Method for measuring tracp-5b and kit for the method

Abstract

Provided is a method for selectively detecting TRACP-5b in a TRACP-5b assay, which further reduces the influence of coexisting TRACP-5a. The method is a method for detecting TRACP-5b in the presence of an organic sulfonic acid, a sulfate, or a salt thereof, wherein the density of a sulfo group (-S(O)2OH), a sulfate group (-OS(O)2OH), and / or a salt thereof in the organic sulfonic acid, the sulfate, or the salt thereof is 6.5 mmol / g or more.

Description

TECHNICAL FIELD

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

[0002] Acid phosphatase (ACP) is an enzyme having hydrolytic activity of organic monophosphate under acidic conditions. There are multiple isozymes of ACP, which are distributed in various tissues. As ACPs present in serum, there are those from platelets, from red blood cells, from prostate, from osteoclasts, from macrophages, and the like. These isozymes are separated by polyacrylamide gel electrophoresis, and ACPs from osteoclasts and from macrophages are located at the 5th position from the origin, have resistance to tartaric acid, and thus are 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 and Lam W.K.W. et al., Clin. Biochem. 14, 177-181, 1981, etc.). TRACP-5 is classified into TRACP-5a having more sugar chains and sialic acid bound thereto and TRACP-5b having less sugar chains and sialic acid bound thereto, 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 for diagnosis aid and therapeutic effect determination of metabolic bone diseases and bone metastatic cancer. Therefore, a method for selectively measuring TRACP-5b from ACPs in serum is being studied.

[0003] In the past, as a method for measuring TRACP-5, there is a method in which a synthetic substrate such as p-nitrophenyl phosphate is used for enzyme reaction in the presence of tartaric acid, and the enzyme activity is calculated by colorimetrically quantifying the generated reaction product. However, tartaric acid can inhibit ACP from prostate, but since TRACP-5a, ACP from red blood cells, and ACP from platelets have resistance to tartaric acid, this method also measures these isozymes, and cannot specifically measure TRACP-5b.

[0004] As a method for improving the specificity of TRACP-5b in the above method, there is known a method in which a serum diluted 5-fold is incubated at 37°C for 1 hour, and then the enzyme activity is measured in the same manner (Non-Patent Literature 3). In this method, in addition to the ACP from the prostate, the influence of the ACP from red blood cells can also be avoided, but the influence of TRACP-5a and the ACP from platelets cannot be avoided.

[0005] As another method for specifically measuring the activity of TRACP-5b, there is a method in which the activity of TRACP-5b is calculated by subtracting the ACP activity measured in the presence of tartrate and fluoride from the ACP activity measured in the presence of tartrate (Patent Literature 1). In this method, the influence of the ACP from red blood cells and the ACP from platelets can be avoided using the difference in sensitivity to fluoride. However, the influence of TRACP-5a cannot be excluded, and the activity of TRACP-5b is calculated by subtraction, which is problematic in terms of precision.

[0006] As still another method, there is reported a method in which the activity of TRACP-5b is specifically measured using an acid mucopolysaccharide as an inhibitor of acid phosphatase other than TRACP-5b (Patent Literature 2). However, the inhibitory effect of the acid mucopolysaccharide on the activity of TRACP-5a is insufficient, and further improvement in specificity for TRACP-5b is required.

[0007] As a method for measuring the activity of TRACP-5b using a chemiluminescent substrate, there is known a method in which TRACP-5b is allowed to react with a chemiluminescent 1,2-dioxetane compound under acidic conditions, and then the reaction solution is made alkaline to measure the luminescence intensity (Patent Literature 3). In addition, there is reported a method in which a specific buffer such as MES is used during the enzyme reaction, or the luminescence intensity ratio of TRACP-5b to TRACP-5a is made larger by adding manganese ions or cobalt ions, thereby specifically measuring TRACP-5b (Patent Literature 4). However, these methods are limited to activity measurement using a chemiluminescent substrate, and a more inexpensive method for specifically measuring TRACP-5b is required.

[0008] As an immunoassay method for TRACP-5b using an antibody, there is, for example, a method in which an antibody specific to TRACP-5 is used to specifically measure TRACP-5b by utilizing the difference in the optimum pH of both TRACP-5a and TRACP-5b after binding both of them (Patent Literature 5). However, the specificity for TRACP-5b is insufficient only by the difference in the optimum pH, and since the amount of TRACP-5b is calculated by subtraction, there is also a problem in terms of precision.

[0009] Alternatively, there is a method that uses an antibody with a certain selectivity for TRACP-5b, and then uses 2-chloro-4-nitrophenyl phosphate as a chromogenic substrate for activity assay after antibody binding, thereby achieving a more specific assay for TRACP-5b (Patent Documents 6 and 7). However, it is also desirable to further improve the selectivity of this antibody for TRACP-5b.

[0010] In addition, there is a method for quantifying the protein content of TRACP-5b using monoclonal antibodies with high specificity against TRACP-5b (Patent Document 8).

[0011] Existing technical documents

[0012] Patent documents

[0013] Patent Document 1: Japanese Patent Application Publication No. 10-337198

[0014] Patent Document 2: Japanese Patent Application Publication No. 2001-231595

[0015] Patent Document 3: Japanese Patent Application Publication No. 2011-024470

[0016] Patent Document 4: Japanese Patent Application Publication No. 2022-174015

[0017] Patent Document 5: Japanese Patent Application Publication No. 2002-510050

[0018] Patent Document 6: Japanese Patent Application Publication No. 2004-026805

[0019] Patent Document 7: WO2004 / 077059

[0020] Patent Document 8: WO2016 / 027697

[0021] Non-patent literature

[0022] Non-patent literature 1: Clin. Chem. 33: 458-462. 1987 Summary of the Invention

[0023] The problem that the invention aims to solve

[0024] Compared with the prior art, the purpose of the present invention is to provide a method for selectively detecting TRACP-5b that can further reduce the influence of coexisting TRACP-5a in the determination of TRACP-5b.

[0025] Methods for solving problems

[0026] The inventors have studied a method for selectively detecting TRACP-5b and found that by having compounds with a certain density of sulfonyl (-S(O)2OH), sulfate (-OS(O)2OH) and / or their salts present in the reaction system where TRACP-5a may be present, the influence of TRACP-5a can be significantly reduced, thereby enabling the selective detection of TRACP-5b.

[0027] Specifically, the present invention provides the following method for determining TRACP-5b and a kit for determining TRACP-5b.

[0028] [1] A method for determining TRACP-5b, which is a method for determining tartrate-resistant acid phosphatase 5b (TRACP-5b) in a sample, comprising a step of performing a reaction to detect TRACP-5b in the presence of an organic sulfonic acid, a sulfate ester or its salt, wherein the density of the sulfonyl group (-S(O)2OH), sulfate group (-OS(O)2OH) and / or its salt in the organic sulfonic acid, sulfate ester or its salt is 6.5 mmol / g or higher.

[0029] [2] The method for determining tartrate-resistant acid phosphatase 5b (TRACP-5b) in the specimen as described in [1] includes a step of performing an enzymatic reaction or an antigen-antibody reaction in the presence of organic sulfonic acids, sulfates and / or their salts, wherein each unit of the organic sulfonic acid, sulfate and / or their salt has a structure of S(O)2O. - The conversion value has 50% by mass or more of sulfonyl (-S(O)2OH), sulfate (-OS(O)2OH) and / or their salts; or

[0030] Each unit structure of the organic sulfonic acid, sulfate ester, or their salt is S(O)2O - The conversion value has more than 50% by mass of sulfonyl (-S(O)2OH), sulfate (-OS(O)2OH) and / or their salts.

[0031] [3] According to the method of [1] or [2], wherein the organic sulfonic acid, sulfate ester or salt thereof:

[0032] It can be expressed by equation (1),

[0033] RYS(O)2OX…(1)

[0034] In formula (1), R is an alkyl, alkenyl or alkynyl group with 1 to 3 carbon atoms, Y is a bond or oxygen atom, and X is hydrogen or alkali metal;

[0035] or,

[0036] Having repeating units of equation (2),

[0037]

[0038] In formula (2), Y is a bond or oxygen atom, n is an integer from 2 to 5000, and X is hydrogen or an alkali metal;

[0039] or,

[0040] Repeating units with a structure including formula (3),

[0041]

[0042] (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 ones are OS(O)2OX or S(O)2OX (where X is hydrogen or an alkali metal), R 1 ~R 5 None of them are OX or C(O)OX (where X is hydrogen or an alkali metal).

[0043] [4] According to the method of [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.

[0044] [5] The method according to any one of [1] to [4], wherein the organic sulfonic acid, sulfate ester or salt thereof:

[0045] 1) Added independently to the reaction system or the sample, or

[0046] It is contained in 2) the sample diluent, 3) the substrate-containing reagent, or 4) the reagent containing anti-TRACP-5b antibody (typically an anti-TRACP-5b monoclonal antibody).

[0047] Therefore, the reaction for detecting TRACP-5b is carried out in the presence of the said organic sulfonic acid, sulfate ester or their salt.

[0048] [6] The method according to any one of [1] to [5], wherein the determination is performed by the following methods: enzymatic method, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA) or latex agglutination method.

[0049] [7] The method according to any one of [1] to [6], wherein the specimen is blood, serum or plasma.

[0050] [8] A TRACP-5b assay kit containing organic sulfonic acids, sulfates and / or their salts, wherein the density of the sulfonyl (-S(O)2OH), sulfate (-OS(O)2OH) and / or their salts is greater than 6.5 mmol / g.

[0051] [9] The TRACP-5b assay kit according to [8] contains organic sulfonic acids, sulfates and / or their salts, each unit of said organic sulfonic acids, sulfates and / or their salts having a structure of S(O)2O. - The conversion value has 50% or more of sulfonyl (-S(O)2OH), sulfate (-OS(O)2OH) or their salts; or

[0052] Each unit structure of the organic sulfonic acid, sulfate ester and / or their salts is S(O)2O - The conversion value has 50% or more of sulfonyl (-S(O)2OH), sulfate (-OS(O)2OH) or their salts.

[0053]

[10] The kit according to [8] or [9], wherein the organic sulfonic acid, sulfate ester and / or their salts:

[0054] It can be expressed by equation (1),

[0055] RYS(O)2OX…(1)

[0056] In formula (1), R is an alkyl, alkenyl or alkynyl group with 1 to 5 carbon atoms, Y is a bond or oxygen atom, and X is hydrogen or alkali metal;

[0057] or,

[0058] Having a repeating structure as in equation (2),

[0059]

[0060] In formula (2), Y is a bond or oxygen atom, n is an integer from 2 to 5000, and X is hydrogen or an alkali metal;

[0061] or,

[0062] A repeating structure containing equation (3),

[0063]

[0064] (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 ones are OS(O)2OX or S(O)2OX (where X is hydrogen or an alkali metal), R 1 ~R5 None of them are OX or C(O)OX (where X is hydrogen or an alkali metal).

[0065]

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

[0066]

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

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

[0067]

[13] The kit according to

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

[0068]

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

[11] measures TRACP-5b by enzyme immunoassay (EIA).

[0069] The kit comprises: a solid support, an anti-TRACP-5b monoclonal antibody adsorbed on the solid support, a substrate of the TRACP-5b, the organic sulfonic acid, sulfate ester or salt thereof, optional reagent for sample dilution, and optional washing solution.

[0070]

[15] The kit according to

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

[0071]

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

[11] measures TRACP-5b by enzyme-linked immunosorbent assay (ELISA).

[0072] 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 bound to TRACP-5b with an epitope different from that of the first anti-TRACP-5b monoclonal antibody and labeled with a substance detectable in an enzyme reaction; a reagent for enabling the label to be detectable in an enzyme reaction; the organic sulfonic acid, sulfate ester, or salt thereof; optionally a reagent for diluting the sample; and optionally a washing solution.

[0073]

[17] The kit according to

[16] contains the organic sulfonic acid, sulfate ester or salt thereof in the reagent containing the second anti-TRACP-5b monoclonal antibody, the reagent for detecting the marker in an enzyme reaction or the reagent for diluting the sample.

[0074]

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

[11] measures TRACP-5b by chemiluminescent enzyme immunoassay (CLEIA method).

[0075] 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 ester or salt thereof, optional reagent for sample dilution, and optional washing solution.

[0076]

[19] The kit according to

[18] contains the organic sulfonic acid, sulfate ester or salt thereof in the reagent containing the chemiluminescent substrate of the TRACP-5b, the reagent containing the anti-TRACP-5b monoclonal antibody or the reagent for diluting the sample.

[0077]

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

[11] measures TRACP-5b by chemiluminescent immunoassay (CLIA method).

[0078] 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 bound to TRACP-5b with an epitope different from that of the first anti-TRACP-5b monoclonal antibody and labeled with a substance detectable in chemiluminescence; a reagent for enabling the label to be detectable in chemiluminescence; the organic sulfonic acid, sulfate ester, or salt thereof; optional reagent for sample dilution; and optional washing solution.

[0079]

[21] The kit according to

[20] contains the organic sulfonic acid, sulfate ester or salt thereof in the reagent containing the first anti-TRACP-5b monoclonal antibody, the reagent containing the second anti-TRACP-5b monoclonal antibody, the reagent for detecting the marker in chemiluminescence or the reagent containing the sample dilution reagent.

[0080]

[22] The kit described in any one of [8] to

[11] is used to determine TRACP-5b by latex agglutination.

[0081] The kit comprises: latex particles, an anti-TRACP-5b antibody adsorbed onto the latex particles, the organic sulfonic acid, sulfate ester or salt thereof, and optional reagents for sample dilution.

[0082]

[23] The kit according to

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

[0083]

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

[23] is used to measure blood, serum or plasma.

[0084] Invention Effects

[0085] According to the present invention, a means (method) for selectively detecting TRACP-5b is provided, which can reduce the influence of tartrate-resistant acid phosphatase 5b (TRACP-5b). Therefore, it is useful in diagnosing diseases that use TRACP-5b as a marker. Detailed Implementation

[0086] The embodiments of the present invention will be described in detail below. However, the present invention should not be construed as being limited to the following embodiments.

[0087] One embodiment of the present invention provides a method for determining TRACP-5b in a sample, comprising the following steps: performing a tartrate-resistant acid phosphatase 5b (TRACP-5b) detection reaction in the presence of a specific organic sulfonic acid, sulfate ester or salt thereof.

[0088] Another embodiment of the present invention provides a kit for determining TRACP-5b, the kit comprising a specific organic sulfonic acid, sulfate ester, or salt thereof.

[0089] These implementation methods are described in detail below.

[0090] 1. Determination method of TRACP-5b

[0091] 1-1. Organic sulfonic acids, sulfates, or their salts

[0092] In this assay, TRACP-5a is inhibited and TRACP-5b is selectively determined by presenting an organic sulfonic acid, a sulfate ester, or its salt in the reaction system, and by ensuring that the density of the sulfonyl group (-S(O)2OH), sulfate group (-OS(O)2OH), and / or its salt in the organic sulfonic acid, sulfate ester, or its salt is 6.5 mmol / g or higher.

[0093] As shown in the examples described later, the effect of inhibiting TRACP-5a increases depending on the density of the sulfonate, sulfate, or their salts. In a preferred embodiment, the density of the sulfonate, sulfate, 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.

[0094] Furthermore, in a preferred embodiment, each unit of such organic sulfonic acid, sulfate ester, or salt thereof has a structure of S(O)₂O. - The converted value has 50% or more of sulfonyl, sulfate and / or their salts, more preferably 60% or more of sulfonyl, sulfate and / or their salts, and even more preferably 70% or more of sulfonyl, sulfate and / or their salts.

[0095] In this application, the term "unit structure" refers to the structure of the repeating unit when the target compound has a repeating structure, and to the structure of the compound as a whole when the target compound does not have a repeating structure. Furthermore, when the unit structure is a salt of sulfonyl (-S(O)2OH) or sulfate (-OS(O)2OH), the molecular weight of the unit structure is calculated based on the sulfonyl (-S(O)2OH) or sulfate (-OS(O)2OH) group.

[0096] As a specific embodiment of such organic sulfonic acids, sulfates, or their salts, compounds represented by formula (1) can be cited:

[0097] [Chemistry 7]

[0098] RYS(O)2OX…(1)

[0099] In formula (1), R is an alkyl, alkenyl or alkynyl group with 1 to 3 carbon atoms, preferably an alkenyl group with 1 to 3 carbon atoms.

[0100] Y is a bond or an oxygen atom, preferably a bond.

[0101] X is hydrogen or an alkali metal, preferably hydrogen or Na.

[0102] Examples of compounds used in this embodiment include, for example, methanesulfonic acid or its salts (e.g., Na salts), vinylsulfonic acid or its salts (e.g., Na salts), or vinyl sulfate or its salts.

[0103] Other specific embodiments of the above-mentioned organic sulfonic acids, sulfates, or their salts include compounds having repeating units of formula (2):

[0104] [Chemistry 8]

[0105]

[0106] In equation (2), Y is a bond or an oxygen atom, preferably a bond.

[0107] n is an integer from 2 to 5000, preferably from 10 to 500, and more preferably from 20 to 100.

[0108] X is hydrogen or an alkali metal, preferably hydrogen or Na.

[0109] Preferred examples of compounds for this embodiment include, for example, polyvinylsulfonic acid or its salts (e.g., Na salts), or polyvinylsulfonic acid or its salts (e.g., Na salts), preferably polyvinylsulfonic acid or its salts having 2 to 5000, more preferably 10 to 500, and even more preferably 20 to 100 or fewer repeating structures.

[0110] [Chemistry 9]

[0111]

[0112] (In formula (2-1), X is hydrogen or an alkali metal, preferably hydrogen or Na.)

[0113] Such polyvinyl sulfonic acid or its salts typically have a molecular weight of 200 to 500,000, preferably 1,000 to 50,000, and more preferably 4,000 to 6,000.

[0114] Here, in this application, the terms "molecular weight" or "average molecular weight" refer to weight-average molecular weight (Mw) unless used in a particularly different sense. Furthermore, weight-average molecular weight (Mw) refers to a value determined using liquid chromatography via gel permeation chromatography (GPC).

[0115] As another specific embodiment of the above-mentioned organic sulfonic acids, sulfates, or their salts,

[0116] Examples of compounds having repeating units comprising formula (3) can be given:

[0117] [Chemistry 10]

[0118]

[0119] In formula (3), Y is O or CH2, preferably O.

[0120] R 1 ~R 5 Two of them are O- or bonds, and at least two of the remaining ones are OS(O)2OX or S(O)2OX (where X is hydrogen or an alkali metal (e.g., Na)), R 1 ~R 5None of them are OX or C(O)OX (where X is hydrogen or an alkali metal (e.g., Na), preferably not C(O)OX (where X is hydrogen or an alkali metal (e.g., Na)). Preferably, R 1 ~R 5 Two of them are O- or bonds, and the rest are OS(O)2OX (X is hydrogen or an alkali metal (e.g., Na)).

[0121] Additionally, examples of repeating units containing this structure include:

[0122] [Chemistry 11]

[0123]

[0124] In formula (3-1), Y is O or CH2, preferably O.

[0125] L 1 and L 4 Each can be an O or a bond independently, preferably one is an O- and the other is a bond.

[0126] L 2 and L 3 Each can be independently O, CH2 or bonded, preferably one is O and the other is CH2 or bonded.

[0127] R 1 ~R 3 At least two of them are independently OS(O)₂OX or S(O)₂OX (where X is hydrogen or an alkali metal (e.g., Na)). If present, the remainder are OX or C(O)OX (where X is hydrogen or an alkali metal (e.g., Na)), preferably C(O)OX (where X is hydrogen or an alkali metal (e.g., Na)). R 1 ~R 3 Preferably, each is independently OS(O)2OX or S(O)2OX (X is hydrogen or an alkali metal (e.g., Na)), more preferably, each is independently OS(O)2OX (X is hydrogen or an alkali metal (e.g., Na)).

[0128] R 4 ~R 6 At least two of them are independently OS(O)₂OX or S(O)₂OX (where X is hydrogen or an alkali metal (e.g., Na)). If present, the remainder are OX or C(O)OX (where X is hydrogen or an alkali metal (e.g., Na)), preferably C(O)OX (where X is hydrogen or an alkali metal (e.g., Na)). R 4 ~R 6 Preferably, each is independently OS(O)2OX or S(O)2OX (X is hydrogen or an alkali metal (e.g., Na)), more preferably, each is independently OS(O)2OX (X is hydrogen or an alkali metal (e.g., Na)).

[0129] n is 2 to 1000, preferably 3 to 700, more preferably 4 to 200, and even more preferably 5 to 70.

[0130] Furthermore, as a preferred example of the compound for this embodiment, examples include dextran sulfate or its salt (e.g., Na salt), and more preferably, dextran sulfate or its salt (e.g., Na salt) having 2 to 1000, more preferably 3 to 700, and more preferably 5 to 70 or less repeating structures.

[0131] [Chemistry 12]

[0132]

[0133] (In formula (3-2), X is hydrogen or an alkali metal (e.g., Na))

[0134] Such sulfated dextran or its salts typically have a molecular weight of 1,500 to 1,000,000, preferably 2,000 to 550,000, and more preferably 4,000 to 55,000.

[0135] In the method of the present invention, the properties of the coexisting TRACP-5a antigen are altered and its activity is inhibited by presenting the aforementioned organic sulfonic acids, sulfates, or their salts in the reaction system. These TRACP-5a inhibitors can be present in the reaction system in various forms. For example, these TRACP-5a inhibitors or solutions thereof can be added separately to the reaction solution used for detecting TRACP-5b; they can also be pre-dissolved in a sample diluent before being added to the test sample; or, depending on the reaction system, they can be pre-added to reagents containing enzyme substrates or reagents containing anti-TRACP-5b antibodies, and the reactions (e.g., enzyme reactions or antigen-antibody reactions) can be carried out using these reagents.

[0136] As illustrated in the examples described later, the inhibitory activity of the aforementioned organic sulfonic acids, sulfates, or their salts increases depending on the density of the sulfonyl groups, sulfate groups, and their salts, as well as their average molecular weight. The lower limit of the concentration of these compounds in the reaction solution required to exert TRACP-5a inhibitory activity decreases depending on this density and average molecular weight. For any compound, as the concentration in the reaction solution increases from this lower limit, the inhibitory activity increases with concentration within a certain range. However, above a certain concentration, the inhibitory activity no longer increases with concentration but remains at a certain level, gradually decreasing after reaching that level. Even when the inhibitory activity plateaus, high inhibitory activity is maintained over a wide range of concentrations. In practice, the inhibitory activity is often used at concentrations higher than the concentration at which it no longer increases with concentration in the reaction solution.

[0137] Therefore, the concentration of the aforementioned organic sulfonic acids, sulfate esters, or their salts in the reaction solution is preferably determined taking these factors into account.

[0138] For example, when using dextran sulfate or its salts with an average molecular weight of 4000–6000, the preferred concentration in the reaction solution is 0.03 × 10⁻⁶. -3 ~10.0% by mass, more preferably 0.15×10 -3 ~5.0% by mass, more preferably 0.3 × 10⁻⁶. -3 ~1.0% by mass. Furthermore, when using dextran sulfate or its salts with a molecular weight of 40,000 to 60,000, the concentration in the reaction solution is preferably 0.03 × 10⁻⁶. -4 ~5.0% by mass, more preferably 0.15×10 -4 ~1.0% by mass, more preferably 0.5 × 10⁻⁶ -4 ~0.5% by mass. Furthermore, when using dextran sulfate or its salts with a molecular weight of 400,000 to 600,000, the concentration in the reaction solution is preferably 0.01 × 10⁻⁶. -5 ~1.0% by mass, more preferably 0.3×10 -4 ~0.1% by mass, more preferably 0.5 × 10⁻⁶. -5 ~0.05% by mass.

[0139] Furthermore, when using vinyl sulfonic acid or a salt thereof, the concentration in the reaction solution is preferably 0.012 to 15.0% by mass, more preferably 0.020 to 5.0% by mass, and even more preferably 0.10 to 3.0% by mass. Additionally, for example, when using polyvinyl sulfonic acid or a salt thereof with an average molecular weight of 4000 to 6000, the concentration in the reaction solution is preferably 0.08 × 10⁻⁶. -3 ~5.0% by mass, more preferably 0.3×10 -3 ~1.0% by mass, more preferably 0.3 × 10⁻⁶. -2 ~0.3% by mass.

[0140] In the assay method, TRACP-5a is inhibited by presenting the aforementioned organic sulfonic acid, sulfate ester, or their salt in the reaction system for detecting TRACP-5b. Examples of such "reaction systems" include enzyme reactions and antigen-antibody reactions. The TRACP-5a inhibitor used in the method of the present invention can be used in any of these reaction systems.

[0141] In this application, "enzyme reaction" refers to the reaction between TRACP-5b and its substrate. TRACP-5b substrates include, for example, p-nitrophenyl phosphate or its salts, 2-chloro-4-nitrophenyl phosphate or its salts, 1,2-dioxane derivatives, acridine derivatives, and 4-methylumbelliferyl phosphate (4-methylumbelliferyl phosphate). Additionally, in this specification, "antigen-antibody reaction" refers to the reaction between TRACP-5b and its corresponding antibody. Antibodies for TRACP-5b include, for example, monoclonal antibodies, polyclonal antibodies, and antigen-binding fragments. 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 commissioned designations IPOD FERM BP-7889, IPOD FERM BP-8249, or IPOD FERM BP-7890.

[0142] Various assay methods utilize enzyme reactions and antigen-antibody reactions, such as: enzymatic methods, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), and latex agglutination assay.

[0143] In this application, the enzymatic method is a method for determining the enzyme activity of TRACP-5b. For example, TRACP-5b in the sample is reacted with an enzyme substrate of TRACP-5b, such as p-nitrophenyl phosphate or a salt thereof, and the enzyme activity of TRACP-5b is measured. More specifically, a chromogenic substrate solution containing tartaric acid or a salt thereof (e.g., 2-chloro-4-nitrophenyl phosphate solution) is added to the sample to initiate an enzyme reaction, and the enzyme activity is determined based on the change in absorbance between specific time points. In this enzymatic method, the aforementioned organic sulfonic acids, sulfates, or their salts can be added independently to the reaction solution, to the sample, or to the chromogenic substrate solution.

[0144] In this application, the enzyme immunoassay (EIA) is an immunoassay that detects the enzyme activity of TRACP-5b. For example, TRACP-5b in the sample is bound to an anti-TRACP-5b monoclonal antibody, and a TRACP-5b enzyme substrate, such as p-nitrophenyl phosphate or its salt, reacts with the bound TRACP-5b to measure its enzyme activity. More specifically, first, the sample is contacted with an anti-TRACP-5b monoclonal antibody adsorbed on a solid support, allowing the TRACP-5b in the sample to bind to the immobilized antibody. Next, the solid support is washed with a washing solution to remove components from the sample that have not been adsorbed by the antibody. Then, a TRACP-5b enzyme substrate, such as p-nitrophenyl phosphate or its salt, is added to the reaction system, causing the antibody-bound TRACP-5b to react with the substrate. The enzyme reaction is stopped with a reaction stop solution, and the absorbance of the phenolic compound produced, such as p-nitrophenol, is measured at a specified wavelength, such as 390 nm to 450 nm, preferably 400 nm to 430 nm. Since the absorbance reflects the TRACP-5b enzyme activity, this value can be used to determine the presence of TRACP-5b in the sample. In this enzyme immunoassay (EIA), the aforementioned organic sulfonic acids, sulfates, or their salts can be added independently to the support, to the sample or sample diluent, to a reagent containing anti-TRACP-5b monoclonal antibody, or to a chromogenic substrate solution.

[0145] In this application, the enzyme-linked immunosorbent assay (ELISA) is a method for determining TRACP-5b by measuring enzyme activity using two different antibodies against TRACP-5b (one of which is an antibody labeled with a substance detectable by an enzyme reaction). For example, the sample is contacted with a first anti-TRACP-5b monoclonal antibody adsorbed on a solid support, causing TRACP-5b in the sample to bind to the first antibody. Then, a second anti-TRACP-5b monoclonal antibody labeled with a substance detectable by an enzyme reaction is used to contact the immobilized TRACP-5b, causing the second antibody to bind to TRACP-5b. The labeling of the second antibody is used to determine TRACP-5b by an enzyme reaction.

[0146] More specifically, firstly, a first anti-TRACP-5b monoclonal antibody is adsorbed onto a solid support, and the sample is brought into contact with the fixed first antibody, allowing TRACP-5b in the sample to bind to the first antibody. Next, the solid support is washed with a washing solution to remove components from the sample that were not adsorbed by the antibody. Then, the fixed TRACP-5b is brought into contact with a second anti-TRACP-5b monoclonal or polyclonal antibody labeled with a substance detectable by an enzymatic reaction, such as an enzyme or biotin, and binding to TRACP-5b at an epitope different from that of the first antibody, allowing TRACP-5b to bind to the second antibody. Next, depending on the labeling of the second antibody, for example, biotin reacts with streptavidin labeled with peroxidase followed by a peroxidase enzymatic reaction, or the labeled enzyme reacts with its substrate, a chromogenic substance is generated. TRACP-5b can be measured by determining the absorbance of this chromogenic substance. Examples of enzymes that can be detected by enzyme reactions include peroxidase and alkaline phosphatase. Examples of substances that can be detected by enzyme reactions other than enzymes include avidin and substrates of various enzymes. When using avidin, the ABC colorimetric method can be used.

[0147] In this enzyme-linked immunosorbent assay (ELISA), the aforementioned organic sulfonic acids, sulfates, or their salts can be added independently to the support, to the sample or sample diluent, to a reagent containing a first antibody and / or a second antibody, or to a chromogenic substrate solution.

[0148] In this application, the chemiluminescent enzyme immunoassay (CLEIA method) is an immunoassay that detects the enzyme activity of TRACP-5b through chemiluminescence. For example, the sample is contacted with an anti-TRACP-5b monoclonal antibody adsorbed on a solid support such as beads, causing TRACP-5b in the sample to bind to the antibody. The chemiluminescent substrate of TRACP-5b is then contacted with the immobilized TRACP-5b, and the activity of TRACP-5b is determined by measuring the generated chemiluminescence. More specifically, firstly, the sample is contacted with an anti-TRACP-5b monoclonal antibody adsorbed on a solid support such as magnetic particles, causing TRACP-5b in the sample to bind to the immobilized antibody. Next, the fixed solid support obtained by magnetically immobilizing magnetic particles is washed with a washing solution to remove components from the sample that have not been adsorbed by the antibody. Then, a chemiluminescent substrate for TRACP-5b, such as 2-chloro-5-{4-methoxyspiro[1,2-dioxane-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 allow the antibody-bound TRACP-5b to react with the substrate, and the luminescence intensity is measured. Since this luminescence intensity reflects the TRACP-5b enzyme activity, the TRACP-5b in the sample can be determined based on its value.

[0149] In this chemiluminescent enzyme immunoassay (CLEIA method), the aforementioned organic sulfonic acids, sulfates, or their salts can be added independently to the support, to the sample or sample diluent, to a reagent containing anti-TRACP-5b monoclonal antibody, or to a chromogenic substrate solution.

[0150] The chemiluminescent enzyme immunoassay (CLEIA method) can also be described as follows: The sample is contacted with a first anti-TRACP-5b monoclonal antibody immobilized on a solid support, causing TRACP-5b in the sample to bind to the first antibody. Next, the immobilized TRACP-5b is contacted with a second anti-TRACP-5b monoclonal antibody labeled with an epitope different from that of the first antibody and labeled with a substance detectable by chemiluminescence substrate or chemiluminescence, causing TRACP-5b to bind to the second antibody. Chemiluminescence is generated based on the labeling of the second antibody or by utilizing this labeling, and the intensity of the luminescence is measured, thereby determining TRACP-5b. More specifically, firstly, the first anti-TRACP-5b monoclonal antibody is adsorbed onto a solid support, such as magnetic particles, and the sample is contacted with the immobilized first antibody, causing TRACP-5b in the sample to bind to the first antibody. Next, the fixed solid support obtained by magnetically immobilizing magnetic particles is washed with a washing solution to remove components from the sample that are not adsorbed by the antibody. Then, the fixed TRACP-5b is brought into contact with a second anti-TRACP-5b monoclonal or polyclonal antibody labeled with a substance that can generate chemiluminescence using an enzyme or enzyme reaction and which binds to TRACP-5b with an epitope different from that of the first antibody. This allows TRACP-5b to bind to the second antibody. Then, based on the labeling of the second antibody or the chemiluminescence generated by the labeling, the intensity of the luminescence is measured, thereby determining TRACP-5b.

[0151] In this chemiluminescent enzyme immunoassay (CLEIA method), the aforementioned organic sulfonic acids, sulfates, or their salts can be added independently to the support, to the sample, or to a reagent containing a first antibody and / or a second antibody.

[0152] In this application specification, the latex agglutination method is a method for determining TRACP-5b by using the turbidity generated by the agglutination of latex particles as an absorbance measurement. For example, the sample is contacted with an anti-TRACP-5b polyclonal antibody adsorbed on latex particles, causing the latex particles to agglutinate due to TRACP-5b, and the resulting turbidity (absorbance) is measured to determine TRACP-5b. In this latex agglutination method, the aforementioned organic sulfonic acids, sulfates, or their salts can be added independently to the reaction solution, to the sample, or to a reagent containing latex particles.

[0153] The specimens used for TRACP-5b assays can be from any organism, typically from mammals, and most often from humans. Furthermore, the specimens can be from any tissue or bodily fluid, with blood, serum, and plasma being the most common sources.

[0154] 2. TRACP-5b assay kit

[0155] Another embodiment of the present invention relates to a kit for the determination of TRACP-5b, characterized in that it contains the aforementioned organic sulfonic acids, sulfates and / or their salts.

[0156] Depending on the kit's composition and assay principle, organic sulfonic acids, sulfates, or their salts may be included in the kit as follows: 1) as a standalone solution, 2) in a sample diluent, 3) in a substrate-containing reagent, or 4) in a reagent containing anti-TRACP-5b antibody.

[0157] The concentration of the organic sulfonic acid, sulfate ester or their salt in each solution or reagent is preferably adjusted according to the density of the sulfonyl group (-S(O)2OH), sulfate group (-OS(O)2OH) and / or their salt of the organic sulfonic acid, sulfate ester or their salt, the molecular weight of these compounds and the assay procedure (measurement conditions) (in particular the dilution factor (vol) / volume of solution or reagent in which organic sulfonic acid, sulfate ester or their salt is dissolved or added) to achieve the concentration in the reaction solution as described above.

[0158] For example, when using dextran sulfate or its salts with an average molecular weight of 4000–6000, the concentration (C) in the solution or reagent is preferably 0.03 × 10⁻⁶. -3 ~10.0% by mass, more preferably 0.15×10 -3 ~5.0% by mass, more preferably 0.3 × 10⁻⁶. -3 ~1.0% by mass. Furthermore, when using dextran sulfate or its salts with a molecular weight of 40,000 to 60,000, the concentration in the solution or reagent is preferably 0.03 × 10⁻⁶. -4 ~5.0% by mass, more preferably 0.15×10 -4 ~1.0% by mass, more preferably 0.5 × 10⁻⁶ -4 ~0.5% by mass. Furthermore, when using dextran sulfate or its salts with a molecular weight of 400,000 to 600,000, the concentration (C) in the solution or reagent is preferably 0.01 × 10⁻⁶. -5 ~1.0% by mass, more preferably 0.3×10 -5 ~0.1% by mass, more preferably 0.5 × 10⁻⁶. -5 ~0.05% by mass.

[0159] Furthermore, when vinyl sulfonic acid or its salts are used, for example, 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. Additionally, when polyvinyl sulfonic acid or its salts with an average molecular weight of 4000 to 6000 are used, the concentration (C) in the solution or reagent is preferably 0.08 × 10⁻⁶. -3 ~5.0% by mass, more preferably 0.3×10 -3 ~1.0% by mass, more preferably 0.3 × 10⁻⁶. -2 ~0.3% by mass.

[0160] As mentioned above, TRACP-5b can be determined by various methods, and the TRACP-5b assay kits also use different reagents depending on the assay method.

[0161] For example, a kit for the enzymatic determination of TRACP-5b includes a substrate for TRACP-5b, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof, and optional reagents for sample dilution. Alternatively, in this kit, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof may be contained in a separate solution or in a reagent containing the TRACP-5b substrate or a reagent for sample dilution. The aforementioned organic sulfonic acid, sulfate ester, or salt thereof can be dissolved in the separate solutions and reagents for sample dilution included in the kit, for example in a buffer such as tris(hydroxymethyl)aminomethane (Tris).

[0162] A kit for the determination of TRACP-5b by enzyme immunoassay (EIA) comprises: a solid support such as a multi-well plate, an anti-TRACP-5b monoclonal antibody adsorbed on the solid support, a substrate for TRACP-5b, the aforementioned organic sulfonic acid, sulfate ester or salt thereof, optional reagent for sample dilution, and optional washing solution.

[0163] In addition to being adsorbed onto a solid support, anti-TRACP-5b monoclonal antibodies can also be included in the kit as an antibody solution. In this case, the antibody is adsorbed onto the solid support during the TRACP-5b assay. After the TRACP-5b in the sample is bound to the antibody, unadsorbed components from the sample are removed using a washing buffer. For example, a tris(hydroxymethyl)aminomethane (Tris) buffer containing a surfactant can be used as the washing buffer.

[0164] Specimen diluents are used to adjust the concentration of the specimen; for example, buffers such as tris(hydroxymethyl)aminomethane (Tris) can be used. Additionally, chelating agents such as EDTA-2Na and inorganic salts such as sodium chloride can be added to the buffer as needed.

[0165] Examples of materials that can be used as solid supports include: polystyrene, polypropylene, polycarbonate, polyethylene, nylon, and methacrylate. Examples of shapes that can be used as solid supports include: plates, (magnetic) beads (particles), etc.

[0166] In this kit, the aforementioned organic sulfonic acids, sulfates, or their salts may be contained in separate solutions or in reagents containing TRACP-5b substrates, reagents containing anti-TRACP-5b monoclonal antibodies, or reagents for sample dilution. The aforementioned organic sulfonic acids, sulfates, or their salts may be dissolved in the separate solutions and reagents for sample dilution included in the kit, for example in buffers such as tris(hydroxymethyl)aminomethane (Tris).

[0167] A kit for measuring TRACP-5b by enzyme-linked immunosorbent assay (ELISA) comprises: a solid support, a primary anti-TRACP-5b monoclonal antibody adsorbed on the solid support, a secondary anti-TRACP-5b monoclonal antibody bound to TRACP-5b with an epitope different from that of the primary anti-TRACP-5b monoclonal antibody and labeled with a substance detectable in an enzyme reaction, a reagent for enabling the label of the secondary anti-TRACP-5b monoclonal antibody to be detectable in an enzyme reaction, an organic sulfonic acid, a sulfate ester or a salt thereof, optional reagents for sample dilution, and optional washing solution.

[0168] The first anti-TRACP-5b monoclonal antibody can be included in the kit either as an antibody solution or adsorbed onto a solid support. In this case, the first antibody is adsorbed onto the solid support during the TRACP-5b assay. After the TRACP-5b in the sample binds to the first or second antibody, a washing buffer is used to remove any components that have not bound to these antibodies. For example, a tris(hydroxymethyl)aminomethane (Tris) buffer containing a surfactant can be used as a washing buffer.

[0169] Specimen diluents are used to adjust the concentration of the specimen; for example, buffers such as tris(hydroxymethyl)aminomethane (Tris) can be used. Additionally, chelating agents such as EDTA-2Na and inorganic salts such as sodium chloride can be added to the buffer as needed.

[0170] In this kit, the aforementioned organic sulfonic acids, sulfates, or their salts may be contained in separate solutions, or in labeled reagents that can detect the second anti-TRACP-5b monoclonal antibody in an enzymatic reaction, reagents containing the first or second antibody, or reagents for sample dilution. The aforementioned organic sulfonic acids, sulfates, or their salts may be dissolved in the separate solutions and reagents for sample dilution included in the kit, for example in a buffer such as tris(hydroxymethyl)aminomethane (Tris).

[0171] Examples of substances that can be detected in enzyme reactions include: enzymes other than ACP, substrates of enzymes other than ACP, or biotin.

[0172] Furthermore, when the labeled substance is biotin, the reagent used to detect the labeled substance by an enzyme reaction is, for example, a reagent containing streptavidin labeled with peroxidase, a peroxidase substrate of tetramethylbenzidine, and hydrogen peroxide. Additionally, when the labeled substance is an enzyme, the reagent used to detect the labeled substance by an enzyme reaction is, for example, its substrate; and when the labeled substance is alkaline phosphatase, the reagent used to detect the labeled substance by an enzyme reaction is, for example, p-nitrophenyl phosphate, which is a substrate of that enzyme.

[0173] Examples of materials that can be used as solid supports include: polystyrene, polypropylene, polycarbonate, polyethylene, nylon, and methacrylate. Examples of shapes that can be used as solid supports include: plates, (magnetic) beads (particles), etc.

[0174] A kit for the determination of TRACP-5b by chemiluminescent enzyme immunoassay (CLEIA method) includes, for example, a solid support, an anti-TRACP-5b monoclonal antibody adsorbed on the solid support, a chemiluminescent substrate of TRACP-5b, the aforementioned organic sulfonic acid, sulfate ester or salt thereof, optional reagent for sample dilution, and optional washing solution.

[0175] In addition to being adsorbed onto a solid support, anti-TRACP-5b monoclonal antibodies can also be included in the kit as an antibody solution. In this case, the antibody is adsorbed onto the solid support when TRACP-5b is measured.

[0176] Examples of chemiluminescent substrates include:

[0177] (1) Disodium salts of 3-(4-methoxyspiro{1,2-dioxane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane}-4-yl)phenyl phosphate, 4-chloro-3-(methoxyspiro{1,2-dioxane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane}-4-yl)phenyl phosphate, 3-(4-methoxyspiro{1,2-dioxane-3,2'-tricyclo[3.3.1.1(3,7)]decane}-4-yl)phenyl phosphate, 4-methoxy-4-(3-phenyl phosphate)spiro[1,2-dioxane-3,2'-adamantane]-disodium salt, and other 1,2-dioxane derivatives; or,

[0178] (2) Acridinium derivatives such as [10-methyl-9(10H)-acridinylindene]phenoxymethyl phosphate disodium salt.

[0179] Specimen diluents are used to adjust the concentration of the specimen. For example, buffers selected from the following can be used: 2-(N-morpholinyl)ethanesulfonic acid (MES) buffer, 3-(N-morpholinyl)propanesulfonic acid (MOPSO) buffer, 3-morpholinylpropanesulfonic 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-acetamide)iminodiacetic acid (ADA) buffer, N-(2-acetamide)-2-aminoethanesulfonic acid (ACES) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, and phosphate buffer. Additionally, cobalt or manganese ions can be added to the buffer as metal ions as needed.

[0180] After binding TRACP-5b to the antibody in the sample, a washing solution is used to remove unadsorbed components from the sample. For example, the aforementioned buffer containing a surfactant can be used as the washing solution.

[0181] In this kit, the aforementioned organic sulfonic acids, sulfates, or their salts may be contained in separate solutions or in reagents containing TRACP-5b chemiluminescent substrates, reagents containing anti-TRACP-5b monoclonal antibodies, or reagents for sample dilution. The aforementioned organic sulfonic acids, sulfates, or their salts may be dissolved in the separate solutions and reagents for sample dilution included in the kit, for example, in the aforementioned buffer solutions.

[0182] A kit for determining TRACP-5b by chemiluminescent immunoassay (CLIA) comprises: a solid support, a first anti-TRACP-5b monoclonal antibody adsorbed on the solid support, a second anti-TRACP-5b monoclonal antibody labeled with a substance that binds to TRACP-5b with an epitope different from that of the first anti-TRACP-5b monoclonal antibody and is detectable by chemiluminescence, a labeling reagent that can be detected by chemiluminescence, the aforementioned organic sulfonic acid, sulfate ester or salt thereof, optional reagent for sample dilution, and optional washing solution.

[0183] The first anti-TRACP-5b monoclonal antibody can be included in the kit either as an antibody solution or adsorbed onto a solid support. In this case, the first antibody is adsorbed onto the solid support when TRACP-5b is measured. After TRACP-5b in the sample is bound to the first or second antibody, a washing solution is used to remove any components that are not bound to these antibodies.

[0184] The cleaning solution and sample diluent are the same as described above.

[0185] When labeled as biotin, the labeled reagent can be detected by chemiluminescence, for example, a reagent containing streptavidin labeled with peroxidase, a chemiluminescent substrate of peroxidase, and a reagent containing hydrogen peroxide. Similarly, when labeled as an enzyme such as alkaline phosphatase, the labeled reagent can be detected by chemiluminescence, for example, a substrate of that enzyme, such as p-nitrophenyl phosphate. The chemiluminescent substrate is the same as described above.

[0186] Examples of materials that can be used as solid supports include: polystyrene, polypropylene, polycarbonate, polyethylene, nylon, and methacrylate. Examples of shapes that can be used as solid supports include: plates, (magnetic) beads (particles), etc.

[0187] In this kit, the aforementioned organic sulfonic acids, sulfates, or their salts may be contained in separate solutions, or in reagents that can be detected by chemiluminescence, reagents containing a first antibody or a second antibody, or reagents for sample dilution. The aforementioned organic sulfonic acids, sulfates, or their salts may be dissolved in the separate solutions and reagents for sample dilution included in the kit, for example, in the aforementioned buffer solutions.

[0188] A kit for the determination of TRACP-5b by latex agglutination assay comprises: latex particles, anti-TRACP-5b polyclonal antibody or anti-TRACP-5b monoclonal antibody adsorbed onto the latex particles, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof, and optional reagents for sample dilution. In this kit, the aforementioned organic sulfonic acid, sulfate ester, or salt thereof may be contained in a separate solution or in a reagent containing latex particles or a reagent for sample dilution. The aforementioned organic sulfonic acid, sulfate ester, or salt thereof may be dissolved in the separate solutions and reagents for sample dilution included in the kit, for example, in the aforementioned buffer solution.

[0189] In addition, examples of buffer solutions 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-acetamide)iminodiacetic acid (ADA) buffer, N-(2-acetamide)-2-aminoethanesulfonic acid (ACES) buffer, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, and phosphate buffer.

[0190] Example

[0191] The present invention will be described in more detail below through embodiments, but the present invention is not limited thereto.

[0192] The abbreviations used in the embodiments are explained below.

[0193] DSS Na 5k Dextran sulfate sodium with average molecular weight 5000 DSS Na 50k Dextran sulfate sodium with average molecular weight 50000 DSS Na 500k Dextran sulfate sodium with average molecular weight 500000 PVSA Na 5k Polyvinyl sulfate sodium with average molecular weight 5000 VSA Na Vinyl sulfate sodium β-CyD-S Na β-Cyclodextrin sulfate sodium PAA 5k Polyacrylic acid with average molecular weight 5000 Poly-Glu Na Polyglutamic acid sodium Chon S Na Chondroitin sulfate sodium PEG 6k Polyethylene glycol with average molecular weight 6000

[0194] Experiment 1. Evaluation of TRACP-5a inhibitors by enzymatic method

[0195] 1-1. Evaluation of compounds

[0196] The inhibitory effects of TRACP-5a on the following compounds were evaluated using an enzymatic method.

[0197]

[0198]

[0199]

[0200] 1-2. Sample Measurement

[0201] TRACP-5b (Nittobo Medical) and TRACP-5a (R&D Systems) were diluted with 22 mL MME S buffer (pH 5.0) to prepare 7 U / L TRACP-5b and 7 U / L TRACP-5a solutions, respectively, as the assay samples. Note that the same 7 U / L TRACP-5b and 8 U / L TRACP-5a solutions were used for the determination of sodium hyaluronate.

[0202] 1-3. Assay reagents (colorimetric substrate solution)

[0203] Use a colorimetric substrate solution with the following composition.

[0204]

[0205] 1-4. Determination Methods

[0206] The assay was performed using a general-purpose Hitachi Automated Analytical System Model 7180 (manufactured by Hitachi High Technology Corporation). 200 μL of the chromogenic substrate solution containing the evaluation compound was added to 16 μL of 7 U / L TRACP-5b solution, 7 U / L TRACP-5a solution, or 8 U / L TRACP-5a solution to initiate the enzyme reaction. The absorbance was measured from 340.5 seconds to 516.0 seconds (dominant wavelength 405 nm, secondary wavelength 480 nm). Based on the absorbance changes between the two time points, the change in absorbance per minute (the change in absorbance per unit time during the enzyme reaction) was calculated.

[0207] In addition, serially diluted solutions of TRACP-5b with known activity were prepared using 22 mM MES buffer (pH 5.0) and a blank of 22 mM MES buffer (pH 5.0). Using chromogenic substrate solutions containing each evaluation compound, the absorbance of the serially diluted TRACP-5b solutions was measured to prepare a standard linear curve for TRACP-5b. Similarly, using chromogenic substrate solutions without evaluation compounds, the absorbance of the serially diluted TRACP-5b solutions was measured to prepare another standard linear curve for TRACP-5b. Using these standard linear curves, the activity value was calculated based on the change in absorbance obtained from measuring TRACP-5b and TRACP-5a solutions using chromogenic substrate solutions containing and without each evaluation compound.

[0208] It should be noted that the determination of sodium hyaluronate was performed separately and in batches from other evaluation compounds.

[0209] 1-5. Measurement Results

[0210] [Table 4]

[0211]

[0212] Experiment 2. Study on the added concentration of TRACP-5a inhibitor

[0213] The effects of the concentrations of sodium dextran sulfate (5K), sodium dextran sulfate (50K), sodium polyvinyl sulfonate (5K), and sodium vinyl sulfonate on inhibitory activity were investigated.

[0214] 2-1. Concentration of each evaluation compound

[0215] The concentrations of each evaluation compound in the assay reagent (chromogenic substrate solution) were adjusted as follows.

[0216] [Table 5]

[0217]

[0218] 2-2. Sample Measurement

[0219] In tests using reagents containing sodium dextran sulfate (5K) and sodium dextran sulfate (50K), 7 U / L TRACP-5b and 7 U / L TRACP-5a solutions were used. In tests using reagents containing sodium polyvinyl sulfonate (5K), 7 U / L TRACP-5b and 7 U / L or 8 U / L TRACP-5a solutions were used. In tests using reagents containing sodium vinyl sulfonate, 7 U / L TRACP-5b and 8 U / L TRACP-5a solutions were used.

[0220] 2-3. Determination reagents (chromogenic substrate solution) and determination method

[0221] The concentrations of each evaluation compound were set as described above, except that the chromogenic substrate solution with the same composition as in Experiment 1 was used. Furthermore, the assay was performed in the same order as in Experiment 1.

[0222] 2-4. Measurement Results

[0223] [Table 6]

[0224]

[0225] [Table 7]

[0226]

[0227] Example 4 is another batch of data, but it is recorded with reference to the data in Experiment 1.

[0228] [Table 8]

[0229]

[0230] Example 5 is another batch of data, but it is recorded with reference to the data in Experiment 1.

[0231] [Table 9]

[0232]

[0233] Example 1 is another batch of data, but it is recorded with reference to the data in Experiment 1.

[0234] [Table 10]

[0235]

[0236] Example 2 is another batch of data, but it is recorded with reference to the data in Experiment 1.

[0237] 3. Evaluation of TRACP-5a inhibitors using EIA method

[0238] 3-1. Evaluation of compounds

[0239] The inhibitory effects of TRACP-5a on the following compounds were evaluated by EIA method.

[0240]

[0241]

[0242]

[0243] 3-2. Preparation of Antibody Fixation Plates

[0244] The 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 (ThermoFisher SCIENTIFIC, model 468667). After freezing overnight, 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. The plate was then frozen overnight.

[0245] 3-3. Determination Method

[0246] After removing the TBS containing 1.5% BSA from the prepared antibody fixation plate, add 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 to each well, and react with stirring for 1 hour (25°C, 700 rpm). Wash three times with 300 μL of TBS-T, and then add 100 μL of a chromogenic substrate solution containing the evaluation compound at a concentration of 0.1% (5 mM 2-chloro-4-nitrophenylphosphine, 109 mM MES, 40 mM sodium tartrate, pH 6.6) to each well, and let stand at 37°C for 1 hour. Then, add 50 μL of 0.5N NaOH, and measure the absorbance using a plate reader (dominant wavelength 405 nm, secondary wavelength 490 nm).

[0247] 3-4. Measurement Results

[0248] [Table 14]

[0249]

Claims

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

2. The method of claim 1, wherein, Each unit structure of the organic sulfonic acid, sulfate ester, or their salt is S(O)2O - The conversion value has more than 50% by mass of sulfonyl (-S(O)2OH), sulfate (-OS(O)2OH) and / or their salts.

3. The method of claim 1 or 2, wherein, The organic sulfonic acids, sulfates, or their salts: It can be expressed by equation (1), In formula (1), R is an alkyl, alkenyl or alkynyl group with 1 to 3 carbon atoms, Y is a bond or oxygen atom, and X is hydrogen or alkali metal; or, Having repeating units of equation (2), In formula (2), Y is a bond or oxygen atom, n is an integer from 2 to 5000, and X is hydrogen or an alkali metal; or, Repeating units with a structure including formula (3), In equation (3), Y is O or CH2, and R is... 1 ~R 5 Two of them are O- or bonded ends, and the remaining at least two are OS(O)2OX or S(O)2OX, where X is hydrogen or an alkali metal. The ones that do not belong to any of the above are OX or C(O)OX, where 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 of claim 1 or 2, wherein, The organic sulfonic acids, sulfates, or their salts: 1) Added independently to the reaction system or the sample, or It is contained in 2) the sample diluent, 3) the substrate-containing reagent, or 4) the reagent containing anti-TRACP-5b antibody. Therefore, the reaction for detecting TRACP-5b is carried out in the presence of the said organic sulfonic acid, sulfate ester or their salt.

6. The method of claim 1 or 2, wherein, The following methods can be used for determination: enzymatic method, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay (CLIA), or latex agglutination method.

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

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

9. The kit according to claim 8, wherein, Each unit structure of the organic sulfonic acid, sulfate ester and / or their salts is S(O)2O - The conversion value has 50% or more of sulfonyl (-S(O)2OH), sulfate (-OS(O)2OH) or their salts.

10. The kit according to claim 8 or 9, wherein, The organic sulfonic acids, sulfates, and / or their salts: It can be expressed by equation (1), In formula (1), R is an alkyl, alkenyl or alkynyl group with 1 to 5 carbon atoms, Y is a bond or oxygen atom, and X is hydrogen or alkali metal; or, Having a repeating structure as in equation (2), In formula (2), Y is a bond or oxygen atom, n is an integer from 2 to 5000, and X is hydrogen or an alkali metal; or, A repeating structure containing equation (3), In equation (3), Y is O or CH2, and R is... 1 ~R 5 Two of them are O- or bonded ends, and the remaining at least two are OS(O)2OX or S(O)2OX, where X is hydrogen or an alkali metal. The ones that do not belong to any of the above are OX or C(O)OX, where 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. The kit according to claim 8 or 9, used for the enzymatic determination of TRACP-5b. The kit contains: a substrate for TRACP-5b, the organic sulfonic acid, sulfate ester or salt thereof, and optional reagents for sample dilution.

13. The kit of claim 12, wherein the organic sulfonic acid, sulfate ester or salt thereof is contained in the reagent containing the substrate of said TRACP-5b or the reagent for diluting the sample.

14. The kit according to claim 8 or 9, 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, a substrate of the TRACP-5b, the organic sulfonic acid, sulfate ester or salt thereof, optional reagent for sample dilution, and optional washing solution.

15. The kit of 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 the substrate of the TRACP-5b or the reagent for diluting the sample.

16. The kit of claim 8 or 9, wherein TRACP-5b is measured by enzyme-linked immunosorbent assay (ELISA). 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 bound to TRACP-5b with an epitope different from that of the first anti-TRACP-5b monoclonal antibody and labeled with a substance detectable in an enzyme reaction; a reagent for enabling the label to be detectable in an enzyme reaction; the organic sulfonic acid, sulfate ester, or salt thereof; optionally a reagent for diluting the sample; and optionally a washing solution.

17. The kit of claim 16, wherein the organic sulfonic acid, sulfate ester or salt thereof is contained in the reagent containing the second anti-TRACP-5b monoclonal antibody, the reagent for enabling the marker to be detected in an enzyme reaction, or the reagent for diluting the sample.

18. The kit of claim 8 or 9, 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 ester or salt thereof, optional reagent for sample dilution, and optional washing solution.

19. The kit of claim 18, wherein the organic sulfonic acid, sulfate ester or salt thereof is contained in the reagent containing the substrate of said TRACP-5b, the reagent containing the anti-TRACP-5b monoclonal antibody or the reagent for diluting the sample.

20. The kit of claim 8 or 9, wherein TRACP-5b is measured by chemiluminescent immunoassay (CLIA). 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 labeled with a substance that binds to TRACP-5b with a different epitope than the first anti-TRACP-5b monoclonal antibody and is detectable in chemiluminescence; a reagent that enables the label to be detectable in chemiluminescence; the organic sulfonic acid, sulfate ester, or salt thereof; optionally a reagent for diluting the 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 the reagent containing the first anti-TRACP-5b monoclonal antibody, the reagent containing the second anti-TRACP-5b monoclonal antibody, the reagent enabling the marker to be detected in chemiluminescence, or the reagent containing the reagent for diluting the sample.

22. The kit of claim 8 or 9, wherein TRACP-5b is determined by latex agglutination. The kit comprises: latex particles, an anti-TRACP-5b antibody immobilized on the latex particles, the organic sulfonic acid, sulfate ester or salt thereof, and optional reagents for sample dilution.

23. The kit of 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 sample.

24. The kit of claim 8 or 9, wherein the sample being measured is blood, serum or plasma.

Images