JPH052020A - Oxygen immunity dyeing method and immunity organization dyeing method - Google Patents

Abstract

PURPOSE:To establish a dyeing method which does not induce carcinoma and enables clear and high sensitivity detection and correct diagnosis by using a naphthosultam derivative represented by a specific formula and an aromatic primary amine compound as a chromagen. CONSTITUTION:According to this dyeing method, a naphthosultam derivative represented by a formula and an aromatic primary amine compound are used as the chromagen. In the formula, X is a hdryogen atom, a leaving atom or a leaving radical, and (m) indicates an integer 0-5. The oxygen is a labelling oxygen of the oxygen antibody method, preferably, peroxidase. A specific component to be measured is a substance or a group of substances from which a specific substance to be coupled with the specific component is obtained, e.g. protein, nucleic acid, hormone, etc. For the matrix medium able to be used to carry a generated pigment, a film of cellulose acetate or nitrocellulose, etc., is used.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzyme immunostaining method for a living tissue medium.

[0002]

BACKGROUND OF THE INVENTION Various analytical methods for detecting a specific component such as a biological component have been developed because it is necessary to perform clinical analysis reliably and easily. Among these methods, the most accurate methods include, for example, antigen and antibody, certain sugar chain and lectin, biotin and avidin, protein A and I.
Specific components and substances that can specifically bind to them, such as gG, hormones and receptors, enzymes and substrates, etc.
A method using a specific binding reaction with a specific binding substance) is known.

[0003] In general, a specific binding substance (hereinafter referred to as a labeled substance) labeled with some kind of label is used, and the specific signal is detected by detecting the signal of the labeled substance which changes according to the specific component of interest. The measurement of the components is performed. In particular, a method of reacting a specific component directly or indirectly supported on a support with a label, immobilizing the label as a complex of both, and detecting a signal from the label substantially corresponding to the specific component. Is used as appropriate. For example, a method in which a biological component (specific component) of an electrophoresed protein is transferred from a gel and carried on a nitrocellulose membrane, and this is reacted with a label, for example, an antibody label, to detect a signal, and developed on a TLC plate. A method of detecting a signal by reacting a labeled component with a specific component such as a lipid, a method of detecting a signal by reacting DNA with a labeled cDNA for the DNA on a membrane,
Alternatively, an immunohistological staining method or the like is used.

As the labeling substance for the specific binding substance, for example, a radioisotope, a fluorescent substance, a luminescent substance, an enzyme or the like is used. However, the radioisotope has the problems of decay of radioactivity, disposal of waste, exposure, exposure to equipment cost, and complicated operation. Also, when using a fluorescent substance or a luminescent substance,
Special operation and equipment are required.

On the other hand, when an enzyme is used, the operation is relatively simple, the produced dye can be easily visualized, and the specific component can be quantified. Incidentally, as such a labeling enzyme,
Peroxidase, alkaline phosphatase, β-galactosidase and the like have been used. Enzyme immunostaining,
In particular, the immunohistological staining method for detecting and measuring the antigenic substance on living tissues and cells is a diagnostically useful method. For example, in the immunohistological staining method, information such as the location and state of the target specific component on the tissue can be obtained.

In the immunohistochemical staining method, for example, the chromogen is oxidized by the oxidation catalytic action of peroxidase (POD) to produce a dye, which is then stained, and then stored.
The sample is washed with ethanol and xylene, dehydrated, and then immersed in an encapsulant to encapsulate the sample. By the way, diaminobenzidine (DAB) and aminoethylcarbazole (AEC) are used as chromogens.

However, when DAB is used, the chromosome is polymerized, is insoluble in an organic solvent, and is convenient for encapsulation, but it is a carcinogenic substance and its color tone is yellow to brown, which makes detection and diagnosis difficult. There is. When AEC is used, the dye is red, which is convenient in terms of color tone, but it is soluble in organic solvents such as ethanol and xylene.
Washing, dehydration, and encapsulation operations are extremely difficult, and complicated operations using special reagents are required.

Regarding general enzyme immunostaining methods,
It has been disclosed that it is useful to use a phenol compound (JP-A 63-6462) or an active methylene compound (JP-A 63-71654) in combination with an aromatic amine compound as a chromogen. However, these dyes are also soluble in organic solvents, which hinders encapsulation and storage.

[0009]

DISCLOSURE OF THE INVENTION A first object of the present invention is to provide an enzyme immunostaining method and an immunohistological staining method which are free from the risk of carcinogenicity. A second object of the present invention is to provide an enzyme immunostaining method and an immunohistochemical staining method which are clear and can be detected with higher sensitivity and which makes diagnosis more accurate.

[0010] A third object of the present invention is to provide an immunohistological staining method in which a dye that is hardly soluble in an organic solvent is produced and a simple encapsulation operation is possible. The above object of the present invention is achieved by an enzyme-linked immunostaining method characterized in that a naphthosartan derivative represented by the following general formula (I) and an aromatic primary amine compound are used as chromogens. .

General formula (I)

[0012]

[Chemical 5]

(Wherein, X represents a hydrogen atom, a leaving atom, or a leaving group, R represents a substituent, and m represents an integer of 0 to 5). This is achieved by an immunohistological staining method characterized in that a metal ion is allowed to act after the formation of a dye using a naphthosartan derivative represented by the general formula (I) and an aromatic primary amine compound.

General formula (I)

[0015]

[Chemical 6]

(Wherein, X represents a hydrogen atom, a leaving atom, or a leaving group, R represents a substituent, and m represents an integer of 0 to 5). General formula (I
It is achieved by an enzyme immunostaining method characterized in that a compound represented by I) and an aromatic primary amine compound are used. General formula (II)

[0017]

[Chemical 7]

(Wherein, X represents a hydrogen atom, a leaving atom, or a leaving group, R represents a substituent, and m represents an integer of 0 to 5). General formula (I
It is achieved by an immunohistochemical staining method characterized in that a metal ion is allowed to act after the formation of a dye using the compound represented by I) and an aromatic primary amine compound.

General formula (II)

[0020]

[Chemical 8]

(In the formula, X represents a hydrogen atom, a leaving atom, or a leaving group, R represents a substituent, and m represents an integer of 0 to 5.) The enzyme in the present invention is generally an enzyme. Labeled enzymes of the enzyme antibody method can be mentioned, but peroxidase is preferable. In the enzyme immunostaining method and the immunohistochemical staining method of the present invention, the specific component to be measured is a substance or a group of substances that gives a specific substance that specifically binds to the specific component. Examples thereof include proteins, nucleic acids, hormones, lipids, complex carbohydrates, glycolipids, polysaccharides, enzymes, vitamins, antigens, antibodies and the like.

The specific binding substance that can be used in the enzyme immunostaining method and the immunohistological staining method of the present invention is a substance that can specifically bind to a specific component or another specific binding substance, It can be appropriately selected according to the requirements. For example, proteins, nucleic acids, hormones, lipids, glycoconjugates, glycolipids, polysaccharides, enzymes, vitamins, antigens, antibodies, lectins,
Examples include protein A, avidin, biotin, receptors, coenzymes, enzyme substrates, toxins, complements and complexes thereof.

In the present invention, as a matrix medium which can be used for supporting the produced dye, a membrane such as cellulose acetate or nitrocellulose, a gel-like support such as polyacrylamide, a silica gel carrier such as TLC plate, dextran, agarose, etc. And polysaccharides thereof, plate-shaped or bead-shaped plastics, glass, metals, fibers, activated carbon, hydroxyapatite and the like. In the immunohistological staining method, the tissue itself can be used as the matrix medium.

The above general formula (I) used in the present invention and
And the leaving atom in the compound of the general formula (II),
Examples include halogen atoms such as chlorine and bromine atoms,
Also, as a leaving group, -OR₁, -OCOR₁, -OSO
₂R₁, -SR₁, -OCONHR₁, -OSO₂NH
R₁, -N (COR₁) R₂, -N (SO₂R₁)
R ₂, -N (R₁) R₂, -SCN, bonded at N atom
Examples thereof include a nitrogen-containing heterocycle. Here, R₁, R
₂Is a hydrogen atom, an aliphatic hydrocarbon residue (saturated, unsaturated,
Either linear or branched type may be used, preferably
Alkyl groups such as methyl and ethyl groups, allyl groups, etc.
Alkenyl group), alicyclic compound residue (eg 5, 6
Members, eg cyclopentyl or cyclohexyl
Groups), aryl groups (eg phenyl and naphthyl)
Groups) or heterocyclic residues (eg, pyridyl groups,
Piperazinyl group, quinolyl group, furyl group, thiazolyl
Group, benzimidazolyl group, benzthiazolyl group, etc.)
Is. Incidentally, these groups are, for example, a halogen atom and nitrite.
B group, cyano group, hydroxy group, keto group, carboxyl
Group, sulfo group, amino group, alkyl group, alkenyl group,
Even if it has a substituent such as an aryl group or an alkoxy group
good. Table of R in general formula (I) and general formula (II)
There are no particular restrictions on the substituents, but typical examples
Is an alkyl group, aryl group, anilino group, acyl group
Mino group, sulfonamide group, alkylthio group, aryl
Each group such as thio group, alkenyl group, cycloalkyl group
Can be mentioned. In addition, for example, a halogen atom, cyclo
Alkenyl group, alkynyl group, heterocycle, sulfonyl group,
Sulfanyl group, phosphonyl group, acyl group, carbamoy
Group, sulfamoyl group, cyano group, alkoxy group, sulfur group
Rufonyloxy group, aryloxy group, heterocyclic oxy
Group, siloxy group, acyloxy group, carbamoyloxy
Group, amino group, alkylamino group, imide group, ureido
Group, sulfamoylamino group, alkoxycarbonyl group
Mino group, aryloxycarbonylamino group, alkoxy
Cycarbonyl group, aryloxycarbonyl group, heterocycle
Thio group, thioureido group, carboxyl group, hydroxy
Group, mercapto group, nitro group, sulfonic acid group, etc.
To be In particular, alkylcarbamoyl substituted in the 2-position
Groups, arylcarbamoyl groups, acylamino groups are preferred
It is a good thing.

The following are some typical examples of the naphthsartan derivative represented by the general formula (I).

[0026]

[Chemical 9]

[0027]

[Chemical 10]

[0028]

[Chemical 11]

[0029]

[Chemical 12]

[0030]

[Chemical 13]

[0031]

[Chemical 14]

[0032]

[Chemical 15]

[0033]

[Chemical 16]

Among these compounds, for example, Exemplified Compounds-1 and -6 are synthesized as follows.

[0035]

[Chemical 17]

1,8-Naphthosultam (Exemplified Compound-
20 g of Aldrich Co., Ltd.) was dissolved in 150 ml of DMF. To this, under ice cooling, 14.5 g of N-chlorosuccinimide was added over about 1 hour, and the mixture was further stirred for 3 hours. The reaction solution was poured into 300 ml of ice water, and the precipitated solid was recrystallized with ethanol to obtain 13.6 g of Exemplified Compound-1 as light brown crystals.

13.6 g of this exemplified compound-1 was added to acetic acid 4
After dissolving in 0 ml, 40 ml of sulfuric acid was added, and further 4.2 ml of a nitric acid aqueous solution having a specific gravity of 1.36 was added dropwise over about 1 hour under ice cooling. After stirring for 4 hours, the reaction solution was poured into 400 ml of ice water, and the precipitated solid was recrystallized with a mixed solvent of ethanol / ethyl acetate to obtain 14.1 g of Intermediate 1.
Was obtained.

14.1 g of this intermediate 1 was added to methanol 3
Dissolve in 00 ml, and then palladium on carbon 0.7
0 g was added. Hydrogen gas was introduced into this until its absorption disappeared. After filtration of palladium / carbon, methanol was distilled off under reduced pressure to obtain 12.5 g of Intermediate 2. After dissolving 12.5 g of this intermediate 2 in 100 ml of pyridine, 5.2 g of acetic anhydride was gradually added dropwise. Furthermore, 2 at 15 ℃
After stirring for 1 hour, the mixture was stirred at 50 ° C. for 1 hour, and concentrated hydrochloric acid was added to neutralize. 200 ml of ethyl acetate was added thereto, washed twice with 100 ml of water, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The solid thus obtained was recrystallized from acetonitrile to obtain 8.2 g of pale yellow Exemplified Compound-6.

Further, some typical examples of the compound represented by the general formula (II) are shown below.

[0040]

[Chemical 18]

[0041]

[Chemical 19]

[0042]

[Chemical 20]

[0043]

[Chemical 21]

[0044]

[Chemical formula 22]

[0045]

[Chemical formula 23]

[0046]

[Chemical formula 24]

[0047]

[Chemical 25]

Examples of the aromatic primary amine compound which can be used in the present invention include o- or p-aminophenol compounds, o- or p-phenylenediamine compounds and salts thereof. Preferred are p-phenylenediamine compounds, which are represented by the following general formula (III). General formula (III)

[0049]

[Chemical formula 26]

In the formula (III), A and B represent a hydrogen atom or an alkyl group, A and B may form a heterocycle together with a nitrogen atom, and D, E, G and J represent a hydrogen atom and a halogen atom. Represents a hydroxy group, an amino group, an alkoxy group, an acylamide group, an arylsulfonamide group, an alkylsulfonamide group or an alkyl group. The alkyl group represented by A and B has 1 to 6 carbon atoms.
Those having 1 to 4 are particularly preferable.
Examples thereof include a methyl group, an ethyl group and a butyl group. These alkyl groups may have a substituent, and as the substituent, for example, a hydroxyl group, a ureido group, a tetrahydrofuryl group, a carboxyl group, a methanesulfonamide group, a sulfo group, a methoxy group, an ethoxy group,
Examples thereof include a methoxyethoxy group, a methoxyethoxyethoxy group and a methoxytetraethoxy group. Of these, a hydroxyl group and a methanesulfonamide group are preferred.

D, G and J are preferably a hydrogen atom, an alkoxy group, an alkylsulfonamide group or an arylsulfonamide group, more preferably a hydrogen atom. E is preferably a hydrogen atom, an alkyl group or an acylamido group, more preferably an alkyl group having 1 to 3 carbon atoms, particularly a methyl group. As the salt of the compound represented by the general formula (III), p-trienesulfonic acid,
Sulfonic acid, sulfinic acid, sulfuric acid ester, sulfamic acid, thiosulfuric acid S-ester, carboxylic acid, phosphoric acid ester, amidophosphoric acid, phosphoric acid, phosphorous acid ester, organic boron compound, salts of organic acids or inorganic acids such as hydrochloric acid and sulfuric acid Among them, p-triene sulfonate, hydrochloride and sulfate are particularly preferable.

The following are some typical examples of aromatic primary amine compounds. (3-1) N, N-diethyl-3-methyl-4-aminoaniline (3-2) N, N-diethyl-4-aminoaniline (3-3) N-carbamidomethyl-N-methyl-4- Aminoaniline (3-4) N-carbamidomethyl-N-tetrahydrofurfuryl-3-methyl-4-aminoaniline (3-5) N-ethyl-N-carboxymethyl-3-methyl-4-aminoaniline ( 3-6) N-carbamidomethyl-N-ethyl-3-methyl-4-aminoaniline (3-7) N-ethyl-N-tetrahydrofurfuryl-
3-Methyl-4-aminophenol (3-8) 3-acetylamino-4-aminodimethylaniline (3-9) N-ethyl-N-β-methanesulfonamidoethyl-4-aminoaniline (3-10) N-Ethyl-N-β-methanesulfonamidoethyl-3-methyl-4-aminoaniline (3-11) N-methyl-N-β-sulfoethyl-p-
Phenylenediamine (3-12) N-ethyl-N-hydroxyethyl-3-
Methyl-4-aminoaniline (3-13) N-ethyl-N- [2- (2-methoxyethoxy) ethyl] -3-methyl-4-aminoaniline (3-14) N-ethyl-N- {2 -[2- (2-Methoxyethoxy) ethoxy] ethyl} -3-methyl-4-
Aminoaniline (3-15) N-ethyl-N- [2- {2- [2- [2
-(2-Methoxyethoxy) ethoxy] ethoxy] ethoxy} ethyl] -3-methyl-4-aminoaniline (3-16) N, N-diethyl-3-methanesulfonamidoethyl-4-aminoaniline The above Salts of aromatic primary amine compounds are generally water soluble and can be readily dissolved in water or buffers.

In order to form and deposit the dye on the complex conjugate of the specific component and the peroxidase-labeled substance supported on the matrix medium, the support may be immersed in the color-developing solution. The coloring solution is prepared by dissolving hydrogen peroxide, an aromatic primary amine compound, and a chromogen (compounds represented by the general formulas (I) and (II)) in a buffer solution having an appropriate pH. It
The chromogen may be dissolved in a small amount of a hydrophilic organic solvent such as methanol, ethanol or DMF and added. The molar ratio of the aromatic primary amine compound to the naphthosultan derivative is suitably from 1: 100 to 100: 1, and particularly preferably from 1:10 to 10: 1.

After the pigment is sufficiently formed and deposited on the matrix medium by the enzymatic reaction, the unreacted substances are washed away to stop the reaction. Next, by immersing the dye in an aqueous solution containing metal ions, the metal ions are allowed to act, and the solubility of the dye in an organic solvent is reduced. The metal acting on the matrix medium carrying the dye is a polyvalent metal ion, preferably Fe (III) ³⁺ or Co (III) ³⁺ ion, which may be a complex ion.

In the case of immunohistological staining, dehydration with alcohol or xylene and clearing operation are then carried out. Then, the information about the dyes thus generated can be read visually or using a known method such as a spectrophotometer.

[0056]

【Example】

Example 1 A PVDF membrane (manufactured by Millipore) wetted with methanol was dot-blotted using a blotting device (Biorad) to quantify human IgG.

1% BSA-PBS at 37 ° C.
After blocking for a period of time, it was reacted with a peroxidase-labeled goat anti-human IgG antibody (produced by Kappel, 2000-fold diluted with 1% BSA-PBS) at 4 ° C for 1 hour, and then thoroughly washed with PBS. The membrane after the above-mentioned immunoreaction was immersed in the following color forming liquids A, C and D, and the color forming reaction was carried out at room temperature.

[Coloring liquid A] 15 mg of the naphthosartan derivative represented by the general formula (I) (Exemplified compound 1) was dissolved in 5 ml of methanol, and the aromatic primary amine compound (N-ethyl-N-β was added thereto. -Methanesulfonamidoethyl-
3-Methyl-4-aminoaniline, 3/2 sulfuric acid monohydrate)
50mM Tris-HCl buffer (20mg)
Containing 0 mM NaCl, pH 7.4, hereinafter TBS) 25
ml was further added, and 100 μl of 3% hydrogen peroxide solution was further added, which was designated as color developing solution A (invention).

[Coloring Liquid C] In Coloring Liquid A, 4-chloro- was used instead of the naphthosartan derivative (Exemplified Compound 1).
The same procedure is performed using 1-naphthol, and this is used as the coloring liquid C.
(Comparative example 1). [Coloring liquid D] Coloring liquid C was similarly used without using the aromatic primary amine compound, and this was designated as coloring liquid D (Comparative Example 2).

After 10 minutes of color reaction, the film was taken out of the color developing solution, washed with pure water, air-dried and visually evaluated. As a result, when the color developing solution of the present invention was used, it was possible to detect human IgG in an amount of 0.1 ng. On the contrary,
When the color developing solution of Comparative Example 1 was used, only 0.5 ng of human IgG could be detected, and when the color developing solution of Comparative Example 2 was used, 5 ng of human IgG could be detected. However, the detection sensitivity is far worse than that of the present invention.

Example 2 A paraffin section of human colon cancer prepared by a conventional method was used as a sample, which was deparaffinized and washed with PBS. Then, it was immersed in a 1% hydrogen peroxide-methanol solution for 30 minutes to remove the endogenous peroxidase activity, washed with PBS, and then blocked with 5% goat serum-PBS for 10 minutes.

After washing with PBS, rabbit anti-CEA antibody (manufactured by Cosmo Bio Inc., diluted 200-fold with PBS) and 3 at room temperature
The reaction was allowed for 0 minutes. After washing 3 times with PBS, after reaction with a peroxidase-labeled goat IgG Fab anti-rabbit IgG antibody (manufactured by Kappel, diluted 200-fold with PBS) at room temperature, color reaction was performed in the following color developing solutions B, C and E. . [Coloring liquid B] 15 mg of the naphthosartan derivative represented by the general formula (I) (Exemplified compound 2) was dissolved in 5 ml of methanol, and the aromatic primary amine compound (N-ethyl-
N-β-methanesulfonamidoethyl-3-methyl-4
-Aminoaniline, 3/2 sulfuric acid monohydrate (5 mg) dissolved in 50 mM Tris-HCl buffer (200 mM NaC
1-containing, pH 7.4, 25 ml of TBS below was added, and 100 μl of 3% hydrogen peroxide solution was further added to prepare a color developing solution B (invention).

[Coloring Liquid C] In Coloring Liquid B, 4-chloro- was used instead of the naphthosartan derivative (Exemplified Compound 2).
The same procedure is performed using 1-naphthol, and this is used as the coloring liquid C.
(Comparative example). [Coloring liquid E] 20 ml of TBS containing 0.01% hydrogen peroxide was added to 1 ml of DMF in which 10 mg of 3-aminoethyl-9-carbazole was dissolved, and this was used as a coloring liquid E (comparative example).

After the reaction for 10 minutes, the plate was washed 3 times with PBS and then with pure water, and then the following operation was performed. [Operation a] Still standing in pure water for 10 minutes [Operation b] 10% in 3% (W / V) ferric chloride aqueous solution [Operation c] 3% (W / V) 10% in hexaminecobalt chloride aqueous solution After each standing treatment for a minute, the sample was washed with pure water. As a result, all the samples with the color developing solution B showed a pale blue CEA positive image. The samples of color developing solutions C and E showed red and dark blue CEA positive images.

After the above operations a, b or c, dehydration and penetration operations for encapsulating each sample were performed. That is,
It was dipped in two tanks of 70% ethanol, two tanks of 95% ethanol, two tanks of pure ethanol, and then two tanks of xylene in order. The results are shown in Table-1. Table-1 Coloring liquid (chromogen) operation Dye image Coloring liquid B Operation b Hold present invention Coloring liquid B Operation c Hold present invention Coloring liquid C Operation a disappearance Comparative example Coloring liquid C Operation b disappearance Comparative example Coloring liquid C Operation c disappeared Comparative example Coloring liquid E Operation a disappearance Comparative example Coloring liquid E Operation b disappearance Comparative example Coloring liquid E Operation c disappearance Comparative example According to this, by using the chromogen of the present invention and further treating with a metal salt, an organic solvent resistant dye image can be formed, and a useful immunohistological staining method is provided. I know what I can do.

[Example 3] Using a blotting device (Bio-Rad) on a PVDF membrane (manufactured by Millipore) wetted with methanol, human IgG of which amount was varied was dot-blotted. This is 1% BSA-
After blocking with PBS at 37 ° C. for 1 hour, peroxidase-labeled goat anti-human IgG antibody (manufactured by Kappel,
Diluted 2000 times with 1% BSA-PBS) and 1 at 4 ° C
After reacting for a time, it was thoroughly washed with PBS.

The membrane after the above immunoreaction was treated with the following coloring solution A,
It was immersed in C and D, and the color reaction was carried out at room temperature. [Coloring liquid A] 15 mg of the compound represented by the general formula (II) (Exemplified Compound 1) is dissolved in 5 ml of methanol, and the aromatic primary amine compound (N-ethyl-N-β-methanesulfonamidoethyl) is added to the solution. 50 mM Tris-hydrochloric acid buffer (containing 200 mM NaCl, pH) in which 5 mg of -3-methyl-4-aminoaniline, 3/2 sulfuric acid monohydrate was dissolved
7.4 ml, hereinafter TBS), and then 100 μl of 3% hydrogen peroxide solution were added, and this was used as a coloring solution A (invention).
And

[Coloring Liquid C] The same procedure as in Coloring Liquid A was carried out by using 4-chloro-1-naphthol instead of Exemplified Compound 1 to obtain Coloring Liquid C (Comparative Example 1). [Coloring liquid D] Coloring liquid C was similarly used without using the aromatic primary amine compound, and this was designated as coloring liquid D (Comparative Example 2).

After 10 minutes of color development reaction, the film was taken out from the color developing solution, washed with pure water, air-dried and visually evaluated. As a result, when the color developing solution of the present invention was used, it was possible to detect human IgG in an amount of 0.1 ng. On the contrary,
When the color developing solution of Comparative Example 1 was used, only 0.5 ng of human IgG could be detected, and when the color developing solution of Comparative Example 2 was used, 5 ng of human IgG could be detected. However, the detection sensitivity is far worse than that of the present invention.

Example 4 A paraffin section of human colon cancer prepared by a conventional method was used as a sample, which was deparaffinized and washed with PBS. Then, it was immersed in a 1% hydrogen peroxide-methanol solution for 30 minutes to remove the endogenous peroxidase activity, washed with PBS, and then blocked with 5% goat serum-PBS for 10 minutes.

After washing with PBS, the rabbit anti-CEA antibody (manufactured by Cosmo Bio Inc., diluted 200-fold with PBS) was added at room temperature for 3 times.
The reaction was allowed for 0 minutes. After washing 3 times with PBS, after reaction with a peroxidase-labeled goat IgG Fab anti-rabbit IgG antibody (manufactured by Kappel, diluted 200-fold with PBS) at room temperature, color reaction was performed in the following color developing solutions B, C and E. . [Coloring liquid B] 15 mg of the compound represented by the general formula (II) (Exemplified compound 2) was dissolved in 5 ml of methanol, and the aromatic primary amine compound (N-ethyl-N-β-methanesulfonamidoethyl) was added to the solution. 50 mM Tris-hydrochloric acid buffer (containing 200 mM NaCl, pH) in which 5 mg of -3-methyl-4-aminoaniline, 3/2 sulfuric acid monohydrate was dissolved
7.4 ml (hereinafter, TBS) is added, and 100 μl of 3% hydrogen peroxide solution is further added, which is used as a coloring solution B (present invention).
And

[Coloring Liquid C] The same procedure as in Coloring Liquid B was performed using 4-chloro-1-naphthol instead of Exemplified Compound 2 to obtain Coloring Liquid C (Comparative Example). [Coloring liquid E] 20 ml of TBS containing 0.01% hydrogen peroxide was added to 1 ml of DMF in which 10 mg of 3-aminoethyl-9-carbazole was dissolved, and this was used as a coloring liquid E (comparative example).

After the reaction for 10 minutes, the plate was washed 3 times with PBS and then with pure water, and then the following operation was performed. [Operation a] Still standing in pure water for 10 minutes [Operation b] 10% in 3% (W / V) ferric chloride aqueous solution [Operation c] 3% (W / V) 10% in hexaminecobalt chloride aqueous solution After each standing treatment for a minute, the sample was washed with pure water. As a result, all the samples with the color developing solution B showed a pale blue CEA positive image. The samples of color developing solutions C and E showed red and dark blue CEA positive images.

After the above operations a, b or c, dehydration and penetration operations for encapsulating each sample were performed. That is,
It was dipped in two tanks of 70% ethanol, two tanks of 95% ethanol, two tanks of pure ethanol, and then two tanks of xylene in order. The results are shown in Table-2. Table-2 Coloring liquid (chromogen) operation Dye image Coloring liquid B Operation b Holding invention Coloring liquid B Operation c Holding invention Coloring liquid C Operation a disappearance Comparative example Coloring liquid C Operation b disappearance Comparative example Coloring liquid C Operation c disappeared Comparative example Coloring liquid E Operation a disappearance Comparative example Coloring liquid E Operation b disappearance Comparative example Coloring liquid E Operation c disappearance Comparative example According to this, by using the chromogen of the present invention and further treating with a metal salt, an organic solvent resistant dye image can be formed, and a useful immunohistological staining method is provided. I know what I can do.

[0075]

[Effects] According to the present invention, there is no risk of carcinogenicity, clear detection can be performed with higher sensitivity, and diagnosis can be made more accurate. Also, dyes that are hardly soluble in organic solvents are produced,
The encapsulation operation becomes simple.

Claims (4)

[Claims] 1. An enzyme immunostaining method, which comprises using a naphthosartan derivative represented by the following general formula (I) and an aromatic primary amine compound as a chromogen. General formula (I) (However, in the formula, X represents a hydrogen atom, a leaving atom, or a leaving group, R represents a substituent, and m represents an integer of 0 to 5.) 2. A metal ion is allowed to act after the formation of a dye using a naphthosartan derivative represented by the following general formula (I) and an aromatic primary amine compound as a chromogen. Immunohistochemical staining method. General formula (I) (However, in the formula, X represents a hydrogen atom, a leaving atom, or a leaving group, R represents a substituent, and m represents an integer of 0 to 5.) 3. An enzyme immunostaining method, wherein a compound represented by the following general formula (II) and an aromatic primary amine compound are used as chromogens. General formula (II): (However, in the formula, X represents a hydrogen atom, a leaving atom, or a leaving group, R represents a substituent, and m represents an integer of 0 to 5.) 4. A metal ion is allowed to act after the formation of a dye using a compound represented by the following general formula (II) as a chromogen and an aromatic primary amine compound. Immunohistochemistry. General formula (II): (However, in the formula, X represents a hydrogen atom, a leaving atom, or a leaving group, R represents a substituent, and m represents an integer of 0 to 5.)