JPH02109997A - Determination of specific component using peroxidase enzyme reaction - Google Patents

Abstract

PURPOSE:To determine specific component such as nucleic acid in high sensitivity by carrying a complex bonded body composed of specific component of determining object and labeled body having peroxidase as a label on a support and sedimenting coloring matter on the bonded body. CONSTITUTION:A complex bonded body composed of specific component of determining object and a labeled body having peroxidase as a label is carried on a support. Next, coloring matter is formed by enzyme reaction of peroxidase and sedimented on the complex bonded body of the support, then the support is made into contact with polyvinylpyrrolidone to determine the specific component. Homopolymer of vinylpyrrolidone is preferable as polyvinylpyrrolidone and 0.01-50% is preferable as concentration of the polyvinylpyrrolidone in solution. Concrete examples of the objective specific component are nucleic acid, protein, hormone and lipid, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for measuring a specific component, and more particularly to a method for measuring a specific component using a peroxidase enzymatic reaction, and furthermore to stabilization of the dye formed in the reaction.

[Background of the invention]

Various analytical methods for detecting specific components such as biological components have been developed, and the membranes of these methods are also highly accurate methods for detecting the specific components and substances that can specifically bind to them. (hereinafter referred to as specific binding substances), for example, using specific binding reactions between antigens and antibodies, certain sugar chains and lectins, biotin and avidin, protein A and IgG hormones and receptors, enzymes and substrates, etc. method is known.

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

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

For example, a method in which an electrophoresed protein biological component (specific component) is transferred from a gel onto a nitrocellulose membrane and reacted with a label, such as an antibody label, to detect a signal, TL
A method of detecting a signal by reacting a labeled substance with a specific component such as a lipid developed on a C plate, and detecting a signal between DNA and the DNA on a membrane.
These methods include a method of reacting with labeled complementary DNA to A and detecting a signal, or an immunohistochemical staining method.

These methods make it possible to obtain a great deal of information not only on the quantification of specific components and the reactivity of specific components with specific binding substances, but also on the properties, state of existence, etc. of specific components or specific binding substances. For example, a signal is detected on a protein or nucleic acid transferred and supported on a membrane after electrophoresis, or on a complex of a specific component of a living body such as a lipid component developed on TLC and a label for the specific component. In this method, information such as the molecular weight, isoelectric point, or polarity of a specific component can be obtained from the position and mobility of the signal of the specific component.

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

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

To meet this requirement, radioactive isotopes, fluorescent substances, luminescent substances, enzymes, and the like have been used as labels for specific binding substances.

However, when radioactive isotopes are used, there are problems such as decay of radioactivity, disposal, exposure to radiation, and large investment costs. In some cases, the disadvantage is that exposure and development of photographic materials requires a long time and complicated operations.

Further, when using a fluorescent substance or a luminescent substance, special equipment and equipment are required.

On the other hand, when enzymes are used, the procedure is relatively simple and the produced pigment can be easily visualized and quantified. Conventionally, peroxidase, alkaline phosphatase, β-galactosidase, etc. have been used as labeling enzymes. In the method of producing and depositing a dye by enzymatic reaction on a complex conjugate carried on a support, peroxidase is mainly used as a labeling enzyme, and in this case, as a substrate,
Conventionally, diaminobenzidine, 0-dianisidine, 4-chloro-1-naphthol, etc. have been used.

However, diaminobenzidine and O-dianisidine have the drawback of being highly toxic and prone to background. 4
-Chlor-1-su7tol is slightly more sensitive than others, but it is still sensitive enough to measure trace amounts of specific components or to clearly obtain more information about specific components. do not have.

The present inventor has already demonstrated a method that is simple, has high sensitivity, and has high resolution.
In addition, he developed a quick method for measuring specific components, and
It was disclosed in No.-150723 and No. 61-216299. That is, a measurement in which a complex conjugate consisting of a specific component to be measured and a label having peroxidase as a label is supported on a support, and a dye is formed and deposited on the complex conjugate by an enzymatic reaction of peroxidase. The former method uses hydrogen peroxide, an aromatic primary amine compound, and a phenol compound as substrates for the enzymatic reaction, and the latter method uses hydrogen peroxide, an aromatic primary amine compound, and a phenol compound. This is a measurement method that uses three active methylene compounds.

A specific component in which a complex conjugate consisting of a specific component to be measured and a label having peroxidase as a label is supported on a support, and a dye is formed and deposited on the complex conjugate by an enzymatic reaction of peroxidase. In particular, in the high-sensitivity measurement method for the above-mentioned specific components that uses hydrogen peroxide, aromatic primary amine compounds, and phenolic compounds as substrates for the peroxidase enzymatic reaction, It has become clear that the dyes decomposed over time during long-term storage, especially under the influence of light. Such pigment decomposition poses a major problem in terms of accuracy in measuring specific components and long-term storage of records.

[Purpose of the invention]

In view of the above circumstances, an object of the present invention is to provide a stable and highly sensitive method for measuring a specific component in which decomposition of the produced dye is suppressed.

[Structure of the invention]

The object of the present invention as described above is to support a complex conjugate consisting of a specific component to be measured and a label having peroxidase as a label on a support, and to carry on the complex conjugate by an enzymatic reaction of peroxidase. A method for measuring a specific component that forms or deposits a pigment is achieved by a method for measuring a specific component, which is characterized by including a step of bringing the support into contact with polyvinylpyrrolidone after the enzymatic reaction.

The polyvinylpyrrolidone that can be used in the present invention is preferably a homopolymer of vinylpyrrolidone, but may also be a copolymer with other polymers such as vinyl acetate or styrene. Although the molecular weight is not particularly limited, i
1.000.000 from o and ooo is preferable.

When used in the present invention, polyvinylpyrrolidone is preferably used in a solution state. The solvent may be an organic solvent or a mixed solvent with water as long as it does not dissolve the produced dye, but an aqueous solvent is preferred. The concentration of polyvinylpyrrolidone in the solution is preferably from 0.01% to 50%, particularly preferably from o,i% to 20%.

In the present invention, the specific component may be directly supported on the support by physical adsorption, chemical bond such as ionic bond or covalent bond, or indirectly supported through one or more specific binding substances. may be done.

Further, after the specific component is directly or indirectly supported on the support, the labeled body may be reacted to form the composite conjugate, or after forming the composite conjugate. The composite conjugate may be supported directly or indirectly on a support. Furthermore, the label forms a complex conjugate with the specific component and is supported on the support; in this case, the specific component and the label may be bound directly or via one or more other specific binding substances. may be combined.

Here, the expression "supported on a support" means that it is immobilized on the surface or inside of the support so that it cannot be separated from the support.

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

In the present invention, the target specific component is a substance or a group of substances from which a specific binding substance that specifically binds to the specific component is obtained.

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

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

Supports that can be used in the present invention include membranes such as cellulose acetate, nitrocellulose, and polyvinylidene fluoride;
Examples include gel supports such as polyacrylamide; silica gel carriers such as iTLC plates; polysaccharides and derivatives thereof such as dextran and agarose; plate-shaped and bead-shaped plastics, glass, metals, fibers, and activated carbon. Furthermore, in histochemical staining, the tissue itself can be used as a support.

The enzymatic reaction of peroxidase according to the present invention oxidizes a substrate, which is a chromogen, to produce a pigment through the action of hydrogen peroxide and peroxidase.

The peroxidase substrate that can be used in the present invention may be any compound that can produce an insoluble pigment when oxidized. Substrates commonly used for enzyme immunostaining and histological staining can be used; examples include diaminobenzidine, 0-dianisidine, tetramethylbenzi, zine, 4-chloro-1-naphthol, 3-amino-9 -Ethylcarbazole, etc., and JP-A-61-15
The combination of an aromatic primary amine compound and a phenol compound disclosed in No. 0723, and JP-A-61-216299
Examples include the combination of an aromatic primary amine compound and an active methylene compound disclosed in No. The present invention is particularly effective for measurement methods that use aromatic primary amine compounds and phenol compounds as substrates. In order to generate and deposit a dye on a complex conjugate of a specific component supported on a support with a peroxidase label, the support may be immersed in a coloring substrate reagent solution.

A coloring substrate reagent solution is prepared by dissolving hydrogen peroxide, which is a substrate, and a chromogen in a buffer solution of an appropriate pH.

After the dye is sufficiently generated and deposited on the support by the enzymatic reaction,
Wash away unreacted substances and stop the reaction.

The support and polyvinylpyrrolidone are brought into contact by immersion in a polyvinylpyrrolidone solution, or by spraying. A polyvinylpyrrolidone solution may be used as a cleaning agent during cleaning after color development, or the substrate may be immersed in a polyvinylpyrrolidone solution or sprayed with a polyvinylpyrrolidone solution after cleaning. The support in contact with polyvinylpyrrolidone may be stored in a wet state, but it is more advantageous to store it in a dry state.

Information about the pigment produced can be read visually or using methods known in the art, such as spectrophotometry.

The measurement method of the present invention, which is characterized by contacting with polyvinylpyrrolidone, is able to control the decomposition of the produced dye, especially the decomposition by light, without reducing the detection sensitivity, compared to the conventional method that does not involve this operation. It has become possible to store signals for a long time and analyze accurate information.

〔Example〕

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

Example 1 Phosphate buffered saline (pH 7.4: hereinafter referred to as PBS)
The nitrocellulose membrane soaked in water for 5 minutes was attached to a plotting device (Bio-Rad), and 30 ng of a mouse monoclonal IgM antibody dissolved in PBS was dot plotted on the membrane as an antigen. The antigen-supported membrane was incubated with a 1% bovine serum albumin (BSA)-PBS solution for 4 hours.
Blocking was performed overnight at ℃, and then peroxidase-labeled goat anti-mouse IgM antibody (manufactured by Kappel; 1
% BJA-2000 times diluted with PBS solution) at room temperature for 2 hours. After washing with a 0.05% Tween-20 (manufactured by Wako Pure Chemical Industries, Ltd.)-PBS solution, it was immersed in a coloring substrate reagent solution to perform a coloring reaction. The coloring substrate test solution is 10ml of methanol containing 30g of 4-chloro-1-naphthol.
50mM) lithium-hydrochloric acid buffer (pH 7.4;
00a+M sodium chloride containing) 50-0 was added, and further 200 μa of 3% hydrogen peroxide solution was added.

After 15 minutes, the membrane was taken out of the coloring substrate sample solution, washed three times with pure water for 5 minutes each, and then immersed in polyvinylpyrrolidone (hereinafter referred to as PvP) aqueous solutions of various concentrations for 30 minutes, and then dried. Stability evaluation was performed by exposing the membrane to 7c under an 18W fluorescent lamp.
This was carried out by placing the dye spot at a position separated by m and measuring the reflection density of the dye spot over time at 650 ns+. The results are shown in Table 1.

Table 1 Example 2 Color was developed in the same manner as in Example 1, and after washing three times with pure water for 5 minutes each, it was immersed in a 3% PvP aqueous solution for a certain period of time, and then dried. Evaluation was performed in the same manner as in Example 1. The results are shown in Table 2.

Table 3 shows the results of washing three times with 43% PVP for 5 minutes each.

Table 3 Table 2 Removal of PVP by washing with water tends to reduce the stability effect after drying, and contact with PVP immediately before drying Example 3 After color development as in Example 1, the following various washings After the operation, it was dried and evaluated in the same manner as in Example 1.

Washing operation 1: Washing with pure water three times for 5 minutes each time 2 After washing twice with pure water for 5 minutes each, 3% was confirmed to be advantageous.

Claims (3)

[Claims] (1) A complex conjugate consisting of a specific component to be measured and a label having peroxidase as a label is supported on a support, and a dye is applied onto the complex conjugate by an enzymatic reaction of peroxidase. A method for measuring a specific component formed or deposited, the method comprising the step of contacting a support with polyvinylpyrrolidone after the enzymatic reaction. (2) The method for measuring a specific component according to claim 1, characterized in that hydrogen peroxide, an aromatic primary amine compound, and a phenol compound are used as substrates for the enzymatic reaction of peroxidase. (3) A method for stabilizing a dye formed on a support by an enzymatic reaction of peroxidase by contacting it with polyvinylpyrrolidone.