Reagent for biological component measurement which reduces effects of dobesilic acid

Abstract

Provided is a reagent for biological component measurement which is capable of reducing the effects of dobesilic acid. One embodiment of the present invention is a reagent for biological component measurement containing a nitroxyl radical that has a redox-reactive functional group or does not have a functional group.

Description

Reagent for measuring biological components that reduces the effects of dobesilate

[0001] The present invention relates to a reagent for measuring a biological component that reduces the effects of dobesilic acid.

[0002] In clinical diagnosis, enzymatic methods have been used to measure biological components, and in particular, methods using an oxidase-peroxidase-oxidation-reduction color-developing reagent system, i.e., a method in which a substance to be measured in a sample is subjected to an enzymatic reaction to generate hydrogen peroxide, which is then reacted with a color-developing agent in the presence of peroxidase for colorimetric quantification, have been widely used. This method has been known to have the problem of being susceptible to interferences coexisting in serum, such as biological reducing substances such as ascorbic acid and bilirubin, but various countermeasures have been developed to overcome these interferences.

[0003] On the other hand, Patent Document 1 reports that dobesilic acid is an interfering substance and describes that the interference by dobesilic acid is avoided by using a reagent containing 4-methoxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical.

[0004] Chinese Patent No. 111766234

[0005] However, since the 4-methoxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical used in Patent Document 1 is present at a high concentration of 8 mM (1.49 g / L) or 5 mM (0.93 g / L), it is thought that the manufacturing cost of the measurement reagent will be high.

[0006] Objects of the present invention include providing a reagent for measuring a biological component that reduces the effects of dobesilic acid.

[0007] The present inventors conducted extensive research to solve the problems and found that the effects of dobesilic acid can be reduced even at low doses by incorporating a nitroxy radical, either with or without a redox-reactive functional group, into a reagent for measuring a biological component. Based on this finding, the present inventors conducted further extensive research and have completed the present invention.

[0008] The present invention encompasses the following aspects: (Item 1) A reagent for measuring a biological component that reduces the effects of dobesilic acid, characterized by containing a nitroxy radical that has a redox-reactive functional group or that has no functional group. (Item 2) The reagent for measuring a biological component according to Item 1, wherein the redox-reactive functional group is at least one selected from the group consisting of a hydroxy group, an oxo group, a carbonyl group, a carboxy group, a nitro group, an amino group, an amide group, a nitrile group, a thiol group, and an isothiocyanato group. (Item 3) The reagent for measuring a biological component according to Item 1 or 2, wherein the nitroxy radical having a redox-reactive functional group is at least one selected from the group consisting of 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-oxo-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-amino-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, and 3-carboxy-2,2,5,5-tetramethylpyrrolidine 1-oxyl free radical. (Item 4) The reagent for measuring a biological component according to any one of Items 1 to 4, wherein the nitroxy radical not having a functional group is 2,2,6,6-tetramethylpiperidine 1-oxyl free radical. (Item 5) The reagent for measuring a biological component according to any one of Items 1 to 4, wherein the nitroxy radical is contained in an amount of 0.01 to 2 mmol / L. (Item 6) The reagent for measuring a biological component according to any one of Items 1 to 5, wherein the biological component is creatinine. (Item 7) The reagent for measuring a biological component according to any one of Items 1 to 6, further comprising a chloride salt.(Item 8) The reagent for measuring a biological component according to Item 7, wherein the chloride salt is at least one selected from the group consisting of sodium chloride, niobium pentachloride, lead(II) chloride, potassium chloride, indium(III) chloride, zinc(II), antimony(III), cesium chloride, silver(I), rubidium chloride, cerium(III), cobalt(II), copper(II), gallium(III), lithium chloride, manganese(II), nickel(II), aluminum(III), samarium(III), barium chloride, copper(I), chromium(II), cadmium(II), europium(III), palladium(II), and tin(II). (Item 9) The reagent for measuring a biological component according to Item 7 or 8, wherein the chloride salt is contained at 30 to 70 mmol / L. (Item 10) The reagent for measuring a biological component according to any one of Items 1 to 9, further comprising a cyano complex. (Item 11) Item 11. The reagent for measuring a biological component according to Item 10, wherein the cyano complex is at least one selected from the group consisting of potassium hexacyanoferrate(II) (potassium ferrocyanide), potassium hexacyanoferrate(III) (potassium ferricyanide), potassium tetracyanozincate(II), potassium hexacyanoiridate(III), potassium dicyanoaurate(I), potassium dicyanoargentate(I), potassium tetracyanoaurate(III), sodium tetracyanoaurate(III), potassium hexacyanocobaltate(III), potassium tetracyanomercurate(II), potassium tetracyanonickelate(II), potassium tetracyanoplatinate(II), cesium tetracyanoplatinate(II), sodium tetracyanoplatinate(II), barium tetracyanoplatinate(II), rubidium tetracyanoplatinate(II), potassium tetracyanopalladate(II), and potassium hexacyanorhodiumate(III). (Item 12) The reagent for measuring a biological component according to Item 10 or 11, wherein the cyano complex is potassium hexacyanoferrate(II) (potassium ferrocyanide). (Item 13) The reagent for measuring a biological component according to any one of Items 10 to 12, wherein the cyano complex is contained at 0.1 to 1 mmol / L. (Item 14) A reagent kit for measuring a biological component, comprising at least the following components (1) to (7):(1) a nitroxy radical having or not having a redox-reactive functional group; (2) a chloride salt; (3) a cyano complex; (4) a peroxidase; (5) an enzyme other than peroxidase; (6) a buffer; and (7) a color developer. (Item 15) The reagent kit for measuring a biological component according to Item 14, wherein the enzyme other than peroxidase (5) is at least one selected from the group consisting of ascorbic acid oxidase, sarcosine oxidase, catalase, creatine amidinohydrolase, and creatinine amidohydrolase. (Item 16) The reagent kit for measuring a biological component according to Item 14 or 15, wherein the buffer (6) is at least one selected from the group consisting of Tris buffer, citrate buffer, borate buffer, phosphate buffer, MES, Bis-Tris, ADA, ACES, BES, PIPES, MOPS, TES, HEPES, Tricine, Bicine, POPSO, TAPS, CHES, and CAPS. The color former (7) is N-ethyl-N-(3-sulfopropyl)-m-anisidine, aniline, N,N-dimethylaniline, N,N-diethylaniline, N,N-diethyl-m-toludine, N,N-dimethyl-m-anisidine, N-ethyl-(3-methylphenyl)-N'-acetylethylenediamine, N-ethyl-N-(β-hydroxyethyl)-m-toluidine, N-ethyl-N-(2-hydroxy-3-sulfoethyl)-m-toluidine, N-ethyl-N-sulfopropyl-m-toluidine, 17. The reagent kit for measuring a biological component according to any one of Items 14 to 16, wherein the 4-methoxy-2,2,6,6-tetramethylpiperidine-1-oxyl free radical is at least one selected from the group consisting of 4-methoxy-2,2,6,6-tetramethylpiperidine, N-ethyl-sulfopropyl-3,5-methoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine, phenol, p-chlorophenol, 2,4-dichlorophenol, 2,4-dibromophenol, and 2,3,4-trichlorophenol. (18) The reagent for measuring a biological component according to any one of Items 1 to 13, or the reagent kit for measuring a biological component according to any one of Items 14 to 17, which does not contain 4-methoxy-2,2,6,6-tetramethylpiperidine-1-oxyl free radical.

[0009] According to the present invention, it is possible to provide a reagent for measuring a biological component that is less susceptible to the effects of dobesilic acid.

[0010] The present invention will be described in further detail below while illustrating embodiments of the present invention, but the present invention is not limited thereto. All non-patent documents and patent documents described in this specification are incorporated herein by reference. In addition, the term "to" in this specification means "at least or equal to, at most or equal to," and for example, "X to Y" in this specification means "at least X and at most Y." In addition, the term "and / or" in this specification means any one or any possible combination of two or more of the listed elements. Furthermore, the term "comprise" in this specification encompasses the concepts of "consisting essentially of" and "consisting only of." In addition, in this specification, singular expressions should be understood to include the plural concept as well, unless otherwise specified.

[0011] (Reagent for Measuring Biological Component) In one aspect of the present invention, the reagent for measuring a biological component contains a nitroxy radical, which may or may not have a redox-reactive functional group. The reagent for measuring a biological component can reduce the influence of dobesilic acid. As used herein, the term "influence of dobesilic acid" encompasses the influence of dobesilic acid on biological component measurement, regardless of its form, and includes, for example, not only the influence of dobesilic acid in its free form, but also the influence of dobesilic acid in its salt form (e.g., a metal salt such as calcium salt).

[0012] (Redox-reactive functional group) Examples of the redox-reactive functional group include a hydroxy group (—OH), an oxo group (═O), a carbonyl group (—CO—; including —O—CO— and —CO—O—), a carboxy group (—COOH), a nitro group (—NO ２ ), amino group (-NH ２ ), an amide group (—NH—CO—), a nitrile group (—CN), a thiol group (—SH), an isothiocyanato group (—NCS), etc., and a hydroxy group or a carboxy group is preferable. On the other hand, examples of functional groups that are not redox-reactive functional groups (hereinafter also referred to as "non-redox-reactive functional groups") include an ether group (—O—), a methoxy group (—OCH ３) and other alkoxy groups.

[0013] (Nitroxy radical) Nitroxy radical is N—O ・ There are no particular limitations as long as the nitroxy radical has an atomic group. The nitroxy radical is preferably a substance having a cyclic structure represented by the following formula (1).

[0014] The nitroxy radical represented by formula 1 has an unpaired electron. Substances with unpaired electrons are highly reactive and undergo oxidation-reduction reactions with other atoms or molecules, resulting in stable molecules or ions. Furthermore, the ring size and steric hindrance allow it to exist stably, making it useful as a mild oxidation catalyst for conversion to ketones or carboxylic acids. Examples of the ring in formula 1 include piperidine, pyrrolidine, 2-azaadamantane, 9-azanoradamantane, and 9-azabicyclo[3.3.1]nonane.

[0015] The type of nitroxy radical is not particularly limited as long as it has a redox-reactive functional group or does not have a functional group, and examples thereof include 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-oxo-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-oxo ... Examples of nitroxy radicals include 2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-methyl-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-isothiocyanato-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-cyano-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-glycidyloxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, and 3-carboxy-2,2,5,5-tetramethylpyrrolidine 1-oxyl free radical. Among the above-mentioned nitroxy radicals, the 2,2,6,6-tetramethylpiperidine 1-oxyl free radical corresponds to a "nitroxy radical having no functional group" and has a hydrogen group (-H) instead of a functional group.

[0016] Among these, at least one selected from the group consisting of 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-oxo-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, and 3-carboxy-2,2,5,5-tetramethylpyrrolidine 1-oxyl free radical is preferred, and at least one selected from the group consisting of 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical and 4-carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical is more preferred. These nitroxy radicals are commercially available products, etc.

[0017] The composition of the reagent for measuring a biological component of the present invention is not particularly limited, but it preferably contains a first reagent and a second reagent. The nitroxy radical, which may or may not have a redox-reactive functional group, may be contained in either the first reagent or the second reagent, but is preferably contained in the first reagent.

[0018] Without being limited to a particular theory, it is believed that nitroxy radicals having a redox-reactive functional group are more reactive to redox reactions than nitroxy radicals having a non-redox-reactive functional group. A redox reaction is a reaction in which structure changes through the transfer of electrons. It is assumed that each time such a reaction occurs, the balance of the electronic state within a substance becomes unbalanced, creating an environment in which electron transfer is more likely to occur. Therefore, it is believed that nitroxy radicals having a redox-reactive functional group can reduce the effects of dobesilic acid even at low doses, compared to nitroxy radicals having a non-redox-reactive functional group, such as the 4-methoxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical used in Patent Document 1.

[0019] The concentration of nitroxy radicals in the biological component measuring reagent or first reagent is not particularly limited, but is preferably adjusted to 0.01 to 2 mmol / L, more preferably 0.05 to 1.5 mmol / L, and even more preferably 0.1 to 1.25 mmol / L. A concentration of 0.01 mmol / L or higher can further reduce the effects of dobesilic acid. On the other hand, a concentration of 2 mmol / L or lower can reduce the possibility of falsely high biological component measurements.

[0020] Dobesilic acid (formula 2) has a vascular protective effect and is used to treat diabetic retinopathy, cardiovascular and cerebrovascular diseases, microvascular circulatory disorders, etc.

[0021] Dobesilic acid reacts with hydrogen peroxide produced in the measurement reaction of biological components, for example, in the presence of peroxidase. As a result, hydrogen peroxide reacting with the dye is consumed, resulting in negative errors in the measurement results of biological components. Because nitroxy radicals are oxidizing agents, their reaction with dobesilic acid is thought to eliminate the reactivity of dobesilic acid. As mentioned above, redox-reactive functional groups undergo structural changes through electron transfer. This is thought to lead to an imbalance in the electronic state within the substance, creating an environment that is more conducive to electron transfer. The tendency of this redox-reactive functional group to readily transfer electrons is thought to enhance its reactivity with dobesilic acid (e.g., in the calcium salt form). On the other hand, nitroxy radicals without functional groups (2,2,6,6-tetramethylpiperidine 1-oxyl free radicals) have been found to have stronger oxidizing properties than nitroxy radicals with functional groups. This is thought to be due to the steric hindrance that occurs to the substance when a functional group is added. In other words, it is thought that the effect of nitroxy radicals without functional groups on dobesilic acid is greater than that of nitroxy radicals with redox-reactive functional groups, and that the effect of nitroxy radicals with redox-reactive functional groups is greater than that of nitroxy radicals with non-redox-reactive functional groups.

[0022] (Chloride Salt) In one aspect of the present invention, the reagent for measuring a biological component may further contain a chloride salt.

[0023] When the biological component measurement reagent contains a first reagent and a second reagent, the chloride salt may be contained in either the first reagent or the second reagent, but is preferably contained in the first reagent. Furthermore, when the first reagent contains a nitroxy radical, the chloride salt is also preferably contained in the first reagent.

[0024] The coexistence of chloride salts with nitroxy radicals can reduce the effects of dobesilic acid even at low nitroxy radical doses. Chloride salts can also be used, for example, as stabilizers for sarcosine oxidase. The inclusion of chloride in the reagent can reduce the effects of turbidity in the measurement sample due to M protein and the like. The chloride concentration in the first reagent is preferably, for example, 5 to 70 mmol / L, preferably 20 to 70 mmol / L, and more preferably 30 to 70 mmol / L. A chloride concentration of 5 mmol / L or higher can fully exert the reduction effect.

[0025] The type of chloride salt is not particularly limited, but examples include chloride metal salts, such as sodium chloride, niobium pentachloride, lead(II) chloride, potassium chloride, indium(III) chloride, zinc(II) chloride, antimony(III) chloride, cesium chloride, silver(I) chloride, rubidium chloride, cerium(III) chloride, cobalt(II) chloride, copper(II) chloride, gallium(III) chloride, lithium chloride, manganese(II) chloride, nickel(II) chloride, aluminum(III) chloride, samarium(III) chloride, barium chloride, copper(I) chloride, chromium(II) chloride, cadmium(II) chloride, europium(III) chloride, palladium(II) chloride, and tin(II) chloride. Among these, alkali metal chloride salts such as sodium chloride are preferred. All of these are commercially available.

[0026] (Cyano Complex) In one aspect of the present invention, the reagent for measuring a biological component may further contain a cyano complex, which is a complex in which a cyanide ion serves as a ligand.

[0027] When the biological component measurement reagent contains a first reagent and a second reagent, the cyano complex may be contained in either the first reagent or the second reagent, but is preferably contained in the second reagent. Furthermore, when the nitroxy radical is contained in the first reagent, the cyano complex is preferably contained in the second reagent.

[0028] Examples of cyano complexes include potassium hexacyanoferrate(II) (potassium ferrocyanide), potassium hexacyanoferrate(III) (potassium ferricyanide), potassium tetracyanozincate(II), potassium hexacyanoiridate(III), potassium dicyanoaurate(I), potassium dicyanoargentate(I), potassium tetracyanoaurate(III), sodium tetracyanoaurate(III), potassium hexacyanocobaltate(III), potassium tetracyanomercurate(II), potassium tetracyanonickelate(II), potassium tetracyanoplatinate(II), cesium tetracyanoplatinate(II), sodium tetracyanoplatinate(II), barium tetracyanoplatinate(II), rubidium tetracyanoplatinate(II), potassium tetracyanopalladate(II), and potassium hexacyanorhodium(III). Preferably, at least one selected from the group consisting of potassium hexacyanoferrate(II) (potassium ferrocyanide) and potassium hexacyanoferrate(III) (potassium ferricyanide) is used, and more preferably potassium hexacyanoferrate(II) (potassium ferrocyanide). All of these are commercially available products.

[0029] The concentration of the cyano complex is not particularly limited, but is, for example, 0.1 to 1 mmol / L, preferably 0.1 to 0.8 mmol / L, and more preferably 0.1 to 0.6 mmol / L in the biological component measurement reagent or second reagent. When the concentration of the cyano complex is 1 mmol / L or less, the possibility of negative errors in the biological component measurement value can be reduced. On the other hand, when the concentration of the cyano complex is 0.1 mmol / L or more, the possibility of falsely low values ​​in the biological component measurement value due to in vivo reducing substances such as bilirubin that may coexist with the biological component can be reduced.

[0030] (Biological Component) The biological component to be measured by the biological component measuring reagent of the present invention is not particularly limited, and can be used to measure various biological components. For example, biological components that can be used for biological component measurement include creatinine (CRE), uric acid (UA), neutral fat (triglyceride (TG)), cholesterol (CHO), AST (GOT), ALT (GPT), LDH (lactate dehydrogenase) and its isozymes, ALP (alkaline phosphatase) and its isozymes, CK (creatine kinase) and its isozymes, amylase (Amy) and its isozymes, lipase, γ-GTP (γ-glutamyl transpeptidase), cholinesterase (ChE), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), phosphorus (P) [inorganic phosphorus (IP)], and the like. ) ), iron (Fe), magnesium (Mg), total protein (TP), serum protein fraction (PF), urea nitrogen (BUN), bilirubin (Bil), ammonia, cholesterol, HDL cholesterol (HDL-C, high-density lipoprotein cholesterol), LDL cholesterol (LDL-C, low-density lipoprotein cholesterol), BTR (BTR, total branched-chain amino acid / tyrosine ratio), tyrosine measurement reagent (TYR), blood glucose (BS, GLU), 1,5-anhydro-D-glucitol (1,5-AG), glycated albumin (GA), glycated hemoglobin (HbA1c), etc., but are not limited to these. In a preferred embodiment, the biological component is creatinine (CRE).

[0031] Hydrogen peroxide can be generated by reacting these biological components with any oxidase and, if necessary, other enzymes (e.g., hydrolases). Specific aspects of biological component measurement will be described below for cases where the biological component is, for example, creatinine (CRE), uric acid (UA), triglyceride (TG), or glycated hemoglobin (HbA1c).

[0032] When measuring creatinine (CRE), hydrogen peroxide is not directly produced in the reaction of creatinine amidinohydrolase using creatinine (CRE) as a substrate. Therefore, creatine produced in the reaction of creatinine amidinohydrolase is reacted with creatine amide hydrolase added in advance to the reagent to produce sarcosine, and sarcosine is further produced by sarcosine oxidase (oxidizing enzyme) added in advance to the reagent to produce hydrogen peroxide. By designing a so-called coupled reaction, it becomes possible to quantify the concentration of creatinine (CRE) using a peroxidase-color developer system.

[0033] When measuring uric acid (UA), hydrogen peroxide produced by the reaction of uricase (an oxidase) with uric acid (UA) as a substrate can be quantified using a peroxidase-coloring agent system.

[0034] When measuring triglyceride (TG), hydrogen peroxide is generated using lipoprotein lipase, which uses triglyceride (TG) as a substrate, and glycerol kinase and glycerol-3-phosphate oxidase (oxidase) as conjugated enzymes, thereby enabling the quantification of triglyceride (TG) concentration using a peroxidase-coloring agent system.

[0035] When measuring glycated hemoglobin (HbA1c), hydrogen peroxide produced by the reaction of glycated hemoglobin oxidase (for example, fructosyl amino acid oxidase) using glycated hemoglobin as a substrate can be quantified in a peroxidase-coloring agent system.

[0036] In this way, even if there is no suitable enzyme that catalyzes a reaction that directly oxidizes the target substance to generate hydrogen peroxide, it is possible to measure the concentration or amount of other biological components by appropriately designing a coupled reaction that combines the target substance with an enzyme (which may be a series of enzyme reactions) that catalyzes a reaction that can convert the target substance to a substrate for an oxidase that can generate hydrogen peroxide. Even when measuring other biological components, hydrogen peroxide can be generated by methods well known in the art in the same manner as described above.

[0037] Among the biological components listed above, the contents of biological components such as creatinine (CRE) and glycated hemoglobin (HbA1c) are extremely low, and therefore highly sensitive measurement is particularly required. The present invention is suitable for measuring creatinine or glycated hemoglobin, and is particularly suitable for use in measuring creatinine.

[0038] (Reagent kit for measuring biological components) In one embodiment, the reagent kit for measuring biological components of the present invention includes a reagent for measuring biological components containing a nitroxy radical, which may or may not have a redox-reactive functional group as described above.

[0039] In a further embodiment, the reagent kit for measuring a biological component of the present invention preferably contains at least the following (1) and one or more of the following (2) to (7) as constituent elements, more preferably the following (1) and (2) and one or more of the following (3) to (7) as constituent elements, and even more preferably all of the following (1) to (7) as constituent elements: (1) a nitroxy radical having or not having a redox-reactive functional group, (2) a chloride salt, (3) a cyano complex, (4) a peroxidase, (5) an enzyme other than peroxidase, (6) a buffer, and (7) a color developer.

[0040] In a preferred embodiment, the reagent kit for measuring a biological component of the present invention comprises a first reagent and a second reagent. In this embodiment, the first reagent preferably contains at least the above-mentioned (1), (2), and (5) to (7) as constituent elements, and the second reagent preferably contains at least the above-mentioned (3) to (7) as constituent elements.

[0041] (Biological Component Measurement Method) In one aspect of the present invention, a biological component measurement method is characterized in that a biological component is measured using the aforementioned reagent kit for measuring a biological component.

[0042] (Method for manufacturing a reagent kit for measuring a biological component) In one aspect of the present invention, the method for manufacturing a reagent kit for measuring a biological component preferably includes a step of combining the following (1) with one or more of the following (2) to (7), more preferably a step of combining the following (1) and (2) with one or more of the following (3) to (7), and preferably a step of combining all of the following (1) to (7): (1) a nitroxy radical having or not having a redox-reactive functional group, (2) a chloride salt, (3) a cyano complex, (4) a peroxidase, (5) an enzyme other than peroxidase, (6) a buffer, and (7) a color developer.

[0043] (4) Peroxidase Any type of peroxidase may be used as long as it catalyzes the reaction between hydrogen peroxide and a redox color-developing reagent, and examples thereof include peroxidases derived from plants, bacteria, and basidiomycetes. Among these, peroxidases derived from horseradish, rice, and soybeans are preferred, with horseradish peroxidase being more preferred, for reasons of purity, ease of availability, cost, and the like. Commercially available products that can be suitably used include PEO-131 (manufactured by Toyobo Co., Ltd.), PEO-301 (manufactured by Toyobo Co., Ltd.), and PEO-302 (manufactured by Toyobo Co., Ltd.). There are no particular restrictions on the amount used, the form of addition, and the like.

[0044] (5) Enzymes Other Than Peroxidases An example of an enzyme other than peroxidase is an oxidase. Any oxidase capable of generating hydrogen peroxide from a substrate can be used without limitation depending on the target substance to be measured. Specific examples include, but are not limited to, ascorbic acid oxidase, sarcosine oxidase, uricase, glycerol-3-phosphate oxidase, fructosyl amino acid oxidase, and bilirubin oxidase. Commercially available oxidases suitable for use include UAO-211 (manufactured by Toyobo Co., Ltd.), SAO-351 (manufactured by Toyobo Co., Ltd.), and G3O-311 (manufactured by Toyobo Co., Ltd.). The amount used, the form of addition, and the like are not particularly limited. When sarcosine oxidase is used as the oxidase, it is preferably combined with at least one selected from the group consisting of catalase, creatine amidinohydrolase, and creatinine amidohydrolase. In one embodiment, the enzyme other than peroxidase is preferably at least one selected from the group consisting of ascorbic acid oxidase, sarcosine oxidase, catalase, creatine amidinohydrolase, and creatinine amidohydrolase.

[0045] (6) Buffering Agent The buffering agent is not particularly limited as long as it is a substance having a buffering effect, and examples thereof include GOOD buffer, Tris buffer, citrate buffer, phosphate buffer, borate buffer, carbonate buffer, etc. GOOD buffers include 3-morpholinopropanesulfonic acid (MOPS), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES), 2-morpholinoethanesulfonic acid (MES), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), N-tris(hydroxymethyl)methyl-2-aminomethanesulfonic acid (TES), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), 3-[N,N- Examples of the buffer include]bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS), 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPSO), 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) (POPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO), N-(2-acetamido)iminodiacetic acid (ADA), N,N-bis(2-hydroxyethyl)glycine (Bicine), and N-[tris(hydroxymethyl)methyl]glycine (Tricine). The amount of the buffer used, the set pH, the form of addition, etc. are not particularly limited. The buffering agent is usually used in the form of a buffer solution. All of the buffering agents exemplified above are commercially available.In one embodiment, the buffer is preferably at least one selected from the group consisting of Tris buffer, citrate buffer, borate buffer, phosphate buffer, MES, Bis-Tris, ADA, ACES, BES, PIPES, MOPS, TES, HEPES, Tricine, Bicine, POPSO, TAPS, CHES, and CAPS.

[0046] (7) Color Former An example of a color former is a redox color reagent. Any type of redox color reagent may be used as long as it reacts with hydrogen peroxide to form a dye and develop a color. Examples include a combination of a hydrogen donor and a coupler, a leuco compound, and a tetrazolium salt. From the viewpoint of more effectively achieving the effects of the present invention, it is preferable to use a combination of a hydrogen donor and a coupler as the color former. A typical example using a hydrogen donor and a coupler is the Trinder method, in which a dye is formed by oxidative condensation of a hydrogen donor and a coupler with hydrogen peroxide in the presence of peroxidase. There are no particular restrictions on the amount of the color former used or the form of addition. All of these are commercially available products.

[0047] Examples of leuco compounds include triphenylmethane derivatives, phenothiazine derivatives, diphenylamine derivatives, etc. Specific examples include 4,4'-benzylidenebis(N,N-dimethylaniline), 4,4'-bis[N-ethyl-N-(3-sulfopropylamino)-2,6-dimethylphenyl]methane, 1-(ethylaminothiocarbonyl)-2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-bis(4-diethylaminophenyl)imidazole, 4,4'-bis(dimethylamino)diphenylamine, N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine salt (DA64), and 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine salt (DA67).

[0048] Examples of tetrazolium salts include 2,3,5-triphenyltetrazolium salt, 2,5-diphenyl-3-(1-naphthyl)-2H-tetrazolium salt, 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium] salt, and 3,3'-[3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium] salt. bis(2,5-diphenyl-2H-tetrazolium) salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium salt, 3,3'-(1,1'-biphenyl-4,4'-diyl)-bis(2,5-diphenyl-2H-tetrazolium) salt, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium salt.

[0049] A typical example of a combination of a hydrogen donor and a coupler is a reaction in which a Trinder reagent (hydrogen donor) and a coupler are oxidatively condensed with hydrogen peroxide in the presence of peroxidase to form a dye. Known hydrogen donors used as Trinder reagents include phenol, phenol derivatives, aniline derivatives, naphthol, naphthol derivatives, naphthylamine, and naphthylamine derivatives, and these can be suitably used in the present invention. Aniline derivatives are preferred.

[0050] (Hydrogen Donor) In the biological component measuring method of the present invention, phenol, phenol derivatives, aniline derivatives, naphthol, naphthol derivatives, naphthylamine, naphthylamine derivatives, etc. are used as hydrogen donors for the Trinder method, etc. Examples include N-ethyl-N-sulfopropyl-3-methoxyaniline, N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-3-methylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, N-ethyl-N-(2-hydroxy- Examples of suitable hydrogen donors include aniline derivatives such as N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline, N-sulfopropylaniline, and N-(2-hydroxy-3-sulfopropyl)-2,5-dimethylaniline; N-ethyl-N-(3-methylphenyl)-N'-succinylethylenediamine; and N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine. These hydrogen donors are preferably used in combination with a coupler.

[0051] (Coupler) These hydrogen donors can be used in combination with a coupler. Examples of couplers include aminoantipyrine compounds such as 4-aminoantipyrine (4AA) and aminoantipyrine derivatives; vanillin diamine sulfonic acid compounds such as vanillin diamine sulfonic acid; and methylbenzthiazolinone hydrazone compounds such as methylbenzthiazolinone hydrazone (MBTH) and sulfonated methylbenzthiazolinone hydrazone (SMBTH). 4-Aminoantipyrine is preferred. These couplers may contain impurities. For example, 4-aminoantipyrine may contain impurities such as 4-hydroxyantipyrine. Two or more couplers may be used in combination.

[0052] In one embodiment, the color former is N-ethyl-N-(3-sulfopropyl)-m-anisidine, aniline, N,N-dimethylaniline, N,N-diethylaniline, N,N-diethyl-m-toludine, N,N-dimethyl-m-anisidine, N-ethyl-(3-methylphenyl)-N'-acetylethylenediamine, N-ethyl-N-(β-hydroxyethyl)-m-toluidine, N-ethyl-N-(2-hydroxy-3-sulfoethyl)-m-toluidine, N-ethyl-N- It is preferably at least one selected from the group consisting of sulfopropyl-m-toluidine, N-ethyl-sulfopropyl-3,5-methoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine, phenol, p-chlorophenol, 2,4-dichlorophenol, 2,4-dibromophenol, and 2,3,4-trichlorophenol.

[0053] (Other Components, etc.) In one embodiment of the present invention, the reagent kit for measuring a biological component may contain preservatives, chelating agents, antibiotics, antibacterial agents, salts, enzyme stabilizers, chromogen stabilizers, etc., to the extent that they do not affect the reaction. These can be used to eliminate in vivo reducing substances, such as ascorbic acid and bilirubin, which tend to affect the results of biological component measurement.

[0054] The concentration of the buffer in the reagent for measuring a biological component is not particularly limited, but is preferably adjusted to a concentration of 0 to 500 mM, more preferably 5 to 100 mM, more preferably 10 to 75 mM, and even more preferably 20 to 50 mM.

[0055] There are no particular limitations on the amount used or the form of addition of preservatives, chelating agents, antibiotics, antibacterial agents, salts, enzyme stabilizers, chromogen stabilizers, etc. All of these can be obtained as commercially available products.

[0056] Examples of preservatives include Proclin 150, Proclin 200, Proclin 300, Proclin 950, azides, chelating agents, antibiotics, antibacterial agents, and the like.

[0057] The chelating agent includes ethylenediaminetetraacetic acid and its salts.

[0058] Antibiotics include gentamicin, kanamycin, chloramphenicol, and the like.

[0059] Antibacterial agents include methylisothiazolinone, imidazolidinyl urea, and the like.

[0060] Examples of salts include sodium chloride, potassium chloride, and aluminum chloride.

[0061] Examples of enzyme stabilizers include sucrose, trehalose, cyclodextrin, gluconates, and amino acids.

[0062] Examples of chromogen stabilizers include chelating agents such as ethylenediaminetetraacetic acid and its salts, and cyclodextrin.

[0063] In one aspect of the present invention, the reagent contained in the reagent kit for measuring biological components may be a liquid reagent dissolved in any solvent (e.g., purified water, organic solvent, etc.), or may be a dry powder reagent (e.g., freeze-dried powder) that is dissolved in the same solvent as above before use.

[0064] In a preferred embodiment of the present invention, the reagent or reagent kit for measuring a biological component is a reagent or reagent kit for measuring creatinine, comprising: a first reagent, which is a buffer solution containing a nitroxy radical, which may or may not have a redox-reactive functional group, and optionally a chloride salt and / or a cyano complex; and a second reagent, which is a buffer solution containing creatinine amidohydrolase and a color developer, such as Trinder's reagent. Preferably, the first reagent further contains ascorbic acid oxidase, sarcosine oxidase, and / or creatine amidinohydrolase. The creatine amidinohydrolase contained in the first reagent acts on creatine in the sample to produce sarcosine, and the resulting sarcosine oxidase acts on the sarcosine to produce hydrogen peroxide. Catalase then acts on the resulting hydrogen peroxide to eliminate the creatine-derived hydrogen peroxide, thereby suppressing its effect on creatinine measurement. After the first reagent is added to eliminate the hydrogen peroxide derived from creatine, the second reagent is added to generate hydrogen peroxide derived from creatinine, and the generated hydrogen peroxide is reacted with a color developer, thereby enabling accurate quantification of the hydrogen peroxide derived from creatinine. The reagent kit for measuring creatinine may further include a sample collection tool, an instruction manual, etc., in addition to the bottle containing the first reagent and the bottle containing the second reagent.

[0065] (Measurement Method Using a Biological Component Measurement Reagent Kit) When measuring a biological component using the biological component measurement reagent kit of the present invention, a general-purpose automatic analyzer (e.g., Hitachi 7180 automatic analyzer) can be used. The biological component measurement reagent kit of the present invention may be configured to be compatible with such an automatic analyzer. The form of the biological component measurement kit is not particularly limited, and various forms can be exemplified, such as a kit composed of a liquid reagent, a kit composed of a combination of a dry reagent produced by means such as lyophilization and a dissolving solution, a so-called dry system kit in which an enzyme or the like is supported on a suitable carrier, or a kit in which a sensor is used. A liquid reagent is preferred, such as a liquid reagent divided into two reagent packets (hereinafter also referred to as a two-reagent liquid reagent). In this method, a first type of reagent (also referred to as a first reagent) is first added to a sample and allowed to react for a certain period of time, and then a second type of reagent (also referred to as a second reagent) is further added and allowed to react. The target component can be quantified by measuring the change in absorbance during this period.

[0066] In one embodiment of the present invention, the reagent kit for measuring a biological component may be configured as a kit containing a single reagent or a kit containing two or more individually packaged reagents. When the reagents are individually packaged to form a kit containing two or more individually packaged reagents, the kit may be configured by separately packaging a reagent containing (1) a nitroxy radical with or without a redox-reactive functional group, (2) a chloride salt, (5) one or more enzymes other than peroxidase, (6) a buffer, and (7) a hydrogen donor as a color developer, and a reagent containing (3) a cyano complex, (4) peroxidase, (5) an enzyme other than peroxidase, (6) a buffer, and (7) a color developer.

[0067] Hereinafter, an example of a kit configured with a liquid reagent divided into two packages (hereinafter also referred to as a two-reagent liquid reagent) will be described.

[0068] In a method of analyzing with an automatic analyzer using this type of reagent, a first type of reagent (also referred to as first reagent or R1) is first added to a sample and allowed to react for a certain period of time, and then a second type of reagent (also referred to as second reagent or R2) is further added and allowed to react, and the target component can be quantified by measuring the change in absorbance during this period.

[0069] When the biological component measurement reagent of the present invention is supplied in two or more packets in consideration of application to an automatic analyzer, as described above, it is determined whether the concentrations of the iron-containing substance and other components in each packet of reagent are within the preferred concentration ranges of the respective components.

[0070] (Method for Measuring Biological Component) In one aspect of the present invention, the method for measuring a biological component comprises the following steps (1) to (3): (1) a step of reacting an oxidase with a biological component to generate hydrogen peroxide, (2) a step of reacting a peroxidase with the hydrogen peroxide generated in step (1) to cause oxidative condensation between a hydrogen donor and a coupler, thereby coloring the reaction solution, and (3) a step of colorimetrically quantifying the colored reaction product in step (2).

[0071] The method for measuring a biological component of the present invention is an enzymatic method for measuring a biological component, particularly a method using an oxidase-peroxidase-coloring agent system. Specifically, the measurement principle involves subjecting a biological component in a sample to an enzymatic reaction to generate hydrogen peroxide in proportion to the amount of the biological component, which is then reacted with a coloring agent in the presence of peroxidase to colorimetrically quantify the resulting color. Methods for measuring a biological component using this principle have already been established in the technical field. Therefore, this knowledge can be applied to the present invention to measure the amount or concentration of a biological component in various samples, and the embodiment is not particularly limited.

[0072] (Specimen) Examples of specimens containing biological components that can be used in the biological component measurement of the present invention include biological fluids such as blood (particularly serum, plasma, etc.), urine, ascites, cerebrospinal fluid, etc., as well as beverages, foods, etc. that are ingested by humans. Of these, it is preferable to use human biological fluids (blood-derived samples such as serum, plasma, etc., and urine-derived samples, etc.) as specimens to be measured.

[0073] (Detection Sensitivity of Reagent Kit for Measuring a Biological Component) Hereinafter, the detection sensitivity of the reagent kit for measuring a biological component will be described, taking creatinine as an example of a biological component.

[0074] In recent years, calculation of eGFR (also known as estimated glomerular filtration rate) requires creatinine measurement accuracy to two decimal places, and a minimum detection sensitivity of approximately 0.03 mg / dL in creatinine concentration is required.

[0075] On the other hand, in terms of measurement accuracy of an automated analyzer, the blank variation of a creatinine reagent is σ = approximately 0.045 to 0.114 mABS, and 2.6σ (99.5% normal distribution), which is generally considered to be the minimum detection sensitivity of in vitro diagnostic reagents, is approximately 0.117 to 0.296 mABS. Therefore, if the absorbance is equal to or greater than 0.296 mABS, the maximum value of 2.6σ, that is, approximately 0.3 mABS, it can be detected as a signal, and it is believed that it will be possible to determine the presence or absence of creatinine and to quantify creatinine.

[0076] The present invention is not limited to the configurations described above, and various modifications are possible within the scope of the claims. Embodiments and examples obtained by appropriately combining, modifying, or replacing the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0077] The present invention will be specifically described below with reference to examples, but the present invention is not particularly limited to these examples.

[0078] Example: Reducing the effect of dobesilic acid The following creatinine measurement reagent was prepared and measurements were carried out.

[0079] (Creatinine Measuring Reagent) As creatinine measuring reagents, a first reagent and a second reagent having the following compositions were prepared. First Reagent: MOPS 25.0 mmol / L, Preservative 9.0 g / L, Ascorbic acid oxidase (Toyobo Co., Ltd., ASO-311) 2.5 KU / L, Sarcosine oxidase (Toyobo Co., Ltd., SAO-351) 6.0 KU / L, Creatinine amidohydrolase (Toyobo Co., Ltd., CRH-221) 23.0 KU / L, Catalase (Toyobo Co., Ltd., CAO-519) 120.0 KU / L, N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline 0.14 g / L. Second Reagent: MOPS 50.0 mmol / L, Preservative 1.2 g / L, Potassium ferrocyanide 0.41 mmol / L, Creatine amidinohydrolase (Toyobo Co., Ltd., CNH-311) 160.0 KU / L. Peroxidase (Toyobo Co., Ltd. PEO-301) 8.0 KU / L 4-AA 0.6 g / L

[0080] The first creatinine measurement reagent was added with sodium chloride concentrations of 66.7 mmol / L, 56.5 mmol / L, 35.9 mmol / L, 25.7 mmol / L, and 5.1 mmol / L, respectively, and the study was carried out.

[0081] Furthermore, the first reagent for measuring creatinine was prepared with each substance at the following concentrations: Comparative example: 4-methoxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical 0.54 mmol / L Reagent 1: 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical 0.58 mmol / L Reagent 2: 4-carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical 0.50 mmol / L

[0082] (Measurement Sample) The measurement sample was prepared as follows: Calcium dobesilate Calcium dobesilate was added to liquid Nescol N (Alfresa Pharma) so that the concentration in the sample was 1.5 mg / dL.

[0083] (Measurement Method) Measurements were performed using a Hitachi 7180 automatic analyzer. 120 μL of the first reagent was added to 2.7 μL of sample and incubated for 5 minutes to form the first reaction. 40 μL of the second reagent was then added and incubated for 5 minutes to form the second reaction. The absorbance at 546 nm (main wavelength) and the absorbance at 800 nm (secondary wavelength) were measured using a two-point end method, which calculates the difference between the absorbances of the first and second reactions, corrected for the volume of the liquid. The absorbance was calculated by subtracting the secondary wavelength from the main wavelength. The creatinine concentration of the above-mentioned measurement sample (sample with unknown creatinine concentration) was calculated by comparing it with a calibration curve prepared from the measured absorbances of purified water and a 5 mg / dL creatinine aqueous solution. The deviation from each drug-free sample was calculated using the following formula: Deviation from each drug-free sample = {(creatinine measurement value with drug (mg / dL) - creatinine measurement value without drug (mg / dL)) / creatinine measurement value without drug (mg / dL)} × 100 (%)

[0084] The measurement results for sodium chloride concentrations of 66.7 mmol / L are shown in Table 1, 56.5 mmol / L in Table 2, 35.9 mmol / L in Table 3, 25.7 mmol / L in Table 4, and 5.1 mmol / L in Table 5.

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] The above results demonstrate that the 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical (reagent 1) and 4-carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical (reagent 2) additive reagents can further reduce the effects of dobesilic acid at each chloride concentration.

Claims

1. A reagent for measuring biological components that reduces the effects of dobesilic acid, characterized by containing a nitroxy radical that has either a redox-reactive functional group or no functional group.

2. The reagent for measuring a biological component according to claim 1, wherein the redox-reactive functional group is at least one selected from the group consisting of a hydroxy group, an oxo group, a carbonyl group, a carboxy group, a nitro group, an amino group, an amide group, a nitrile group, a thiol group, and an isothiocyanato group.

3. The reagent for measuring a biological component according to claim 1, wherein the nitroxy radical having a redox-reactive functional group is at least one selected from the group consisting of 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-oxo-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, 4-amino-2,2,6,6-tetramethylpiperidine 1-oxyl free radical, and 3-carboxy-2,2,5,5-tetramethylpyrrolidine 1-oxyl free radical.

4. The reagent for measuring a biological component according to claim 1, wherein the nitroxy radical having no functional group is a 2,2,6,6-tetramethylpiperidine 1-oxyl free radical.

5. The reagent for measuring a biological component according to claim 1 or 3, wherein the nitroxy radical is contained in an amount of 0.01 to 2 mmol / L.

6. The reagent for measuring a biological component according to claim 1, wherein the biological component is creatinine.

7. The reagent for measuring a biological component according to claim 1, further comprising a chloride salt.

8. The reagent for measuring a biological component according to claim 7, wherein the chloride salt is at least one selected from the group consisting of sodium chloride, niobium pentachloride, lead(II) chloride, potassium chloride, indium(III) chloride, zinc(II), antimony(III), cesium chloride, silver(I), rubidium chloride, cerium(III), cobalt(II), copper(II), gallium(III), lithium chloride, manganese(II), nickel(II), aluminum(III), samarium(III), barium chloride, copper(I), chromium(II), cadmium(II), europium(III), palladium(II), and tin(II).

9. The reagent for measuring a biological component according to claim 7 or 8, wherein the chloride salt is contained in an amount of 30 to 70 mmol / L.

10. The reagent for measuring a biological component according to claim 1, further comprising a cyano complex.

11.

11. The reagent for measuring a biological component according to claim 10, wherein the cyano complex is at least one selected from the group consisting of potassium hexacyanoferrate(II) (potassium ferrocyanide), potassium hexacyanoferrate(III) (potassium ferricyanide), potassium tetracyanozincate(II), potassium hexacyanoiridate(III), potassium dicyanoaurate(I), potassium dicyanoargentate(I), potassium tetracyanoaurate(III), sodium tetracyanoaurate(III), potassium hexacyanocobaltate(III), potassium tetracyanomercurate(II), potassium tetracyanonickelate(II), potassium tetracyanoplatinate(II), cesium tetracyanoplatinate(II), sodium tetracyanoplatinate(II), barium tetracyanoplatinate(II), rubidium tetracyanoplatinate(II), potassium tetracyanopalladate(II), and potassium hexacyanorhodiumate(III).

12. The reagent for measuring a biological component according to claim 10 or 11, wherein the cyano complex is potassium hexacyanoferrate (II) (potassium ferrocyanide).

13. The reagent for measuring a biological component according to claim 10, wherein the cyano complex is contained in an amount of 0.1 to 1 mmol / L.

14. A reagent kit for measuring a biological component, containing at least the following components (1) to (7): (1) a nitroxy radical with or without a redox-reactive functional group, (2) a chloride salt, (3) a cyano complex, (4) a peroxidase, (5) an enzyme other than peroxidase, (6) a buffer, and (7) a color developer.

15. A reagent kit for measuring a biological component according to claim 14, wherein the enzyme (5) other than peroxidase is at least one selected from the group consisting of ascorbic acid oxidase, sarcosine oxidase, catalase, creatine amidinohydrolase, and creatinine amidohydrolase.

16. The reagent kit for measuring a biological component according to claim 14, wherein the buffer (6) is at least one selected from the group consisting of Tris buffer, citrate buffer, borate buffer, phosphate buffer, MES, Bis-Tris, ADA, ACES, BES, PIPES, MOPS, TES, HEPES, Tricine, Bicine, POPSO, TAPS, CHES, and CAPS.

17. The color former (7) is N-ethyl-N-(3-sulfopropyl)-m-anisidine, aniline, N,N-dimethylaniline, N,N-diethylaniline, N,N-diethyl-m-toluidine, N,N-dimethyl-m-anisidine, N-ethyl-(3-methylphenyl)-N'-acetylethylenediamine, N-ethyl-N-(β-hydroxyethyl)-m-toluidine, N-ethyl-N-(2-hydroxy-3-sulfoethyl)-m-toluidine, N-ethyl-N-sulfopropyl-m- The reagent kit for measuring a biological component according to claim 14, wherein the reactant is at least one selected from the group consisting of toluidine, N-ethyl-sulfopropyl-3,5-methoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine, phenol, p-chlorophenol, 2,4-dichlorophenol, 2,4-dibromophenol, and 2,3,4-trichlorophenol.

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