JPWO2013038936A1 - Method for stopping peroxidase reaction and reaction stopper - Google Patents

Abstract

  A method for terminating a peroxidase reaction, wherein citric acid is preferably added to a peroxidase reaction solution containing peroxidase and tetraalkylbenzidine or a salt thereof so that the final concentration is preferably 0.125 M or more. According to the present invention, the sensitivity or accuracy of the measurement of peroxidase activity can be improved, and in addition, the effect of improving the safety of the reaction stopping work and the reaction stopping agent can be obtained. It is useful in the fields of genetic engineering, applied life science, and medicine.

Description

  The present invention relates to a method for stopping a color reaction by peroxidase, and a stopper used for the method. More specifically, the present invention relates to a method for stopping a color reaction by a peroxidase using tetraalkylbenzidine or a salt thereof as a color developing substance, and a stopper used in the stopping method.

  As a method for measuring a component in a body fluid, such as an antigen or antibody in blood or urine, an enzyme immunoassay method in which the antigen or antibody is labeled with an enzyme is widely used. As a typical method, there is a sandwich method. In this method, (1) a first antibody against a substance (antigen) to be measured is first solid-phased on a carrier such as a plate or a bead, and an antigen is added thereto. Then, the antigen is bound to the immobilized antibody. (2) Next, a second antibody labeled with an enzyme is added to form a solid-phased antibody-antigen-labeled antibody complex. (3) The enzyme-labeled antibody not bound to the antigen is removed by washing, and the amount of the antigen is determined by measuring the enzyme activity bound to the solid phase. That is, since the enzyme activity is determined by the amount of the enzyme-labeled antibody bound to the solid phase, and the amount of the enzyme-labeled antibody is determined by the amount of the antigen bound to the immobilized antibody, the enzyme activity is correlated with the antigen amount. Here, as the enzyme, peroxidase is widely used because it has a relatively small molecular weight, a fast reaction, and high sensitivity can be expected in a short time.

  Conventionally, peroxidase activity has been measured by various reaction methods. The most commonly used method is the colorimetric determination of a dye by forming a coloring substance by oxidizing a coloring substance in the presence of a peroxide. Yes. Various materials have been used as such coloring substances. Orthophenylenediamine or the like has been used as a typical coloring material, but it is harmful to the human body and requires caution during use. Tetraalkylbenzidine has been developed as a new coloring substance, and 3,3 ′, 5,5′-tetramethylbenzidine (hereinafter referred to as TMBZ), which has extremely sensitive sensitivity and is non-carcinogenic, has been developed (non-patented). It is used in the sandwich method (Patent Document 1) and the test strip (Patent Document 2) because of its usefulness.

JP 62-28666 A Japanese Patent Laid-Open No. 52-21892

Tetrahedron, 30, 3299-3302, 1974

  In order to stop the color reaction by peroxidase, a strong acid such as 0.5 to 2 M sulfuric acid is generally used. However, when sulfuric acid is used to stop the peroxidase reaction using tetraalkylbenzidine as the coloring material, the rate of fading increases as the color intensity increases and the risk of precipitate formation increases, affecting the reliability of the data. It was found that there is a possibility of affecting. Further, since the fading becomes prominent after 1 hour, it has been found that there is a problem in that the time condition of the measurement is remarkably limited.

  In view of the above situation, an object of the present invention is to provide a method for stopping a color reaction with a peroxidase that suppresses fading after the reaction is stopped in a color reaction with a peroxidase, and a stop agent used in the stop method. .

  The inventors of the present invention have made extensive studies in order to solve the above-described problem of improving the color stability after stopping the reaction. As a result, in the peroxidase reaction containing tetraalkylbenzidine as a color-developing substance, it was found that the reaction solution to which a reaction terminator containing citric acid was added had high coloration stability, and the present invention was completed.

That is, when the present invention is outlined, it relates to the following [1] to [11].
[1] A method for stopping a peroxidase reaction, comprising adding citric acid to a peroxidase reaction solution containing a peroxidase and a tetraalkylbenzidine or a salt thereof to stop the reaction.
[2] The method according to [1], wherein the tetraalkylbenzidine or a salt thereof is 3,3 ′, 5,5′-tetramethylbenzidine or a salt thereof.
[3] The method according to [1], wherein citric acid is added to the peroxidase reaction solution so that the final concentration is 0.125 M or more.
[4] The method according to [1], wherein the peroxidase contains a peroxidase-labeled product.
[5] The method according to [4], wherein the peroxidase-labeled product is a peroxidase-labeled antibody or antigen.
[6] The reaction terminator used in the method for terminating the peroxidase reaction according to any one of [1] to [5], containing citric acid as an active ingredient.
[7] A kit for measuring peroxidase activity comprising a peroxidase reaction terminator containing (1-a) tetraalkylbenzidine or a salt thereof and (1-b) citric acid as an active ingredient.
[8] A peroxidase reaction terminator containing citric acid as an active ingredient is added to a reaction solution containing tetraalkylbenzidine or a salt thereof and peroxidase so that the final concentration of citric acid is 0.125 M or more. [7] The kit according to [7].
[9] (2-a) tetraalkylbenzidine or a salt thereof,
(2-b) A kit for an enzyme immunoassay containing a peroxidase-labeled antibody or antigen, and (2-c) a peroxidase reaction terminator containing citric acid as an active ingredient.
[10] A peroxidase reaction terminator containing citric acid as an active ingredient is a reaction solution containing tetraalkylbenzidine or a salt thereof and a peroxidase-labeled antibody or antigen, and the final concentration of citric acid is 0.125 M or more. The kit according to [9], which is a reaction terminator added so that
[11] The kit according to any one of [7] to [10], wherein the tetraalkylbenzidine or a salt thereof is 3,3 ′, 5,5′-tetramethylbenzidine or a salt thereof.

  According to the present invention, in the colorimetric determination of peroxidase activity, the coloration after reaction is stabilized, so that the sensitivity or accuracy of measurement of peroxidase activity is improved. In addition, since no precipitation was observed when the reaction was stopped by sulfuric acid, it was possible to measure in a region where the peroxidase activity was high. In addition, citric acid is not toxic to the human body as it is used as a food additive, so it can be effective in stopping the reaction and improving the safety of the reaction stopper. The problem is less likely to occur. Further, the citric acid solution has a low metal corrosivity, so that it is not likely to corrode the analytical instrument.

FIG. 1 is a graph showing the relationship between the absorbance of a reaction solution after elapse of a color reaction with peroxidase by addition of sulfuric acid or citric acid aqueous solution of each concentration and the elapsed time. FIG. 2 is a graph showing the relationship between the absorbance of the color reaction solution with peroxidase after the reaction is stopped by the addition of sulfuric acid and the PIP concentration. FIG. 3 is a graph showing the relationship between the PIP concentration and the absorbance of the color reaction solution with peroxidase after the reaction was stopped by adding an aqueous citrate solution.

  Peroxidase is an enzyme that reduces peroxides such as peroxides of hydrogen peroxide. Peroxidase is known to be extracted from horseradish, milk, yeast, leukocytes, erythrocytes and the like. In the present invention, peroxidase derived from horseradish, which is widely used as a research reagent and labeling enzyme, is preferred.

  The color reaction by peroxidase as used in the present invention refers to a reaction catalyzed by peroxidase, for example, a color reaction of a dye conjugated to a decomposition reaction of hydrogen peroxide. In the present specification, such a dye is referred to as a coloring substance, and a color reaction by peroxidase is sometimes simply referred to as a peroxidase reaction. For example, in the “Enzyme Immunoassay” edited by Yuji Ishikawa et al., Pages 30-49 (published in 1982), peroxidase-labeled antibodies or antigens are used and the amount of peroxidase activity is peroxidized. An enzyme immunoassay method is described in which measurement is performed using a substrate solution containing a product and a coloring substance. In the present invention, tetraalkylbenzidine or a salt thereof is used as a color developing substance, and therefore any method for measuring the amount of peroxidase activity using a substrate solution containing a color developing substance selected from peroxides and tetraalkylbenzidine or a salt thereof. This method is also applied.

  Specifically, the method of the present invention can be used in a method for detecting a peroxide such as hydrogen peroxide using peroxidase, or a method for detecting and measuring a peroxidase in a sample. Further, as described above, peroxidase is widely used for the detection of substances using antibodies, and the method of the present invention can be suitably used for enzyme immunoassays using peroxidase-labeled antibodies or antigens. In the present invention, a liquid in which a peroxidase reaction is performed in such a method is referred to as a peroxidase reaction liquid, and the reaction liquid contains a peroxide, a peroxidase, and a tetraalkylbenzidine or a salt thereof as a coloring substance. These components may be present in the reaction solution in advance or may be added separately.

  In the method for stopping the peroxidase reaction according to the present invention, the reaction is stopped by adding citric acid to the peroxidase reaction solution. In the present invention, when the final concentration of citric acid in the reaction solution is 0.125 M or more, the color reaction by peroxidase is stably stopped. In addition, when performing absorbance measurement at 450 nm used in the end point assay of a color reaction with peroxidase using 3,3 ′, 5,5′-tetramethylbenzidine as a color substance, the higher the final citric acid concentration, the higher the absorbance. Therefore, it is preferable in terms of measurement sensitivity. Therefore, the citric acid concentration is preferably 0.125M or more, more preferably 0.24M or more, still more preferably 0.5M or more, and even more preferably 1M or more after the final concentration after being added to the peroxidase reaction solution. is there. The concentration of citric acid is not particularly limited, but for example, the final concentration of citric acid after addition to the peroxidase reaction solution is preferably 3M or less, more preferably 1.5M or less. Moreover, the suitable range of the citric acid final concentration in this invention is 0.980M-1.471M (refer Example 2 mentioned later). In this invention, the citric acid solution prepared so that it may become this citric acid final concentration can be used as a reaction terminator.

  The peroxidase in the present invention may be a peroxidase alone or a peroxidase labeled product. Examples of the peroxidase-labeled product include peroxidase-labeled antibodies or antigens, and the labeled product can be prepared according to a known method. The peroxidase-labeled antibody used in the present invention may be either a polyclonal antibody or a monoclonal antibody against the substance to be measured, but a peroxidase-labeled monoclonal antibody is preferred from the viewpoint of measurement reproducibility. Labeling of the antibody with peroxidase can be performed by a known method, for example, a method using glutaraldehyde, a method using periodic acid, or a method using introduction of a maleimide group.

  The tetraalkylbenzidine or a salt thereof used in the present invention is a derivative of benzidine, which is oxidized in combination with the decomposition of hydrogen peroxide by peroxidase to turn into a strong blue compound, and becomes yellow under acidity. Examples of the tetraalkylbenzidine include 3,3 ', 5,5'-tetramethylbenzidine. Examples of the salt include 3,3 ', 5,5'-tetramethylbenzidine dihydrochloride dihydrate.

  Among these, 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ) or a salt thereof preferably used in the present invention can measure peroxidase activity with very sensitive sensitivity and is non-carcinogenic. Is done. The composition of the composition containing TMBZ used in the present invention is not limited as long as it can be used for a color reaction of a dye conjugated to a peroxidase reaction, and a commercial product such as a hydrochloride of TMBZ or a DMSO solution is used. can do.

  TMBZ is oxidized in combination with the decomposition of hydrogen peroxide by peroxidase, and is vividly colored blue-green (λmax = 655 nm). This color development immediately changes to a yellow (λmax = 450 nm) dye under acidic conditions. Therefore, in the method of the present invention in which the color reaction by peroxidase is stopped with citric acid, the reaction can be stopped under acidic conditions as in the conventional method using sulfuric acid, and the colorimetric determination of peroxidase activity can be performed at the same measurement wavelength. it can.

  The temperature at which the peroxidase reaction is stopped is not particularly limited as long as citric acid is sufficiently mixed with the reaction solution, but is preferably 4 to 37 ° C and more preferably 20 to 30 ° C.

  In addition, when using the method of this invention by an immunoassay, there is no limitation in particular in the substance used as a measuring object. Substances that are desired to be measured in fields such as research, diagnosis, and environmental measurement, and for which antibodies that bind to the substances can be obtained can be measured. For example, it includes antigens and allergens derived from human or non-human animal organisms, plants, microorganisms, and viruses.

  Thus, the reaction of peroxidase can be stopped by citric acid, and as a result, the peroxidase activity can be measured by the coloration of tetraalkylbenzidine under acidic conditions. Therefore, as another aspect of the present invention, there is a method for measuring peroxidase activity, characterized in that citric acid is added to a peroxidase reaction solution containing peroxidase and tetraalkylbenzidine or a salt thereof to stop the reaction.

  Moreover, since the peroxidase reaction can be stopped by adding citric acid to the reaction solution as one embodiment of the present invention, a reaction stopper for peroxidase reaction containing citric acid as an active ingredient is provided. The reaction terminator is preferably used in the method for terminating the peroxidase reaction of the present invention.

  The reaction terminator in the present invention contains citric acid as an active ingredient, stops the reaction catalyzed by peroxidase when added to a reaction solution containing tetraalkylbenzidine and peroxidase, and is a yellow pigment produced after the reaction is stopped There is no limitation as long as the absorbance is stably maintained. Specifically, for example, an aqueous citric acid solution can be mentioned.

  The citric acid content in the reaction terminator is not generally determined, and the final concentration of citric acid after being added to the peroxidase reaction solution is preferably 0.125 M or more, more preferably 0.5 M or more, and even more preferably 1 M or more. A citric acid solution prepared to be suitable as a reaction terminator. Although there is no particular upper limit, for example, a reaction terminator prepared so that the final concentration of citric acid after addition to the reaction solution is preferably 3M or less, more preferably 1.5M or less is suitable for the present invention. . If it becomes such a citric acid concentration, the method for preparing the reaction terminator is not particularly limited, and for example, it can be prepared by dissolving citric acid monohydrate in water.

  In the present specification, “containing citric acid as an active ingredient” means containing citric acid as a component that substantially stops the peroxidase reaction, and the reaction terminator of the present invention includes citric acid. In addition, the well-known component which can stop peroxidase reaction may be contained in the range which does not impair the effect of this invention, and the component which does not participate in peroxidase reaction may be contained.

  A kit containing a reaction terminator used in the method for terminating the peroxidase reaction of the present invention is also an aspect of the present invention.

In particular,
(1-a) tetraalkylbenzidine or a salt thereof, and (1-b) a kit for measuring peroxidase activity containing a peroxidase reaction terminator containing citric acid as an active ingredient (peroxidase activity measurement kit) Is mentioned.

  As the (1-a) tetraalkylbenzidine or a salt thereof in the kit, those similar to those used in the method for terminating the peroxidase reaction of the present invention can be used, among which 3, 3 ′, 5, 5′-tetramethylbenzidine or a salt thereof is preferred. The (1-b) reaction terminator is not particularly limited as long as it is the reaction terminator of the present invention.

  The form of the kit and the method of use are not particularly limited. For example, (1-b) a reaction stopper for peroxidase reaction containing citric acid as an active ingredient, and (1-a) a reaction solution containing tetraalkylbenzidine and peroxidase. In addition, the peroxidase activity can be measured by stopping the peroxidase reaction by adding so that the final concentration of citric acid is 0.125 M or more. You may add the component similar to the component contained in the reaction liquid in the stop method of the peroxidase reaction of this invention to this reaction liquid. Moreover, what is necessary is just to add the reaction terminator at the point which wants to stop reaction.

Also,
(2-a) tetraalkylbenzidine or a salt thereof,
A kit for an enzyme immunoassay containing (2-b) peroxidase-labeled antibody or antigen and (2-c) a peroxidase reaction terminator containing citric acid as an active ingredient may be mentioned.

  As the (2-a) tetraalkylbenzidine or a salt thereof in the kit, the same one as used in the method for terminating the peroxidase reaction of the present invention can be used, among which 3, 3 ′, 5, 5′-tetramethylbenzidine or a salt thereof is preferred.

  (2-b) Examples of the peroxidase-labeled antibody or antigen include those described in the peroxidase reaction termination method of the present invention, and the preparation method thereof is also the same.

  (2-c) The reaction terminator is not particularly limited as long as it is the reaction terminator of the present invention.

  The form of the kit and the method of use are not particularly limited. For example, (2-c) a peroxidase reaction terminator containing citric acid as an active ingredient, (2-a) tetraalkylbenzidine or a salt thereof and (2- b) Enzyme immunoassay can be performed by adding peroxidase to a reaction solution containing an antibody or antigen so that the final concentration of citric acid is 0.125 M or more and measuring peroxidase activity. You may add the component similar to the component contained in the reaction liquid in the stop method of the peroxidase reaction of this invention to this reaction liquid. Moreover, what is necessary is just to add the reaction terminator at the point which wants to stop reaction.

More specifically, as an example of the enzyme immunoassay method using the present invention, for example, the measurement in the procedures of the following steps 1) to 5) is exemplified.
1) Capture of a substance to be measured by a solid-phased primary antibody and washing of the solid phase.
2) Binding of peroxidase-labeled secondary antibody to the substance to be measured and washing of the solid phase.
3) Incubation by adding a substrate solution (hydrogen peroxide and TMBZ) (color reaction by peroxidase).
4) Stop the color reaction by peroxidase by adding the reaction terminator of the present invention.
5) Measure absorbance.

  The above steps 1) to 3) can be carried out in exactly the same manner as in the conventional enzyme immunoassay using a peroxidase-labeled antibody. In the present invention, since fading of the coloration that occurs after the reaction is stopped is suppressed, even if it takes time between steps 4) and 5), reproducible data can be obtained. .

  Needless to say, the present invention can be applied to enzyme immunoassays other than the sandwich method as described above (for example, competitive method, Western blot method).

Used for detection and quantification of substances to be measured
(3-a) tetraalkylbenzidine or a salt thereof,
(3-b) a peroxidase-labeled antibody that binds to a measurement target substance, or a peroxidase-labeled measurement target substance, and (3-c) a peroxidase reaction terminator containing citric acid as an active ingredient,
A kit containing the above is also encompassed by the present invention.

As described in detail above, as an aspect of the present invention,
[12] A method for stabilizing a color reaction by peroxidase, comprising adding citric acid to a peroxidase reaction solution containing tetraalkylbenzidine as a color developing substance,
[13] The method according to [12], wherein the tetraalkylbenzidine is 3,3 ′, 5,5′-tetramethylbenzidine.
[14] The method according to [12], wherein citric acid is added to the peroxidase reaction solution so that the final concentration is 0.125 M or more,
[15] The method according to [12], wherein the peroxidase reaction solution is a reaction solution containing a peroxidase-labeled product,
[16] The method according to [15], wherein the peroxidase-labeled product is a peroxidase-labeled antibody or antigen.
[17] A reaction terminator and / or stabilizer used in the method for stabilizing a color reaction with a peroxidase according to any one of [12] to [16], containing citric acid as an active ingredient,
Is also provided.

  EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the scope of the examples. In addition, in this specification, room temperature is 4 to 37 degreeC unless there is particular notice.

Example 1 Termination of Color Reaction by Peroxidase with Reaction Stopper Containing Citric Acid 1.5 mL Micro Centrifuge Tube (manufactured by Tref) and TMB One Component HRP Microwell Substrate (manufactured by SurModics) and 500 μL obtained by the present inventors After adding 20 μL of the anti-Rat Collagen 2 antibody-peroxidase labeled product solution using periodic acid, the mixture was stirred and allowed to stand at room temperature for 8 minutes to perform a color reaction with peroxidase. Next, 500 μL of various reaction stoppers were added to stop the reaction. Dispense 100 μL of each reaction solution after stopping the reaction into each well of 96 Well Micro titer Plate (Thermo Fisher Scientific) and use Thermo Max Micro plate reader (from Molecular Devices 5 immediately after stopping). The absorbance at 450 nm was measured. The reaction was performed in quadruplicate and the average value of the measured values was adopted. Here, the reaction terminator is 0.5 M sulfuric acid prepared by diluting sulfuric acid (manufactured by Wako Pure Chemical Industries) with purified water, or citric acid monohydrate (manufactured by Nacalai Tesque) 0.25 M, 0.5 M 1M, 1.5M, and 2M aqueous solutions were used, and the final concentration of sulfuric acid or citric acid used in each reaction stopper in the reaction solution was 0.245M for sulfuric acid, 0.123M, 0.245M for citric acid, They were 0.490M, 0.735M, and 0.980M.

  The result is shown in FIG. That is, FIG. 1 shows 0.5M sulfuric acid (● in FIG. 1) or citric acid aqueous solution of each concentration (in FIG. 1, 0.25M (×), 0.5M (◇), 1M (Δ), 1.5M ( (□) It is a figure which shows the relationship between the light absorbency of the reaction liquid after reaction stop by addition of 2M ((circle)), and elapsed time. As shown in FIG. 1, when 0.5 M sulfuric acid or an aqueous citric acid solution having various concentrations was added for the purpose of stopping the reaction, no increase in the absorbance of the reaction solution was observed immediately after the addition. From this, it was shown that citric acid can stop the color reaction by peroxidase like sulfuric acid. It was confirmed that even with the dilute aqueous citric acid solution (concentration 0.25 M) used this time, the color reaction by peroxidase after addition did not proceed and the reaction could be stopped. On the other hand, the absorbance at 450 nm of the reaction solution after stopping the reaction with each citric acid aqueous solution compared with the reaction solution after stopping the reaction with sulfuric acid is low when the final concentration of citric acid is low, and as the final concentration of citric acid increases. It became clear that it was necessary to add 2 M or more citric acid aqueous solution, that is, to add citric acid having a final concentration of 1 M or more in order to obtain the same absorbance as that of the reaction solution after stopping the reaction with 0.5 M sulfuric acid.

  Surprisingly, in the present measurement conditions showing an absorbance of around 4, compared to the fact that the reaction solution after the reaction was discolored and the decrease in absorbance was observed under the reaction termination condition with sulfuric acid as shown in FIG. It was found that, under the reaction termination conditions, the fading was suppressed and the absorbance stability was remarkably improved under any citric acid concentration conditions. In addition, the precipitates that were observed after the reaction was stopped under the reaction stop condition with sulfuric acid were not observed under the reaction stop condition with citric acid.

Example 2 Identification of a citric acid aqueous solution concentration at which an absorbance equivalent to that of a reaction solution after stopping the reaction with 0.5 M sulfuric acid was obtained. The same test as in Example 1 was performed, and the absorbance at 450 nm of the reaction solution immediately after stopping the reaction was measured. . The reaction was performed in quadruplicate and the average value of the measured values is shown in Table 1. Here, the reaction terminator used was 0.5 M sulfuric acid or an aqueous citric acid monohydrate solution of 2 M, 2.25 M, 2.4 M, 3 M. The final concentration of sulfuric acid or citric acid in each reaction solution was 0.245 M for sulfuric acid, 0.980 M, 1.103 M, 1.176 M, and 1.471 M for citric acid. As shown in Table 1, the reaction was carried out in the final concentration range of 0.980 to 1.471M.

  As shown in Table 1, it was found that the same absorbance as the reaction solution after stopping the reaction by adding 0.5 M sulfuric acid was stably obtained under the condition of adding 2 M or more citric acid aqueous solution. Therefore, in the following Examples, 50 w / v% citric acid monohydrate aqueous solution was used as a reaction terminator containing citric acid. A 50 w / v% aqueous solution of citric acid monohydrate corresponds to 2.38M.

Example 3 Evaluation of Stability of Color Reaction by Peroxidase The following test was conducted using TaKaRa Procollagen type I C-peptide (PIP) EIA Kit (Precoated) (manufactured by Takara Bio Inc.). After adding 100 μL of peroxidase-labeled anti-PIP monoclonal antibody solution attached to the kit to each well of the anti-PIP monoclonal antibody plate attached to the kit, add 20 μL of the PIP solution of each concentration prepared in advance to each well with a micropipette. The reaction was carried out at 0 ° C. for 3 hours. The PIP solution was immediately added (within 5 minutes). Next, the reaction solution was discarded, and after washing 4 times with PBS, 100 μL of the TMBZ solution supplied with the kit was added to each well with an 8-unit pipette and allowed to react at room temperature (20-30 ° C.) for 15 minutes. Next, 100 μL of a reaction terminator was added to each well in the order of adding the TMBZ solution to stop the reaction and mixed well. Immediately after the reaction was stopped, 1 hour and 3 hours later, the absorbance at 450 nm was measured. During each measurement, the reaction solution after stopping the reaction was allowed to stand at room temperature. The reaction was performed in duplicate and the average value of the measured values was adopted. Here, 0.5 M sulfuric acid or 50 w / v% citric acid monohydrate aqueous solution was used as a reaction terminator. The final concentration of sulfuric acid or citric acid in each reaction solution was 0.25 M for sulfuric acid and 1.19 M for citric acid.

  The results are shown in FIGS. That is, FIG. 2 shows the reaction mixture at 450 nm immediately after the reaction was stopped by addition of 0.5 M sulfuric acid (◯ in FIG. 2), 1 hour later (□ in FIG. 2), and 3 hours later (◇ in FIG. 2). It is a figure which shows the relationship between a light absorbency and PIP density | concentration. FIG. 3 shows a color reaction solution with peroxidase immediately after stopping the reaction by adding 50 w / v% aqueous citrate solution (● in FIG. 3), 1 hour later (■ in FIG. 3), and 3 hours later (♦ in FIG. 3). It is a figure which shows the relationship between the light absorbency in 450 nm, and PIP density | concentration. As is clear from FIG. 2, the reaction solution after the termination of the reaction by addition of 0.5 M sulfuric acid is particularly fading under conditions where the PIP concentration is 640 ng / mL or more, and the degree of decrease in absorbance over time is high, and the reliability of the data. Was damaged. On the other hand, as is apparent from FIG. 3, the reaction solution after the reaction was stopped by addition of citric acid maintained the absorbance curve immediately after the reaction was stopped even after 3 hours from the stop of the reaction. From this, it became clear that the color stability of the reaction solution after the stop of the color reaction by peroxidase was dramatically improved by using the reaction terminator containing citric acid according to the present invention as an active ingredient. This indicates that the accuracy of the measurement is improved and the limitation on the time condition of the measurement is improved.

  In the reaction solution after the reaction was stopped by addition of 0.5 M sulfuric acid, the occurrence of precipitation was observed under the condition that the PIP concentration was 640 ng / mL or more. On the other hand, in the reaction solution after the termination of the reaction by adding 50 w / v% citric acid aqueous solution, precipitation did not occur even when the PIP concentration was 2560 ng / mL, and the absorbance could be measured normally. This indicates that the use of a reaction terminator containing citric acid as an active ingredient according to the present invention has the advantage that the measurable range of the kit, which has been conventionally limited to a PIP concentration of 640 ng / mL or less, is widened. .

  According to the present invention, the sensitivity or accuracy of measurement of peroxidase activity can be improved. Therefore, the present invention is useful in the fields of immunobiochemistry, genetic engineering, applied life science, and medicine.

Claims (11)

  A method for terminating a peroxidase reaction, comprising adding citric acid to a peroxidase reaction solution containing peroxidase and a tetraalkylbenzidine or a salt thereof to stop the reaction.   The method according to claim 1, wherein the tetraalkylbenzidine or a salt thereof is 3,3 ', 5,5'-tetramethylbenzidine or a salt thereof.   The method according to claim 1, wherein citric acid is added to the peroxidase reaction solution so that the final concentration is 0.125M or more.   The method according to claim 1, wherein the peroxidase contains a peroxidase-labeled product.   The method according to claim 4, wherein the peroxidase-labeled product is a peroxidase-labeled antibody or antigen.   The reaction terminator used for the peroxidase reaction termination method according to any one of claims 1 to 5, comprising citric acid as an active ingredient. (1-a) A kit for measuring peroxidase activity containing a peroxidase reaction terminator containing tetraalkylbenzidine or a salt thereof and (1-b) citric acid as an active ingredient.   A reaction stopper in which a peroxidase reaction stopper containing citric acid as an active ingredient is added to a reaction solution containing tetraalkylbenzidine or a salt thereof and peroxidase so that the final concentration of citric acid is 0.125 M or more. The kit according to claim 7, which is an agent. (2-a) tetraalkylbenzidine or a salt thereof,
(2-b) A kit for an enzyme immunoassay containing a peroxidase-labeled antibody or antigen, and (2-c) a peroxidase reaction terminator containing citric acid as an active ingredient.   The peroxidase reaction terminator containing citric acid as an active ingredient is added to the reaction solution containing tetraalkylbenzidine or its salt and peroxidase-labeled antibody or antigen so that the final concentration of citric acid is 0.125 M or more. The kit according to claim 9, which is a reaction terminator added to.   The kit according to any one of claims 7 to 10, wherein the tetraalkylbenzidine or a salt thereof is 3,3 ', 5,5'-tetramethylbenzidine or a salt thereof.

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