Quantitative method and reagent for protein in urine

Abstract

The invention provides a method and a reagent for correcting the obvious reactivity difference between protein types in urine and reducing hemolytic hemoglobin interference at the same time. Specifically, the present invention provides a reagent for measuring a protein in urine, the reagent comprising: a complex in which a metal and a dye are coordinated (wherein when the complex binds to a protein, the dye binds to the metal; ), at least one anionic surfactant selected from the group consisting of sulfonic acid-type anionic surfactants and sulfate-type anionic surfactants, and a polyoxyalkylene-type nonionic surfactant.

Description

Technical Field

[0001] The present invention relates to a method and a reagent for quantifying total protein in urine. More specifically, the present invention relates to a method and a reagent for quantifying protein in urine using a complex formed by coordination of a metal and a pigment. Background Art

[0002] Total protein in urine increases due to kidney and urinary tract system diseases. Therefore, quantification of total protein in urine is widely used for diagnosis of kidney and urinary tract system diseases and confirmation of treatment effects. In addition, in clinical practice, a method using a complex formed by coordination of a metal and a pigment, such as the pyrogallol red-molybdate method, is widely used for quantification of total protein in urine, and a method for measuring protein by using the shift of the absorption wavelength of the complex in the presence of protein is used.

[0003] However, several problems have been pointed out in the method for quantifying protein using such a complex. Among them, 1) negative errors due to significant differences in reactivity between protein species, and 2) positive errors due to hemolyzed hemoglobin.

[0004] Regarding the negative error caused by the significant difference in reactivity between protein species, a method for correcting the significant difference in reactivity between albumin and globulin by adding sodium dodecyl sulfate (SDS) has been reported (Non-Patent Document 1). This report also teaches that by setting the concentration of SDS in the reagent to 25 mg / L, the significant difference in reactivity between albumin and globulin can be eliminated. However, in this report, no study was conducted on the effects of adding SDS on coexisting substances in the sample.

[0005] Interference of hemolyzed hemoglobin in clinical examinations needs to be studied from various viewpoints according to the reaction system, and various methods have been proposed (Patent Documents 1 to 15). For example, in a method for quantifying bilirubin by two-point measurement, a method for suppressing the time-dependent change in absorbance of hemoglobin by an aliphatic higher sulfonic acid or its salt, a sulfate ester having an alkyl group with 11 to 16 carbon atoms, a primary amine having 11 to 16 carbon atoms or its salt, or a quaternary ammonium salt to reduce the influence of hemoglobin has been disclosed (Patent Document 8). In addition, as a method for reducing the influence of hemolyzed hemoglobin and the like in a protein measurement method, a reaction of a two-reagent system in which a reagent containing copper ions at pH 10 to 13 is reacted with a sample and then an alkali solution such as lithium hydroxide is added has been proposed (Patent Document 15). In addition, methods for reducing the influence of hemolyzed hemoglobin and other substances coexisting in the sample (such as chyle and bilirubin) have also been studied (Patent Documents 2, 9, 10, and 12 to 15).

[0006] However, in a urine test sample containing hemolyzed hemoglobin, there is no report on how sodium dodecyl sulfate (SDS) affects the interference of hemolyzed hemoglobin when there is no significant difference in correcting the reactivity between albumin and globulin, nor is there a report on a method for reducing the interference of hemolyzed hemoglobin while correcting the reactivity difference between different types of urinary proteins. Prior art documents

[0007] Patent documents Patent Document 1: Japanese Patent Laid-Open No. 2001-231596 Patent Document 2: Japanese Patent Laid-Open No. 2008-197077 Patent Document 3: WO2010 / 001619 Patent Document 4: Japanese Patent Laid-Open No. 2013-51907 Patent Document 5: Japanese Patent Laid-Open No. 9-84598 Patent Document 6: Japanese Patent Laid-Open No. 2000-189194 Patent Document 7: Japanese Patent Laid-Open No. 2015-29444 Patent Document 8: Japanese Patent Laid-Open No. 60-168050 Patent Document 9: Japanese Patent Laid-Open No. 2022-180380 Patent Document 10: Japanese Patent Laid-Open No. 2006-81471 Patent Document 11: Japanese Patent Laid-Open No. 2008-70346 Patent Document 12: WO2002 / 086151 Patent Document 13: Japanese Patent Laid-Open No. 2000-93200 Patent Document 14: Japanese Patent Laid-Open No. 2000-83698 Patent Document 15: Japanese Patent Laid-Open No. 10-19898

[0008] Non-patent documents Non-patent Document 1: CLIN.CHEM.Vol.35,No.11,2233-2236(1989) Summary of the invention Problems to be solved by the invention

[0009] Therefore, an object of the present invention is to provide a method and a reagent for reducing the interference of hemolyzed hemoglobin while correcting the significant difference in reactivity between different types of urinary proteins. Means for solving the problems

[0010] The present inventors have confirmed that when a prescribed amount of sodium dodecyl sulfate (SDS) is added to a urine sample containing lysohemoglobin to correct the significant difference in the reactivity between albumin and globulin, SDS actually increases the interference of lysohemoglobin. Various studies have been conducted on methods to solve this problem, and it has been found that the problem can be improved by reacting a complex with protein in the presence of a specific anionic surfactant such as SDS and a polyoxyalkylene type nonionic surfactant. That is, the present invention provides the following method and reagent. [1] A method for quantifying protein in urine, comprising using a complex formed by coordination of a metal and a pigment, the absorption wavelength of which shifts when bound to a protein, and reacting the protein with the complex in the presence of the following surfactant: at least one anionic surfactant selected from sulfonic acid type anionic surfactants and sulfate type anionic surfactants, and Polyoxyalkylene type nonionic surfactant. [2] A method for reducing the interference of lysohemoglobin and the apparent difference in reactivity between protein species when quantifying protein in urine, wherein a complex composed of a metal and a pigment whose absorption wavelength shifts when bound to a protein is used, and the protein is reacted with the complex in the presence of the following surfactant: at least one anionic surfactant selected from sulfonic acid type anionic surfactants and sulfate type anionic surfactants, and Polyoxyalkylene type nonionic surfactant. [3] The method according to [1] or [2], wherein the sulfonic acid type anionic surfactant is a sulfonate represented by the following formula: [Chemistry 1] R 1 -SO3X 1 …(1)[In formula (1), X 1 is an alkali metal, ammonium or alkanolamine, R 1 Selected from the following (1) to (5): (1) a hydrocarbon group having 8 to 18 carbon atoms, (2) a monocyclic or bicyclic aromatic hydrocarbon substituted with a hydrocarbon group having 8 to 18 carbon atoms, (3) Succinic acid residues of formula (2), their (di)esters or their salts: [Chemistry 2] (In formula (2), R 2 and R 3 Each independently represents hydrogen, a hydrocarbon group having 8 to 18 carbon atoms, or an alkali metal.) (4) The succinic acid monoamide residue of formula (3), its ester, or a salt thereof: [Chemical formula 3] (In formula (3), R 4 and R 5 each independently represent hydrogen, a hydrocarbon group having 8 to 18 carbon atoms, or an alkali metal.) And (5) The sulfonate salt of the hydrocarbyl-substituted diphenyl ether residue of formula (4): [Chemical formula 4] (In formula (4), R 6 is a hydrocarbon group having 8 to 18 carbon atoms, and X 2 is an alkali metal, ammonium, or alkanolamine.). [4] The method according to [3], wherein R 1 is (1) an alkyl or alkenyl group having 8 to 18 carbon atoms, or (2) a monocyclic or bicyclic aromatic hydrocarbon substituted with an alkyl or alkenyl group having 8 to 18 carbon atoms. [5] The method according to [4], wherein the sulfonic acid type anionic surfactant is an alkane sulfonate having 11 to 16 carbon atoms, or a linear alkylbenzene sulfonate having 10 to 14 carbon atoms. [6] The method according to [1] or [2], wherein the sulfate type anionic surfactant is a sulfate represented by formula (5) or (6): [Chemical formula 5] R 7 O-SO3X 3 …(5) (In formula (5), R 7 is a hydrocarbon group having 8 to 18 carbon atoms or a monocyclic or bicyclic aromatic hydrocarbon substituted with a hydrocarbon group having 8 to 18 carbon atoms, and X 3 is an alkali metal, ammonium, or alkanolamine.) [Chemical formula 6] R 8 O-(CHR 9 CHR 10 O) n -SO3X 4 ···(6) (In formula (6), R 8 is a hydrocarbon group having 8 to 18 carbon atoms, R 9 and R 10 are independently hydrogen or an alkyl group having 1 to 3 carbon atoms, n is 1 to 4, and X 4 is an alkali metal, ammonium, or alkanolamine.) [7] The method according to [6], wherein the sulfate type anionic surfactant is an alkyl sulfate having 11 to 16 carbon atoms. [8] The method according to any one of [1] to [7], wherein the polyoxyalkylene type nonionic surfactant is a polyoxyalkylene compound represented by the formula (7) or (8): [Chemical formula 7] R 11 -Y 1 -((CH2) m O) n -R 12 ···(7) (In the formula (7), Y 1 is O, C(O)O or C(O)NH, R 11 is a linear hydrocarbon group having 8 to 22 carbon atoms, m is an integer of 1 to 4, n is an integer of 2 to 35, and R 12 is hydrogen or an alkyl group having 1 to 3 carbon atoms.) [Chemical formula 8] R 13 -Y 2 -((CH2) m1 O) n1 -((CH2) m2 O) n2 -((CH2) m3 O) n3 -R 14 ···(8) (In the formula (8), Y 2 is O, C(O)O or C(O)NH, m1 and m3 are each independently an integer of 1 to 4, m2 is an integer of 2 to 4, m1 and / or m3 is different from m2, n1, n2 and n3 are each independently an integer of 2 to 200, R 13 is hydrogen or a linear hydrocarbon group having 1 to 30 carbon atoms, and R 14 is hydrogen or an alkyl group having 1 to 3 carbon atoms.) [9] The method according to [8], wherein the polyoxyalkylene type nonionic surfactant is a polyoxyalkylene compound represented by the formula (7). In the formula (7), Y 1 is O or C(O)O, R 11 is a linear alkyl or alkenyl group having 11 to 20 carbon atoms, m is 2, n is an integer of 20 to 35, and R 12 is hydrogen.

[10] The method according to any one of [1] to [9], wherein the concentration of the nonionic surfactant in the reaction solution is 0.0015% by mass to 0.030% by mass, and the concentration of the anionic surfactant in the reaction solution is 0.001% by mass to 0.0035% by mass.

[11] The method according to

[10] , wherein the concentration of the anionic surfactant in the reaction solution is 0.0015 to 0.0030% by mass.

[12] The method according to

[10] or

[11] , wherein the concentration of the nonionic surfactant in the reaction solution is 0.0025 to 0.024% by mass.

[13] The method according to any one of [1] to

[12] , wherein the pigment is pyrogallol red or pyrocatechol violet, and the metal is molybdenum, tin or iron.

[14] A reagent for determining proteins in urine, comprising: A complex formed by coordination of a metal and a pigment, the absorption wavelength of the complex shifting when bound to a protein; At least one anionic surfactant selected from sulfonic acid type anionic surfactants and sulfate ester type anionic surfactants; and A polyoxyalkylene type nonionic surfactant.

[15] The reagent for determining proteins in urine according to

[14] , wherein the sulfonic acid type anionic surfactant is a sulfonate represented by the following formula: [Chemical formula 9] R 1 -SO3X 1 …(1) [In formula (1), X 1 is an alkali metal, ammonium or alkanolamine, R 1 is selected from the following (1) to (5): (1) A hydrocarbon group having 8 to 18 carbon atoms, (2) A monocyclic or bicyclic aromatic hydrocarbon substituted with a hydrocarbon group having 8 to 18 carbon atoms, (3) A succinic acid residue of formula (2), its (di)ester or their salts: [Chemical formula 10] (In formula (2), R 2 and R 3 each independently represent hydrogen, a hydrocarbon group having 8 to 18 carbon atoms or an alkali metal.) (4) A succinic acid monoamide residue of formula (3), its ester, or their salts: [Chemical formula 11] (In formula (3), R 4 and R 5 each independently represent hydrogen, a hydrocarbon group having 8 to 18 carbon atoms or an alkali metal.) And (5) Sulfonates of the hydrocarbyl-substituted diphenyl ether residue of formula (4): [Chemical Formula 12] (In formula (4), R 6 is a hydrocarbyl group having 8 to 18 carbon atoms, and X 2 is an alkali metal, ammonium, or alkanolamine.)

[16] The reagent for measuring proteins in urine as described in

[15] , wherein R 1 is (1) an alkyl or alkenyl group having 8 to 18 carbon atoms, or (2) a monocyclic or bicyclic aromatic hydrocarbon substituted by an alkyl or alkenyl group having 8 to 18 carbon atoms.

[17] The reagent for measuring proteins in urine as described in

[16] , wherein the sulfonic acid type anionic surfactant is an alkane sulfonate having 11 to 16 carbon atoms, or a linear alkyl benzene sulfonate having 10 to 14 carbon atoms.

[18] The reagent for measuring proteins in urine as described in

[14] , wherein the sulfate ester type anionic surfactant is a sulfate ester represented by formula (5) or (6): [Chemical Formula 13] R 7 O-SO3X 3 …(5) (In formula (5), R 7 is a hydrocarbyl group having 8 to 18 carbon atoms, or a monocyclic or bicyclic aromatic hydrocarbon substituted by a hydrocarbyl group having 8 to 18 carbon atoms, and X 3 is an alkali metal, ammonium, or alkanolamine.) [Chemical Formula 14] R 8 O-(CHR 9 CHR 10 O) n -SO3X4…(6) (In formula (6), R 8 is a hydrocarbyl group having 8 to 18 carbon atoms, R 9 and R 10 are independently hydrogen or an alkyl group having 1 to 3 carbon atoms, n is 1 to 4, and X 4 is an alkali metal, ammonium, or alkanolamine.)

[19] The reagent for measuring proteins in urine as described in

[18] , wherein the sulfate ester type anionic surfactant is an alkyl sulfate having 11 to 16 carbon atoms.

[20] The reagent for measuring proteins in urine as described in any one of

[14] to

[19] , wherein the polyoxyalkylene type nonionic surfactant is a polyoxyalkylene compound represented by formula (7) or (8): [Chemical Formula 15] R 11 -Y 1-((CH2) m O) n -R 12 …(7) (In formula (7), Y 1 is O, C(O)O or C(O)NH, R 11 is a linear hydrocarbon group having 8 to 22 carbon atoms, m is an integer of 1 to 4, n is an integer of 2 to 35, and R 12 is hydrogen or an alkyl group having 1 to 3 carbon atoms.) [Chemical formula 16] R 13 -Y 2 -((CH2) m1 O) n1 -((CH2) m2 O) n2 -((CH2) m3 O) n3 -R 14 ···(8) (In formula (8), Y 2 is O, C(O)O or C(O)NH, m1 and m3 are each independently an integer of 1 to 4, m2 is an integer of 2 to 4, m1 and / or m3 is different from m2, n1, n2 and n3 are each independently an integer of 2 to 200, R 13 is hydrogen or a linear hydrocarbon group having 1 to 30 carbon atoms, and R 14 is hydrogen or an alkyl group having 1 to 3 carbon atoms.)

[21] The reagent for measuring urinary protein as described in

[20] , wherein the polyoxyalkylene type nonionic surfactant is a polyoxyalkylene compound represented by formula (7), and in formula (7), Y 1 is O or C(O)O, R 11 is a linear alkyl or alkenyl group having 11 to 20 carbon atoms, m is 2, n is an integer of 20 to 35, and R 12 is hydrogen.

[22] The reagent for measuring urinary protein as described in any one of

[14] to

[21] , wherein the concentration of the nonionic surfactant is 0.0015% by mass to 0.030% by mass, and the concentration of the anionic surfactant is 0.001% by mass to 0.0035% by mass.

[23] The reagent for measuring urinary protein as described in

[22] , wherein the concentration of the anionic surfactant is 0.0015% by mass to 0.0030% by mass.

[24] The reagent for measuring urinary protein as described in

[22] or

[23] , wherein the concentration of the nonionic surfactant is 0.0025 to 0.024% by mass.

[25] The reagent for determining urinary proteins according to any one of

[14] to

[24] , wherein the pigment is pyrogallol red or pyrocatechol violet, and the metal is molybdenum, tin or iron.

[26] The reagent for determining urinary proteins according to any one of

[14] to

[25] , which is used for determination as a single reagent.

[27] A method for manufacturing a reagent for determining urinary proteins, which comprises dissolving the following substances in a buffer solution: a pigment selected from pyrocatechol violet, pyrogallol red, bromopyrogallol red, o-hydroxyhydroquinone phthalide and gallein, an oxygen-containing acid salt, halide, complex salt, or organic or inorganic acid salt of a metal selected from molybdenum, tin and iron, at least one anionic surfactant selected from sulfonic acid type anionic surfactants and sulfate type anionic surfactants, and a polyoxyalkylene type nonionic surfactant. Effects of the Invention

[0011] In the quantification of total protein in urine using a complex formed by coordination of a metal and a pigment such as pyrogallol red-molybdenum complex, while correcting the significant differences in reactivity among urinary protein species, it is also possible to reduce the interference of hemolyzed hemoglobin. Detailed Description of the Invention

[0012] The embodiments of the present invention will be described in detail. However, the present invention should not be construed as being limited to the following embodiments.

[0013] The protein quantification method of the present invention is characterized in that when determining proteins in urine using a complex formed by coordination of a metal and a pigment (hereinafter sometimes simply referred to as "metal-pigment complex" or "complex"), the protein is reacted with the complex in the presence of a specific anionic surfactant and a specific nonionic surfactant. In addition, the reagent for determining urinary proteins of the present invention is characterized in that the "metal-pigment complex" and the "specific anionic surfactant and specific nonionic surfactant" are combined in such a way that at least these two coexist in the reaction solution.

[0014] As the pigment constituting the metal-pigment complex, any pigment can be used as long as it coordinates with a metal to form a complex and the absorption wavelength of the complex is shifted by binding to a protein. Examples include pyrocatechol violet, pyrogallol red, bromopyrogallol red, o-hydroxyhydroquinone phthalein, and galligen. In addition, as the metal constituting the metal-pigment complex, any metal can be used as long as it coordinates with the pigment to form a complex and the absorption wavelength is shifted by the binding of the protein to the complex. Examples include molybdenum, tin, iron, and aluminum. Specific examples of the metal-pigment complex include pyrocatechol-tin(IV) complex, pyrogallol red-molybdenum complex, bromopyrogallol red-molybdenum complex, o-hydroxyhydroquinone phthalein-iron(III) complex, o-hydroxyhydroquinone phthalein-aluminum(III) complex, and galligen-molybdenum complex. The concentration of the metal-pigment complex in the reaction solution is set to be a sufficient amount relative to the protein concentration in the assumed sample. Usually, the concentration of the metal-pigment complex in the reaction solution may be 0.030 to 0.090 mM, preferably 0.037 to 0.057 mM. In addition, the concentration of the metal-pigment complex in the assay reagent is designed to be the concentration of the metal-pigment complex in such a reaction solution. Usually, the concentration of the metal-pigment complex in the assay reagent is adjusted to 0.030 to 0.090 mM, preferably adjusted to 0.037 to 0.057 mM.

[0015] The reagent containing the metal-pigment complex is prepared by dissolving the above pigment and metal in a buffer solution adjusted to a specified pH according to the type of the complex. The metal is usually mixed with the above pigment in the form of an oxygen-containing acid salt, halide, complex salt, or organic or inorganic acid salt. Examples of such forms include molybdates (alkali metal salts, ammonium salts, etc.), stannates (alkali metal salts, ammonium salts, etc.), tin chloride, tin sulfate, ferrites (alkali metal salts, ammonium salts, etc.), and ferric chloride. When preparing the reagent, the addition amounts of the pigment and the metal in the buffer solution are determined according to the concentration of the formed complex. The addition amount of the pigment is usually 0.040 to 0.180 mM in the assay reagent, preferably 0.054 to 0.081 mM. In addition, the addition amount of the metal is usually 0.030 to 0.090 mM in the assay reagent, preferably 0.037 to 0.057 mM.

[0016] The method of the present invention is carried out by bringing the metal-pigment complex into contact with the protein in the sample in the presence of at least one anionic surfactant selected from sulfonic acid type anionic surfactants and sulfate ester type anionic surfactants and a polyoxyalkylene type nonionic surfactant. Due to the presence of these surfactants, while correcting the reactivity differences among urinary protein species, the interference of hemolyzed hemoglobin is also reduced.

[0017] In this specification, the "sulfonic acid type anionic surfactant" refers to an anionic surfactant having one or more sulfonic acid groups or their salts. In a preferred embodiment, the sulfonic acid type anionic surfactant is a sulfonate represented by the formula (1): [Chemical formula 17] R 1 -SO3X 1 ···(1) [In formula (1), X 1 is an alkali metal, ammonium or alkanolamine (preferably an alkanolamine having 1 to 3 carbon atoms), preferably Na, and R 1 is selected from the following (1) to (5): (1) A hydrocarbon group, preferably a hydrocarbon group having 8 to 18 carbon atoms, more preferably an alkyl or alkenyl group having 8 to 18 carbon atoms, particularly preferably a straight-chain alkyl group having 11 to 16 carbon atoms. (2) A monocyclic or bicyclic aromatic hydrocarbon substituted with a hydrocarbon group, preferably a monocyclic or bicyclic aromatic hydrocarbon substituted with a hydrocarbon group having 8 to 18 carbon atoms, more preferably a 5- or 6-membered monocyclic aromatic hydrocarbon or a 10- to 12-membered bicyclic aromatic hydrocarbon substituted with a hydrocarbon group having 8 to 18 carbon atoms, further preferably a 5- or 6-membered monocyclic aromatic hydrocarbon or a 10- to 12-membered bicyclic aromatic hydrocarbon substituted with an alkyl or alkenyl group having 8 to 18 carbon atoms, particularly preferably a benzene ring substituted with a straight-chain alkyl group having 10 to 14 carbon atoms (it should be noted that in the bond between the hydrocarbon group and the aromatic ring, the bonding position of the two is any position possible chemically). (3) A succinic acid residue of formula (2), its (di)ester or their salts: [Chemical formula 18] (In formula (2), R 2 and R 3 each independently represent hydrogen, a hydrocarbon group or an alkali metal, preferably a hydrocarbon group having 8 to 18 carbon atoms or Na, more preferably a straight-chain alkyl group having 8 to 16 carbon atoms.) (4) A succinic acid monoamide residue of formula (3), its ester, or their salts: [Chemical formula 19] (In formula (3), R 4 and R 5 each independently represent hydrogen, a hydrocarbon group or an alkali metal, preferably a hydrocarbon group having 8 to 18 carbon atoms or Na, more preferably a straight-chain alkyl group having 11 to 16 carbon atoms.) (5) A sulfonate of a hydrocarbon group-substituted diphenyl ether residue of formula (4): [Chemical formula 20] (In formula (4), R6 is a hydrocarbon group having 8 to 18 carbon atoms, preferably an alkyl or alkenyl group having 8 to 18 carbon atoms, particularly preferably a straight-chain alkyl group having 11 to 16 carbon atoms, X 2 is an alkali metal, ammonium or alkanolamine (preferably an alkanolamine having 1 to 3 carbon atoms), preferably Na.)

[0018] More preferred sulfonic acid type anionic surfactants are alkanesulfonates having 11 to 16 carbon atoms or linear alkylbenzenesulfonates having 10 to 14 carbon atoms, and their Na salts are particularly preferred.

[0019] In this specification, the "sulfate type anionic surfactant" is an anionic surfactant having one or more structures represented by the formula: -O-SO3 - For example, alkyl sulfates, polyoxyethylene alkyl ether sulfates and their salts can be cited. In a preferred embodiment, the sulfate type anionic surfactant is a sulfate salt represented by the following formula (5) or (6): [Chemical formula 21] R 7 O-SO3X 3 ···(5) (In formula (5), R 7 is a hydrocarbon group having 8 to 18 carbon atoms, or a monocyclic or bicyclic aromatic hydrocarbon substituted with a hydrocarbon group having 8 to 18 carbon atoms. The hydrocarbon group having 8 to 18 carbon atoms is preferably an alkyl or alkenyl group having 8 to 18 carbon atoms, more preferably an alkyl group having 11 to 16 carbon atoms. The monocyclic or bicyclic aromatic hydrocarbon substituted with a hydrocarbon group having 8 to 18 carbon atoms is preferably a 5- or 6-membered monocyclic aromatic hydrocarbon or a 10- to 12-membered bicyclic aromatic hydrocarbon substituted with a hydrocarbon group having 8 to 18 carbon atoms, more preferably a 5- or 6-membered monocyclic aromatic hydrocarbon or a 10- to 12-membered bicyclic aromatic hydrocarbon substituted with an alkyl or alkenyl group having 8 to 18 carbon atoms, particularly preferably a benzene ring substituted with a straight-chain alkyl group having 10 to 14 carbon atoms (it should be noted that in the bond between the hydrocarbon group and the aromatic ring, the bonding position of the two is any position chemically possible).). X 3 is an alkali metal, ammonium or alkanolamine (preferably an alkanolamine having 1 to 3 carbon atoms), preferably Na.) [Chemical formula 22] R 8 O-(CHR 9 CHR 10 O) n -SO3X 4 ···(6) (In formula (6), R 8 is a hydrocarbon group having 8 to 18 carbon atoms (including alkyl, alkenyl), preferably an alkyl group having 11 to 16 carbon atoms, R 9 and R 10Independently is hydrogen or an alkyl group having 1 to 3 carbon atoms, preferably hydrogen, n is 1 to 10, preferably 1 to 4, X 4 is an alkali metal, ammonium or alkanolamine (preferably an alkanolamine having 1 to 3 carbon atoms), preferably Na.).

[0020] A more preferred sulfate-type anionic surfactant is an alkyl sulfate having 11 to 16 carbon atoms, and its Na salt is particularly preferred.

[0021] The concentration of the sulfate-type anionic surfactant or sulfonic acid-type anionic surfactant in the reaction solution can be determined according to applicable measurement parameters so as to be able to cooperate with the polyoxyalkylene-type nonionic surfactant to correct the significant difference in reactivity among urinary protein species, while reducing the interference of hemolyzed hemoglobin. The concentration of the sulfate-type anionic surfactant or sulfonic acid-type anionic surfactant in the reaction solution is preferably 0.001% by mass to 0.004% by mass, more preferably 0.001% by mass to 0.0035% by mass, further preferably 0.0015% by mass to 0.0030% by mass, and particularly preferably 0.0015% by mass to 0.0025% by mass. In addition, the concentration of the sulfate-type anionic surfactant or sulfonic acid-type anionic surfactant in the measurement reagent can be designed to achieve the desired concentration in the reaction solution, preferably 0.001% by mass to 0.004% by mass, more preferably 0.001% by mass to 0.0035% by mass, further preferably 0.0015% by mass to 0.003% by mass, and particularly preferably 0.0015% by mass to 0.0025% by mass.

[0022] In the present application, the so-called "polyoxyalkylene-type nonionic surfactant" refers to an anionic surfactant containing a structure of the formula: R-Y-(alkylene-O) n -(R is a linear hydrocarbon group, Y is O, C(O)O or C(O)NH, and n is an integer of 1 or more and 50 or less). In a preferred embodiment, the polyoxyalkylene-type nonionic surfactant is a polyoxyalkylene compound represented by the following formula (7) or (8).

[0023] [Chemical formula 23] R 11 -Y 1 -((CH2) m O) n -R 12 ···(7) (In formula (7), Y 1 is O, C(O)O or C(O)NH, preferably O or C(O)O. R 11is a linear hydrocarbon group having 8 to 22 carbon atoms, preferably an alkyl or alkenyl group having 8 to 22 carbon atoms, more preferably a straight-chain alkyl or alkenyl group having 11 to 20 carbon atoms. m is an integer of 1 to 4, preferably 2. n is an integer of 2 to 35, preferably an integer of 15 to 35, more preferably an integer of 20 to 35. R 12 is hydrogen or an alkyl group having 1 to 3 carbon atoms, preferably hydrogen.)

[0024] [Chemical formula 24] R 13 -Y 2 -((CH2) m1 O) n1 -((CH2) m2 O) n2 -((CH2) m3 O) n3 -R 14 ···(8) (In formula (8), Y 2 is O, C(O)O or C(O)NH, preferably O or C(O)O, more preferably O. m1 and m3 are each independently an integer of 1 to 4, preferably 2. m2 is an integer of 2 to 4, preferably 3. m1 or m3 is different from m2, preferably m1 and m3 are different from m2. n1, n2 and n3 are each independently an integer of 2 to 200, preferably an integer of 2 to 100, more preferably an integer of 2 to 70. R 13 is hydrogen or a linear hydrocarbon group having 1 to 30 carbon atoms, preferably a linear hydrocarbon group having 8 to 22 carbon atoms, more preferably an alkyl or alkenyl group having 8 to 22 carbon atoms, particularly preferably an alkyl or alkenyl group having 11 to 20 carbon atoms. R 14 is hydrogen or an alkyl group having 1 to 3 carbon atoms, preferably hydrogen.)

[0025] As the polyoxyalkylene type nonionic surfactant, a surfactant having an HLB value of 13 to 19 is preferred, and a surfactant having an HLB value of 15 to 19 is more preferred. It should be noted that the HLB value in the specification of this application refers to the value calculated according to the Griffin formula (Surfactant Handbook, published by Kogyo Tosho Co., Ltd., 1987, pages 234-235).

[0026] The concentration of the polyoxyalkylene type nonionic surfactant in the reaction solution can be determined according to applicable measurement parameters so as to correct the reactivity difference among urinary protein species in cooperation with the sulfate type anionic surfactant or the sulfonic acid type anionic surfactant, and at the same time reduce the interference of hemolyzed hemoglobin. The concentration of the polyoxyalkylene type nonionic surfactant in the reaction solution is preferably 0.0015% by mass to 0.03% by mass, more preferably 0.0025% by mass to 0.024% by mass, still more preferably 0.003% by mass to 0.012% by mass, and particularly preferably 0.004% by mass to 0.008% by mass. In addition, the concentration of the polyoxyalkylene type nonionic surfactant in the measurement reagent can be designed to achieve a desired concentration in the reaction solution, but is preferably 0.0015% by mass to 0.03% by mass, more preferably 0.0025% by mass to 0.024% by mass, still more preferably 0.003% by mass to 0.012% by mass, and particularly preferably 0.004% by mass to 0.008% by mass.

[0027] In the present invention, the above-mentioned anionic surfactant and nonionic surfactant only need to be present during the reaction between the metal-pigment complex and the protein. These surfactants can be added to the reagent solution containing the metal-pigment complex, or can be added to a specimen such as urine, and further, these surfactants can also be added when the measurement reagent and the specimen are mixed. From a practical aspect, it is convenient to pre-add these surfactants to the reagent containing the metal-pigment complex. The quantification of urinary proteins mostly uses a reagent of a single reagent system. In this case, these surfactants and other components can be contained simultaneously.

[0028] In the method and reagent of the present invention, there are no particular limitations on the components other than the above-mentioned metal-pigment complex, anionic surfactant, and nonionic surfactant, and other components used in the reagent and method for quantifying proteins by the reaction between the metal-pigment complex and urinary proteins can be contained. For example: other components that reduce the influence of hemolyzed hemoglobin, components that reduce coexisting substances other than hemolyzed hemoglobin, etc. These components are disclosed in, for example, Patent Documents 1 to 18 (the contents of these documents are incorporated herein by reference).

[0029] Examples of the components that reduce coexisting substances include polyhydric alcohols, chelating agents, etc.

[0030] Examples of the polyhydric alcohols include diols, sugar alcohols, etc., and specifically, mannitol, sorbitol, dulcitol, glycerol, polyglycerol, etc. These polyhydric alcohols can be used alone or in combination of two or more.

[0031] Examples of the chelating agent include: ethylenediaminetetraacetic acid (EDTA), hydroxyethylethylenediaminetriacetic acid (EDTA-OH), ethylenediaminediacetic acid (EDDA), iminodiacetic acid (IDA), nitrilopropionic acid (NTP), nitrilotriacetic acid (NTA), hydroxyethyliminodiacetic acid (HIDA), citric acid, tartaric acid, oxalic acid, 1-hydroxyethane-1,1-diphosphonic acid, pyrophosphoric acid, hexametaphosphoric acid, tripolyphosphoric acid, metaphosphoric acid, and salts thereof. These chelating agents can be used alone or in combination of two or more. In addition, examples of the salt include: alkali metal salts such as sodium, potassium, and lithium, and ammonium salts.

[0032] The assay reagent of the present invention can be prepared by dissolving the above components in a buffer solution having a specified pH value. The buffer solution is selected according to the metal-pigment complex used. For example, it includes: glycine buffer solution, maleic acid buffer solution, succinic acid buffer solution, citric acid buffer solution, oxalic acid buffer solution, phosphate buffer solution. In addition, regarding the pH of the assay reagent (buffer solution), according to the metal-pigment complex used, a pH that appropriately causes complex formation and absorption wavelength shift is selected. For example, in the case of pyrogallol-molybdenum complex, a pH of 2 to 2.5, preferably pH 2.2, is selected.

[0033] In the quantification of urinary proteins in the present invention, in addition to carrying out the reaction of the metal-pigment complex with proteins in the presence of the above anionic surfactant and nonionic surfactant, it can be carried out in the same manner as the conventional method using a metal-pigment complex. If an example is shown, the assay reagent containing the above components is mixed with a sample for quantifying protein concentration, and at a constant temperature from room temperature to 37 °C, the reaction is carried out for a certain time (for example, about 3 to 15 minutes). The sample for quantifying protein concentration can also be appropriately diluted as needed, and the diluted sample is assayed. When proteins bind to the metal-pigment complex, the absorption wavelength of the complex shifts. Therefore, the vicinity of the maximum absorption wavelength after the shift is used as the main wavelength, and a sub-wavelength is selected as needed to measure the absorbance. On the other hand, instead of the sample, a standard sample with a known protein concentration is used, and the same operation is carried out to measure the absorbance to prepare a standard curve. By comparing this standard curve with the absorbance obtained from the above sample, the protein concentration in the sample can be determined.

[0034] The method of the present invention can be simply carried out in a short time by colorimetry and is applicable to the determination using an automatic analyzer. In addition, it is also applicable to the protein determination using a test strip method. Examples

[0035] Hereinafter, the present invention will be described more specifically by way of examples, but the present invention is not limited thereto.

[0036] 1. Evaluation method In order to evaluate the effect of correcting the significant difference in reactivity between protein species in urine and the effect of reducing the interference of hemolyzed hemoglobin by the reagent of the present invention, the following tests were conducted on the reagents of the following Examples and Comparative Examples.

[0037] 1-1. Test for evaluating the significant difference in reactivity between protein species in urine Using the measurement reagents of each Example and Comparative Example, the total protein concentration of albumin solution and γ-globulin solution with known concentrations was quantified, and the analytical values were compared with the known concentrations to evaluate the reactivity to the two protein species.

[0038] (1) Preparation of human serum albumin solution and human serum γ-globulin solution Weighed human serum albumin (manufactured by SCRIPPS) and human serum γ-globulin (manufactured by SIGMA), dissolved them in ion-exchanged water, and prepared solutions with concentrations of 0 mg / dl, 120 mg / dl, and 600 mg / dl, respectively.

[0039] (2) Quantification of total protein In a Hitachi 7180 automatic analyzer (Hitachi, Ltd.), using the measurement reagents of each Example and Comparative Example, the total protein in human serum albumin solution and human serum γ-globulin solution at each concentration was quantified. The calibrator was prepared by weighing human serum albumin (manufactured by SCRIPPS), dissolving it in ion-exchanged water to a concentration of 100 mg / dl, and the measurement was performed using the following measurement parameters.

[0040] 1-2. Effect of reducing the interference of hemolyzed hemoglobin Using the measurement reagents of each Example and Comparative Example, the total protein concentration of urine samples containing a specified concentration of hemolyzed hemoglobin was quantified to evaluate the influence of hemolyzed hemoglobin on the analytical value.

[0041] (1) Preparation of hemolyzed hemoglobin solution Using "Interference Check·A+" (manufactured by SYSMEX), the blank (hemoglobin concentration: 0 mg / dL) and 1000 mg / dL hemolyzed hemoglobin solution contained therein were each diluted 10 times with a urine control sample (Liquichek2, manufactured by Bio-rad) to prepare urine base samples with hemoglobin concentrations of 0 mg / dL and 100 mg / dL, respectively. (2) Quantification of total protein The total protein in each solution was quantified under the same conditions as those for the quantification of total protein when evaluating the significant difference in reactivity between protein species in urine, using the same apparatus.

[0042] 2. Research on the Types of Surfactants

[0043] 2-1. Preparation of Reagents [Example 1] Add 0.068 mM pyrogallol red, 0.047 mM ammonium molybdate, 12.65 mM sodium benzoate, 13.47 mM D-mannitol, 0.81 mM sulfanilic acid, 0.04% by mass of EDTA-OH, 0.002% by mass of sodium dodecyl sulfate (SDS), and 0.008% by mass of Brij58 to 100 mM glycine buffer, mix, and adjust the pH to 2.2 to prepare a measurement reagent containing a complex of pyrogallol red and molybdenum. Sodium dodecyl sulfate (SDS) [Chemical Formula 25] Brij58 Polyoxyethylene (20) cetyl ether [Chemical Formula 26] CH3(CH2) 15 O((CH2)2O) 20 H

[0044] [Comparative Example 1] Without adding sodium dodecyl sulfate (SDS) and Brij58, perform the same operations as in Example 1 to prepare a measurement reagent. [Comparative Example 2] Without adding Brij58, perform the same operations as in Example 1 to prepare a measurement reagent. [Comparative Example 3] Without adding sodium dodecyl sulfate (SDS), perform the same operations as in Example 1 to prepare a measurement reagent.

[0045] [Example 2] and [Comparative Example 4] Instead of 0.002% by mass of sodium dodecyl sulfate (SDS), add linear alkylbenzene sulfonate and sodium cholate at the same concentration respectively, and perform the same operations as in Example 1 to prepare measurement reagents respectively. Linear alkylbenzene sulfonate [Chemical Formula 27] Sodium cholate [Chemical Formula 28]

[0046] [Comparative Examples 5 - 7] Instead of 0.002% by mass of sodium dodecyl sulfate (SDS), octylamine hydrochloride, benzylhexadecyldimethylammonium chloride, and lauryldimethylaminoacetate betaine were added at the same concentration, respectively. Except for this, the operation was the same as in Example 1 to prepare the assay reagent. n-Octylamine hydrochloride [Chemical formula 29] Benzyl hexadecyl dimethyl ammonium chloride [Chemical formula 30] Lauryl dimethylamino acetic betaine [Chemical formula 31]

[0047] [Examples 3 - 5] Instead of 0.008% by mass of Brij58, Brij 35, nonionic E - 230, and nonionic S15.4 were added at the same concentration, respectively. Except for this, the operation was the same as in Example 1 to prepare the assay reagent. Brij35 Polyoxyethylene (23) lauryl ether [Chemical formula 32] CH3(CH2) 11 O((CH2)2O) 23 H Non-ionic E-230 Polyoxyethylene (30) oleyl ether [Chemical formula 33] CH3(CH2)7CH=CH(CH2)8O((CH2)2O) 30 H Non-ionic S15.4 Polyethylene glycol (35) monostearate [Chemical formula 34] CH3(CH2) 16 C(O)O((CH2)2O) 35 H

[0048] [Comparative Examples 8 - 10] Instead of 0.008% by mass of Brij58, Triton X - 100, BPSH - 25, and Tween 20 were added at the same concentration, respectively. Except for this, the operation was the same as in Example 1 to prepare the assay reagent. TritonX-100 [Chemical formula 35] BPSH-25 [Chemical Formula 36] Tween 20 [Chemical Formula 37]

[0049] The following is a summary of the compositions of the measurement reagents for Examples 1 to 5 and Comparative Examples 1 to 10. [Table 1] Comparative Example 1 Comparative Example 2 Comparative Example 3 Example 1 Example 2 Comparative Example 4 Glycine 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM Pyrogallol red 0.068 mM 0.068 mM 0.068 mM 0.068 mM 0.068 mM 0.068 mM Ammonium molybdate 0.047 mM 0.047 mM 0.047 mM 0.047 mM 0.047 mM 0.047 mM D-Mannitol 13.47 mM 13.47 mM 13.47 mM 13.47 mM 13.47 mM 13.47 mM p-Aminobenzenesulfonic acid 0.81 mM 0.81 mM 0.81 mM 0.81 mM 0.81 mM 0.81 mM Sodium benzoate 12.65 mM 12.65 mM 12.65 mM t2.65 mM 12.65 mM 12.65 mM EDTA-OH 0.04％ 0.04％ 0.04％ 0.04％ 0.04％ 0.04％ SDS - 0.002％ - 0.002％ - - Linear alkylbenzene sulfonate - - - - 0.002％ - Sodium cholate - - - - - 0.002％ Brij58 - - 0.008％ 0.008％ 0.008％ 0.008％ [Table 2] [Table 3] Example 3 Example 4 Example 5 Glycine 100 nlm 100 mM 100 mM Pyrogallol red 0.068 mM 0.068 nlm 0.068 mM Ammonium molybdate 0.047 mM 0.047 mM 0.047 mM D-Mannitol 13.47 mM 13.47 mM 13.47 mM p-Aminobenzenesulfonic acid 0.81 mM 0.81 mM 0.81 mM Sodium benzoate 12.65 mM 12.65 mM 12.65 mM EDTA-OH 0.04％ 0.04％ 0.04％ SDS 0.002％ 0.002％ 0.002％ Brii35 0.008％ - - Non-ionic E-230 - 0.008％ - Non-ionic S15.4 - - 0.008％ [Table 4] Comparative Example 8 Comparative Example 9 Comparative Example 10 Glycine 100 mM 100 mM 100 mM Pyrogallol red 0.068 mM 0.068 mM 0.068 mM Ammonium molybdate 0.047 mM 0.047 mM 0.047 mM D-Mannitol 13.47 mM 13.47 mM 13.47 mM p-Aminobenzenesulfonic acid 0.81 mM 0.81 mM 0.81 mM Sodium benzoate 12.65 mM 12.65 mM 12.65 mM EDTA-OH 0.04％ 0.04％ 0.04％ SDS 0.002％ 0.002％ 0.002％ TritonX-100 0.008％ - - BPSH-25 - 0.008％ - Tween 20 - - 0.008％

[0050] 2-2. Measurement Results The measurement results are as follows. [Table 5] The sensitivity is calculated by subtracting the measurement value of the blank (0 mg / dL) from the measurement value of each protein solution. [Table 6] The sensitivity is calculated by subtracting the measurement value of the blank (0 mg / dL) from the measurement value of each protein solution. The values of Comparative Example 1 and Example 1 are different from those in Table 5 because the reagent with the same composition was measured again together with the reagents of Comparative Example 5, etc. [Table 7] The sensitivity is calculated by subtracting the measurement value of the blank (0 mg / dL) from the measurement value of each protein solution. [Table 8] The sensitivity is calculated by subtracting the measurement value of the blank (0 mg / dL) from the measurement value of each protein solution.

[0051] As shown in the above table, compared with the reagent without surfactant (Comparative Example 1) and the reagent with only Brij58 added (Comparative Example 3), in the reagents with SDS and Brij58 added (Example 1), the reagent with linear alkylbenzene sulfonate and Brij58 added (Example 2), the reagent with SDS and Brij 35 added (Example 3), the reagent with SDS and nonionic E-230 added (Example 4), and the reagent with SDS and nonionic S15.4 added (Example 5), the significant difference in reactivity between protein species (globulin sensitivity / albumin sensitivity) was greatly reduced, and the influence of hemolyzed hemoglobin was also greatly reduced. Compared with the reagent with only SDS added (Comparative Example 2), the significant difference in reactivity between protein species (globulin sensitivity / albumin sensitivity) was at the same level or reduced, and the influence of hemolyzed hemoglobin was greatly reduced. In addition, in the reagents with Brij58 and respective sodium cholate, octylamine hydrochloride, benzylhexadecyldimethylammonium chloride, or lauryldimethylaminobetaine added (Comparative Examples 4 to 7), no significant improvement was found in either the significant difference in reactivity between protein species (globulin sensitivity / albumin sensitivity) or the influence of hemolyzed hemoglobin. In addition, in the reagents with SDS and respective Triton X-100, BPSH-25, or Tween 20 added (Comparative Examples 8 to 10), although an improvement was found in the significant difference in reactivity between protein species (globulin sensitivity / albumin sensitivity), no significant improvement was found in the influence of hemolyzed hemoglobin.

[0052] 3. Study on the concentration of polyoxyalkylene type nonionic surfactant 3-1. Preparation of reagents [Examples 6-8] The concentration of Brij58 was changed to 0.002%, 0.004%, and 0.016% respectively, and otherwise, the same operations as in Example 1 were performed to prepare the assay reagents.

[0053] The compositions of the assay reagents of Example 1, Comparative Examples 1 and 2, and the assay reagents of Examples 6 to 8 are summarized below.

[0054] [Table 9] Comparative Example 1 Comparative Example 2 Example 6 Example 7 Example 1 Example 8 Glycine 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM Pyrogallol red 0.068 mM 0.068 mM 0.068 mM 0.068 mM 0.068 mM 0.068 mM Ammonium molybdate 0.047 mM 0.047 mM 0.047 mM 0.047 mM 0.047 mM 0.047 mM D-Mannitol 13.47 mM 13.47 mM 13.47 mM 13.47 mM 13.47 mM 13.47 mM p-Aminobenzenesulfonic acid 0.81 mM 0.81 mM 0.81 mM 0.81 mM 0.81 mM 0.81 mM Sodium benzoate 12.65 mM 12.65 mM 12.65 mM 12.65 mM 12.65 mM 12.65 mM EDTA-OH 0.04％ 0.04％ 0.04％ 0.04％ 0.04％ 0.04％ SDS - 0.002％ 0.002％ 0.002％ 0.002％ 0.002％ Brij58 - - 0.002％ 0.004％ 0.008％ 0.016％

[0055] 3-2. Measurement results Using the reagents of Example 1, 7 to 11 and Comparative Example 1, the test results on the effect of improving the significant difference in reactivity between albumin and γ-globulin and the interference of hemolyzed hemoglobin, together with the measurement results of the reagent of Comparative Example 2 shown in Table 5, are summarized in the following table. [Table 10] The sensitivity was calculated by subtracting the measured value of the blank (0 mg / dL) from the measured value of each protein solution. The values of Comparative Example 1 and Example 1 are different from those in Tables 5 and 6 because the reagents of the same composition were measured again together with the reagents of Comparative Example 7 etc.

[0056] As shown in the above table, when Brij58 is 0.002% to 0.016% by mass (Examples 6, 7, 1, and 8), the reactivity difference between protein species and the influence of hemolyzed hemoglobin are both improved.

[0057] 4. Study on the concentration of sulfonic acid type anionic surfactant or sulfate type anionic surfactant 4-1. Preparation of reagents [Examples 9 to 11] The concentration of sodium dodecyl sulfate (SDS) was changed to 0.001%, 0.003%, and 0.0035% respectively, and otherwise, the same operations as in Example 1 were performed to prepare the measurement reagents.

[0058] Hereinafter, the compositions of the measurement reagents of Example 1, Comparative Examples 1 and 3, and the measurement reagents of Examples 9 to 11 are summarized and shown. [Table 11] Comparative Example 1 Comparative Example 3 Example 9 Example 1 Example 10 Example 11 Glycine 100 mM 100 mM 100 mM 100 mM 100 mM 100 mM Pyrogallol red 0.068 mM 0.068 mM 0.068 mM 0.068 mM 0.068 mM 0.068 mM Ammonium molybdate 0.047 mM 0.047 mM 0.047 mM 0.047 mM 0.047 mM 0.047 mM D-Mannitol 13.47 mM 13.47 mM 13.47 mM 13.47 mM 13.47 mM 13.47 mM p-Aminobenzenesulfonic acid 0.81 mM 0.81 mM 0.81 mM 0.81 mM 0.81 mM 0.81 mM Sodium benzoate 12.65 mM 12.65 mM 12.65 mM 12.65 mM 12.65 mM 12.65 mM EDTA-OH 0.04％ 0.04％ 0.04％ 0.04％ 0.04％ 0.04％ SDS - - 0.001％ 0.002％ 0.003％ 0.0035％ Brij58 - 0.008％ 0.008％ 0.008％ 0.008％ 0.008％

[0059] 4-2. Measurement results Using the reagents of Example 1, Examples 9 to 11, and Comparative Examples 1 and 3, the test results on the significant difference in improving the reactivity between albumin and γ-globulin and the effect of hemolyzed hemoglobin interference are summarized in the following table. [Table 12] The sensitivity was calculated by subtracting the measured value of the blank (0 mg / dL) from the measured value of each protein solution. The values of Comparative Examples 1 and 3 and Example 1 are different from those in Tables 5 and 6 because the reagents of the same composition were measured again together with the reagents of Example 9 etc.

[0060] As shown in the above table, when SDS is 0.001% to 0.0035% by mass (Examples 9, 1, 10, and 11), the reactivity difference between protein species and the influence of hemolyzed hemoglobin are both improved.

Claims

1. A method for quantifying proteins in urine, wherein, A complex formed by coordination of a metal and a pigment, which exhibits a shift in absorption wavelength when binding to a protein, is used to react the protein with the complex in the presence of the following surfactants: At least one anionic surfactant selected from sulfonic acid type anionic surfactants and sulfate ester type anionic surfactants, and A polyoxyalkylene type nonionic surfactant.

2. A method for reducing the interference of hemolyzed hemoglobin and significantly reducing the obvious differences in reactivity among protein species when quantifying proteins in urine, wherein, A complex formed by coordination of a metal and a pigment, which exhibits a shift in absorption wavelength when binding to a protein, is used to react the protein with the complex in the presence of the following surfactants: At least one anionic surfactant selected from sulfonic acid type anionic surfactants and sulfate ester type anionic surfactants, and A polyoxyalkylene type nonionic surfactant.

3. The method according to claim 1 or 2, wherein The sulfonic acid type anionic surfactant is a sulfonate represented by the following formula: R 1 -SO3X 1 ···(1) In formula (1), X 1 is an alkali metal, ammonium or alkanolamine, R 1 selected from the following (1) to (5): (1) A hydrocarbon group having 8 to 18 carbon atoms, (2) A monocyclic or bicyclic aromatic hydrocarbon substituted with a hydrocarbon group having 8 to 18 carbon atoms, (3) A succinic acid residue of formula (2), its (di)ester or their salts: In formula (2), R 2 and R 3 each independently represents hydrogen, a hydrocarbon group having 8 to 18 carbon atoms, or an alkali metal, (4) A succinic acid monoamide residue of formula (3), its ester, or their salts: In formula (3), R 4 and R 5 each independently represents hydrogen, a hydrocarbon group having 8 to 18 carbon atoms, or an alkali metal, And (5) A sulfonate of a hydrocarbon group-substituted diphenyl ether residue of formula (4): In formula (4), R 6 is a hydrocarbon group having 8 to 18 carbon atoms, and X 2 is an alkali metal, ammonium or alkanolamine.

4. The method according to claim 3, wherein R 1 is (1) an alkyl or alkenyl group having 8 to 18 carbon atoms, or (2) a monocyclic or bicyclic aromatic hydrocarbon substituted with an alkyl or alkenyl group having 8 to 18 carbon atoms.

5. The method according to claim 4, wherein, The sulfonic acid type anionic surfactant is an alkane sulfonate having 11 to 16 carbon atoms or a linear alkylbenzene sulfonate having 10 to 14 carbon atoms.

6. The method according to claim 1 or 2, wherein The sulfate ester type anionic surfactant is a sulfate ester represented by formula (5) or (6): R 7 O-SO3X 3 ···(5) In formula (5), R 7 is a hydrocarbon group having 8 to 18 carbon atoms, or a monocyclic or bicyclic aromatic hydrocarbon substituted with a hydrocarbon group having 8 to 18 carbon atoms, and X 3 is an alkali metal, ammonium or alkanolamine. R 8 O-(CHR 9 CHR 10 O) n -SO3X 4 ···(6) In formula (6), R 8 is a hydrocarbon group having 8 to 18 carbon atoms, R 9 and R 10 are independently hydrogen or an alkyl group having 1 to 3 carbon atoms, n is 1 to 4, and X 4 is an alkali metal, ammonium, or alkanolamine.

7. The method according to claim 6, wherein, The sulfate ester type anionic surfactant is an alkyl sulfate having 11 to 16 carbon atoms.

8. The method according to any one of claims 1 to 7, wherein The polyoxyalkylene type nonionic surfactant is a polyoxyalkylene compound represented by formula (7) or (8): R 11 -Y 1 ((CH2) m O) n -R 12 ···(7) In formula (7), Y 1 is O, C(O)O or C(O)NH, R 11 is a linear hydrocarbon group having 8 to 22 carbon atoms, m is an integer of 1 to 4, n is an integer of 2 to 35, R 12 is hydrogen or an alkyl group having 1 to 3 carbon atoms. R 13 -Y 2 -((CH2) m1 O) n1 -((CH2) m2 O) n2 -((CH2) m3 O) n3 -R 14 …(8) In formula (8), Y 2 is O, C(O)O or C(O)NH, m1 and m3 are each independently an integer from 1 to 4, m2 is an integer from 2 to 4, m1 and / or m3 are different from m2, n1, n2 and n3 are each independently an integer from 2 to 200, R 13 is hydrogen or a linear hydrocarbon group having 1 to 30 carbon atoms, R 14 is hydrogen or an alkyl group having 1 to 3 carbon atoms.

9. The method according to claim 8, wherein, The polyoxyalkylene type nonionic surfactant is a polyoxyalkylene compound represented by the formula (7), and in the formula (7), Y 1 is O or C(O)O, R 11 is a linear alkyl or alkenyl group having 11 to 20 carbon atoms, m is 2, n is an integer of 20 to 35, and R 12 is hydrogen.

10. The method according to any one of claims 1 to 9, wherein The concentration of the nonionic surfactant in the reaction solution is 0.0015% to 0.03% by mass, and the concentration of the anionic surfactant in the reaction solution is 0.001% to 0.0035% by mass.

11. The method according to claim 10, wherein, The concentration of the anionic surfactant in the reaction solution is 0.0015% to 0.003% by mass.

12. The method according to claim 10 or 11, wherein The concentration of the nonionic surfactant in the reaction solution is 0.0025% to 0.024% by mass.

13. The method according to any one of claims 1 to 12, wherein The pigment is pyrogallol red or pyrocatechol violet, and the metal is molybdenum, tin or iron.

14. A reagent for determining proteins in urine, which contains: A complex formed by coordination of a metal and a pigment, whose absorption wavelength shifts when binding to a protein; At least one anionic surfactant selected from sulfonic acid type anionic surfactants and sulfate ester type anionic surfactants; and A polyoxyalkylene type nonionic surfactant.

15. The reagent for measuring proteins in urine according to claim 14, wherein, The sulfonic acid type anionic surfactant is a sulfonate represented by the following formula: R 1 -SO3X 1 …(1) In formula (1), X 1 is an alkali metal, ammonium or alkanolamine, R 1 Selected from the following (1) to (5): (1) A hydrocarbon group having 8 to 18 carbon atoms, (2) A monocyclic or bicyclic aromatic hydrocarbon substituted with a hydrocarbon group having 8 to 18 carbon atoms, (3) A succinic acid residue of formula (2), its (di)ester or their salts: In formula (2), R 2 and R 3 each independently represent hydrogen, a hydrocarbon group having 8 to 18 carbon atoms, or an alkali metal, (4) A succinic acid monoamide residue of formula (3), its ester, or their salts: In formula (3), R 4 and R 5 each independently represents hydrogen, a hydrocarbon group having 8 to 18 carbon atoms, or an alkali metal. And (5) A sulfonate of a hydrocarbon group-substituted diphenyl ether residue of formula (4): In formula (4), R 6 is a hydrocarbon group having 8 to 18 carbon atoms, and X 2 is an alkali metal, ammonium or alkanolamine.

16. The reagent for measuring proteins in urine according to claim 15, wherein, R 1 is (1) an alkyl or alkenyl group having 8 to 18 carbon atoms, or (2) a monocyclic or bicyclic aromatic hydrocarbon substituted with an alkyl or alkenyl group having 8 to 18 carbon atoms.

17. The reagent for measuring proteins in urine according to claim 16, wherein, The sulfonic acid type anionic surfactant is an alkane sulfonate having 11 to 16 carbon atoms or a linear alkylbenzene sulfonate having 10 to 14 carbon atoms.

18. The reagent for measuring proteins in urine according to claim 14, wherein, The sulfate type anionic surfactant is a sulfate represented by formula (5) or (6): R 7 O-SO3X3…(5) In formula (5), R 7 is a hydrocarbon group having 8 to 18 carbon atoms, or a monocyclic or bicyclic aromatic hydrocarbon substituted with a hydrocarbon group having 8 to 18 carbon atoms, and X 3 is an alkali metal, ammonium or alkanolamine. R 8 O-(CHR 9 CHR 10 O) n -SO3X 4 …(6) In formula (6), R 8 is a hydrocarbon group having 8 to 18 carbon atoms, R 9 and R 10 are independently hydrogen or an alkyl group having 1 to 3 carbon atoms, n is 1 to 4, and X 4 is an alkali metal, ammonium, or alkanolamine.

19. The reagent for measuring proteins in urine according to claim 18, wherein, The sulfate type anionic surfactant is an alkyl sulfate having 11 to 16 carbon atoms.

20. The reagent for measuring proteins in urine according to any one of claims 14 to 19, wherein, The polyoxyalkylene type nonionic surfactant is a polyoxyalkylene compound represented by formula (7) or (8): R 11 -Y 1 -((CH2) m O) n -R 12 …(7) In formula (7), Y 1 is O, C(O)O or C(O)NH, and R 11 is a linear hydrocarbon group having 8 to 22 carbon atoms, m is an integer of 1 to 4, n is an integer of 2 to 35, and R 12 is hydrogen or an alkyl group having 1 to 3 carbon atoms. R 13 -Y 2 -((CH2) m1 O) n1 -((CH2) m2 O) n2 -((CH2) m3 O) n3 -R 14 …(8) In formula (8), Y 2 is O, C(O)O or C(O)NH, m1 and m3 are each independently an integer from 1 to 4, m2 is an integer from 2 to 4, m1 and / or m3 is different from m2, n1, n2 and n3 are each independently an integer from 2 to 200, R 13 is hydrogen or a linear hydrocarbon group having 1 to 30 carbon atoms, R 14 is hydrogen or an alkyl group having 1 to 3 carbon atoms.

21. The reagent for measuring proteins in urine according to claim 20, wherein, The polyoxyalkylene type nonionic surfactant is a polyoxyalkylene compound represented by the formula (7). In the formula (7), Y 1 is O or C(O)O, R 11 is a linear alkyl or alkenyl group having 11 to 20 carbon atoms, m is 2, n is an integer of 20 to 35, and R 12 is hydrogen.

22. The reagent for measuring proteins in urine according to any one of claims 14 to 21, wherein, The concentration of the nonionic surfactant is 0.0015% by mass to 0.03% by mass, and the concentration of the anionic surfactant is 0.001% by mass to 0.0035% by mass.

23. The reagent for measuring proteins in urine according to claim 22, wherein, The concentration of the anionic surfactant is 0.0015% by mass to 0.003% by mass.

24. The reagent for determining proteins in urine according to claim 22 or 23, wherein, The concentration of the nonionic surfactant is 0.0025% by mass to 0.024% by mass.

25. The reagent for measuring proteins in urine according to any one of claims 14 to 24, wherein, The pigment is pyrogallol red or pyrocatechol violet, and the metal is molybdenum, tin or iron.

26. The reagent for measuring urinary protein according to any one of claims 14 to 25, which is used for measurement as a single reagent.

27. A method for producing a reagent for measuring urinary protein, which comprises dissolving the following substances in a buffer solution: a pigment selected from pyrocatechol violet, pyrogallol red, bromopyrogallol red, o-hydroxyhydroquinone phthalide and galligen; an oxygen-containing acid salt, halide, complex salt, or organic or inorganic acid salt of a metal selected from molybdenum, tin and iron; at least one anionic surfactant selected from a sulfonic acid type anionic surfactant and a sulfate type anionic surfactant; and a polyoxyalkylene type nonionic surfactant.

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