Method for stabilizing a leuco dye

Abstract

To provide a stable leuco dye-containing reagent by suppressing the non-specific color development over time of a leuco dye.SOLUTION: Provided is a method for suppressing a non-specific color reaction of a leuco dye, which is characterized by the coexistence of a chelating agent in a reagent containing the leuco dye. The method for suppressing the non-specific color development reaction of the present invention is effective for suppressing the non-specific color development over time of the leuco dye promoted by a component capable of generating at least one metal ion selected from the group consisting of calcium ion, manganese ion, iron ion and copper ion.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for stabilizing a chromogenic substrate solution used in the field of clinical tests and the like, and a stabilized chromogenic substrate solution.

Background Art

[0002] In the field of clinical tests, various items are measured by the enzyme method. For example, a method is conventionally known in which hydrogen peroxide is generated using an oxidase such as glucose oxidase, and the absorbance after color development is measured in the presence of peroxidase and a dye to quantify the concentration of a substance. As the types of dyes used at this time, for example, there are dyes called Trinder reagents or dyes called leuco dyes. A Trinder reagent is a dye composed of two types, a coupler such as 4-aminoantipyrine and various Trinder reagents. This Trinder reagent is a dye that is stable to light and heat and is known to react with two molecules of hydrogen peroxide. On the other hand, as leuco dyes, 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium (DA-67), N-(carboxymethylaminocarbonyl)-4,4-bis(dimethylamino)biphenylamine (DA-64), etc. are known. Although high-sensitivity measurement is possible because they react with one molecule of hydrogen peroxide, there are problems such as a high blank before measurement because they are unstable to light and heat. Therefore, various studies have been conducted on methods for enhancing the stability of leuco dyes.

[0003] So far, as a method for stabilizing a substrate solution containing a chromogenic substrate used for peroxidase activity measurement, a method of adding a reducing agent is known (Patent Document 1). Also, as a method for reducing the non-specific color development reaction of leuco dyes, a method of co-existing 1) a leuco dye having a maximum absorption wavelength in the range of 400 nm to 550 nm and not reacting with hydrogen peroxide and another dye, and 2) cyclodextrins is known (Patent Document 2). However, the technology for suppressing the coloring of a chromogenic substrate solution containing a leuco dye is not yet sufficient.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] As described above, the chromogenic substrate solution containing a leuco dye is very unstable and is easily oxidized and colored, so that the substrate solution containing the chromogenic substrate cannot be stored for a long time. In addition, during the development of the chromogenic substrate solution containing a leuco dye, the present inventor has experienced that the self-color development (non-specific color reaction) of the leuco dye may be significantly deteriorated depending on the storage conditions. And it has been found that the increase in self-color development of unknown cause caused by this storage situation is remarkable when the chromogenic substrate solution contains a component capable of generating metal ions (particularly, calcium ions, manganese ions, iron ions, or copper ions). The present invention has been made to solve the newly found drawbacks described above, and particularly aims to improve the stability of a chromogenic substrate solution in which metal ions and a leuco dye coexist, and to provide a method for suppressing coloring due to the non-specific color reaction of the leuco dye, as well as a substrate solution that is stabilized and has suppressed coloring.

Means for Solving the Problems

[0006] As a result of intensive studies to solve the above problems, the present inventor has found that the self-color development of a reagent containing a leuco dye as described above, particularly the non-specific self-color development of the leuco dye promoted by the coexistence of the leuco dye and metal ions, can be highly suppressed by the addition of a chelating agent, and the stability of the reagent containing the leuco dye can be significantly improved, and thus the present invention has been completed.

[0007] That is, a typical aspect of the present invention has the following configuration. [Item 1] A method for suppressing the non-specific color development reaction of a leuco dye, characterized by co-existing a chelating agent in a reagent containing the leuco dye. [Item 2] The method according to Item 1, for suppressing the non-specific color development reaction of a leuco dye caused by a component capable of generating metal ions present in the reagent. [Item 3] The method according to Item 2, wherein the metal ion is at least one selected from the group consisting of calcium ion, manganese ion, iron ion, and copper ion. [Item 4] The method according to Item 2 or 3, wherein the concentration of the component capable of generating calcium ions present in the reagent is 0.4 to 150 mM. [Item 5] The method according to Item 2 or 3, wherein the concentration of the component capable of generating manganese ions present in the reagent is 0.05 to 5 μM. [Item 6] The method according to Item 2 or 3, wherein the concentration of the component capable of generating iron ions present in the reagent is 0.3 to 30 μM. [Item 7] The method according to Item 2 or 3, wherein the concentration of the component capable of generating copper ions present in the reagent is 0.8 to 80 μM. [Item 8] The method according to any one of Items 1 to 7, wherein the chelating agent is at least one selected from the group consisting of ethylenediaminetetraacetic acid, glycol ether diamine tetraacetic acid, citric acid, and salts thereof. [Item 9] The method according to any one of Items 1 to 8, wherein the leuco dye is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium. [Item 10] The method according to any one of Items 1 to 9, wherein the reagent is a reagent for measuring glycated protein. [Item 11] The method according to Item 10, wherein the glycated protein is glycated hemoglobin. [Item 12] The method according to any one of Items 1 to 11, wherein the reagent containing the leuco dye contains neither protease, nor glycated peptide oxidase, nor peroxidase. [Item 13] A biological component measurement reagent containing a leuco dye and a chelating agent, in which the non-specific color development reaction of the leuco dye is suppressed. [Item 14] The biocomponent measurement reagent according to item 13, wherein the non-specific color development reaction of the leuco dye is a non-specific color development reaction of the leuco dye caused by a component capable of generating metal ions present in the reagent. [Item 15] The biocomponent measurement reagent according to item 13 or 14, wherein the non-specific color development reaction of the leuco dye is suppressed even after storage for 7 days. [Item 16] The biocomponent measurement reagent according to any one of items 13 to 15, which is a reagent for measuring glycated protein. [Item 17] The biocomponent measurement reagent according to item 16, wherein the glycated protein is glycated hemoglobin. [Item 18] The biocomponent measurement reagent according to any one of items 13 to 17, which does not contain protease, glycated peptide oxidase, or peroxidase. [Advantages of the Invention]

[0008] Leuco dyes are more sensitive than other dyes and are expected to be used in measurement systems that require higher accuracy. However, the problem has been the low storage stability due to self-color development. According to the present invention, for example, even in a chromogenic substrate solution in which metal ions coexist, the self-color development of the leuco dye can be effectively suppressed over a long period of time, and the performance of the dye can be stably exhibited. By applying the present invention, for example, a leuco dye can be stably used in a chromogenic substrate solution used in the field of biochemical analysis that requires highly accurate measurement such as glycated hemoglobin, contributing to more accurate biochemical analysis measurement. [Modes for Carrying Out the Invention]

[0009] Hereinafter, the present invention will be described in more detail while showing embodiments of the present invention.

[0010] [1. Method for Suppressing Non-Specific Color Development Reaction of Leuco Dye] One aspect of the present invention is a method for suppressing the non-specific color development reaction of a leuco dye, which is characterized by co-existing a chelating agent in a reagent containing the leuco dye. By co-existing the leuco dye and the chelating agent in a reagent solution such as a chromogenic substrate solution, for example, even when a component capable of generating metal ions such as calcium ions, manganese ions, iron ions, or copper ions may be present in the reagent solution containing the leuco dye, it is possible to highly suppress the self-color development of the leuco dye over time during storage.

[0011] The leuco dye that can be used in the present invention is not particularly limited. For example, phenothiazine-based dyes, triphenylmethane-based dyes, diphenylamine-based dyes, o-phenylenediamine, hydroxypropionic acid, diaminobenzidine, tetramethylbenzidine, etc. can be mentioned. From the viewpoint that the effects of the present invention are more easily obtained, as the leuco dye, phenothiazine-based dyes, triphenylmethane-based dyes, and diphenylamine-based dyes are preferred, phenothiazine-based dyes and diphenylamine-based dyes are more preferred, and phenothiazine-based dyes are even more preferred.

[0012] Examples of phenothiazine-based dyes include 10-N-carboxymethylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (MCDP), 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium (DA-67), etc. Also, as the phenothiazine-based dye, a compound having a 10-(aminocarbonyl)-3,7-bis(dialkylamino)phenothiazine skeleton as described in JP-A-2018-141115 can also be used. In this specification, the content described in JP-A-2018-141115 is incorporated by reference. Examples of triphenylmethane dyes include N,N,N’,N’,N’’,N’’-hexa(3-sulfopropyl)-4,4’,4’’-triaminotriphenylmethane (TPM-PS), 4,4’-benzylidenebis(N,N-dimethylaniline) (LMG), 4,4’,4’’-methylidynetris(N,N-dimethylaniline), etc. Examples of diphenylamine dyes include N-(carboxymethylaminocarbonyl)-4,4’-bis(dimethylamino)diphenylamine sodium salt (DA-64), 4,4’-bis(dimethylamino)diphenylamine, bis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine (BCMA), etc. The leuco dye may be used in the form of a salt. The salt is not particularly limited, and examples thereof include sodium salt and potassium salt.

[0013] When the method of the present invention is used in a peroxidase measurement system such as a reagent for measuring glycated hemoglobin, among the above-mentioned leuco dyes, a leuco dye of a phenothiazine dye or a diphenylamine dye is preferable, a leuco dye of a phenothiazine dye is more preferable, and 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium (DA-67) is particularly preferable. By using a leuco dye as described above, there are advantages such as a high molar absorption coefficient, a maximum absorption wavelength on the high wavelength side, the ability to avoid the wavelengths of coexisting substances in blood, and easy detection of the wavelength of the dye.

[0014] The concentration of the leuco dye in the reagent is not particularly limited as long as the effects of the present invention can be achieved, and can be appropriately adjusted according to the type of the leuco dye used, the types and concentrations of other compounding components, the degree of the desired non-specific reaction inhibitory effect, etc. Usually, the concentration of the leuco dye in the reagent (for example, the chromogenic substrate solution) is about 0.001 to 1 mM, preferably about 0.005 to 0.5 mM, more preferably about 0.01 to 0.1 mM. By using a reagent (for example, a chromogenic substrate solution) containing a leuco dye at such a concentration, it is possible to exhibit sufficient sensitivity as a measurement reagent in the field of biochemistry where highly sensitive analysis such as glycated hemoglobin is required, while being stable over time and suppressing self-color development even during reagent storage.

[0015] In the method for suppressing the non-specific color development reaction of the leuco dye of the present invention, in the reagent, the leuco dye is coexisted with one or more chelating agents. Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraminehexaacetic acid (TTHA), 1,3-propanediaminetetraacetic acid (PDTA), 1,3-diamino-2-hydroxypropanetetraacetic acid (DTPA-OH), hydroxyethyliminodiacetic acid (HIDA), dihydroxyethylglycine (DHEG), glycol ether diamine tetraacetic acid (EGTA), dicarboxymethylglutamic acid (CMGA), (S,S)-ethylenediaminedisuccinic acid (EDDS), hydroxyethylidene diphosphonic acid (HEDP), nitrilotris(methylenephosphonic acid) (NTMP), phosphonobutane tricarboxylic acid (PBTC), ethylenediaminetetra(methylenephosphonic acid) (EDTMP), gluconic acid, citric acid, O,O'-bis(2-aminophenyl)ethylene glycol-N,N,N'-tetraacetic acid, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), N,N-bis(2-hydroxyethyl)glycine (Bicine), trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, trans-1,2-cyclohexanediaminetetraacetic acid monohydrate (CyDTA), iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), or salts thereof (for example, disodium ethylenediaminetetraacetate, trisodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, etc.). In one preferred embodiment, when the leuco dye is coexisted with metal ions (for example, when components capable of generating metal ions such as calcium ions, manganese ions, iron ions, or copper ions are coexisted), preferably, ethylenediaminetetraacetic acid, glycol ether diamine tetraacetic acid, citric acid, or salts thereof are used. Here, examples of the salt include sodium salt, potassium salt, etc., but are not limited thereto.In the present invention, such a chelating agent may be used alone or in combination of two or more kinds.

[0016] In the reagent of the present invention (for example, the chromogenic substrate solution), the blending amount of such a chelating agent is not particularly limited as long as the effects of the present invention are achieved. For example, in a reagent containing a leuco dye, it can be about 0.01 to 10 mM, preferably about 0.1 to 10 mM, more preferably about 0.1 to 5 mM. By allowing the chelating agent to coexist in the reagent at such a concentration, non-specific self-color development of the leuco dye can be highly suppressed.

[0017] The solvent used for preparing the reagent of the present invention (for example, the chromogenic substrate solution) is not particularly limited as long as it does not inhibit the effects of the present invention. For example, solvents such as water or an appropriate buffer solution (for example, Good buffer solution, carbonate buffer solution, phosphate buffer solution, acetate buffer solution, borate buffer solution, tartrate buffer solution) can be used. As an example, when water is used as the solvent, it is preferably water that substantially or completely does not contain metal ions, and it is more preferable to use distilled water, purified water, deionized water, ion-exchanged water, pure water, ultrapure water, MilliQ water, biomedical water, etc.

[0018] As the Good buffer solution used in the present invention, any Good buffer solution known in the art can be widely used as long as the effects of the present invention are achieved. For example, as the Good buffer solution, 2-morpholinoethanesulfonic acid (MES) at a concentration of 10 to 200 mM, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 3-morpholinopropanesulfonic acid (MOPS), etc. are used, but it is not particularly limited.

[0019] In still other specific embodiments, a solvent such as water or a suitable buffer that may contain metal ions may be used. According to the present invention, even when metal ions such as calcium ions, manganese ions, iron ions, and copper ions coexist, there is an advantage that the self-color development of the reagent containing the leuco dye can be suppressed, and the reagent can be made stable over time during storage. Therefore, even when using a solvent that may contain such metal ions, a reagent containing a leuco dye that is stable over a long period of time can be obtained.

[0020] In one embodiment, the reagent containing the leuco dye of the present invention can be a reagent containing a component capable of generating metal ions. As shown in the results of the test examples described later, it has been found that the leuco dye promotes non-specific self-color development when coexisting with metal ions. In particular, it has also been confirmed that such non-specific self-color development is remarkable when calcium ions, manganese ions, iron ions, or copper ions coexist as metal ions. According to the present invention, even when a component capable of generating such metal ions coexists, it may be possible to suppress the non-specific self-color development of the leuco dye. The component capable of generating metal ions that may coexist in the reagent containing the leuco dye of the present invention is not particularly limited, and examples thereof include components capable of generating calcium ions, manganese ions, iron ions, copper ions, sodium ions, zinc ions, chromium ions, aluminum ions, and lead ions. In one embodiment, the reagent containing the leuco dye can be one containing a component capable of generating calcium ions, manganese ions, iron ions, and / or copper ions as a component capable of generating metal ions.

[0021] In one embodiment, the component capable of generating iron ions (also referred to as an iron-containing substance) that the reagent containing the leuco dye of the present invention may contain is not particularly limited, and examples thereof include iron-containing compounds containing iron ions, iron proteins, iron oxide-based pigments, and the like. In this specification, the iron ion refers to an ion containing iron in its composition, and includes, for example, a concept including ferricyanide ions and / or ferrocyanide ions. Further, the iron ion is a divalent iron ion (Fe 2+) or trivalent iron ions (Fe 3+ ) may also be used. For example, as components capable of generating iron ions, FeBr2, FeBr3, Fe3C, FeCl2, FeCl3, FeF2, FeF3, FeH2, FeH3, FeI2, FeI3, FeN, Fe3N, Fe3N2, Fe(N3)2, FeO, Fe2O3, Fe3O4, FeS, FeS2, Fe2S3, Fe3S4, FeSe, Fe2Se3, FeSi2, Fe(C5H5)2, Fe(ClO3)3, Fe(ClO4)2, Fe(ClO4)3, Fe(CN)2, Fe(CN)3, FeCO3, Fe(CO)5, FeC2O4, Fe2(CO3)3, Fe2(CO)9, Fe2(C2O4)3, Fe3(CO) 12 , Fe2(CrO4)3, Fe2(Cr2O7)3, Fe5(IO6)2, FeMnO4, FeMoO4, Fe(NO3)2, Fe(NO3)3, Fe(OH)2, Fe(OH)3, FeO(OH), FePO4, Fe3(PO4)2, FeSeO4, FeSO3, FeSO4, Fe2(SO4)3, H2FeO4, BaFeO4, K2FeO4, Fe(IO3)2, Fe(IO3)3, FeWO4, [Fe(C5H5)2]BF4, Fe(CH3COO)2, Fe(CH3COO)3, Fe(HCOO)2, Fe(OCN)2, Fe(SCN)2, Fe(SCN)3, H3[Fe(CN)6], H4[Fe(CN)6], (NH4)2Fe(SO4)2 and other iron-containing compounds; iron proteins such as hemoglobin, myoglobin, catalase, cytochrome, hemopexin, transferrin, hemosiderin, ferritin; iron oxide-based pigments such as red iron oxide (α-Fe2O3 is the main component), ferric yellow (α-FeOOH), iron black (Fe3O4 (FeOFe2O3)), yellow-brown pigment (ZnOFe2O3, MgOFe2O3), transparent iron oxide (α-Fe2O3, α-FeOOH), etc. can be mentioned, but are not limited thereto.

[0022] The components capable of generating these iron ions can be appropriately selected according to, for example, the biological components or biological samples to be measured, and added to the reagent containing the leuco dye. From the viewpoint that it is not only excellent in handling convenience but also expected that the present invention can more highly suppress the non-specific color development of the leuco dye, as the component (iron-containing substance) capable of generating iron ions, iron(II) chloride (FeCl2), iron(III) chloride (FeCl3), ferrocyanide (H3[Fe(CN)6]), ferricyanide (H4[Fe(CN)6]), catalase, etc. are preferably used, and iron(II) chloride and iron(III) chloride are more preferred.

[0023] When the reagent of the present invention (for example, the chromogenic substrate solution) contains an iron-containing substance, the compounding amount of the iron-containing substance in the reagent is not particularly limited as long as the effects of the present invention are achieved. Preferably, the concentration of the iron-containing substance in the reagent containing the leuco dye is about 0.1 to 40 μM, and preferably about 0.3 to 30 μM. Even when an iron-containing substance of such a concentration coexists in the reagent, according to the present invention, the non-specific color development of the leuco dye can be highly suppressed to obtain a reagent that is stable over time.

[0024] In one embodiment, the component capable of generating copper ions (also referred to as a copper-containing substance) that the reagent containing the leuco dye of the present invention may contain is not particularly limited, and examples thereof include copper salts containing copper ions, copper proteins containing copper ions as cofactors, pigments containing copper ions, and the like. For example, as the component capable of generating copper ions, copper salts such as CuBr, CuBr2, CuC2, Cu2C2, CuCl, CuCl2, CuF, CuF2, CuH, CuI, CuI2, CuN3, Cu(N3)2, CuO, CuO2, Cu2O, Cu3P, CuP2, CuS, Cu2S, Cu3(AsO4)2, Cu(BF4)2, Cu(ClO3)2, Cu(ClO4)2, CuCN, Cu(CN)2, CuCrO4, Cu(IO3)2, Cu(IO4)2, Cu(NO3)2, CuOH, Cu(OH)2, Cu3(PO4)2, CuSO4, Cu2SO4, Cu(CH3COO), Cu(CH3COO)2, CuCO3·Cu(OH)2, CuSCN, Cu(SCN)2; mixtures containing copper salts such as Proclin 200 (a preservative containing 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, magnesium salt, copper salt, and water); copper proteins such as ascorbic acid oxidase (also referred to as ascorbate oxidase), azurin, stellacyanin, plastocyanin, quercetin-2,3-dioxygenase, rusticyanin, pseudoazurin, laccase, copper-containing nitrite reductase, superoxide dismutase, galactose oxidase, dopamine-β-hydroxylase, dopamine-β-monooxygenase, peptidylglycine α-amidating monooxygenase, membrane-bound methane monooxygenase, copper amine oxidase, galactose oxidase, tyrosinase, catechol oxidase, ceruloplasmin, cytochrome c oxidase, hemocyanin, zincated nitrite reductase;Examples of pigments containing copper ions include, but are not limited to, CuCO3·Cu(OH)2 (malachite), CuSO4·3Cu(OH)2 (brochantite), Cu2(OH)3Cl (atacamite), Cu2(OH)3Cl (paratacamite), Cu(C2H3O2)2·3Cu(AsO2)2 (scherbenite), CuCO3·Cu(OH)2 (malachite), (Cu,Zn)2(CO3)(OH)2 (rosasite), (Zn,Cu)2(CO3)(OH)2 (zincrosasite), (Zn,Cu)2(AsO4)(OH)2 (adamite), (Cu,Zn)6(AsO4,PO4)2(OH)6·H2O (philipsburgite), etc.;

[0025] Components that generate these copper ions can be appropriately selected according to, for example, biological components or biological samples to be measured, and added to a reagent containing a leuco dye. From the perspective that it is not only excellent in handling convenience but also expected that the present invention can more highly suppress the non-specific color development of the leuco dye, as components capable of generating copper ions, it is preferable to use copper(I) chloride (CuCl), copper(II) chloride (CuCl2), copper acetate, copper sulfate, copper nitrate, procryne 200, ascorbate oxidase, more preferably copper(I) chloride, copper(II) chloride, copper acetate, copper sulfate, copper nitrate, and even more preferably copper(I) chloride and copper(II) chloride.

[0026] When the reagent of the present invention (for example, the chromogenic substrate solution) contains a copper-containing substance, the compounding amount of the copper-containing substance in the reagent is not particularly limited as long as the effects of the present invention are achieved. Preferably, the concentration of the copper-containing substance in the reagent containing the leuco dye is about 0.5 to 100 μM, preferably about 0.8 to 80 μM. Even when a copper-containing substance of such a concentration coexists in the reagent, according to the present invention, non-specific color development of the leuco dye can be highly suppressed, and a reagent that is stable over time can be obtained.

[0027] In one embodiment, the component that can generate calcium ions (also referred to as calcium-containing substance) that the reagent containing the leuco dye of the present invention may contain is not particularly limited, and examples thereof include calcium salts containing calcium ions. For example, as the component that generates calcium ions, CaO, CaO2, Ca(OH)2, CaF2, CaCl2, CaBr2, CaI2, CaH2, CaC2, Ca3P2, CaCO3, Ca(HCO3)2, Ca(NO3)2, CaSO4, CaSO3, CaSiO3, Ca3(PO4)2, Ca2O7P2, Ca(ClO3)2, Ca(BrO3)2, CaCrO4, Ca(CH3COO)2, C 12 H 22 CaO 14 、C6H 10 CaO6、C 14 H 10 CaO4、Ca(C 17 H 35 COO)2 etc. can be mentioned, but are not limited thereto.

[0028] These components that generate calcium ions can be appropriately selected according to, for example, the biological component or biological sample to be measured, and added to the reagent containing the leuco dye. From the viewpoint that it is expected that not only the handling convenience is excellent, but also the non-specific coloring of the leuco dye can be more highly suppressed in the present invention, as the component (calcium-containing substance) that can generate calcium ions, calcium chloride, calcium hydroxide, calcium acetate, and calcium phosphate are preferably used.

[0029] When the reagent of the present invention (for example, the chromogenic substrate solution) contains a calcium-containing substance, the blending amount of the calcium-containing substance in the reagent is not particularly limited as long as the effects of the present invention are exhibited. Preferably, the concentration of the calcium-containing substance in the reagent containing the leuco dye is about 0.1 to 200 mM, preferably about 0.4 to 150 mM. Even when a calcium-containing substance of such a concentration coexists in the reagent, according to the present invention, non-specific coloring of the leuco dye can be highly suppressed to obtain a reagent that is stable over time.

[0030] In one embodiment, the component capable of generating manganese ions (also referred to as a manganese-containing substance) that the reagent containing the leuco dye of the present invention may contain is not particularly limited, and examples thereof include manganese salts containing manganese ions. For example, as the component capable of generating manganese ions, MgCl2, KMnO4, MnO2, MnCO3, Mn(NO3)2, MnSO4, Mn(OH)2, Mn(OCOCH3)2, C 10 H 14 MnO2 and the like can be mentioned, but are not limited thereto.

[0031] These components capable of generating manganese ions can be appropriately selected according to, for example, the biological component or biological sample to be measured, and added to the reagent containing the leuco dye. From the viewpoint that it is not only excellent in handling convenience but also expected that the present invention can more highly suppress the non-specific color development of the leuco dye, it is preferable to use manganese chloride, manganese acetate, or manganese sulfate as the component capable of generating manganese ions.

[0032] When the reagent of the present invention (for example, the chromogenic substrate solution) contains a manganese-containing substance, the blending amount of the manganese-containing substance in the reagent is not particularly limited as long as the effects of the present invention are exhibited. Preferably, the concentration of the manganese-containing substance in the reagent containing the leuco dye is about 0.01 to 10 μM, and preferably about 0.05 to 5 μM. Even when a manganese-containing substance having such a concentration coexists in the reagent, according to the present invention, non-specific color development of the leuco dye can be highly suppressed to obtain a reagent that is stable over time.

[0033] The method for suppressing the non-specific color reaction of the present invention can be used in any reagent for measuring biological components that uses a leuco dye. Examples of the biological components to be measured include glycated hemoglobin, glucose, free cholesterol, total cholesterol, HDL (high-density lipoprotein)-cholesterol, LDL (low-density lipoprotein)-cholesterol, triglyceride, phospholipid, uric acid, monoamine oxidase, etc. in a biological sample, and items that have been conventionally measured by the enzyme method (H2O2 generation system). In one preferred embodiment, as an index for diabetes, more sensitive measurement is required, and the present invention can be preferably implemented in a reagent for measuring glycated proteins that highly suppresses the increase in the blank value based on the self-color development of the leuco dye.

[0034] Glycated proteins are those formed by the binding of sugars such as glucose or fructose to the amino groups of proteins, and examples include glycated hemoglobin and glycated albumin. It is known that the degree of protein glycation in the living body is proportional to the blood glucose concentration over time. For example, by measuring the ratio of glycated proteins such as glycated hemoglobin and glycated albumin in the blood, it becomes possible to grasp the blood glucose control state of diabetes. Among glycated proteins, measuring glycated hemoglobin (especially hemoglobin A1c) with high precision is particularly important in the diagnosis of diabetes. The present invention can make it possible to suppress the self-color development of the measurement reagent over time in the measurement of glycated hemoglobin that requires highly sensitive measurement, enabling more accurate measurement.

[0035] In one embodiment, the reagent containing the leuco dye of the present invention can further contain any component well-known in the art as needed in addition to the chelating agent. Examples include enzymes, preservatives, antioxidants, salts, stabilizers, etc. used in a reaction system for measuring biological components by detection with a leuco dye.

[0036] The enzyme that can be incorporated into the reagent containing the leuco dye of the present invention is not particularly limited, and any enzyme known in the art (for example, catalase, ascorbic acid oxidase, etc.) can be incorporated. For example, in the case of a chromogenic substrate solution used for measuring glycated proteins, it may or may not contain protease, peroxidase, glycated peptide oxidase.

[0037] In this specification, protease is not particularly limited, and examples thereof include aminopeptidase, dipeptidase, dipeptidyl peptidase, tripeptidyl peptidase, peptidyl dipeptidase, serine carboxypeptidase, proline carboxypeptidase, alanine carboxypeptidase, metalloprotease, cysteine carboxypeptidase, omega peptidase, serine endopeptidase, cysteine protease, aspartic protease, neutral protease, threonine endopeptidase, and the like.

[0038] In this specification, peroxidase is not particularly limited as long as it can generate a chromogenic dye by bringing hydrogen peroxide into contact with a chromogenic substrate. Preferred examples include those derived from horseradish and microorganisms. Among them, peroxidase derived from horseradish is preferred. The peroxidase is commercially available in high purity and at low cost.

[0039] In this specification, glycated peptide oxidase means an enzyme that can catalyze a reaction of oxidizing an Amadori compound to generate hydrogen peroxide. Glycated peptide oxidase is also sometimes called glycated amino acid oxidase, fructosyl amino acid oxidase, fructosyl peptide oxidase, fructosylamine oxidase, Amadoriase, ketoamine oxidase, glycated hexapeptide oxidase, glycated hemoglobin oxidase, and the like.

[0040] Generally, many enzymatic reactions are known to require metal ions as cofactors for promoting the reaction. For example, peroxidase requires iron, and metalloprotease, which is a type of protease, is known to require zinc, calcium, etc. as cofactors. Since the reagent containing the leuco dye of the present invention is characterized by coexisting with a chelating agent, it is desirable not to coexist such metal ion-requiring enzymes in the reagent of the present invention containing the leuco dye. In addition, in a reagent for measuring glycated proteins based on the enzymatic method (H2O2 generation system), due to the characteristics of the enzymatic reaction, etc., the reagent configuration is often such that glycated peptide oxidase is added substantially simultaneously with peroxidase. Therefore, a reagent containing glycated peptide oxidase together with peroxidase may be preferably provided in an embodiment prepared separately from the reagent containing the leuco dye, and the reagent containing the leuco dye is provided in an embodiment that does not contain peroxidase or glycated peptide oxidase.

[0041] From the above viewpoints, in one embodiment of the present invention, it is preferable that the reagent containing the leuco dye is a reagent that does not contain enzymes such as protease, glycated peptide oxidase, peroxidase, etc. In this case, enzymes such as protease, glycated peptide oxidase, peroxidase, etc. can be prepared as separate reagents from the reagent containing the leuco dye, and can be provided in the form of a kit comprising a plurality of reagents to be combined at the time of use.

[0042] The preservative that can be incorporated into the reagent containing the leuco dye of the present invention is not particularly limited, and any preservative known in the art can be incorporated. For example, specific examples of the preservative include various procurines (registered trademark, Spellco), azides, antibacterial agents, etc. Examples of the antibacterial agent include gentamicin, kanamycin, chloramphenicol, piperacillin, imidazolidinyl urea, etc. Incorporating a preservative has the merit of preventing the reagent from spoiling.

[0043] When a preservative is incorporated into the reagent containing the leuco dye of the present invention, its concentration is not particularly limited. For example, the concentration of the preservative in the reagent is preferably about 0.005 to 1 w / v%, more preferably about 0.01 to 0.1 w / v%. Further, the reagent containing the leuco dye of the present invention can also be in a form substantially free of a preservative. Here, being substantially free means that the concentration of the preservative in the chromogenic substrate solution is less than 0.005 w / v%.

[0044] When salts are incorporated into the reagent containing the leuco dye of the present invention, their types and concentrations are not particularly limited. Examples include sodium chloride, sodium acetate, sodium phosphate, sodium hydroxide, potassium chloride, potassium acetate, potassium phosphate, potassium hydroxide, aluminum chloride, aluminum acetate, aluminum phosphate, and the like. By incorporating such salts, a more stable reagent can be obtained.

[0045] The properties of the reagent (e.g., chromogenic substrate solution) containing the leuco dye of the present invention are usually adjusted to a range of about pH 4 to 9, more preferably about pH 6 to 8. To adjust the pH of the reagent containing the leuco dye of the present invention, for example, it can be adjusted by appropriately adding a buffer solution.

[0046] By allowing a chelating agent to coexist in the reagent containing the leuco dye of the present invention, non-specific color development that occurs over time during reagent storage can be effectively suppressed. As an example, in the reagent containing the leuco dye of the present invention, non-specific color development of the leuco dye is suppressed even after storage for 1 day after reagent preparation, preferably, non-specific color development of the leuco dye is suppressed even after storage for 7 days after reagent preparation, and more preferably, non-specific color development of the leuco dye is suppressed even after storage for 8 days after reagent preparation. The storage conditions are not particularly limited. For example, storage under refrigeration (about 1 to 8 °C) conditions may be possible, or storage under room temperature (about 1 to 30 °C) or normal temperature (about 15 to 25 °C) conditions may be possible, or storage under higher temperature (about 35 °C) conditions may be possible. For example, even after storage for 8 days under refrigeration conditions or after storage for 7 days at 35 °C, it is possible to suppress the non-specific color development reaction of the reagent containing the leuco dye according to the present invention.

[0047] When the method of the present invention is applied to the measurement of glycated proteins, the operation can be carried out in the same manner as the conventional method. For example, after mixing a sample containing glycated proteins and an enzyme solution containing an enzyme for glycated protein measurement, a chromogenic substrate solution containing the leuco dye of the present invention is mixed, and the mixture is reacted at a predetermined temperature (usually about 37°C) for a predetermined time (usually about 10 minutes). Then, by measuring the absorbance, the reaction amount of the chromogenic substrate can be determined. Furthermore, based on the obtained change amount of absorbance, by comparing it with a calibration curve prepared in advance, the concentration (amount) of the substance to be measured in the sample can be determined.

[0048] [2. Reagent for Measuring Biological Components] As described above, it has been found that by using a reagent in which a leuco dye and a chelating agent coexist, the non-specific self-coloring reaction of the leuco dye can be suppressed, and a reagent that is stable over time can be obtained. Therefore, from another aspect, the present invention further provides a reagent for measuring biological components containing a leuco dye and a chelating agent, in which the non-specific coloring reaction of the leuco dye is suppressed. The types and amounts of the leuco dye and the chelating agent used in this reagent for measuring biological components, and the types and amounts of components that can generate metal ions that may coexist or other components, etc. can be the same as those described in the method for suppressing the non-specific coloring reaction of the leuco dye in 1. above. The reagent for measuring biological components of the present invention can be preferably used, for example, for the measurement of glycated proteins such as glycated hemoglobin and glycated albumin, for which high-precision measurement is required.

Examples

[0049] Hereinafter, the present invention will be described in more detail based on test examples, but the present invention is not limited to these examples.

[0050] [Test Example 1: Evaluation of the Influence When Various Metal Ions are Added] In order to investigate the cause of the non-specific self-coloring of the reagent containing the leuco dye, the following tests were conducted. First, a 50 mM PIPES solution (pH 6.3) containing 0.06 mM DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium) as a leuco dye was prepared. Metal salts shown in Table 1 below were added to this leuco dye-containing solution so as to have a predetermined final concentration. The solution thus prepared was allowed to stand and stored at 4°C for 1 day or 8 days. As a comparative example, a DA-67-containing solution prepared in the same manner as above except that no metal salt was added was prepared and stored under the same conditions. The degree of coloring due to non-specific self-coloring of the solution containing the leuco dye was determined by measuring the absorbance at 660 nm after storage of these solutions. [Table 1]

[0051] The results are shown in Table 2 below. As shown in Table 2, it was found that even in the case of a leuco dye-containing reagent without a metal salt, the absorbance increased over time, indicating that the leuco dye gradually self-colored non-specifically. And it was recognized that this non-specific coloring of the leuco dye was not much affected by the coexistence of the metal salt during relatively short-term storage in the refrigerator (storage at 4°C for 1 day), or if there was an effect, it was only limited to the case where a high-concentration metal salt was added. On the other hand, when stored for a longer period (storage at 4°C for 8 days), it was found that the coexistence of the metal salt promoted the non-specific self-coloring of the leuco dye and the degree of coloring deteriorated significantly. [Table 2]

[0052] [Test Example 2: Inhibitory effect of non-specific coloring reaction caused by calcium-containing substances] In order to evaluate the effect of suppressing the non-specific coloring reaction caused when a leuco dye coexists with a calcium-containing substance, the following test was conducted. First, a 50 mM PIPES solution (pH 6.3) containing 0.06 mM DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium) as a leuco dye was prepared. Chelating agents A to C shown in Table 3 below were added to this leuco dye-containing solution so that the final concentration became 1 mM. Next, metal salt a (calcium chloride) described in Table 1 of Test Example 1 was added to the solution containing this leuco dye and chelating agent so as to reach a predetermined final concentration. The solution thus prepared was allowed to stand and stored at 4°C for 8 days. After storage for 8 days, the absorbance at 660 nm was measured for these solutions to determine the degree of coloring due to non-specific self-color development of the solution containing the leuco dye. [Table 3]

[0053] The results are shown in Table 4 below. As shown in Table 4, by adding 1 mM of chelating agents (EDTA·3Na, EGTA, sodium citrate), the non-specific self-color development reaction of the leuco dye (DA-67) caused by the coexistence of calcium-containing substances was suppressed, and it was found that a stable reagent could be obtained even after long-term storage for 8 days. [Table 4]

[0054] [Test Example 3: Inhibitory effect on non-specific color development reaction caused by manganese-containing substances and iron-containing substances] In order to evaluate the effect of suppressing the non-specific color development reaction caused when the leuco dye coexists with manganese-containing substances and iron-containing substances, the following test was conducted. First, a 50 mM PIPES solution (pH 6.3) containing 0.06 mM DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium) as a leuco dye was prepared. Chelating agent B (EGTA) shown in Table 3 of Test Example 2 above was added to this leuco dye-containing solution to a final concentration of 1 mM. Next, metal salt b (manganese chloride) or metal salt c (iron(II) chloride) described in Table 1 of Test Example 1 was added to the solution containing the leuco dye and the chelating agent to a predetermined final concentration. The solution thus prepared was allowed to stand and stored at 4°C for 8 days. After storage for 8 days, the absorbance at 660 nm of these solutions was measured to determine the degree of coloration due to non-specific self-coloration of the solution containing the leuco dye.

[0055] The results are shown in Table 5 below. As shown in Table 5, by adding 1 mM of the chelating agent (EGTA), the non-specific self-coloration reaction of the leuco dye (DA-67) caused by the coexistence of the manganese-containing substance (manganese chloride) or the iron-containing substance (iron(II) chloride) was suppressed, and it was found that a stable reagent could be obtained even after long-term storage for 8 days. [Table 5]

[0056] [Test Example 4: Suppression effect of non-specific coloration reactions caused by various iron-containing substances] In order to evaluate the effect of suppressing the non-specific coloration reaction caused when the leuco dye coexists with various iron-containing substances, the following test was conducted. First, a 50 mM PIPES solution (pH 6.3) containing 0.06 mM DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium) as a leuco dye was prepared. Chelating agent C (sodium citrate) shown in Table 3 of Test Example 2 above was added to this leuco dye-containing solution so that the final concentration was 1 mM. Subsequently, metal salt c (iron(II) chloride) or metal salt d (iron(III) chloride) described in Table 1 of Test Example 1 was added to the solution containing this leuco dye and chelating agent so as to reach a predetermined final concentration. The solution thus prepared was allowed to stand and stored at 4°C for 8 days or at 35°C for 7 days. Since the evaluation at 35°C corresponds to an accelerated test under refrigerated storage conditions, the effects when stored over a longer period can be evaluated. After storage of the various mixtures, the absorbance at 660 nm of these solutions was measured to determine the degree of coloring due to non-specific self-coloring of the solution containing the leuco dye.

[0057] The results are shown in Table 6 below. As shown in Table 6, by adding 1 mM of the chelating agent (sodium citrate), not only can the non-specific self-coloring reaction of the leuco dye (DA-67) caused by the coexistence of various iron-containing substances (iron(II) chloride or iron(III) chloride) be suppressed when stored at refrigerated conditions for 8 days, but it was also confirmed that it can be effectively suppressed even when stored for a long period of 7 days at 35°C under accelerated test conditions. [Table 6]

[0058] [Test Example 5: Suppression effect on non-specific coloring reaction caused by copper-containing substances] In order to evaluate the effect of suppressing the non-specific coloring reaction caused when a leuco dye coexists with a copper-containing substance, the following test was conducted. First, a 50 mM PIPES solution (pH 6.3) containing 0.06 mM DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium) as a leuco dye was prepared. Chelating agent A (EDTA·3Na) or chelating agent B (EGTA) shown in Table 3 of Test Example 2 above was added to this leuco dye-containing solution so that the final concentration was 1 mM. Next, metal salt e (copper(II) chloride) described in Table 1 of Test Example 1 was added to the solution containing the leuco dye and the chelating agent so as to reach a predetermined final concentration. The solution thus prepared was allowed to stand and stored at 4°C for 8 days or at 35°C for 7 days. After storage, the absorbance at 660 nm of these solutions was measured to determine the degree of coloration due to non-specific self-coloration of the solution containing the leuco dye.

[0059] The results are shown in Table 7 below. As shown in Table 7, by adding 1 mM of a chelating agent (EDTA·3Na or EGTA), not only can the non-specific self-coloration reaction of the leuco dye (DA-67) caused by the coexistence of a copper-containing substance (copper(II) chloride) be suppressed when stored at 4°C for 8 days, but it can also be effectively suppressed when stored for a long time at 35°C for 7 days under accelerated test conditions.

Table 7

[0060] [Test Example 6: Suppression effect of non-specific coloration reaction caused by manganese-containing substances] In order to further evaluate the effect of suppressing the non-specific coloration reaction caused when a leuco dye coexists with a manganese-containing substance, the following test was conducted. First, a 50 mM PIPES solution (pH 6.3) containing 0.06 mM DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium) as a leuco dye was prepared. Chelating agent B (EGTA) or chelating agent C (sodium citrate) shown in Table 3 of Test Example 2 above was added to this leuco dye-containing solution so that the final concentration was 1 mM. Next, metal salt b (manganese chloride) described in Table 1 of Test Example 1 was added to the solution containing this leuco dye and chelating agent so as to reach a predetermined final concentration. The solution thus prepared was allowed to stand and stored at 35°C for 7 days. After storage, the absorbance at 660 nm of these solutions was measured to determine the degree of coloring due to non-specific self-color development of the solution containing the leuco dye.

[0061] The results are shown in Table 8 below. As shown in Table 8, it was confirmed that by adding 1 mM of a chelating agent (EGTA or sodium citrate), the non-specific self-color development reaction of the leuco dye (DA-67) caused by the coexistence of a manganese-containing substance (manganese chloride) can be suppressed even when stored for a long period of 7 days at 35°C. [Table 8]

[0062] [Test Example 7: Suppression effect on non-specific color development reaction caused by calcium-containing substance] In order to further evaluate the effect of suppressing the non-specific color development reaction caused when a leuco dye coexists with a manganese-containing substance, the following test was conducted. First, a 50 mM PIPES solution (pH 6.3) containing 0.06 mM DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium) as a leuco dye was prepared. Chelating agent A (EDTA·3Na), chelating agent B (EGTA), or chelating agent C (sodium citrate) shown in Table 3 of Test Example 2 above was added to this leuco dye-containing solution so that the final concentration was 1 mM. Next, metal salt a (calcium chloride) described in Table 1 of Test Example 1 was added to the solution containing the leuco dye and the chelating agent so that the final concentration was 149.7 mM. The solution thus prepared was allowed to stand and stored at 35°C for 7 days. After storage, the absorbance at 660 nm of these solutions was measured to determine the degree of coloring due to non-specific self-color development of the solution containing the leuco dye.

[0063] The results are shown in Table 9 below. As shown in Table 9, it was confirmed that by adding 1 mM of a chelating agent (EDTA·3Na, EGTA, or sodium citrate), the non-specific self-color development reaction of the leuco dye (DA-67) caused by the coexistence of a calcium-containing substance (calcium chloride) can be suppressed even when stored for a long period of 7 days at 35°C.

Table 9

[0064] [Test Example 8: Suppression effect of non-specific color development reaction not caused by metal ions] The effect of suppressing the non-specific color development reaction that occurs when a solution containing a leuco dye does not contain metal ions was evaluated as follows. First, a 50 mM PIPES solution (pH 6.3) containing 0.06 mM DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium) as a leuco dye was prepared. Chelating agent A (EDTA·3Na), chelating agent B (EGTA), or chelating agent C (sodium citrate) shown in Table 3 of Test Example 2 above was added to this leuco dye-containing solution so that the final concentration was 1 mM. These solutions were allowed to stand and stored at 35°C for 7 days. As a comparative example, a DA-67-containing solution prepared in the same manner as above except that it did not contain a chelating agent was prepared and stored under the same conditions. After storage, the absorbance at 660 nm of these solutions was measured to determine the degree of coloring due to non-specific self-coloring of the solution containing the leuco dye.

[0065] The results are shown in Table 10 below. As shown in Table 10, it was shown that by adding 1 mM of a chelating agent (EDTA·3Na, EGTA, or sodium citrate), the non-specific color development over time of the leuco dye-containing solution in the case of not containing components capable of generating metal ions can also be suppressed.

Table 10

[0066] From the results of Test Examples 1 to 8 above, it can be seen that the reagent containing a leuco dye is colored over time, this non-specific color development is promoted when metal ions coexist, and the non-specific self-coloring of this leuco dye can be effectively suppressed by coexisting a chelating agent.

Industrial Applicability

[0067] The present invention can suppress the non-specific color development over time of a reagent containing a leuco dye (for example, a chromogenic substrate solution), and can be preferably implemented in reagents used in various inspection methods using a leuco dye.

Claims

1. Characterized in that a chelating agent coexists in a reagent containing a leuco dye (provided that it does not contain N,N,N',N',N'',N''-hexa(3-sulfopropyl)-4,4',4''-triaminotriphenylmethane (TPM-PS) and protease), The chelating agent is at least one selected from the group consisting of ethylenediaminetetraacetic acid, glycol ether diamine tetraacetic acid, citric acid, and salts thereof, The leuco dye is sodium 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine, A method for suppressing the non-specific color development reaction of a leuco dye, wherein the concentration of a component capable of generating metal ions present in the reagent satisfies any one of the following (a) to (d). (a) The concentration of a component capable of generating calcium ions present in the reagent is 0.4 to 150 mM (b) The concentration of a component capable of generating manganese ions present in the reagent is 0.05 to 5 μM (c) The concentration of a component capable of generating iron ions present in the reagent is 0.3 to 30 μM (d) The concentration of a component capable of generating copper ions present in the reagent is 0.8 to 80 μM

2. The method according to claim 1, wherein the reagent is a reagent for measuring glycated protein.

3. The method according to claim 2, wherein the glycated protein is glycated hemoglobin.

Images