JP2759800B2 - Substrate reagent and method of stabilizing detection system - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for stabilizing a detection system utilizing an oxidation reaction of an enzyme or a mimetic enzyme thereof, and a stable substrate reagent useful for the detection system.

[Prior art and its problems] In the field of clinical tests and diagnostics, measurement methods using enzymatic reactions are widely used for qualitative and quantitative determination of various in vivo substances.

For example, in an enzyme immunoassay utilizing an antigen-antibody reaction, an enzyme is used as a label, and the amount of an analyte or a substance to be analyzed in a sample is measured using the amount of activity or a change in the amount of activity as an index. As an enzyme used as a label, peroxidase (peroxidase) is most widely used in terms of economy and sensitivity. In addition, measurement of certain biological components, for example, cholesterol, glucose, monoamine, etc., is performed by cholesterol oxidase, a corresponding oxidase,
It is carried out by quantifying the amount of hydrogen peroxide generated by the action of glucose oxidase, monoamine oxidase or the like using a peroxidase.

For the measurement of the activity of peroxidase and the quantification of hydrogen peroxide, the enzyme reaction is used in a detection system comprising a peroxidase, a peroxide substance such as hydrogen peroxide and a substrate. The enzymatic reaction is generally performed in an aqueous solvent, and the measurement is performed by analyzing the degree to which the substrate is oxidized according to the amount of the peroxidase or the peroxidant.

The substrate to be used is a compound that provides an analysis means (generally, a substance) by an oxidizing action of a peroxidase, and an aromatic amine compound or a phenolic compound which usually produces a color dye is known. Aromatic amine compounds, such as o-phenylenediamine, can obtain high sensitivity,
Most widely used.

However, in general, the substrate, including o-phenylenediamine, is often not sufficiently stable, and is non-enzymatically oxidized by light, oxygen, etc., resulting in a high background concentration in the detection system or the detection value over time. This instability caused a measurement error in the actual measurement, such as swinging to a larger value. Therefore, handling in a dark place, time limitation from preparation to use of a substrate-containing reagent solution, and the like are imposed, and there has been a strong demand for a method of stabilizing a substrate and a detection system using the substrate.

[Object of the invention]

In view of the above circumstances, an object of the present invention is to provide a stable substrate reagent in which non-enzymatic oxidation is suppressed and a method for stabilizing a detection system.

[Configuration of the invention]

An object of the present invention is to provide a condensed phosphorus oxo acid derivative comprising three or more phosphorus oxo acid units in a detection system using an enzyme having an oxidizing action or a mimetic enzyme thereof and a substrate which provides analysis means by being oxidized. And a substrate reagent containing the substrate and the condensed phosphorus oxo acid derivative.

In the embodiment of the method for stabilizing the substrate reagent and the detection system of the present invention, the substrate preferably provides a coloring dye, a luminescent substance or a fluorescent substance, and is preferably an aromatic amine compound, especially o-phenylenediamine, o-dianisidine, 3,3 ',
Preferably it is 5,5'-tetramethylbenzidine.

Preferably, the enzyme or its pseudo-enzyme is a peroxidase or its peroxidase-like enzyme. Here, the pseudo-enzyme refers to a substance that is considered to have the same effect as the above-mentioned enzyme.

Further, the above-mentioned condensed phosphorus oxo acid derivative has an oxidation number of 3 or more and 5 or more.
Preferred are those comprising a phosphorus oxo acid unit of the formula (I), and preferred are condensed phosphorus oxo acids or compounds in which some or all of the condensed phosphorus oxo acids are salts or mixtures thereof. Further, alkali metal polyphosphates, alkali metal metaphosphates Salts or alkali metal ultraphosphates are preferred.

 Next, the present invention will be described in detail.

The condensed phosphorus oxo acid derivative according to the present invention may be composed of three or more phosphorus oxo acid units, and preferably has three to six phosphorus oxo acid units bonded.

Further, each unit phosphorous oxoacid may be any phosphoric acid having an oxidation number of 2 to 5, but it is preferable that all the unit phosphorous oxoacids be phosphoric acid having an oxidation number of 5.

The condensed phosphorus oxo acid derivatives according to the present invention include those having various structures depending on the bonding mode of the unit phosphorus oxo acid, that is, those having a chain structure, a cyclic structure or a cross-linked structure, and a structure having a coexistence of those structures. A metaphosphoric acid derivative or an ultraphosphoric acid derivative, or a derivative coexisting with a bond thereof is preferable.

Examples of the derivative include condensed phosphorus oxoacids or condensed phosphorus oxoacid inorganic salts, for example, alkali metal salts such as sodium, potassium and lithium, alkaline earth metal salts such as magnesium and calcium, ammonium salts, and condensed phosphorus oxoacid organic salts. For example, pyridium salts and the like can be mentioned. Also, organic phosphorus oxo acid derivatives such as condensed phosphorus oxo acid ester, condensed phosphorus oxo acid amide, and those in which an organic substituent is directly bonded to phosphorus can be used. In addition, a free acid unit, a unit forming a salt, and a unit to which an organic substituent is bonded may be mixed in one molecule,
It is preferred that the condensed phosphorus oxo acid or a part or all of the condensed phosphorus oxo acid be a salt.

Further, in the present invention, two or more kinds of condensed phosphorus oxo acid derivatives may be used as a mixture.

Next, specific examples of the condensed phosphorus oxo acid derivative according to the present invention will be described, but the present invention is not limited thereto.

In the alkyl substituent, an alkyl group having 1 to 8 carbon atoms is preferable.

Tripolyphosphate pentasodium tripolyphosphate trisodium tripolyphosphate pentapotassium tripolyphosphate pentaammonium tripolyphosphate tetrasodium tetrapolyphosphate hexasodium pentapolyphosphate pentasodium pentapolyphosphate hexasodium hexapolyphosphate hexasodium octapolyphosphate octasodium pentapolyphosphate trisodium trimetaphosphate trisodium trimetaphosphate trimetaphosphate Triammonium trimetaphosphate tetrametaphosphate tetrasodium tetrametaphosphate tetrapotassium tetrametaphosphate pentasodium pentametaphosphate hexametaphosphate hexasodium hexametaphosphate Graham salt Disodium tripolyphosphate pentaalkyl Rutripolyphosphoric acid Monoalkyltetrapolyphosphoric acid Tetraalkyltetrapolyphosphoric acid Monoalkyltrimetaphosphate disodium Trialkyltrimetaphosphate Triphenyltripolyphosphoric acid The substrate according to the present invention provides an analytical means by being oxidized. For example, a compound that produces a coloring dye, a luminescent substance or a fluorescent substance may be used, and generally includes a substrate used in a detection system utilizing an enzymatic reaction of a peroxidase and hydrogen peroxide. Representative examples include aromatic amine compounds and phenol compounds.

Specific examples include o-phenylenediamine, m-phenylenediamine, p-phenylenediamine, benzidine, and o-phenylenediamine.
Dianididine, o-tolidine, 3,3 ', 5,5'-tetramethylbenzidine, diaminobenzidine, dicarboxyzine,
2,2'-azino-bis (3-ethylbenzothiazoline-
6-sulfonic acid), 3-amino-9-ethylcarbazole, 4-chloro-1-naphthol, pyrogallol, 4-amino-antipyrine, 4-amino-N, N-dimethylaniline, 4-aminoantipyrine and dimethylaniline, 4-aminoantipyrine and dimethylaniline mixture, 4-aminoantipyrine and dimethylananiline mixture, 4-aminoacetipirin and phenol mixture, pyrogallol and p-
A mixture of phenylenediamine, a mixture of 4-chloro-1-naphthol and N-ethyl-N'-hydroxyethyl-3-methyl-4-aminoaniline, 5-aminosalicylic acid, 3-methyl-2-benzothiazolinone hydrazone And dimethylaniline, 4-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) propionic acid, luminol and the like, and salts thereof are also useful.

The enzyme or pseudoenzyme according to the present invention is not particularly limited as long as it catalyzes a reaction that oxidizes a substrate and provides an analysis means, but a peroxidase or a pseudoperoxidase is preferable. A peroxidant is required for the oxidation reaction by peroxidase or pseudoperoxidase. The peroxide may be any peroxide, such as an organic peroxide,
Hydrogen peroxide is preferred.

As the peroxidase, for example, horseradish peroxidase, lactoperoxidase, myeloperoxidase, glutathione peroxidase, cytochrome C peroxidase, etc. can be used. As the pseudoperoxidase, for example, metals such as hemoglobin, iron, gold, silver and the like, Compounds can be used.

It is sufficient that the substrate reagent of the present invention contains at least a substrate that provides an analysis means by being oxidized and a condensed phosphorus oxoacid derivative composed of three or more phosphorus oxoacid units.
In addition, a peroxide substance such as hydrogen peroxide or a peroxidase may be contained, and other substances such as a stabilizer may be contained as necessary.

The substrate reagent of the present invention includes various forms such as solid, powder, and solution, and is in the form of a solution that can be used as it is in a detection system utilizing an enzyme reaction (hereinafter referred to as a substrate reagent reagent). Is also included. If necessary, these can be prepared into a solution of the substrate reagent usable in the detection system by a suitable operation such as dissolution in a solvent.

The detection system according to the present invention is a system in which the substrate or the mimetic enzyme thereof is used to oxidize the substrate, and the degree of the oxidation is detected, for example, by optical means. It is possible to obtain the amount of the analytical substance in the corresponding sample. Generally, the oxidation reaction of the enzyme or mimetic enzyme is performed in a solution in which the substrate is dissolved.

In general, an aqueous solvent is preferable as a solvent used for the substrate reagent reagent or the detection system, but an organic solvent or a mixed solvent of an organic solvent and an aqueous solvent may be used as long as it does not extremely inhibit the enzymatic oxidation reaction. The solvent preferably has a pH value suitable for the enzymatic reaction, and is preferably prepared with a commonly used buffer. When using peroxidase, pH
3 to 9 are preferred.

The order of adding each component to the solvent for preparing the substrate reagent solution is not particularly limited.

The concentration of the condensed phosphorus oxoacid derivative according to the present invention contained in the substrate reagent reagent solution or the detection system, which is one form of the substrate reagent of the present invention, takes into account the degree of inhibition in the enzymatic reaction and the degree of inhibition of non-enzymatic oxidation. It is determined, and varies slightly depending on the type, but is usually 0.01 to 1000 mM, preferably 0.1
~ 300mM. More preferably, for example, about 0.3 to about 50 mM for pentapotassium tripolyphosphate, about 0.3 to 50 mM for hexasodium tetrapolyphosphate, about 0.3 to about 50 mM for disodium tetraultraphosphate, and hexasodium hexametaphosphate. About 1 to 100 mM. Further, the condensed phosphorus oxoacid derivative according to the present invention has a buffering ability, and can be used as a buffer for a substrate reagent test solution by adjusting the pH to an appropriate value by adding an appropriate acid or base. Further, the concentration of the substrate is suitably 0.1 to 100 mM.

A detection system in which the substrate reagent of the present invention is particularly usefully applied, and in which the stabilization method of the present invention is particularly effective, comprises a peroxidant, a peroxidase, and a substrate that produces an optically detectable substance by an oxidizing action. It is a detection system, and the present invention enables stable and accurate measurement of the amount of hydrogen peroxide or the activity of peroxidase.

Also in this case, the order of adding each component to the solvent at the time of preparing the substrate reagent test solution is not particularly limited. For example, regarding the preparation of a substrate reagent reagent solution for identifying peroxidase, the advantage of storage stability of hydrogen peroxide is that the condensed phosphorus oxoacid derivative has the effect of suppressing the decomposition of peroxides, particularly hydrogen peroxide. Considering, for example, first, a solution in which hydrogen peroxide and a condensed phosphorus oxo acid derivative are dissolved in a solvent is used as a storage solution,
By adding the substrate to this just before use in the detection system,
It is also possible to prepare a target substrate reagent reagent solution.

For example, the detection system is performed as follows. That is, 0.1 mM
Substrate at a concentration of ~ 100mM and 0.01 ~ 1000m as stabilizer
A buffer having an arbitrary pH in the range of pH 3 to 9 containing the condensed phosphorus oxoacid derivative of the present invention at a concentration of M, preferably 0.01 to 300 mM is prepared. To measure the activity of peroxidase, hydrogen peroxide is further added, and to measure the amount of hydrogen peroxide, a certain amount of peroxidase is further added to prepare a substrate reagent test solution.
The substrate reagent reagent solution and the sample are mixed, an enzyme reaction is performed at a temperature of 2 to 50 ° C., and after a certain period of time, an acid such as sulfuric acid, a fluorine compound, sodium azide, and the like are added to stop the reaction. The substrate is oxidized according to the oxygen peroxide activity or the amount of hydrogen peroxide in the sample, and the resulting substance is optically detected.

If the produced substance is a coloring dye, for example, quantitative measurement can be performed by measuring the absorbance at the maximum absorption wavelength and comparing with a calibration curve prepared by performing the same operation on a known amount of peroxidase or hydrogen peroxide. It becomes possible.

The present invention relates to any enzyme immunoassay, for example, a homogeneous assay or a homogeneous assay, a one-antibody assay or a two-antibody assay, an enzyme-linked immunoabsorbent assay or an enzyme immunostaining. Is also applicable.

In the detection system using the method of the present invention or the detection system to which the substrate reagent of the present invention is applied, the non-enzymatic oxidation reaction of the substrate is suppressed without inhibiting the enzymatic reaction, and is extremely stabilized. In addition, an increase in the amount of product due to the non-enzymatic oxidation reaction after the stop of the enzymatic reaction is suppressed, and an accurate value is maintained.

According to the present invention, it has become possible to make the measurement of the enzyme activity or the amount of hydrogen peroxide and also the amount of the analysis target substance corresponding thereto extremely accurate.

〔Example〕

Hereinafter, the present invention will be described in more detail by examples,
These examples do not limit the scope of the invention in any way.

Example 1 A 50 mM citric acid-100 mM phosphate buffer (pH 4.9) containing o-phenylenediamine at a concentration of 3 mg / ml was prepared by adding pentasodium tripolyphosphate and without adding pentasodium tripolyphosphate. To a concentration of 0.02%. If the pH is shifted, the pH is adjusted to 4.9 with hydrochloric acid or sodium hydroxide.
And used as a substrate reagent test solution. The substrate reagent TS was dispensed into two 0.5 ml portions, one of which was added 10 μl of a horseradish peroxidase-conjugated goat anti-mouse immunoglobulin antibody (manufactured by Kappel) at a concentration of 0.5 μg / ml, and the other was added. Did not. Each was allowed to react in a dark place at 37 ° C. for 30 minutes. Then, 2 ml of 1NH ₂ SO ₄ was added to the mixture to stop the reaction, and the absorbance at 492 nm was measured. Table 1 shows the results.

The enzyme activity value was represented by the difference between the value when the enzyme-conjugated antibody was added and the value when no enzyme-conjugated antibody was added.

From Table 1, pentasodium tripolyphosphate was added from 0.3 mM to 50 mM.
It is evident that the addition within the above range significantly inhibits the non-enzymatic oxidation reaction without substantially inhibiting the enzymatic reaction and stabilizes the detection system.

Example 2 Example 2 was repeated except that pentapotassium tripolyphosphate was used instead of pentasodium tripolyphosphate as a condensed phosphorus oxoacid derivative to be added. Table 2 shows the results.

From Table 2, it is clear that the addition of pentapotassium tripolyphosphate in the range of about 0.3 to 50 mM hardly inhibits the enzymatic reaction, significantly suppresses the non-enzymatic oxidation reaction, and stabilizes the detection system. is there.

Example 3 The procedure of Example 1 was repeated, except that hexasodium tetrapolyphosphate was used instead of pentasodium tripolyphosphate as the condensed phosphorus oxoacid derivative to be added.

 Table 3 shows the results.

Table 3 shows that hexapolysodium tetrapolyphosphate was added at about 0.3 to 50 mM.
It is evident that the addition within the above range significantly suppresses the non-enzymatic oxidation reaction without substantially inhibiting the enzymatic reaction, and stabilizes the detection system.

Example 4 The same measurement as in Example 1 was performed except that hexasodium hexametaphosphate was used instead of pentasodium tripolyphosphate as a condensed phosphorus oxoacid derivative to be added.

 Table 4 shows the results.

From Table 4, hexasodium hexametaphosphate is about 1 to about 100m
It is clear that the addition in the range of M significantly suppresses the non-enzymatic oxidation reaction with almost no inhibition of the enzymatic reaction and stabilizes the detection system.

Example 5 The same operation as in Example 1 was carried out except that disodium tetraultraphosphate (molecular formula: Na ₂ P ₄ O ₁₁ ) was used instead of pentasodium tripolyphosphate as a condensed phosphorus oxoacid derivative to be added. Table 5 shows the results.

From Table 5, the disodium tetraultraphosphate was added to about 1 to
It is clear that the addition in the range of about 50 mM significantly suppresses the non-oxygenous oxidation reaction without substantially inhibiting the oxygen reaction, and stabilizes the detection system.

Example 6 Various condensed phosphoric acid derivatives were added to a 50 mM citric acid-100 mM phosphoric oxoacid buffer (pH 4.9) adjusted to a concentration of orthophenylenediamine of 3 mg / ml and a concentration of hydrogen peroxide of 0.02%, and Adjust the additive-free one, and use it as a substrate reagent TS. After dispensing 0.5 ml each and leaving under various conditions, 1NH ₂ S
2 ml of O ₄ was added, and the absorbance at 492 nm was measured. Table 6 shows the results.

As shown in Table 6, non-enzymatic oxidation reaction is suppressed by adding a condensed phosphorus oxo acid derivative such as pentasodium tripolyphosphate, pentapotassium tripolyphosphate, hexasodium tetrapolyphosphate, hexasodium hexametaphosphate, disodium tetraultraphosphate, and the aging is stable. It is clear that the sex is increasing.

Example 7 A substrate reagent TS was prepared in the same manner as in Example 6, allowed to stand in a dark place at 4 ° C. for 24 hours, and then 10 μl of a horseradish-bound goat anti-mouse immunoglobulin antibody (manufactured by Kappel) having a concentration of 0.5 μg / ml was prepared. The reaction was carried out at 37 ° C. for 30 minutes. 1NH ₂ SO
₄ Add 2 ml to stop the reaction, measure the absorbance at 492 nm, the case of no addition of the condensed phosphorus oxo acid derivative as a control,
The relative enzyme activity was determined.

 Table 7 shows the results.

Table 7 shows that the substrate reagent test solution containing the condensed phosphorus oxoacid derivative of the present invention is stable even during storage over time without inhibiting the enzyme reaction.

Example 8 In Examples 1 to 5, the substrate reagent solution in which sulfuric acid was added to stop the reaction was left at room temperature in a dark place for 24 hours, and then 492 nm
Was measured, and the relative value was determined using the case immediately after the stop as a control. Table 8 shows the results.

From Table 8, it is clear that the stability of the substrate reagent TS after stopping the enzyme reaction is improved by containing the condensed phosphorus oxosulfate derivative.

Example 9 100 mM phosphate buffer solution (pH 5.0) containing o-phenylenediamine at a concentration of 3 mg / ml was prepared with and without various condensed phosphorus oxoacid derivatives, and hydrogen peroxide was further reduced to 0.01%. In addition, a substrate reagent test solution was prepared. The substrate reagent test solution was dispensed into two 0.5 ml portions each, and 20 μl of a horseradish peroxidase-conjugated goat anti-mouse immunoglobulin antibody (manufactured by Kappel) at a concentration of 0.1 μg / ml was added to one, and to the other was added Did not. Each at 37 ° C
The reaction was allowed to proceed for 30 minutes, and then the reaction was stopped by adding sodium azide. Using the excitation wavelength at 410 nm, the fluorescence intensity at 550 nm was measured. Table 9 shows the results.

From Table 9, it can be seen that even in the detection system for measuring the enzyme activity by fluorescence, when the substrate reagent TS containing the condensed phosphorus oxoacid derivative of the present invention was used, the enzyme reaction was almost inhibited as compared with the substrate reagent TS not containing the same. It is clear that the non-enzymatic oxidation reaction is suppressed without being performed.

Example 10 12 ml of 50 mM citric acid-100 mM phosphate buffer (pH 4.9) containing pentasodium tripolyphosphate at various concentrations in 3 ml of dimethyl sulfoxide containing o-dianisidine at a concentration of 5 mg / ml
Was added, and hydrogen peroxide was further added to a concentration of 0.02% to prepare a substrate reagent test solution.

After dispensing 5 ml of the substrate reagent solution into 2 ml aliquots, 100 μl of a horseradish peroxidase-conjugated goat anti-mouse immunoglobulin antibody (manufactured by Kappel) at a concentration of 0.5 μg / ml was added to each, and the other one was added. At 37 ° C. in a dark place for 1 hour, and the difference in absorbance at 496 nm was defined as the enzyme activity.

In addition, the absorbance was 0 in all cases where no enzyme-linked antibody was added. After leaving the remaining three tubes in a light place at room temperature for 48 hours, the absorbance of one was measured as it was, and the enzyme activity was measured in the same manner as described above using the remaining two tubes.

Relative enzyme activity was determined using the case without pentasodium tripolyphosphate as a control.

 Table 10 shows the results.

From Table 10, even when o-dianisidine was used as the substrate, pentasodium tripolyphosphate was added in an amount of about 0.3 to about 30 m.
By adding in the range of M, it is clear that the non-enzymatic oxidation reaction is remarkably suppressed without substantially inhibiting the enzymatic reaction, the detection system is stabilized, and the storage stability of the substrate reagent test solution is apparent.

Example 11 The same operation as in Example 10 was carried out except that disodium tetraultraphosphate was used instead of pentasodium tripolyphosphate as the condensed phosphorus oxoacid derivative.

 Table 11 shows the results.

From Table 11, even when o-dianisidine was used as the substrate, disodium tetraultraphosphate was added to about 0.3 mM.
The same effects as in Example 10 can be obtained by using in the range of from about 50 mM to about 50 mM.

 If the amount added exceeds 100 mM, precipitation occurs.

Example 12 50 mM citric acid-100 mM phosphate buffer (pH 4) containing various concentrations of pentasodium tripolyphosphate in 0.5 ml of N-methylpyrrolidone containing 3,3 ', 5,5'-tetramethylbenzidine at a concentration of 6 mg / ml .9) Add 3.5 ml, and add 0.02% hydrogen peroxide
In addition, a substrate reagent test solution was prepared. Substrate reagent solution
After dispensing 5 bottles each to 0.05 ml, one of them was added with 10 μl of a horseradish peroxidase-conjugated goat anti-mouse immunoglobulin antibody (manufactured by Kappel) at a concentration of 0.5 μg / ml, and the other was incubated at 37 ° C. Allowed to react for 30 minutes in the dark
₂ ml of 1NH ₂ SO ₄ was added, and the difference in absorbance at 450 nm was defined as the enzyme activity.

In addition, the absorbance was 0 in all cases where no enzyme-linked antibody was added. The remaining three tubes were allowed to stand at room temperature and in a light place for 24 hours, and the absorbance of one was measured as it was, and the enzyme activity of the other two tubes was measured in the same manner as described above. Relative enzyme activity was determined using a control without pentasodium tripolyphosphate as a control.

 The progress is shown in Table 12.

According to Table 12, even when 3,3 ', 5,5'tetramethylbenzidine is used as the substrate, the enzyme reaction is almost inhibited by adding pentasodium tripolyphosphate in the range of about 1 mM to about 100 mM. Thus, the non-enzymatic oxidation reaction is remarkably suppressed and the detection system is stabilized, and the storage stability of the substrate reagent test solution is clear.

Example 13 The same operation as in Example 10 was performed except that hexasodium hexametaphosphate was used instead of pentasodium tripolyphosphate as the condensed phosphorus oxoacid derivative. Table 13 shows the results.

From Table 13, it can be seen that even when 3,3'5,5'tetramethylbenzidine is used as a substrate, hexasodium hexametaphosphate is used in the range of about 3 mM to about 100 mM to give an example.
The same effect as 11 appears.

Example 14 The effects of the present invention were examined using substrate reagent test solutions prepared by various methods.

Method 1 30 mg of o-phenylenediamine was added to 10 ml of a 50 mM citric acid-100 mM phosphate buffer (pH 4.9) (hereinafter referred to as a buffer for a test solution), and 10 mg of a condensed phosphorus oxoacid derivative was further added to completely dissolve.

 Then, 6% of 33% aqueous hydrogen peroxide was added to prepare.

Method 2 O-phenylenediamine 30 mg, 33 ml
6% aqueous hydrogen peroxide was added, and 10 mg of a condensed phosphorus oxo acid derivative was further added.

Method 3 30 mg of o-phenylenediamine and condensed phosphorus oxo acid derivative
10 mg was dissolved in 3 ml of pure water, frozen at −20 ° C., and lyophilized under reduced pressure. After storing in the dark at 37 ° C for one week,
It was prepared by mixing with 10 ml of a buffer solution for test solution to which 6 μm of 33% hydrogen peroxide solution was added.

Method 4 To 10 ml of a buffer solution for the test solution, 6 μm of 33% hydrogen peroxide and 10 mg of a condensed phosphorus oxo acid derivative were added, and the mixture was stored at 37 ° C. for one week in a dark place, followed by addition of 30 mg of o-phenylenediamine.

Method 5 30 mg of o-phenylenediamine and condensed phosphorus oxo acid derivative
5 mg was dissolved in 3 ml of pure water, frozen at −20 ° C., freeze-dried under reduced pressure, and stored at 37 ° C. for one week in a dark place. To 10 ml of the buffer for the test solution, 6 μm of 33% aqueous hydrogen peroxide and 5 mg of a condensed phosphorus oxoacid derivative were added, and the mixture was similarly stored at 37 ° C. for one week in a dark place. It was prepared by mixing both.

As a condensed phosphorus oxo acid derivative, pentasodium tripolyphosphate or disodium tetraultraphosphate was used. Each evaluation was performed in the same manner as in Example 1. In Comparative Example 1, a method in which a condensed phosphorus oxo acid derivative was not added in Method 1 was used.

 Table 14 shows the results.

The effects of the present invention are clear regardless of the order of addition of the condensed phosphorus oxoacid derivative during preparation of the substrate reagent test solution.

Claims (2)

(57) [Claims] 1. A substrate reagent comprising a substrate which provides an analysis means by being oxidized by a peroxidase and a condensed phosphorus oxo acid derivative comprising three or more phosphorus oxo acid units. 2. A condensed phosphorus oxo acid comprising three or more phosphorus oxo acid units in a peroxidase or pseudoperoxidase, a substrate that provides an analysis means by being oxidized, and a detection system using hydrogen peroxide. A method for stabilizing a detection system containing a derivative.