TRACP-5b MEASUREMENT METHOD AND KIT THEREFOR

Abstract

Provided is a measurement method capable of selectively detecting TRACP-5b by further reducing the influence of co-existing TRACP-5a in the measurement of TRACP-5b.　A reaction for detecting TRACP-5b is performed in the presence of an organic sulfonic acid, a sulfuric acid ester, or a salt thereof, which has a sulfo group (-S(O)2OH), a sulfuric acid group (-OS(O)2OH), and / or a salt thereof at a density of at least 6.5 mmol / g.

Description

METHOD FOR MEASURING TRACP-5b AND KIT THEREFOR

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

[0002] Acid phosphatase (ACP) is an enzyme that has the activity of hydrolyzing organic monophosphate esters under acidic conditions. ACP exists in multiple isozymes and is distributed in various tissues. ACPs known to exist in serum are derived from platelets, erythrocytes, prostates, osteoclasts, macrophages, and the like. These isozymes are separated by polyacrylamide gel electrophoresis, and osteoclast- and macrophage-derived ACPs are located fifth from the origin and are resistant to tartaric acid, and are therefore also called Band-5 tartrate-resistant acid phosphatase (TRACP-5) (Patent Document 5, Lam W.K.W. et al., Clin. Chem. 24(7):1105-1108, 1978, Lam W.K.W. 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 its sugar chain, and TRACP-5b, which has a low degree of sialic acid binding; 5a is said to be derived from macrophages, and 5b is derived from osteoclasts. Because serum TRACP-5b concentrations sensitively reflect osteoclast activity, they are used as a bone resorption marker to aid in the diagnosis of metabolic bone diseases and bone metastatic cancers, as well as to assess the effectiveness of treatment. Therefore, methods for selectively measuring TRACP-5b from among the ACPs in serum have been investigated.

[0003] Conventional methods for measuring TRACP-5 include performing an enzymatic reaction using a synthetic substrate such as p-nitrophenyl phosphate in the presence of tartaric acid, and then determining the enzymatic activity by colorimetrically quantifying the resulting reaction product. However, although tartaric acid can inhibit prostate-derived ACP, TRACP-5a and ACPs derived from erythrocytes and platelets are resistant to tartaric acid. Therefore, this method also measures these isozymes, making it impossible to specifically measure TRACP-5b.

[0004] A known method for improving the TRACP-5b specificity of the above method involves incubating a 5-fold diluted serum solution at 37°C for 1 hour and then measuring the enzyme activity in the same manner (Non-Patent Document 3). This method can avoid the effects of not only prostate-derived ACP but also erythrocyte-derived ACP, but it still cannot avoid the effects of TRACP-5a and platelet-derived ACP.

[0005] Another method for specifically measuring TRACP-5b activity is to calculate TRACP-5b activity by subtracting ACP activity measured in the presence of tartaric acid and fluoride from ACP activity measured in the presence of tartaric acid (Patent Document 1). This method can avoid the influence of red blood cell-derived and platelet-derived ACPs by utilizing the difference in sensitivity to fluoride. However, it is not possible to eliminate the influence of TRACP-5a, and TRACP-5b activity is determined by subtraction, which has problems with accuracy.

[0006] Another method has been reported in which TRACP-5b activity is specifically measured using acidic mucopolysaccharides as inhibitors of acid phosphatases other than TRACP-5b (Patent Document 2). However, the inhibitory effect of acidic mucopolysaccharides on TRACP-5a activity is insufficient, and further improvement in the specificity for TRACP-5b is desired.

[0007] A known method for measuring TRACP-5b activity using a chemiluminescent substrate involves reacting TRACP-5b with a chemiluminescent 1,2-dioxetane compound under acidic conditions, followed by measuring the luminescence intensity by changing the reaction solution to alkaline conditions (Patent Document 3). Additionally, a method has been reported for specifically measuring TRACP-5b by utilizing the fact that the luminescence intensity ratio of TRACP-5b to TRAPC-5a is increased by using a specific buffer such as MES during the enzyme reaction or by adding manganese ions or cobalt ions (Patent Document 4). However, these methods are limited to activity measurements using chemiluminescent substrates, and there is a need for a less expensive method that can specifically measure TRACP-5b.

[0008] An example of an antibody-based immunoassay for TRACP-5b is a method in which an antibody specific to TRACP-5 is used to bind both TRACP-5a and TRACP-5b, 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 determined by subtraction, which poses a problem in terms of accuracy.

[0009] There is also a method for more specifically measuring TRACP-5b by using an antibody that has a certain selectivity for TRACP-5b and then measuring the activity using 2-chloro-4-nitrophenyl phosphate as a chromogenic substrate after antibody binding (Patent Documents 6 and 7). However, it is desirable 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 clones of monoclonal antibodies that have high specificity for TRACP-5b (Patent Document 8).

[0011] JP 10-337198, JP 2001-231595, JP 2011-024470, JP 2022-174015, JP 2002-510050, JP 2004-026805, WO 2004 / 077059, WO 2016 / 027697

[0012] Non-patent literature Clin. Chem. 33:458-462.1987

[0013] In contrast to the above-mentioned conventional techniques, the present invention aims to provide a method for measuring TRACP-5b that can selectively detect TRACP-5b while further reducing the influence of coexisting TRACP-5a.

[0014] The present inventors have investigated a method for selectively detecting TRACP-5b, and have found that a sulfo group (-S(O) ２ OH), sulfate group (-OS(O) ２ They found that the presence of a compound having TRACP-5b (H) and / or a salt thereof at a certain density or higher can significantly reduce the influence of TRACP-5a, thereby enabling selective detection of TRACP-5b.

[0015] That is, the present invention provides the following method for measuring TRACP-5b and kit for measuring TRACP-5b: [1] A method for measuring tartrate-resistant acid phosphatase 5b (TRACP-5b) in a sample, comprising: ２ OH), sulfate group (-OS(O) ２ 1. A method for measuring TRACP-5b, comprising the 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 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: ２ OH), sulfate group (-OS(O) ２ OH) and / or salts thereof with S(O) ２ O － a method for measuring TRACP-5b, the method 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, the organic sulfonic acid, the sulfate ester, or the salt thereof having 50 mass % or more of a sulfo group (—S(O) ２ OH), sulfate group (-OS(O) ２OH) and / or salts thereof with S(O) ２ O － The method according to [1], wherein the content is 50% by 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): (In formula (1), R is alkyl, alkenyl, or alkynyl having 1 to 3 carbon atoms, Y is a bond or an oxygen atom, and X is hydrogen or an alkali metal.) or represented by formula (2): (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 (In 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 (wherein X is hydrogen or an alkali metal), and those that do not fall into either of the above categories are OX or C(O)OX (wherein X is hydrogen or an alkali metal). The method according to [1] or [2], wherein the repeating unit includes a structure of

[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 specimen or a reaction system, or is contained in 2) a specimen 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 carrying out 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], wherein the measurement is performed 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 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 greater.

[0022] [9] A kit for measuring TRACP-5b, comprising: a unit structure having a sulfo group (—S(O) ２ OH), sulfate group (-OS(O) ２ OH) or a salt thereof, ２ O － a kit containing an organic sulfonic acid, a sulfate ester, and / or a salt thereof having 50% by mass or more of a sulfonic acid, a sulfate ester, and / or a salt thereof in terms of a sulfo group (—S(O) ２ OH), sulfate group (-OS(O) ２ OH) or a salt thereof, ２ O － 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 is represented by the formula (1): (wherein R is alkyl, alkenyl, or alkynyl having 1 to 5 carbon atoms, Y is a bond or an oxygen atom, and X is hydrogen or an alkali metal), or represented by formula (2): (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 (In 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 of the above categories, it is OX or C(O)OX (X is hydrogen or an alkali metal). The kit according to [8] or [9],

[0024]

[11] The kit according to [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, comprising a substrate for TRACP-5b, the organic sulfonic acid, sulfate ester, or a 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 reagent for diluting a specimen contains the organic sulfonic acid, sulfate ester, or salt thereof.

[0027]

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

[11] , for measuring TRACP-5b by enzyme immunoassay (EIA), 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 a salt thereof, optionally a reagent for diluting a specimen, and optionally a washing solution.

[15] The kit according to

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

[0028]

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

[11] for measuring TRACP-5b by enzyme-linked immunosorbent assay (ELISA), 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 enzymatic reaction; a reagent for detecting the labeled substance by enzymatic reaction; the organic sulfonic acid, sulfate ester, or salt thereof; optionally, a specimen dilution reagent; and optionally, a washing solution.

[17] The kit according to

[16] , wherein the reagent containing the secondary anti-TRACP-5b monoclonal antibody, the reagent for detecting the labeled substance by enzymatic reaction, or the specimen dilution reagent contains the organic sulfonic acid, sulfate ester, or salt thereof.

[0029]

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

[11] , which measures TRACP-5b by chemiluminescent enzyme immunoassay (CLEIA), 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 a salt thereof, optionally a sample dilution reagent, and optionally a wash solution.

[19] The kit according to

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

[0030]

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

[11] , for measuring TRACP-5b by chemiluminescence immunoassay (CLIA), 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 according to

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

[0031]

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

[11] for measuring TRACP-5b by latex agglutination, comprising latex particles, an anti-TRACP-5b antibody adsorbed to the latex particles, the organic sulfonic acid, sulfate ester, or a salt thereof, and optionally a sample dilution reagent.

[23] The kit according to

[22] , wherein the reagent comprising the anti-TRACP-5b antibody or the sample dilution reagent comprises 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.

[0033] According to the present application, a means is provided that enables selective detection of tartrate-resistant acid phosphatase 5b (TRACP-5b) by reducing the influence of TRACP-5b, which is therefore useful in diagnosing diseases using TRACP-5b as a marker.

[0034] The present invention will be described in detail below with reference to the preferred embodiments, but 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 the step of carrying out a detection reaction for TRACP-5b in the presence of a specific organic sulfonic acid, sulfate ester, or salt thereof. In another embodiment, the present invention provides a kit for measuring TRACP-5b, comprising the specific organic sulfonic acid, sulfate ester, or salt thereof. These embodiments are described in detail below.

[0036] 1. Measurement method for TRACP-5b 1-1. Organic sulfonic acid, sulfate ester, or salt thereof In this measurement method, a sulfo group (-S(O) ２ OH), sulfate group (-OS(O) ２ The presence of an organic sulfonic acid, sulfate ester, or salt thereof having a sulfo group, sulfate group, and / or salt thereof at a density of 6.5 mmol / g or more in the reaction system inhibits TRACP-5a, enabling selective measurement of TRACP-5b. As shown in the Examples below, the effect of inhibiting TRACP-5a increases depending on the density of the sulfo group, sulfate group, or salt thereof. 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 8.5 mmol / g or more.

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

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

[0039] Other specific embodiments of the above organic sulfonic acids, sulfates, or salts thereof include those represented by formula (2): In formula (2), Y is a bond or an oxygen atom, and preferably a bond. n is an integer of 2 to 5000, preferably 10 to 500, and more preferably 20 to 100. X is hydrogen or an alkali metal, and 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 sulfate or a salt thereof (e.g., Na salt), and more preferably compounds having the following repeating structure: (In formula (2-1), X represents hydrogen or an alkali metal, and preferably hydrogen or Na.) A polyvinyl sulfonic acid or a salt thereof having a molecular weight of 2 to 5,000, more preferably 10 to 500, and even more preferably 20 to 100. Such polyvinyl sulfonic acid or a salt thereof typically has a molecular weight of 200 to 500,000, preferably 1,000 to 50,000, and more preferably 4,000 to 6,000. Herein, when the terms "molecular weight" or "average molecular weight" are used, they refer to the weight-average molecular weight (Mw) unless otherwise specified. Furthermore, the weight-average molecular weight (Mw) refers to a value measured by gel permeation chromatography (GPC) using a liquid chromatograph.

[0040] Still another specific embodiment of the above-mentioned organic sulfonic acid, sulfate ester, or salt thereof is a compound represented by formula (3): In formula (3), Y is O or CH ２ and preferably O. １ ~R ５ Among them, two are O— or a bond, 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 (e.g., Na)), and if it does not fall into either category, it 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 is O-S(O) ２ OX (X is hydrogen or an alkali metal (e.g., Na)).

[0041] In addition, examples of repeating units containing this structure include: In formula (3-1), Y is O or CH ２ and preferably O. １ and L ４are each independently O or a bond, and preferably one is O— and the other is a bond. ２ and L ３ are each independently O, CH ２ or a bond, preferably one is O and the other is CH ２ or a bond. １ ~R ３ At least two of the groups are independently O—S(O) ２ OX or S(O) ２ OX (X is hydrogen or an alkali metal (e.g., Na)), and the remainder, 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 O—S(O) ２ OX or S(O) ２ OX (wherein X is hydrogen or an alkali metal (e.g., Na), and more preferably, each X is independently O—S(O) ２ OX (X is hydrogen or an alkali metal (e.g., Na)). R ４ ~R ６ At least two of the groups are independently O—S(O) ２ OX or S(O) ２ OX (X is hydrogen or an alkali metal (e.g., Na)), and the remainder, 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 O—S(O) ２ OX or S(O) ２ OX (wherein X is hydrogen or an alkali metal (e.g., Na), and more preferably, each X is independently O—S(O) ２OX (X is hydrogen or an alkali metal (e.g., Na)). n is 2 to 1000, preferably 3 to 700, more preferably 4 to 200, and even more preferably 5 to 70).

[0042] Preferred examples of the compound of this embodiment include dextran sulfate or a salt thereof (e.g., Na), and more preferably, a compound having the following repeating structure: (In formula (3-2), X is hydrogen or an alkali metal (e.g., Na).) Examples thereof include dextran sulfate or a salt thereof (e.g., Na) having a molecular weight of 2 to 1,000, preferably 3 to 700, and more preferably 5 to 70. Such dextran sulfate or a salt thereof typically 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, the presence of the above-mentioned organic sulfonic acid, sulfate ester, or salt thereof in a reaction system modifies the antigenic properties of the coexisting TRACP-5a and inhibits 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 may be added separately to a reaction solution for detecting TRACP-5b, or may be dissolved in a sample diluent and then added to a measurement sample. Alternatively, they may be added to a reagent containing an enzyme substrate or a reagent containing an anti-TRACP-5b antibody, depending on the reaction system, and a reaction (e.g., an enzyme reaction or an antigen-antibody reaction) may be carried out using these reagents.

[0044] As shown in the Examples below, the TRACP-5a inhibitory activity of the aforementioned organic sulfonic acids, sulfate esters, or salts thereof increases depending on the density and average molecular weight of the sulfo group, sulfate group, and salts thereof, 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 in a concentration-dependent manner within a certain range. However, at concentrations above a certain level, the inhibitory activity no longer increases in a concentration-dependent manner and remains at a constant level, and once the concentration reaches a certain level, the inhibitory activity gradually decreases. However, even when the inhibitory activity plateaus, high inhibitory activity is still maintained over a wide range of concentrations. In practice, compounds are often used at concentrations higher than the concentration at which the inhibitory activity no longer increases in a concentration-dependent manner in the reaction solution. For this reason, it is preferable to determine the concentration of the aforementioned organic sulfonic acids, sulfate esters, or salts thereof in the reaction solution taking these factors into consideration.

[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 0.03 × 10 －３ Preferably, the content is 0.15×10 －３ More preferably, up to 5.0 mass%, 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 in the reaction solution is preferably 0.03 × 10 －４ Preferably, the content is up to 5.0 mass%, and more preferably 0.15 × 10 －４ More preferably, up to 1.0 mass%, 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 in the reaction solution is preferably 0.01 × 10 to 0.5% by mass. －５ Preferably, the content is up to 1.0 mass%, and more preferably 0.3 × 10 －４ More preferably, up to 0.1 mass%, 0.5 × 10 －５ More preferably, it is up to 0.05% by mass.

[0046] When vinyl sulfonic acid or a salt thereof is used, the concentration thereof 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 thereof in the reaction solution is preferably 0.08×10 －３ Preferably, the content is 0.3×10 to 5.0% by mass. －３ More preferably, up to 1.0 mass%, 0.3 × 10 －２ More preferably, it is up to 0.3 mass %.

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

[0048] As used herein, the term "enzyme reaction" refers to the reaction between TRACP-5b and its substrate, and examples of TRACP-5b substrates include 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. Furthermore, as used herein, the term "antigen-antibody reaction" refers to the reaction between TRACP-5b and an antibody thereagainst, and examples of antibodies thereagainst include monoclonal antibodies, polyclonal antibodies, and antigen-binding fragments. Examples of the monoclonal antibody or antigen-binding fragment thereof include the anti-TRACP-5b monoclonal antibody or antigen-binding fragment thereof described in Patent Documents 6 and 7, etc., and more specifically, examples thereof include antibodies or antigen-binding fragments thereof produced by hybridomas with accession numbers IPOD FERM BP-7889, IPOD FERM BP-8249, and IPOD FERM BP-7890.

[0049] There are various methods utilizing enzyme reactions and antigen-antibody reactions, including, for example, enzyme methods, enzyme immunoassays (EIA), enzyme-linked immunosorbent assays (ELISA), chemiluminescent enzyme immunoassays (CLEIA), chemiluminescent immunoassays (CLIA), and latex agglutination assays.

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

[0051] As used herein, enzyme immunoassay (EIA) refers to an immunoassay that utilizes the enzymatic 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 a TRACP-5b enzyme substrate, such as p-nitrophenyl phosphate or a salt thereof, to measure the enzymatic activity of TRACP-5b. More specifically, the sample is first contacted with an anti-TRACP-5b monoclonal antibody adsorbed to a solid support, allowing TRACP-5b in the sample to bind to the immobilized antibody. Next, the solid support is washed with a washing solution to remove sample-derived components that have not adsorbed to the antibody, and then a TRACP-5b enzyme substrate, such as p-nitrophenyl phosphate or a salt thereof, is added to the reaction system, allowing the TRACP-5b bound to the antibody to react with the substrate. After the enzymatic reaction is terminated with a reaction stop solution, the absorbance of the phenols produced 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 this 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 chromogenic substrate solution.

[0052] As used herein, enzyme-linked immunosorbent assay (ELISA) refers to a method for measuring TRACP-5b by using two different antibodies against TRACP-5b, one of which is labeled with a substance that enables detection by enzymatic reaction, and measuring the activity of the enzyme. For example, a sample is contacted with a primary anti-TRACP-5b monoclonal antibody adsorbed to a solid support to allow TRACP-5b in the sample to bind to the primary antibody. Next, a secondary anti-TRACP-5b monoclonal antibody, which binds to TRACP-5b via an epitope different from that of the primary antibody and is labeled with a substance that enables detection by enzymatic reaction, is contacted with the immobilized TRACP-5b to allow the secondary antibody to bind to TRACP-5b, and RACP-5b is measured by an enzymatic reaction utilizing the label of the secondary antibody. More specifically, a primary anti-TRACP-5b monoclonal antibody is first adsorbed onto a solid support, and a sample is contacted with the immobilized primary antibody to allow TRACP-5b in the sample to bind to the primary antibody. The solid support is then washed with a wash solution to remove sample-derived components that have not been adsorbed to the antibody. The immobilized TRACP-5b is then labeled with a substance that enables detection by an enzymatic reaction, such as an enzyme or biotin, and contacted with a secondary anti-TRACP-5b monoclonal or polyclonal antibody that binds to TRACP-5b via an epitope different from that of the primary antibody to allow the secondary antibody to bind to TRACP-5b. Next, TRACP-5b can be measured by reacting the label of the secondary antibody, for example, biotin, with peroxidase-labeled streptavidin, followed by a peroxidase enzymatic reaction, or by reacting the labeling enzyme with its substrate, to produce a chromogenic substance, and measuring the absorbance of the chromogenic substance. Enzymes that can be used as substances that enable detection by enzymatic reaction include peroxidase and alkaline phosphatase, and substances that enable detection by enzymatic reaction other than enzymes include avidin and substrates for various enzymes. When avidin is used, ABC-based 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 chromogenic substrate solution.

[0053] As used herein, chemiluminescent enzyme immunoassay (CLEIA) is an immunoassay that detects the enzymatic activity of TRACP-5b by chemiluminescence. For example, TRACP-5b activity is measured by contacting a specimen with an anti-TRACP-5b monoclonal antibody adsorbed to a solid support such as beads to allow TRACP-5b in the specimen to bind to the antibody, contacting a chemiluminescent substrate for TRACP-5b with the immobilized TRACP-5b, and measuring the generated chemiluminescence. More specifically, the specimen is first contacted with an anti-TRACP-5b monoclonal antibody adsorbed to a solid support such as magnetic particles, allowing TRACP-5b in the specimen to bind to the immobilized antibody. Next, the solid support on which the magnetic particles have been immobilized, for example by magnetic immobilization, is washed with a washing solution to remove components derived from the sample that have not adsorbed to the antibody. 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 then added to the reaction system, allowing the TRACP-5b bound to the antibody to react with the substrate, and the luminescence intensity is measured. The luminescence intensity reflects the TRACP-5b enzyme activity, and the TRACP-5b in the sample can be measured from this 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] Chemiluminescent enzyme immunoassay (CLEIA) can also be used to measure TRACP-5b by contacting a sample with a primary anti-TRACP-5b monoclonal antibody immobilized on a solid support to allow TRACP-5b in the sample to bind to the primary antibody, then contacting the immobilized TRACP-5b with 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 enables chemiluminescence detection, to allow TRACP-5b to bind to the secondary antibody, and then generating chemiluminescence from or utilizing the label of the secondary antibody, and measuring the luminescence intensity. More specifically, the primary anti-TRACP-5b monoclonal antibody is first adsorbed to a solid support, such as magnetic particles, and the sample is then contacted with the immobilized primary antibody to allow TRACP-5b in the sample to bind to the primary antibody. Next, the solid-phase support on which the magnetic particles have been immobilized, for example by magnetic immobilization, is washed with a washing solution to remove components derived from the sample that have not been adsorbed to the antibody. Then, the immobilized TRACP-5b is contacted with a secondary anti-TRACP-5b monoclonal or polyclonal antibody that is labeled with an enzyme or a substance capable of generating chemiluminescence by an enzymatic reaction and that binds to TRACP-5b at an epitope different from that of the primary antibody, allowing the secondary antibody to bind to TRACP-5b. Subsequently, chemiluminescence is generated from or by the label of the secondary antibody, and the luminescence intensity is measured, thereby enabling TRACP-5b to be measured. 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 the primary antibody and / or the 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 aggregation of latex particles as absorbance. For example, TRACP-5b is measured by contacting a specimen with an anti-TRACP-5b polyclonal antibody adsorbed to latex particles to cause agglutination of the latex particles via TRACP-5b, and then measuring the resulting turbidity (absorbance). In this latex agglutination method, the above-mentioned organic sulfonic acid, sulfate ester, or salt thereof may be added independently to a reaction solution, to a specimen, or to a reagent containing latex particles.

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

[0057] 2. TRACP-5b Assay Kit Another embodiment of the present invention relates to a TRACP-5b assay kit, which is characterized by comprising the aforementioned organic sulfonic acid, sulfate ester, and / or salt thereof. Depending on the kit configuration and assay principle, the organic sulfonic acid, sulfate ester, or salt thereof can be included in the kit 1) as an independent solution, 2) contained in a sample dilution solution, 3) contained in a substrate-containing reagent, or 4) contained in a reagent containing an anti-TRACP-5b antibody.

[0058] The concentrations of the organic sulfonic acid, sulfate ester, or salts thereof in each solution or reagent are adjusted by adjusting the sulfo group (—S(O) ２ OH), sulfate group (-OS(O) ２ It is preferable to adjust the concentration of the organic sulfonic acid, sulfuric acid ester, or salt thereof according to the density of the organic sulfonic acid, sulfuric acid ester, or salt thereof, the molecular weight of the compound, and the measurement protocol (particularly, the dilution ratio (volume (vol) of reaction solution / volume (vol) of solution or reagent in which the organic sulfonic acid, sulfuric acid ester, or salt thereof is dissolved or added)).

[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 0.03 × 10 －３ Preferably, the content is 0.15×10 －３ More preferably, up to 5.0 mass%, 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 in the solution or reagent is preferably 0.03 × 10 －４ Preferably, the content is up to 5.0 mass%, and more preferably 0.15 × 10 －４ More preferably, up to 1.0 mass%, 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 Preferably, the content is up to 1.0 mass%, and more preferably 0.3 × 10 －４ More preferably, up to 0.1 mass%, 0.5 × 10 －５ More preferably, it is 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 Preferably, the content is 0.3×10 to 5.0% by mass. －３ More preferably, up to 1.0 mass%, 0.3 × 10 －２ More preferably, it is up to 0.3 mass %.

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

[0062] For example, a kit for measuring TRACP-5b by an enzymatic method comprises a substrate for TRACP-5b, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof, and, optionally, a reagent for diluting a specimen. In this kit, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof can be contained in an independent solution, or in a reagent containing a substrate for TRACP-5b or in a reagent for diluting a specimen. In the solution and the reagent for diluting a specimen that are independently contained in the kit, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof can be dissolved in a buffer solution such as Tris.

[0063] A kit for measuring TRACP-5b by enzyme immunoassay (EIA) comprises a solid support such as a multiwell plate, an anti-TRACP-5b monoclonal antibody adsorbed to the solid support, a TRACP-5b substrate, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof, optionally a sample dilution reagent, and optionally a wash solution. The anti-TRACP-5b monoclonal antibody may be included in the kit as an antibody solution separate from the solid support. In this case, the antibody is adsorbed to the solid support when measuring TRACP-5b. The wash solution is used to remove unadsorbed components from the sample after TRACP-5b in the sample has bound to the antibody. Examples of the wash solution include a Tris buffer solution containing a surfactant. A sample dilution solution is used to adjust the concentration of the sample, and can be, for example, a Tris buffer solution. Furthermore, a chelating agent such as EDTA-2Na or an inorganic salt such as sodium chloride may be added to the buffer solution, if necessary. Examples of materials for the solid support include polystyrene, polypropylene, polycarbonate, polyethylene, nylon, and methacrylate. Examples of the form of the solid support include plates and (magnetic) beads (particles). In this kit, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof can 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 diluting a specimen. In the solutions and reagents for diluting a specimen that are independently included in the kit, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof can 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 enzymatic reaction, a reagent for detecting the label of the secondary anti-TRACP-5b monoclonal antibody by enzymatic reaction, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof, optionally a reagent for diluting a sample, and optionally a wash 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, the primary antibody is adsorbed to the solid support when measuring TRACP-5b. The wash solution is used to remove components that do not bind to the primary or secondary antibody after TRACP-5b in the sample has bound to these antibodies. For example, a Tris buffer solution containing a surfactant can be used as the wash solution. The sample dilution solution is used to adjust the concentration of the sample and can be, for example, a Tris buffer solution. Furthermore, a chelating agent such as EDTA-2Na or an inorganic salt such as sodium chloride can be added to the buffer solution as needed. In this kit, the organic sulfonic acid, sulfate ester, or salt thereof can be contained in an independent solution, or in the reagent for making the label of the secondary anti-TRACP-5b monoclonal antibody detectable by enzymatic reaction, the reagent containing the primary or secondary antibody, or the sample dilution reagent. In the solution independently included in the kit and the sample dilution reagent, the organic sulfonic acid, sulfate ester, or salt thereof can be dissolved in, for example, a Tris buffer solution. Examples of substances that enable detection by enzymatic reaction include enzymes other than ACP, substrates of enzymes other than ACP, and biotin. Furthermore, when the labeling substance is biotin, 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.Furthermore, when the labeling substance is an enzyme, it is its substrate, for example, in the case of alkaline phosphatase, it is p-nitrophenyl phosphate, which is a substrate of the enzyme. Materials for the solid support include polystyrene, polypropylene, polycarbonate, polyethylene, nylon, methacrylate, etc. Shapes of the solid support include plates, (magnetic) beads (particles), etc.

[0065] A kit for measuring TRACP-5b by chemiluminescent enzyme immunoassay (CLEIA) comprises, 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 a 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 which case the antibody is adsorbed to the solid support when measuring TRACP-5b.

[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) Acridan derivatives such as [10-methyl-9(10H)-acridinylidene]phenoxymethylphosphate disodium salt.

[0067] Sample diluents are used to adjust the concentration of a sample, and examples of such diluents 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- A buffer selected from the group consisting of N-(2-hydroxyethyl)-1-piperazinylethanesulfonic 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 can be used. Furthermore, cobalt ions or manganese ions may be added to the buffer as metal ions, if necessary. The wash solution is used to remove unadsorbed components from the sample after TRACP-5b in the sample is bound to the antibody. For example, the above-mentioned buffer containing a surfactant can be used as the wash solution. 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 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, for example, the buffer solution described above.

[0068] A kit for measuring TRACP-5b by chemiluminescence immunoassay (CLIA) 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 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 makes the label detectable by chemiluminescence, the aforementioned 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 antibody after TRACP-5b in the sample has bound to these antibodies. The wash solution and specimen 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. When the label is an enzyme, such as alkaline phosphatase, the reagent is a substrate of that enzyme, such as p-nitrophenyl phosphate. The chemiluminescent substrate is the same as that described above. Materials for the solid support include polystyrene, polypropylene, polycarbonate, polyethylene, nylon, and methacrylate. The solid support may take the form of a plate, a (magnetic) bead (particle), or the like. In this kit, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof can be contained in an independent solution, or in the reagent for making the label detectable by chemiluminescence, the reagent containing the primary or secondary antibody, or the specimen dilution reagent. For the solutions and specimen dilution reagent independently included in the kit, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof can be dissolved in, for example, the buffer solution described above.

[0069] A kit for measuring TRACP-5b by latex agglutination comprises latex particles, an anti-TRACP-5b polyclonal antibody or an anti-TRACP-5b monoclonal antibody adsorbed to the latex particles, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof, and, optionally, a sample dilution reagent. In this kit, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof can be contained in an independent solution, or in the reagent containing the latex particles or the sample dilution reagent. In the solution and the sample dilution reagent independently contained in the kit, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof can be dissolved in, for example, a buffer solution. Examples of buffer solutions 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)-2-aminoethanesulfonic acid] buffer solution, Examples of suitable buffers include N-(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.

[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 meanings of the abbreviations used in the examples are as follows:

[0071] Test 1. Evaluation of TRACP-5a inhibitors by enzymatic method 1-1. Compounds evaluated The TRACP-5a inhibitory effect of the following compounds was evaluated by enzymatic method.

[0072] 1-2. Measurement Samples TRACP-5b manufactured by Nittobo Medical Co., Ltd. and TRACP-5a manufactured by R&D Systems Co., Ltd. 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, which were used as measurement samples. For the measurement of sodium hyaluronate, similarly prepared 7 U / L TRACP-5b solution and 8 U / L TRACP-5a solution were used.

[0073] 1-3. Measurement Reagent (Chromogenic Substrate Solution) A chromogenic substrate solution with the following composition was used: 5 mM 2-chloro-4-nitrophenyl phosphate, 109 mM MES, 40 mM sodium tartrate, 0.1% by mass of the compound to be evaluated, pH 6.6

[0074] 1-4. Measurement Method: Measurements were performed using a general-purpose Hitachi 7180 automatic analyzer (Hitachi High-Tech). 200 μL of a chromogenic substrate solution containing the compound to be evaluated was added to 16 μL of a 7 U / L TRACP-5b solution, or a 7 U / L or 8 U / L TRACP-5a solution to initiate the enzyme reaction. The absorbance (dominant wavelength: 405 nm, minor wavelength: 480 nm) was measured from 340.5 seconds to 516.0 seconds after the start of the reaction. The change in absorbance between these two points was used to calculate the change in absorbance per minute (the change in absorbance per unit time during the enzyme reaction). In addition, serial dilutions of TRACP-5b solutions with known activity in 22 mM MES buffer (pH 5.0), as well as 22 mM MES buffer (pH 5.0) as a blank, were prepared, and serial dilutions of TRACP-5b solutions were measured using a chromogenic substrate solution containing each evaluation compound to prepare a standard line for TRACP-5b. Similarly, serial dilutions of TRACP-5b solutions were measured using a chromogenic substrate solution not containing the evaluation compound to prepare a standard line for TRACP-5b. Using these standard lines, activity values ​​were calculated from the change in absorbance obtained by measuring TRACP-5b solutions and TRACP-5a solutions using chromogenic substrate solutions containing each evaluation compound and chromogenic substrate solutions not containing the evaluation compound. Note that measurements using sodium hyaluronate were performed in a separate batch from the other evaluation compounds.

[0075] 1-5. Measurement results

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

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

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

[0079] 2-3. Measurement Reagent (Chromogenic Substrate Solution) and Measurement Method A chromogenic substrate solution with the same composition as in Test 1 was used, except that the concentrations of each evaluation compound were as described above. The measurement method was carried out using the same procedure as in Test 1.

[0080] 2-4. Measurement results Example 4 is data from a different batch, but the data from Test 1 is listed for reference. Example 5 is data from a different batch, but the data from Test 1 is listed for reference. Although Example 1 is data from a different batch, the data from Test 1 is listed for reference. Example 2 is data from a different batch, but the data from Test 1 is listed for reference.

[0081] 3. Evaluation of TRACP-5a Inhibitors by EIA 3-1. Compounds to be Evaluated The TRACP-5a inhibitory effect of the following compounds was evaluated by EIA.

[0082] 3-2. Preparation of antibody-immobilized plate 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 of this solution 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), after which 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 the 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 incubated for 1 hour with stirring (25°C, 700 rpm). After washing three times with 300 μL of TBS-T, 100 μL of a chromogenic substrate solution (5 mM 2-chloro-4-nitrophenyl phosphate, 109 mM MES, 40 mM sodium tartrate, pH 6.6) containing the compound to be evaluated at a concentration of 0.1% by mass was added to each well and the plate was left to stand at 37°C for 1 hour. Thereafter, 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

Claims

1. A method for measuring tartrate-resistant acid phosphatase 5b (TRACP-5b) in a sample, comprising: 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 has the formula (1): (In formula (1), R is an alkyl, alkenyl, or alkynyl having 1 to 3 carbon atoms, Y is a bond or an oxygen atom, and X is hydrogen or an alkali metal.) or represented by 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 has a repeating unit of formula (3): (In the formula (3), Y is O or CH ２ and R １ ~R ５ Of these, two are O- or a bond, and at least two of the remaining are O-S(O) ２ OX or S(O) ２ 3. The method according to claim 1, having a repeating unit including a structure of: C(O)OX (X is hydrogen or an alkali metal); and if not, OX or C(O)OX (X is hydrogen or an alkali metal).

4. 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 any one of claims 1 to 4, 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 any one of claims 1 to 5, wherein the measurement is performed 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 claims 1 to 6, wherein the sample is blood, serum or plasma.

8. A kit for measuring TRACP-5b, comprising a 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 is represented by the formula (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 represented by 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 (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 10. The kit according to claim 8 or 9, having a repeating structure including the structure: C(O)OX (X is hydrogen or an alkali metal), and if not, OX or C(O)OX (X is hydrogen or an alkali metal).

11. 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. A kit according to any one of claims 8 to 11 for measuring TRACP-5b by an enzymatic method, comprising a substrate for TRACP-5b, the organic sulfonic acid, sulfate ester or salt thereof, and optionally a reagent for diluting a sample.

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

14. A kit according to any one of claims 8 to 11 for measuring TRACP-5b by enzyme immunoassay (EIA), 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 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. A kit according to any one of claims 8 to 11 for measuring TRACP-5b by enzyme-linked immunosorbent assay (ELISA), 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 at 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 enabling detection of 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 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 labeled substance by an enzyme reaction, or a reagent for diluting a specimen.

18. The kit according to any one of claims 8 to 11, for measuring TRACP-5b by chemiluminescent enzyme immunoassay (CLEIA), 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 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. A kit according to any one of claims 8 to 11 for measuring TRACP-5b by chemiluminescence immunoassay (CLIA), 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 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 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 of 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 chemiluminescently detectable label, or the specimen dilution reagent.

22. A kit according to any one of claims 8 to 11 for measuring TRACP-5b by a latex agglutination method, 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 reagent for diluting the 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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