Method for measuring TRACP-5b and kit for same

Abstract

The present invention provides a measurement method with which it is possible to selectively detect TRACP-5b in the measurement of TRACP-5b, said method further reducing the influence of TRACP-5a that coexists. In the method, a reaction for detecting TRACP-5b is performed in the presence of an organic sulfonic acid, a sulfate ester or a salt thereof, and the density of a sulfo group (-S (O) 2OH), a sulfo group (-OS (O) 2OH) and / or a salt thereof in the organic sulfonic acid, the sulfate ester or the salt thereof is 6.5 mmol / g or more.

Description

Technical Field

[0001] The present invention relates to a method for measuring TRACP-5b and a kit used in the method, and more particularly, to a method for selectively measuring TRACP-5b without being affected by TRACP-5a and a kit used in the method. Background Art

[0002] Acid phosphatase (ACP) is an enzyme that has the hydrolysis activity of organic monophosphate under acidic conditions. There are multiple isozymes in ACP, which are distributed in various tissues. As ACP present in serum, it is known that it comes from platelets, red blood cells, prostate, osteoclasts, macrophages, etc. These isozymes are separated by polyacrylamide gel electrophoresis, and the ACP from osteoclasts and macrophages is located at the 5th position of the origin, and has resistance to tartaric acid, so it is also called Band-5 tartrate-resistant acid phosphatase (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 divided into TRACP-5a, which has more sugar chains and sialic acid, and TRACP-5b, which has less sugar chains and sialic acid. 5a is derived from macrophages, and 5b is derived from osteoclasts. Since the concentration of TRACP-5b in serum can sensitively reflect the activity of osteoclasts, it is used as a bone resorption marker to assist in the diagnosis of metabolic bone diseases and bone metastatic cancer and to determine the therapeutic effect. Therefore, a method for selectively measuring TRACP-5b from ACP in serum is being studied.

[0003] Conventionally, there is a method for measuring TRACP-5, which involves performing an enzyme reaction using a synthetic substrate such as p-nitrophenyl phosphate in the presence of tartaric acid, and determining the enzyme activity by colorimetrically quantifying the generated reaction product. However, although tartaric acid can inhibit prostate-derived ACP, since TRACP-5a, erythrocyte-derived ACP, and platelet-derived ACP are resistant to tartaric acid, this method also measures these isozymes, and cannot specifically measure TRACP-5b.

[0004] As a method for improving the TRACP-5b specificity of the above method, there is known a method in which a liquid diluted 5 times serum is incubated at 37°C for 1 hour and the enzyme activity is similarly measured (Non-patent Document 3). In this method, in addition to avoiding ACP from the prostate, the influence of ACP from erythrocytes can also be avoided, but the influence of TRACP-5a and ACP from platelets cannot be avoided.

[0005] As other methods for specifically measuring the activity of TRACP-5b, there is a method for calculating the activity of TRACP-5b by subtracting the ACP activity measured in the coexistence of tartaric acid and fluoride from the ACP activity measured in the presence of tartaric acid (patent document 1). In this method, the difference in sensitivity to fluoride can be used to avoid the influence of ACP from red blood cells and ACP from platelets. However, the influence of TRACP-5a cannot be excluded, and there is a problem in accuracy in obtaining the activity of TRACP-5b by subtraction.

[0006] As another method, a method of using acidic mucopolysaccharide as an inhibitor of acid phosphatase other than TRACP-5b to specifically measure TRACP-5b activity has been reported (Patent Document 2). However, the inhibitory effect of acidic mucopolysaccharide on TRACP-5a activity is insufficient, and further improvement of the specificity for TRACP-5b is required.

[0007] As a method for measuring the activity of TRACP-5b using a chemiluminescent substrate, it is known that TRACP-5b is reacted with a chemiluminescent 1,2-dioxetane compound under acidic conditions, and then the reaction solution is set to alkaline conditions to measure the luminescence intensity (Patent Document 3). In addition, a method for specifically measuring TRACP-5b by using a specific buffer such as MES during an enzyme reaction, or by adding manganese ions or cobalt ions, making the luminescence intensity ratio of TRACP-5b relative to TRAPC-5a larger, has been reported (Patent Document 4). However, these methods are limited to activity measurements using chemiluminescent substrates, and a cheaper method is required to specifically measure TRACP-5b.

[0008] As an immunoassay method for TRACP-5b using an antibody, for example, there is a method in which TRACP-5a and TRACP-5b are bound using an antibody specific to TRACP-5, and then TRACP-5b is specifically measured using the difference in the optimal pH between the two (Patent Document 5). However, the specificity for TRACP-5b is insufficient only by the difference in the optimal pH, and the amount of TRACP-5b is determined by subtraction, so there is also a problem in terms of accuracy.

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

[0010] In addition, there is also a method for quantifying the amount of TRACP-5b protein using two clones of monoclonal antibodies that are highly specific to TRACP-5b (Patent Document 8). Prior art literature

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

[0012] Non-patent document 1: Clin. Chem. 33: 458-462.1987 Summary of the invention Problems to be solved by the invention

[0013] With respect to the above-mentioned prior art, an object of the present invention is to provide a measurement method capable of further reducing the influence of coexisting TRACP-5a in the measurement of TRACP-5b and selectively detecting TRACP-5b. Means of solving problems

[0014] The present inventors have studied an assay method capable of selectively detecting TRACP-5b and have discovered that by allowing compounds having a sulfone group (-S(O)2OH), a sulfate group (-OS(O)2OH) and / or their salts to have a density above a certain level in a reaction system where TRACP-5a may be present, the influence of TRACP-5a can be greatly reduced, thereby enabling selective detection of TRACP-5b.

[0015] That is, the present invention provides the following TRACP-5b measurement method and TRACP-5b measurement kit. [1] A method for measuring TRACP-5b, which is a method for measuring tartrate-resistant acid phosphatase 5b (TRACP-5b) in a specimen, 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, wherein the density of the sulfonic group (-S(O)2OH), the sulfate group (-OS(O)2OH) and / or the salt thereof in the organic sulfonic acid, the sulfate ester or the salt thereof is greater than 6.5 mmol / g.

[0016] [2] The method for measuring tartrate-resistant acid phosphatase 5b (TRACP-5b) in a sample according to [1], comprising the step of performing an enzyme reaction or an antigen-antibody reaction in the presence of an organic sulfonic acid, a sulfate ester and / or a salt thereof, wherein each unit structure of the organic sulfonic acid, the sulfate ester and / or the salt thereof is S(O)2O - Containing 50% by mass or more of sulfonic groups (-S(O)2OH), sulfuric acid groups (-OS(O)2OH) and / or salts thereof; or Each unit structure of the organic sulfonic acid, sulfate or salt thereof is S(O)2O - The converted value contains 50 mass % or more of sulfonic groups (-S(O)2OH), sulfuric acid groups (-OS(O)2OH) and / or salts thereof.

[0017] [3] The method according to [1] or [2], wherein the organic sulfonic acid, sulfuric acid ester or a salt thereof: It is expressed by formula (1): RYS(O)2OX…(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; or, A repeating unit having formula (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 repeating unit having a structure comprising formula (3), (In formula (3), Y is O or CH2, R 1 ~R 5 Two of them are O- or bonds, and at least two of the remaining two are OS(O)2OX or S(O)2OX (X is hydrogen or an alkali metal), R 1 ~R 5 None of them is OX or C(O)OX (X is hydrogen or alkali metal). )

[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 sulfuric acid 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, sulfuric acid ester or a salt thereof: 1) Added independently to the reaction system or sample, or The method is contained in 2) a sample diluent, 3) a reagent containing a substrate, or 4) a reagent containing an anti-TRACP-5b antibody (typically an anti-TRACP-5b monoclonal antibody), Thus, the reaction for detecting TRACP-5b is carried out in the presence of the organic sulfonic acid, sulfate ester or a salt thereof.

[0020] [6] The method according to any one of [1] to [5], wherein the determination is performed using an enzyme method, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA method), chemiluminescent immunoassay (CLIA method) or 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 TRACP-5b assay kit comprising an organic sulfonic acid, a sulfate ester and / or their salts, wherein the density of the sulfonic group (-S(O)2OH), the sulfate group (-OS(O)2OH) and / or their salts in the organic sulfonic acid, the sulfate ester and / or their salts is greater than 6.5 mmol / g.

[0022] [9] The TRACP-5b assay kit according to [8], comprising an organic sulfonic acid, a sulfate ester and / or a salt thereof, wherein each unit structure of the organic sulfonic acid, the sulfate ester and / or the salt thereof is S(O)2O - Containing 50% by mass or more of sulfonic acid groups (-S(O)2OH), sulfuric acid groups (-OS(O)2OH) or salts thereof; or Each unit structure of the organic sulfonic acid, sulfate and / or salt thereof is S(O)2O - The converted value contains 50 mass % or more of sulfonic acid group (-S(O)2OH), sulfuric acid group (-OS(O)2OH) or a salt thereof.

[0023]

[10] The kit according to [8] or [9], wherein the organic sulfonic acid, sulfate ester and / or salt thereof: It is expressed by formula (1): RYS(O)2OX…(1) 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; or, Having a repeating structure of formula (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 repeating structure comprising a structure of formula (3), (In formula (3), Y is O or CH2, R 1 ~R 5 Two of them are O- or bonds, and at least two of the remaining two are OS(O)2OX or S(O)2OX (X is hydrogen or an alkali metal), R 1 ~R 5 None of them is OX or C(O)OX (X is hydrogen or alkali metal). )

[0024]

[11] The kit according to [8] or [9], wherein the organic sulfonic acid, sulfate ester or salt thereof is vinyl sulfate or its salt, vinyl sulfonic acid or its salt, polyvinyl sulfonic acid or its salt, polyvinyl sulfuric acid or its salt, methanesulfonic acid or its salt, or dextran sulfate or its salt.

[0025]

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

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

[0026]

[13] The kit according to

[12] , wherein the organic sulfonic acid, sulfuric acid ester or a salt thereof is contained in a reagent containing the substrate of TRACP-5b or the reagent for diluting the specimen.

[0027]

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

[11] , wherein TRACP-5b is measured by enzyme immunoassay (EIA), The kit comprises: a solid support, an anti-TRACP-5b monoclonal antibody adsorbed on the solid support, the TRACP-5b substrate, the organic sulfonic acid, sulfate or salt thereof, an optional sample dilution reagent, and an optional cleaning solution.

[15] The kit according to

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

[0028]

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

[11] , wherein TRACP-5b is measured by enzyme-linked immunosorbent assay (ELISA), The kit comprises: a solid phase support, a first anti-TRACP-5b monoclonal antibody adsorbed on the solid phase support, a second anti-TRACP-5b monoclonal antibody that binds to TRACP-5b with an epitope different from that of the first anti-TRACP-5b monoclonal antibody and is labeled with a substance that can be detected in an enzyme reaction, a reagent for making the labeled substance detectable in an enzyme reaction, the organic sulfonic acid, sulfate or salt thereof, an optional reagent for sample dilution, and an optional cleaning solution.

[17] The kit according to

[16] , wherein the organic sulfonic acid, sulfate ester or salt thereof is contained in a reagent containing the second anti-TRACP-5b monoclonal antibody, a reagent for detecting the labeling substance in an enzyme reaction or a reagent for sample dilution.

[0029]

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

[11] , wherein TRACP-5b is measured by chemiluminescent enzyme immunoassay (CLEIA method), The kit comprises: a solid support, an anti-TRACP-5b monoclonal antibody adsorbed on the solid support, a chemiluminescent substrate of TRACP-5b, the organic sulfonic acid, sulfate or salt thereof, an optional sample dilution reagent, and an optional cleaning solution.

[19] The kit according to

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

[0030]

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

[11] , wherein TRACP-5b is measured by chemiluminescent immunoassay (CLIA method), The kit comprises: a solid support, a first anti-TRACP-5b monoclonal antibody adsorbed on the solid support, a second anti-TRACP-5b monoclonal antibody that binds to TRACP-5b with an epitope different from that of the first anti-TRACP-5b monoclonal antibody and is labeled with a substance that can be detected in chemiluminescence, a reagent for making the labeled substance detectable in chemiluminescence, the organic sulfonic acid, sulfate or salt thereof, an optional reagent for sample dilution, and an optional cleaning solution.

[21] According to the kit described in

[20] , the organic sulfonic acid, sulfate ester or their salts are contained in a reagent containing the first anti-TRACP-5b monoclonal antibody, a reagent containing the second anti-TRACP-5b monoclonal antibody, a reagent for detecting the marker by chemiluminescence, or a reagent containing the specimen dilution reagent.

[0031]

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

[11] , wherein TRACP-5b is measured by a latex agglutination method, The kit comprises latex particles, anti-TRACP-5b antibodies adsorbed on the latex particles, the organic sulfonic acid, sulfate or salt thereof, and an optional reagent for sample dilution.

[23] The kit according to

[22] , wherein the organic sulfonic acid, sulfuric acid ester or a salt thereof is contained in the reagent containing the anti-TRACP-5b antibody or the reagent for sample dilution.

[0032]

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

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

[0033] According to the present invention, a means (method) is provided for selectively detecting tartrate-resistant acid phosphatase 5b (TRACP-5b) by reducing the influence of TRACP-5b, which is useful for diagnosing diseases for which TRACP-5b is used as a marker. DETAILED DESCRIPTION

[0034] 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.

[0035] One embodiment of the present invention provides a method for measuring TRACP-5b in a sample, comprising the following steps: performing a detection reaction of tartrate-resistant acid phosphatase 5b (TRACP-5b) in the presence of a specific organic sulfonic acid, sulfate ester or a salt thereof. Another embodiment of the present invention provides a kit for measuring TRACP-5b, wherein the kit comprises a specific organic sulfonic acid, sulfate ester or a salt thereof. These embodiments are described in detail below.

[0036] 1. TRACP-5b determination method 1-1. Organic sulfonic acid, sulfuric acid ester or their salts In this determination method, by allowing an organic sulfonic acid, sulfate ester or their salts to be present in the reaction system and making the density of the sulfone group (-S(O)2OH), sulfate group (-OS(O)2OH) and / or their salts in the organic sulfonic acid, sulfate ester or their salts to be above 6.5 mmol / g, TRACP-5a can be inhibited and TRACP-5b can be selectively determined. As shown in the examples described below, the effect of inhibiting TRACP-5a increases depending on the density of the sulfonic group, sulfate group or their salts. In a preferred embodiment, the density of the sulfonic group, sulfate group and / or their salts in the organic sulfonic acid, sulfate ester or their salts is 7.5 mmol / g or more, more preferably 8.5 mmol / g or more.

[0037] In a preferred embodiment, each unit structure of such an organic sulfonic acid, sulfuric acid ester or salt thereof is S(O)2O - The converted value contains 50 mass % or more of sulfonic groups, sulfate groups and / or salts thereof, more preferably 60 mass % or more of sulfonic groups, sulfate groups and / or salts thereof, and further preferably 70 mass % or more of sulfonic groups, sulfate groups and / or salts thereof. Here, when used in the specification of this application, the so-called "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, in the case of a salt having a sulfonic group (-S(O)2OH) or a sulfate group (-OS(O)2OH) in the unit structure, the molecular weight of the unit structure is calculated based on the sulfonic group (-S(O)2OH) or the sulfate group (-OS(O)2OH).

[0038] As a specific embodiment of such an organic sulfonic acid, sulfuric acid ester or a salt thereof, a compound represented by formula (1) can be mentioned: [Chemistry 7] RYS(O)2OX…(1) In the formula (1), R is an alkyl group, alkenyl group 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, and is preferably a bond. X is hydrogen or an alkali metal, preferably hydrogen or Na. Examples of the compound of this embodiment include methanesulfonic acid or a salt thereof (eg, Na salt), vinylsulfonic acid or a salt thereof (eg, Na salt), or vinyl sulfate or a salt thereof.

[0039] As other specific embodiments of the above-mentioned organic sulfonic acid, sulfuric acid ester or salt thereof, compounds having repeating units of formula (2) can be mentioned: [Chemistry 8] In formula (2), Y is a bond or an oxygen atom, and is preferably a bond. n is an integer of 2-5000, preferably 10-500, more preferably 20-100. X is hydrogen or an alkali metal, preferably hydrogen or Na. Preferred examples of the compound of this embodiment include polyvinyl sulfonic acid or a salt thereof (e.g., Na salt), or polyvinyl sulfuric acid or a salt thereof (e.g., Na salt), preferably polyvinyl sulfonic acid or a salt thereof having 2 to 5000, more preferably 10 to 500, and further preferably 20 to 100 or less repeating structures. [Chemistry 9] (In formula (2-1), X is hydrogen or an alkali metal, preferably hydrogen or Na.) Such polyvinylsulfonic acid or a salt thereof generally has a molecular weight of 200 to 500,000, preferably 1,000 to 50,000, and more preferably 4,000 to 6,000. Here, in the present application, "molecular weight" or "average molecular weight" refers to the weight average molecular weight (Mw) unless it is used in a particularly different sense. In addition, the weight average molecular weight (Mw) refers to the value measured by gel permeation chromatography (GPC method) using liquid chromatography.

[0040] As another specific embodiment of the above-mentioned organic sulfonic acid, sulfate or their salts, Examples include compounds having a repeating unit having a structure of formula (3): [Chemistry 10] In formula (3), Y is O or CH2, preferably O. R 1 ~R 5 Two of them are O- or bonds, and at least two of the remaining two are OS(O)2OX or S(O)2OX (X is hydrogen or an alkali metal (such as Na)), R 1 ~R 5 are not OX or C(O)OX (X is hydrogen or an alkali metal (such as Na), preferably not C(O)OX (X is hydrogen or an alkali metal (such as Na)). Preferably, R 1 ~R 5Two of them are O- or bonds, and the rest are OS(O)2OX (X is hydrogen or an alkali metal (such as Na)).

[0041] In addition, examples of repeating units including this structure include: [Chemistry 11] In formula (3-1), Y is O or CH2, preferably O. L 1 and L 4 Each is independently O or a bond, preferably one is O- and the other is a bond. L 2 and L 3 Each is independently O, CH2 or a bond, preferably one is O and the other is CH2 or a bond. R 1 ~R 3 At least two of them are independently OS(O)2OX or S(O)2OX (X is hydrogen or an alkali metal (such as Na)). If present, the rest are OX or C(O)OX (X is hydrogen or an alkali metal (such as Na)), preferably C(O)OX (X is hydrogen or an alkali metal (such as Na)). R 1 ~R 3 Preferably, each independently is OS(O)2OX or S(O)2OX (X is hydrogen or an alkali metal (eg, Na)), and more preferably each independently is OS(O)2OX (X is hydrogen or an alkali metal (eg, Na)). R 4 ~R 6 At least two of them are independently OS(O)2OX or S(O)2OX (X is hydrogen or an alkali metal (such as Na)). If present, the rest are OX or C(O)OX (X is hydrogen or an alkali metal (such as Na)), preferably C(O)OX (X is hydrogen or an alkali metal (such as Na)). R 4 ~R 6 Preferably, each independently is OS(O)2OX or S(O)2OX (X is hydrogen or an alkali metal (eg, Na)), and more preferably each independently is OS(O)2OX (X is hydrogen or an alkali metal (eg, Na)). n is 2-1000, preferably 3-700, more preferably 4-200, further preferably 5-70.

[0042] Preferred examples of the compound of this embodiment include dextran sulfate or its salt (eg, Na salt), and preferably dextran sulfate or its salt (eg, Na salt) having 2 to 1000, preferably 3 to 700, more preferably 5 to 70 repeating structures or less. [Chemistry 12] (In formula (3-2), X is hydrogen or an alkali metal (such as Na)) Such dextran sulfate or a salt thereof generally has a molecular weight of 1,500 to 1,000,000, preferably 2,000 to 550,000, and more preferably 4,000 to 55,000.

[0043] In the method of the present invention, by allowing the above-mentioned organic sulfonic acid, sulfate or their salt to exist in the reaction system, the characteristics of the coexisting TRACP-5a antigen are changed and its activity is inhibited. 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 to the reaction solution used to detect TRACP-5b; they can also be pre-dissolved in a specimen diluent and then added to the measurement sample; they can also be pre-added to a reagent containing an enzyme substrate or a reagent containing an anti-TRACP-5b antibody according to the reaction system, and these reagents can be used to react (for example, an enzyme reaction or an antigen-antibody reaction).

[0044] As shown in the examples described later, the above-mentioned organic sulfonic acid, sulfate or salt thereof increases the TRACP-5a inhibitory activity depending on the density and average molecular weight of the sulfonic 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 exert the TRACP-5a inhibitory activity depends on the density and average molecular weight. Regardless of the compound, when the concentration in the reaction solution is increased from the lower limit, the inhibitory activity increases within a certain range depending on the concentration, but when a certain concentration is reached, the inhibitory activity no longer increases depending on the concentration, but is maintained at a certain level. When a certain level of concentration is reached, the inhibitory activity gradually decreases. However, even when the inhibitory activity reaches a plateau, a high inhibitory activity is still maintained at a wide range of concentrations. In practice, the inhibitory activity is often used at a concentration higher than the concentration that no longer increases depending on the concentration in the reaction solution. Therefore, the concentration of the organic sulfonic acid, sulfuric acid ester or salt thereof in the reaction solution is preferably determined in consideration of these factors.

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

[0046] When vinyl sulfonic acid or its salt is used, the concentration in the reaction solution is preferably 0.012 to 15.0 mass %, more preferably 0.020 to 5.0 mass %, and even more preferably 0.10 to 3.0 mass %. When polyvinyl sulfonic acid or its salt having an average molecular weight of 4000 to 6000 is used, the concentration in the reaction solution is preferably 0.08×10 -3 ~5.0% by mass, more preferably 0.3×10 -3 ~1.0 mass%, more preferably 0.3×10 -2 ~0.3 mass %.

[0047] In the determination method, TRACP-5a is inhibited by allowing the above-mentioned organic sulfonic acid, sulfate or salt thereof to exist in the reaction system for detecting TRACP-5b. Examples of the "reaction system" include enzyme reaction and antigen-antibody reaction. 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 application, "enzyme reaction" refers to the reaction between TRACP-5b and its substrate, and the substrate of TRACP-5b includes, for example, p-nitrophenyl phosphate or its salt, 2-chloro-4-nitrophenyl phosphate or its salt, 1,2-dioxetane derivatives, acridane derivatives, 4-methylumbelliferyl phosphate (lin acid 4-methylumbelliferyl). In addition, in the present application specification, "antigen-antibody reaction" refers to the reaction between TRACP-5b and its corresponding antibody, and the antibody for TRACP-5b includes, for example, monoclonal antibody, polyclonal antibody, and antigen-binding fragment. Examples of monoclonal antibodies or antigen-binding fragments thereof include anti-TRACP-5b monoclonal antibodies or antigen-binding fragments thereof described in Patent Documents 6 and 7, etc. More specifically, examples include antibodies or antigen-binding fragments thereof produced by hybridomas with entrusted number IPOD FERM BP-7889, entrusted number IPOD FERM BP-8249, or entrusted number IPOD FERM BP-7890.

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

[0050] In the present application specification, the enzymatic method is a method for measuring the enzymatic activity of TRACP-5b, for example, the TRACP-5b in the specimen is reacted with an enzyme substrate of TRACP-5b, such as p-nitrophenyl phosphate or a salt thereof, to measure the enzymatic activity of TRACP-5b. More specifically, a chromogenic substrate solution (e.g., 2-chloro-4-nitrophenyl phosphate solution) containing tartaric acid or a salt thereof is added to the specimen to initiate the enzyme reaction, and the enzyme activity is obtained based on the change in absorbance between specific moments. In the enzymatic method, the above-mentioned organic sulfonic acid, sulfate or their salt can be added to the reaction solution independently, can be added to the specimen, and can also be added to the chromogenic substrate solution.

[0051] In the present application specification, enzyme immunoassay (EIA) is an immunoassay that utilizes the enzymatic activity of TRACP-5b for detection, for example, TRACP-5b in the specimen is bound to an anti-TRACP-5b monoclonal antibody, an enzyme substrate of TRACP-5b, such as p-nitrophenyl phosphate or a salt thereof, is reacted with the bound TRACP-5b, and the enzyme activity of TRACP-5b is measured. More specifically, first, the specimen is contacted with an anti-TRACP-5b monoclonal antibody adsorbed on a solid support, and TRACP-5b in the specimen is bound to the fixed antibody. Next, the solid support is cleaned with a cleaning solution, and after removing the components from the specimen that are not adsorbed by the antibody, an enzyme substrate of TRACP-5b, such as p-nitrophenyl phosphate or a salt thereof, is added to the reaction system, and TRACP-5b bound to the antibody is reacted with the substrate. The enzyme reaction is stopped with a reaction stopping solution, and then the absorbance of the phenols generated by the reaction, such as p-nitrophenol, is measured at a specified wavelength, such as 390nm to 450nm, preferably 400 to 430nm. Since the magnitude of the absorbance reflects the TRACP-5b enzyme activity, the TRACP-5b in the specimen can be measured by this value. In the enzyme immunoassay (EIA), the above-mentioned organic sulfonic acid, sulfate or salt thereof can be added to the support independently, can be added to the specimen or the specimen diluent, can be added to the reagent containing the anti-TRACP-5b monoclonal antibody, and can be added to the color substrate solution.

[0052] In the present application, enzyme-linked immunosorbent assay (ELISA) is a method for measuring the activity of an enzyme to determine TRACP-5b using two different antibodies against TRACP-5b (one of which is an antibody labeled with a substance detectable by an enzyme reaction). For example, a specimen is brought into contact with a first anti-TRACP-5b monoclonal antibody adsorbed on a solid support, TRACP-5b in the specimen is allowed to bind to the first antibody, then TRACP-5b is bound to an epitope different from that of the first antibody, a second anti-TRACP-5b monoclonal antibody labeled with a substance detectable by an enzyme reaction is brought into contact with the fixed TRACP-5b, the second antibody is allowed to bind to TRACP-5b, and TRACP-5b is determined by an enzyme reaction using the label of the second antibody. More specifically, first, the first anti-TRACP-5b monoclonal antibody is adsorbed on a solid support, and the specimen is brought into contact with the fixed first antibody, so that TRACP-5b in the specimen is bound to the first antibody. Next, the solid support is washed with a washing solution to remove components from the specimen that are not adsorbed by the antibody, and then the fixed TRACP-5b is brought into contact with a second anti-TRACP-5b monoclonal antibody or polyclonal antibody that is labeled with a substance detectable by an enzyme reaction, such as an enzyme or biotin, and binds to TRACP-5b at an epitope different from that of the first antibody, so that TRACP-5b is bound to the second antibody. Next, according to the labeling of the second antibody, for example, biotin is reacted with peroxidase-labeled streptavidin, and then an enzyme reaction of peroxidase is performed, or a labeled enzyme is reacted with its substrate, etc., to generate a color-developing substance, and TRACP-5b can be measured by measuring the absorbance of the color-developing substance. Enzymes used as substances detectable by enzyme reaction include peroxidase, alkaline phosphatase, etc., and substances other than enzymes detectable by enzyme reaction include avidin and substrates of various enzymes. When avidin is used, ABC-based color development can be utilized. In the enzyme-linked immunosorbent assay (ELISA), the above-mentioned organic sulfonic acid, sulfate or their salts can be added to the support independently, can be added to the specimen or specimen diluent, can be added to the reagent containing the first antibody and / or the second antibody, and can also be added to the color developing substrate solution.

[0053] In the present application, the chemiluminescent enzyme immunoassay (CLEIA method) is an immunoassay for detecting the enzymatic activity of TRACP-5b by chemiluminescence, for example, a specimen is brought into contact with an anti-TRACP-5b monoclonal antibody adsorbed on a solid support such as beads, TRACP-5b in the specimen is bound to the antibody, a chemiluminescent substrate of TRACP-5b is brought into contact with the fixed TRACP-5b, and the activity of TRACP-5b is measured by measuring the generated chemiluminescence. More specifically, first, a specimen is brought into contact 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 fixed antibody. Next, the fixed solid phase support obtained by fixing magnetic particles with magnetic force is washed with a washing solution to remove the components from the specimen that are not adsorbed by the antibody, and then a chemiluminescent substrate of 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}phenyl phosphate-disodium salt reaction (CDP-Star, registered trademark) is added to the reaction system to react TRACP-5b bound to the antibody with the substrate, and the luminescence intensity is measured. Since the luminescence intensity reflects the TRACP-5b enzyme activity, the TRACP-5b in the specimen can be measured based on its value. In the chemiluminescent enzyme immunoassay (CLEIA method), the above-mentioned organic sulfonic acid, sulfate or their salts can be added to the support independently, can be added to the specimen or specimen diluent, can be added to the reagent containing anti-TRACP-5b monoclonal antibody, and can also be added to the colorimetric substrate solution.

[0054] The chemiluminescent enzyme immunoassay (CLEIA method) can also be described as follows: a specimen is brought into contact with a first anti-TRACP-5b monoclonal antibody immobilized on a solid support, TRACP-5b in the specimen is allowed to bind to the first antibody, then the immobilized TRACP-5b is brought into contact with a second anti-TRACP-5b monoclonal antibody that binds to TRACP-5b with an epitope different from that of the first antibody and is labeled with a chemiluminescent substrate or a substance detectable by chemiluminescence, TRACP-5b is allowed to bind to the second antibody, chemiluminescence is generated according to the label of the second antibody or by utilizing the label, and its luminescence intensity is measured, thereby determining TRACP-5b. More specifically, first, the first anti-TRACP-5b monoclonal antibody is adsorbed on a solid support such as magnetic particles, the specimen is brought into contact with the immobilized first antibody, and TRACP-5b in the specimen is allowed to bind to the first antibody. Next, the fixed solid phase support obtained by fixing magnetic particles with magnetic force is washed with a washing solution to remove components from the specimen that are not adsorbed by the antibody, and then the fixed TRACP-5b is brought into contact with a second anti-TRACP-5b monoclonal antibody or polyclonal antibody that is labeled with a substance that can produce chemiluminescence using an enzyme or enzyme reaction and binds to TRACP-5b with an epitope different from that of the first antibody, so that TRACP-5b is bound to the second antibody, and then the luminescence intensity is measured based on the label of the second antibody or the chemiluminescence produced by the label, thereby determining TRACP-5b. In the chemiluminescent enzyme immunoassay (CLEIA method), the organic sulfonic acid, sulfuric acid ester or salt thereof may be added independently to a support, added to a specimen, or added to a reagent containing the first antibody and / or the second antibody.

[0055] In the present application, the latex agglutination method is a method for measuring TRACP-5b by measuring the turbidity generated by the agglutination of latex particles as absorbance, for example, a specimen is brought into contact with an anti-TRACP-5b polyclonal antibody adsorbed on latex particles, the latex particles are agglutinated via TRACP-5b, and the turbidity (absorbance) thus generated is measured to measure TRACP-5b. In the latex agglutination method, the organic sulfonic acid, sulfuric acid ester or salt thereof may be added independently to the reaction solution, may be added to the specimen, or may be added to a reagent containing latex particles.

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

[0057] 2. TRACP-5b assay kit Another embodiment of the present invention relates to a TRACP-5b measurement kit, which is characterized by comprising the above-mentioned organic sulfonic acid, sulfate ester and / or salts thereof. Depending on the composition of the kit and the assay principle, the organic sulfonic acid, sulfate ester or their salts can be included in the kit in the following ways: 1) as an independent solution, 2) contained in a specimen diluent, 3) contained in a reagent containing a substrate, or 4) contained in a reagent containing an anti-TRACP-5b antibody.

[0058] The concentration of the organic sulfonic acid, sulfate or their salts in each solution or reagent is preferably adjusted based on the density of the sulfonic group (-S(O)2OH), sulfate group (-OS(O)2OH) and / or their salts of the organic sulfonic acid, sulfate or their salts, the molecular weight of these compounds and the measurement scheme (measurement conditions) (especially the dilution factor (amount (vol) of the reaction solution / amount (vol) of the solution or reagent in which the organic sulfonic acid, sulfate or their salt is dissolved or added)) to achieve the above-mentioned concentration in the reaction solution.

[0059] For example, when dextran sulfate 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 0.03×10 -3 ~10.0% by mass, more preferably 0.15×10 -3 ~5.0 mass %, more preferably 0.3×10 -3 When dextran sulfate or a salt thereof having a molecular weight of 40,000 to 60,000 is used, the concentration thereof in the solution or reagent is preferably 0.03×10 -4 ~5.0 mass %, more preferably 0.15×10 -4 ~1.0 mass%, more preferably 0.5×10 -4 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 ~1.0 mass%, more preferably 0.3×10 -5 ~0.1 mass%, more preferably 0.5×10 -5 ~0.05 mass%.

[0060] In addition, for example, when vinyl sulfonic acid or its salt is used, the concentration (C) in the solution or reagent is preferably 0.012 to 15.0 mass %, more preferably 0.020 to 5.0 mass %, and further preferably 0.10 to 3.0 mass %. In addition, for example, when polyvinyl sulfonic acid or its salt having an average molecular weight of 4000 to 6000 is used, the concentration (C) in the solution or reagent is preferably 0.08×10 -3~5.0 mass %, more preferably 0.3×10 -3 ~1.0 mass%, more preferably 0.3×10 -2 ~0.3 mass %.

[0061] As described above, TRACP-5b can be measured by various methods, and a TRACP-5b measurement kit is also composed of different reagents depending on the measurement method.

[0062] For example, a kit for measuring TRACP-5b by an enzymatic method includes a substrate for TRACP-5b, the above-mentioned organic sulfonic acid, sulfate or salt thereof, and an optional reagent for sample dilution. In addition, in the kit, the above-mentioned organic sulfonic acid, sulfate or salt thereof may be contained in an independent solution or in a reagent containing the substrate for TRACP-5b or a reagent for sample dilution. In the independent solution and the reagent for sample dilution contained in the kit, the above-mentioned organic sulfonic acid, sulfate or salt thereof may be dissolved in a buffer such as tris(hydroxymethyl)aminomethane (Tris).

[0063] The kit for measuring TRACP-5b by enzyme immunoassay (EIA) comprises: a solid support such as a porous plate, an anti-TRACP-5b monoclonal antibody adsorbed on the solid support, a TRACP-5b substrate, the above-mentioned organic sulfonic acid, sulfate or their salts, an optional specimen dilution reagent, and an optional cleaning solution. In addition to being adsorbed on a solid support, the anti-TRACP-5b monoclonal antibody can also be included in the kit as an antibody solution. In this case, when measuring TRACP-5b, the antibody is adsorbed on the solid support. After the TRACP-5b in the specimen is bound to the antibody, a cleaning solution is used to remove the components in the specimen that are not adsorbed. As the cleaning solution, for example, a tris (hydroxymethyl) aminomethane (Tris) buffer containing a surfactant can be used. The sample diluent is used to adjust the concentration of the sample, and for example, a buffer such as tris(hydroxymethyl)aminomethane (Tris) can be used. In addition, a chelating agent such as EDTA-2Na or an inorganic salt such as salt can be added to the buffer as needed. Examples of the material of the solid support include polystyrene, polypropylene, polycarbonate, polyethylene, nylon, methacrylate, etc. Examples of the shape of the solid support include plates, (magnetic) beads (particles), and the like. In the kit, the organic sulfonic acid, sulfate or salt thereof may be contained in an independent solution or in a reagent containing a TRACP-5b substrate, a reagent containing an anti-TRACP-5b monoclonal antibody or a reagent for sample dilution. The organic sulfonic acid, sulfate or salt thereof may be dissolved in a buffer such as tris(hydroxymethyl)aminomethane (Tris) or the like in an independent solution or a reagent for sample dilution contained in the kit.

[0064] The kit for measuring TRACP-5b by enzyme-linked immunosorbent assay (ELISA) comprises: a solid phase support, a first anti-TRACP-5b monoclonal antibody adsorbed on the solid phase support, a second anti-TRACP-5b monoclonal antibody that binds to TRACP-5b with an epitope different from that of the first anti-TRACP-5b monoclonal antibody and is labeled with a substance that can be detected in an enzyme reaction, a reagent for making the label of the second anti-TRACP-5b monoclonal antibody detectable in an enzyme reaction, an organic sulfonic acid, a sulfate ester or a salt thereof, an optional reagent for sample dilution, and an optional cleaning solution. The first anti-TRACP-5b monoclonal antibody can be included in the kit as an antibody solution in addition to being adsorbed on a solid support. In this case, when measuring TRACP-5b, the first antibody is adsorbed on a solid support. After TRACP-5b in the specimen is bound to the first antibody or the second antibody, a cleaning solution is used to remove components that are not bound to these antibodies. As a cleaning solution, for example, a tris (hydroxymethyl) aminomethane (Tris) buffer containing a surfactant can be used. The sample diluent is used to adjust the concentration of the sample, and for example, a buffer such as tris(hydroxymethyl)aminomethane (Tris) can be used. In addition, a chelating agent such as EDTA-2Na or an inorganic salt such as salt can be added to the buffer as needed. In the kit, the organic sulfonic acid, sulfate or salt thereof may be contained in an independent solution, or in a labeled reagent capable of detecting the second anti-TRACP-5b monoclonal antibody in an enzyme reaction, a reagent containing the first antibody or the second antibody, or a reagent for sample dilution. In the independent solution and the reagent for sample dilution contained in the kit, the organic sulfonic acid, sulfate or salt thereof may be dissolved in a buffer such as tris(hydroxymethyl)aminomethane (Tris). Examples of substances that can be detected in an enzyme reaction include enzymes other than ACP, substrates of enzymes other than ACP, and biotin. In addition, when the labeling substance is biotin, the reagent for detecting the labeling substance by enzyme reaction is, for example, a reagent containing peroxidase-labeled streptavidin, a peroxidase substrate of tetramethylbenzidine, and hydrogen peroxide. In addition, when the labeling substance is an enzyme, the reagent for detecting the labeling substance by enzyme reaction is, for example, its substrate, and when the labeling substance is alkaline phosphatase, the reagent for detecting the labeling substance by enzyme reaction is, for example, p-nitrophenyl phosphate, which is a substrate of the enzyme. Examples of the material of the solid support include polystyrene, polypropylene, polycarbonate, polyethylene, nylon, methacrylate, etc. Examples of the shape of the solid support include plates, (magnetic) beads (particles), and the like.

[0065] A kit for determining TRACP-5b by chemiluminescent enzyme immunoassay (CLEIA method) includes, for example: a solid phase support, an anti-TRACP-5b monoclonal antibody adsorbed on the solid phase support, a chemiluminescent substrate for TRACP-5b, the above-mentioned organic sulfonic acid, sulfate ester or their salts, an optional reagent for sample dilution, and an optional cleaning solution. The anti-TRACP-5b monoclonal antibody may be contained in the kit as an antibody solution in addition to being adsorbed on a solid support. In this case, when measuring TRACP-5b, the antibody is adsorbed on a 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)phenyl phosphate-disodium salt, 4-chloro-3-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane}-4-yl)phenyl phosphate-disodium salt, 3-(4-methoxyspiro{1,2-dioxetane-3,2'-tricyclo[3.3.1.1(3,7)]decane}-4-yl)phenyl phosphate-disodium salt, and 4-methoxy-4-(3-phenyl phosphate)spiro[1,2-dioxetane-3,2'-adamantane]-disodium salt; or (2) Acridinium derivatives such as [10-methyl-9(10H)-acridinylindene]phenoxymethylphosphonic acid disodium salt

[0067] The sample diluent is used when adjusting the concentration of the sample, and for example, a buffer selected from the following can be used: 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-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer, N-(2-acetamido)iminodiacetic acid (ADA) buffer, N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, and phosphate buffer. In addition, cobalt ions or manganese ions can be added to the buffer as metal ions as needed. After TRACP-5b in the sample is bound to the antibody, the non-adsorbed components in the sample are removed using a washing solution. As the washing solution, for example, the above-mentioned buffer solution containing a surfactant can be used. In the kit, the organic sulfonic acid, sulfate or salt thereof may be contained in an independent solution, or in a reagent containing a TRACP-5b chemiluminescent substrate, a reagent containing an anti-TRACP-5b monoclonal antibody or a reagent for sample dilution. In the independent solution and the reagent for sample dilution contained in the kit, for example, the organic sulfonic acid, sulfate or salt thereof may be dissolved in the buffer.

[0068] The kit for measuring TRACP-5b by chemiluminescent immunoassay (CLIA method) includes: a solid phase support, a first anti-TRACP-5b monoclonal antibody adsorbed on the solid phase support, a second anti-TRACP-5b monoclonal antibody that binds to TRACP-5b with an epitope different from that of the first anti-TRACP-5b monoclonal antibody and is labeled with a substance detectable by chemiluminescence, a reagent that can detect the label by chemiluminescence, the above-mentioned organic sulfonic acid, sulfate or salt thereof, an optional reagent for sample dilution, and an optional cleaning solution. The first anti-TRACP-5b monoclonal antibody may be adsorbed on a solid support or contained in the kit as an antibody solution. In this case, when measuring TRACP-5b, the first antibody is adsorbed on a solid support. After TRACP-5b in the specimen is bound to the first antibody or the second antibody, a washing solution is used to remove components not bound to these antibodies. The washing solution and the specimen diluent are the same as above. When the label is biotin, the reagent that can detect the label by chemiluminescence is, for example, a reagent containing peroxidase-labeled streptavidin, a chemiluminescent substrate of peroxidase, and hydrogen peroxide. In addition, when the label is an enzyme such as alkaline phosphatase, the reagent that can detect the label by chemiluminescence is, for example, a substrate of the enzyme, such as p-nitrophenyl phosphate, etc. The chemiluminescent substrate is the same as described above. Examples of the material of the solid support include polystyrene, polypropylene, polycarbonate, polyethylene, nylon, methacrylate, etc. Examples of the shape of the solid support include plates, (magnetic) beads (particles), and the like. In the kit, the organic sulfonic acid, sulfate or salt thereof may be contained in an independent solution, or in a reagent that can detect a label by chemiluminescence, a reagent containing the first antibody or the second antibody, or a reagent for sample dilution. In the independent solution and the reagent for sample dilution contained in the kit, for example, the organic sulfonic acid, sulfate or salt thereof may be dissolved in the buffer.

[0069] The kit for measuring TRACP-5b by latex agglutination method comprises: latex particles, anti-TRACP-5b polyclonal antibodies or anti-TRACP-5b monoclonal antibodies adsorbed on the latex particles, the above-mentioned organic sulfonic acid, sulfate ester or their salts, and an optional sample dilution reagent. In the kit, the above-mentioned organic sulfonic acid, sulfate ester or their salts can be contained in an independent solution or in a reagent containing latex particles or a sample dilution reagent. In the independent solution and sample dilution reagent contained in the kit, the above-mentioned organic sulfonic acid, sulfate ester or their salts can be dissolved in, for example, the above-mentioned buffer. In addition, examples of the buffer 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-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer, N-(2-acetamido)iminodiacetic acid (ADA) buffer, N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, and phosphate buffer. Example

[0070] Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto. In addition, the meanings of the abbreviations used in the examples are as follows. DSS Na 5k Dextran sulfate sodium with average molecular weight of 5000 DSS Na 50k Dextran sulfate sodium with average molecular weight of 50,000 DSS Na 500k Dextran sulfate sodium with average molecular weight of 500000 PVSA Na 5k Polyvinyl sodium sulfate with an average molecular weight of 5000 VSA Sodium vinyl sulfate β-CyD-S Na β-Cyclodextrin Sodium Sulfate PAA 5k Polyacrylic acid with an average molecular weight of 5000 Poly-Glu Na Sodium Polyglutamate Chon S Na Chondroitin Sulfate Sodium PEG 6k Polyethylene glycol with an average molecular weight of 6000

[0071] Experiment 1. Evaluation of TRACP-5a inhibitors by enzymatic method 1-1. Evaluation compounds The following compounds were evaluated for their TRACP-5a inhibitory effects by an enzymatic method.

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

[0073] 1-3. Assay reagent (chromogenic substrate solution) A chromogenic substrate solution of the following composition was used.

[0074] 1-4. Measurement method The determination was performed using a general-purpose Hitachi automatic analyzer 7180 (manufactured by Hitachi High-Tech Co., Ltd.). 200 μL of a chromogenic substrate solution containing the evaluation compound was added to 16 μL of a 7U / L TRACP-5b solution or a 7U / L TRACP-5a solution or an 8U / L TRACP-5a solution, and the enzyme reaction was started, and the absorbance (main wavelength 405 nm, secondary wavelength 480 nm) from 340.5 seconds to 516.0 seconds was measured. Based on the change in absorbance between the two time points, the change in absorbance per minute (the change in absorbance per unit time in the enzyme reaction) was calculated. In addition, a TRACP-5b solution with known activity was prepared by serial dilution with 22 mM MES buffer (pH 5.0) and a blank solution of 22 mM MES buffer (pH 5.0), and the serial dilution of the TRACP-5b solution was measured using a chromogenic substrate solution containing each evaluation compound to prepare a standard straight line for TRACP-5b, and a chromogenic substrate solution not containing the evaluation compound was used to measure the serial dilution of the TRACP-5b solution in the same manner to prepare a standard straight line for 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. In addition, the measurement using sodium hyaluronate was carried out separately from the other evaluation compounds in batches.

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

[0076] Experiment 2. Study on the added concentration of TRACP-5a inhibitor The effects of the added concentrations of sodium dextran sulfate (5K), sodium dextran sulfate (50K), sodium polyvinyl sulfonate (5K) and sodium vinyl sulfonate on the inhibitory activity were studied.

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

[0078] 2-2. Measurement samples In the test using the reagent containing sodium dextran sulfate (5K) and sodium dextran sulfate (50K), 7U / L TRACP-5b solution and 7U / L TRACP-5a solution were used, in the test using the reagent containing sodium polyvinyl sulfonate (5K), 7U / L TRACP-5b solution and 7U / L or 8U / L TRACP-5a solution were used, and in the test using the reagent containing sodium vinyl sulfonate, 7U / L TRACP-5b solution and 8U / L TRACP-5a solution were used.

[0079] 2-3. Assay reagent (chromogenic substrate solution) and assay method A chromogenic substrate solution having the same composition as that in Test 1 was used except that the concentration of each evaluation compound was set as above. The measurement method was carried out in accordance with the same procedure as that in Test 1.

[0080] 2-4. Measurement results [Table 6] [Table 7] Example 4 is data from another batch, but is described with reference to the data in Experiment 1. [Table 8] Example 5 is data from another batch, but is described with reference to the data in Experiment 1. [Table 9] Example 1 is data from another batch, but is described with reference to the data in Experiment 1. [Table 10] Example 2 is data from another batch, but is described with reference to the data in Experiment 1.

[0081] 3. Evaluation of TRACP-5a inhibitors by EIA 3-1. Evaluation compounds The following compounds were evaluated for their TRACP-5a inhibitory effects by EIA.

[0082] 3-2. Preparation of antibody-immobilized plate TRACP-5b monoclonal antibody (trust 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 (manufactured by ThermoFisher SCIENTIFIC, model 468667). After standing overnight in a freezer, 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 then 300 μL of TBS containing 1.5% BSA was added, and the plate was stood overnight in a freezer.

[0083] 3-3. Measurement method After removing TBS containing 1.5% BSA from the prepared 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 the mixture was reacted for 1 hour (25°C, 700 rpm) while stirring. The mixture was washed 3 times with 300 μL of TBS-T, and then 100 μL of a chromogenic substrate solution (5 mM 2-chloro-4-nitrophenyl phosphate, 109 mM MES, 40 mM sodium tartrate, pH 6.6) to which the evaluation compound was added at a concentration of 0.1% by mass was added to each well, and the mixture was allowed to stand at 37°C for 1 hour. Then, 50 μL of 0.5 N NaOH was added, and the absorbance was measured with a plate reader (main wavelength 405 nm, secondary wavelength 490 nm).

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

Claims

1. A method for determining TRACP-5b, which is a method for determining tartrate-resistant acid phosphatase 5b (TRACP-5b) in a specimen, comprising a step of conducting a reaction to detect TRACP-5b in the presence of an organic sulfonic acid, sulfate ester or a salt thereof, wherein the density of the sulfone group (-S(O)2OH), sulfate group (-OS(O)2OH) and / or a salt thereof in the organic sulfonic acid, sulfate ester or a salt thereof is greater than 6.5 mmol / g.

2. The method of claim 1, wherein: The organic sulfonic acid, sulfate ester or salt thereof has 50% by mass or more of sulfonic groups (-S(O)2OH), sulfate groups (-OS(O)2OH) and / or salts thereof per unit structure in terms of S(O)2O- conversion value.

3. The method according to claim 1 or 2, wherein The organic sulfonic acid, sulfate or their salts: It is expressed by formula (1): RYS(O)2OX…(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; or, A repeating unit having formula (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 repeating unit having a structure comprising formula (3), In formula (3), Y is O or CH2, R 1 ~R 5 Two of them are O- or bonds, and at least two of the remaining two are OS(O)2OX or S(O)2OX, where X is hydrogen or an alkali metal, and R 1 ~R 5 None of them is OX or C(O)OX, wherein X is hydrogen or an alkali metal.

4. The method according to claim 1 or 2, wherein The organic sulfonic acid, sulfuric acid ester or salt thereof is vinylsulfonic acid or salt thereof, vinyl sulfate or salt thereof, polyvinylsulfonic acid or salt thereof, polyvinylsulfonic acid or salt thereof, methanesulfonic acid or salt thereof, or dextran sulfate or salt thereof.

5. The method according to any one of claims 1 to 4, wherein The organic sulfonic acid, sulfate or their salts: 1) Added independently to the reaction system or sample, or Contained in 2) a sample diluent, 3) a reagent containing a substrate, or 4) a reagent containing an anti-TRACP-5b antibody, Thus, the reaction for detecting TRACP-5b is carried out in the presence of the organic sulfonic acid, sulfate ester or a salt thereof.

6. The method according to any one of claims 1 to 5, wherein The assay was performed by the following methods: enzyme method, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA method), chemiluminescent immunoassay (CLIA method) or latex agglutination method.

7. The method according to any one of claims 1 to 6, wherein The sample is blood, serum or plasma.

8. A TRACP-5b assay kit comprising an organic sulfonic acid, a sulfate ester and / or their salts, wherein the density of the sulfonic group (-S(O)2OH), the sulfate group (-OS(O)2OH) and / or their salts in the organic sulfonic acid, the sulfate ester and / or their salts is greater than 6.5 mmol / g.

9. The kit according to claim 8, wherein The organic sulfonic acid, sulfuric acid ester and / or their salts have 50% by mass or more of sulfonic groups (-S(O)2OH), sulfuric acid groups (-OS(O)2OH) or their salts per unit structure in terms of S(O)2O- conversion value.

10. The kit according to claim 8 or 9, wherein The organic sulfonic acid, sulfate and / or their salts: It is expressed by formula (1): RYS(O)2OX…(1) 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; or, Having a repeating structure of formula (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 repeating structure comprising a structure of formula (3), In formula (3), Y is O or CH2, R 1 ~R 5 Two of them are O- or bonds, and at least two of the remaining two are OS(O)2OX or S(O)2OX, where X is hydrogen or an alkali metal, and R 1 ~R 5 None of them is OX or C(O)OX, wherein X is hydrogen or an alkali metal.

11. The kit according to claim 8 or 9, wherein The organic sulfonic acid, sulfuric acid ester or salt thereof is vinylsulfonic acid or salt thereof, vinyl sulfate or salt thereof, polyvinylsulfonic acid or salt thereof, polyvinylsulfonic acid or salt thereof, methanesulfonic acid or salt thereof, or dextran sulfate or salt thereof.

12. The kit according to any one of claims 8 to 11, which is used for measuring TRACP-5b by an enzymatic method. The kit comprises: a substrate of TRACP-5b, the organic sulfonic acid, sulfate ester or salt thereof, and an optional reagent for sample dilution. 13 . The kit according to claim 12 , wherein the organic sulfonic acid, sulfuric acid ester or a salt thereof is contained in a reagent containing the substrate of TRACP-5b or the reagent for diluting the specimen.

14. The kit according to any one of claims 8 to 11, which measures TRACP-5b by enzyme immunoassay (EIA), The kit comprises: a solid support, an anti-TRACP-5b monoclonal antibody adsorbed on the solid support, the TRACP-5b substrate, the organic sulfonic acid, sulfate or salt thereof, an optional sample dilution reagent, and an optional cleaning solution. 15 . The kit according to claim 14 , wherein the organic sulfonic acid, sulfuric acid ester or salt thereof is contained in a reagent containing the anti-TRACP-5b monoclonal antibody, a reagent containing the substrate of TRACP-5b or the reagent for sample dilution.

16. The kit according to any one of claims 8 to 11, wherein TRACP-5b is measured by enzyme-linked immunosorbent assay (ELISA), The kit comprises: a solid phase support, a first anti-TRACP-5b monoclonal antibody adsorbed on the solid phase support, a second anti-TRACP-5b monoclonal antibody that binds to TRACP-5b with an epitope different from that of the first anti-TRACP-5b monoclonal antibody and is labeled with a substance that can be detected in an enzyme reaction, a reagent for making the labeled substance detectable in an enzyme reaction, the organic sulfonic acid, sulfate or salt thereof, an optional reagent for sample dilution, and an optional cleaning solution.

17. The kit according to claim 16, wherein the organic sulfonic acid, sulfuric acid ester or salt thereof is contained in a reagent containing the second anti-TRACP-5b monoclonal antibody, a reagent for making the label detectable in an enzyme reaction or a reagent for sample dilution.

18. The kit according to any one of claims 8 to 11, which measures TRACP-5b by chemiluminescent enzyme immunoassay (CLEIA method), The kit comprises: a solid support, an anti-TRACP-5b monoclonal antibody adsorbed on the solid support, a chemiluminescent substrate of TRACP-5b, the organic sulfonic acid, sulfate or salt thereof, an optional sample dilution reagent, and an optional cleaning solution. 19 . The kit according to claim 18 , wherein the organic sulfonic acid, sulfuric acid ester or salt thereof is contained in a reagent containing a chemiluminescent substrate of TRACP-5b, a reagent containing an anti-TRACP-5b monoclonal antibody or a reagent for diluting a specimen.

20. The kit according to any one of claims 8 to 11, which measures TRACP-5b by chemiluminescent immunoassay (CLIA method), The kit comprises: a solid phase support, a first anti-TRACP-5b monoclonal antibody adsorbed on the solid phase support, a second anti-TRACP-5b monoclonal antibody that binds to TRACP-5b with an epitope different from that of the first anti-TRACP-5b monoclonal antibody and is labeled with a substance that can be detected in chemiluminescence, a reagent that enables the labeled substance to be detected in chemiluminescence, the organic sulfonic acid, sulfate or salt thereof, an optional reagent for sample dilution, and an optional cleaning solution.

21. The kit according to claim 20, which contains the organic sulfonic acid, sulfate or salt thereof in a reagent containing the first anti-TRACP-5b monoclonal antibody, a reagent containing the second anti-TRACP-5b monoclonal antibody, a reagent that enables the marker to be detected by chemiluminescence or a reagent containing the specimen dilution reagent.

22. The kit according to any one of claims 8 to 11, wherein TRACP-5b is measured by latex agglutination method. The kit comprises latex particles, anti-TRACP-5b antibodies immobilized on the latex particles, the organic sulfonic acid, sulfate or salt thereof, and an optional reagent for sample dilution. 23 . The kit according to claim 22 , wherein the organic sulfonic acid, sulfuric acid ester or a salt thereof is contained in a reagent containing the anti-TRACP-5b antibody or the reagent for diluting a specimen.

24. The kit according to any one of claims 8 to 23, wherein the sample to be measured is blood, serum or plasma.

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