Methods and reagents for quantifying proteins

Abstract

The present invention addresses the problem of pseudo-increase in a method for quantifying a protein using a complex in which a metal and a dye are coordinated. In a method for quantifying a protein in a sample using a complex in which a metal and a dye are coordinated, the protein and the complex are reacted in the presence of an aromatic carboxylic acid or a salt thereof.

Description

Technical Field

[0001] The present invention relates to a method and a reagent for quantifying proteins, and more specifically, to a method and a reagent for quantifying proteins using a complex formed by coordination of a metal and a pigment. Background Art

[0002] Conventionally, in the field of clinical examinations and the like, in order to obtain information related to various diseases or symptoms, the total protein concentration in a specimen (test sample) is usually measured. As methods for measuring proteins, the Kjeldahl method, the Lowry method, the biuret method, the Coomassie brilliant blue method, etc. are known. However, in these methods, the following problems have been found: not suitable for routine examinations using automated analysis, affected by amino acids, not suitable for measuring proteins in urine containing a large amount of proteins, and insufficient sensitivity when measuring trace amounts of proteins in urine or cerebrospinal fluid. In the quantification of proteins in specimens such as urine and cerebrospinal fluid, a method using a complex formed by coordination of a metal and a pigment such as pyrogallol red-molybdate complex and measuring the protein by using the shift of the absorption wavelength of the complex in the presence of the protein has been used in many examination rooms.

[0003] However, several problems have also been found in the method for quantifying proteins using such a complex. Tokuda et al. pointed out the following problem: in the method using a complex containing a pigment such as molybdenum and pyrogallol red, oxalic acid, citric acid, phosphoric acid or their salts present in urine bind to molybdenum, causing a negative error and becoming negative values in the urine of healthy people (Patent Document 1). As a method for solving this problem, Tokuda et al. proposed adding a chelating agent that binds to molybdenum in advance to the reagent, or adding metal ions that do not react with the pigment but can bind to oxalic acid, citric acid, phosphoric acid or their salts coexisting in the sample (Patent Document 1).

[0004] Tokuda et al. also pointed out that in the method for analyzing proteins in urine, nitrite ions cause a negative error, and to solve this problem, they proposed adding a specific nitrogen-containing organic compound such as a specific aniline derivative or its soluble salt to the analytical reagent (Patent Document 2).

[0005] In addition, Kishi et al. pointed out the problem that the method of Tokuda et al. combining a chelating agent that binds to molybdenum or a metal ion that can bind to a chelating component coexisting in the sample with a pyrogallol red-molybdate complex has a long measurement time and is not suitable for automated analysis, and proposed a method of reacting a pyrogallol red-molybdate complex with a protein in the presence of a polyol (Patent Document 3).

[0006] These reports all found that the common components in urine react with the metal or pigment forming the complex, hindering the formation of the complex, thus leading to negative errors, especially negative values in the range of low protein concentrations. Therefore, components that reduce the influence of such co-existing substances are added to the assay reagent. Prior art documents

[0007] Patent documents Patent document 1: Japanese Patent Application Laid-Open No. 61-155757 Patent document 2: Japanese Patent Application Laid-Open No. 63-235865 Patent document 3: Japanese Patent Application Laid-Open No. 4-361160 Summary of the invention Problems to be solved by the invention

[0008] In response to this, the present inventors evaluated the performance of a method for measuring proteins using a complex formed by coordination of a metal and a pigment, using patient specimens including specimens showing abnormally high values of protein concentration. As a result, a new phenomenon of pseudo-high value formation was found. The object of the present invention is to provide a method and a reagent for improving this problem of pseudo-high value formation. Means for solving the problems

[0009] The present inventors conducted various studies on means for solving this problem, and as a result, found that reacting the complex with a protein in the presence of an aromatic carboxylic acid or its salt can improve the problem of pseudo-high value formation. That is, the present invention provides the following method and reagent. [1] A method for quantifying a protein in a specimen using a complex formed by coordination of a metal and a pigment whose absorption wavelength shifts when bound to a protein, wherein the protein is reacted with the complex in the presence of an aromatic carboxylic acid or its salt. [2] A method for improving pseudo-high value formation when quantifying a protein in a specimen using a complex formed by coordination of a metal and a pigment whose absorption wavelength shifts when bound to a protein, wherein the protein is reacted with the complex in the presence of an aromatic carboxylic acid or its salt. [3] The method according to [1] or [2], wherein the aromatic carboxylic acid or its salt is a monocyclic or bicyclic aromatic carboxylic acid or its salt, having 2 or less carboxyl groups, having no other substituents, or the aromatic ring is substituted with a sulfonic acid group, a hydroxyl group, a halogen, or an alkyl group having 1 to 4 carbon atoms. [4] The method according to [1] or [2], wherein the aromatic carboxylic acid or its salt comprises one or more selected from benzoic acid, hydroxybenzoic acid, aminobenzoic acid, salicylic acid, phthalic acid, and their salts. [5] The method according to [4], wherein the aromatic carboxylic acid or its salt comprises one or more selected from benzoic acid, hydroxybenzoic acid, salicylic acid, phthalic acid, and their salts. [6] The method according to any one of [1] to [5], wherein the aromatic carboxylic acid or its salt is contained in the reaction solution at a concentration of 4.2 mM or more. [7] The method according to any one of [1] to [6], wherein the protein is reacted with the complex in the presence of the aromatic carboxylic acid or its salt and the following substances: (1) An aliphatic amine or its salt (R-NH-R) having at least one substituent selected from an alkyl group having 1 to 4 carbon atoms, a hydroxyamino group having 1 to 4 carbon atoms, and an aminoalkyl group having 1 to 4 carbon atoms, (2) An aniline derivative or its salt (R-NH-substituted or unsubstituted phenyl or phenylene-R) having at least one substituent selected from a sulfonic acid group, a hydroxy group, a sulfonamido group, a halogen, an alkyl group having 1 to 4 carbon atoms, a hydroxyamino group having 1 to 4 carbon atoms, and an amino group, (3) Hydrazine or a hydrazine derivative in which hydrazine is substituted by an alkyl group having 1 to 4 carbon atoms, (4) Thiourea or a thiourea derivative in which thiourea is substituted by an alkyl group having 1 to 4 carbon atoms, (5) A polyol, and / or (6) A chelating agent. [8] The method according to any one of [1] to [7], wherein the pigment is pyrogallol red or catechol violet, and the metal is molybdenum, tin, or iron. [9] The method according to any one of [1] to [8], wherein the specimen is urine or bone marrow fluid.

[10] A reagent for protein determination, which is a complex formed by coordination of a metal and a pigment, and contains a complex whose absorption wavelength shifts when bound to a protein and an aromatic carboxylic acid or its salt.

[11] The reagent for protein determination according to

[10] , wherein the aromatic carboxylic acid or its salt is a monocyclic or bicyclic aromatic carboxylic acid or its salt, has 2 or less carboxyl groups, has no other substituents, or the aromatic ring is substituted by a sulfonic acid group, a hydroxy group, a halogen, or an alkyl group having 1 to 4 carbon atoms.

[12] The reagent for protein determination according to

[10] , wherein the aromatic carboxylic acid or its salt contains one or more selected from benzoic acid, hydroxybenzoic acid, aminobenzoic acid, salicylic acid, phthalic acid, and their salts.

[13] The reagent for protein determination according to

[12] , wherein the aromatic carboxylic acid or its salt contains one or more selected from benzoic acid, hydroxybenzoic acid, salicylic acid, phthalic acid, and their salts.

[14] The reagent for protein determination according to any one of

[10] to

[13] , which contains the aromatic carboxylic acid or its salt at a concentration of 4.2 mM or more.

[15] The protein assay reagent according to any one of

[10] to

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

[16] The protein assay reagent according to any one of

[10] to

[15] , further comprising: (1) an aliphatic amine (R-NH-R) having at least one substituent selected from an alkyl group having 1 to 4 carbon atoms, a hydroxyamino group having 1 to 4 carbon atoms, and an aminoalkyl group having 1 to 4 carbon atoms, or a salt thereof, (2) aniline, an aniline derivative (R-NH-substituted or unsubstituted phenyl or phenylene residue-R) in which aniline is substituted with at least one substituent selected from a hydroxy group, a hydroxy group, a sulfonamido group, a halogen, an alkyl group having 1 to 4 carbon atoms, a hydroxyamino group having 1 to 4 carbon atoms, and an amino group, or a salt thereof, (3) hydrazine, a hydrazine derivative (R-NH-NH 2 ) in which hydrazine is substituted with an alkyl group having 1 to 4 carbon atoms, or a salt thereof, (4) thiourea, a thiourea derivative (R-NH-C(=S)-N(-R)-R) in which thiourea is substituted with an alkyl group having 1 to 4 carbon atoms, or a salt thereof, (5) a polyol, and / or (6) a chelating agent.

[17] The protein assay reagent according to any one of

[10] to

[16] , which is used for assaying proteins in urine or marrow.

[18] A method for producing a protein assay reagent, comprising: 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 salt, halide, complex salt, or organic or inorganic acid salt of a metal selected from molybdenum, tin, and iron, and an aromatic carboxylic acid or a salt thereof. Advantages of the Invention

[0010] In a method for assaying the total protein concentration in a sample using a complex formed by coordinating a metal such as a pyrogallol red-molybdenum complex with a pigment, when the assay is carried out in the presence of an aromatic carboxylic acid, the problem of false high values can be improved. Thus, it is possible to more accurately quantify the total protein, particularly in the high value range, and to improve the problem of false positives that may be a problem in conventional reagents. Detailed Description of the Invention

[0011] 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.

[0012] The protein quantification method of the present invention is characterized in that when measuring a protein in a sample, a complex formed by coordinating a metal with a pigment (hereinafter sometimes simply referred to as "metal-pigment complex" or "complex") reacts with the protein in the presence of an aromatic carboxylic acid or its salt. In addition, the protein assay reagent of the present invention is characterized in that the metal-pigment complex and the aromatic carboxylic acid are combined in such a way that at least the two coexist in the reaction solution.

[0013] As the pigment constituting the metal-pigment complex, any pigment can be used as long as it forms a complex by coordinating with a metal and can cause a shift in the absorption wavelength of the complex by binding to a protein. For example, catechol violet, pyrogallol red, bromopyrogallol red, o-hydroxyhydroquinone phthalein, galligen, etc. can be cited. In addition, as the metal constituting the metal-pigment complex, any metal can be used as long as it forms a complex by coordinating with the pigment and can cause a shift in the absorption wavelength by the binding of the protein to the complex. For example, molybdenum, tin, iron, aluminum, etc. can be cited. Specific examples of the metal-pigment complex include catechol violet-tin(IV) complex, pyrogallol red-molybdenum complex, bromopyrogallol red-molybdenum complex, o-hydroxyhydroquinone phthalein-iron(III) complex, o-hydroxyhydroquinone phthalein-aluminum(III), galligen-molybdenum complex. The concentration of the metal-pigment complex in the reaction solution is set to a sufficient amount relative to the expected protein concentration in the sample. Usually, the concentration of the metal-pigment complex in the reaction solution is preferably 0.037 to 0.057 mM, as long as it is 0.030 to 0.090 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.

[0014] 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 salt, halide, complex salt, or organic acid salt or inorganic acid salt. As such a form, for example, molybdate (alkali metal salt, ammonium salt, etc.), stannate (alkali metal salt, ammonium salt, etc.), tin chloride, tin sulfate, ferrate (alkali metal salt, ammonium salt, etc.), iron chloride, etc. can be cited. When preparing the reagent, the amounts of the pigment and the metal added to the buffer solution are determined according to the concentration of the formed complex. Usually, the added amount of the pigment in the assay reagent is preferably 0.054 to 0.081 mM, as long as it is 0.040 to 0.180 mM. In addition, the added amount of the metal in the assay reagent is preferably 0.037 to 0.057 mM, as long as it is 0.030 to 0.090 mM.

[0015] In the method of the present invention, a metal-pigment complex is brought into contact with a protein in a sample in the presence of an aromatic carboxylic acid or a salt thereof to perform the determination of the protein. Due to the presence of the aromatic carboxylic acid, the problem of false high values is improved. The aromatic carboxylic acid is an aromatic hydrocarbon having one or more carboxyl groups, preferably a monocyclic and bicyclic aromatic hydrocarbon having one or more carboxyl groups. In addition, the aromatic carboxylic acid is preferably an aromatic carboxylic acid having two or less carboxylic acids. Further, the aromatic carboxylic acid may have a substituent other than a carboxylic acid, and examples thereof include anionic functional groups such as a sulfonic acid group, a hydroxyl group, and a halogen, cationic functional groups such as a nitro group and an amino group, and an alkyl group (preferably an alkyl group having 1 to 4 carbon atoms). As the aromatic carboxylic acid used in the present invention, from the viewpoint of a large effect of improving false high values, an aromatic carboxylic acid having no substituent other than a carboxylic acid or substituted with an anionic functional group is preferred, and an aromatic carboxylic acid having no substituent other than a carboxylic acid is particularly preferred.

[0016] Examples of the salt of the aromatic carboxylic acid include alkali metal salts such as a sodium salt and a potassium salt.

[0017] Specific examples of the aromatic carboxylic acid and its salt used in the present invention include benzoic acid, methylbenzoic acid, hydroxybenzoic acid, 2-hydroxybenzoic acid, aminobenzoic acid, 4-aminobenzoic acid, salicylic acid, phthalic acid, isophthalic acid, terephthalic acid, mellitic acid, trimellitic acid, cinnamic acid, gallic acid, p-hydroxybenzoic acid (hybenzic acid), anthranilic acid, nicotinic acid, dimethylbenzoic acid, acid, cumic acid, phenylacetic acid, atropic acid, hydrocinnamic acid, linic acid, biphenic acid, pyromellitic acid, 2,3-dimethylbenzoic acid, duric acid, α-isoduric acid, γ-isoduric acid, 2,3,4-trimethylbenzoic acid, cuminic acid, uvatic acid, semi acid, trimellitic acid, pyromellitic acid, anisic acid, leptonic acid or their salts, etc., and one or more of them may be contained. As the aromatic carboxylic acid used in the present invention, it is preferred to contain one or more selected from benzoic acid, hydroxybenzoic acid, salicylic acid, phthalic acid and their salts, more preferably to contain one or more selected from benzoic acid, hydroxybenzoic acid and their salts, and particularly preferably to contain one or more selected from benzoic acid and its salts.

[0018] The concentration of the aromatic carboxylic acid or its salt in the reaction solution can be determined according to applicable measurement parameters to obtain the desired improvement effect of pseudo-high value, as confirmed by the following examples. The improvement effect of pseudo-high value increases depending on the concentration of the aromatic carboxylic acid. Therefore, according to the applicable measurement parameters, it can be appropriately set to a concentration of 4.2 mM or more, usually, the concentration of the aromatic carboxylic acid in the reaction solution is preferably 7.0 mM or more, more preferably 9.8 mM or more. In addition, the concentration of the aromatic carboxylic acid or its salt in the measurement reagent is designed to be the desired concentration of the aromatic carboxylic acid in the reaction solution, usually adjusted to a concentration of 4.2 mM or more, preferably adjusted to 7.0 mM or more, more preferably adjusted to 9.8 mM or more.

[0019] On the other hand, depending on the aromatic carboxylic acid or its salt used, considering its solubility in the measurement reagent, it is preferable to determine the upper limit of the concentration in the reaction solution and the measurement reagent. The concentration of the aromatic carboxylic acid in the reaction solution is preferably 20.0 mM or less, more preferably 17.0 mM or less, particularly preferably 15.0 mM or less. Similarly, the concentration of the aromatic carboxylic acid in the measurement reagent is preferably 20.0 mM or less, more preferably 17.0 mM or less, further preferably 15.0 mM or less. Therefore, the concentration of the aromatic carboxylic acid in the reaction solution is preferably 4.2 mM to 20.0 mM, more preferably 7.0 mM to 17.0 mM, particularly preferably 9.8 mM to 15.0 mM. Similarly, the concentration of the aromatic carboxylic acid in the measurement reagent is preferably 4.2 mM to 20.0 mM, more preferably 7.0 mM to 17.0 mM, particularly preferably 9.8 mM to 15.0 mM.

[0020] In the present invention, the aromatic carboxylic acid or its salt only needs to be present when the metal-pigment complex reacts with the protein. The aromatic carboxylic acid or its salt can be added to the reagent solution containing the metal-pigment complex, or can be added to a sample such as urine, can be added to the sample diluent, or can be added when the measurement reagent and the sample are mixed. From a practical aspect, it is convenient to pre-add the aromatic carboxylic acid or its salt to the reagent solution containing the metal-pigment complex.

[0021] In the method and reagent according to the present invention, in the presence of the aromatic carboxylic acid or its salt, by carrying out the reaction of the metal-pigment complex with the protein, the pseudo-high value can be improved in a relatively high range of protein concentrations. In addition, in order to reduce the negative error at a low range of protein concentrations, the following substances can also be used together with the aromatic carboxylic acid: aliphatic amines or their salts, aniline, aniline derivatives or their salts, hydrazine, hydrazine derivatives or their salts, thiourea, thiourea derivatives or their salts, polyhydric alcohols, and / or chelating agents.

[0022] As the aliphatic amine, examples thereof include: an aliphatic amine having at least one substituent selected from an alkyl group having 1 to 4 carbon atoms, a hydroxyamino group having 1 to 4 carbon atoms, and an aminoalkyl group having 1 to 4 carbon atoms (R-NH-R, wherein two Rs are each independently a hydrogen atom or the above substituent, and at least one R is not a hydrogen atom). As the salt thereof, examples include: hydrochloride, sulfate, etc. As specific compounds, examples include: N-methyl-N-ethanolamine, 2-amino-2-methyl-1-propanol, 1-aminopropanol, and their hydrochloride or sulfate. As the aniline derivative, examples thereof include: a compound in which aniline is substituted with at least one substituent selected from a sulfonic acid group, a hydroxy group, a sulfonamido group, a halogen, an alkyl group having 1 to 4 carbon atoms, a nitro group, a hydroxyamino group having 1 to 4 carbon atoms, and an amino group (R-NH-a substituted or unsubstituted phenyl or phenylene residue-R, wherein two Rs are each independently a hydrogen atom or the above substituent, and at least one R is not a hydrogen atom), preferably: the NH of aniline 2 is unsubstituted or substituted with an alkyl group having 1 to 4 carbon atoms, a hydroxyamino group having 1 to 4 carbon atoms, or an amino group, and the phenyl group of aniline is unsubstituted or substituted with any one of a sulfonic acid group, a hydroxy group, a sulfonamido group, a halogen, an alkyl group having 1 to 4 carbon atoms, and a nitro group. As the salt thereof, examples include: hydrochloride, sulfate, etc. In addition, as specific compounds, examples include: p-aminobenzenesulfonic acid, p-aminobenzenesulfonamide, aniline-2,5-disulfonic acid, 2-aminophenol-4-sulfonic acid, 2-chloroaniline-4-sulfonic acid, 4-chloroaniline-3-sulfonic acid, 2,5-dichloroaniline-4-sulfonic acid, p-aminophenol, p-chloroaniline, hydroxyethylaniline, 3-hydroxy-4-nitroaniline, and their hydrochloride or sulfate. As the hydrazine derivative, examples thereof include: a compound in which hydrazine is substituted with an alkyl group having 1 to 4 carbon atoms (R-NH-NH 2 , wherein R is an alkyl group having 1 to 4 carbon atoms). As the salt thereof, examples include: hydrochloride, sulfate, etc. In addition, as specific compounds, examples include: phenylhydrazine, hydrazine hydrochloride. As the thiourea derivative, examples thereof include: a compound in which thiourea is substituted with an alkyl group having 1 to 4 carbon atoms or a phenyl group (R-NH-C(=S)-N(-R)-R, wherein three Rs are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, or a phenyl group, and at least one R is not a hydrogen atom). As the salt thereof, examples include: hydrochloride, sulfate, etc. As specific compounds, examples include: phenylthiourea, hydroxyethylaniline, 3-hydroxy-4-nitroaniline, and their hydrochloride or sulfate. The concentration of an aliphatic amine or its salt, aniline, an aniline derivative or their salts, hydrazine and hydrazine derivatives or their salts in the reaction solution can be determined according to the concentration of nitrite ions in the sample assumed. Usually, the concentration in the reaction solution is 0.05 to 300 mM, preferably 0.24 to 60 mM. Therefore, the concentration of an aliphatic amine or its salt, aniline, an aniline derivative or their salts, hydrazine and hydrazine derivatives or their salts in the assay reagent is usually 0.05 to 300 mM, preferably 0.24 to 60 mM. It is sufficient that an aliphatic amine or its salt, aniline, an aniline derivative or their salts, hydrazine and hydrazine derivatives or their salts are present during the reaction of the metal-pigment complex with the protein. These compounds can also be added to the assay reagent containing the metal-pigment complex, or can be added to a sample such as urine. They can be added to the sample diluent or added when the assay reagent is mixed with the sample. From a practical point of view, it is convenient to pre-add these compounds to the assay reagent containing the metal-pigment complex.

[0023] Examples of the polyol include diols, sugar alcohols, etc. Specifically, mannitol, sorbitol, dulcitol, glycerol, polyglycerol, etc. can be mentioned. These polyols can be used alone or in combination of two or more. The concentration of the polyol in the reaction solution can be determined according to the content of the chelating component in the sample assumed. Usually, the concentration in the reaction solution is 2 to 100 mM, preferably 5 to 50 mM. Therefore, the concentration of the polyol in the assay reagent is usually 2 to 100 mM, preferably 5 to 50 mM. It is sufficient that the polyol is present during the reaction of the metal-pigment complex with the protein. The polyol can be added to the assay reagent containing the metal-pigment complex, or can be added to a sample such as urine, or can also be added when the assay reagent is mixed with the sample. From a practical point of view, it is convenient to pre-add the polyol to the assay reagent containing the metal-pigment complex.

[0024] Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA), hydroxyethylethylenediaminetriacetic acid (EDTA-OH), ethylenediaminediacetic acid (EDDA), iminodiacetic acid (IDA), nitrilotripropionic acid (NTP), nitrilotriacetic acid (NTA), hydroxyethyliminodiacetic acid (HIDA), citric acid, tartaric acid, oxalic acid, 1-hydroxyethylidene-1,1-diphosphonic acid, pyrophosphoric acid, hexametaphosphoric acid, tripolyphosphoric acid, metaphosphoric acid and their salts. 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, lithium and ammonium salts. The concentration of the chelating agent in the reaction solution and the assay reagent varies depending on the chelating agent used. The concentration of the chelating agent in the reaction solution is usually 0.13 to 31 mM, preferably 0.25 to 2.5 mM. In addition, the concentration of the chelating agent in the assay reagent is usually 0.13 to 31 mM, preferably 0.25 to 2.5 mM.

[0025] In the present invention, in addition to the above components, components commonly used for protein determination may also be contained. For example, various surfactants are used to prevent contamination of reaction tubes, measurement cells, etc. or to reduce the influence of hemolysis, chylomicrons, etc., but the reagent of the present invention may also contain various surfactants according to the purpose. The surfactant is usually added to the measurement reagent at a concentration of about 0.01 to 2% by mass.

[0026] The measurement reagent of the present invention can be prepared by dissolving the above components in a buffer solution adjusted to a specified pH. The buffer solution is selected according to the metal-pigment complex used. For example, glycine buffer solution, maleic acid buffer solution, succinic acid buffer solution, citric acid buffer solution, oxalic acid buffer solution, phosphate buffer solution can be cited. In addition, the pH of the measurement reagent (buffer solution) is selected according to the metal-pigment complex used, and the pH at which the complex is formed and the absorption wavelength is appropriately shifted is selected. For example, for the pyrrole-molybdenum complex, pH 2 to 2.5 is selected, preferably pH 2.2.

[0027] The method of the present invention can be carried out in the same manner as the conventional method using a metal-pigment complex except that the reaction between the metal-pigment complex and the protein is carried out in the presence of an aromatic carboxylic acid or its salt. If an example is shown, the measurement reagent containing the above components is mixed with a sample for quantifying the protein concentration, and the reaction is carried out at a constant temperature of room temperature to 37°C for a certain time (for example, about 3 to 15 minutes). The sample for quantifying the protein concentration can be appropriately diluted as needed, and the diluted sample is subjected to measurement. When the protein binds 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 the 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 calibration curve. By comparing this calibration curve with the absorbance obtained from the above sample, the protein concentration in the sample can be determined.

[0028] The measurement method of the present invention can be used for the quantification of proteins in various samples, but is suitable for the measurement of samples with generally low protein concentrations such as urine and spinal fluid with high sensitivity. In addition, it can be simply measured by colorimetry in a short time, and is suitable for measurement using an automatic analyzer. In addition, it can also be applied to the measurement of proteins by the dipstick method. Examples

[0029] Hereinafter, the present invention will be described more specifically by way of examples, but the present invention is not limited thereto.

[0030] 1. Study on the effect of improving false high values by adding aromatic carboxylic acids 1-1. Preparation of reagents [Example 1] 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% EDTA-OH, and 0.01% surfactant were added to 100 mM glycine buffer, mixed, and the pH was adjusted to 2.2 to prepare a measurement reagent containing a complex of pyrogallol red and molybdenum.

[0031] [Examples 2 to 4] Potassium benzoate (Example 2), p-hydroxybenzoic acid (Example 3), and p-aminobenzoic acid (Example 4) were added respectively instead of 12.65 mM sodium benzoate, and otherwise, the measurement reagent was prepared in the same manner as in Example 1.

[0032] [Comparative Example 1] 12.65 mM sodium benzoate was not added, and otherwise, the measurement reagent was prepared in the same manner as in Example 1.

[0033] The compositions of the measurement reagents of Examples 1 to 4 and Comparative Example 1 are summarized below. [Table 1] Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Glycine 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 Ammonium Molybdate 0.047 mM 0.047 mM 0.047 mM 0.047 mM 0.047 mM Sodium Benzoate 12.65 mM - - - - Potassium Benzoate - 12.65 mM - - p-Hydroxybenzoic Acid - - 12.65 mM - - p-Aminobenzoic Acid- - - - 12.65 mM D-Mannitol 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 EDTA-OH 0.04％ 0.04％ 0.04％ 0.04％ 0.04％ Surfactant 0.01％ 0.01％ 0.01％ 0.01％ 0.01％

[0034] 1-2. Evaluation by spike recovery test For the measurement reagents of Examples 1 to 4 and Comparative Example 1, the improvement effect of false high value due to addition of aromatic carboxylic acid was evaluated by spike recovery test. In this test, 4 urine specimens were used, a human serum albumin (HSA) solution with a known concentration was added thereto, the protein concentrations before and after were measured using each reagent, and the recovery rate was calculated from the measured values. Specifically, it is as follows.

[0035] (1) Preparation of human serum albumin (HSA) stock solution Human serum albumin (HSA) was weighed and dissolved in ion-exchanged water to prepare a 1500 mg / dl HSA solution. However, since HSA preparations usually contain about 20% water, the weighed value is not the correct amount of HSA. Therefore, the HSA concentration of the prepared HSA stock solution was quantified by HPLC under the following conditions, and it was accurately confirmed that the HSA concentration was 1311 mg / dl. <Measurement conditions> HPLC (Agilent OpenLAB CDS) Eluent flow path pump: G7129A 1260 Vialsampler Detector: G7114A 1260VWD Chromatographic column: Yarratm 3μm SEC-2000, LC Column 300×7.8mm, Ea Eluent: 100mM P04 buffer, pH 7.0 Column temperature: Flow rate: 35°C: 1.00 mL / min Standard: NIST 7% BSA

[0036] (2) Preparation of test samples Four urine specimens with different protein concentrations were used, and HSA stock solution was added to these specimens in amounts such that the HSA concentration became 100 mg / dl and 300 mg / dl based on the weighed values. The HSA concentrations based on the weighed values were calculated to be 87.4 mg / dl and 262.2 mg / dl respectively from the HSA quantitative values in the HSA stock solution by the above HPLC.

[0037] (3) Quantification of total protein Using a Hitachi 7180 automatic analyzer (Hitachi, Ltd.), the total protein in four urine specimens and the samples to which HSA stock solution was added in amounts such that the HSA concentration became 87.4 mg / dl and 262.2 mg / dl was quantified. The calibrator used was an HSA solution quantified by HPLC, and the following measurement parameters were used.

[0038] The measurement results are as follows. [Table 2]

[0039] [Table 3]

[0040] (4) Calculation of recovery rate Based on the total protein concentration of each sample measured using each reagent, the recovery rate was calculated according to the following formula. · Recovery rate of the sample with 87.4 mg / dl HSA added [(Total protein concentration in the sample with 87.4 mg / dl HSA added - Total protein concentration in the sample before HSA addition) / 87.4]×100 · Recovery rate of the sample with 262.2 mg / dl HSA added [(Total protein concentration in the sample with 262.2 mg / dl HSA added - Total protein concentration in the sample before HSA addition) / 262.2]×100

[0041] The recovery rates of each sample calculated from the total protein concentration (mg / dL) of the urine specimens measured by each reagent are as follows.

[0042] [Table 4] [Table 5]

[0043] As described above, in the case of measurement with the reagent of Comparative Example 1, the recovery rate greatly exceeded 100%, and it was confirmed that the degree of deviation tended to increase as the protein concentration increased. In contrast, when measured with the reagents of Examples 1 to 4, the recovery rate was about 100%, and a significant improvement was confirmed. From the above, it can be understood that by including an aromatic carboxylic acid in the reagent, false high values can be reduced, and the total protein can be quantified more accurately.

[0044] 2. Study on the effective concentration of aromatic carboxylic acid 2-1. Preparation of reagents [Examples 5 to 9] Sodium benzoate was added at concentrations of 2.8 mM, 5.6 mM, 8.4 mM, 11.2 mM, or 14.0 mM, respectively, instead of 12.65 mM sodium benzoate, and the measurement reagent was prepared in the same manner as in Example 1 except for this.

[0045] 2-2. Measurement of total protein Using the reagents of Examples 5 to 9 and Comparative Example 1, the total protein concentration (mg / dL) in two urine specimens (Specimens 3 and 4) and samples to which an HSA stock solution was added in an amount such that the HSA concentration became 87.4 mg / dl and 262.2 mg / dl was quantified in the same manner as in Example 1. The measurement results are summarized in the following table.

[0046] [Table 6]

[0047] 2-3. Calculation of recovery rate Based on the total protein concentration of each sample measured using each reagent, the recovery rate was calculated according to the above formula. The calculated recovery rates of each sample are summarized in the following table.

[0048] [Table 7] As described above, compared with the case of measurement using the reagent of Comparative Example 1 with a benzoic acid sodium concentration of 0 mM, for the case of measurement using the reagent of Example 5 containing 2.8 mM benzoic acid sodium, the recovery rate when adding benzoic acid sodium increased by several percentage points, and for the case of measurement using the reagents of Examples 6 to 9 containing 5.6 mM or more of benzoic acid sodium, the recovery rate when adding benzoic acid sodium increased by more than 10%.

Claims

1. A method for quantifying protein in a sample using a complex formed by coordination of a metal and a dye, the absorption wavelength of which shifts when bound to the protein, wherein: The protein is reacted with the complex in the presence of an aromatic carboxylic acid or a salt thereof.

2. A method for improving false high values ​​when quantifying proteins in a sample using a complex formed by coordination of a metal and a dye whose absorption wavelength shifts when bound to a protein, wherein: The protein is reacted with the complex in the presence of an aromatic carboxylic acid or a salt thereof.

3. The method according to claim 1 or 2, wherein: The aromatic carboxylic acid or its salt is a monocyclic or bicyclic aromatic carboxylic acid having two or less carboxyl groups and no other substituents, or the aromatic ring is substituted by a sulfonic acid group, a hydroxyl group, a halogen or an alkyl group having 1 to 4 carbon atoms.

4. The method according to claim 1 or 2, wherein: The aromatic carboxylic acid or its salt includes one or more selected from benzoic acid, hydroxybenzoic acid, aminobenzoic acid, salicylic acid, phthalic acid and salts thereof.

5. The method according to claim 4, wherein: The aromatic carboxylic acid or its salt includes one or more selected from benzoic acid, hydroxybenzoic acid, salicylic acid, phthalic acid and salts thereof.

6. The method according to any one of claims 1 to 5, wherein: The aromatic carboxylic acid or a salt thereof is contained in the reaction solution at a concentration of 4.2 mM or more.

7. The method according to any one of claims 1 to 6, wherein: The protein is reacted with the complex in the presence of the aromatic carboxylic acid or a salt thereof and: (1) an aliphatic amine or a salt thereof (R—NH—R) having at least one substituent selected from an alkyl group having 1 to 4 carbon atoms, a hydroxyamino group having 1 to 4 carbon atoms, and an aminoalkyl group having 1 to 4 carbon atoms, (2) an aniline derivative or a salt thereof having at least one substituent selected from the group consisting of a sulfonic acid group, a hydroxyl group, a sulfonylamino group, a halogen group, an alkyl group having 1 to 4 carbon atoms, a hydroxyamino group having 1 to 4 carbon atoms, and an amino group (R-NH-substituted or unsubstituted phenyl or phenylene-R), (3) hydrazine or a hydrazine derivative substituted with an alkyl group having 1 to 4 carbon atoms, (4) thiourea or thiourea derivatives in which thiourea is substituted with an alkyl group having 1 to 4 carbon atoms, (5) polyols, and / or (6) Chelating agent.

8. The method according to any one of claims 1 to 7, wherein: The pigment is pyrogallol red or pyrogallol violet, and the metal is molybdenum, tin or iron.

9. The method according to any one of claims 1 to 8, wherein: The sample is urine or spinal fluid.

10. A reagent for measuring protein, comprising a complex formed by coordination of a metal and a dye, and an aromatic carboxylic acid or a salt thereof, wherein the absorption wavelength of the complex shifts when it binds to a protein.

11. The protein assay reagent according to claim 10, wherein The aromatic carboxylic acid or its salt is a monocyclic or bicyclic aromatic carboxylic acid having two or less carboxyl groups and no other substituents, or the aromatic ring is substituted by a sulfonic acid group, a hydroxyl group, a halogen or an alkyl group having 1 to 4 carbon atoms.

12. The protein assay reagent according to claim 10, wherein The aromatic carboxylic acid or its salt includes one or more selected from benzoic acid, hydroxybenzoic acid, aminobenzoic acid, salicylic acid, phthalic acid and salts thereof.

13. The protein assay reagent according to claim 12, wherein The aromatic carboxylic acid or its salt includes one or more selected from benzoic acid, hydroxybenzoic acid, salicylic acid, phthalic acid and salts thereof. 14 . The protein assay reagent according to claim 10 , comprising the aromatic carboxylic acid or a salt thereof at a concentration of 4.2 mM or more.

15. The protein assay reagent according to any one of claims 10 to 14, wherein The pigment is pyrogallol red or pyrogallol violet, and the metal is molybdenum, tin or iron.

16. The protein assay reagent according to any one of claims 10 to 15, further comprising: (1) an aliphatic amine (R—NH—R) having at least one substituent selected from an alkyl group having 1 to 4 carbon atoms, a hydroxyamino group having 1 to 4 carbon atoms, and an aminoalkyl group having 1 to 4 carbon atoms, or a salt thereof; (2) aniline, aniline derivatives in which aniline is substituted with at least one substituent selected from the group consisting of hydroxyl, hydroxyl, sulfonylamino, halogen, alkyl having 1 to 4 carbon atoms, hydroxyamino having 1 to 4 carbon atoms, and amino (R-NH-substituted or unsubstituted phenyl or phenylene residue -R), or salts thereof, (3) hydrazine, hydrazine derivatives (R-NH-NH2) in which hydrazine is substituted with an alkyl group having 1 to 4 carbon atoms, or salts thereof, (4) thiourea, thiourea derivatives in which thiourea is substituted with an alkyl group having 1 to 4 carbon atoms (R-NH-C(=S)-N(-R)-R), or salts thereof, (5) polyols, and / or (6) Chelating agent. 17 . The protein measurement reagent according to claim 10 , which is used for measuring protein in urine or a sample derived from urine.

18. A method for producing a protein assay reagent, comprising: Dissolve the following in buffer: A pigment selected from catechol purple, pyrogallol red, bromopyrogallol red, o-hydroxyhydroquinone phthalide and galloin, Oxygen-containing salts, halides, complex salts or organic or inorganic acid salts of metals selected from molybdenum, tin and iron, and Aromatic carboxylic acid or its salt.

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