(Wherein, X ₁ represents a hydrogen atom or a leaving group, Y ₁ and Y ₂ represent an electron-withdrawing substituent, and R ₁ represents an alkyl group, an aryl group, or a heterocyclic group);

(Wherein X ₂ represents a hydrogen atom or a leaving group, R ₂ represents an acylamino group, a ureido group or a urethane group, R ₃ represents a hydrogen atom, an alkyl group or an acylamino group, R ₄ represents a hydrogen atom, or Represents a substituent, and R ₃ and R ₄ may be linked to each other to form a ring);

(Wherein X ₃ represents a hydrogen atom or a leaving group, R ₅ represents a carbamoyl group or a sulfamoyl group, and R ₆ represents a hydrogen atom or a substituent);

(Wherein X ₄ represents a hydrogen atom or a leaving group, R ₇ represents an alkyl group, an aryl group or a heterocyclic group, and R ₈ represents a substituent);

(Wherein X ₅ represents a hydrogen atom or a leaving group, R ₉ represents an alkyl group, an aryl group or a heterocyclic group, and R ₁₀ represents a substituent);

(Wherein X ₆ represents a hydrogen atom or a leaving group, R ₁₁ represents an alkyl group, an aryl group, an acylamino group or an anilino group, and R ₁₂ represents an alkyl group, an aryl group or a heterocyclic group);

(Wherein X ₇ represents a hydrogen atom or a leaving group, R ₁₃ represents an alkyl group, an aryl group, or an indoleenyl group, and R ₁₄ represents an aryl group or a heterocyclic group);

(Wherein X ₈ represents a hydrogen atom or a leaving group, Z represents a divalent group necessary to form a 5- to 7-membered ring, and R ₁₅ represents an aryl group or a heterocyclic group.) ;

(In the formula, X ₉ represents a hydrogen atom or a leaving group, R ₁₆ , R ₁₇ and R ₁₈ each represent a substituent, n represents an integer of 0 to 4, and m represents an integer of 0 to 5. When n or m is 2 or more, the plurality of R ₁₆ and R ₁₇ may be the same group or different groups.
<11> The step of forming the complex further includes a second protein (X) capable of specifically recognizing the analyte in the specimen, and the second protein (X) is the analyte and <1> to <10>, wherein the first protein (Y) is allowed to act simultaneously or stepwise to form a complex containing the X, the analyte and Y. The immunological detection method according to any one of the above.
<12> The immunological detection according to any one of <1> to <11>, wherein the analyte is an antigen, and a protein capable of specifically recognizing the analyte is an antibody. Method.
<13> The immunological detection method according to <11>, wherein the antibody is a monoclonal antibody.
<14> The immunological detection method according to any one of <11> to <13>, wherein the second protein (X) is supported on an insoluble carrier.

According to the present invention, there is provided an immunological detection method capable of detecting an analyte contained in a sample easily, quickly and with high sensitivity. In particular, an immunological detection method for detecting an analyte with high sensitivity using an amplified color reaction is provided.
The present invention utilizes an antigen-antibody reaction to amplify an oxidation-reduction reaction using an antibody or a label immobilized on an antigen as a catalyst by an organic peracid generated by a reaction between an organic peracid precursor and a peroxide, By detecting a colored color by inducing a coloring reaction, it is easily detected with high sensitivity. Alternatively, one of the proteins capable of specifically recognizing the analyte can be immobilized on a substrate and detected more easily and with high sensitivity by chromatography.

The immunological detection method of the present invention includes the following steps.
a) A first protein (Y) capable of specifically recognizing an analyte in a specimen modified with a label containing a metal is allowed to act on the specimen or a sample containing the specimen, and the analyte and Y are Forming a composite comprising

In this step, a first protein (Y) labeled with a protein that can specifically recognize the analyte is prepared. As a protein that can specifically recognize an analyte, an antibody is preferably used when an antigen-antibody reaction is used. In the present invention, a metal label that can function as a catalyst for a redox reaction of peroxide is used as the label. As such a metal label, a metal selected from metals such as silver, gold and platinum, or metal salts such as silver halide is used.
When the first protein (Y) is allowed to act on a specimen or a measurement sample including a specimen in which the specimen is dispersed in a buffer solution or the like, the analyte and the first protein (Y) are caused by a specific interaction such as an antigen-antibody reaction. ) Is formed.

b) A step of separating the complex from the first protein (Y) that does not form the complex. The separation step includes separating the formed complex from the first complex that does not form a complex. It is the process of isolate | separating from protein (Y). That is, in the separation step, an amount of the complex corresponding to the amount of the analyte present in the sample is separated.

c) Coloring step in which the organic peracid produced by the reaction of the organic peracid precursor and the peroxide and the color former is allowed to act on the separated complex The organic peracid is formed by the reaction of the organic peracid precursor and the peroxide. Is done. When the generated organic peracid, reducing agent, and coloring material are allowed to act on the composite, a metal-containing label serves as a catalyst, causing a chain reaction via the organic peracid, oxidizing the reducing agent, and oxidizing the reducing agent. Generate a body. Subsequently, a coloring reaction occurs between the oxidized oxidant of the reducing agent and the coloring material to produce a coloring dye. The amount of dye produced depends on the amount of complex and therefore corresponds to the amount of analyte.
In general, an organic peracid is an unstable compound and decomposes with time. Therefore, it is preferably used promptly within 1 hour after the formation of the organic peracid.

d) A step of detecting the dye produced by the color developing step The amount of the produced dye can be detected by various known means. For example, the presence or absence of an analysis object can be determined instantaneously by visually sensory evaluation of the presence or absence of color development. It is also possible to quantitatively measure the amount of dye using a densitometer or a spectrophotometer.

  As a means for separating the complex that can be used in the separation step, centrifugation or ultracentrifugation using a difference in specific gravity, precipitation using a difference in solubility, salting out, filtration, dialysis, Conventionally known separation means such as a filtration method and a separation method using magnetism can be used. In the present invention, the separation means is particularly effective in that a protein capable of specifically recognizing an analyte is preliminarily membrane or chromatographed. Immobilize on an insoluble carrier such as a fixed layer of the graph, and after forming the complex, wash away and wash away the components in the sample that have not formed the complex or the components used for sample preparation, or This is a method of separation by chromatography.

  In addition, the amount of the free first protein (Y), which is the other separated component, corresponds to the amount obtained by subtracting the amount of the first protein (Y) initially contained to form the complex. This amount also reflects the amount of the analyte present in the sample. Therefore, both the amount of complex and the amount of free first protein (Y) can be used for detecting the amount of the analyte. A means for directly detecting the complex is preferable for highly accurate and simple detection.

  Means for generating organic peracid by reaction of organic peracid precursor and peroxide, and means for allowing organic peracid, reducing agent and coloring material to act on the separated complex include organic peracid precursor, peroxide In addition, it is possible to use a method in which each of the reducing agent and the coloring material is contained or a mixture or a solution containing two or more of them is allowed to act in the state of a dispersion or dispersion. As another embodiment, a color developing sheet containing at least one of an organic peracid precursor, a peroxide, a reducing agent (CD) and a color developing material (Cp) is prepared, and the color developing sheet is superimposed on the separated composite. Then, the composite and the color former are brought into contact with each other to cause a color development reaction. Preferably, the coloring sheet contains a reducing agent and a coloring material, and a solution in which an organic peracid precursor and a peroxide are mixed is supplied to the superposed body.

  The organic peracid precursor used in the present invention is preferably a compound having an alkanoylamino group or an alkanoyloxy group. More preferably, the alkanoyl in the alkanoylamino group or alkanoyloxy group is acetyl.

In the present invention, the label is preferably silver particles or silver halide grains.
In the present invention, a coupling reaction between an oxidized form of a reducing agent and a coupler is preferably used for the color development reaction. In particular, the reducing agent represented by the general formula (I) and the general formulas (C-1) and (C-2) capable of efficiently performing a coupling reaction by heat development in a heat-developable recording material using an organic silver salt. ), (C-3), (M-1), (M-2), (M-3), (Y-1), (Y-2) and at least selected from the group consisting of (Y-3). One type of coupler is preferably used.

  A preferred embodiment of the present invention further includes a second protein (X) capable of specifically recognizing the analyte in the specimen, and the second protein (X) is the analyte and the first protein. (Y) is allowed to act simultaneously or stepwise to form a complex containing X, the analyte, and Y.

Preferably, the analyte is an antigen, and the protein capable of specifically recognizing the analyte is an antibody. More preferably, the antibody is a monoclonal antibody.
Furthermore, a preferred embodiment of the present invention is an immunochromatograph in which the second protein (X) is carried on an insoluble carrier and a complex is separated and detected by chromatography.

  The antigen-antibody reaction constituting the immune reaction stage usable in the present invention is arbitrary, and any conventionally known reaction can be adopted.

In the present invention, examples of the insoluble carrier used in the separation step include polymer compounds such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, crosslinked dextran, and polysaccharides, glass, metal, and magnetic particles. And combinations thereof. The insoluble carrier can be used in various shapes such as a tray shape, a spherical shape, a fiber shape, a rod shape, a disk shape, a container shape, a cell, a microplate, and a test tube.
Furthermore, the immobilization method of the antigen or antibody to these insoluble carriers is arbitrary, but a physical adsorption method, a covalent bond method, an ionic bond method or the like can be used.

The antibodies used in the present invention (the first protein (X) and the second protein (Y) can be appropriately selected depending on the analysis target. Here, monoclonal antibodies and polyclonal antibodies can be used as the antibodies. Any antibody can be used, and the form thereof can be a whole antibody or a fragment such as F (ab ′) ₂ , Fab, etc. Although the origin of the antibody is arbitrary, mouse, rat, Antibodies derived from rabbits, sheep, goats, chickens, etc. are preferably used, wherein both or one of the first protein and the second protein is a monoclonal antibody, or F (ab ′) ₂ or F (ab ′ ) Is preferable.

  The binding between the antibody of the present invention and the protein is usually performed in a buffer solution. As a buffer solution used here, a Tris buffer solution, a phosphate buffer solution, a borate buffer solution, a carbonate buffer solution, a glycine-sodium hydroxide buffer solution and the like can be arbitrarily used. The concentration of these buffers is desirably used in the range of 1 mM to 1M. Moreover, additives, such as surfactant and a chelating agent, can be arbitrarily used at the time of reaction.

The immunological detection method according to the present invention includes a complex formation step by an interaction between an analyte and a protein that can specifically recognize the analyte, a separation step of the complex, It has a color developing step in which a peracid, a reducing agent (CD) and a color forming material (Cp) are allowed to act to develop color. The temperature and time required for the complex formation step vary depending on the combination of the analyte to be used and the protein, but are generally 0 ° C. to 50 ° C. and 10 seconds to 1 hour. The organic peracid generated by mixing the organic peracid precursor and the peracid is preferably allowed to act on the composite within 1 hour, more preferably within 10 minutes. The temperature and time required for the color development step vary depending on the combination of the composite used, the reducing agent (CD) and the color development material (Cp), but are generally 5 ° C. to 60 ° C. and 30 seconds to 30 minutes.
In the separation process of the complex, a preferable process time is set for each separation method.
The determination time of color development is arbitrary, but it is desirable to select a time during which the color density is maintained high and color development is stable after the color development step. The color determination time is generally preferably 0 to 24 hours, more preferably 0 to 2 hours, and particularly preferably 0 to 30 minutes after the color forming step. If the time after the color development step is too long, discoloration or fading of the colored pigment occurs, which is not preferable.

In the present invention, as a means for amplifying and detecting an immunological reaction such as an antigen-antibody reaction, a redox reaction of a peroxide centered on a metal is used. The metal oxide-centered peroxide redox reaction is generally known. For example, US Pat. No. 4,414,305 discloses a processing method including a color developer and H ₂ O ₂ and processing with a developing assisting solution. Has been.

As the metal, gold, silver, platinum and alloys thereof, or silver halide (silver chloride, silver bromide, silver iodide, silver chlorobromide, silver iodobromide, silver chloroiodobromide, etc.) are used. . Moreover, the metal known to decompose | disassemble peroxides, such as tin and iron, can also be used. In the present invention, gold or silver is particularly preferable. Although the metal particle size is not particularly limited, the sphere equivalent particle size is preferably 1 nm to 1 μm, and more preferably 10 nm to 100 nm.
When a peroxide comes into contact with these metals, an active oxidation component is generated from the peroxide with the metal as a reaction center. The active oxidation component acts on the reducing agent (CD), oxidizes the CD, generates an oxidant (CDox), and regenerates the peroxide from the active oxidation component. Since the regenerated peroxide again generates an active oxidizing component, a series of cycles proceeds repeatedly. The oxidant (CDox) forms a dye by a coupling reaction of the color former (Cp). Since the oxidant (CDox) is amplified and formed by chain reaction, it is amplified to produce a dye. Therefore, the detection accuracy can be remarkably improved as a result of greatly amplifying a small amount of antibody by modifying the antibody with a metal label.

(Organic peracid precursor)
The organic peracid precursor used in the present invention is not particularly limited as long as it is a compound that reacts with a peroxide to produce an organic peracid. A preferred organic peracid precursor is a compound having an alkanoylamino group or an alkanoyloxy group.
The compound having an alkanoylamino group is preferably a compound represented by the following general formula (1), and the compound having an alkanoyloxy group is preferably a compound represented by the following general formula (2).

In General Formula (1) or (2), R ₁ is an alkyl group or an aryl group. As an alkyl group, Preferably it is a C1-C8 alkyl group, More preferably, it is a methyl group. The aryl group is a substituted or unsubstituted phenyl group, and the substituent is preferably an acyl group.
R ₂ and R ₃ each independently represent a hydrogen atom or a substituent, and may be bonded to each other to form a ring structure. As the substituent, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted acyl group is preferable. More preferably, they are bonded to each other to form a ring structure, and particularly preferably a group that forms an imidazole group together with a nitrogen atom.

R ₄ represents a hydrogen atom or a substituent. A hydrogen atom, a substituted or unsubstituted alkyl group, or a substituted or unsubstituted aryl group is preferable, and a hydrogen atom is particularly preferable.

  Although the specific example of the organic peracid precursor used for this invention is given to the following, this application is not necessarily limited to these compounds.

  The amount of the organic peracid precursor used is preferably from 0.1 mol to 1000 mol, more preferably from 1 mol to 100 mol, per 1 mol of peroxide.

(Peroxide)
As the peroxide of the present invention, an inorganic peroxide having a chemical formula of a metal salt of hydrogen peroxide, a peroxo acid compound, or an organic peroxide having a percarboxylic acid can be used. Compounds that release are preferred. As the compound that releases hydrogen peroxide, perboric acid, percarbonate and the like are preferable. Of these, hydrogen peroxide is particularly preferred. The amount used is preferably from the same number of moles to about 1 million times with respect to Cp that develops color. More preferably 10,000 times from the same number of moles.

(Reducing agent)
The reducing agent used in the present invention is a reducing agent capable of forming a dye by coupling its oxidant with a coloring material.

  One embodiment of a preferable reducing agent used in the present invention is a reducing agent represented by the following general formula (I).

In the formula, R ₁ , R ₂ , R ₃ and R ₄ each independently represent a hydrogen atom or a substituent, and R ₅ and R ₆ each independently represent an alkyl group, an aryl group, a heterocyclic group, an acyl group, or Represents a sulfonyl group, and R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₂ and R ₅ , and R ₄ and R ₆ are bonded to each other to form a 5-membered, 6-membered or A 7-membered ring may be formed.

Below, the compound represented by general formula (I) of this invention is demonstrated in detail. R ₁ , R ₂ , R ₃ and R ₄ each independently represents a hydrogen atom or a substituent. Examples of the substituent represented by R ₁ , R ₂ , R ₃ and R ₄ include a halogen atom, an alkyl group (including a cycloalkyl group and a bicycloalkyl group), and an alkenyl group (including a cycloalkenyl group and a bicycloalkenyl group). , Alkynyl group, aryl group, heterocyclic group, cyano group, hydroxyl group, nitro group, carboxyl group, alkoxy group, aryloxy group, silyloxy group, heterocyclic oxy group, acyloxy group, carbamoyloxy group, alkoxycarbonyloxy group, Aryloxycarbonyloxy, amino group (including anilino group), acylamino group, aminocarbonylamino group, alkoxycarbonylamino group, aryloxycarbonylamino group, sulfamoylamino group, alkyl and arylsulfonylamino group, mercapto group, alkyl Thio group, arylthio group, heterocyclic thio group, sulfamoyl group, sulfo group, alkyl and arylsulfinyl group, alkyl and arylsulfonyl group, acyl group, aryloxycarbonyl group, alkoxycarbonyl group, carbamoyl group, aryl and heterocyclic azo group Imide group, phosphino group, phosphinyl group, phosphinyloxy group, phosphinylamino group, and silyl group.

  More specifically, a halogen atom (for example, a chlorine atom, a bromine atom, an iodine atom), an alkyl group [represents a linear, branched, or cyclic substituted or unsubstituted alkyl group. An alkyl group (preferably an alkyl group having 1 to 30 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, t-butyl, n-octyl, eicosyl, 2-chloroethyl, 2-cyanoethyl or 2-ethylhexyl), cyclo An alkyl group (preferably a substituted or unsubstituted cycloalkyl group having 3 to 30 carbon atoms, such as cyclohexyl, cyclopentyl, or 4-n-dodecylcyclohexyl), a bicycloalkyl group (preferably a substituent having 5 to 30 carbon atoms) Alternatively, an unsubstituted bicycloalkyl group, that is, a monovalent group obtained by removing one hydrogen atom from a bicycloalkane having 5 to 30 carbon atoms, for example, bicyclo [1,2,2] heptan-2-yl, bicyclo [ 2,2,2] octane-3-yl), tricyclo structures with more ring structures, etc. It encompasses.

  An alkyl group (for example, an alkyl group of an alkylthio group) in the substituents described below also represents such an alkyl group. ], An alkenyl group [represents a linear, branched or cyclic substituted or unsubstituted alkenyl group. An alkenyl group (preferably a substituted or unsubstituted alkenyl group having 2 to 30 carbon atoms, such as vinyl, allyl, prenyl, geranyl, or oleyl), a cycloalkenyl group (preferably a substituted or unsubstituted group having 3 to 30 carbon atoms) That is, a monovalent group in which one hydrogen atom of a cycloalkene having 3 to 30 carbon atoms has been removed (eg, 2-cyclopenten-1-yl, 2-cyclohexen-1-yl), bicycloalkenyl A group (a substituted or unsubstituted bicycloalkenyl group, preferably a substituted or unsubstituted bicycloalkenyl group having 5 to 30 carbon atoms, that is, a monovalent group obtained by removing one hydrogen atom of a bicycloalkene having one double bond) For example, bicyclo [2,2,1] hept-2-en-1-yl, bicyclo [2,2 2] oct-2-en-4-yl)], an alkynyl group (preferably a substituted or unsubstituted alkynyl group having 2 to 30 carbon atoms, such as ethynyl, propargyl, trimethylsilylethynyl group), or an aryl group (Preferably a substituted or unsubstituted aryl group having 6 to 30 carbon atoms, such as phenyl, p-tolyl, naphthyl, m-chlorophenyl, or o-hexadecanoylaminophenyl), a heterocyclic group (preferably 5 or 6 members) A monovalent group obtained by removing one hydrogen atom from a substituted or unsubstituted aromatic or non-aromatic heterocyclic compound, more preferably a 5- or 6-membered aromatic group having 3 to 30 carbon atoms. Heterocyclic groups such as 2-furyl, 2-thienyl, 2-pyrimidinyl, or 2-benzothiazolyl), cyano groups, hydrides A xyl group, a nitro group, a carboxyl group, or an alkoxy group (preferably a substituted or unsubstituted alkoxy group having 1 to 30 carbon atoms such as methoxy, ethoxy, isopropoxy, t-butoxy, n-octyloxy, or 2 -Methoxyethoxy),

An aryloxy group (preferably a substituted or unsubstituted aryloxy group having 6 to 30 carbon atoms such as phenoxy, 2-methylphenoxy, 4-t-butylphenoxy, 3-nitrophenoxy, or 2-tetradecanoylamino; Phenoxy), a silyloxy group (preferably a silyloxy group having 3 to 20 carbon atoms, such as trimethylsilyloxy, t-butyldimethylsilyloxy), a heterocyclic oxy group (preferably a substituted or unsubstituted group having 2 to 30 carbon atoms) Heterocyclic oxy group, 1-phenyltetrazol-5-oxy, or 2-tetrahydropyranyloxy), acyloxy group (preferably formyloxy group, substituted or unsubstituted alkylcarbonyloxy group having 2 to 30 carbon atoms, carbon number 6 to 30 substituted or unsubstituted aryl A rubonyloxy group such as formyloxy, acetyloxy, pivaloyloxy, stearoyloxy, benzoyloxy, or p-methoxyphenylcarbonyloxy), a carbamoyloxy group (preferably a substituted or unsubstituted carbamoyloxy group having 1 to 30 carbon atoms) , For example, N, N-dimethylcarbamoyloxy, N, N-diethylcarbamoyloxy, morpholinocarbonyloxy, N, N-di-n-octylaminocarbonyloxy, or Nn-octylcarbamoyloxy), alkoxycarbonyloxy group (Preferably, a substituted or unsubstituted alkoxycarbonyloxy group having 2 to 30 carbon atoms such as methoxycarbonyloxy, ethoxycarbonyloxy, t-butoxycarbonyloxy, or n-octylca Bonyloxy), an aryloxycarbonyloxy group (preferably a substituted or unsubstituted aryloxycarbonyloxy group having 7 to 30 carbon atoms, such as phenoxycarbonyloxy, p-methoxyphenoxycarbonyloxy, or pn-hexadecyloxy Phenoxycarbonyloxy),

An amino group (preferably an amino group, a substituted or unsubstituted alkylamino group having 1 to 30 carbon atoms, a substituted or unsubstituted anilino group having 6 to 30 carbon atoms, such as amino, methylamino, dimethylamino, anilino, N-methyl-anilino or diphenylamino), acylamino group (preferably formylamino group, substituted or unsubstituted alkylcarbonylamino group having 1 to 30 carbon atoms, substituted or unsubstituted arylcarbonyl group having 6 to 30 carbon atoms) An amino group such as formylamino, acetylamino, pivaloylamino, lauroylamino, benzoylamino, or 3,4,5-tri-n-octyloxyphenylcarbonylamino), an aminocarbonylamino group (preferably having 1 to 30 carbon atoms) Substituted or unsubstituted aminocar Nylamino, such as carbamoylamino, N, N-dimethylaminocarbonylamino, N, N-diethylaminocarbonylamino, or morpholinocarbonylamino), an alkoxycarbonylamino group (preferably a substituted or unsubstituted alkoxycarbonylamino having 2 to 30 carbon atoms) Groups such as methoxycarbonylamino, ethoxycarbonylamino, t-butoxycarbonylamino, n-octadecyloxycarbonylamino, or N-methyl-methoxycarbonylamino), aryloxycarbonylamino groups (preferably having 7 to 30 carbon atoms) A substituted or unsubstituted aryloxycarbonylamino group, for example, phenoxycarbonylamino, p-chlorophenoxycarbonylamino, or mn-octyloxyphenoxy Boniruamino),

Sulfamoylamino group (preferably a substituted or unsubstituted sulfamoylamino group having 0 to 30 carbon atoms, such as sulfamoylamino, N, N-dimethylaminosulfonylamino, or Nn-octylaminosulfonyl Amino), alkyl and arylsulfonylamino groups (preferably substituted or unsubstituted alkylsulfonylamino having 1 to 30 carbon atoms, substituted or unsubstituted arylsulfonylamino having 6 to 30 carbon atoms, such as methylsulfonylamino, butylsulfonyl) Amino, phenylsulfonylamino, 2,3,5-trichlorophenylsulfonylamino, or p-methylphenylsulfonylamino), mercapto group, alkylthio group (preferably a substituted or unsubstituted alkylthio group having 1 to 30 carbon atoms, such as Tilthio, ethylthio, or n-hexadecylthio), arylthio group (preferably a substituted or unsubstituted arylthio group having 6 to 30 carbon atoms, such as phenylthio, p-chlorophenylthio, or m-methoxyphenylthio), heterocyclic thio group (Preferably a substituted or unsubstituted heterocyclic thio group having 2 to 30 carbon atoms, such as 2-benzothiazolylthio, 1-phenyltetrazol-5-ylthio), a sulfamoyl group (preferably a substituent having 0 to 30 carbon atoms) Or an unsubstituted sulfamoyl group such as N-ethylsulfamoyl, N- (3-dodecyloxypropyl) sulfamoyl, N, N-dimethylsulfamoyl, N-acetylsulfamoyl, N-benzoylsulfamoyl, N- (N′-phenylcarbamoyl) sulfamoy ), A sulfo group, an alkyl and arylsulfinyl group (preferably a substituted or unsubstituted alkylsulfinyl group having 1 to 30 carbon atoms, a substituted or unsubstituted arylsulfinyl group having 6 to 30 carbon atoms, such as methylsulfinyl, ethylsulfinyl, Phenylsulfinyl or p-methylphenylsulfinyl),

Alkyl and arylsulfonyl groups (preferably substituted or unsubstituted alkylsulfonyl groups having 1 to 30 carbon atoms, substituted or unsubstituted arylsulfonyl groups having 6 to 30 carbon atoms, such as methylsulfonyl, ethylsulfonyl, phenylsulfonyl, or p) -Methylphenylsulfonyl), acyl group (preferably formyl group, substituted or unsubstituted alkylcarbonyl group having 2 to 30 carbon atoms, substituted or unsubstituted arylcarbonyl group having 7 to 30 carbon atoms, such as acetyl, pivaloyl, 2-chloroacetyl, stearoyl, benzoyl, or pn-octyloxyphenylcarbonyl), an aryloxycarbonyl group (preferably a substituted or unsubstituted aryloxycarbonyl group having 7 to 30 carbon atoms, such as phenoxycarbonyl, o - B phenoxycarbonyl, m- nitrophenoxy carbonyl, or p-t-butyl-phenoxycarbonyl),

An alkoxycarbonyl group (preferably a substituted or unsubstituted alkoxycarbonyl group having 2 to 30 carbon atoms, such as methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, or n-octadecyloxycarbonyl), a carbamoyl group (preferably having a carbon number) 1 to 30 substituted or unsubstituted carbamoyl such as carbamoyl, N-methylcarbamoyl, N, N-dimethylcarbamoyl, N, N-di-n-octylcarbamoyl, N- (methylsulfonyl) carbamoyl), aryl and hetero A cyclic azo group (preferably a substituted or unsubstituted arylazo group having 6 to 30 carbon atoms, a substituted or unsubstituted heterocyclic azo group having 3 to 30 carbon atoms, such as phenylazo, p-chlorophenylazo, or 5-ethylthio- 1,3,4 Asiazol-2-ylazo), imide group (for example, N-succinimide, N-phthalimide), phosphino group (preferably a substituted or unsubstituted phosphino group having 2 to 30 carbon atoms, such as dimethylphosphino, diphenylphosphine) Fino, or methylphenoxyphosphino), a phosphinyl group (preferably a substituted or unsubstituted phosphinyl group having 2 to 30 carbon atoms, such as phosphinyl, dioctyloxyphosphinyl, or diethoxyphosphinyl), phosphinyl An oxy group (preferably a substituted or unsubstituted phosphinyloxy group having 2 to 30 carbon atoms such as diphenoxyphosphinyloxy or dioctyloxyphosphinyloxy), a phosphinylamino group (preferably C2-C30 substituted or non-substituted photo A finylamino group such as dimethoxyphosphinylamino, dimethylaminophosphinylamino), a silyl group (preferably a substituted or unsubstituted silyl group having 3 to 30 carbon atoms such as trimethylsilyl, t-butyldimethylsilyl, or Phenyldimethylsilyl).

When the group represented by R ₁ to R ₄ is a group capable of being further substituted, the group represented by R ₁ to R ₄ may further have a substituent group, preferred substituents in this case is R ₁ ~R represent the same meanings of the group as the substituent described in the explanation of _4. When substituted with two or more substituents, these substituents may be the same or different.

R ₅ and R ₆ each independently represents an alkyl group, an aryl group, a heterocyclic group, an acyl group, an alkylsulfonyl group, or an arylsulfonyl group, and the alkyl group, aryl group, heterocyclic group, acyl group, alkylsulfonyl group The preferred range of the group and the arylsulfonyl group includes the alkyl group, aryl group, heterocyclic group, acyl group, alkylsulfonyl group, and arylsulfonyl group described for the substituent represented by R _{1 to} R ₄ above. Represents a group having the same meaning. When the group represented by R ₅ and R ₆ is a group capable of being further substituted, the group represented by R ₅ and R ₆ may further have a substituent group, preferred substituents in this case is R ₁ ~R represent the same meanings of the group as the substituent described in the explanation of _4. When substituted with two or more substituents, these substituents may be the same or different.

R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₂ and R ₅ , and / or R ₄ and R ₆ are bonded to each other to form a 5-membered, 6-membered or 7-membered ring. May be.

Below, the preferable range of the compound represented by general formula (I) is demonstrated. R _{1 to} R ₄ are hydrogen atom, halogen atom, alkyl group, aryl group, acylamino group, alkyl and arylsulfonylamino group, alkoxy group, aryloxy group, alkylthio group, arylthio group, acyl group, alkoxycarbonyl group, aryloxy A carbonyl group, a carbamoyl group, a cyano group, a hydroxyl group, a carboxyl group, a sulfo group, a nitro group, a sulfamoyl group, an alkylsulfonyl group, an arylsulfonyl group, and an acyloxy group are preferred, and a hydrogen atom, a halogen atom, an alkyl group, an acylamino group, Alkyl and arylsulfonylamino groups, alkoxy groups, alkylthio groups, arylthio groups, alkoxycarbonyl groups, carbamoyl groups, cyano groups, hydroxyl groups, carboxyl groups, sulfo groups, nitro groups, sulfamoys Group, an alkylsulfonyl group, or an arylsulfonyl group. Particularly preferably, among R _{1 to} R ₄ , either R ₁ or R ₃ is a hydrogen atom.

R ₅ and R ₆ are preferably an alkyl group, an aryl group, and a heterocyclic group, and most preferably an alkyl group.

  Specific examples of the compound represented by formula (I) in the present invention are shown below, but the present invention is not limited thereto.

  The oxidation potential of the compound represented by the general formula (I) in water at pH 10 is preferably 5 mV or less (vs SCE) from the viewpoint of achieving both color developability and storage stability.

(Reducing agent addition method)
In the present invention, the reducing agent can be used as an aqueous solution or a solution dissolved in an organic solvent, an emulsified dispersion, or a solid dispersion.

  The number average particle size when used in the fine particle dispersion is preferably 0.001 μm to 5 μm, and more preferably 0.001 μm to 0.5 μm.

  The preferable range of the amount of the reducing agent used in the present invention is preferably from the same mole to 1000 times mole, more preferably from the same mole to 100 times mole, with respect to 1 mole of the coloring material used.

(Coloring material)
The coloring material that can be used in the present invention is preferably a compound known as a coupler in color photographic applications.
The coupler may have any structure as long as it is a compound capable of forming a dye having absorption in the visible region by coupling with the oxidized oxidant of the reducing agent of the present invention. Such compounds are well known in color photographic systems, and typical examples thereof include pyrrolotriazole couplers, phenol couplers, naphthol couplers, pyrazolotriazole couplers, pyrazolone couplers, acylacetanilide couplers, and the like. Can be mentioned.

  Preferred couplers in the present invention are represented by the general formulas (C-1), (C-2), (C-3), (M-1), (M-2), (M-3), (Y-1), A coupler having a structure represented by (Y-2) or (Y-3).

In the formula, X ₁ represents a hydrogen atom or a leaving group, Y ₁ and Y ₂ represent an electron-withdrawing substituent, and R ₁ represents an alkyl group, an aryl group, or a heterocyclic group.

In the formula, X ₂ represents a hydrogen atom or a leaving group, R ₂ represents an acylamino group, a ureido group or a urethane group, R ₃ represents a hydrogen atom, an alkyl group or an acylamino group, and R ₄ represents a hydrogen atom or a substituted group Represents a group. R ₃ and R ₄ may be connected to each other to form a ring.

In the formula, X ₃ represents a hydrogen atom or a leaving group, R ₅ represents a carbamoyl group or a sulfamoyl group, and R ₆ represents a hydrogen atom or a substituent.

In the formula, X ₄ represents a hydrogen atom or a leaving group, R ₇ represents an alkyl group, an aryl group or a heterocyclic group, and R ₈ represents a substituent.

In the formula, X ₅ represents a hydrogen atom or a leaving group, R ₉ represents an alkyl group, an aryl group or a heterocyclic group, and R ₁₀ represents a substituent.

In the formula, X ₆ represents a hydrogen atom or a leaving group, R ₁₁ represents an alkyl group, an aryl group, an acylamino group or an anilino group, and R ₁₀ represents an alkyl group, an aryl group or a heterocyclic group.

In the formula, X ₇ represents a hydrogen atom or a leaving group, R ₁₃ represents an alkyl group, an aryl group, or an indoleenyl group, and R ₁₄ represents an aryl group or a heterocyclic group.

In the formula, X ₈ represents a hydrogen atom or a leaving group, Z represents a divalent group necessary to form a 5- to 7-membered ring, and R ₁₅ represents an aryl group or a heterocyclic group.

In the formula, X ₉ represents a hydrogen atom or a leaving group, R ₁₆ , R ₁₇ and R ₁₈ each represent a substituent, n represents an integer of 0 to 4, and m represents an integer of 0 to 5. When n or m is 2 or more, the plurality of R ₁₆ and R ₁₇ may be the same group or different groups.

In General Formula (C-1), X ₁ represents a hydrogen atom or a leaving group, Y ₁ and Y ₂ represent an electron-withdrawing substituent, and R ₁ represents an alkyl group, an aryl group, or a heterocyclic group. These groups may have a substituent.
X ₁ is a hydrogen atom or a leaving group, preferably a leaving group.
The leaving group referred to in the present invention means a group capable of leaving from the mother nucleus when it is coupled with an oxidant of a reducing agent to form a dye. Examples of the leaving group include a halogen atom, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an acyloxy group, a carbamoyloxy group, an imide group, a methylol group, and a heterocyclic group. X1 is more preferably a carbamoyloxy group or a benzoyloxy group. Y ₁ and Y ₂ represent an electron withdrawing group. Specific examples include cyano group, nitro group, acyl group, oxycarbonyl group, carbamoyl group, sulfonyl group, sulfoxide group, oxysulfonyl group, sulfamoyl group, heterocyclic group, trifluoromethyl group, and halogen atom. Among these, a cyano group, an oxycarbonyl group, and a sulfonyl group are preferable, and a cyano group and an oxycarbonyl group are more preferable. It is more preferable that either Y ₁ or Y ₂ is a cyano group, and it is particularly preferable that Y ₁ is a cyano group. Y ₂ is preferably an oxycarbonyl group, particularly preferably an oxycarbonyl group substituted with a bulky group (for example, 2,6-di-t-butyl-4-methylpiperazinyloxycarbonyl group). R ₁ is preferably an alkyl group or an aryl group, and these groups optionally have a substituent. The alkyl group is preferably a secondary or tertiary alkyl group, more preferably a tertiary alkyl group. As an alkyl group, it is preferable that the sum of carbon number is the range of 3-12, and it is more preferable that it is the range of 4-8. The aryl group is preferably a phenyl group and may have a substituent, but the sum of the carbon numbers is preferably in the range of 6 to 16, more preferably in the range of 6 to 12. The coupler of general formula (C-1) preferably has a molecular weight of 700 or less, more preferably 650 or less, and even more preferably 600 or less.

In General Formula (C-2), X ₂ represents a hydrogen atom or a leaving group, R ₂ represents an acylamino group, a ureido group, or a urethane group, R ₃ represents a hydrogen atom, an alkyl group, or an acylamino group, R ₄ Represents a hydrogen atom or a substituent. R ₃ and R ₄ may be connected to each other to form a ring.
X ₂ is a hydrogen atom or a leaving group similar to X ₁ , but X ₂ is preferably a halogen atom, an aryloxy group, an alkoxy group, an arylthio group or an alkylthio group, more preferably a halogen atom or an aryloxy group. R ₂ is preferably an acylamino group or a ureido group. R ₂ preferably has a total carbon number of 2 to 12, more preferably 2 to 8. R ₃ is preferably an alkyl group having 1 to 4 carbon atoms or an acylamino group having 2 to 12 carbon atoms, and more preferably an alkyl group having 2 to 4 carbon atoms or an acylamino group having 2 to 8 carbon atoms. R ₄ is preferably a halogen atom, an alkoxy group, an acylamino group or an alkyl group, more preferably a halogen atom or an acylamino group, and particularly preferably a chlorine atom. The coupler of the general formula (C-2) preferably has a molecular weight of 500 or less, more preferably 450 or less, and even more preferably 400 or less.

In general formula (C-3), X ₃ is a hydrogen atom or a leaving group similar to X ₁ , but X ₃ is preferably a halogen atom, an aryloxy group, an alkoxy group, an arylthio group, or an alkylthio group. A group or an alkylthio group is more preferable. R ₅ is preferably an acyl group, an oxycarbonyl group, a carbamoyl group or a sulfamoyl group, and more preferably a carbamoyl group or a sulfamoyl group. R ₅ is preferably a group having 1 to 12 carbon atoms, more preferably 2 to 10 carbon atoms. R ₆ is a hydrogen atom or a substituent, and the substituent is preferably an amide group, a sulfonamide group, a urethane group or a ureido group, more preferably an amide group or a urethane group. The substitution position is preferably the 5th or 8th position of the naphthol ring, and more preferably the 5th position. R ₆ is preferably a group having 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms. The coupler of the general formula (C-2) preferably has a molecular weight of 550 or less, more preferably 500 or less, and even more preferably 450 or less.

In general formula (M-1), X ₄ is a hydrogen atom or a leaving group similar to X ₁ , but X ₄ is preferably a halogen atom, an aryloxy group, an alkoxy group, an arylthio group, an alkylthio group, or a heterocyclic group. , A halogen atom, an aryloxy group, an arylthio group or a heterocyclic group is more preferable. The heterocyclic group is preferably an azole group such as a pyrazole group, an imidazole group, a triazole group, a tetrazole group, a benzimidazole group, or a benzotriazole group, and more preferably a pyrazole group. R ₇ is an alkyl group, an aryl group, or a heterocyclic group, and these groups optionally have a substituent. A secondary or tertiary alkyl group or an aryl group is preferred. The alkyl group is preferably a group having 2 to 14 carbon atoms, more preferably a group having 3 to 10 carbon atoms. The aryl group is preferably a group having 6 to 18 carbon atoms, more preferably a group having 6 to 14 carbon atoms. R ₈ is preferably an alkyl group, an aryl group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, or a heterocyclic ring, and these groups optionally have a substituent. The alkyl group is preferably a secondary or tertiary alkyl group, more preferably a tertiary alkyl group. As an alkyl group, it is preferable that the sum of carbon number is the range of 3-12, and it is more preferable that it is the range of 4-8. The aryl group is preferably a phenyl group and may have a substituent, but the sum of the carbon numbers is preferably in the range of 6 to 16, more preferably in the range of 6 to 12. As an alkoxy group, a C1-C8 alkoxy group is preferable, More preferably, it is a C1-C4 alkoxy group. As the aryloxy group, an aryloxy group having 6 to 14 carbon atoms is preferable, and a group having 6 to 10 carbon atoms is more preferable. The alkylthio group and the arylthio group are preferably groups having the same carbon number as the alkoxy group and the aryloxy group, respectively. The coupler of general formula (M-1) preferably has a molecular weight of 600 or less, more preferably 550 or less, and even more preferably 500 or less.

The groups represented by X ₅ , R ₉ and R ₁₀ of the coupler of the general formula (M-2) are the same as the groups represented by X ₄ , R ₇ and R ₈ of the coupler of the general formula (M-1), respectively. The preferred range of each group is the same as that of the coupler of general formula (M-1).

In general formula (M-3), X ₆ is a hydrogen atom or a leaving group similar to X ₁ , but X ₆ is preferably an alkylthio group, an arylthio group or a heterocyclic group, more preferably an arylthio group or a heterocyclic ring. It is a group. The arylthio group is preferably a phenyl group, particularly preferably an arylthio group substituted with an alkoxy group or an amide group at the 2-position. The sum of the carbon number of the arylthio group is preferably in the range of 6 to 16, and more preferably in the range of 7 to 12. The heterocyclic group is preferably an azole group such as a pyrazole group, an imidazole group, a triazole group, a tetrazole group, a benzimidazole group, or a benzotriazole group, and more preferably a pyrazole group. R ₁₁ is preferably an alkyl group, an aryl group, an acylamino group or an anilino group, more preferably an acylamino group or an anilino group. Most preferred is an anilino group. The alkyl group is preferably an alkyl group having 1 to 8 carbon atoms, and the aryl group is preferably an aryl group having 6 to 14 carbon atoms. The acylamino group is preferably a group having 2 to 14 carbon atoms, and more preferably a group having 2 to 10 carbon atoms. The anilino group is preferably a group having 6 to 16 carbon atoms, and more preferably a group having 6 to 12 carbon atoms. As the substituent of the anilino group, a halogen atom and an acylamino group are preferable. The coupler of general formula (M-3) preferably has a molecular weight of 700 or less, more preferably 650 or less, and even more preferably 600 or less.

In the general formula (Y-1), X ₇ is a hydrogen atom or a leaving group similar to X ₁ , but X ₇ is preferably an aryloxy group, an imide group or a heterocyclic group. The aryloxy group is preferably an aryloxy group substituted with an electron withdrawing group. As the imide group, a cyclic imide group is preferable, and a hydantoin group, a 1,3-oxazolidine-2,5-dione group, and a succinimide group are particularly preferable. The total carbon number of the imide group is preferably in the range of 3 to 15, more preferably in the range of 4 to 11, and still more preferably in the range of 5 to 9. As the heterocyclic group, a pyrazole group, an imidazole group, a triazole group, a benzimidazole group, and a benzotriazole group are preferable, and an imidazole group is more preferable. The total number of carbon atoms of the azole group is preferably in the range of 3 to 12, more preferably in the range of 3 to 10, and still more preferably in the range of 3 to 8. R ₁₃ is preferably a secondary or tertiary alkyl group, an aryl group or a heterocyclic group. The alkyl group may be a cycloalkyl group or a bicycloalkyl group, and a tertiary alkyl group is more preferable. A 1-alkylcyclopropyl group, a bicycloalkyl group, and an adamantyl group are particularly preferable. R ₁₄ is an aryl group or a heterocyclic group, more preferably an aryl group. Of these, a phenyl group substituted with a halogen atom, an alkoxy group, an aryloxy group, an alkylthio group or an arylthio group at the 2-position is particularly preferred. The total carbon number of R ₁₄ is preferably in the range of 6-18, more preferably in the range of 7-16, and even more preferably in the range of 8-14. The coupler of general formula (Y-1) preferably has a molecular weight of 700 or less, more preferably 650 or less, and even more preferably 600 or less.

The groups represented by X ₈ and R ₁₅ of the coupler of the general formula (Y-2) are the same groups as the groups represented by X ₇ and R ₁₄ of the coupler of the general formula (Y-1), respectively. The preferred range of the group is the same as that of the coupler of the general formula (Y-1). Z represents a divalent group necessary for forming a 5- to 7-membered ring, and this ring may have a substituent, or another ring may be condensed.

Of the general formula (Y-2), a coupler represented by the general formula (Y-3) is preferable.
In the coupler of formula (Y-3), _{X 9} is a _{X 7} the same meaning as the general formula (Y-1), and preferred ranges are also the same. R ₁₆ is preferably a halogen atom, alkyl group, alkoxy group, acyl group, acyloxy group, acylamino group, alkoxycarbonyl group, sulfonamide group, cyano group, sulfonyl group, sulfamoyl group, carbamoyl group or alkylthio group, 4 is more preferable. n is preferably an integer of 0 to 3, more preferably 0 to 2, still more preferably 0 to 1, and most preferably 0. R ₁₇ is preferably the same group as R ₁₆ , more preferably a halogen atom, an alkyl group, an alkoxy group, an acylamino group, a sulfonamide group, an alkoxycarbonyl group, a sulfamoyl group or a sulfonyl group. In particular, it is more preferred that R ₁₇ which is a halogen atom, an alkoxy group or an alkylthio group is substituted in the ortho position with respect to the NH group. Most preferred is an alkylthio group. The coupler of general formula (Y-3) preferably has a molecular weight of 750 or less, more preferably 700 or less, and even more preferably 650 or less.
Specific examples of the coupler of the present invention are given below, but the present invention is not limited thereto.

  In addition to the above, the compound No. described in JP-A-2004-4439. CP101 to CP117, CP201 to CP220, CP301 to CP331, and the like can also be used in the present invention.

The coupler of the present invention can be used as an oil emulsion or a solid fine particle dispersion, for example, as a coating film using gelatin or polymer latex as a binder. The coating amount at this time is preferably 1 × 10 ⁻⁶ mol / m ² to 1 × 10 ⁻² mol / m ² , more preferably 1 × 10 ⁻⁵ mol / m ² to 1 × 10 ⁻³ mol. / M ² for use.

(Inactive carrier)
The inert carrier (substrate) used in the present invention does not diffuse into the membrane or the fixed layer containing the antibody protein to be carried, and does not prevent the generated complex from being fixed or diffused in the membrane or the fixed layer. Inert materials are preferred.
Examples of the inert carrier used in the present invention include nitrocellulose.
When the inert carrier used in the present invention serves as a background for observing the colored pigment, it is preferably a white light-reflective substrate, and the colored pigment is observed through the inert carrier. In this case, a transparent substrate is preferable.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

Example 1
1. Production of Colored Sheet Coupler (CP-1) 4.5 g, Color Image Stabilizer (ST-1) 1.3 g, Color Image Stabilizer (ST-2) 0.22 g, and Color Image Stabilizer (ST-3) 1 .4 g was dissolved in 6.7 g of the solvent (SOL-1), 6.7 g of the solvent (SOL-2) and 19 g of the solvent (ethyl acetate), and this liquid was dissolved in 25% by mass containing 1.0 g of sodium dodecylbenzenesulfonate. Was mixed with 84 mL of an aqueous gelatin solution, and emulsified and dispersed with a high-speed stirring emulsifier (dissolver).
An aqueous gelatin solution is added to the emulsified dispersion A, a gelatin crosslinking agent (H-1) is added, and a coating solution is prepared so as to have the following composition, which is coated on a triacetylcellulose (TAC) support. Dried. This was cut into a size of 5 mm × 18 mm to produce a coloring sheet.

(G / m ² )
CP-1 0.27
ST-1 0.079
ST-2 0.0133
ST-3 0.085
SOL-1 0.41
SOL-2 0.41
Gelatin 2.2
H-1 0.026

2. Preparation of hCG detection immunochromatography kit (2-1) Preparation of anti-hCG antibody-modified silver colloid 90 mL of a silver colloid solution having a diameter of 80 nm (product number: EM.SC80, manufactured by BBI) was centrifuged at 8000 G, 4 ° C. for 15 minutes (himacCF16RX, Hitachi) and concentrated to 9 mL by removing the upper fine. To the silver colloid solution adjusted to pH by adding 1 mL of 20 mM borate buffer (pH 9.0) to this silver colloid solution, 60 μg / mL anti-hCG monoclonal antibody (product name: Anti-hCG 5008 SP-5, Medix Biochemica) Manufactured, isoelectric point = 7.4) 1 mL of the solution was added and stirred. After standing for 10 minutes, 550 μL of a 1% by weight polyethylene glycol (PEG Mw. 20000, product number 168-11285, manufactured by Wako Pure Chemical Industries) aqueous solution was added and stirred, followed by 10% by weight bovine serum albumin (BSA Fraction V, product number A- 7906, SIGMA) aqueous solution (1.1 mL) was added and stirred. After centrifuging this solution at 8000 G, 4 ° C. for 30 minutes (himacCF16RX, Hitachi), the supernatant was removed leaving about 1 mL, and the silver colloid was redispersed with an ultrasonic cleaner. Thereafter, 20 mL of silver colloid preservation solution (20 mM trishydroxymethylaminomethane hydrochloride buffer (pH 8.2), 0.05 mass% PEG (Mw. 20000), 150 mM NaCl, 1 mass% BSA, 0.1 mass%) Disperse in NaN ₃ ), centrifuge again at 8000 G, 4 ° C. for 30 minutes, remove the supernatant, leaving about 1 mL, redisperse the silver colloid with an ultrasonic washer, and antibody-modified silver colloid (80 nm) solution Got.

(2-2) Preparation of silver colloid antibody retaining pad Each antibody-modified silver colloid prepared in (2-1) was mixed with a silver colloid coating solution (20 mM Tris-HCl buffer (pH 8.2), 0.05 mass% PEG ( Mw. 20000), 5% by mass sucrose) and water. At that time, the optical density was measured with monochromatic light of 455 nm under the condition of an optical path length of 1 cm, and diluted to 1.25. This solution is uniformly applied to each glass fiber pad (product name: Glass Fiber Conjugate Pad, manufactured by Millipore) 0.8 mm cut into 8 mm × 150 mm, and dried overnight under reduced pressure to obtain a silver colloid antibody holding pad. It was.

(2-3) Preparation of antibody-immobilized membrane (chromatographic carrier) A nitrocellulose membrane cut into 25 mm x 200 mm (with plastic lining, product name: HiFlow Plus HF120, manufactured by Millipore) is used to produce an antibody by the following method. An antibody-immobilized membrane was prepared by immobilization. The anti-hCG monoclonal antibody for immobilization (Product name: Anti-Alpha subunit 6601 SPR-5, Medix) prepared so as to be 0.5 mg / mL at a position 8 mm from the bottom of the membrane with the long side down. Biochemica, isoelectric point = 7.4) The solution was applied in a line shape having a width of about 1 mm using an ink jet type applicator (BioDot). Similarly, an anti-mouse IgG antibody for control (anti-mouse IgG (H + L), rabbit F (ab ′) 2, product number 656-70621, Wako Jun) prepared to be 0.5 mg / mL at a position of 12 mm from the bottom. The drug solution was applied in a line. The coated membrane was dried at 50 ° C. for 30 minutes with a hot air dryer. 500 mL of blocking solution (0.5% by mass casein (milk-derived, product number 030-01505, manufactured by Wako Pure Chemical Industries) containing 50 mM borate buffer (pH 8.5)) was placed in a vat and allowed to stand for 30 minutes. Thereafter, the sample was transferred to 500 mL of a washing / stabilizing solution (50 mM Tris-HCl (pH 7.5) buffer containing 0.5% by mass of sucrose and 0.05% by mass of sodium cholate) in a similar vat, and immersed in the same. Let stand for a minute. The membrane was removed from the solution and dried overnight at room temperature to obtain an antibody-immobilized membrane.

(2-4) Preparation of immunochromatography kit The antibody-immobilized membrane prepared in (1-3) was attached to a back adhesive sheet (ARcare9020, Nipple Technocluster). At that time, the anti-hCG antibody line side of the long side of the membrane is the lower side. The silver colloid antibody holding pad prepared in (2-2) is attached to the lower side of the antibody-immobilized membrane so as to overlap about 2 mm, and the sample addition pad (18 mm × 18 mm) is attached to the lower side of the silver colloid antibody holding pad so as to overlap about 4 mm. A glass fiber pad (product name: Glass Fiber Conjugate Pad, manufactured by Millipore) cut to 150 mm was laminated and pasted. Further, an absorption pad (cellulose glass film (product number: CF6, manufactured by Whatman)) cut to 80 mm × 150 mm) was overlaid on the upper side of the antibody-immobilized membrane so as to be overlapped by about 5 mm. By cutting these laminated members with a guillotine cutter (product number: CM4000, manufactured by NIPPN Technocluster Co., Ltd.) in parallel with the short side so that the long side of the member is 5 mm wide, it is 5 mm × 55 mm. An immunochromatographic strip was prepared. These were placed in a plastic case (Nippon Techno Cluster Co., Ltd.) to prepare an immunochromatography kit for testing.

(2-5) Preparation of Coloring Solution To 800 mL of an aqueous buffer solution consisting of 0.4 mL of 8N potassium hydroxide and 26.4 g of potassium bicarbonate, 75 g of N-acetylimidazole, 4 g of potassium sulfite and 3.2 g of 17D are added as an organic peracid precursor. Then, after dissolution, the solution was diluted with water so that the total liquid volume became 1000 mL to prepare a color developing solution.

3. Signal amplification at detection line (3-1) Adsorption treatment of antibody-modified metal colloid at detection line Dissolve hCG (recombinant hCG R-506, manufactured by Rohto Pharmaceutical Co., Ltd.) in PBS buffer containing 1% by mass BSA Then, as shown in Table 1, test hCG solutions having various concentrations were prepared. 100 μL of this test hCG solution was dropped onto the sample addition pad of each test immunochromatography kit and allowed to stand for 10 minutes.

(3-2) Signal amplification by color developing sheet and color developing solution After removing the membrane from the case of the immunochromatography kit after finishing the processing of (3-1), and further removing the water absorbing pad, 5 mm × 20 mm as a new water absorbing pad. Three pieces were pasted with cellophane tape on the place where (Product name: Cellulose Fiber Sample Pad, manufactured by Millipore) was removed. A color developing sheet was superimposed on the detection line portion, and the overlapped portion was sandwiched and fixed between two acrylic plates. A specimen dropping part (sample addition pad) is immersed in a microtube (BM equipment, product number: BM4020) in which 200 μL of a 30% by mass hydrogen peroxide solution mixed with 9 mL of coloring solution is added to this membrane. I stood up. At this time, the time from the mixing of the coloring solution and the hydrogen peroxide solution to the immersion of the sample addition pad was 5 minutes. The standing point was taken as 0 minutes, and it was taken out after 10 minutes. The coloring sheet was peeled off and immersed in a 1% acetic acid solution for 1 minute to stop the coloring reaction.

Example 2
In Example 1, the time from the mixing of the color developing solution and the hydrogen peroxide solution described in (3-2) to the immersion of the sample addition pad was set to 1 hour.

(Comparative Example 1)
The test was conducted in the same manner as in Example 1 except for N-acetylimidazole in Example (2-5).

(Comparative Example 2)
The test was conducted in the same manner as in Example 1 except that 75 g of N-acetylimidazole in the item (2-5) was replaced with 41 g of acetic acid in Example 1.

(Evaluation methods)
Visually observe the portion of the color developing sheet in contact with the membrane detection line obtained in Examples 1 and 2 and Comparative Examples 1 and 2 or the membrane detection line in a room illuminated with a fluorescent lamp of normal brightness. Then, the performance of each immunochromatography kit was evaluated. The evaluation criteria are shown below.
○: Blue (650 nm) color was observed, clearly distinguishable from the control (hCG concentration 0), and the presence of hCG in the specimen could be detected.
X: Indistinguishable from control.

  The obtained results are shown in Table 1.

As clearly shown in Table 1, it can be seen that a trace amount of antigen (hCG) can be detected by using the color developing sheet of the present invention (detection sensitivity has been remarkably improved).
Among them, the detection sensitivity was particularly high in Example 1 in which the time from the mixing of the color developing solution and the hydrogen peroxide solution to the immersion of the sample addition pad was short.

Claims (14)

An immunological detection method comprising:
a) A first protein (Y) capable of specifically recognizing an analyte in a specimen modified by a label containing a metal is allowed to act on the specimen or a sample containing the specimen, and the analyte and Y are Forming a complex comprising:
b) a step of separating the complex from the first protein (Y) not forming the complex;
c) a color developing step in which the organic peracid produced by the reaction of the organic peracid precursor and peroxide, and the color former act on the separated complex;
d) An immunological detection method comprising a step of detecting color development produced by the color development step.   The immunological detection method according to claim 1, wherein the organic peracid precursor is a compound having an alkanoylamino group or an alkanoyloxy group.   The immunological detection method according to claim 2, wherein the alkanoyl in the alkanoylamino group or alkanoyloxy group is acetyl.   The immunological detection method according to any one of claims 1 to 3, wherein the color development step is performed within 1 hour from the reaction between the organic peracid precursor and the peroxide.   The immunological detection method according to any one of claims 1 to 4, wherein the color former comprises a reducing agent and a color former.   The immunological detection method according to any one of claims 1 to 5, wherein the metal-containing label contains at least one of silver, gold, or platinum atoms.   The immunological detection method according to claim 6, wherein the label containing the metal is silver.   The immunological detection method according to claim 6, wherein the label containing the metal is silver halide. The immunological detection method according to any one of claims 5 to 8, wherein the reducing agent is a compound represented by the following general formula (I):

(Wherein R ₁ , R ₂ , R ₃ and R ₄ each independently represents a hydrogen atom or a substituent, and R ₅ and R ₆ each independently represents an alkyl group, an aryl group, a heterocyclic group, an acyl group, Or a sulfonyl group, which may have a substituent, and R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₂ and R ₅ , or R ₄ and R ₆ are bonded to each other. And may form a 5- to 7-membered ring). The coloring material is represented by the following general formulas (C-1), (C-2), (C-3), (M-1), (M-2), (M-3), (Y-1), ( The immunological detection method according to any one of claims 5 to 9, which is at least one compound selected from the group consisting of Y-2) and (Y-3):

(Wherein, X ₁ represents a hydrogen atom or a leaving group, Y ₁ and Y ₂ represent an electron-withdrawing substituent, and R ₁ represents an alkyl group, an aryl group, or a heterocyclic group);

(Wherein X ₂ represents a hydrogen atom or a leaving group, R ₂ represents an acylamino group, a ureido group or a urethane group, R ₃ represents a hydrogen atom, an alkyl group or an acylamino group, R ₄ represents a hydrogen atom, or Represents a substituent, and R ₃ and R ₄ may be linked to each other to form a ring);

(Wherein X ₃ represents a hydrogen atom or a leaving group, R ₅ represents a carbamoyl group or a sulfamoyl group, and R ₆ represents a hydrogen atom or a substituent);

(Wherein X ₄ represents a hydrogen atom or a leaving group, R ₇ represents an alkyl group, an aryl group or a heterocyclic group, and R ₈ represents a substituent);

(Wherein X ₅ represents a hydrogen atom or a leaving group, R ₉ represents an alkyl group, an aryl group or a heterocyclic group, and R ₁₀ represents a substituent);

(Wherein X ₆ represents a hydrogen atom or a leaving group, R ₁₁ represents an alkyl group, an aryl group, an acylamino group or an anilino group, and R ₁₂ represents an alkyl group, an aryl group or a heterocyclic group);

(Wherein X ₇ represents a hydrogen atom or a leaving group, R ₁₃ represents an alkyl group, an aryl group, or an indoleenyl group, and R ₁₄ represents an aryl group or a heterocyclic group);

(Wherein X ₈ represents a hydrogen atom or a leaving group, Z represents a divalent group necessary to form a 5- to 7-membered ring, and R ₁₅ represents an aryl group or a heterocyclic group.) ;

(In the formula, X ₉ represents a hydrogen atom or a leaving group, R ₁₆ , R ₁₇ and R ₁₈ each represent a substituent, n represents an integer of 0 to 4, and m represents an integer of 0 to 5. When n or m is 2 or more, the plurality of R ₁₆ and R ₁₇ may be the same group or different groups.   The step of forming the complex further includes a second protein (X) capable of specifically recognizing the analyte in the specimen, and the second protein (X) is converted into the analyte and the first analyte. The protein (Y) is allowed to act simultaneously or stepwise to form a complex containing X, the analyte and Y. 11. The immunological detection method according to Item.   The immunological detection method according to any one of claims 1 to 11, wherein the analyte is an antigen, and a protein capable of specifically recognizing the analyte is an antibody. .   The immunological detection method according to claim 12, wherein the antibody is a monoclonal antibody.   The immunological detection method according to any one of claims 11 to 13, wherein the second protein (X) is supported on an insoluble carrier.