JPH0669397B2 - Measurement method using peroxidase labeling - Google Patents

Description

Detailed Description of the Invention [Industrial applications]

The present invention relates to a method for measuring a specific component, and more particularly to a method for measuring a specific component using an enzymatic reaction of peroxidase.

2. Description of the Related Art Various analytical methods for detecting a specific component such as a biological component have been developed. The most accurate method among these methods is to bind the specific component and the specific binding thereto. Possible substances (hereinafter referred to as specific binding substances), such as antigens and antibodies, certain sugar chains and lectins, biotin and avidin, protein A and IgG, hormones and receptors, enzymes and substrates, etc. The method used is known. In general, a specific component is measured by using a specific binding substance having a label (hereinafter referred to as a labeled substance) and detecting a signal of the label which is changed according to the specific component. 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, TLC, which is a method for transferring a biological component (specific component) of an electrophoresed protein from a gel onto a nitrocellulose membrane and reacting it with a label such as an antibody label, and detecting a signal.
A method of detecting a signal by reacting a label with a specific component such as a lipid spread on a plate, a method of detecting a signal by reacting DNA with a labeled complementary DNA to the DNA, or immunohistochemical staining Such as the law. By these methods, not only the quantitative determination of the specific component and the reactivity of the specific component with the specific binding substance, but also a great deal of information on the property, the state of existence of the specific component or the specific binding substance can be obtained. For example, after electrophoresis, a signal is detected on a complex in which a specific component of a living body such as a protein or nucleic acid transferred or carried on a membrane, or a lipid component developed on TLC and a label for the specific component are bound. In the method, information such as the molecular weight, isoelectric point or polarity of the specific component can be obtained from the position and mobility of the signal of the specific component. Further, in the immunohistochemical staining method, information such as the location and state of the target specific component on the tissue can be obtained.

[Problems to be Solved by the Invention]

In the above-described measurement of the specific component, which substantially detects a signal according to the amount of the specific component on the complex conjugate directly or indirectly supported on the support, the label is labeled because the specific component of interest is a trace amount. It is essential that detection is performed with high sensitivity, and that the method for detecting the label has high resolution in order to obtain more information on a specific component. Radiolabels, fluorescent substances, luminescent substances, enzymes and the like are used as labels for specific binding substances. Radioisotopes have problems such as attenuation of radioactivity, disposal, exposure, and huge cost for installation, and further, there is a drawback that a complicated operation for detecting a signal on a label carried on a support is required. Fluorescent substances or luminescent substances require special equipment and facilities. On the other hand, when an enzyme is used, the operation is relatively simple, and the produced dye can be easily visualized and quantified. Conventionally, peroxidase, alkaline phosphatase, β-galactosidase and the like have been used as labeling enzymes. Peroxidase is mainly used as a labeling enzyme in the method of producing and depositing a dye by an enzymatic reaction on a complex conjugate supported on a support, and in that case, as a substrate,
Conventional diaminobenzidine, o-dianididine, 4-chloro-
1-naphthol and the like have been used. Diaminobenzidine and o-dianididine have the drawback of being highly toxic and easily causing a background. Although 4-chloro-naphthol is slightly more sensitive than others, it is not sensitive enough to measure smaller amounts of specific components or to obtain more information about specific components. Further, according to Japanese Patent Laid-Open No. 61-10772, it is described that when 4-methoxy-1-naphthol is used as a substrate, the analytical sensitivity and resolution are increased by one digit or more as compared with 4-chloro-1-naphthol. There is. However, this method is a special method for developing color in the gel support, and is insufficient in analysis sensitivity and resolution. Therefore, an object of the present invention is to provide a simple, high-sensitivity, high-resolution and rapid method for measuring a specific component.

[Means for solving problems]

As a result of various examinations in line with the above purpose, a specific component to be measured is measured by using a labeled substance having peroxidase as a label, and measuring the amount of a dye produced by an enzymatic reaction of peroxidase. In the method, the problem is solved by using hydrogen peroxide and 4-alkoxy-1-naphthol (wherein the alkoxy group is an ethoxy group, a propoxy group, an isopropoxy group) as a substrate for the enzymatic reaction. The purpose was achieved. Next, the present invention will be described in detail. In the present invention, the specific component may be directly supported on the support by physical adsorption, chemical bonding or the like, or may be indirectly supported via one or more specific binding substances. Alternatively, the specific component may be directly or indirectly supported on a support and then the label may be reacted to form the complex conjugate, or the complex conjugate may be formed and then the complex bond may be formed. The body may be supported directly or indirectly on a support. Further, the label forms a complex conjugate with the specific component,
Although carried on a support, the specific component and the label may be directly bound or may be bound via one or more other specific binding substances. Further, in the present invention, the labeled substance may be one in which the specific binding substance is overlapped and labeled with peroxidase and anti-peroxidase antibody. Hydrogen peroxide and 4-alkoxy-1-naphthol are used as substrates for the enzymatic reaction of peroxidase in the complex conjugate. Due to the action of peroxidase and hydrogen peroxide, 4-alkoxy-1-naphthol self-couples to form a dye. Analytical biochemistry (Analytic) using conventional hydrogen peroxide and 4-chloro-1-naphthol as substrates
al Biochemistry) 119 , 142-147 (1982), the method of the present invention shortened the color development time, the spot was clear, and the sensitivity was increased 10 times or more. As a result, the amount of peroxidase-labeled product can be reduced, which is advantageous in terms of cost. In the present invention, the specific component of interest is a substance or a group of substances from which a specific binding substance that specifically binds to the specific component is obtained. 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 the specific component or another specific binding substance, and can be appropriately selected according to the specific component. 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. As the support that can be used in the present invention, cellulose, acetate, a membrane such as nitrocellulose, a gel-like support such as polyacrylamide, a silica gel carrier such as a TLC plate,
Examples thereof include plate-shaped and bead-shaped plastics, glass, metal, fibers and the like. In the histochemical staining, the tissue itself can also be used as a support. The 4-alkoxy-1-naphthol used in the present invention can be synthesized according to the method described in Journal of Organic Synthetic Chemistry, Vol. 17, No. 12, PP27-30 (1959). In order to form the dye on the complex conjugate of the specific component and the peroxidase-labeled substance carried on the support, the support may be dipped in the substrate reagent for color development. Substrate reagent for color development is hydrogen peroxide, 4-alkoxy-
Prepared by dissolving 1-naphthol. 4-alkoxy-1-
Naphthol may be added after dissolving it in a small amount of a hydrophilic organic solvent such as methanol, ethanol or DMF. After the dye is formed on the support by the enzymatic reaction, the reaction is stopped by washing off the unreacted materials. Information about the dye formed can be read visually or using methods known in the art, such as a spectrophotometer. Alternatively, the information may be read after adding an appropriate organic solvent to dissolve the dye.

【Example】

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited by the Examples. Example 1 Measurement of Antigen on Nitrocellulose Membrane: After washing with pure water, air-dried nitrocellulose membrane (Bio-Rad; pore size 0.45 μm) was treated with a phosphate buffer (hereinafter referred to as PBS) 1 μ of diluted goat IgG was spotted. After air drying, 4 with 1% bovine serum albumin (BSA) -PBS solution
Blocked overnight at ℃, then peroxidase-labeled rabbit anti-goat IgG antibody (Kappel; 1% BSA-PSA)
The solution was diluted 1500 times with the solution) and reacted at 4 ° C. for 2 hours. It was washed 5 times with 0.05% Tween 20 (polyoxyethylene sorbitan monolaurate; manufactured by Wako Pure Chemical Industries, Ltd.)-PBS solution and immersed in a substrate reagent for color development. The following three types were used as the substrate reagents for color development for comparison. (1): Dissolve 3 mg of 4-chloro-1-naphthol in 1 ml of methanol, and add 5 ml of 0.05M Tris-HCl buffer (pH7.4,200m).
MNacl-containing; hereinafter referred to as TBS) is added, and further 20% of 3% hydrogen peroxide is added. (2): Dissolve 3 mg 4-methoxy-1-naphthol in 0.5 ml DMF, add 5.5 ml TBS, and add 3% hydrogen peroxide 20μ
Add. (3); 3 mg of 4-ethoxy-1-naphthol was dissolved in 0.5 ml of DMF, 5.5 ml of TBS was added, and 3% hydrogen peroxide 20 μm was added.
Add. The color-developing substrate reagent solution (2) was colored blue over time, but the color-developing substrate reagent solution (3) of the present invention was not colored and was stable. After reacting for 15 minutes, it was thoroughly washed with water and air dried. When the substrate reagent for color development (1) was used, the spot was gray purple, and the detection limit of goat IgG that was the antigen was 10 ng. When the substrate reagent for color development (2) was used, the spot was blue and the detection limit was Is 1n
It was g. On the other hand, when the color-developing substrate reagent solution (3) of the present invention was used, the spot was a bright blue color and had a high resolution. The detection limit was less than 0.5 ng. Also,
In (3), instead of 4-ethoxy-1-naphthol
4-propoxy-1-naphthol or 4-isopropoxy-1-
The same high resolving power and sensitivity were exhibited when naphthol was used. Example 2: Screening of antibodies and hybridomas: Balb / C mice (female, 6 weeks old) were intraperitoneally injected with 50 μg of α ₁ -antitrypsin together with Freund's complete adjuvant. 3 weeks later, further α ₁ -antitrypsin 50μ
g was injected intraperitoneally with Freund's incomplete adjuvant, and 2 weeks later, 30 μg of α ₁ -antitrypsin was added to PB.
Three days after the final immunization, the spleen cells were dissolved in S and injected intravenously, and the spleen cells were taken out and mouse myeloma cells x 63.
Merged with 6.5.3. It was distributed to 5 96-well plates and cultured in HAT selection medium. Three weeks after the fusion, the wells in which hybridoma colonies were formed were measured for antibodies by the following method. The nitrocellulose was cut into squares of 4 mm square, and each spot was spotted with 1 μ of α ₁ -antitrypsin 500 μg / ml PBS solution. 96 holes Microtiter plate 1 per hole
They were added one by one and blocked with a 1% BSA-PBS solution at 4 ° C. overnight. After washing with PBS, hybridoma culture supernatant 4
0 μm was added, and the mixture was repelled at room temperature for 2 hours. 0.05% Tween
After washing three times with -20-PBS solution, peroxidase-labeled goat anti-mouse immunoglobulin antibody (Kappel; 1
% Diluted with a BSA-PBS solution (1,500 times) was added to the mixture (40 μ), and the mixture was reacted at room temperature for 2 hours. After washing three times with a 0.05% Tween-20-PBS solution, a substrate reagent for color development was added, and a dye formed on nitrocellulose was determined to be positive for antibody activity.
Two types of (1): the same as (1) of Example 1 (2): the same as (3) of Example 1 were used as substrate reagents for color development for comparison. Of the 350 wells measured, when the reagent of (1) was used, antibody activity positive hybridomas could only be detected in 15 wells, but when the reagent of (2) of the present invention was used, antibody activity positive hybridomas were detected in 23 wells. did it.

Claims (1)

[Claims] 1. A method for measuring a specific component to be measured by using a labeled product having peroxidase as a label and measuring the amount of a dye produced by the enzymatic reaction of peroxidase in the enzymatic reaction. A method for measuring the specific component, wherein hydrogen peroxide and 4-alkoxy-1-naphthol (wherein the alkoxy group is an ethoxy group, a propoxy group, an isopropoxy group) are used as a substrate.