JP2005110507A - Method for stabilizing reagent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for stabilizing TPM-PS [N,N,N',N',N",N"-hexa(3-sulfopropyl)-4,4',4"-triaminotriphenylmethane hexasodium salt] in a solution state. <P>SOLUTION: The TPM-PS is made to coexist with Bis-Tris [bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane], ADA [N-(2-acetamido)iminodiacetic acid], TEA (triethanolamine), PIPES [piperazine-1,4-bis(2-ethanesulfonic acid)], MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid), BES [N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid], MOPS (3-morpholinopropanesulfonic acid), TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid], HEPES ä2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid}, DIPSO ä3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid}, POPSO [pierazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) dihydrate], HEPPSO ä2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid monohydrate}, EPPS ä3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid}, Tris [2-amino-2-hydroxymethyl-1,3-propanediol or tris(hydroxymethyl)aminomethane], Tricine äN-[tris(hydroxymethyl)methyl]glycine}, CyDTA (trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), EDDP (ethylenediamine-N,N'-dipropionic acid dihydrochloride), EGTA [O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid], HIDA [N-(2-hydroxyethyl)iminodiacetic acid], IDA (iminodiacetic acid), NTA (nitrilotriacetic acid), TTHA (triethylenetetramine-N,N,N',N",N''', N'''-hexaacetic acid), EDTA (ethylenediaminetetraacetic acid), a salt thereof and a hydroxide thereof and DPTA-OH (1,3-diamino-2-hydroxypropane-N,N,N',N'-tetraacetic acid) in a solution. Thereby, the TPM-PS can be stabilized to prevent nonenzymic self-coloring. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an oxidizing color former N, N, N ′, N ′, N ″, N ″ -hexa (3-sulfopropyl) -4,4 ″ -triaminotriphenylmethane salt in a solution. It is related with the method of stabilizing with.

  In the analysis of various substances including biological components, the enzyme method for measuring absorbance is known as an extremely important method. Among the enzyme methods, for example, a method of widely generating hydrogen peroxide as an intermediate metabolite from an analysis target component, measuring the amount of hydrogen peroxide, and converting the amount of the analysis target component from the amount of hydrogen peroxide is widely adopted. Has been. The amount of hydrogen peroxide is generally reduced by oxidation of the chromogenic substrate by reducing the hydrogen peroxide with peroxidase (POD) in the presence of a substrate (chromogenic substrate) that develops color by oxidation of a Trinder reagent or the like. The degree of color development is measured and calculated from the measured value.

In such an enzymatic method, if the amount of hydrogen peroxide can be detected with high sensitivity, component analysis can be performed even with a trace amount of components. Therefore, establishment of a highly sensitive measurement method, particularly a highly sensitive chromogenic substrate. Is required. However, since the conventional chromogenic substrate, in particular, the above-mentioned Trinder reagent has a molar extinction coefficient in terms of hydrogen peroxide of 2 × 10 ^{4 to} 3 × 10 ⁴ , the measurable hydrogen peroxide is on the order of μmol / L, which is high. It was insufficient to realize a sensitive measurement.

Therefore, in recent years, N, N, N ', N', N '', N ''-hexa (3-sulfopropyl) -4,4 ''-triaminotriphenylhexa A sodium salt (hereinafter referred to as “TPM-PS”) has been developed. Since this substrate has a color development sensitivity 5 to 10 times that of the above-mentioned Trinder reagent, it is considered that, for example, it is possible to analyze components that could not be analyzed by a conventional enzymatic method. Moreover, since it is highly water-soluble, it has the advantage that it is excellent also in the handleability as a substrate reagent (for example, refer patent document 1 and patent document 2).
JP-A-6-197795 Japanese Patent Laid-Open No. 3-206869

  However, when TPM-PS is applied to the enzymatic method, it is necessary to use the TPM-PS as a solution reagent. In this case, it has become clear that the following problems occur. That is, because TPM-PS becomes unstable when dissolved in water, even if it is prepared at the time of use, it develops non-enzymatic color due to dissolved oxygen in water, etc., and the background increases during absorbance measurement, Measurement accuracy is reduced or measurement is impossible. In addition, the chromogenic substrate used in such an enzymatic method is usually stored in a solution state for easy measurement, but becomes unstable in a solution state as described above. In particular, the non-enzymatic color development increases, making it impossible to use it as a reagent.

  Further, when a mixed reagent of TPM-PS and POD is prepared for easy measurement, TPM-PS is further destabilized, for example, 6 to 10 times that in the absence of POD. It was also found that the color development occurred. In recent years, as a method of analyzing glycated proteins such as glycated hemoglobin, fructosyl amino acid oxidase (hereinafter referred to as “FAOD”) is allowed to act on the glycated portion of the protein, and hydrogen peroxide generated upon the release of sugar An enzymatic method for measuring a glycated protein by measuring using the POD has been put into practical use. In this case as well as POD, it is possible to prepare a mixed reagent of TPM-PS, which is a chromogenic substrate, and FAOD. However, even when FAOD coexists, TPM-PS is further destabilized and non-enzymatic. It was also found that typical color development occurs.

  Accordingly, an object of the present invention is to provide a method and a solution reagent for stabilizing TPM-PS in a solution.

  In order to achieve the above object, the TPM-PS of the present invention (N, N, N ′, N ′, N ″, N ″ -hexa (3-sulfopropyl) -4,4 ″ -triaminotrimethyl) The following four methods are listed as methods for stabilizing the phenylmethane salt).

The first stabilization method of the present invention is a method of stabilizing TPM-PS in a solution,
In the solution, TPM-PS, Bis-Tris, ADA (N- (2-Acetamido) iminodiacetic acid), TEA, PIPES (Piperazine-1,4-bis (2-ethanesulfonic acid)), MOPSO (2- Hydroxy-3-morpholinopropanesulfonic acid), BES (N, N-Bis (2-hydroxyethyl) -2-aminoethanesulfonic acid), MOPS (3-Morpholinopropanesulfonic acid), TES (N-Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid) , HEPES (2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid), DIPSO (3- [N, N-Bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid), POPSO (Piperazine- 1,4-bis (2-hydroxy-3-propanesulfonic acid), dihydrate), HEPPSO (2-Hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid, monohydrate), EPPS (3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid), Tris (2-Amino-2-hydroxymethyl-1,3-propanediol Tris (hydroxymethyl) aminomethane), Tricine (N- [Tris (hydroxymethyl) methyl] glycine), TAPS (N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid), CyDTA (trans-1,2-diaminocyclohexane-N, N, N ', N'-teto Laacetic acid), DTPA (diethylenetriaminepentaacetic acid), EDDP (Tthylenediamine-N, N'-dipropionic acid, dihydrochloride), EGTA (O, O'-Bis (2-aminoethyl) ethyleneglycol-N, N, N ', N' -tetraacetic acid), HIDA (N- (2-Hydroxyethyl) iminodiacetic acid), IDA (Iminodiacetic acid), NTA (nitrilotriacetic acid), TTHA (Triethylenetetramine-N, N, N ', N'',N''', N '''-hexaacetic acid), EDTA (ethylenediaminetetraacetic acid), their salts (eg EDTA-2K) and their hydroxides (eg EDTA-OH), and DPTA-OH (1,3-Diamino -2-hydroxypropane-N, N, N ', N'-tetraacetic acid), and at least one stabilizer selected from the group consisting of:

  The stabilization method of the second aspect of the present invention is a method of stabilizing TPM-PS in a solution in the presence of POD, wherein TPM-PS and POD, Bis-Tris, ADA, TEA , PIPES, MOPSO, BES, MOPS, HEPES, Tricine, Bicine (N, N-Bis (2-hydroxyethyl) glycine), MES, DIPSO, TAPSO, TAPS, CyDTA, DTPA, EDDP, EGTA, HIDA, IDA, NTA, TTHA, EDTA, a salt thereof (for example, EDTA-2K) and a hydroxide thereof (for example, EDTA-OH), and at least one stabilizer selected from the group consisting of DPTA-OH It is characterized by.

  A third stabilization method of the present invention is a method of stabilizing TPM-PS in a solution in the presence of POD and fructosyl amino acid oxidase (hereinafter referred to as “FAOD”), wherein -PS, POD and FAOD, MES, ADA, PIPES, MOPSO, HEPES, MOPS, TAPSO, Tricine, Bicine, CyDTA, DTPA, EDDP, EGTA, HIDA, IDA, NTA, TTHA, EDTA, their salts (for example, EDTA-2K) and their hydroxides (for example, EDTA-OH), and at least one stabilizer selected from the group consisting of DPTA-OH is characterized by coexistence.

  The fourth stabilization method of the present invention is a method of stabilizing TPM-PS in a solution in the presence of FAOD, wherein TPM-PS and FAOD are mixed with MES, ADA, PIPES, MOPSO in the solution. , MOPS, HEPES, DIPSO, TAPSO, Tricine, TAPS, Bicine, CyDTA, DTPA, EDDP, EGTA, HIDA, IDA, NTA, TTHA, EDTA, their salts (for example, EDTA-2K) and their hydroxides ( For example, it is characterized by coexisting with at least one stabilizer selected from the group consisting of EDTA-OH) and DPTA-OH.

  Furthermore, the solution reagent of the present invention includes the following four reagents.

  The solution reagent of the first aspect of the present invention is a solution reagent containing TPM-PS, and further includes Bis-Tris, ADA, TEA, PIPES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, POPSO, HEPPSO, EPPS , Tris, Tricine, CyDTA, DTPA, EDDP, EGTA, HIDA, IDA, NTA, TTHA, EDTA, their salts (eg EDTA-2K) and their hydroxides (eg EDTA-OH), and DPTA- It contains at least one stabilizer selected from the group consisting of OH.

  The solution reagent of the second aspect of the present invention is a solution reagent containing POD and TPM-PS, and further, Bis-Tris, ADA, TEA, PIPES, MOPSO, BES, MOPS, HEPES, Tricine, Bicine, MES, DIPSO , TAPSO, TAPS, CyDTA, DTPA, EDDP, EGTA, HIDA, IDA, NTA, TTHA, EDTA, their salts (eg EDTA-2K) and their hydroxides (eg EDTA-OH), and DPTA- It contains at least one stabilizer selected from the group consisting of OH.

  The solution reagent of the third aspect of the present invention is a solution reagent containing POD, FAOD and TPM-PS, and further includes MES, ADA, PIPES, MOPSO, HEPES, MOPS, TAPSO, Tricine, Bicine, CyDTA, DTPA, EDDP EGTA, HIDA, IDA, NTA, TTHA, EDTA, their salts (eg, EDTA-2K) and their hydroxides (eg, EDTA-OH), and at least one selected from the group consisting of DPTA-OH It contains two stabilizers.

  The solution reagent of the fourth aspect of the present invention is a solution reagent containing FAOD and TPM-PS, and further includes MES, ADA, PIPES, MOPSO, MOPS, HEPES, DIPSO, TAPSO, Tricine, TAPS, Bicine, CyDTA, DTPA EDDP, EGTA, HIDA, IDA, NTA, TTHA, EDTA, their salts (eg, EDTA-2K) and their hydroxides (eg, EDTA-OH), and DPTA-OH It contains at least one stabilizer.

  Thus, if TPM-PS coexists with various stabilizers, the mechanism is unknown, but non-enzymatic spontaneous color development of TPM-PS in the solution state can be suppressed. Even in the case of using natural color, it is possible to prevent a decrease in measurement accuracy of the color development reaction due to natural color development. In addition, storage in a liquid state is also possible. For this reason, TPM-P, which was difficult to put into practical use despite its extremely excellent measurement sensitivity, can be used in the enzyme method as described above in a solution state, so that the measurement operation is easy and simple, Measurement sensitivity can be improved. Therefore, it is possible to provide a TPM-PS liquid reagent that is particularly useful for analyzing trace components.

  The stabilization method and solution reagent of the present invention will be specifically described below. In the present invention, the TPM-PS includes, for example, N, N, N ′, N ′, N ″, N ″ -hexa (3-sulfopropyl) -4,4 ″ -triaminotrimethyl Phenylmethane hexasodium salt (for example, trade name TPM-PS; manufactured by Dojin Chemical Co., Ltd.) can be used, but is not limited to sodium salt. Since TPM-PS develops color by oxidation and the color development has absorption at a wavelength of 591 nm, the degree of color development can be measured by measuring the absorbance at the wavelength.

  First, the first stabilization method and the first solution reagent of the present invention will be described. As described above, the first stabilization method is a method of stabilizing TPM-PS in a solution, and the various stabilizers may coexist with TPM-PS in the solution. For example, It can be carried out by preparing a TPM-PS-containing solution in which TPM-PS and the stabilizer are dissolved in an aqueous solvent. When the stabilizer is present, TPM-PS is stabilized and extremely non-enzymatic color development can be suppressed as compared with a solution in which only TPM-PS is dissolved in water. For this reason, for example, it is possible to suppress an increase in background during the color development reaction, and it is also possible to store in a solution state. Therefore, TPM-PS stabilized by such a method is useful as a solution reagent.

  For example, when the color development degree (for example, absorbance at 591 nm) of a solution in which only TPM-PS is dissolved in water (hereinafter referred to as “TPM-PS aqueous solution”) is 100%, the color development degree is about 5 to 90%. Can be suppressed. Specifically, when left at 0 to 25 ° C. for 1 year, the color development can be suppressed to about 5 to 60% compared to the TPM-PS aqueous solution. In addition, when preserve | saving, the temperature in particular is not restrict | limited, For example, it is 0-10 degreeC, Preferably it is 0-4 degreeC.

  Bis-Tris, ADA, TEA, PIPES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, POPSO, HEPPSO, EPPS, Tris, Tricine, TAPS, CyDTA, DTPA, EDDP, EGTA, HIDA, Among IDA, NTA, TTHA, EDTA, salts thereof (for example, EDTA-2K, etc.), hydroxides (for example, EDTA-OH, etc.), DPTA-OH, preferably ADA, PIPES, MOPSO, BES, MOPS, DIPSO, POPSO, HEPPSO, EPPS, Tricine, TAPS, DTPA, EGTA, HIDA, IDA, NTA, TTHA, EDTA, EDTA-2K, DPTA-OH, EDTA-OH, more preferably ADA, PIPES, MOPSO , BES, MOPS, DIPSO, Tricine, TAPS, DTPA, EGTA, HIDA, TTHA, DPTA-OH. In addition, any one of these stabilizers may be used, or two or more kinds may be used in combination.

  The aqueous solvent is preferably water. For example, it is preferable to prepare a solution in which the various stabilizers are dissolved in water in advance and dissolve TPM-PS in this solution.

  The TPM-PS concentration in the solution is, for example, in the range of 0.03 to 1.0 mM, and preferably in the range of 0.125 to 0.25 mM. On the other hand, the concentration of various stabilizers can be appropriately determined according to, for example, the TPM-PS concentration and the type of stabilizer (particularly whether the stabilizer belongs to a chelating agent or a buffer). For example, the range is 25 to 300 mM. Specifically, when the stabilizer belongs to, for example, a chelating agent, it is preferably in the range of 25 to 300 mM, more preferably 100 to 300 mM, and particularly preferably 300 mM in the solution. Moreover, when a stabilizer belongs to a chelating agent, for example, the range of 10-100 mM is preferable, and about 25-50 mM is especially preferable.

  The ratio (molar ratio A: B) of TPM-PS (A) and stabilizer (B) in the solution is, for example, in the range of 1:25 to 1: 10,000, preferably 1: 100 to 1. : In the range of 10,000, more preferably in the range of 1: 100 to 1: 2400. Specifically, when the TPM-PS concentration is 0.25 mM, the stabilizer ADA is preferably 300 mM, and the stabilizer DTPA is preferably 25 to 50 mM.

  The pH of the solution is not particularly limited and can be appropriately determined according to the type of the stabilizer. For example, the pH is 7 to 10, and preferably 7 to 9.

  Since the solution can further improve the stabilization, it is preferable that potassium thiocyanate (KSCN) coexist as a stabilization accelerator. As a result, the color development with only the stabilizer (no potassium thiocyanate) can be further suppressed by about 70% to 90%. The KSCN concentration in the solution is, for example, in the range of 10 to 200 mM, preferably in the range of 10 to 100 mM, more preferably in the range of 20 to 50 mM when the TPM-PS concentration is in the range of 0.125 to 0.25 mM. It is. The ratio (molar ratio B: C) of the stabilizer (B) to the stabilization promoter (C) is, for example, in the range of 30: 1 to 3: 1, preferably 15: 1 to 6: 1. Range. Moreover, the ratio (molar ratio A: C) of TPM-PS (A) and stabilization promoter (C) is, for example, in the range of 1:80 to 1: 400.

  It is preferable that sodium azide coexist in the solution since the measurement sensitivity of TPM-PS can be improved in the enzyme reaction using POD as described above. Thus, when a TPM-PS-containing solution is used as an enzyme reaction in the present invention, the measurement sensitivity can also be improved. The same applies to the second to fourth stabilization methods described later.

  Thus, in order to stabilize TPM-PS, a solution in which TPM-PS and the stabilizing agent coexist in an aqueous solvent is the first solution reagent of the present invention. The concentration of each component in this solution reagent is, for example, in the same range as described above, and can be appropriately diluted according to the conditions of the enzyme reaction when used. Moreover, this solution reagent can be used similarly to the chromogenic substrate reagent in the conventionally known enzymatic method, and its use and handling are not particularly limited. The same applies to second to fourth solution reagents described later.

  Next, the second stabilization method and the second solution reagent of the present invention will be specifically described. In addition, unless otherwise indicated, it can carry out on the same conditions as the said 1st stabilization method. In the second stabilization method, as described above, various stabilizers as described above may be allowed to coexist with TPM-PS and POD in a solution. For example, TPM-PS may be added to an aqueous solvent. It can be carried out by preparing a TPM-PS-containing solution in which POD and the stabilizer are dissolved. According to such a method, for example, when the color development degree (for example, the absorbance at 591 nm) of the TPM-PS aqueous solution in which only TPM-PS is dissolved is 100%, the color development degree is about 10 to 65%. Can be suppressed. Specifically, when left at 0 to 4 ° C. for 1 year, the color development can be suppressed to about 10 to 65% compared to the TPM-PS aqueous solution.

  Bis-Tris, ADA, TEA, PIPES, MOPSO, BES, MOPS, HEPES, Tricine, Bicine, MES, DIPSO, TAPSO, TAPS, CyDTA, DTPA, EDDP, EGTA, HIDA, IDA, NTA, the stabilizer Among TTHA, EDTA, salts thereof (for example, EDTA-2K, etc.), hydroxides (for example, EDTA-OH, etc.), DPTA-OH, preferably Bis-Tris, ADA, TEA, PIPES, MOPSO, BES, MOPS, HEPES, Tricine, Bicine, MES, CyDTA, DTPA, EGTA, IDA, NTA, TTHA, EDTA-2K, DPTA-OH, EDTA-OH, more preferably ADA, PIPES, MOPSO, BES, MOPS , HEPES, Tricine, DTPA, IDA, NTA, DPTA-OH. In addition, any one of these stabilizers may be used, or two or more kinds may be used in combination.

  The POD concentration in the solution is, for example, in the range of 10 to 100 KU / L, preferably in the range of 50 to 70 KU / L, and more preferably 67 KU / L. Moreover, the addition ratio with respect to TPM-PS 0.25 mM is the range of 10-100KU, for example, Preferably it is the range of 50-70KU.

  Since the solution can further improve the stabilization, it is preferable that NaCl, sodium nitrate, sodium sulfate, potassium thiocyanate (KSCN), or the like be further present as a stabilization accelerator. This can further suppress non-enzymatic color development by only the stabilizer (the absence of the stabilization promoter) by about 30% to 50%. Of these, NaCl and KSCN are preferable. The concentration of the stabilization promoter in the solution is not particularly limited. For example, in the case of NaCl or sodium nitrate, a range of 300 to 1000 mM is preferable, and a range of 300 to 700 mM is more preferable. The range of 200 mM is preferable, the range of 10 to 100 mM is more preferable, and the range of 20 to 50 mM is particularly preferable. Moreover, in the case of sodium sulfate, the range of 100-500 mM is preferable, for example. Any of these stabilization promoters may be used, or two or more of them may be used in combination. The same applies to the third and fourth stabilization methods described later.

  Thus, in order to stabilize TPM-PS, a solution in which TPM-PS, POD and the stabilizer coexist in an aqueous solvent is the second solution reagent of the present invention. The concentration of each component in this solution reagent is, for example, in the same range as described above, and can be appropriately diluted according to the conditions of the enzyme reaction when used.

  Next, the third stabilization method of the present invention will be specifically described. Unless otherwise indicated, the reaction can be performed under the same conditions as in the first and second stabilization methods. In the third stabilization method, as described above, various stabilizers as described above may be allowed to coexist with TPM-PS, POD, and FAOD in a solution. For example, a TPM is added to an aqueous solvent. It can be carried out by preparing a TPM-PS-containing solution in which -PS, POD, FAOD and the stabilizer are dissolved. According to such a method, for example, when the color development degree (for example, the absorbance at 591 nm) of the TPM-PS aqueous solution in which only TPM-PS is dissolved is 100%, the color development degree is about 5 to 80%. Can be suppressed. Specifically, when left at 0 to 4 ° C. for 1 year, the color development can be suppressed to about 5 to 40% compared to the TPM-PS aqueous solution.

  The stabilizers MES, ADA, PIPES, MOPSO, HEPES, MOPS, TAPSO, Tricine, Bicine, CyDTA, DTPA, EDDP, EGTA, HIDA, IDA, NTA, TTHA, EDTA, and salts thereof (for example, EDTA- 2K, etc.), these hydroxides (for example, EDTA-OH, etc.), among DPTA-OH, preferably MES, ADA, PIPES, MOPSO, HEPES, DTPA, IDA, TTHA, EDTA-2K, DPTA-OH, EDTA-OH. In addition, any one of these stabilizers may be used, or two or more kinds may be used in combination.

  The POD concentration in the solution is, for example, the same as in the second stabilization method, while the FAOD concentration in the solution can be set, for example, by the ratio with the POD. The ratio of POD (E) to FAOD (F) (activity ratio E: F) is, for example, in the range of 10:18 to 100: 18, and preferably in the range of 50:18 to 70:18. Specifically, the FAOD concentration in the solution is preferably 10 to 50 KU / L, more preferably about 18 KU / L.

  The FAOD may be any enzyme that catalyzes the reaction of the following formula (1), and the origin thereof is not particularly limited. For example, the commercial names FAOX-E (manufactured by Kikkoman), FOD (Asahi Kasei) Can be used.

R ¹ —CO—CH ₂ —NH—R ² + H ₂ O + O ₂
→ R ¹ —CO—CHO + NH ₂ —R ² + H ₂ O ₂ (1)
In the formula (1), R ¹ represents a hydroxyl group or a residue (sugar residue) derived from a sugar before saccharification reaction. The sugar residue (R ¹ ) is an aldose residue when the sugar before the reaction is aldose, and is a ketose residue when the sugar before the reaction is ketose. For example, when the sugar before the reaction is glucose, the structure after the reaction takes a fructose structure due to the Amadori rearrangement. In this case, the sugar residue (R ¹ ) becomes a glucose residue (aldose residue). The sugar residue (R ¹ ) can be represented by, for example, — [CH (OH)] _n —CH ₂ OH, and n is an integer of 0 to 6.

In the formula (1), R ² is not particularly limited. For example, in the case of a glycated amino acid, a glycated peptide or a glycated protein, the α-amino group is glycated and the other amino groups are glycated. It differs depending on the case.

In the formula (1), when the α-amino group is glycated, R ² is an amino acid residue or a peptide residue represented by the following formula (2).

—CHR ³ —CO—R ⁴ (2)
In the formula (2), R ³ represents an amino acid side chain group. R ⁴ represents a hydroxyl group, an amino acid residue, or a peptide residue, and can be represented by, for example, the following formula (3). In the following formula (3), n is an integer of 0 or more, and R ³ represents an amino acid side chain group as described above.

^{- (NH-CHR 3 -CO)} n -OH ... (3)
In the formula (1), when an amino group other than the α-amino group is saccharified (the amino acid side chain group is saccharified), R ² can be represented by the following formula (4).

—R ⁵ —CH (NH—R ⁶ ) —CO—R ⁷ (4)
In the formula (4), R ⁵ is the amino acid side chain, showing a portion other than the glycated amino group. For example, when the glycated amino acid is lysine, R ⁵ is —CH ₂ —CH ₂ —CH ₂ —CH ₂ —,
For example, when the glycated amino acid is arginine, R ⁵ is
_{-CH 2 -CH 2 -CH 2 -NH-} CH (NH 2) - a.

In the formula (4), R ⁶ is hydrogen, an amino acid residue or a peptide residue, and can be represented by, for example, the following formula (5). In the following formula (5), n is an integer of 0 or more, and R ³ represents an amino acid side chain group as described above.

^{- (CO-CHR 3 -NH)} n -H ... (5)
In the formula (4), R ⁷ is a hydroxyl group, an amino acid residue or a peptide residue, and can be represented by the following formula (6), for example. In the following formula (6), n is an integer of 0 or more, and R ³ represents an amino acid side chain group as described above.

— (NH—CHR ³ —CO) _n —OH (6)
Thus, in order to stabilize TPM-PS, a solution in which TPM-PS, POD, FAOD and the stabilizer coexist in an aqueous solvent is the third solution reagent of the present invention. The concentration of each component in this solution reagent is, for example, in the same range as described above, and can be appropriately diluted according to the conditions of the enzyme reaction when used.

  Since the third solution reagent contains FAOD in addition to POD, it is particularly useful for measuring glycated proteins such as glycated hemoglobin and glycated albumin. For example, if the third solution reagent is added to glycated protein, sugar is released from the glycated protein and hydrogen peroxide oxidation is generated by FAOD in the reagent, and POD reduces the generated hydrogen peroxide. As a result, TPM-PS develops color by oxidation. By measuring the degree of color development, the amount of protein saccharification can be determined.

  Next, the fourth stabilization method of the present invention will be specifically described. In addition, unless otherwise indicated, it can carry out on the conditions similar to the said 1st-3rd stabilization method. In the fourth stabilization method, as described above, various stabilizers as described above may be allowed to coexist with TPM-PS and FAOD in a solution. For example, TPM-PS may be added to an aqueous solvent. It can be carried out by preparing a TPM-PS-containing solution in which FAOD and the stabilizer are dissolved. According to such a method, for example, when the color development degree of the TPM-PS aqueous solution in which only TPM-PS is dissolved (for example, the absorbance at 591 nm) is 100%, the color development degree is about 2 to 30%. Can be suppressed. Specifically, when left at 0 to 4 ° C. for 1 year, the color development can be suppressed to about 2 to 10% compared to the TPM-PS aqueous solution.

  The FAOD concentration in the solution is, for example, in the range of 10-50 KU / L, and preferably in the range of 15-20 KU / L. Moreover, the addition ratio with respect to TPM-PS 0.25 mM is the range of 10-50KU, for example, Preferably it is the range of 15-20KU.

  The stabilizers MES, ADA, PIPES, MOPSO, MOPS, HEPES, DIPSO, TAPSO, Tricine, TAPS, Bicine, CyDTA, DTPA, EDDP, EGTA, HIDA, IDA, NTA, TTHA, EDTA, and their salts ( For example, EDTA-2K etc.), hydroxides thereof (eg EDTA-OH etc.), DPTA-OH, preferably ADA, DTPA, HIDA, IDA, NTA, TTHA, DPTA-OH, EDTA-OH is there.

  Thus, in order to stabilize TPM-PS, a solution in which TPM-PS and the stabilizer are present together in an aqueous solvent is the fourth solution reagent of the present invention. The concentration of each component in this solution reagent is, for example, in the same range as described above, and can be appropriately diluted according to the conditions of the enzyme reaction when used.

  Since the third solution reagent contains FAOD in addition to POD, it is particularly useful for measuring glycated proteins such as glycated hemoglobin and glycated albumin. For example, if the third solution reagent is added to glycated protein, sugar is released from the glycated protein and hydrogen peroxide oxidation is generated by FAOD in the reagent, and POD reduces the generated hydrogen peroxide. As a result, TPM-PS develops color by oxidation. By measuring the degree of color development, the amount of protein saccharification can be determined.

  In addition, although the stabilization method of this invention is further demonstrated using the following examples and comparative examples below, this invention is not limited only to these Examples.

  In this example, suppression of an increase in absorbance due to non-enzymatic oxidation of TPM-PS was examined for a TPM-PS reagent (TPM-PS only) to which various stabilizers were added. As the stabilizer, buffers described in Tables 1 and 2 below were used. Moreover, TPM-PS used the brand name TPM-PS (made by Dojin Chemical Co., Ltd.) (hereinafter the same).

  Using water as a solvent, TPM-PS reagent samples (TPM-PS concentrations 150 mM: stabilizer concentrations 150 mM, 300 mM) shown in Tables 1 and 2 below were prepared and stored for 7 days. The storage temperature was 25 ° C. for a stabilizer concentration of 150 mM and 4 ° C. for a stabilizer concentration of 300 mM. And about the said TPM-PS reagent, the light absorbency at the time of a storage start and 7 days after a preservation | save was each measured (measurement wavelength 571 nm), and the light absorbency change amount was calculated | required.

  The absorbance measurement was performed by the following method using an automatic analyzer (trade name Bio Majesty 8: manufactured by JEOL Ltd.). First, after automatically adding 75.6 μL of distilled water to 8.4 μL of the solution to be measured (distilled water) and incubating at 37 ° C. for 5 minutes, 18.9 μL of the TPM-PS reagent sample after the storage Was added. This reaction solution was incubated at 37 ° C., and the absorbance of the reaction solution was measured 2.5 minutes after the addition of the sample. On the other hand, the absorbance measurement was similarly performed for the unstored TPM-PS reagent sample, and the absorbance for the unstored sample was subtracted from the absorbance for the sample after storage, and the change in absorbance due to storage for 7 days was obtained. The amount. In this method, the TPM-PS reagent sample is diluted 5.44 times. Further, an aqueous TPM-PS solution in which only TPM-PS was dissolved in water was prepared, and the absorbance was measured in the same manner. Then, the amount of change in absorbance due to storage for 7 days was multiplied by 365/7, and the amount of increase in absorbance for one year was converted. Absorbance increase of each sample when 1 year increase in absorbance (converted value) and 1 year increase in absorbance (converted value) for TPM-PS aqueous solution in which only TPM-PS is dissolved in water The increase ratio of the amount (converted value) is shown in Tables 1 and 2 below. Table 1 below shows the results when the stabilizer concentration in the TPM-PS reagent sample is 150 mM and the storage temperature is 25 ° C. Table 2 below shows the results when the stabilizer concentration is 300 mM and the storage temperature is 4 ° C. .

(Table 1)
Sample Stabilizer pH = pKa Increase in absorbance Increase rate
(150mM) (1 year)
1-1 Bis-Tris 6.65 0.447 0.54
1-2 ADA 6.77 0.445 0.53
1-3 TEA 6.99 0.441 0.53
1-4 PIPES 7.0 0.392 0.47
1-5 MOPSO 7.12 0.482 0.58
1-6 BES 7.25 0.379 0.45
1-7 MOPS 7.28 0.423 0.51
1-8 TES 7.6 0.751 0.90
1-9 HEPES 7.68 0.297 0.36
1-10 DIPSO 7.74 0.630 0.76
1-11 POPSO 8.0 0.160 0.19
1-12 HEPPSO 8.05 0.135 0.19
1-13 EPPS 8.22 0.245 0.29
1-14 Tris 8.36 0.451 0.54
1-15 Tricine 8.25 0.322 0.39
1-16 Bicine 8.48 0.044 0.05
1-17 MES 6.3 2.821 3.38
1-18 ACES 6.98 1.096 1.31
1-19 Imidazole 7.15 5.446 6.53
1-20 TAPSO 7.82 1.049 1.26
Water-0.834 1.0

(Table 2)
Sample Stabilizer pH = pKa Increase in absorbance Increase rate
(300mM) (1 year)
1-21 ADA 7.0 0.033 0.10
1-22 PIPES 7.0 0.049 0.16
7.5 0.109 0.34
1-23 MOPSO 7.0 0.090 0.28
7.5 0.046 0.14
1-24 BES 7.0 0.028 0.09
7.5 0.034 0.11
1-25 MOPS 7.5 0.032 0.10
8.0 0.029 0.09
1-26 DIPSO 8.0 0.024 0.08
1-27 Tricine 8.5 0.019 0.06
1-28 TAPS 8.5 0.037 0.12
1-29 MES 7.0 0.453 1.43
1-30 ACES 8.0 1.926 6.06
Water-0.318 1.0

As shown in Table 1, samples (1-1) to (1-16) are examples, and the increase in absorbance due to non-enzymatic color development is extremely suppressed compared to the TPM-PS aqueous solution using water. It was. On the other hand, Samples (1-17) to (1-20) which are comparative examples showed a significant increase in absorbance. Among samples (1-1) to (1-16), particularly when POPSO, HEPPSO, EPPS, and Bicine were used, excellent increase in absorbance was shown. Furthermore, Table 1 shows the results of storage under the severe conditions of a storage temperature of 25 ° C., and it was found that the absorbance can be suppressed even under such conditions. From the viewpoint of storage, for example, it is desirable to improve the stability at around 0 to 4 ° C. However, TPM-PS also has a problem of an increase in background during the enzyme reaction. The fact that the increase in absorbance is suppressed even at an enzyme reaction temperature of 25 ° C. indicates that the TPM-PS reagent of the present invention is extremely useful. Further, as shown in Table 2, samples (1-21) to (1-28) are examples, and the increase in absorbance due to non-enzymatic oxidation is extremely higher than that of TPM-PS aqueous solution using water. Suppressed. On the other hand, samples (1-29) to (1-30), which are comparative examples, showed a significant increase in absorbance. Among samples (1-21) to (1-28), particularly when ADA, BES, MOPS, DIPSO, and Tricine were used, excellent suppression of increase in absorbance was shown.

  In this example, suppression of an increase in absorbance due to non-enzymatic oxidation of TPM-PS was examined for a TPM-PS reagent (TPM-PS + POD) to which various stabilizers were added. As the stabilizer, buffers described in Tables 3 and 4 below were used.

  Except that water was used as a solvent and the TPM-PS reagent samples (POD concentration 67KU / L: TPM-PS concentration 0.25 mM: stabilizer concentration 150 mM, 300 mM) shown in Tables 3 and 4 below were prepared. In the same manner as in Example 1, the sample was stored and the absorbance was measured. The storage temperature was 25 ° C. when the stabilizer concentration was 150 mM, and 4 ° C. when the stabilizer concentration was 300 mM. The amount of change for one year converted from the amount of change in absorbance by storage for 7 days, and the amount of increase in absorbance for 1 year (converted value) for a TPM-PS aqueous solution in which only TPM-PS is dissolved in water Tables 3 and 4 below show the rate of increase in absorbance (converted value) of each sample. Table 3 below shows the results when the stabilizer concentration in the sample is 150 mM, and Table 4 below shows the results when the stabilizer concentration is 300 mM.

  Further, using the TPM-PS reagent sample at the time of preparation, a POD reaction was performed on the hydrogen peroxide solution, and the absorbance of the reaction solution was measured. Specifically, in the method for measuring absorbance described in Example 1, the absorbance was measured in the same manner except that 8.4 μL of 14 μM hydrogen peroxide solution was used instead of 8.4 μL of distilled water as the solution to be measured. Tables 3 and 4 below show the absorbance obtained by subtracting a blank (absorbance when 8.4 μL of distilled water is used) from this absorbance. The final concentration of hydrogen peroxide during the reaction was 1 μM.

(Table 3)
Sample Stabilizer pH = pKa Increase in absorbance Increase rate H ₂ O ₂ measured value
(150mM) (1 year)
2-1 Bis-Tris 6.65 3.65 0.45 0.123
2-2 ADA 6.77 1.37 0.17 0.105
2-3 TEA 6.99 2.18 0.25
2-4 PIPES 7.0 2.07 0.25 0.156
2-5 MOPSO 7.12 3.46 0.42 0.152
2-6 BES 7.25 2.92 0.36 0.143
2-7 MOPS 7.28 2.86 0.35 0.159
2-8 HEPES 7.68 3.71 0.45 0.148
2-9 Tricine 8.25 5.34 0.65 0.153
2-10 MES 6.3 8.10 0.99
2-11 ACES 6.98 9.78 1.19 0.158
2-12 Imidazole 7.15 9.27 1.13
2-13 TES 7.6 9.98 1.22 0.162
2-14 POPSO 8.0 72.99 8.91 0.046
2-15 HEPPSO 8.05 58.21 7.10 0.073
2-16 EPPS 8.22 60.97 7.44 0.116
2-17 Tris 8.36 14.49 1.77
2-18 TAPSO 8.57 11.24 1.37
Water-8.19 1.0 0.161

(Table 4)
Sample Stabilizer pH = pKa Increase in absorbance Increase rate H ₂ O ₂ measured value
(300mM) (1 year)
2-19 MES 7.0 0.382 0.23 0.119
2-20 ADA 7.0 0.384 0.23 0.045
2-21 PIPES 7.0 0.367 0.22 0.113
7.5 0.172 0.10 0.106
2-22 MOPSO 7.0 0.195 0.11 0.116
7.5 0.264 0.16 0.111
2-23 BES 7.0 0.188 0.11 0.097
7.5 0.183 0.11 0.079
2-24 MOPS 7.5 0.202 0.12 0.110
2-25 HEPES 8.0 0.209 0.12 0.065
2-26 DIPSO 8.0 0.213 0.13 0.034
2-27 TAPSO 8.0 1.097 0.65 0.112
2-28 Tricine 8.5 0.202 0.12 0.093
2-29 TAPS 8.5 0.503 0.30 0.111
Water-1.699 1.0 0.128

In Table 3, samples (2-1) to (2-10) are examples, and other samples are comparative examples. In Table 4, samples (2-19) to (2-29) are implemented. It is an example.

  When TPM-PS coexists with POD alone in water, TPM-PS becomes extremely unstable, so when dissolved in water, it is about 5 to 10 times more non-enzymatic than TPM-PS alone. Coloration occurs. However, as shown in Tables 3 and 4, according to each Example, an increase in absorbance was suppressed as compared with the TPM-PS aqueous solution using water. On the other hand, the comparative example showed a significant increase in absorbance. Moreover, as shown in Table 4, it can be said that the increase in absorbance was further suppressed by increasing the concentration of various stabilizers. Among the samples (2-1) to (2-10), ADA, PIPES, MOPSO, BES, MOPS, and Tricine show excellent effects, and among the samples (2-19) to (2-29) In particular, PIPES (pH 7.5), MOPSO (pH 7.0), BES (pH 7.0), MOPS, and Tricine showed excellent effects. In addition, from the results of the absorbance measurement on the hydrogen peroxide solution, hydrogen peroxide can be measured with sufficient sensitivity by the enzymatic method, so that the TPM-PS reagent of the present invention is sufficient as a coloring reagent for the enzymatic method. It turns out that it is applicable. In addition, although the light absorbency about a hydrogen oxide solution showed the result using the TPM-PS reagent sample at the time of preparation, this result did not change with storage days (the same applies to other examples).

  In this example, suppression of an increase in absorbance due to non-enzymatic oxidation of TPM-PS was examined for a TPM-PS reagent (TPM-PS + POD + FAOD) to which various stabilizers were added. As the stabilizer, buffers described in Tables 5 and 6 below were used.

  Using water as the solvent, TPM-PS reagent sample TPM-PS reagent sample shown in Tables 5 and 6 below (POD concentration 67KU / L: FAOD concentration 18KU / L: TPM-PS concentration 0.25mM: Stabilizer concentration 150mM, The sample was stored and the absorbance was measured in the same manner as in Example 1 except that 300 mM) was prepared. The storage temperature was 25 ° C. when the stabilizer concentration was 150 mM, and 4 ° C. when the stabilizer concentration was 300 mM. In addition, the trade name PAODL (manufactured by Kikkoman) was used for FAOD (hereinafter the same). The amount of change for one year converted from the amount of change in absorbance by storage for 7 days, and the amount of increase in absorbance for 1 year (converted value) for a TPM-PS aqueous solution in which only TPM-PS is dissolved in water Tables 5 and 6 below show the rate of increase in absorbance (converted value) of each sample. Table 5 below shows the results when the stabilizer concentration is 150 mM, and Table 6 below shows the results when the stabilizer concentration is 300 mM. Further, the absorbance was measured in the same manner as in Example 2 using a hydrogen peroxide solution. These results are shown in Tables 5 and 6 below.

  Furthermore, using the TPM-PS reagent samples (3-6) to (3-16) at the time of preparation, an enzyme reaction was performed on the FV (fructosyl valine) solution, and the absorbance of the reaction solution was measured. Specifically, in the method for measuring absorbance described in Example 1, the absorbance was measured in the same manner except that 8.4 μL of 14 μM FV solution was used instead of 8.4 μL of distilled water as the solution to be measured. The absorbance obtained by subtracting a blank (absorbance when 8.4 μL of distilled water is used as the measurement target solution) from this absorbance is shown in Table 6 below. The final concentration of FV during the reaction was 1 μM.

(Table 5)
Sample Stabilizer pH = pKa Increase in absorbance Increase rate H ₂ O ₂ measured value
(150mM) (1 year)
3-1 MES 6.65 7.02 0.50 0.123
3-2 ADA 6.77 3.31 0.23 0.105
3-3 PIPES 6.99 5.53 0.39
3-4 MOPSO 7.0 6.20 0.44 0.156
3-5 HEPES 7.12 9.17 0.65 0.152
Water-14.15 1.0 0.161

(Table 6)
Sample Stabilizer pH = pKa Increase in absorbance Increase rate H ₂ O ₂ FV
(300mM) (1 year) Measured value Measured value
3-6 MES 7.0 1.951 0.20 0.115 0.075
3-7 ADA 7.0 0.348 0.04 0.046 0.045
3-8 PIPES 7.0 1.223 0.13 0.107 0.081
7.5 1.119 0.11 0.103 0.088
3-9 MOPSO 7.0 2.268 0.23 0.107 0.073
7.5 1.896 0.19 0.104 0.100
3-10 MOPS 7.5 2.810 0.29 0.113 0.097
3-11 HEPES 8.0 1.353 0.14 0.076 0.068
3-12 TAPSO 8.0 4.924 0.51 0.118 0.106
3-13 Tricine 8.5 4.795 0.49 0.097 0.083
3-14 BES 7.0 30.028 3.08 0.097 0.058
7.5 26.830 2.75 0.085 0.079
3-15 DIPSO 8.0 23.392 2.40 0.040 0.041
3-16 TAPS 8.5 63.207 6.48 0.120 0.099
Water-9.749 1.0 0.128 0.071

When TPM-PS coexists with POD alone in water, TPM-PS becomes extremely unstable, so when dissolved in water, it is about 5 to 10 times more non-enzymatic than TPM-PS alone. Coloration occurs. However, as shown in Tables 5 and 6, according to each Example, an increase in absorbance was suppressed as compared with the TPM-PS aqueous solution using water. Further, as shown in Table 6, it can be said that the increase in absorbance was further suppressed by increasing the concentration of various stabilizers. In addition, from the results of the absorbance measurement for hydrogen peroxide solution and FV solution, since the measurement of hydrogen peroxide and FV can be performed with sufficient sensitivity by the enzyme method, the TPM-PS reagent of the present invention is a coloring reagent. It can be seen that the method is sufficiently applicable to enzymatic methods. In addition, although the light absorbency about FV solution showed the result using the TPM-PS reagent sample at the time of preparation, this result did not change with storage days (the same applies to other examples).

  In this example, suppression of an increase in absorbance due to non-enzymatic oxidation of TPM-PS was examined for a TPM-PS reagent (TPM-PS + FAOD) to which various stabilizers were added. As the stabilizer, buffers described in Table 7 below were used.

  Except that water was used as a solvent and a TPM-PS reagent sample (FAOD concentration 18KU / L: TPM-PS concentration 0.25 mM: stabilizer concentration 300 mM) shown in Table 7 below was prepared, the same procedure as in Example 1 was performed. The sample was stored (storage temperature 4 ° C.) and the absorbance was measured. The amount of change for one year converted from the amount of change in absorbance by storage for 7 days, and the amount of increase in absorbance for 1 year (converted value) for a TPM-PS aqueous solution in which only TPM-PS is dissolved in water Table 7 below shows the rate of increase in absorbance (converted value) for each sample. Further, in the same manner as in Example 3, an enzyme reaction was performed on the FV solution using the sample, and the absorbance of the reaction solution was measured. These results are also shown in Table 7 below.

(Table 7)
Sample Stabilizer pH = pKa Increase in absorbance Increase rate FV measurement
(150mM) (1 year)
4-1 MES 7.0 0.558 0.60 0.079
4-2 ADA 7.0 0.256 0.27 0.043
4-3 PIPES 7.0 0.614 0.66 0.081
7.5 0.476 0.51 0.087
4-4 MOPSO 7.0 0.605 0.65 0.071
7.5 0.412 0.44 0.100
4-5 MOPS 7.5 0.482 0.52 0.095
4-6 HEPES 8.0 0.212 0.23 0.065
4-7 DIPSO 8.0 0.307 0.33 0.039
4-8 TAPSO 8.0 0.156 0.17 0.080
4-9 Tricine 8.5 0.289 0.31 0.078
4-10 TAPS 8.5 0.802 0.86 0.090
4-11 BES 7.0 1.683 1.80 0.057
7.5 0.979 1.05 0.076
Water-0.936 1.0 0.088

When TPM-PS is allowed to coexist with FAOD alone in water, TPM-PS becomes extremely unstable, and when dissolved in water, remarkably non-enzymatic coloring occurs as compared to TPM-PS alone. However, as shown in Table 7, if the stabilizers of samples (4-1) to (4-10) are used, compared to the TPM-PS aqueous solution using water and the comparative example of sample (4-11) As a result, the increase in absorbance was suppressed. In particular, as shown in Example 1 above, TAPSO cannot suppress an increase in absorbance in the reagent of TPM-PS alone, but in the case of a TPM-PS reagent containing FAOD, the increase in absorbance is caused by coexistence with FAOD. The effect that it can suppress was shown. Further, from the results of the absorbance measurement with respect to the FV solution, since the FV measurement can be performed with sufficient sensitivity by the enzyme method, the TPM-PS reagent of the present invention is sufficiently applicable to the enzyme method as a coloring reagent. I understand that.

  In this example, suppression of an increase in absorbance due to non-enzymatic oxidation of TPM-PS was examined for a TPM-PS reagent (TPM-PS only) to which various stabilizers were added. As the stabilizer, chelating agents described in Table 8 below were used.

  Example 1 except that water was used as a solvent and a TPM-PS reagent sample (TPM-PS concentration 0.25 mM: stabilizer concentration 25 mM) shown in Table 8 below was prepared and stored at 4 ° C. for 7 days. Similarly, the sample was stored and the absorbance was measured. The amount of change for one year converted from the amount of change in absorbance by storage for 7 days, and the amount of increase in absorbance for 1 year (converted value) for a TPM-PS aqueous solution in which only TPM-PS is dissolved in water Table 8 below shows the rate of increase in absorbance (converted value) of each sample.

(Table 8)
Sample Stabilizer pH = pKa Increase in absorbance Increase rate
(25mM) (1 year)
5-1 ADA 8.0 0.073 0.22
5-2 Bicine 7.0 0.047 0.15
5-3 CyDTA 7.0 0.142 0.43
5-4 DPTA-OH 7.0 0.122 0.37
8.0 0.043 0.13
9.0 0.051 0.16
5-5 DTPA 8.0 0.024 0.07
9.0 0.050 0.15
5-6 EDDP 7.0 0.099 0.30
5-6 EDTA-OH 7.0 0.064 0.19
5-7 EGTA 9.0 0.045 0.14
5-8 HIDA 8.0 0.035 0.11
5-9 IDA 7.0 0.040 0.12
5-10 NTA 9.0 0.055 0.17
5-11 TTHA 7.0 0.036 0.11
5-12 EDTA-2K 7.0 0.067 0.21
5-13 EDTA 9.0 0.058 0.18
Water-0.327 1.0

As shown in Table 8, according to samples (5-1) to (5-13), an increase in absorbance due to extremely non-enzymatic oxidation was suppressed as compared with the TPM-PS aqueous solution using water. Moreover, compared with the various stabilizer in Example 1, the inhibitory effect of sufficient increase in absorbance was shown at a low concentration. In particular, DTPA (pH 8.0), HIDA, and TTHA showed excellent effects.

  In this example, suppression of an increase in absorbance due to non-enzymatic oxidation of TPM-PS was examined for a TPM-PS reagent (TPM-PS + POD) to which various stabilizers were added. As the stabilizer, chelating agents described in Table 9 below were used.

  Except that water was used as a solvent and a TPM-PS reagent sample (POD concentration 67KU / L: TPM-PS concentration 0.25 mM: chelating agent concentration 25 mM) shown in Table 9 below was prepared, the same procedure as in Example 5 was performed. The sample was stored and the absorbance was measured. The amount of change in absorbance after storage for 7 days, the amount of change for one year converted from the amount of change, and the amount of increase in absorbance (converted value) for one year for a TPM-PS aqueous solution in which only TPM-PS is dissolved in water are 1 Table 9 below shows the increase ratio of the absorbance increase (converted value) of each sample. Further, in the same manner as in Example 2, the POD reaction was performed on the hydrogen peroxide solution (1 μM), and the absorbance was measured. The results are also shown in Table 9 below.

(Table 9)
Sample Stabilizer pH = pKa Increase in absorbance Increase rate H ₂ O ₂ measured value
(25mM) (1 year)
6-1 ADA 7.0 0.338 0.20 0.114
8.0 0.329 0.19 0.113
6-2 Bicine 7.0 0.605 0.35 0.046
6-3 CyDTA 7.0 0.291 0.17 0.086
6-4 DPTA-OH 7.0 0.266 0.15 0.080
8.0 0.133 0.08 0.096
9.0 0.080 0.05 0.064
6-5 DTPA 8.0 0.126 0.07 0.103
9.0 0.090 0.05 0.069
6-6 EDDP 7.0 0.351 0.20 0.116
6-6 EDTA-OH 7.0 0.300 0.17 0.097
6-7 EGTA 9.0 0.085 0.05 0.049
6-8 HIDA 8.0 0.316 0.18 0.090
6-9 IDA 7.0 0.191 0.11 0.057
6-10 NTA 9.0 0.115 0.07 0.087
6-11 TTHA 7.0 0.259 0.15 0.105
6-12 EDTA-2K 7.0 0.276 0.16 0.115
6-13 EDTA 9.0 0.076 0.04 0.031
Water-1.721 1.0 0.127

As shown in Table 9, according to each sample using the stabilizer, an increase in absorbance was suppressed as compared with the TPM-PS reagent using water. In particular, DPTA-OH (pH8.0), DTPA (pH8.0), IDA, NTA, TTHA, EDTA-2K show excellent effects, among them DPTA-OH (pH8.0), TTHA (pH7.0) Showed a very good effect without a decrease in measurement sensitivity. In addition, from the results of the absorbance measurement on the hydrogen peroxide solution, hydrogen peroxide can be measured with sufficient sensitivity by the enzymatic method, so that the TPM-PS reagent of the present invention is sufficient as a coloring reagent for the enzymatic method. It turns out that it is applicable.

  In this example, suppression of an increase in absorbance due to non-enzymatic oxidation of TPM-PS was examined for a TPM-PS reagent (TPM-PS + POD + FAOD) to which various stabilizers were added. As the stabilizer, chelating agents described in Table 10 below were used.

  Except that water was used as a solvent and the TPM-PS reagent samples shown in Table 10 below were prepared (POD concentration 67KU / L: FAOD concentration 18KU / L: TPM-PS concentration 0.25 mM: chelant concentration 25 mM). In the same manner as in Example 5, the sample was stored and the absorbance was measured. The amount of change for one year converted from the amount of change in absorbance by storage for 7 days, and the amount of increase in absorbance for 1 year (converted value) for a TPM-PS aqueous solution in which only TPM-PS is dissolved in water Table 10 below shows the rate of increase in absorbance (converted value) of each sample. In addition, the POD reaction is performed on the hydrogen peroxide solution in the same manner as in Example 2, the absorbance of the reaction solution is measured, and the enzyme reaction is performed on the FV solution in the same manner as in Example 3. The absorbance of the liquid was measured. These results are also shown in Table 10 below.

(Table 10)
Sample Stabilizer pH = pKa Increase in absorbance Increase rate H ₂ O ₂ FV
(25mM) (1 year) Measured value Measured value
7-1 ADA 8.0 2.83 0.29 0.115 0.109
7-2 CyDTA 7.0 3.22 0.33 0.098 0.083
7-3 DPTA-OH 7.0 1.88 0.19 0.092 0.078
8.0 2.30 0.23 0.109 0.080
7-4 DTPA 8.0 1.67 0.17 0.115 0.076
7-5 EDDP 7.0 7.35 0.74 0.131 0.071
7-6 EDTA-OH 7.0 1.38 0.14 0.111 0.079
7-7 HIDA 8.0 2.48 0.25 0.101 0.094
7-8 IDA 7.0 0.484 0.05 0.065 0.049
7-9 TTHA 7.0 1.30 0.13 0.121 0.103
Water-9.89 1.0 0.120 0.078

As shown in Table 10, according to the sample using each stabilizer, an increase in absorbance was suppressed as compared to the TPM-PS aqueous solution using water. In addition, from the results of the absorbance measurement for hydrogen peroxide solution and FV solution, since the measurement of hydrogen peroxide and FV can be performed with sufficient sensitivity by the enzyme method, the TPM-PS reagent of the present invention is a coloring reagent. It can be seen that the method is sufficiently applicable to enzymatic methods.

  In this example, suppression of an increase in absorbance due to non-enzymatic oxidation of TPM-PS was examined for a TPM-PS reagent (TPM-PS + FAOD) to which various stabilizers were added. As the stabilizer, chelating agents described in Table 11 below were used.

  Except that water was used as a solvent and a TPM-PS reagent sample (FAOD concentration 18KU / L: TPM-PS concentration 0.25 mM: chelating agent concentration 25 mM) shown in Table 11 below was prepared, the same procedure as in Example 5 was performed. The sample was stored and the absorbance was measured. The amount of change for one year converted from the amount of change in absorbance by storage for 7 days, and the amount of increase in absorbance for 1 year (converted value) for a TPM-PS aqueous solution in which only TPM-PS is dissolved in water Table 11 shows the rate of increase in absorbance (converted value) of each sample. Further, the enzyme reaction was performed on the FV solution in the same manner as in Example 3, and the absorbance was measured. These results are shown in Table 11 below.

(Table 11)
Sample Stabilizer pH = pKa Increase in absorbance Increase rate FV measurement
(25mM) (1 year)
8-1 ADA 8.0 0.029 0.03 0.113
8-2 CyDTA 7.0 0.110 0.13 0.086
8-3 DPTA-OH 7.0 0.152 0.18 0.080
8.0 0.044 0.05 0.082
8-4 DTPA 8.0 0.026 0.03 0.079
8-5 EDDP 7.0 0.239 0.28 0.074
8-6 EDTA-OH 7.0 0.030 0.04 0.082
8-7 HIDA 8.0 0.027 0.03 0.097
8-9 IDA 7.0 0.089 0.10 0.050
8-10 TTHA 7.0 0.062 0.07 0.106
Water-0.852 1.0 0.082

As shown in Table 11, when each stabilizer was used, an increase in absorbance was suppressed as compared with a TPM-PS aqueous solution using water. In particular, ADA, DPTA-OH (pH 8.0), DTPA (pH 8.0), EDTA-OH, and HIDA showed excellent effects. In addition, the increase in absorbance could be further suppressed as compared with Example 4. Further, from the results of the absorbance measurement with respect to the FV solution, since the FV measurement can be performed with sufficient sensitivity by the enzyme method, the TPM-PS reagent of the present invention is sufficiently applicable to the enzyme method as a coloring reagent. I understand that.

  In this example, the improvement of suppression of non-enzymatic color development was examined by further coexisting a stabilizing accelerator.

  Except that a TPM-PS reagent having the following composition was prepared and stored at 4 ° C. for 7 days, the absorbance of each reagent was measured in the same manner as in Example 1, and the increase in absorbance for one year was converted. Further, when the absorbance increase amount of the TPM-PS reagent having an ADA concentration of 300 mM is 1, the increase ratio of the absorbance increase amount of each reagent was determined. These results are shown in Table 12 below. Further, using the TPM-PS reagent immediately after preparation, an enzyme reaction was performed on the FV solution in the same manner as in Example 3, and the absorbance was measured. These results are shown in Table 12 below.

(TPM-PS reagent)
ADA 300 mM
POD 67 KU / L
FAOD 18 KU / L
TPM-PS 0.25 mM
Additive 0, 50 mM, 100 mM

(Table 12)
Stabilization accelerator Absorbance increase Increase ratio FV measurement
(1 year)
No additive (0mM) 0.365-0.047
100 mM Na salt NaCl 0.298 0.817 0.048
Nitric acid Na 0.309 0.846 0.047
Sodium sulfate 0.311 0.851 0.046
50 mM K salt thiocyanate K 0.289 0.791 0.035

As shown in Table 12, it was found that not only ADA was added to the TPM-PS reagent but also non-enzymatic color development could be further suppressed by coexisting Na salt or K salt as a stabilization promoter. It was. Specifically, the color development degree could be further suppressed by about 20% compared to the case where no stabilization accelerator was added. In particular, the TPM-PS reagent using Na salt, as shown in the results of absorbance measurement with respect to the FV solution in Table 12, shows no decrease in measurement sensitivity even when a stabilization accelerator is added. By adding a stabilization accelerator, the stability of TPM-PS can be further improved, and the TPM-PS reagent can be used sufficiently as a color-developing reagent.For example, by adding an appropriate concentration of the stabilization accelerator, It can be said that stabilization can be improved.

  This example is an example in which improvement of non-enzymatic color suppression was examined by further adding NaCl.

  Except that TPM-PS reagents A, B and C having the following composition were prepared and stored at 4 ° C. for 7 days, the absorbance of each reagent was measured in the same manner as in Example 1, and the absorbance increased for one year. Was converted. Further, when the absorbance increase amount of the TPM-PS reagent (NaCl 0 mM) having an ADA concentration of 300 mM is 1, the increase ratio of the absorbance increase amount of each reagent was determined. These results are shown in Table 13 and Table 14 below. Further, using the TPM-PS reagent A immediately after preparation, an enzyme reaction was performed on the FV solution in the same manner as in Example 3, and the absorbance was measured. These results are shown in Table 13 and Table 14 below. Table 13 shows the results of TPM-PS reagent A, and Table 14 shows the results of TPM-PS reagents B and C.

(TPM-PS Reagent A)
ADA 300 mM, 200 mM, 100 mM
POD 67 KU / L
FAOD 18 KU / L
TPM-PS 0.25 mM
NaCl 0, 300mM, 500mM, 700mM, 1000mM

(TPM-PS Reagent B)
ADA 300 mM, 200 mM, 100 mM
POD 67 KU / L
TPM-PS 0.25 mM
NaCl 0, 500mM, 700mM

(TPM-PS Reagent C)
ADA 300 mM, 200 mM, 100 mM
FAOD 18 KU / L
TPM-PS 0.25 mM
NaCl 0, 500mM, 700mM

(Table 13)
TPM-PS Reagent A
ADA concentration NaCl concentration Increase in absorbance Increase rate FV measurement
(mM) (mM) (1 year)
300 mM 0 0.394 1.0 0.046
200 mM 0 0.449 1.14 0.060
300 0.301 0.76 0.049
500 0.272 0.69 0.044
700 0.261 0.66 0.041
1000 0.251 0.64 0.037
100 mM 0 1.003 2.55 0.075
500 0.296 0.75 0.060
700 0.265 0.67 0.056
1000 0.248 0.63 0.051

(Table 14)
TPM-PS Reagent B TPM-PS Reagent C
ADA concentration NaCl concentration Increase in absorbance Increase rate Increase in absorbance Increase rate
(mM) (mM) (1 year) (1 year)
300 mM 0 0.382 1.0 0.256 1.0
200 mM 500 0.234 0.612 0.241 0.943
100 mM 700 0.222 0.581 0.228 0.889

As shown in Tables 13 and 14, it was found that not only ADA was added to the TPM-PS reagent but also non-enzymatic color development could be further suppressed by coexisting NaCl. In particular, TPM-PS Reagent A containing POD and FAOD showed particularly excellent effects when the NaCl concentration was 500 mM for the ADA concentration of 200 mM and the NaCl concentration was 700 mM for the ADA concentration of 100 mM. Similar effects were obtained when Na nitrate, Na sulfate and K chloride were added instead of NaCl.

  In this example, the improvement in non-enzymatic color suppression was further investigated by the addition of KSCN.

  Except for the preparation of TPM-PS Reagent D, TPM-PS Reagent E, and TPM-PS Reagent F having the following composition and storage at 4 ° C. for 7 days, the absorbance of each reagent was measured in the same manner as in Example 1. The increase in absorbance over one year was converted. Further, when the absorbance increase amount of the TPM-PS reagent (KSCN 0 mM) having an ADA concentration of 300 mM was 1, the increase ratio of the absorbance increase amount of each reagent was determined. These results are shown in Table 15 and Table 16 below. Further, using the TPM-PS reagent D immediately after preparation, an enzyme reaction was performed on the FV solution in the same manner as in Example 3, and the absorbance of the reaction solution was measured. Further, using the TPM-PS reagent D immediately after preparation, the POD reaction was performed on the hydrogen peroxide solution in the same manner as in Example 2, and the absorbance of the reaction solution was measured. These results are shown in Table 15 below.

(TPM-PS Reagent D)
ADA 300 mM, 200 mM (only for ADA 200 mM, also contains 500 mM NaCl)
POD 67 KU / L
FAOD 18 KU / L
TPM-PS 0.25 mM
KSCN 0, 10mM, 20mM, 30mM, 40mM, 50mM

(TPM-PS Reagent E)
ADA 300 mM, 200 mM (only for ADA 200 mM, also contains 500 mM NaCl)
TPM-PS 0.25 mM
KSCN 0, 20mM, 30mM, 50mM

(TPM-PS Reagent F)
ADA 300 mM, 200 mM (only for ADA 200 mM, also contains 500 mM NaCl)
POD 67 KU / L
TPM-PS 0.25 mM
KSCN 0, 20mM, 30mM, 50mM

(Table 15)
TPM-PS Reagent D
ADA concentration KSCN concentration Increase in absorbance Increase rate H ₂ O ₂ FV
(mM) (mM) (1 year) Measured value Measured value
300 mM 0 0.346 1.0 0.049 0.046
200 mM 0 0.240 0.694 0.049 0.046
10 0.228 0.660 0.045 0.041
20 0.214 0.618 0.042 0.038
30 0.203 0.588 0.037 0.032
40 0.206 0.596 0.035 0.030
50 0.208 0.600 0.032 0.028

(Table 16)
TPM-PS Reagent E TPM-PS Reagent F
ADA concentration KSCN concentration Increase in absorbance Increase rate Increase in absorbance Increase rate
(mM) (mM) (1 year) (1 year)
300 mM 0 0.039 1.0 0.321 1.0
200 mM 20 0.036 0.915 0.180 0.560
30 0.034 0.870 0.173 0.540
50 0.031 0.802 0.183 0.571

As shown in Tables 15 and 16, it was found that not only the addition of ADA to the TPM-PS reagent but also the coexistence of KSCN can further suppress non-enzymatic color development. In particular, TPM-PS reagent A containing POD and FAOD showed particularly excellent effects. In addition, the stability of TPM-PS can be improved at a low KSCN concentration of 20 to 50 mM, preferably 30 mM, based on the balance between the measurement results of H ₂ O ₂ and FV and the increase in absorbance in Table 15. I found out that Note that at a preferable KSCN concentration of 30 mM in terms of stability improvement, as shown in Table 15, a slight decrease in measurement sensitivity is observed, but in a range where there is no practical problem in measurement. The same effect was obtained when Na nitrate, Na sulfate and K chloride were added instead of KSCN.

  In this example, improvement in the measurement sensitivity of TPM-PS by adding sodium azide to a TPM-PS reagent containing TPM-PS and a stabilizer was examined.

A TPM-PS reagent (pH 7.0) having the following composition was prepared, and an enzyme reaction was performed on the FV solution in the same manner as in Example 3 using the TPM-PS reagent immediately after the preparation, and the absorbance of the reaction solution was measured. did. Further, using the TPM-PS reagent immediately after the preparation, a POD reaction was performed on the hydrogen peroxide solution in the same manner as in Example 2, and the absorbance of the reaction solution was measured. These results are shown in Table 15 below. In Table 15 below, “DW measurement value” is the absorbance of a blank when 8.4 μL of distilled water is used as the solution to be measured, and “FV measurement value” and “H ₂ O ₂ measurement value” Is the absorbance of the reaction solution before subtracting the absorbance of, and “ΔFV” and “ΔH ₂ O ₂ ” are values obtained by subtracting the absorbance of the blank from the absorbance of the reaction solution. The final concentration of azide Na in the reaction solution was 0, 0.15 g / L, 0.37 g / L, and 0.73 g / L, respectively, and the final concentration of the substrate (FV) in the reaction solution was 1.63 μM. .

(TPM-PS reagent)
ADA 200 mM
NaCl 500 mM
KSCN 30 mM
POD 67 KU / L
FAOD 18 KU / L
TPM-PS 0.25 mM
Na azide 0, 0.2g / L, 0.5g / L, 1.0g / L

(Table 17)
NaN ₃ concentration DW FV ΔFV H ₂ O ₂ ΔH ₂ O ₂
(g / L) Measured value Measured value (1.63μM) Measured value (1.63μM)
0 0.0146 0.0687 0.0541 0.0741 0.0595
0.2 0.0152 0.0757 0.0605 0.0761 0.0609
0.5 0.0159 0.0784 0.0625 0.0781 0.0622
1.0 0.0165 0.0786 0.0621 0.0783 0.0618

As shown in Table 17, an increase in absorbance was observed by adding sodium azide, and thus it was found that measurement sensitivity by TPM-PS can be improved by sodium azide.

  In this example, suppression of absorbance change due to non-enzymatic spontaneous color development was examined for a TPM-PS reagent containing TPM-PS and a stabilizer.

  Using water as a solvent, TPM-PS reagent samples G, H, I, and J having the following compositions were prepared and diluted 5.44 times with purified water. While the diluted solution was incubated at 30 ° C., the change in absorbance was measured over time. In addition, four kinds of TPM-PS aqueous solutions similar to the reagent samples G, H, I, and J were prepared except that no stabilizer was added, and the absorbance was measured in the same manner. These results are shown in FIGS. FIG. 1 is a graph showing the change in absorbance over time for TPM-PS reagent G, FIG. 2 is a graph showing the change in absorbance over time for TPM-PS reagent H, and FIG. FIG. 4 is a graph showing changes in absorbance over time for PS reagent I, and FIG. 4 is a graph showing changes in absorbance over time for TPM-PS reagent J.

(TPM-PS Reagent G)
TPM-PS Reagent G-1
TPM-PS 0.25mM
Tricine 300mM (pH8.5)
TPM-PS Reagent G-2
TPM-PS 0.25mM
BES 300mM (pH7.0)

(TPM-PS Reagent H)
TPM-PS Reagent H-1
TPM-PS 0.25mM
POD 67KU / L
PIPES 300mM (pH7.5)
TPM-PS Reagent H-2
TPM-PS 0.25mM
POD 67KU / L
ADA 300mM (pH7.0)

(TPM-PS Reagent I)
TPM-PS Reagent I-1
TPM-PS 0.25mM
POD 67KU / L
FAOD 18KU / L
ADA 300mM (pH7.0)
TPM-PS Reagent I-2
TPM-PS 0.25mM
POD 67KU / L
FAOD 18KU / L
PIPES 300mM (pH7.5)

(TPM-PS Reagent J)
TPM-PS Reagent J-1
TPM-PS 0.25mM
FAOD 18KU / L
TAPSO 300mM (pH8.0)
TPM-PS Reagent J-2
TPM-PS 0.25mM
FAOD 18KU / L
ADA 300mM (pH7.0)

As shown in each figure, as compared with the aqueous TPM-PS solution, the TPM-PS reagent containing various stabilizers could suppress the increase in absorbance over time. That is, it can be said that destabilization of TPM-PS was suppressed and non-enzymatic natural color development was prevented.

  In this example, suppression of an increase in absorbance due to non-enzymatic oxidation of TPM-PS was examined for a TPM-PS reagent (TPM-PS + POD) combined with two kinds of stabilizers.

  Use TPM-PS reagent sample (POD concentration 67KU / L: TPM-PS concentration 0.25mM) containing two kinds of stabilizers (Stabilizer 1 + Stabilizer 2) shown in Table 18 below using water as a solvent. The absorbance was measured in the same manner as in Example 1 except that the sample was prepared and stored at 4 ° C. for 45 days. The amount of change in absorbance due to storage for 45 days was multiplied by 365/45, and the amount of increase in absorbance for one year was converted. Further, the absorbance of the prepared reagent sample was measured using a hydrogen peroxide solution in the same manner as in Example 2. These results are shown in Table 18 below.

(Table 18)
Sample Stabilizer 1 pH Stabilizer 2 pH Absorbance increase H ₂ O ₂
(300mM) (10mM) (1 year) Measured value
8-1 Tricine 8.5 DPTA-OH 8.0 0.081 0.082
8-2 Tricine 8.5 DTPA 8.0 0.143 0.089
8-3 PIPES 7.0 IDA 7.0 0.078 0.074
8-4 MOPSO 7.0 IDA 7.0 0.085 0.084
8-5 BES 7.0 IDA 7.0 0.102 0.065
8-6 MOPS 7.5 IDA 7.0 0.156 0.080
8-7 Tricine 8.5 IDA 7.0 0.097 0.069
8-8 PIPES 7.0 TTHA 7.0 0.086 0.102
8-9 MOPSO 7.0 TTHA 7.0 0.051 0.111
8-10 BES 7.0 TTHA 7.0 0.115 0.083
8-11 MOPS 7.5 TTHA 7.0 0.035 0.103
8-12 Tricine 8.5 TTHA 7.0 0.081 0.083
8-13 PIPES 7.0 EDTA-2K 7.0 0.088 0.106
8-14 MOPSO 7.0 EDTA-2K 7.0 0.069 0.114
8-15 BES 7.0 EDTA-2K 7.0 0.094 0.088
8-16 MOPS 7.5 EDTA-2K 7.0 0.052 0.107
8-17 Tricine 8.5 EDTA-2K 7.0 0.069 0.092

As shown in Table 18, the stabilization of TPM-PS was further improved by using a stabilizer in combination. Specifically, when compared with the results shown in Table 4 in Example 2 using only one type of stabilizer, the amount of increase in absorbance per year was 0.202 for Tricine alone (2-29), and MOPSO alone ( 2-23) was 0.195, BES alone (2-24) was 0.188, and MOPS alone (2-25) was 0.202, but DPTA-OH, DTPA, IDA, TTHA, and EDTA-2K It was possible to reduce the increase to 0.035 to 0.156 only by adding 10 mM further. Further, by using two kinds of stabilizers in combination, there is no practical decrease in measurement accuracy. In Table 18, Samples 8-9, 8-11 and 8-16 showed excellent effects.

  In this example, suppression of an increase in absorbance due to non-enzymatic oxidation of TPM-PS was examined for a TPM-PS reagent (TPM-PS + POD + FAOD) combined with two kinds of stabilizers.

  TPM-PS reagent sample using water as a solvent and containing two kinds of stabilizers (Stabilizer 1 + Stabilizer 2) shown in Table 19 below (POD concentration 67KU / L: FAOD concentration 18KU / L: TPM- Absorbance was measured in the same manner as in Example 1 except that a PS concentration of 0.25 mM was prepared and stored at 4 ° C. for 45 days. The amount of change in absorbance due to storage for 45 days was multiplied by 365/45, and the amount of increase in absorbance for one year was converted. In addition, the absorbance of the prepared reagent sample was measured using a hydrogen peroxide solution in the same manner as in Example 2, and the absorbance was measured using an FV solution in the same manner as in Example 3. . These results are shown in Table 18 below.

(Table 19)
Sample Stabilizer 1 pH Stabilizer 2 pH Absorbance increase H ₂ O ₂ FV
(300mM) (10mM) (1 year) Measurement value Measurement value
9-1 MES 7.0 DPTA-OH 7.0 0.419 0.0958 0.0706
9-2 ADA 7.0 DPTA-OH 7.0 0.278 0.0421 0.0378
9-3 MOPSO 7.5 DPTA-OH 7.0 0.414 0.0701 0.0734
9-4 HEPES 8.0 DPTA-OH 7.0 0.256 0.0500 0.0426
9-5 HEPES 8.0 DTPA 8.0 0.264 0.0526 0.0464
9-6 MES 7.0 IDA 7.0 0.270 0.0898 0.0672
9-7 ADA 7.0 IDA 7.0 0.150 0.0422 0.0368
9-8 PIPES 7.5 IDA 7.0 0.197 0.0690 0.0565
9-9 MOPSO 7.5 IDA 7.0 0.288 0.0815 0.0554
9-10 HEPES 8.0 IDA 7.0 0.266 0.0508 0.0414
9-11 MES 7.0 TTHA 7.0 0.453 0.1145 0.0816
9-12 ADA 7.0 TTHA 7.0 0.281 0.0460 0.0412
9-13 PIPES 7.5 TTHA 7.0 0.309 0.0899 0.0728
9-14 MOPSO 7.5 TTHA 7.0 0.463 0.1046 0.0851
9-15 HEPES 8.0 TTHA 7.0 0.299 0.0511 0.0425

As shown in Table 19, the stabilization of TPM-PS was further improved by using a stabilizer in combination. Specifically, as shown in Table 6 in Example 3 using only one type of stabilizer, the amount of increase in absorbance per year was 1.951 for MES alone (3-6), ADA alone (3 -7) 0.348, PIPES alone (3-8) 1.223, MOPSO alone (3-9) 2.268, Tricine alone (3-13) 4.795, DPTA-OH, DTPA, Only by adding 10 mM of either TTHA or EDTA-2K, the amount could be reduced to 0.150 to 0.463. Further, by using two kinds of stabilizers in combination, there is no practical decrease in measurement accuracy. In Table 19, 9-6 to 9-10 to which sample IDA was added and 9-4, 9-5, 9-10 and 9-15 to which HEPES was added showed excellent effects.

  As described above, according to the stabilization method of the present invention, TPM-PS can be stably stored in a solution state. For this reason, it has become possible to use TPM-PS, which has heretofore been difficult to use despite being extremely excellent in sensitivity due to non-enzymatic natural color development, as a liquid reagent.

FIG. 1 is a graph showing changes in absorbance over time of a TPM-PS reagent in an example of the present invention. FIG. 2 is a graph showing changes in absorbance over time of the TPM-PS reagent in other examples of the present invention. FIG. 3 is a graph showing changes in absorbance of the TPM-PS reagent over time in still another example of the present invention. FIG. 4 is a graph showing changes in absorbance of the TPM-PS reagent over time in still another example of the present invention.

Claims (5)

A method of stabilizing N, N, N ', N', N '', N ''-hexa (3-sulfopropyl) -4,4 ''-triaminotriphenylmethane salt in solution,
In the solution, N, N, N ′, N ′, N ″, N ″ -hexa (3-sulfopropyl) -4,4 ″ -triaminotriphenylmethane salt, Bis-Tris, ADA , TEA, PIPES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, POPSO, HEPPSO, EPPS, Tris, Tricine, TAPS, CyDTA, DTPA, EDDP, EGTA, HIDA, IDA, NTA, TTHA, EDTA, and their salts N, N, N ', N', N '', N ''-hexa (3) coexisting with these hydroxides and at least one stabilizer selected from the group consisting of DPTA-OH Method for stabilizing -sulfopropyl) -4,4 ''-triaminotriphenylmethane salt. The stabilization method according to claim 1, wherein potassium thiocyanate (KSCN) is further allowed to coexist in the solution as a stabilization accelerator. Stabilizes N, N, N ', N', N '', N ''-hexa (3-sulfopropyl) -4,4 ''-triaminotriphenylmethane salt in solution in the presence of peroxidase A method,
In the solution, N, N, N ′, N ′, N ″, N ″ -hexa (3-sulfopropyl) -4,4 ″ -triaminotriphenylmethane salt and peroxidase, and Bis-Tris , ADA, TEA, PIPES, MOPSO, BES, MOPS, HEPES, Tricine, Bicine, MES, DIPSO, TAPSO, TAPS, CyDTA, DTPA, EDDP, EGTA, HIDA, IDA, NTA, TTHA, EDTA, their salts and these N, N, N ', N', N '', N ''-hexa (3-sulfo) in the presence of at least one stabilizer selected from the group consisting of Propyl) -4,4 ''-triaminotriphenylmethane salt stabilization method. Solution of N, N, N ', N', N '', N ''-hexa (3-sulfopropyl) -4,4 ''-triaminotriphenylmethane salt in the presence of peroxidase and fructosyl amino acid oxidase A way to stabilize in,
N, N, N ′, N ′, N ″, N ″ -hexa (3-sulfopropyl) -4,4 ″ -triaminotriphenylmethane salt, peroxidase and fructosyl amino acid oxidase in the solution MES, ADA, PIPES, MOPSO, HEPES, MOPS, TAPSO, Tricine, Bicine, CyDTA, DTPA, EDDP, EGTA, HIDA, IDA, NTA, TTHA, EDTA, their salts and their hydroxides and DPTA- N, N, N ', N', N '', N ''-hexa (3-sulfopropyl) -4,4 '' coexisting with at least one stabilizer selected from the group consisting of OH -Method for stabilizing triaminotriphenylmethane salt. N, N, N ', N', N '', N ''-hexa (3-sulfopropyl) -4,4 ''-triaminotriphenylmethane salt in solution in the presence of fructosyl amino acid oxidase A method of stabilizing,
In said solution, N, N, N ′, N ′, N ″, N ″ -hexa (3-sulfopropyl) -4,4 ″ -triaminotriphenylmethane salt and fructosyl amino acid oxidase; MES, ADA, PIPES, MOPSO, MOPS, HEPES, DIPSO, TAPSO, Tricine, TAPS, Bicine, CyDTA, DTPA, EDDP, EGTA, HIDA, IDA, NTA, TTHA, EDTA, their salts and their hydroxides, And N, N, N ′, N ′, N ″, N ″ -hexa (3-sulfopropyl) -4, in the presence of at least one stabilizer selected from the group consisting of and DPTA-OH A method for stabilizing 4 ''-triaminotriphenylmethane salt.

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