Method for measuring TRACP-5b and kit therefor

Abstract

The present invention provides a method for measuring TRACP-5b, which is capable of selectively detecting TRACP-5b while further reducing the influence of coexisting TRACP-5a. The reaction to detect TRACP-5b is carried out in the presence of an organic sulfonic acid, a sulfate ester, or a salt thereof having a sulfo group (-S(O)2OH), a sulfate group (-OS(O)2OH), and / or a salt thereof at a density of 6.5 mmol / g or more.

Description

[Technical field]

[0001] The present invention relates to a method for measuring TRACP-5b and a kit therefor, and more specifically to a method for selectively measuring TRACP-5b without being affected by TRACP-5a and a kit therefor. [Background technology]

[0002] Acid phosphatase (ACP) is an enzyme that has hydrolytic activity of organic monophosphate esters under acidic conditions. ACP has multiple isozymes and is distributed in various tissues. ACPs present in serum are known to be derived from platelets, erythrocytes, prostates, osteoclasts, macrophages, etc. These isozymes are separated by polyacrylamide gel electrophoresis, and ACPs derived from osteoclasts and macrophages are located fifth from the origin and are resistant to tartaric acid, so they are also called Band-5 tartrate-resistant acid phosphatase (TRACP-5) (Patent Document 5, Lam WKW et al., Clin. Chem. 24(7):1105-1108, 1978 and Lam WKW et al., Clin. Biochem. 14,177-181, 1981, etc.). TRACP-5 is classified into TRACP-5a, which has a high degree of sialic acid binding to the glycan, and TRACP-5b, which has a low degree of sialic acid binding, with 5a derived from macrophages and 5b derived from osteoclasts. Since the concentration of TRACP-5b in serum sensitively reflects the activity of osteoclasts, it is used as a bone resorption marker to aid in the diagnosis of metabolic bone diseases and bone metastatic cancer and to evaluate the effectiveness of treatment. Therefore, a method for selectively measuring TRACP-5b from among ACPs in serum is being investigated.

[0003] Conventionally, the method for measuring TRACP-5 involves performing an enzyme reaction using a synthetic substrate such as p-nitrophenyl phosphate in the presence of tartaric acid, and measuring the resulting reaction product by colorimetry to determine the enzyme activity. However, although tartaric acid can inhibit prostate-derived ACP, TRACP-5a, red blood cell-derived ACP, and platelet-derived ACP are resistant to tartaric acid, so this method also measures these isoenzymes and cannot specifically measure TRACP-5b.

[0004] As a method for improving the TRACP-5b specificity of the above method, a method is known in which serum is diluted 5-fold, incubated at 37° C. for 1 hour, and then the enzyme activity is measured in the same manner (Non-Patent Document 3). In this method, the effects of prostate-derived ACP as well as red blood cell-derived ACP can be avoided, but the effects of TRACP-5a and platelet-derived ACP cannot be avoided.

[0005] Another method for specifically measuring TRACP-5b activity is to calculate the activity of TRACP-5b by subtracting the ACP activity measured in the presence of tartaric acid and fluoride from the ACP activity measured in the presence of tartaric acid (Patent Document 1). In this method, the influence of red blood cell-derived and platelet-derived ACPs can be avoided by utilizing the difference in sensitivity to fluoride. However, the influence of TRACP-5a cannot be eliminated, and the activity of TRACP-5b is calculated by subtraction, which has a problem in terms of accuracy.

[0006] As another method, a method for specifically measuring TRACP-5b activity using acid mucopolysaccharides as inhibitors of acid phosphatases other than TRACP-5b has been reported (Patent Document 2). However, the inhibitory effect of acid mucopolysaccharides on TRACP-5a activity is not sufficient, and there is a need to further improve the specificity for TRACP-5b.

[0007] As a method for measuring the activity of TRACP-5b using a chemiluminescent substrate, a method is known in which TRACP-5b is reacted with a chemiluminescent 1,2-dioxetane compound under acidic conditions, and then the reaction solution is made alkaline to measure the luminescence intensity (Patent Document 3). In addition, a method has been reported in which a specific buffer such as MES is used during the enzyme reaction, or manganese ions or cobalt ions are added to increase the luminescence intensity ratio of TRACP-5b to TRAPC-5a, thereby specifically measuring TRACP-5b (Patent Document 4). However, these methods are limited to activity measurements using chemiluminescent substrates, and there is a demand for a cheaper method to specifically measure TRACP-5b.

[0008] As an example of an immunoassay method for TRACP-5b using an antibody, there is a method in which TRACP-5a and TRACP-5b are bound to each other using an antibody specific to TRACP-5, and then TRACP-5b is specifically measured by utilizing the difference in the optimum pH between the two (Patent Document 5). However, the difference in the optimum pH alone is not sufficient to provide specificity for TRACP-5b, and the amount of TRACP-5b is calculated by subtraction, which has a problem in terms of accuracy.

[0009] In addition, there is a method for more specifically measuring TRACP-5b by using an antibody having a certain selectivity for TRACP-5b and then measuring the activity using 2-chloro-4-nitrophenyl phosphate as a color-developing substrate after antibody binding (Patent Documents 6 and 7). However, it is desired to further improve the TRACP-5b selectivity of this antibody as well.

[0010] There is also a method for quantifying the amount of TRACP-5b protein using two monoclonal antibodies with high specificity to TRACP-5b (Patent Document 8). [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Patent Publication No. 10-337198 [Patent Document 2] Patent Publication No. 2001-231595 [Patent Document 3] Patent Publication No. 2011-024470 [Patent Document 4] Patent Publication No. 2022-174015 [Patent Document 5] Special table 2002-510050 [Patent Document 6] Patent Publication No. 2004-026805 [Patent Document 7] WO2004 / 077059 [Patent Document 8] WO2016 / 027697 [Non-patent literature]

[0012] [Non-Patent Document 1] Non-patent literature Clin.Chem.33:458-462.1987 Summary of the Invention [Problem to be solved by the invention]

[0013] In contrast to the above-mentioned conventional techniques, an object of the present invention is to provide a measurement method capable of selectively detecting TRACP-5b while further reducing the influence of coexisting TRACP-5a in the measurement of TRACP-5b. [Means for solving the problem]

[0014] The inventors have investigated a measurement method capable of selectively detecting TRACP-5b and have found that by adding a compound having a sulfo group (-S(O)2OH), a sulfate group (-OS(O)2OH) and / or their salts at a certain density or higher to a reaction system in which TRACP-5a may be present, the influence of TRACP-5a can be significantly reduced and TRACP-5b can be selectively detected.

[0015] That is, the present invention provides the following methods for measuring TRACP-5b and kits for measuring TRACP-5b. [1] A method for measuring tartrate-resistant acid phosphatase 5b (TRACP-5b) in a sample, comprising: A method for measuring TRACP-5b, comprising a step of carrying out a reaction to detect TRACP-5b in the presence of an organic sulfonic acid, a sulfate ester, or a salt thereof having a sulfo group (-S(O)2OH), a sulfate group (-OS(O)2OH), and / or a salt thereof at a density of 6.5 mmol / g or more.

[0016] [2] A method for measuring tartrate-resistant acid phosphatase 5b (TRACP-5b) in a sample, comprising: Sulfo group (-S(O)2OH), sulfate group (-OS(O)2OH) and / or their salts are added to each unit structure as S(O)2O - A method for measuring TRACP-5b, comprising a step of carrying out an enzyme reaction or an antigen-antibody reaction in the presence of an organic sulfonic acid, a sulfate ester and / or a salt thereof having a converted value of 50% by mass or more; or The organic sulfonic acid, sulfate ester, or salt thereof has a sulfo group (-S(O)2OH), a sulfate group (-OS(O)2OH), and / or a salt thereof per unit structure, - The method according to [1], wherein the content is 50 mass% or more in terms of the converted value.

[0017] [3] The organic sulfonic acid, sulfate ester, or salt thereof is represented by the formula (1): [ka] (In formula (1), R is an alkyl, alkenyl, or alkynyl group having 1 to 3 carbon atoms, Y is a bond or an oxygen atom, and X is hydrogen or an alkali metal.) Is it expressed as Formula (2): [ka] (In formula (2), Y is a bond or an oxygen atom, n is an integer of 2 to 5000, and X is hydrogen or an alkali metal.) or having a repeat unit of or Formula (3): [ka] (In formula (3), Y is O or CH2, and R 1 ~R 5 Among these, two are O- or bonds, and at least two of the remaining are OS(O)2OX or S(O)2OX (X is hydrogen or an alkali metal). If none of these apply, it is OX or C(O)OX (X is hydrogen or an alkali metal). The method according to [1] or [2], having a repeating unit comprising the structure:

[0018] [4] The method according to [1] or [2], wherein the organic sulfonic acid, sulfate ester, or salt thereof is vinyl sulfonic acid or a salt thereof, vinyl sulfate or a salt thereof, polyvinyl sulfonic acid or a salt thereof, polyvinyl sulfate or a salt thereof, methanesulfonic acid or a salt thereof, or dextran sulfate or a salt thereof.

[0019] [5] The method according to any one of [1] to [4], wherein the organic sulfonic acid, sulfate ester or salt thereof is 1) added independently to a sample or a reaction system, or is contained in 2) a sample dilution solution, 3) a substrate-containing reagent, or 4) a reagent containing an anti-TRACP-5b antibody (typically an anti-TRACP-5b monoclonal antibody), thereby causing a reaction to detect TRACP-5b in the presence of the organic sulfonic acid, sulfate ester or salt thereof.

[0020] [6] The method according to any one of [1] to [5], in which the antibody is measured by an enzyme method, an enzyme immunoassay (EIA), an enzyme-linked immunosorbent assay (ELISA), a chemiluminescent enzyme immunoassay (CLEIA), a chemiluminescent immunoassay (CLIA), or a latex agglutination method. [7] The method according to any one of [1] to [6], wherein the sample is blood, serum or plasma.

[0021] [8] A kit for measuring TRACP-5b, comprising: A kit comprising an organic sulfonic acid, a sulfate ester, and / or a salt thereof having a sulfo group (-S(O)2OH), a sulfate group (-OS(O)2OH), and / or a salt thereof at a density of 6.5 mmol / g or more.

[0022] [9] A kit for measuring TRACP-5b, comprising: Per unit structure, sulfo group (-S(O)2OH), sulfate group (-OS(O)2OH) or their salts are - A kit containing organic sulfonic acids, sulfates and / or salts thereof, with a converted value of 50% by mass or more; or The organic sulfonic acid, sulfate ester and / or salt thereof may have a sulfo group (-S(O)2OH), a sulfate group (-OS(O)2OH) or a salt thereof per unit structure, - The kit according to [8], having a converted value of 50% by mass or more.

[0023]

[10] The organic sulfonic acid, sulfate ester and / or salt thereof may be represented by the formula (1): [ka] (In formula (1), R is an alkyl, alkenyl, or alkynyl group having 1 to 5 carbon atoms, Y is a bond or an oxygen atom, and X is hydrogen or an alkali metal.) Is it expressed as Formula (2): [ka] (In formula (2), Y is a bond or an oxygen atom, n is an integer of 2 to 5000, and X is hydrogen or an alkali metal.) or Formula (3): [ka] (In formula (3), Y is O or CH2, and R 1 ~R 5 Among these, two are O- or bonds, and at least two of the remaining are OS(O)2OX or S(O)2OX (X is hydrogen or an alkali metal). If none of these apply, it is OX or C(O)OX (X is hydrogen or an alkali metal). The repeat structure includes the structure The kit according to [8] or [9].

[0024]

[11] The kit described in [8] or [9], wherein the organic sulfonic acid, sulfate ester, or salt thereof is vinyl sulfate or a salt thereof, vinyl sulfonic acid or a salt thereof, polyvinyl sulfonic acid or a salt thereof, polyvinyl sulfate or a salt thereof, methanesulfonic acid or a salt thereof, or dextran sulfate or a salt thereof.

[0025]

[12] The kit according to any one of [8] to

[11] , for measuring TRACP-5b by an enzymatic method, A kit comprising a substrate for TRACP-5b, the organic sulfonic acid, sulfate ester or salt thereof, and optionally a reagent for diluting a sample.

[0026]

[13] The kit according to

[12] , wherein the reagent containing a substrate for TRACP-5b or the sample dilution reagent contains the organic sulfonic acid, sulfate ester, or a salt thereof.

[0027]

[14] The kit according to any one of [8] to

[11] , which measures TRACP-5b by enzyme immunoassay (EIA), A kit comprising a solid support, an anti-TRACP-5b monoclonal antibody adsorbed to the solid support, a substrate for TRACP-5b, the organic sulfonic acid, sulfate ester or salt thereof, optionally a reagent for diluting a sample, and optionally a washing solution.

[15] The kit according to

[14] , wherein the reagent containing the anti-TRACP-5b monoclonal antibody, the reagent containing a substrate for TRACP-5b, or the sample dilution reagent contains the organic sulfonic acid, sulfate ester, or a salt thereof.

[0028]

[16] The kit according to any one of [8] to

[11] , which measures TRACP-5b by enzyme-linked immunosorbent assay (ELISA), A kit comprising a solid support, a primary anti-TRACP-5b monoclonal antibody adsorbed to the solid support, a secondary anti-TRACP-5b monoclonal antibody that binds to TRACP-5b via an epitope different from that of the primary anti-TRACP-5b monoclonal antibody and is labeled with a substance that enables detection by enzyme reaction, a reagent for detecting the labeled substance by enzyme reaction, the organic sulfonic acid, sulfate ester or salt thereof, optionally a reagent for diluting a sample, and optionally a washing solution.

[17] The kit described in

[16] , wherein the organic sulfonic acid, sulfate ester, or salt thereof is contained in a reagent containing the secondary anti-TRACP-5b monoclonal antibody, a reagent for detecting the labeled substance by an enzymatic reaction, or a reagent for diluting the sample.

[0029]

[18] The kit according to any one of [8] to

[11] , which measures TRACP-5b by chemiluminescence enzyme immunoassay (CLEIA), A kit comprising a solid support, an anti-TRACP-5b monoclonal antibody adsorbed to the solid support, a chemiluminescent substrate for TRACP-5b, the organic sulfonic acid, sulfate ester or salt thereof, optionally a reagent for diluting a sample, and optionally a washing solution.

[19] The kit described in

[18] , wherein the organic sulfonic acid, sulfate ester, or salt thereof is contained in a reagent containing a chemiluminescent substrate for TRACP-5b, a reagent containing an anti-TRACP-5b monoclonal antibody, or a sample dilution reagent.

[0030]

[20] The kit according to any one of [8] to

[11] , which measures TRACP-5b by a chemiluminescence immunoassay (CLIA method), A kit comprising a solid support, a primary anti-TRACP-5b monoclonal antibody adsorbed to the solid support, a secondary anti-TRACP-5b monoclonal antibody that binds to TRACP-5b via an epitope different from that of the primary anti-TRACP-5b monoclonal antibody and is labeled with a substance that enables detection by chemiluminescence, a reagent for detecting the labeled substance by chemiluminescence, the organic sulfonic acid, sulfate ester or salt thereof, optionally a reagent for diluting a sample, and optionally a washing solution.

[21] The kit described in

[20] , wherein the organic sulfonic acid, sulfate ester, or salt thereof is contained in a reagent comprising the primary anti-TRACP-5b monoclonal antibody, a reagent comprising the secondary anti-TRACP-5b monoclonal antibody, a reagent for detecting the labeled substance by chemiluminescence, or a reagent for diluting the sample.

[0031]

[22] The kit according to any one of [8] to

[11] , wherein TRACP-5b is measured by a latex agglutination method. A kit comprising latex particles, an anti-TRACP-5b antibody adsorbed to the latex particles, the organic sulfonic acid, sulfate ester or salt thereof, and optionally a reagent for diluting a sample.

[23] The kit according to

[22] , wherein the reagent containing the anti-TRACP-5b antibody or the sample dilution reagent contains the organic sulfonic acid, sulfate ester, or a salt thereof.

[0032]

[24] The kit according to any one of [8] to

[23] , wherein the sample to be measured is blood, serum or plasma. Effect of the Invention

[0033] According to the present application, tartrate-resistant acid phosphatase 5 a (TRACP-5 a The present invention provides a means for selectively detecting TRACP-5b while reducing the influence of TRACP-5b. This is therefore useful for diagnosing diseases using TRACP-5b as a marker. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present invention will be described in detail below with reference to the preferred embodiments. However, the present invention should not be understood as being limited to the following embodiments.

[0035] In one embodiment, the present invention provides a method for measuring tartrate-resistant acid phosphatase 5b (TRACP-5b) in a sample, comprising a step of carrying out a detection reaction for TRACP-5b in the presence of a specific organic sulfonic acid, sulfate ester, or a salt thereof. In another embodiment, the present invention provides a kit for measuring TRACP-5b, which comprises a specific organic sulfonic acid, sulfate ester, or a salt thereof. These embodiments are described in detail below.

[0036] 1. How to measure TRACP-5b 1-1. Organic sulfonic acids, sulfates, or their salts In this measurement method, TRACP-5a is inhibited by the presence of an organic sulfonic acid, a sulfate ester, or a salt thereof having a sulfo group (-S(O)2OH), a sulfate group (-OS(O)2OH), and / or a salt thereof at a density of 6.5 mmol / g or more in the reaction system, thereby enabling the selective measurement of TRACP-5b. As shown in the examples described below, the effect of inhibiting TRACP-5a increases depending on the density of the sulfo group, sulfate group, or salt thereof, and in a preferred embodiment, the organic sulfonic acid, sulfate ester, or salt thereof has a sulfo group, sulfate group, and / or salt thereof at a density of 7.5 mmol / g or more, more preferably at a density of 8.5 mmol / g or more.

[0037] In a preferred embodiment, the organic sulfonic acid, sulfate ester, or salt thereof has a sulfo group, sulfate group, and / or salt thereof per unit structure, as represented by S(O)O - In terms of converted value, it has 50% by mass or more, more preferably 60% by mass or more, and further preferably 70% by mass or more. Here, when used in the present specification, the term "unit structure" refers to the structure of the repeating unit when the target compound has a repeating structure, and refers to the structure of the entire compound when the target compound does not have a repeating structure. In addition, when the unit structure has a salt of a sulfo group (-S(O)2OH) or a sulfate group (-OS(O)2OH), the molecular weight of the unit structure is calculated as the sulfo group (-S(O)2OH) or the sulfate group (-OS(O)2OH).

[0038] A specific embodiment of such an organic sulfonic acid, sulfate ester, or salt thereof is Formula (1): [ka] Examples of the compound include compounds represented by the following formula: In formula (1), R is an alkyl, alkenyl, or alkynyl group having 1 to 3 carbon atoms, and is preferably an alkenyl group having 1 to 3 carbon atoms. Y is a bond or an oxygen atom, preferably a bond. X is hydrogen or an alkali metal, preferably hydrogen or Na. Exemplary compounds of this embodiment include, for example, methanesulfonic acid or a salt thereof (eg, the Na salt), vinylsulfonic acid or a salt thereof (eg, the Na salt), or vinyl sulfate or a salt thereof.

[0039] Other specific embodiments of the above-mentioned organic sulfonic acids, sulfates or salts thereof include: Formula (2): [ka] Examples of the compound include a compound having a repeating unit of the formula: In formula (2), Y is a bond or an oxygen atom, and is preferably a bond. n is an integer of 2 to 5,000, preferably 10 to 500, and more preferably 20 to 100. X is hydrogen or an alkali metal, preferably hydrogen or Na. Preferred examples of the compound of this embodiment include, for example, polyvinyl sulfonic acid or a salt thereof (e.g., Na salt) or polyvinyl sulfate or a salt thereof (e.g., Na salt), more preferably, a compound having the following repeating structure: [ka] (In formula (2-1), X is hydrogen or an alkali metal, and preferably hydrogen or Na.) Examples of such polyvinyl sulfonic acids or salts thereof include polyvinyl sulfonic acids or salts thereof having a molecular weight of 2 to 5,000, more preferably 10 to 500, and further preferably 20 to 100. Such polyvinyl sulfonic acids or salts thereof usually have a molecular weight of 200 to 500,000, preferably a molecular weight of 1,000 to 50,000, and more preferably a molecular weight of 4,000 to 6,000. In this specification, the term "molecular weight" or "average molecular weight" refers to the weight average molecular weight (Mw) unless otherwise specified. The weight average molecular weight (Mw) refers to a value measured by gel permeation chromatography (GPC) using a liquid chromatograph.

[0040] Further specific embodiments of the above-mentioned organic sulfonic acid, sulfate ester, or salt thereof include: Formula (3): [ka] Examples of the compound include a compound having a repeating unit containing the structure: In formula (3), Y is O or CH2, and preferably O. R 1 ~R 5 Among these, two are O- or a bond, and at least two of the remaining are OS(O)OX or S(O)OX (X is hydrogen or an alkali metal (e.g., Na)). Any other than these is OX or C(O)OX (X is hydrogen or an alkali metal (e.g., Na), preferably C(O)OX (X is hydrogen or an alkali metal (e.g., Na). Preferably, R 1 ~R 5 Of these, two are O- or a bond, and the remaining one is OS(O)OX (wherein X is hydrogen or an alkali metal (e.g., Na).

[0041] In addition, examples of repeating units containing this structure include: [ka] Examples include: In formula (3-1), Y is O or CH2, and preferably O. L 1 and L 4 are each independently O or a bond, and preferably, one is O- and the other is a bond. L2 and L 3 are each independently O, CH2 or a bond, and preferably, one is O and the other is CH2 or a bond. R 1 ~R 3 At least two of the groups are independently OS(O)OX or S(O)OX (X is hydrogen or an alkali metal (e.g., Na)), and the remaining group, if present, is OX or C(O)OX (X is hydrogen or an alkali metal (e.g., Na), preferably C(O)OX (X is hydrogen or an alkali metal (e.g., Na). R 1 ~R 3 are preferably each independently OS(O)OX or S(O)OX (X is hydrogen or an alkali metal (e.g., Na), more preferably each independently OS(O)OX (X is hydrogen or an alkali metal (e.g., Na). R 4 ~R 6 At least two of the groups are independently OS(O)OX or S(O)OX (X is hydrogen or an alkali metal (e.g., Na)), and the remaining group, if present, is OX or C(O)OX (X is hydrogen or an alkali metal (e.g., Na), preferably C(O)OX (X is hydrogen or an alkali metal (e.g., Na). R 4 ~R 6 are preferably each independently OS(O)OX or S(O)OX (X is hydrogen or an alkali metal (e.g., Na), more preferably each independently OS(O)OX (X is hydrogen or an alkali metal (e.g., Na). n is an integer of 2 to 1,000, preferably an integer of 3 to 700, more preferably an integer of 4 to 200, and further preferably an integer of 5 to 70.

[0042] A preferred example of the compound of this embodiment is dextran sulfate or a salt thereof (e.g., Na), more preferably having the following repeating structure: [ka] (In formula (3-2), X is hydrogen or an alkali metal (e.g., Na).) Examples of such dextran sulfate or a salt thereof (e.g., Na) include dextran sulfate or a salt thereof having a molecular weight of 2 to 1000, preferably 3 to 700, and more preferably 5 to 70. Such dextran sulfate or a salt thereof usually has a molecular weight of 1500 to 1000000, preferably a molecular weight of 2000 to 550000, and more preferably a molecular weight of 4000 to 55000.

[0043] In the method of the present invention, the above-mentioned organic sulfonic acid, sulfuric acid ester or their salts are present in the reaction system, thereby modifying the coexisting TRACP-5a antigen characteristics and inhibiting its activity.These TRACP-5a inhibitors can be present in the reaction system in various forms, for example, these TRACP-5a inhibitors or their solutions can be added separately to the reaction solution for detecting TRACP-5b, or can be dissolved in a sample dilution solution and added to the measurement sample, or can be added to a reagent containing an enzyme substrate or a reagent containing anti-TRACP-5b antibody according to the reaction system, and can be used to carry out a reaction (for example, an enzyme reaction or an antigen-antibody reaction).

[0044] As shown in the examples below, the above-mentioned organic sulfonic acid, sulfate ester or salt thereof increases TRACP-5a inhibitory activity depending on the density and average molecular weight of the sulfo group, sulfate group, and salt thereof of these compounds, and the lower limit of the concentration of these compounds in the reaction solution required to exhibit TRACP-5a inhibitory activity decreases depending on the density and average molecular weight.For any compound, when the concentration in the reaction solution is increased from this lower limit, the inhibitory activity increases depending on the concentration within a certain range, but when the concentration reaches a certain level or more, the inhibitory activity does not increase depending on the concentration and maintains a certain level, and when the concentration reaches a certain level, the inhibitory activity gradually decreases.However, even when the inhibitory activity reaches a plateau, it still maintains a high inhibitory activity in a wide range of concentrations, and in practice, it is often used at a concentration higher than the concentration at which the inhibitory activity does not increase depending on the concentration in the reaction solution. Therefore, it is preferable to determine the concentration of the above-mentioned organic sulfonic acid, sulfate ester or salt thereof in the reaction solution taking these points into consideration.

[0045] For example, when dextran sulfate or its salt having an average molecular weight of 4000 to 6000 is used, the concentration in the reaction solution is 0.03 × 10 -3 Up to 10.0% by mass is preferable, and 0.15×10 -3 More preferably, the percentage is 0.3×10 -3 In addition, when dextran sulfate or a salt thereof having a molecular weight of 40,000 to 60,000 is used, the concentration in the reaction solution is preferably 0.03×10 -4 Up to 5.0% by mass is preferable, and 0.15×10 -4 More preferably, the percentage is 0.5×10 -4 In addition, when dextran sulfate or a salt thereof having a molecular weight of 400,000 to 600,000 is used, the concentration in the reaction solution is preferably 0.01×10 -5 Up to 1.0% by mass is preferable, and 0.3×10 -4 More preferably, up to 0.1% by mass, and up to 0.5×10 -5 It is more preferably up to 0.05% by mass.

[0046] When vinyl sulfonic acid or a salt thereof is used, the concentration in the reaction solution is preferably 0.012 to 15.0% by mass, more preferably 0.020 to 5.0% by mass, and even more preferably 0.10 to 3.0% by mass. When polyvinyl sulfonic acid or a salt thereof having an average molecular weight of 4000 to 6000 is used, the concentration in the reaction solution is preferably 0.08×10 -3 Preferably, the content is 0.3×10 -3 More preferably, the percentage is 0.3×10 -2 It is more preferably up to 0.3% by mass.

[0047] In the measurement method, TRACP-5a is inhibited by making the above-mentioned organic sulfonic acid, sulfate ester or their salt present in a reaction system for detecting TRACP-5b. Examples of this "reaction system" include enzyme reactions and antigen-antibody reactions, and the TRACP-5a inhibitor used in the method of the present invention can be used in any of these reaction systems.

[0048] In the present specification, the term "enzyme reaction" refers to the reaction between TRACP-5b and its substrate, and examples of the substrate of TRACP-5b include, for example, p-nitrophenyl phosphate or a salt thereof, 2-chloro-4-nitrophenyl phosphate or a salt thereof, 1,2-dioxetane derivatives, acridan derivatives, and 4-methylumbelliferyl phosphate. In addition, in the present specification, the term "antigen-antibody reaction" refers to the reaction between TRACP-5b and an antibody against it, and examples of the antibody against TRACP-5b include, for example, a monoclonal antibody, a polyclonal antibody, and an antigen-binding fragment. Examples of the monoclonal antibody or an antigen-binding fragment thereof include the anti-TRACP-5b monoclonal antibody or an antigen-binding fragment thereof described in Patent Documents 6 and 7, and more specifically, examples of the antibody or an antigen-binding fragment thereof produced by the hybridoma with Accession No. IPOD FERM BP-7889, Accession No. IPOD FERM BP-8249, or Accession No. IPOD FERM BP-7890 are included.

[0049] There are various methods utilizing enzyme reactions and antigen-antibody reactions, including, for example, the enzyme method, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescence enzyme immunoassay (CLEIA), chemiluminescence immunoassay (CLIA), and latex agglutination assay.

[0050] In the present specification, the enzyme method is a method for measuring the enzyme activity of TRACP-5b, for example, by reacting TRACP-5b in a sample with an enzyme substrate of TRACP-5b, such as P-nitrophenyl phosphate or its salt, to measure the enzyme activity of TRACP-5b. More specifically, a chromogenic substrate solution containing tartaric acid or its salt (e.g., 2-chloro-4-nitrophenyl phosphate solution) is added to the sample to initiate the enzyme reaction, and the enzyme activity is determined from the change in absorbance during a specific time period. In this enzyme method, the above-mentioned organic sulfonic acid, sulfate ester, or salt thereof may be added independently to the reaction solution, to the sample, or to the chromogenic substrate solution.

[0051] In the present specification, enzyme immunoassay (EIA) is an immunoassay that uses the enzyme activity of TRACP-5b to detect TRACP-5b. For example, TRACP-5b in a sample is bound to an anti-TRACP-5b monoclonal antibody, and the bound TRACP-5b is reacted with an enzyme substrate of TRACP-5b, such as p-nitrophenyl phosphate or its salt, to measure the enzyme activity of TRACP-5b. More specifically, the sample is first contacted with an anti-TRACP-5b monoclonal antibody adsorbed on a solid support, and TRACP-5b in the sample is bound to the immobilized antibody. Next, the solid support is washed with a washing solution to remove components derived from the sample that are not adsorbed to the antibody, and then an enzyme substrate of TRACP-5b, such as p-nitrophenyl phosphate or its salt, is added to the reaction system, and the TRACP-5b bound to the antibody is reacted with the substrate. After stopping the enzyme reaction with a reaction stop solution, the absorbance of phenols generated by the reaction, such as p-nitrophenol, is measured at a predetermined wavelength, for example, 390 nm to 450 nm, preferably 400 to 430 nm. The magnitude of the absorbance reflects the TRACP-5b enzyme activity, and the TRACP-5b in the sample can be measured from the absorbance value. In this enzyme immunoassay (EIA), the above-mentioned organic sulfonic acid, sulfate ester, or salt thereof may be added independently to the support, to the sample or sample dilution solution, to a reagent containing an anti-TRACP-5b monoclonal antibody, or to a color-developing substrate solution.

[0052] In the present specification, enzyme-linked immunosorbent assay (ELISA) is a method for measuring TRACP-5b by using two different antibodies against TRACP-5b, one of which is labeled with a substance that allows detection by enzyme reaction, and measuring the activity of the enzyme. For example, a sample is contacted with a first anti-TRACP-5b monoclonal antibody adsorbed on a solid support to bind TRACP-5b in the sample to the first antibody, and then a second anti-TRACP-5b monoclonal antibody that binds to TRACP-5b at an epitope different from that of the first antibody and is labeled with a substance that allows detection by enzyme reaction is contacted with the immobilized TRACP-5b to bind the second antibody to TRACP-5b, and RACP-5b is measured by enzyme reaction using the label of the second antibody. More specifically, first, a primary anti-TRACP-5b monoclonal antibody is adsorbed on a solid support, and a specimen is contacted with the immobilized primary antibody to allow TRACP-5b in the specimen to bind to the primary antibody.Then, the solid support is washed with a washing solution to remove the components derived from the specimen that are not adsorbed to the antibody, and then the immobilized TRACP-5b is contacted with a substance that enables detection by enzyme reaction, such as an enzyme or a secondary anti-TRACP-5b monoclonal antibody or polyclonal antibody that is labeled with a different epitope from the primary antibody and binds to TRACP-5b, allowing the secondary antibody to bind to TRACP-5b.Then, from the label of the secondary antibody, for example, biotin is reacted with peroxidase-labeled streptavidin, followed by a peroxidase enzyme reaction, or by reacting the labeling enzyme with its substrate, etc., to generate a color-developing substance, and the absorbance of this color-developing substance is measured, thereby measuring TRACP-5b. Enzymes used as substances that enable detection by enzymatic reactions include peroxidase, alkaline phosphatase, etc., and substances that enable detection by enzymatic reactions other than enzymes include avidin and substrates for various enzymes. When avidin is used, ABC color development can be utilized. In this enzyme-linked immunosorbent assay (ELISA), the above-mentioned organic sulfonic acid, sulfate ester or salt thereof may be added independently to the support, to the specimen or specimen dilution solution, to a reagent containing a primary antibody and / or a secondary antibody, or to a color-developing substrate solution.

[0053] In the present specification, the chemiluminescence enzyme immunoassay (CLEIA method) is an immunoassay method for detecting the enzyme activity of TRACP-5b by chemiluminescence, for example, a specimen is contacted with an anti-TRACP-5b monoclonal antibody adsorbed on a solid support such as beads to bind TRACP-5b in the specimen to the antibody, a chemiluminescence substrate for TRACP-5b is contacted with the immobilized TRACP-5b, and the generated chemiluminescence is measured to measure TRACP-5b activity. More specifically, a specimen is first contacted with an anti-TRACP-5b monoclonal antibody adsorbed on a solid support such as magnetic particles, and TRACP-5b in the specimen is bound to the immobilized antibody. Next, the solid support on which the magnetic particles are immobilized is washed with a washing solution to remove components from the sample that were not adsorbed to the antibody, and then a chemiluminescent substrate for TRACP-5b, such as 2-chloro-5-{4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl}phenylphosphate, disodium salt (CDP-Star, registered trademark), is added to the reaction system, and the TRACP-5b bound to the antibody is reacted with the substrate to measure the luminescence intensity. The luminescence intensity reflects the TRACP-5b enzyme activity, and the TRACP-5b in the sample can be measured from the value. In this chemiluminescent enzyme immunoassay (CLEIA), the above-mentioned organic sulfonic acid, sulfate ester, or salt thereof may be added independently to the support, to the specimen or specimen dilution solution, to a reagent containing an anti-TRACP-5b monoclonal antibody, or to a color-developing substrate solution.

[0054] Chemiluminescence enzyme immunoassay (CLEIA) can also be used by contacting a specimen with a primary anti-TRACP-5b monoclonal antibody immobilized on a solid support to allow TRACP-5b in the specimen to bind to the primary antibody, then contacting a secondary anti-TRACP-5b monoclonal antibody, which binds to TRACP-5b at an epitope different from that of the primary antibody and is labeled with a chemiluminescent substrate or a substance that allows detection by chemiluminescence, with the immobilized TRACP-5b to allow TRACP-5b to bind to the secondary antibody, and measuring the luminescence intensity by generating chemiluminescence from or using the label of the secondary antibody. More specifically, first, the primary anti-TRACP-5b monoclonal antibody is adsorbed to a solid support, for example, magnetic particles, and the specimen is contacted with the immobilized primary antibody to allow TRACP-5b in the specimen to bind to the primary antibody. Next, the solid support on which the magnetic particles have been fixed, for example by magnetic force, is washed with a washing solution to remove components from the sample that have not been adsorbed to the antibody, and the fixed TRACP-5b is then contacted with a secondary anti-TRACP-5b monoclonal or polyclonal antibody that is labeled with an enzyme or a substance that can produce chemiluminescence using an enzyme reaction and binds to TRACP-5b at an epitope different from that of the primary antibody, allowing the secondary antibody to bind to TRACP-5b. TRACP-5b can then be measured by producing chemiluminescence from or using the label of the secondary antibody and measuring the luminescence intensity. In this chemiluminescent enzyme immunoassay (CLEIA), the above-mentioned organic sulfonic acid, sulfate ester or salt thereof may be added independently to the support, to the sample, or to a reagent containing a primary antibody and / or a secondary antibody.

[0055] In the present specification, the latex agglutination method is a method for measuring TRACP-5b by measuring the turbidity caused by the agglutination of latex particles as absorbance, for example, by contacting a specimen with an anti-TRACP-5b polyclonal antibody adsorbed to latex particles to cause agglutination of latex particles via TRACP-5b, and measuring the turbidity (absorbance) caused thereby to measure TRACP-5b. In this latex agglutination method, the above-mentioned organic sulfonic acid, sulfuric acid ester, or salt thereof may be added independently to a reaction solution, may be added to a specimen, or may be added to a reagent containing latex particles.

[0056] The sample to be measured for TRACP-5b may be from any living organism, but is usually from a mammal, and in most cases from a human. The sample may be from any tissue or body fluid, but in most cases blood, serum, and plasma are used.

[0057] 2.TRACP-5b measurement kit Another embodiment of the present invention relates to a kit for measuring TRACP-5b, which is characterized in that the kit contains the above-mentioned organic sulfonic acid, sulfate ester and / or salt thereof. Depending on the kit configuration and measurement principle, the organic sulfonic acid, sulfate ester, or salt thereof may be included in the kit 1) as a separate solution, 2) contained in the sample diluent, 3) contained in the substrate-containing reagent, or 4) contained in the reagent containing the anti-TRACP-5b antibody.

[0058] The concentration of the organic sulfonic acid, sulfate ester, or salt thereof in each solution or reagent is preferably adjusted according to the density of the sulfo group (-S(O)OH), sulfate group (-OS(O)OH) and / or salt thereof of the organic sulfonic acid, sulfate ester, or salt thereof, the molecular weight of these compounds, and the measurement protocol (particularly, the dilution factor (volume (vol) of reaction solution / volume (vol) of solution or reagent in which the organic sulfonic acid, sulfate ester, or salt thereof is dissolved or added)) so as to achieve the concentration in the reaction solution described above.

[0059] For example, when using dextran sulfate or its salt with an average molecular weight of 4000 to 6000, the concentration (C) in the solution or reagent is 0.03 × 10 -3 Up to 10.0% by mass is preferable, and 0.15×10 -3 More preferably, the percentage is 0.3×10 -3 In addition, when dextran sulfate or a salt thereof having a molecular weight of 40,000 to 60,000 is used, the concentration in the solution or reagent is preferably 0.03×10 -4 Up to 5.0% by mass is preferable, and 0.15×10 -4 More preferably, the percentage is 0.5×10 -4 In addition, when dextran sulfate or a salt thereof having a molecular weight of 400,000 to 600,000 is used, the concentration (C) in the solution or reagent is preferably 0.01×10 -5 Up to 1.0% by mass is preferable, and 0.3×10 -4 More preferably, up to 0.1% by mass, and up to 0.5×10 -5 It is more preferably up to 0.05% by mass.

[0060] When vinyl sulfonic acid or a salt thereof is used, the concentration (C) in the solution or reagent is preferably 0.012 to 15.0% by mass, more preferably 0.020 to 5.0% by mass, and even more preferably 0.10 to 3.0% by mass. When polyvinyl sulfonic acid or a salt thereof having an average molecular weight of 4000 to 6000 is used, the concentration (C) in the solution or reagent is preferably 0.08×10 -3 Up to 5.0% by mass is preferable, and 0.3×10 -3 More preferably, the percentage is 0.3×10 -2 It is more preferably up to 0.3% by mass.

[0061] As mentioned above, TRACP-5b can be measured by various methods, and TRACP-5b measurement kits also have different reagent compositions depending on the measurement method.

[0062] For example, a kit for measuring TRACP-5b by enzyme method comprises the substrate of TRACP-5b, the above-mentioned organic sulfonic acid, sulfate ester or their salt, and optionally a sample dilution reagent.In addition, in this kit, the above-mentioned organic sulfonic acid, sulfate ester or their salt can be contained in an independent solution, or in a reagent containing the substrate of TRACP-5b, or in a sample dilution reagent.In the solution and sample dilution reagent independently contained in the kit, the above-mentioned organic sulfonic acid, sulfate ester or their salt can be dissolved in a buffer solution such as Tris.

[0063] A kit for measuring TRACP-5b by enzyme immunoassay (EIA) includes a solid support such as a multiwell plate, an anti-TRACP-5b monoclonal antibody adsorbed to the solid support, a substrate for TRACP-5b, the aforementioned organic sulfonic acid, sulfate ester or salt thereof, optionally a reagent for diluting a sample, and optionally a washing solution. The anti-TRACP-5b monoclonal antibody may be included in the kit as an antibody solution separate from the solid support, and in this case, when measuring TRACP-5b, the antibody is adsorbed to the solid support. The washing solution is used to remove components in the sample that have not been adsorbed after TRACP-5b in the sample is bound to the antibody. As the washing solution, for example, a Tris buffer solution containing a surfactant can be used. A sample diluent is used to adjust the concentration of a sample, and can be, for example, a buffer solution such as Tris. If necessary, a chelating agent such as EDTA·2Na or an inorganic salt such as table salt can be added to the buffer solution. Examples of materials for the solid support include polystyrene, polypropylene, polycarbonate, polyethylene, nylon, methacrylate, etc. Examples of shapes of the solid support include plates, (magnetic) beads (particles), etc. In this kit, the organic sulfonic acid, sulfate ester, or salt thereof may be contained in an independent solution, or in a reagent containing a substrate for TRACP-5b, a reagent containing an anti-TRACP-5b monoclonal antibody, or a reagent for diluting a specimen. In the solutions and reagents for diluting a specimen that are independently contained in the kit, the organic sulfonic acid, sulfate ester, or salt thereof may be dissolved in a buffer solution such as Tris.

[0064] A kit for measuring TRACP-5b by enzyme-linked immunosorbent assay (ELISA) comprises a solid support, a primary anti-TRACP-5b monoclonal antibody immobilized on the solid support, a secondary anti-TRACP-5b monoclonal antibody that binds to TRACP-5b via an epitope different from that of the primary anti-TRACP-5b monoclonal antibody and is labeled with a substance that enables detection by enzyme reaction, a reagent for detecting the labeled substance of the secondary anti-TRACP-5b monoclonal antibody by enzyme reaction, the above-mentioned organic sulfonic acid, sulfate ester or salt thereof, optionally a reagent for diluting a sample, and optionally a washing solution. The primary anti-TRACP-5b monoclonal antibody may be included in the kit as an antibody solution separate from the solid support, in which case the primary antibody is adsorbed to the solid support when measuring TRACP-5b. The washing solution is used to remove components that have not bound to the primary or secondary antibodies after TRACP-5b in the sample is bound to these antibodies. For example, a Tris buffer solution containing a surfactant can be used as the washing solution. A sample diluent is used to adjust the concentration of a sample, and can be, for example, a buffer solution such as Tris. If necessary, a chelating agent such as EDTA·2Na or an inorganic salt such as table salt can be added to the buffer solution. In this kit, the organic sulfonic acid, sulfate ester, or salt thereof may be contained in an independent solution, or in a reagent for making the label of the secondary anti-TRACP-5b monoclonal antibody detectable by an enzyme reaction, a reagent containing a primary antibody or a secondary antibody, or a reagent for diluting a specimen. In the solution and the reagent for diluting a specimen that are independently contained in the kit, the organic sulfonic acid, sulfate ester, or salt thereof may be dissolved in a buffer solution such as Tris. Substances that enable detection in an enzyme reaction include enzymes other than ACP, substrates for enzymes other than ACP, and biotin. Furthermore, the reagent for detecting the labeling substance by an enzyme reaction is, for example, a reagent containing peroxidase-labeled streptavidin, a peroxidase enzyme substrate of tetramethylbenzidine, and hydrogen peroxide when the labeling substance is biotin, or a substrate thereof when the labeling substance is an enzyme, such as p-nitrophenyl phosphate, which is a substrate of the enzyme, when the labeling substance is alkaline phosphatase. Examples of materials for the solid support include polystyrene, polypropylene, polycarbonate, polyethylene, nylon, methacrylate, etc. Examples of shapes of the solid support include plates, (magnetic) beads (particles), etc.

[0065] A kit for measuring TRACP-5b by chemiluminescent enzyme immunoassay (CLEIA) contains, for example, a solid support, an anti-TRACP-5b monoclonal antibody adsorbed to the solid support, a chemiluminescent substrate for TRACP-5b, the aforementioned organic sulfonic acid, sulfate ester or salt thereof, optionally a reagent for diluting a sample, and optionally a washing solution. The anti-TRACP-5b monoclonal antibody may be contained in the kit as an antibody solution separate from the solid support. In this case, when TRACP-5b is measured, the antibody is adsorbed to the solid support.

[0066] Examples of chemiluminescent substrates include: (1) 1,2-dioxetane derivatives such as 3-(4-methoxyspiro{1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane}-4-yl)phenylphosphate disodium salt, 4-chloro-3-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane}-4-yl)phenylphosphate disodium salt, 3-(4-methoxyspiro{1,2-dioxetane-3,2'-tricyclo[3.3.1.1(3,7)]decane}-4-yl)phenylphosphate disodium salt, and 4-methoxy-4-(3-phenylphosphate)spiro[1,2-dioxetane-3,2'-adamantane] disodium salt; or (2) Acridan derivatives such as [10-methyl-9(10H)-acridinylidene]phenoxymethyl phosphate disodium salt Examples include:

[0067] Sample diluents are used to adjust the concentration of samples. Examples of sample diluents include 2-(N-morpholino)ethanesulfonic acid (MES) buffer, 3-(N-morpholino)propanesulfonic acid (MOPSO) buffer, 3-morpholinopropanesulfonic acid (MOPS) buffer, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer, 2-[4-(2- A buffer solution selected from the group consisting of N-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer solution, N-(2-acetamido)iminodiacetic acid (ADA) buffer solution, N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer solution, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) buffer solution, tris(hydroxymethyl)aminomethane (Tris) buffer solution, and phosphate buffer solution can be used. Cobalt ions or manganese ions can be added as metal ions to the buffer solution, if necessary. The washing solution is used to remove unadsorbed components from the sample after TRACP-5b in the sample is bound to the antibody. As the washing solution, for example, the above-mentioned buffer solution containing a surfactant can be used. In this kit, the organic sulfonic acid, sulfate ester, or salt thereof may be contained in an independent solution, or in a reagent containing a chemiluminescent substrate for TRACP-5b, a reagent containing an anti-TRACP-5b monoclonal antibody, or a reagent for diluting a specimen. In the solution and the reagent for diluting a specimen that are independently contained in the kit, the organic sulfonic acid, sulfate ester, or salt thereof may be dissolved in, for example, the buffer solution described above.

[0068] A kit for measuring TRACP-5b by chemiluminescence immunoassay (CLIA method) also includes a solid support, a primary anti-TRACP-5b monoclonal antibody adsorbed to the solid support, a secondary anti-TRACP-5b monoclonal antibody that binds to TRACP-5b at an epitope different from that of the primary anti-TRACP-5b monoclonal antibody and is labeled with a substance that enables detection by chemiluminescence, a reagent that makes the label detectable by chemiluminescence, the above-mentioned organic sulfonic acid, sulfate ester or salt thereof, optionally a reagent for diluting a sample, and optionally a washing solution. The primary anti-TRACP-5b monoclonal antibody may be included in the kit as an antibody solution separate from the solid support. In this case, when measuring TRACP-5b, the primary antibody is adsorbed to the solid support. The washing solution is used to remove components that do not bind to the primary or secondary antibodies after TRACP-5b in the sample is bound to these antibodies. The washing solution and the sample dilution solution are the same as those described above. The reagent for making the label detectable by chemiluminescence is, for example, a reagent containing peroxidase-labeled streptavidin, a peroxidase chemiluminescent substrate, and hydrogen peroxide when the label is biotin, and a substrate for the enzyme, for example, p-nitrophenyl phosphate, when the label is an enzyme, for example, alkaline phosphatase. The chemiluminescent substrate is the same as described above. Examples of materials for the solid support include polystyrene, polypropylene, polycarbonate, polyethylene, nylon, methacrylate, etc. Examples of shapes of the solid support include plates, (magnetic) beads (particles), etc. In this kit, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof can be contained in an independent solution, or in a reagent that renders the label detectable by chemiluminescence, a reagent containing a primary antibody or a secondary antibody, or a reagent for diluting a specimen. In the solutions independently contained in the kit and the reagent for diluting a specimen, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof can be dissolved in, for example, the above-mentioned buffer solution.

[0069] A kit for measuring TRACP-5b by latex agglutination includes latex particles, anti-TRACP-5b polyclonal antibody or anti-TRACP-5b monoclonal antibody adsorbed to the latex particles, the above-mentioned organic sulfonic acid, sulfate ester or salt thereof, and optionally a sample dilution reagent.In this kit, the above-mentioned organic sulfonic acid, sulfate ester or salt thereof can be contained in an independent solution, or in a reagent containing latex particles, or a sample dilution reagent.In the solution and sample dilution reagent independently contained in the kit, the above-mentioned organic sulfonic acid, sulfate ester or salt thereof can be dissolved in, for example, a buffer solution. Examples of the buffer solution include 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution, 3-(N-morpholino)propanesulfonic acid (MOPSO) buffer solution, 3-morpholinopropanesulfonic acid (MOPS) buffer solution, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer solution, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) buffer solution, 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer solution, N-(2-acetamido)iminodiacetic acid (ADA) buffer solution, N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer solution, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) buffer solution, tris(hydroxymethyl)aminomethane (Tris) buffer solution, and phosphate buffer solution. EXAMPLES

[0070] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. The abbreviations used in the examples have the following meanings: TIFF0007653596000013.tif68148

[0071] Study 1. Evaluation of TRACP-5a inhibitors using the enzymatic method 1-1. Compounds to be evaluated The following compounds were evaluated for their TRACP-5a inhibitory effects using an enzymatic method. [Table 1] [Table 2] [Table 3]

[0072] 1-2. Measurement sample Nittobo Medical's TRACP-5b and R&D Systems' TRACP-5a were diluted with 22mM MES buffer (pH 5.0) to prepare 7U / L TRACP-5b solution and 7U / L TRACP-5a solution, which were used as measurement samples. For the measurement of sodium hyaluronate, 7U / L TRACP-5b solution and 8U / L TRACP-5a solution were used, which were prepared in the same manner.

[0073] 1-3. Measurement reagent (chromogenic substrate solution) A color-developing substrate solution having the following composition was used. 5mM 2-chloro-4-nitrophenyl phosphate 109mM MES 40mM Sodium Tartrate 0.1% by mass of evaluation compound pH 6.6

[0074] 1-4.Measurement method A general-purpose Hitachi automatic analyzer, model 7180 (Hitachi High-Technologies) was used for the measurements. 200 μL of a color-developing substrate solution containing the evaluation compound was added to 16 μL of 7 U / L TRACP-5b solution, or 7 U / L TRACP-5a solution or 8 U / L TRACP-5a solution to initiate the enzyme reaction, and the absorbance (main wavelength 405 nm, secondary wavelength 480 nm) was measured from 340.5 seconds to 516.0 seconds. The amount of absorbance change per minute (the amount of absorbance change per unit time during the enzyme reaction) was calculated from the change in absorbance between the two points in time. In addition, a serial dilution solution of a known activity TRACP-5b solution in 22 mM MES buffer (pH 5.0), as well as 22 mM MES buffer (pH 5.0) as a blank were prepared, and a serial dilution solution of the TRACP-5b solution was measured using a chromogenic substrate solution containing each evaluation compound to prepare a standard straight line of TRACP-5b. A serial dilution solution of the TRACP-5b solution was similarly measured using a chromogenic substrate solution not containing the evaluation compound to prepare a standard straight line of TRACP-5b. Using these standard straight lines, the activity value was calculated from the absorbance change obtained by measuring the TRACP-5b solution and the TRACP-5a solution using the chromogenic substrate solution containing each evaluation compound and the chromogenic substrate solution not containing the evaluation compound. The measurement using sodium hyaluronate was carried out in a batch separate from that for the other evaluation compounds.

[0075] 1-5.Measurement results [Table 4]

[0076] Study 2. Examination of the concentration of TRACP-5a inhibitor The effect of the concentration of dextran sulfate (5K), dextran sulfate (50K), polyvinyl sulfonate (5K), and vinyl sulfonate (sodium) on the inhibitory activity was examined.

[0077] 2-1. Concentration of each evaluation compound The concentrations of each evaluation compound in the measurement reagent (color-developing substrate solution) were adjusted as follows. [Table 5]

[0078] 2-2. Measurement sample In studies using reagents containing sodium dextran sulfate (5K) and sodium dextran sulfate (50K), 7 U / L TRACP-5b solution and 7 U / L TRACP-5a solution were used; in studies using reagents containing sodium polyvinylsulfonate (5K), 7 U / L TRACP-5b solution and 7 U / L or 8 U / L TRACP-5a solution were used; in studies using reagents containing sodium vinylsulfonate, 7 U / L TRACP-5b solution and 8 U / L TRACP-5a solution were used.

[0079] 2-3. Measurement reagent (color substrate solution) and measurement method Except for the above-mentioned concentrations of each evaluation compound, a color-developing substrate solution having the same composition as in Test 1 was used. The measurement method was carried out in the same manner as in Test 1.

[0080] 2-4.Measurement results [Table 6] [Table 7] Example 4 is data from a different batch, but the data from Test 1 is included for reference. [Table 8] Example 5 is data from a different batch, but the data from Test 1 is included for reference. [Table 9] Example 1 is data from a different batch, but the data from Test 1 is listed for reference. [Table 10] Example 2 is data from a different batch, but the data from Test 1 is included for reference.

[0081] 3. Evaluation of TRACP-5a inhibitors using EIA 3-1. Compounds to be evaluated The following compounds were evaluated for their TRACP-5a inhibitory effects by EIA. [Table 11] [Table 12] [Table 13]

[0082] 3-2. Preparation of antibody-immobilized plates TRACP-5b monoclonal antibody (accession number IPOD FERM BP-7890) was diluted to 10 μg / mL with PBS (8 mM disodium hydrogen phosphate, 1.5 mM sodium dihydrogen phosphate, 2.7 mM KCl, 137 mM NaCl, pH 7.5) and 100 μL was added to each well of an ELISA plate (Thermo Fisher SCIENTIFIC, product number 468667). After standing overnight in a refrigerator, the plate was washed three times with 300 μL of TBS-T (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.5), and 300 μL of 1.5% BSA-containing TBS was added and the plate was left standing overnight in a refrigerator.

[0083] 3-3.Measurement method After removing 1.5% BSA-containing TBS from the antibody-immobilized plate, 100 μL of TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) and 50 μL of 7 U / L TRACP-5b solution or 7 U / L TRACP-5a solution were added to each well and reacted for 1 hour with stirring (25 ° C, 700 rpm). After washing three times with 300 μL of TBS-T, 100 μL of a color-developing substrate solution (5 mM 2-chloro-4-nitrophenyl phosphate, 109 mM MES, 40 mM Na tartrate, pH 6.6) containing the evaluation compound at a concentration of 0.1% by mass was added to each well and left to stand at 37 ° C for 1 hour. Then, 50 μL of 0.5 N NaOH was added, and the absorbance (main wavelength 405 nm, secondary wavelength 490 nm) was measured using a plate reader.

[0084] 3-4.Measurement results [Table 14]

Claims

1. A method for measuring tartrate-resistant acid phosphatase 5b (TRACP-5b) in a sample, comprising: Sulfo group (-S(O) ２ OH), sulfate group (-OS(O) ２ The present invention relates to a method for measuring TRACP-5b, the method comprising the step of carrying out a reaction for detecting TRACP-5b in the presence of an organic sulfonic acid, a sulfate ester, or a salt thereof having a density of 6.5 mmol / g or more.

2. The organic sulfonic acid, sulfate ester, or salt thereof has a sulfo group (-S(O) ２ OH), sulfate group (-OS(O) ２ OH) and / or salts thereof, ２ O － The method according to claim 1 , wherein the content is 50% by mass or more in terms of the converted value.

3. The organic sulfonic acid, sulfate ester, or salt thereof is represented by the formula (1): 【Chemistry 1】 (In formula (1), R is an alkyl, alkenyl, or alkynyl group having 1 to 3 carbon atoms, Y is a bond or an oxygen atom, and X is hydrogen or an alkali metal.) Is it expressed as Formula (2): 【Chemistry 2】 (In formula (2), Y is a bond or an oxygen atom, n is an integer from 2 to 5000, and X is hydrogen or an alkali metal.) or having a repeat unit of or Formula (3): 【Chemistry 3】 (In the formula (3), Y is O or CH ２ and R １ ~R ５ Among these, two are O- or bonds, and at least two of the remaining are O-S(O) ２ OX or S(O) ２ OX (X is hydrogen or an alkali metal), and if it does not fall into either category, it is OX or C(O)OX (X is hydrogen or an alkali metal). The method according to claim 1 or 2, having a repeating unit comprising the structure:

4. 3. The method of claim 1 or 2, wherein the organic sulfonic acid, sulfate ester or salt thereof is vinyl sulfonic acid or a salt thereof, vinyl sulfate or a salt thereof, polyvinyl sulfonic acid or a salt thereof, polyvinyl sulfate or a salt thereof, methanesulfonic acid or a salt thereof, or dextran sulfate or a salt thereof.

5. The method according to claim 1 or 2, wherein the organic sulfonic acid, sulfate ester, or salt thereof is 1) added independently to a reaction system or a specimen, or is contained in 2) a specimen dilution solution, 3) a substrate-containing reagent, or 4) a reagent containing an anti-TRACP-5b antibody, thereby carrying out a reaction to detect TRACP-5b in the presence of the organic sulfonic acid, sulfate ester, or salt thereof.

6. The method according to claim 1 or 2, wherein the measurement is carried out by an enzyme method, an enzyme immunoassay (EIA), an enzyme-linked immunosorbent assay (ELISA), a chemiluminescent enzyme immunoassay (CLEIA), a chemiluminescent immunoassay (CLIA), or a latex agglutination method.

7. The method of claim 1 or 2, wherein the sample is blood, serum or plasma.

8. A kit for measuring TRACP-5b, comprising: Sulfo group (-S(O) ２ OH), sulfate group (-OS(O) ２ 1. A kit comprising an organic sulfonic acid, a sulfate ester and / or a salt thereof having a density of 6.5 mmol / g or more, and / or a salt thereof.

9. The organic sulfonic acid, sulfate ester and / or salt thereof has a sulfo group (-S(O) ２ OH), sulfate group (-OS(O) ２ OH) or a salt thereof, ２ O － The kit according to claim 8 , having a converted value of 50% by mass or more.

10. The organic sulfonic acid, sulfate ester and / or salt thereof may be represented by the formula (1): 【Chemistry 4】 (In formula (1), R is an alkyl, alkenyl, or alkynyl group having 1 to 5 carbon atoms, Y is a bond or an oxygen atom, and X is hydrogen or an alkali metal.) Is it expressed as Formula (2): 【Chemistry 5】 (In formula (2), Y is a bond or an oxygen atom, n is an integer from 2 to 5000, and X is hydrogen or an alkali metal.) or Formula (3): 【Chemistry 6】 (In the formula (3), Y is O or CH ２ and R １ ~R ５ Among these, two are O- or bonds, and at least two of the remaining are O-S(O) ２ OX or S(O) ２ OX (X is hydrogen or an alkali metal), and if it does not fall into either category, it is OX or C(O)OX (X is hydrogen or an alkali metal). The repeat structure includes the structure The kit according to claim 8 or 9.

11. 10. The kit according to claim 8 or 9, wherein the organic sulfonic acid, sulfate ester or salt thereof is vinyl sulfonic acid or a salt thereof, vinyl sulfate or a salt thereof, polyvinyl sulfonic acid or a salt thereof, polyvinyl sulfate or a salt thereof, methanesulfonic acid or a salt thereof, or dextran sulfate or a salt thereof.

12. The kit according to claim 8 or 9 for measuring TRACP-5b by an enzymatic method, A kit comprising a substrate for TRACP-5b, the organic sulfonic acid, sulfate ester or salt thereof, and optionally a reagent for diluting a specimen.

13. The kit according to claim 12, wherein the organic sulfonic acid, sulfate ester, or salt thereof is contained in the reagent containing a substrate for TRACP-5b or the specimen dilution reagent.

14. The kit according to claim 8 or 9, which measures TRACP-5b by enzyme immunoassay (EIA), A kit comprising a solid support, an anti-TRACP-5b monoclonal antibody adsorbed to the solid support, a substrate for TRACP-5b, the organic sulfonic acid, sulfate ester or salt thereof, optionally a reagent for diluting a specimen, and optionally a washing solution.

15. The kit according to claim 14, wherein the organic sulfonic acid, sulfate ester, or salt thereof is contained in the reagent containing the anti-TRACP-5b monoclonal antibody, the reagent containing a substrate for TRACP-5b, or the specimen dilution reagent.

16. The kit according to claim 8 or 9, which measures TRACP-5b by enzyme-linked immunosorbent assay (ELISA), A kit comprising: a solid support; a primary anti-TRACP-5b monoclonal antibody adsorbed to the solid support; a secondary anti-TRACP-5b monoclonal antibody that binds to TRACP-5b via an epitope different from that of the primary anti-TRACP-5b monoclonal antibody and is labeled with a substance that enables detection by an enzyme reaction; a reagent for enabling detection of the labeled substance by an enzyme reaction; the organic sulfonic acid, sulfate ester or salt thereof; optionally a reagent for diluting a sample; and optionally a washing solution.

17. The kit according to claim 16, wherein the organic sulfonic acid, sulfate ester, or salt thereof is contained in a reagent containing the secondary anti-TRACP-5b monoclonal antibody, a reagent for enabling detection of the labeling substance by an enzyme reaction, or a reagent for diluting a specimen.

18. The kit according to claim 8 or 9, which measures TRACP-5b by chemiluminescence enzyme immunoassay (CLEIA), A kit comprising a solid support, an anti-TRACP-5b monoclonal antibody adsorbed to the solid support, a chemiluminescent substrate for TRACP-5b, the organic sulfonic acid, sulfate ester or salt thereof, optionally a reagent for diluting a specimen, and optionally a washing solution.

19. The kit according to claim 18, wherein the organic sulfonic acid, sulfate ester, or salt thereof is contained in the reagent containing a substrate for TRACP-5b, the reagent containing an anti-TRACP-5b monoclonal antibody, or the specimen dilution reagent.

20. The kit according to claim 8 or 9, which measures TRACP-5b by a chemiluminescence immunoassay (CLIA method), A kit comprising: a solid support; a primary anti-TRACP-5b monoclonal antibody adsorbed to the solid support; a secondary anti-TRACP-5b monoclonal antibody that binds to TRACP-5b via an epitope different from that of the primary anti-TRACP-5b monoclonal antibody and is labeled with a substance that enables detection by chemiluminescence; a reagent that enables the labeled substance to be detected by chemiluminescence; the organic sulfonic acid, sulfate ester or salt thereof; optionally a reagent for diluting a sample; and optionally a washing solution.

21. The kit according to claim 20, wherein the organic sulfonic acid, sulfate ester, or salt thereof is contained in a reagent comprising the first anti-TRACP-5b monoclonal antibody, the second anti-TRACP-5b monoclonal antibody, the label detectable by chemiluminescence, or the sample dilution reagent.

22. The kit according to claim 8 or 9, which comprises measuring TRACP-5b by a latex agglutination method, A kit comprising latex particles, an anti-TRACP-5b antibody immobilized on the latex particles, the organic sulfonic acid, sulfate ester or salt thereof, and optionally a reagent for diluting a specimen.

23. The kit according to claim 22, wherein the organic sulfonic acid, sulfate ester, or salt thereof is contained in the reagent containing the anti-TRACP-5b antibody or the specimen dilution reagent.

24. The kit according to claim 8 or 9, wherein the sample to be measured is blood, serum or plasma.