JPH052018A - Oxygen immunity dyeing method - Google Patents

Abstract

PURPOSE:To provide an immunity dyeing method which is superior in stability and achieves clear and easy diagnosis and detection by using an aromatic amino compound selected from a group of orthophenylenediamine, paraphenylenediamine and 1,2,4-triaminobenzene, and a condensed phosphoric acid derivative. CONSTITUTION:An aromatic amino compound selected from a group of orthophenylenediamine, paraphenylenediamine and 1,2,4-triaminobenzene and, a condensed phosphoric acid derivative are used. The orthophenylenediamine, paraphenylenediamine and 1,2,4-triaminobenzene are chromagens form coloring matters when oxidized, for example, they may be hydrochloride or the like salt. For the oxygen, such one that oxidizes the chromagen and catalyzes a reaction to obtain an analyzing means is used, and oxygen peroxide is preferable. The coloring reagent is enough to include the chromagen to obtain the analyzing means when oxidized and, a condensed phosphoric acid derivative such as an ultra phosphoric acid alkali metallic salt, etc., or may include a peroxide substance such as hydrogen peroxide or the like, or peroxide oxygen.

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 biological composition medium, and more particularly to an enzyme immunostaining method for forming a dye that is poorly soluble in an organic solvent.

[0002]

BACKGROUND OF THE INVENTION Various analytical methods for detecting specific components such as biological components have been developed because it is necessary to perform clinical analysis reliably and easily. Of these methods, the most accurate method is to use a specific component and a substance that can specifically bind to it (hereinafter, a specific binding substance), such as an antigen and an antibody, a certain sugar chain and a lectin, and biotin. Avidin,
Methods using a specific binding reaction between protein A and IgG, hormone and receptor, enzyme and substrate, etc. are known.

In general, a specific binding substance (hereinafter referred to as a labeled substance) labeled with some kind of label is used, and the specific component can be measured by detecting the signal of the labeled substance which varies according to the specific component of interest. Done. In particular, a method of reacting a specific component directly or indirectly supported on a matrix medium 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 for transferring a biological component (specific component) of an electrophoresed protein from a gel onto a nitrocellulose membrane and supporting it, and reacting this with a label, for example, an antibody label, and detecting a signal, TLC plate A method of detecting a signal by reacting a labeled substance with a specific component such as a lipid developed on the above, a method of detecting a signal by reacting with complementary DNA labeled to DNA on a membrane, an immunohistological staining method, etc. Is.

In the immunohistological staining method, information such as the location and state of the target specific component on the tissue can be obtained. Radiolabels, fluorescent substances, luminescent substances, enzymes and the like have been conventionally used as labels for specific binding substances.
Among these, the use of radioisotopes has the drawbacks that the decay of radioactivity, disposal, exposure to radiation, or equipment requires enormous cost, and further requires a long time and complicated operations. Further, when using a fluorescent substance or a luminescent substance, a special device or equipment is 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. Conventionally, as such a labeling enzyme,
Peroxidase, alkaline phosphatase, β-galactosidase and the like have been used. In particular, peroxidase has been widely used because of its ease of use and stability.

[0006] The enzyme immunostaining method, particularly the immunohistostaining method for detecting and measuring the antigenic substances on the produced tissue and cells is a diagnostically useful method. As a method commonly used in the immunohistochemical staining method, the chromogen is oxidized by the oxidation catalytic action of peroxidase (POD) to produce a dye and stained, and then the sample is washed with ethanol and xylene for sample storage. Then, after dehydration, it is immersed in an encapsulant and encapsulated.

As a chromogen, diaminobenzidine (DA
B) and aminoethylcarbazole (AEC) are commonly used. When the above-mentioned DAB is used, the dye is polymerized and insoluble in an organic solvent, which is convenient for encapsulation, but it is a carcinogenic substance, and the color tone is yellow or brown, which is useful for detection and diagnosis. Have difficulty. 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, and washing, dehydration, and encapsulation operations are extremely difficult. Cumbersome operations are required using special reagents.

Regarding the general peroxidase enzyme immunostaining method, a phenol compound (JP-A 63-6462) or an active methylene compound (JP-A 63-462) is used in combination with an aromatic amine compound as a chromogen. 7
1654) is useful, but these dyes are also soluble in an organic solvent and have a problem in encapsulation and storage.

[0009]

DISCLOSURE OF THE INVENTION The first object of the present invention is to provide an enzyme immunostaining method having excellent stability. A second object of the present invention is to provide an enzyme immunostaining method which is clear and easy to diagnose and detect.

A third object of the present invention is to provide an enzyme immunostaining method which is free from the risk of carcinogenicity. A fourth object of the present invention is to provide an enzyme-linked immunostaining method with a simple encapsulation operation. The above-mentioned objects of the present invention are ortho-phenylenediamine, para-phenylenediamine, 1,2,4-
It is achieved by an enzyme immunostaining method characterized by using an aromatic amine compound selected from the group of triaminobenzene and a condensed phosphoric acid derivative.

Ortho-phenylenediamine, para-phenylenediamine, and 1,2,4-triaminobenzene are chromogens that produce dyes by being oxidized, and these are in the form of salts such as hydrochloride. It may be one. And, a phosphoric acid derivative having a phosphoric acid oxidation number of 5 in the condensed phosphoric acid derivative is particularly selected from the group of ultraphosphoric acid or ultraphosphate, and most preferably an alkali metal ultraphosphate (sodium). , Potassium, lithium) salts are preferred.

The enzyme (including a pseudoenzyme) is not particularly limited as long as it catalyzes a reaction that oxidizes a chromogen (aromatic amine compound) and provides an analysis means, but a peroxidase is preferable. A peroxidative substance is required for the oxidation reaction by a peroxidase or a peroxidative pseudoenzyme. The peroxide substance may be an inorganic or organic peroxide substance, for example, an organic peroxide substance, but hydrogen peroxide is preferable. As the peroxidase, for example, horseradish peroxidase, lactoperoxidase, myeloperoxidase, glutathione peroxidase, cytochrome C peroxidase or the like can be used, and as the peroxidative pseudoenzyme, for example, hemoglobin or iron,
Metals and metal compounds such as gold and silver are used.

It is sufficient that the color-forming reagent contains at least a chromogen that gives an analysis means by being oxidized and a condensed phosphoric acid derivative such as an alkali metal salt of ultraphosphoric acid. It may contain a peroxide substance or a peroxidase, and may further contain other substances such as a stabilizer, if necessary. The color-forming reagent includes various forms such as solid, powder and solution.The color-forming reagent in various forms such as solid, powder and solution is subjected to an appropriate operation such as dissolution in a solvent. As described above, it is possible to prepare a coloring reagent solution in the form of a solution that can be used in the detection system. The detection system targeted for stabilization is to cause an oxidation reaction of the chromogen by the enzyme or its pseudoenzyme,
It is a system that detects the degree by optical means,
By analyzing the results, it is possible to obtain the target information, for example, the substance to be analyzed in the sample corresponding to the enzyme activity. Generally, the oxidation reaction of the enzyme or the pseudoenzyme is carried out in a solution in which the chromogen is dissolved.

The color reagent solution or the solvent used in the detection system is preferably an aqueous solvent, but may be an organic solvent or a mixed solvent of an organic solvent and an aqueous solvent as long as it does not extremely inhibit the enzymatic oxidation reaction. . Generally water, optionally organic solvents such as methanol, ethanol, N, N-
A mixed solvent of dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, dioxane and an aqueous solvent is used. It is desirable that the solvent has a pH value suitable for the enzymatic reaction, and it is preferable that the solvent is prepared by using a commonly used buffer. When using a peroxidase, pH 3 to pH 9 is preferred.

In order to include a condensed phosphoric acid derivative such as an alkali metal salt of ultraphosphoric acid in the color reagent solution, it may be added to a solvent in which the chromogen is dissolved, and the chromogen is first dissolved in the solvent. It may be added, or the mixture of the chromogen and the condensed phosphoric acid derivative or the lyophilized product is simultaneously added to the solvent,
It may be dissolved. Further, the solution containing the chromogen and the solution containing the condensed phosphoric acid derivative may be mixed.

The concentration of the condensed phosphoric acid derivative contained in the color reagent solution or the detection system is determined in consideration of the degree of inhibition in the enzyme reaction and the degree of inhibition of non-enzymatic oxidation.
It is preferably 0.01 to 1000 mM, preferably 0.1 to 500 mM, more preferably 1 to 100 mM. The chromogen concentration is 0.1 to 50.
0 mM is preferred.

For example, a pH 3 to pH 9 buffer solution containing a chromogen at a concentration of 0.1 to 500 mM and a condensed phosphoric acid derivative at a concentration of 0.01 to 1000 mM is prepared to prepare a color reagent solution. The color reagent solution and the sample are mixed, the enzyme reaction is carried out at a temperature of 2 to 50 ° C., and after a certain period of time, the reaction is stopped by a treatment such as washing with water.
The chromogen is oxidized in response to the peroxidase activity in the sample, and the resulting material settles on the matrix medium and is optically detected. The produced dye is insoluble in organic solvents and can be applied to immunohistological staining.

In the present invention, the specific component to be measured is a substance or a group of substances that gives a specific binding 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 present invention is a substance that can specifically bind to a specific component or another specific binding substance, and can be appropriately selected according to the specific component to be measured. For example, protein, nucleic acid, hormone, lipid, complex carbohydrate, glycolipid, polysaccharide, enzyme, vitamin, antigen, antibody, lectin, protein A, avidin, biotin, receptor, coenzyme, enzyme substrate, toxin, complement And composites thereof and the like.

The enzyme immunostaining method of the present invention can be applied to membranes such as cellulose acetate and nitrocellulose, gel supports such as polyacrylamide, silica gel carriers such as TLC plates, and polysaccharides such as dextran and agarose. And its derivatives, plate-like or bead-like plastics, glass, metals, fibers, activated carbon, matrix media such as hydroxyapatite. As a matrix medium, a chemically active group (for example, −
SH, —NH ₂ , —COOH, etc.) are preferred. Further, the immunohistochemical staining method can be applied to the tissue itself.

[0020]

【Example】

Example 1 100 containing 0.02% hydrogen peroxide
mM citrate-phosphate buffer (pH 5.0) and 0.02
% Of 100mM ultra phosphoric acid containing hydrogen peroxide (manufactured by Kanto Chemical Co., Inc., N ₂ P ₄ O ₁₁₎ buffer (pH 5.0)
And were mixed in a desired amount, and then the chromogen was added and dissolved at 5 mg / 10 ml to prepare a color developing solution shown in Table 1 below.

Then, a PVDF (polyvinyl difluoride manufactured by Millipore) membrane on which 0.5 μg of peroxidase was blotted was dipped in each color-developing solution, reacted for 10 minutes and washed with water. Table 1 Coloring liquid Chromogen Citrate buffer / Ultraphosphate buffer A-1 o-phenylenediamine 1/0 A-2 〃 0.98 / 0.02 A-3 〃 0.8 / 0.2 A -4 〃 0/1 B-1 p-phenylenediamine 1/0 B-2 〃 0.98 / 0.02 B-3 〃 0.8 / 0.2 B-4 〃 0/1 C-1 1. 2.4-triaminobenzene 1/0 C-2 〃 0.98 / 0.02 C-3 〃 0.8 / 0.2 C-4 〃 0/1 As a result, A-1, B-1, When the color developing solution of C-1 was used, the produced dye dissolved in water, but the others did not dissolve, and a dye spot appeared.

[Example 2] A PVDF membrane blotted with serially diluted peroxidase was treated with the same A-
3, B-3, C-3 and comparative coloring solution D
After reacting for 0 minutes, it was washed with water. The comparative coloring liquid D is 3 mg.
4-chloro-1-naphthol was dissolved in 1 ml of methanol, and 0.024% hydrogen peroxide-containing Tris-HCl buffer (pH 7.4, containing 200 mM sodium chloride)
It was prepared by adding 5 ml.

As a result, 0.2 ng of peroxidase was detected in A-3 and B-3, and 0.1 ng of peroxidase was detected in C-3, whereas the color development of comparative color was detected. In liquid D, only 1 ng of peroxidase was detectable. Also, the obtained P
When the VDF film was sequentially immersed in 70% ethanol and pure ethanol, the dye spots using the color developing solutions A-3, B-3, and C-3 were retained, but the comparative color developing solution D was used. The dye spot dissolved and disappeared.

Example 3 A paraffin section of a gastric cancer tissue was blocked with a 10% goat serum / PBS solution according to a conventional method and then reacted with an anti-CEA mouse monoclonal antibody (Cosmo Bio Inc.). After 15 minutes, the plate was washed with PBS and further reacted with a peroxidase-labeled goat anti-mouse immunoglobulin antibody (Kappel).

After 15 minutes, the plate was washed with PBS and then reacted in the following coloring solution for 10 minutes. Coloring liquid A-5 o-phenylenediamine 2 mg / ml
Dissolve in a 100 mM ultraphosphate buffer (pH 5.0) containing 0.02% hydrogen peroxide so that the coloring solution C-5 1.2.4-triaminobenzene is 2 m.
10% containing 0.02% hydrogen peroxide to achieve g / ml
Dissolved in 0 mM ultraphosphate buffer (pH 5.0) Comparative coloring solution E 5 mg of 3-amino-9-ethylcarbazole was dissolved in 1 ml of DMF, and a tris-hydrochloric acid buffer solution containing 0.02% hydrogen peroxide ( pH 7.4) 9 ml
After the reaction for 10 minutes after adjustment, the product was washed with water, and a black-brown CEA-positive image was obtained in the case where the coloring solution A-5 and the coloring solution C-5 were used, and a red color was obtained in the case where the comparative coloring solution E was used. CEA
A positive image was visible under the microscope.

Then, the mixture was immersed in 70% ethanol three times, pure ethanol three times, and xylene three times to carry out a dehydration clearing operation. As a result, CEA was obtained in the case where the color developing solutions A-5 and C-5 were used. Although a positive image was seen, the CEA positive image disappeared in the case where the comparative color developing solution E was used.

[0027]

[Effect] The present invention is an enzyme immunostaining method which is excellent in stability, clear, easy to diagnose and detect, has no risk of carcinogenicity, and has a simple encapsulation operation.

Claims (2)

[Claims] 1. An enzyme immunostaining method comprising using an aromatic amine compound selected from the group consisting of orthophenylenediamine, paraphenylenediamine and 1,2,4-triaminobenzene and a condensed phosphoric acid derivative. 2. The enzyme immunostaining method according to claim 1, wherein the condensed phosphoric acid derivative is ultraphosphoric acid or ultraphosphate.