JPH0755159B2 - Peroxidase activity assay - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention has a peroxidase (hereinafter abbreviated as POD) activity, which is used as a labeling enzyme in, for example, an immunostaining method or an enzyme immunoassay method. Regarding new measurement methods.

[Background of the Invention]

In the field of clinical examination, POD is generally used as an oxidation catalyst for an enzyme analysis method in a hydrogen peroxide (hereinafter, abbreviated as H ₂ O ₂ ) production system using an oxidase and the like. In recent years, it has come to be widely used as a labeling enzyme in immunostaining and enzyme immunoassay. More recently, by combining the electrophoresis method with a blotting operation, the enzyme (POD
Etc.) A method of specifically detecting a trace amount of protein in a living body using a labeled antibody has been attracting attention as an epoch-making protein analysis method.
Because these immunological methods have excellent specificity,
The range of use tends to expand more and more in the future.

As an immunostaining method using POD, for example, PAP (Peroxidase-antiperoxidase comple) which is widely used particularly in immunohistological staining method which is an application of immunoreaction to a tissue section.
x) method and ABC (Avidin-biotinylated peroxidase comple
The x) method and the like can be mentioned as typical ones, but in all of these, the final staining is based on the color reaction by the H ₂ O ₂ -oxidizable color reagent system utilizing POD activity.

Further, the enzyme immunoassay method using POD, the POD activity of the POD-labeled antibody bound to the substance to be measured is measured using an H ₂ O ₂ -oxidizable color reagent, and the concentration of the substance to be measured is determined from the activity value. It is what you want.

All of these methods are oxidizable color reagents such as o-
Phenylenediamine, 3,3'-diaminobenzidine, 4
-Chlor-1-naphthol, 3-amino-9-ethylcarbazole and the like are used. However,
The method using such an oxidizable color reagent is, for example, in the determination of trace components in a biological sample, due to the influence of ascorbic acid present in the sample, a bioreducing substance such as bilirubin, a measured value. Often caused a negative error in. Therefore, in order to remove these interfering substances, ascorbic acid oxidase, ascorbic acid is removed with iodate, or the effect of bilirubin is removed with potassium ferrocyanide or bilirubin oxidase.
Various efforts have been made, but each has advantages and disadvantages.

On the other hand, also in the detection and quantification methods of various trace proteins that combine electrophoresis with blotting, the activity of POD, which is a labeling enzyme, can be measured by H ₂ O ₂ and 3,3′-diaminobenzidine or 4-chloro-1- It is performed using an oxidizable color reagent represented by naphthol, but it is hard to say that it is sufficiently satisfactory in terms of sensitivity and color tone.

In addition, many of these oxidative colored products are relatively unstable compounds, and particularly in the immunohistological staining method, there is a drawback that the stained specimen is discolored during storage.

[Object of the Invention]

The present invention, for example, in an immunostaining method or an enzyme immunoassay method, a POD activity measuring method with less influence of reducing substances such as ascorbic acid and bilirubin coexisting in a sample, and an immunostaining method, It is an object of the present invention to provide a dyeing method capable of obtaining a dyed product having good stability without fading inside.

[Structure of Invention]

 In order to achieve the above object, the present invention has the following configuration.

"Measure the degree of coloration of a coloring compound produced by reducing a reductive color-reactive reagent in response to peroxidase activity in the presence of hydrogen peroxide, reduced coenzyme and amines or phenols (or naphthols) A method for measuring peroxidase activity, which comprises measuring the peroxidase activity according to the above. ”The present inventors have previously described the presence of reduced coenzyme, POD, and amines or phenols (or naphthols), H ₂
We filed a patent application (Japanese Patent Application No. 60-071632) for a method for quantifying H ₂ O ₂ by measuring the degree of coloration of a colored compound produced by reducing a reducible color-forming reagent according to the amount of O _2. However, the present invention utilizes this reaction system. That is,
In the method for measuring POD activity, the present inventors developed a coloring compound produced by oxidation of an oxidizable color reagent in response to POD activity in the presence of H ₂ O ₂ and an oxidizable color reagent. As long as this is done by the method of measuring the degree, the influence of reducing substances such as ascorbic acid and bilirubin coexisting in the sample is unavoidable, and when performing immunostaining, as long as an oxidizable color reagent is used. , It is considered that the disadvantage that the colored material fades during storage cannot be avoided, and instead of measuring the coloration degree of the oxidizable coloration reagent, by measuring the coloration degree of the reducible coloration reagent. The inventors have earnestly researched how to do this, and arrived at the present invention. That is, the inventors of the present invention include tetrazolium salts that are generally used as a color reagent that is reduced to give a color, that is, as a reducible color reagent, such as ascorbic acid and bilirubin from the relationship of redox potential. Intensive research has been conducted with the inspiration that some are difficult to be affected and that among the formazan and diformazan produced by the reduction of the tetrazolium salt, many are poorly soluble in water and have strong dyeing properties. As a result, the present invention has been completed.

In order to measure POD activity by the method of the present invention, a coloring reagent containing a reduced coenzyme, amines or phenols (or naphthols), a reducible color reagent and optionally a surfactant is added to a sample. Then, after adding H ₂ O ₂ solution and reacting for a certain period of time at a constant temperature, stop the reaction with an appropriate reaction terminator (eg, acids such as hydrochloric acid, sulfuric acid, sodium dodecyl sulfate, etc.) and measure the coloration degree. do it. Also,
In the case of the immunostaining method, the same operation staining is performed, followed by washing,
The color tone of the dyed portion may be observed with the naked eye, a microscope, or the like.

The fact that the method of the present invention is not simply the reductive color development of a reducible color reagent by a reduced coenzyme means that an electron carrier such as diaphorase or phenazine methosulfate (PMS), 1-methoxyphenazine methosulfate (1-methoxy PMS) is used. It is clear from the fact that it does not exist in this reaction system, and that it does not develop color when it is reacted with a color reagent solution from which amines or phenols (or naphthols) have been removed, and H ₂ O ₂ solution, and it is also superoxide. Since addition of dismutase does not inhibit the reaction, it is clear that it is not due to superoxide ion.

As described above, according to the method of the present invention, it is completely unexpected that the reducible color-forming reagent is quantitatively reduced and develops color depending on the POD activity.

Examples of the reduced coenzyme used in the present invention include reduced nicotinamide adenine dinucleotide (NA
DH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), and the like, but are not limited thereto. The concentration used is not particularly limited,
Usually, 0.01 to 20 mmol / l is preferably used.

As the amines, any of the usual organic amines can be used, and primary, secondary or tertiary amines can be used. Further, generally, an aromatic amine is more effective than a fatty amine with a small amount used. Specific examples of these amines include, for example,
Aniline, N-methylaniline, N-ethylaniline,
N, N-dimethylaniline, N, N-diethylaniline, N, N
-Diethyl-m-toluidine, N-ethyl-N- (β-
Hydroxyethyl) -m-toluidine, 3,5-dimethoxy-N-ethyl-N- (2-hydroxy-3-sodiumsulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) Examples include, but are not limited to, -m-toluidine sodium (hereinafter abbreviated as TOOS) and the like.

As the phenols, in addition to phenol itself, for example, alkyl-substituted phenols such as methyl, ethyl and propyl, alkoxy-substituted phenols such as methoxy, ethoxy and propoxy, and various derivatives such as halogen-substituted phenols such as chlorine, bromine, fluorine and iodine are used. it can. In addition to naphthol itself, naphthol derivatives such as naphthol sulfonic acids and naphthol carboxylic acids can be used as naphthols.

These amines, phenols and naphthols may be used alone or in any combination of two or more. Also, for example, 1-N, N-dimethylamino-4-
It is also possible to use compounds in which one compound is both a phenol and an amine, such as naphthol and 4-N, N-diethylaminosalicylic acid. However, when the substrate is amine or L-amino acid and the oxidase is amine oxidase or L-amino acid oxidase respectively, it is natural to use phenols instead of amines.

The concentration of these amines, phenols and naphthols used is not particularly limited, but normally, a molar ratio of about 0.08 to 160 times that of the reduced coenzyme is preferably used.

The concentration of hydrogen peroxide used in the present invention is not particularly limited, but it is usually 1.0 molar ratio to the reduced coenzyme.
About 7.5 times is preferably used.

Examples of the reducing-type coloring reagent used in the present invention include all existing and unknown compounds that are colored by reduction regardless of unknown ones. For example, 3- (p-iodophenyl)
-2- (p-nitrophenyl) -5-phenyl-2H tetrazolium chloride (INT), 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium bromide (MTT), 3, 3 '-(4,4'-biphenylene) -bis (2,5-diphenyl-2H tetrazolium chloride) (Neo-TB), 3,3'-(3,3'-dimethoxy-4,4'-biphenylene) -Bis [2- (p-
Nitrophenyl) -5-phenyl-2H tetrazolium chloride] (nitro-TB), 3,3 '-(3,3'-dimethoxy-4,4'-biphenylene) -bis (2,5-diphenyl-2H tetrazolium chloride ) (TB), 3,3 ′
Examples thereof include known tetrazolium salts such as-(3,3'-dimethoxy-4,4'-biphenylene) -bis [2,5-bis (p-nitrophenyl) -2H tetrazolium chloride] (TNTB). In addition, it is a newly developed water-soluble tetrazolium salt. 2- (2-benzothiazolyl) -3- (o-carboxyphenyl) -5- [p-
{Hydroxy poly (oxy-1,2-ethanediyl)}
It goes without saying that phenyl] -2H tetrazolium chloride and the like can be used as well.

The POD activity measuring method of the present invention can be used very effectively in, for example, immunostaining methods such as PAP method and ABC method and enzyme immunoassay methods (EIA method).

The immunostaining method utilizing the method of the present invention is an immunostaining method known per se, instead of using H ₂ O ₂ and an oxidizable color reagent, H ₂ O ₂ , reduced coenzyme, amines Alternatively, it suffices to carry out this in accordance with an immunostaining method known per se except that phenols (or naphthols) and a reducible color reagent are used. The EIA method utilizing the method of the present invention is also an EIA method known per se, instead of using H ₂ O ₂ and an oxidizable color reagent, H ₂ O ₂ , reduced coenzyme, amines or It is sufficient to carry out this in accordance with the EIA method known per se except that phenols (or naphthols) and a reducible color reagent are used.

That is, according to the present invention, which was conventionally performed using an oxidizable color reagent, the reductive property of the tetrazolium compound or the like in the presence of a reduced coenzyme and amines or phenols (or naphthols) was observed. The formazan compound produced by reducing the tetrazolium salt is a yellow to blue color due to its structure, and is often sparingly soluble in water. Therefore, it is extremely advantageous for the immunostaining method. Further, in the case of colorimetric determination with an aqueous solution as in the enzyme immunoassay, the formazan compound can be solubilized by adding gelatin or a surfactant, and there is no problem in practical use. In this case, it is of course possible to use a tetrazolium salt which produces water-soluble formazan, if necessary.

Although the measuring method of the present invention can be carried out in a wide pH range from acidic to alkaline, usually, considering the stability of enzymes,
pH 4-9 are preferably used.

As a measurement target that can be measured by the EIA method utilizing the method of the present invention, a physiologically active substance that can be measured by the EIA method known per se, for example, α-fetoprotein, CEA (carcinoembryonic antigen), a protein such as IgG, insulin, growth hormone, HCG (human chorionic gonadotropin), thyroxine, hormones such as thyroid stimulating hormone, antibodies such as HB _S antibodies, antigens such as HB _S antigen, digoxin, haptens such as morphine and the like all.

As the measurement object that can be measured by the immunostaining method using the method of the present invention, antigens that can be stained by the immunostaining method known per se, for example, various immunoglobulins, lymphoid cell membrane antigens, hormone-producing cells, intracellular Enzymes, fetus-derived antigens, viral antigens and the like are all mentioned.

Since the present invention can measure a peroxidase-like substance, it can also be used for quantifying hemoglobin.

Examples will be given below, but the present invention is not limited to these examples.

〔Example〕

Example 1 Measurement of POD activity (Preparation of reagent solution) Color reagent solution 0.02 with nitro-TB in 0.05M phosphate buffer (pH 6.0)
%, NADH was 0.67 mmol / l, phenol was 0.02%, and Triton x100 was dissolved to a concentration of 0.05% to obtain a color test solution.

H ₂ O ₂ test solution An aqueous solution of H ₂ O ₂ 2.94 mmol / l was prepared.

POD standard solution POD Type III (120U / mg) manufactured by Toyobo Co., Ltd. was added to 0.06,0.1
An aqueous solution containing 2,0.18,0.24,0.3 mg / dl was prepared.

Reaction stop solution A 2% aqueous solution of sodium dodecyl sulfate was prepared.

(Measurement method) Take 20μ of POD standard solution, add 3ml of coloring solution, and then add H ₂ O ₂
0.1 ml of the test solution was added and heated at 37 ° C for 10 minutes. Reaction stop solution
After adding 1.0 ml and mixing, the absorbance at a wavelength of 560 nm was measured using the reagent blank as a control.

Figure 1 shows the relationship between POD concentration and absorbance. As is clear from FIG. 1, the calibration curve connecting the absorbance plotted against each POD concentration (mg / dl) shows good quantification.

In addition, even if phenol 0.02% in the coloring reagent was replaced with TOOS 0.03%, the same result was obtained.

Example 2 Substrate staining 0.06% of nitro-TB in 0.05M phosphate buffer (pH 6.0),
NADPH 1.3m mol / l, phenol 0.005%, H ₂ O ₂ 0.00
It was dissolved to have a concentration of 8% and used as a substrate staining solution.

For the purpose of staining the nerve cells of the supraoptic nucleus using an anti-vasopressin antibody, a section of the hypothalamic pituitary pituitary tissue that has undergone PAP treatment was immersed in the above substrate staining solution, and reacted at room temperature for 30 minutes. After that, the section was washed with distilled water to prepare a stained sample. The dyed specimen thus obtained showed good dyeing property and no fading over time was observed.

Further, in this example, 0.005% of phenol was added to 0.008% of TOOS.
Or, even if the amount of α-naphthol was changed to 0.008%, exactly the same result was obtained.

Example 3 Method for measuring POD activity on nitrocellulose membrane (Preparation of test solution) Staining test solution 0.03 of nitro-TB was added to 0.05M phosphate buffer (pH 7.0).
%, NADH was dissolved at a concentration of 1.4 mmol / l, and phenol was dissolved at a concentration of 0.025% to prepare a dyeing test solution.

H ₂ O ₂ test solution An aqueous solution of H ₂ O ₂ 2.94 mol / l was prepared and used as a H ₂ O ₂ test solution.

POD standard solution Horseradish-derived POD binding affinity-purified anti-rabbit IgG
(H + L) (goat, manufactured by Bio-Rad, abbreviated as IgG-HRP hereinafter) containing 20 mol / l Tris / HCl buffer (pH 7.5) containing 0.5 mol / l NaCl (hereinafter abbreviated as Tris buffer) The IgG-HRP concentration is 0.0625, 0.125, 0.25, 0.5.
POD standard solutions of 0, 0.75, 1.0 μ / ml were prepared.

(Measurement method) 400μ of each POD standard solution in each hole of Bio-Dot (manufactured by Bio-Rad) equipped with a nitrocellulose membrane equilibrated with Tris buffer.
Each was infused and passed naturally. After washing with Tris buffer,
Remove the nitrocellulose membrane from Bio-Dot and use H ₂ O ₂ test solution.
The reaction was initiated by immersing in 10 ml of a staining reagent solution containing 20 μm. After reacting at 25 ° C for 30 minutes, the nitrocellulose membrane washed with purified water and dried was measured with a decalin-treated densitometer (Bio-Rad Model 1650).

FIG. 2 shows the relationship between the POD (IgG-HRP) concentration (μ / ml) and the peak height of the obtained densitogram. As is clear from FIG. 2, the calibration curve connecting the heights of the peaks plotted with respect to each POD (IgG-HRP) concentration shows good quantification. In addition, 0.025% of phenol in the dyeing test solution was added to T
Even if it replaced with OOS0.05%, the same result was obtained.

Example 4 Quantitative method for α-fetoprotein (hereinafter abbreviated as AFP) (Preparation of test solution) Staining test solution 0.03 nitro-TB was added to 0.05M phosphate buffer (pH 7.0).
%, NADH was dissolved at a concentration of 2.8 mmol / l, and phenol was dissolved at a concentration of 0.020% to prepare a dyeing test solution.

H ₂ O ₂ test solution An aqueous solution of H ₂ O ₂ 2.94 mol / l was prepared and used as a H ₂ O ₂ test solution.

AFP standard solution Japanese AFP standard solution manufactured by Japan Bio-Test (1000ng / ml)
Is diluted with 0.05M phosphate buffer (pH7.2) containing 0.15M NaCl and 0.3% bovine albumin to give 3.125,6.25,12.5,25,50,100,200
An AFP standard solution having a concentration of ng / ml was prepared.

Preparation of anti-human AFP antibody (horse) -immobilized nitrocellulose membrane (hereinafter abbreviated as anti-human AFP-NC membrane).

Nitrocellulose membrane (5 x 9.2c) in Tris buffer containing 10 μg / ml of affinity purified anti-human AFP antibody (horse).
m) was soaked for 30 minutes to equilibrate. Then, the nitrocellulose membrane from which excess water was removed with paper was left in steam of glutaraldehyde for 30 minutes to perform immobilization operation, and then 0.02%
Neutralization treatment with Tris buffer containing NaBH ₄ and washing with Tris buffer were performed. Further, it was immersed in a Tris buffer containing 0.5% Tween 20 for 30 minutes for block treatment, and then washed again with Tris buffer to obtain an anti-human AFP-NC membrane.

(Measurement method) B equipped with anti-human AFP-NC membrane equilibrated with Tris buffer
Add 40% AFP standard solution of each concentration to each hole of io-Dot (Bio-Rad).
I injected μ and passed naturally. After washing with Tris buffer, remove the membrane.
It was taken out from the Bio-Dot and washed twice with 10 ml of Tris buffer containing 0.05% Tween 20 (5 minutes × 2). Anti-human AFP diluted 500 times with Tris buffer containing 1% gelatin [rabbit,
DAKO is used as F (ab ') ₂ ] The membrane is immersed in 10 ml of the solution for 30 minutes, and 10 ml of Tris buffer containing 0.05% Tween 20.
It was washed twice (10 minutes x 2) with. Furthermore, IgG-HRP (Bio-based) diluted 1000 times with Tris buffer containing 1% gelatin.
(Manufactured by Rad) for 30 minutes, and washed twice with 10 ml of Tris buffer containing 0.05% Tween 20 (10 minutes × 2).

The film thus obtained was dyed in the same manner as in Example 3 and measured in the same manner.

Fig. 3 shows the relationship between the AFP concentration (ng / ml) and the height of the peak of the obtained densitogram (indicated by ○-○).

Comparative Example 1 Dyeing and measurement were carried out in the same manner as in Example 4 except that the dyeing reagent solution and H ₂ O ₂ reagent solution in Example 4 were replaced with the following dyeing reagent solutions.

Staining reagent (including H ₂ O ₂ reagent) 0.05M Tris-HCl buffer (pH 7.6) with 0.03% of 3,3′-diaminobenzidine and 0.005% of H ₂ O ₂ Was dissolved into a dyeing test solution.

The results are shown in Fig. 3 (indicated by XX).

Comparative Example 2 A dyeing reagent solution (H ₂ O ₂ reagent solution was prepared in the same manner as in Example 4 except that the dyeing reagent solution and the H ₂ O ₂ reagent solution in Example 4 were replaced with the following dyeing reagent solution). Including 0.05M Tris-HCl buffer (pH 7.6) 4-chloro-1-
Naphthol was dissolved in 0.05% and H ₂ O ₂ was dissolved in 0.015% to prepare a dyeing test solution (methanol was used as a dissolution aid).

The results are shown in Fig. 3 (indicated by △-△).

As is apparent from FIG. 3, the method of Example 4 according to the present invention is remarkably superior in measurement sensitivity and linearity to the methods of Comparative Examples 1 and 2 which are conventional methods.

〔The invention's effect〕

As described above, the present invention conventionally measures the coloration degree of a coloring compound produced by oxidation of an oxidizable color reagent according to POD activity in the presence of H ₂ O ₂ and an oxidizable color reagent. measurement of POD activity was done only by the method, H ₂ O _2, reduced coenzyme, amines or phenols (or naphthols)
The present invention provides a method for doing this by measuring the coloration degree of a coloring compound produced by reducing a color-reducing reagent in accordance with POD activity in the presence of, ascorbic acid coexisting in a sample. The point that the effect of reducing substances such as bilirubin can be further reduced, and that, for example, when immunostaining is performed by the method of the present invention, the dyeing property is extremely good and the dyed part may also fade during storage. It can be stored for a long time without using the
This invention has a remarkable effect in that the sensitivity is several times higher than that of 3,3'-diaminobenzidine and 4-chloro-1-naphthol, and is a great contribution to the art.

[Brief description of drawings]

FIG. 1 shows the calibration curve obtained in Example 1, in which the absorbance obtained for each POD concentration (mg / dl) on the horizontal axis is plotted along the vertical axis. . FIG. 2 shows the calibration curve obtained in Example 3, with the vertical axis representing the peak height of the densitogram obtained for each POD (IgG-HRP) concentration (μ / ml) on the horizontal axis. It is the connection of the points plotted along. FIG. 3 shows the calibration curves obtained in Example 4 and Comparative Examples 1 and 2, wherein the vertical axis represents the peak height of the densitogram obtained for each AFP concentration (ng / ml) on the horizontal axis. The points plotted along are connected. In the figure, ○-○ indicates Example 4.
, × − × shows the calibration curve of Comparative Example 1, and Δ−Δ shows the calibration curve of Comparative Example 2, respectively.

Claims (6)

[Claims] 1. Coloration of a coloring compound produced by reducing a color-reducing reagent in accordance with peroxidase activity in the presence of hydrogen peroxide, a reduced coenzyme and amines or phenols (or naphthols). A method for measuring peroxidase activity, which comprises measuring peroxidase activity by measuring the degree. 2. The measuring method according to claim 1, wherein the measurement of peroxidase activity is used in an immunostaining method. 3. The measuring method according to claim 2, wherein the immunostaining method is an immunohistological staining method. 4. The measuring method according to claim 1, wherein the measurement of peroxidase activity is used in an enzyme immunoassay. 5. The measuring method according to any one of claims 1 to 4, wherein the reducible color reagent is a tetrazolium compound. 6. A tetrazolium compound is 3,3 '-(3,3'-
Dimethoxy-4,4'-biphenylene) -bis [2- (p
-Nitrophenyl) -5-phenyl-2H tetrazolium chloride].