JPS636462A - Method for measuring specific component by using peroxidase enzyme reaction - Google Patents

Abstract

PURPOSE:To permit easy and quick measurement with high sensitivity by using a hydrogen peroxide, arom. primary amine and phenolic compd. as the substrate of an enzyme reaction in measurement of a specific component by using the enzyme reaction of peroxidase. CONSTITUTION:The substrate soln. for color formation consisting of the three; hydrogen peroxide, arom. primary amine and phenolic compd. is acted on a composite conjugate consisting of the specific component such as vital component transferred or deposited on a film or developed on a TLC plate after electrophoresis and a labeling body having the peroxidase as a label. The arom. primary amine is oxidized by the reaction of the peroxidase and the hydrogen peroxide and is then coupled with the phenolic compd. to form and settle a bright blue dye. The specific material is quantitatively determined from the degree of the coloration by the formed dye.

Description

[Detailed description of the invention] [Industrial application field]

The present invention relates to a method for measuring a specific component, and particularly to a method for measuring a specific component using a peroxidase enzyme reaction.

[Prior art]

Various analytical methods have been developed to detect specific components such as biological components, and these methods are highly accurate even when measuring medium amounts. (hereinafter referred to as specific binding substances), such as antigens and antibodies, certain sugar chains and lectins, biotin and avinone, protein A and IgG, hormones and receptors, enzymes and substrates, etc. Are known. - Generally, a specific component is measured by using a specific binding substance (hereinafter referred to as a label) attached with some kind 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 and supported on 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 label with a specific component such as a lipid placed on a C plate, and DNA
A method of detecting a signal by reacting the DNA with a labeled complementary DNA, or an immunoffi chemical staining method, etc. With these methods, it is 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 and state of existence of specific components or specific binding substances. In a method of detecting a signal on a complex in which a specific component of a living body, such as a protein or nucleic acid developed or supported on a membrane, or a lipid component developed on a TLC, is bound to a label for the specific component. 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.

[Problems to be solved by the invention]

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. Radioactive isotopes, fluorescent substances, luminescent substances, enzymes, etc. are used as labels for specific binding substances. Radioactive isotopes have problems such as attenuation of radioactivity, disposal, exposure to radiation, and the high cost of membrane tubes.Furthermore, when detecting a signal on a label supported on a support, the exposure of the photographic light-sensitive material, It has the disadvantage that it requires a long time and complicated operations such as development. Fluorescent or luminescent substances require special equipment and equipment. On the other hand, W! When dyes are used, the procedure is relatively simple, and the resulting dyes can be easily visualized and quantified. Conventionally,
As labeling enzymes, peroxidase, 7-lucali-7-o-7-atase, β-lactosigase, etc. have been used. 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, conventional noami/bentunone, 0-noanidinone, 4 −
Chloro-1-su7toll and the like have been used. Noaminobentunone and 0-noanidinone have the drawback of being highly toxic and prone to background. 4-chloro-1-
Although S7 Toll is slightly more sensitive than others, it cannot be said to be sensitive enough to measure trace amounts of specific components or to clearly obtain more information about specific components. Therefore, an object of the present invention is to provide a method for measuring a specific component simply, with high sensitivity, high resolution, and quickly.

[Means to solve the problem]

As a result of various studies in line with the above objectives, we supported a complex conjugate consisting of a specific component to be measured and a label having peroxidase as a label on a support, and applied an enzymatic reaction of peroxidase onto the complex conjugate. Forms pigment,
In a method for measuring a specific component deposited, the problems are solved by a method using hydrogen peroxide, an aromatic primary amine compound, and a phenol compound as substrates for the enzyme reaction, and the above-mentioned object of the present invention is achieved. is achieved. Next, the present invention will be explained in detail. In the present invention, the specific component may be directly supported on the support by physical adsorption, chemical bonding, etc., or indirectly supported via one or more specific binding substances. Further, after the specific component is directly or indirectly supported on the support, the Ha body may be reacted to form the composite conjugate, or the composite conjugate may be formed and then the specific component may be supported directly or indirectly on the support. The composite conjugate may be supported directly or indirectly on a support. Furthermore, the PX:f& body forms a complex binding body with the specific component and is supported on the support, but the specific component and the label may be bound directly or via one or more other specific binding substances. May be combined. Further, in the present invention, the labeled body may be one in which a specific binding substance is labeled with peroxidase and an anti-peroxygose antibody. Hydrogen peroxide, an aromatic primary amine compound, and a heptanoyl compound are used as peroxidase substrates for the enzymatic reaction of peroxidase in the complex conjugate. Aromatic primary amine compounds are oxidized by the action of peroxidase and hydrogen peroxide, and then coupled with phenolic compounds to produce and deposit a bright blue pigment. Analytical Biochemistry Using Conventional Hydrogen Peroxide and 4-Aa-1-67 Tol as Substrates
Compared to the method described in Try) Sato, 142-147 (1982), the method of the present invention shortened the color development time, produced clear spots, and increased sensitivity by more than 10 times. Furthermore, as a result, the amount of peroxygase label can be reduced, which is advantageous in terms of cost. 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 A1 avidin, biotin, receptors, cofactors, enzyme substrates, toxins, and complements. and complexes thereof. Supports that can be used in the present invention include membranes such as cellulose acetate and nitrocellulose, delta supports such as polyacrylamide, silica sol carriers such as TLC plates,
Plate-shaped, bead-shaped plastic, glass, metal,
JaJll, etc. 6 In addition, mm itself can be used as a support in composite fiber chemical dyeing. Examples of the aromatic primary amine compound that can be used in the present invention include 0- or p-7 minophenol compounds, 0- or p-phenylenediamine compounds, and salts thereof. Preferred is a p-phenylenediamine compound represented by the following general formula (1). -General formula [(] In the formula, A and B represent a hydrogen atom or an alkyl group, and A
and B may form a heterocycle together with the nitrogen atom, D,
E, G and J represent a hydrogen atom, a halogen atom, a hydroxyl group, an amide group, an alkoxy group, an acylamido group, an arylsulfonamide group, a furkylsulfonamide group or an alkyl group. The alkyl group represented by A and B has 1 carbon atom
The number of groups is preferably 6 to 6, and 1 to 4 is particularly preferable. Examples include methyl, ethyl, and butyl groups. These alkyl groups may have a substituent, and examples of the substituent include a ureido group, a tetrahydro-7lyl group, a carboxyl group, a methanesulfonamide group,
Examples include sulfo group, methoxy group, ethoxy group, methoxyethoxy group, /toxyethoxyethoxy group, and methoxytetraethoxy group. D, G and J are preferably a hydrogen atom, an alkoxy group, a furkylsulfonamide group, or an arylsulfonamide group, and 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, especially a methyl group. - Salts of the compound represented by the general formula (1) include p-toluenesulfonic acid, sulfonic acid, sulfinic acid, sulfuric acid ester, sulfamic acid, thiosulfuric acid S-ester, carboxylic acid, phosphoric acid ester, amidophosphoric acid,
Mention may be made of salts of organic or inorganic acids such as phosphoric acid, phosphite esters, organic boron compounds, hydrochloric acid and sulfuric acid,
Particularly preferred are p-)luenesulfonate, hydrochloride and sulfate. Typical specific examples of the aromatic primary amine compound according to the present invention are shown below, but the present invention is not limited thereto. Exemplary compound (1-1) N,N-diethyl-3-methyl-4-fuminoaniline (1-2) N,N-diethyl-4-fuminoaniline (1
-3) N-carbamidomethyl-N-methyl-4-7minoaniline (1-4) N-carbamidomethyl-N-tetrahydrofurfuryl-3-methyl-4-7minoaniline (1-5)
N-ethyl-N-carboxymethyl-3-methyl-4-
7 Minoaniline (1-6) N-carbamidomethyl-N-ethyl-3-methyl-4-7 Minoaniline (1-7) N-ethyl-N-tetrahydrofur 7ly-3-methyl-4-7 Minophenol (1 -8) 3-7cetylamino-4-aminomethylaniline (1-9)N-ethyl-N-β-methanesulfonamidoethyl-4-7minoaniline (1-10)N-ethyl-N-β-7thanesulfone Amidoethyl-3-methyl-4-aminoaniline (1-11
) N-methyl-N-βsulfoethyl-p-phenylenediamine (1-12) N-ethyl-N-hydroquinethyl-3-
Methyl-4-aminoaniline (1-13)N-ethyl-N-(2-(2-methoxyethoxy)ethyl)-3-methyl-4-ami7aniline (1-14)N-ethyl-N-12- C2-<2-methoxyethoxy)ethoxy]ethyl)-3-methyl-4-
7minoaniline (1-15)N-ethyl-N-(2-12-C2-C2
-(2-nodoxyethoxyethoxy)ethoxy]ethoxy]ethoxy)ethyl]-3-methyl-4-ami/aniline (1-16)N,N-noethyl-3,methanesulfonamidoethyl-4-7minoaniline. - The salt of the compound represented by the general formula (11) is generally water-soluble and can be easily dissolved in water or a buffer solution. It is a compound that produces a dye by coupling with an oxidized product of a group primary amine compound, and is a phenol compound substituted with an appropriate substituent on the benzene ring in order to eliminate the solubility of the produced dye in water. Among the phenol compounds, preferred are groups that are unsubstituted at the 4-position of the hydroxyl group or groups that can leave during the coupling reaction with the oxidized product of the aromatic primary amine compound (hereinafter referred to as leaving groups); It is a phenol compound substituted with an atom (hereinafter referred to as a leaving atom). The 7-er/-el compound advantageously used in the present invention is represented by the following general formula [[[].General formula (II) O1+ In the formula, R3 represents a monovalent organic group or atom, 2 represents a hydrogen atom, a leaving group, or a leaving atom, and k represents an integer of 1 to 4. The leaving atom represented by Z includes a halogen atom, for example. ,
Examples include chlorine atom and bromine atom. The leaving group represented by Z is, for example, -0R2゜-0C
OR2, -0SO□R2, -SR2, -0CON)IR
,, -0SO□NllR2゜, where R2 and R1
is a hydrogen atom, an aliphatic hydrocarbon residue, an alicyclic compound residue,
The atom represented by 6R, which represents an aryl group or a heterocyclic residue, includes a halogen atom, such as a chlorine atom and a bromine atom. Examples of the monovalent organic group represented by R include aliphatic hydrocarbon residues, alicyclic compound residues, heterocyclic residues, aryl groups, -5CN, -OR,, -0COR,, -0S
O□R,, -SR,. R1 and R5 represent a hydrogen atom, an aliphatic hydrocarbon residue, an alicyclic compound residue, an aryl group, or a heterocyclic residue. The aliphatic hydrocarbon residues represented by R, R, R), R4 and R5 may be either saturated or unsaturated, and may be linear or branched. Preferably, an alkyl group (e.g., methyl group, ethyl group, propyl group, isopropyl group, butyl group, butyl group, isobutyl group, dodecyl group, octadecyl group), alkenyl group (e.g., allyl group, octenyl group, etc.) each group). Examples of the alicyclic compound residues represented by RzRz, RitR- and R1 include five to six groups, such as a cyclopentyl group and a cyclohexyl group. The heterocyclic residues represented by R+, R2, R*+R+ and R5 include bilinonyl group, bilanonyl group, biligtunyl group, x7lyl group, pyrrolinol group, 7ralyl group, thienyl group, piperidyl group, pyrrolyl group, pyrrolinyl group , a tetrazolyl group, a thiazonyl group, an imidazolyl group, a morpholyl group, a furyl group, an oxasilyl group, a thiazolyl group, a benzimidazolyl group, a benzoxasilyl group, a benzthiazolyl group, and the like. Typical aryl groups represented by R, R, R, R4 and R9 are phenyl and naphthyl groups. Examples of the non-aromatic ring fused to the benzene ring formed by the above-mentioned two R3s include those having five to six shells, such as a cyclopentane ring, a cyclohexane ring, and a cyclohexene ring. The aromatic ring fused with the benzene ring formed by bonding the above two R1s, particularly R2 at the 5th and 6th positions, includes 5- to 6-membered rings such as phenyl group, bilinonyl group, pyrazinyl group, and biligtunyl group. , pyrrolidyl group, 7ralyl group, thienyl group, piperinol group, pyrrolyl group, pyrrolinyl group, thiazinyl group, imidazolyl group, furyl group, oxacylyl group, thiazolyl group, and the like. The aliphatic hydrocarbon residues, alicyclic compound residues, aryl groups, heterocyclic residues represented by R, R2, R, R, and R1 above, and the two R1s described above are bonded together. The non-aromatic ring and aromatic ring formed may have a substituent. Examples of such substituents include halogen atoms (e.g. chlorine atoms, fluorine atoms), nitro groups, cyano groups, hydroxy groups, keto groups, carboxyl groups, sulfo groups, amine groups (
For example, amino, alkylamino, dialkylami/,
aniso/, N-furkyaniso/), alkyl groups (e.g. methyl, propyl, isopropyl, butyl, octadecyl, cyanoalkyl, haloalkyl, aralkyl), alkenyl groups, aryl groups (e.g. phenyl, tolyl, acetylamino7), enyl, 4-lauroylaminophenyl, ethoxyphenyl) heterocyclic residues, alkoxy groups (e.g. ethoxy, 7e/xy, methoxy,
(tetradecyloxy), aryloxy groups (e.g., phenoxy, 2,4-not-7 mil7e/oxy, p-t
-butylphenoquine, 4-n-dodecyloxy-7enoxy, 4-hydroquine-3-t-butylphenoxy, 4-
hydroxy-3-n-butylphenoxy), arylthio group, amide group (e.g. acetamide, methanesulfonamide, p-dodecylbenzenesulfonamide), carbamoyl group (e.g. N-p-carboxynatoxyphenylcarbamoyl, N,N -) Hequinyl lupamoyl, N-benzylcarbamoyl, N-ethylcarbamoyl, N-7toxyethylcarbamoyl), sulf77moyl group (e.g. N,N-noethylsulfamoyl), furkylsulfonyl group, 7lylsulfonyl i (e.g. benzenesulfonyl, cuchlorobenzenesulfonyl),
Acyl groups (e.g. acetyl, p-chlorobenzoyl,
benzoyl), acyloxy groups (e.g. acetyloxy, m-talolopenzoyloxy), acyloxycarbonyl groups and alkoxycarbonyl groups (e.g. N-methquinethylcarbamoylmethoxycarbonyl, ethoxycarbonyl, methoxycarbonyl, triethoxycarbonyl), aryl oxycarbonyl group (e.g. phenoxycarbonyl, p-nitrophenoxycarbonyl),
Examples include a ruthiocarbonyl group (eg, phenylthiocarbonyl) and an imide group (eg, succinimide, octadecylsuccinimide). Typical specific examples of the 71/- compound of the present invention are shown below, but the compounds applicable to the present invention are not limited thereto. Exemplary compound (2-1) 2-Benzu-4-chlorophenol (
2-2) N-benzoyl-4,6-fuchloro-5-
Methyl-2-fuminophenol (2-3) 2-benzoylamino-5-7cetamin-4-chloro7-ethyl (2-4) 4-chloro-1-su7tol (2-5
) 4-Methoxy-1-7toll (2-6)
2.4-dichloro-1-su7toll (2-7) 1-
Hydroxy-4-promo N-ethyl-2-su7tamide (2-8) 1-Hydroxy-4-methoxy-N-propyl-2-su7tamide (2-9) 2.6-nopromo1,5 -Nohydroquina 7talene (2-10) 1-Hydroxy-5-phenylsulfonamide naphthol (2-11) 1-Hydroxy-2,4-dichloro-
To generate and deposit a dye on a complex of a specific component and a peroxyguse label supported on a 5-nitronaphthol support, 1. Substrate for color development. 6. Substrate for color development. 6. Substrate for color development. 6. Substrate for color development. A test solution is prepared by dissolving hydrogen peroxide, an aromatic primary amine compound, and a 7-el compound in a buffer solution of an appropriate pH. The phenol compound may be added after being dissolved in a small amount of a hydrophilic organic solvent such as methanol, ethanol, DMF, or the like. The appropriate molar ratio of the aromatic primary amine compound to the 7-dinoyl compound is 1:100 to 100:1;
1ON1 from is preferred. After the dye is sufficiently generated and deposited on the support by the enzymatic reaction,
Wash away unreacted substances and stop the reaction. Information about the pigment formed can be read visually or using methods known in the art, such as spectrophotometry.

【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. Measurement of antigen on nitrocellulose membrane in Example: After washing with pure water, air-dried nitrocellulose membrane (manufactured by Bio-Rad; thickness 0.45 μm scraps) was coated with phosphate buffer (hereinafter referred to as PBS). 1 μl of serially diluted goat IgG was added to the tube. After air-drying, blocking was performed overnight at 4°C with a 1% bovine serum albumin (BAS)-PBS solution, and then anti-goat IgG antibody (manufactured by Kappel:
The mixture was reacted with 1% OS8-P diluted 1500 times with an IS solution at 4°C for 2 hours. 0.05%Tu+ee
n20 (polyoxyethylene sorbitan mo/Laurey) (manufactured by Wako Pure Chemical Industries, Ltd.) - washed five times with a PBS solution and immersed in a coloring substrate reagent solution. The following three types of coloring substrate test solutions were used and compared. . (1): Dissolve 3 mg of 1-4-chloro-1-su7tol in 1 zl of methanol, and add 5 zl of 0.05M
) Lis-HCl buffer (pH 7, 4, 200xHNaCl
(hereinafter referred to as TBS) and further add 20 μl of 3% hydrogen peroxide. (2)=11 g of N-ethyl-N-β-methanesulfonamidoethyl-3-methyl-4-ami/aniline 3/2 sulfuric acid monohydrate was added to the test solution of (1). (3): To the test solution of (1), 1 and 2 parts of N,N-diethyl-3-methyl-4-7minoaniline hydrochloride was added. After reacting for 15 minutes, it was thoroughly washed with water and air-dried. When using the coloring substrate test solution (1), the spot is grayish-purple;
Oz g of goat IgG L could not be detected, but when coloring substrate test solutions (2) and (3) were used, the spot was bright blue and 1 ag of goat IgG could be detected.
'Example (2): Measurement of glycolipid antigen on a TLC plate: GM isolated and purified from bovine brain on a TLC plate (Polygram, manufactured by Mattieri-Nardel) was mixed with chloroform, chloroform, and glioside in different amounts. After dissolving in a methanol mixture and spotting, it was developed with a developing solution of chloroform, methanol, and 0.5% calcium chloride aqueous solution (55:45:10 V/V). 1% polyvinylpyrrolidone after air drying, 1%
Blocking was performed at 4° C. overnight with an opalpmin-PBS solution, and the cells were prepared in rabbits. Anti-GM ganglioside antiserum (1% polyvinylpyrrolidone, 1% opalpmin-P
500 times diluted with BS solution) at 37°C for 2 hours. After washing 3 sides with 0.05% Tween-20-PBS solution, peroxyguse-labeled goat anti-mouse immunoglobulin antibody (Cubbell Co., Ltd.: 3% polyvinylpyrrolidone-PB)
(diluted 1,500 times with S solution) at 37°C for 2 hours. The following three types of substrate reagents for color development, which were washed three times with 0.05% Tween-20-PBSS and then immersed in the substrate reagent solution for color development, were compared. (1): Same as (1) of Example 1 (2): In (1), N-ethyl-N-hydroxylethyl-3-methyl-4-7 minoaniline sulfur filtrate f
g was added. (3): In the test solution of (2), 4-methoxy-1-su7thol was used instead of 4-chloro-1-naphthol. After reacting for 15 minutes, it was thoroughly washed with water and air-dried. When the coloring substrate reagent solution (1) was used, the spot was grayish-purple and 4H CM and y glioside could not be detected, but when the reagent solutions (2) and (3) were used, the spot was gray-purple. /) was bright blue and detectable up to 0.2xg. Example 3: Screening for antibodies and hybridomas: α-biaftitrypsin 50μ was injected intraperitoneally with total adjuvant into Bajjb/C mice (female, 6 weeks old). Three weeks later, 50 μg of α-pianantitrypsin was injected intraperitoneally together with a 70-diamond incomplete 7-day band, and after another 2 weeks, 30 μg of α1-antitrib F was dissolved in PBS and intravenously injected. 3 days after the final immunization # Take out the visceral cells and follow the standard method.
It was fused with Matus myeloma cell X 63.6.5.3. The cells were divided into five 96-well plates and cultured in HAT selection medium. Three weeks after the fusion, antibodies were measured in the wells in which hybridoma colonies had formed using the following method. Nitrocellulose was cut into 4λl squares, and 1μi of α-biaphtitrypsin 500μy/zIPBS solution was spotted on each square. 96-hole microtiter plate) 16 wells) Add 1 plate at a time, add 1% IIIAs-P
The solution was blocked overnight at 4°C. After washing with PBS, 140 μl of ff was added to the hybridoma culture and allowed to react at room temperature for 2 hours. 0.05% Tween-20
After washing three times with -P[lS solution, 40 μl of Baroxyguse-labeled goat anti-mouse immunoglobulin antibody (manufactured by Cafbel; diluted 1500 times with 1% BAS-PBS solution) was added to the solution. and allowed to react at room temperature for 2 hours.
After washing three times with 05% T*eeII-20-PBS solution, a coloring substrate reagent solution is added, and if a dye is formed on the nitrocellulose, it is considered positive for antibody activity and is used as a r20 coloring substrate reagent solution (1): Same as (1) of Example 1 (2): Comparison was made using two types of charts: (1) with one portion of N,N to noethyl-4-aminoaniline sulfate added.

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 formed on the complex conjugate by an enzymatic reaction of peroxidase. . A method for measuring a specific component deposited, the method comprising using hydrogen peroxide, an aromatic primary amine compound, and a phenol compound as substrates for the enzyme reaction. (2) The specific component according to claim 1, wherein the specific component is directly or indirectly supported on a support, and then the label is reacted to form a complex conjugate. How to measure. (3) The method for measuring a specific component according to claim 1, characterized in that, after forming the composite conjugate, the composite conjugate is directly or indirectly supported on a support.