Method for measuring antigen contained in test sample, and kit for measuring human chorionic gonadotropin or estradiol

Abstract

The present invention addresses the problem of providing a measurement method and a measurement kit which, in the measurement of hCG or E2, enable the concentration at the upper limit of measurement to be extended while enabling luminescence measurement at the concentration at the lower limit of measurement. The present invention provides a method for measuring an antigen contained in a test sample, the antigen being human chorionic gonadotropin or estradiol, and the method including causing the following (1), (2) and (3) to react in the presence of one or more compounds represented by formula (1): (1) an antigen to which peroxidase is bound via a binding substance that recognizes the abovementioned antigen, or an antigen to which peroxidase is bound via a binding substance that recognizes a complex of the abovementioned antigen and a binding substance that recognizes said antigen; (2) a chemiluminescent substance; and (3) an oxidizing agent. In the formula, X is a phenyl group, a pyridyl group, an oxazolyl group, a thiophenyl group, or a pyrrolyl group, each of which may have a substituent.

Description

Method for measuring antigens contained in test samples, and kit for measuring human chorionic gonadotropin or estradiol

[0001] The present invention relates to a method for measuring human chorionic gonadotropin or estradiol using chemiluminescence, and a kit for measuring human chorionic gonadotropin or estradiol using chemiluminescence.

[0002] In a chemiluminescence measurement system using luminol or the like, it is known to use TP (thiazolephenol) or BiPCA (4-(4-hydroxyphenyl)benzoic acid) as an enhancer for increasing the amount of chemiluminescence.

[0003] Patent Document 1 describes BiPCA as an enhancer, but hCG and E2 are not included as measurement targets. The effects of BiPCA are described as maintaining chemiluminescence and extending the measurement time. Patent Document 2 also describes BiPCA as an enhancer. However, antigen measurement using BiPCA has not been performed.

[0004] US Patent Publication US2015 / 0346202 JP 7-311197

[0005] The inventors' studies on human chorionic gonadotropin (hCG) and estradiol (E2) in the presence of TP, a conventional enhancer, revealed a problem specific to these antigens: although the chemiluminescence intensity is enhanced, it is not possible to measure the entire range of antigen concentrations required. Specifically, in the measurement of hCG and E2 using TP as an enhancer, the luminescence intensity is enhanced at the lower limit of the measurement, but the enhancement is weaker at the upper limit of the measurement. Therefore, it was found that it is not possible to extend the upper limit of the measurement while still enabling luminescence measurement at the lower limit of the measurement (i.e., the luminescence intensity increases with increasing measurement concentration from the lower limit to the upper limit). Patent Documents 1 and 2 do not mention the problem of extending the upper limit of the measurement while still enabling luminescence measurement at the lower limit of the measurement.

[0006] An object of the present invention is to provide a method for measuring hCG or E2 that enables luminescence measurement at the lower limit of measurement while extending the upper limit of measurement.A further object of the present invention is to provide a kit for measuring hCG or E2 that enables luminescence measurement at the lower limit of measurement while extending the upper limit of measurement.

[0007] The present inventors have conducted extensive research to solve the above problems and screened various compounds, and as a result, have found that the above problems can be solved by using BiPCA or the like as an enhancer in the measurement of hCG or E2. The present invention was completed based on the above findings. That is, the present invention provides the following inventions:

[0008] <1> A method for measuring an antigen contained in a test sample, wherein the antigen is human chorionic gonadotropin or estradiol, the method comprising reacting (1) an antigen bound to peroxidase via a binding substance that recognizes the antigen, or an antigen bound to peroxidase via a binding substance that recognizes a complex of the antigen and a binding substance that recognizes the antigen, (2) a chemiluminescent substance, and (3) an oxidizing agent, in the presence of one or more compounds represented by the following formula (1): In the formula, X is an unsubstituted or substituted phenyl group, an unsubstituted or substituted pyridyl group, an unsubstituted or substituted oxazolyl group, an unsubstituted or substituted thiophenyl group, or an unsubstituted or substituted pyrrolyl group, wherein the substituent is a carboxyl group, an amide group, a halogen atom, an alkoxy group having 1 to 5 carbon atoms, or an alkyl group having 1 to 5 carbon atoms. <2> The measurement method according to <1>, wherein X is a phenyl group having a carboxyl group, an amide group, a halogen atom, or an alkoxy group having 1 to 5 carbon atoms; an unsubstituted pyridyl group or an alkyl group having 1 to 5 carbon atoms or an alkoxy group having 1 to 5 carbon atoms; an unsubstituted oxazolyl group or an alkyl group having 1 to 5 carbon atoms or an alkoxy group having 1 to 5 carbon atoms; an unsubstituted thiophenyl group or a carboxyl group; or an unsubstituted pyrrolyl group or an alkyl group having 1 to 5 carbon atoms or an alkoxy group having 1 to 5 carbon atoms. <3> The measurement method according to <1> or <2>, wherein X is a carboxyl group, an amide group, a halogen atom, or a phenyl group having an alkoxy group having 1 to 5 carbon atoms. <4> The measurement method according to any one of <1> to <3>, wherein the antigen to which peroxidase is bound via a binding substance that recognizes the antigen is obtained by reacting the antigen with a first binding substance that recognizes the antigen to form a complex, and then reacting the complex with a second binding substance that recognizes the antigen and is bound to peroxidase. <5> The measurement method according to any one of <1> to <3>, wherein the antigen to which peroxidase is bound via a binding substance that recognizes a complex of the antigen and a binding substance that recognizes the antigen is obtained by reacting the antigen with a first binding substance that recognizes the antigen to form a complex, and then reacting the complex with a "third binding substance that recognizes the complex of the antigen and the first binding substance that recognizes the antigen" to which peroxidase is bound. <6> The method according to any one of <1> to <5>, wherein the concentration of the compound represented by formula (1) is 10 μmol / L or more and 500 μmol / L or less.<7> The method according to any one of <1> to <6>, wherein the chemiluminescent substance is luminol, a luminol derivative, 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione sodium salt, or a salt thereof. <8> The method according to <7>, wherein the concentration of the chemiluminescent substance is 0.1 mmol / L or more and 10 mmol / L or less. <9> The method according to any one of <1> to <8>, wherein the oxidizing agent is hydrogen peroxide. <10> A kit for measuring human chorionic gonadotropin or estradiol, comprising a peroxidase bound to a binding substance that recognizes an antigen, a chemiluminescent substance, an oxidizing agent, and one or more compounds represented by the following formula (1): In the formula, X represents an unsubstituted or substituted phenyl group, an unsubstituted or substituted pyridyl group, an unsubstituted or substituted oxazolyl group, an unsubstituted or substituted thiophenyl group, or an unsubstituted or substituted pyrrolyl group, wherein the substituent is a carboxyl group, an amide group, a halogen atom, an alkoxy group having 1 to 5 carbon atoms, or an alkyl group having 1 to 5 carbon atoms. <11> The kit according to <10>, further comprising a binding substance that recognizes the antigen to be measured, different from the binding substance bound to the peroxidase, or a solid-phase support to which the binding substance is immobilized. <12> A kit for measuring human chorionic gonadotropin or estradiol, comprising peroxidase bound to a binding substance that recognizes a complex between an antigen and the binding substance that recognizes the antigen, a chemiluminescent substance, an oxidizing agent, and one or more compounds represented by the following formula (1): In the formula, X represents an unsubstituted or substituted phenyl group, an unsubstituted or substituted pyridyl group, an unsubstituted or substituted oxazolyl group, an unsubstituted or substituted thiophenyl group, or an unsubstituted or substituted pyrrolyl group, wherein the substituent is a carboxyl group, an amide group, a halogen atom, an alkoxy group having 1 to 5 carbon atoms, or an alkyl group having 1 to 5 carbon atoms. <13> The kit according to <12>, further comprising a binding substance that recognizes the antigen to be measured, different from the binding substance bound to peroxidase, or a solid phase support to which the binding substance is immobilized.

[0009] According to the method and kit of the present invention, in the measurement of hCG or E2, it is possible to extend the upper limit of the measurement concentration while enabling luminescence measurement at the lower limit of the measurement concentration.

[0010] The results of measuring the amount of luminescence at each E2 concentration in Example 2 and Comparative Example 2 are shown.

[0011] The present invention will be described in detail below. In this specification, mM means mmol / L. <Method for Measuring Antigen> A first aspect of the present invention is a method for measuring an antigen contained in a test sample, wherein the antigen is human chorionic gonadotropin or estradiol, and the method comprises reacting (1) an antigen bound to peroxidase via a binding substance that recognizes the antigen, or an antigen bound to peroxidase via a binding substance that recognizes a complex of the antigen and a binding substance that recognizes the antigen, (2) a chemiluminescent substance, and (3) an oxidizing agent, in the presence of one or more compounds represented by the following formula (1): In the formula, X represents an unsubstituted or substituted phenyl group, an unsubstituted or substituted pyridyl group, an unsubstituted or substituted oxazolyl group, an unsubstituted or substituted thiophenyl group, or an unsubstituted or substituted pyrrolyl group, and the substituent is a carboxyl group, an amide group, a halogen atom, an alkoxy group having 1 to 5 carbon atoms, or an alkyl group having 1 to 5 carbon atoms.

[0012] The test sample may include, but is not limited to, biological fluids such as serum, blood, plasma, and urine, as well as biological samples such as lymph, blood cells, and various types of cells. In the present invention, the antigen contained in the test sample is human chorionic gonadotropin or estradiol.

[0013] Preferably, in formula (1), X is a phenyl group having a carboxyl group, an amide group, a halogen atom, or an alkoxy group having 1 to 5 carbon atoms; an unsubstituted pyridyl group or a pyridyl group having an alkyl group having 1 to 5 carbon atoms or an alkoxy group having 1 to 5 carbon atoms; an oxazolyl group or a pyridyl group having an alkyl group having 1 to 5 carbon atoms or an alkoxy group having 1 to 5 carbon atoms; an unsubstituted thiophenyl group or a pyrrolyl group having an alkyl group having 1 to 5 carbon atoms or an alkoxy group having 1 to 5 carbon atoms.

[0014] More preferably, in formula (1), X is a phenyl group having a carboxyl group, an amide group, a halogen atom, or an alkoxy group having 1 to 5 carbon atoms. Examples of the halogen atom include fluorine, chlorine, bromine, and iodine, and preferably fluorine or chlorine. Examples of the alkoxy group having 1 to 5 carbon atoms include a methoxy group, an ethoxy group, a propoxy group, a butoxy group, and a pentyloxy group, which may be linear or branched. A preferred example of the alkoxy group having 1 to 5 carbon atoms is a methoxy group. Examples of the alkyl group having 1 to 5 carbon atoms include a methyl group, an ethyl group, a propyl group, a butyl group, and a pentyl group, which may be linear or branched. A preferred example of the alkyl group having 1 to 5 carbon atoms is a methyl group.

[0015] Specific examples of the group represented by X are shown below. Among the following, the groups enclosed in a square frame are particularly preferred as the group represented by X. * indicates the bonding position to the benzene ring.

[0016] (1) Substituted phenyl group

[0017] (2) Unsubstituted or substituted pyridyl group

[0018] (3) Unsubstituted or substituted oxazolyl group

[0019] (4) Unsubstituted or substituted thiophenyl group

[0020] (5) Unsubstituted or substituted pyrrolyl group

[0021] Specific examples of the compound represented by formula (1) are shown below.

[0022]

[0023] The concentration of the compound represented by formula (1) is preferably from 10 μmol / L to 500 μmol / L, more preferably from 50 μmol / L to 500 μmol / L, even more preferably from 75 μmol / L to 400 μmol / L, and particularly preferably from 100 μmol / L to 300 μmol / L. The concentration referred to here is the concentration in a reaction solution when an antigen bound to a peroxidase via a binding substance that recognizes the antigen, a chemiluminescent substance, and an oxidizing agent are reacted in the presence of one or more compounds represented by formula (1).

[0024] In the present invention, an antigen to which peroxidase is bound via a binding substance that recognizes the antigen, a chemiluminescent substance, and an oxidizing agent are reacted in the presence of one or more compounds represented by the above formula (1).

[0025] The chemiluminescent substrate is preferably a nitrogen-containing heterocyclic compound having an amino group or a salt thereof, and examples thereof include a 2,3-dihydro-1,4-phthalazinedione compound having an amino group, and 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione sodium salt (L-012) or a salt thereof.

[0026] The structure of L-012 is shown below.

[0027] Examples of the 2,3-dihydro-1,4-phthalazinedione compound that can be used include known 2,3-dihydro-1,4-phthalazinedione compounds described in JP-A Nos. 2-291299, 10-319015, and 2000-279196, as well as mixtures thereof. Of these, luminol, luminol derivatives (e.g., isoluminol, N-aminohexyl-N-ethylisoluminol (AHEI), N-aminobutyl-N-ethylisoluminol (ABEI)), and metal salts thereof (alkali metal salts, etc.) are preferred, luminol and metal salts thereof are more preferred, and luminol sodium salt is particularly preferred.

[0028] The concentration of the chemiluminescent substance is preferably 0.1 mmol / L to 10 mmol / L, more preferably 0.2 mmol / L to 7.5 mmol / L, even more preferably 0.25 mmol / L to 5.0 mmol / L, and particularly preferably 0.25 mmol / L to 2.5 mmol / L. The concentration referred to here is the concentration in a reaction solution when an antigen bound to a peroxidase via a binding substance that recognizes the antigen, a chemiluminescent substance, and an oxidizing agent are reacted in the presence of one or more compounds represented by formula (1).

[0029] Examples of the oxidizing agent include aqueous solutions of known oxidizing agents such as those described in JP-A-8-261943 and JP-A-2000-279196 (inorganic peroxides (such as hydrogen peroxide, sodium perborate, and potassium perborate), organic peroxides (such as dialkyl peroxides and acyl peroxides), and peroxyacid compounds (such as peroxosulfuric acid and peroxophosphoric acid). Among these, from the viewpoint of storage stability and the like, hydrogen peroxide, sodium perborate, and potassium perborate are preferred, and hydrogen peroxide is more preferred.

[0030] The concentration of the oxidizing agent is appropriately set depending on the type thereof and the measurement method and conditions to be used, but is preferably 0.5 mmol / L to 20 mmol / L, more preferably 1 mmol / L to 10 mmol / L, and even more preferably 1.4 mmol / L to 3.5 mmol / L, from the viewpoint of the chemiluminescence enhancement effect, etc. The concentration referred to here is the concentration in a reaction solution when an antigen bound to a peroxidase via a binding substance that recognizes the antigen, a chemiluminescent substance, and an oxidizing agent are reacted in the presence of one or more compounds represented by formula (1).

[0031] In one example of the present invention, an antigen bound to peroxidase via a binding substance that recognizes the antigen may be obtained by reacting the antigen with a first binding substance that recognizes the antigen to form a complex, and then reacting the complex with a second binding substance that recognizes the antigen and has peroxidase bound thereto.

[0032] In one example of the present invention, an antigen to which peroxidase is bound via a binding substance that recognizes a complex of an antigen and a first binding substance that recognizes the antigen may be obtained by reacting an antigen with a first binding substance that recognizes the antigen to form a complex, and then reacting the complex with a "third binding substance that recognizes the complex of an antigen and the first binding substance that recognizes the antigen" to which peroxidase is bound.

[0033] In the present invention, an antigen in a sample can be measured using a solid phase carrier, such as magnetic silica particles, on the surface of which a first binding substance that recognizes the antigen is immobilized.

[0034] Examples of binding substances that recognize an antigen (first binding substance that recognizes an antigen or second binding substance that recognizes an antigen) include substances that bind to the measurement target substance, human chorionic gonadotropin or estradiol, through interactions such as an "antigen"-"antibody" reaction, a "sugar chain"-"protein" reaction, a "sugar chain"-"lectin" reaction, a "protein"-"peptide chain" reaction, a "protein"-"protein" reaction, and a "protein"-"nucleotide chain" reaction.

[0035] Examples of binding substances that recognize the above complexes include those that bind to the above complexes through interactions such as an "antigen"-"antibody" reaction, a "sugar chain"-"protein" reaction, a "sugar chain"-"lectin" reaction, a "protein"-"peptide chain" reaction, a "protein"-"protein" reaction, and a "protein"-"nucleotide chain" reaction.

[0036] The method for measuring a substance to be measured in the present invention may be carried out in accordance with the sandwich method described in literature commonly used in the field [for example, Enzyme Immunoassay, 2nd Edition (edited by Ishikawa Eiji et al., Igaku Shoin), 1982].

[0037] In the sandwich method, a first binding substance that recognizes an antigen is immobilized on the surface of a solid-phase carrier, such as magnetic silica particles. A sample containing the antigen is mixed with the solid-phase carrier and a second binding substance that recognizes the antigen and is labeled with peroxidase. The immobilized first binding substance is brought into contact with the antigen in the sample and the second binding substance. This allows a peroxidase-labeled complex to be formed, which is a complex of the immobilized first binding substance, the antigen in the sample, and the second binding substance labeled with peroxidase. The solid-phase carrier carrying the peroxidase-labeled complex is then subjected to B / F separation, the amount of peroxidase in the peroxidase-labeled complex is measured, and the amount of antigen in the sample is determined based on the amount of peroxidase in the peroxidase-labeled complex.

[0038] Alternatively, for example, a first binding substance that recognizes an antigen is immobilized on the surface of a solid-phase carrier such as magnetic silica particles. A sample containing the antigen is mixed with the solid-phase carrier and a peroxidase-labeled "third binding substance that recognizes a complex between the antigen and the first binding substance that recognizes the antigen." The immobilized first binding substance is then contacted with the antigen in the sample and the third binding substance. This allows the formation of a peroxidase-labeled complex, which is a complex between the immobilized first binding substance, the antigen in the sample, and the peroxidase-labeled third binding substance. The solid-phase carrier carrying the peroxidase-labeled complex is then subjected to B / F separation, the amount of peroxidase in the peroxidase-labeled complex is measured, and the amount of antigen in the sample is determined based on the amount of peroxidase in the peroxidase-labeled complex.

[0039] The solid phase carrier can be any support (particularly an insoluble support) used in conventional immunoassays, and examples thereof include organic substances such as polystyrene, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylamide, polyglycidyl methacrylate, polypropylene, polyolefin, polyimide, polyurethane, polyester, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, silicone rubber, agarose, dextran, and ethylene-maleic anhydride copolymer; inorganic substances such as glass, silicon oxide, diatom, porous glass, ground glass, alumina, silica gel, and metal oxides; magnetic substances such as iron, cobalt, nickel, magnetite, and chromite; and substances prepared from alloys of these magnetic substances.

[0040] Immobilization can be performed on a solid phase (for example, any surface such as beads, magnetic beads, membranes, plates, etc.). Various commercially available magnetic beads can be used. For example, the beads disclosed in WO2012 / 173002A may be used as magnetic beads.

[0041] Specifically, for example, an antigen in a sample is contacted with a first binding substance that recognizes the antigen and is immobilized on the surface of a solid-phase carrier such as magnetic silica particles, thereby forming a complex between the first binding substance that recognizes the antigen immobilized on the surface of the solid-phase carrier and the antigen in the sample. Next, this complex is contacted with a complex of a second binding substance labeled with peroxidase, thereby forming a complex (peroxidase-labeled complex) between the first binding substance that recognizes the antigen immobilized on the solid-phase carrier, the antigen in the sample, and the second binding substance labeled with peroxidase. The peroxidase-labeled complex is then subjected to B / F separation, and the amount of peroxidase in the peroxidase-labeled complex is measured. The amount of antigen in the sample can be determined based on the amount of peroxidase in the peroxidase-labeled complex.

[0042] In the above-described method, the antigen in the sample is reacted with a first binding substance that recognizes the immobilized antigen, and then a second binding substance labeled with peroxidase is reacted therewith. Alternatively, the second binding substance labeled with peroxidase may be reacted with the antigen in the sample, and then the first binding substance that recognizes the immobilized antigen may be reacted therewith, or these three may be reacted simultaneously.

[0043] The B / F separation (bond / free separation) in the sandwich method refers to the separation of a substance supported on a solid phase carrier from other substances. That is, the B / F separation in the present invention includes: (1) the separation of a "complex of a first binding substance that recognizes an antigen immobilized on the surface of a solid phase carrier and an antigen in a sample" from the antigen that was not involved in the formation of the complex and other substances in the reaction system, and (2) the separation of a peroxidase-labeled complex from a second binding substance labeled with peroxidase and other substances in the reaction system that were not involved in the formation of the peroxidase-labeled complex.

[0044] The sandwich method can also be performed using a microfluidic chip (micro-total analysis systems; μTAS). The microfluidic chip includes a flow channel with a sample inlet. For example, at the sample inlet, a first binding substance that recognizes an antigen and that is bound to a polycation or polyanion (e.g., DNA) is contacted with the antigen in the sample to form a complex between the first binding substance that recognizes the antigen and the antigen. Next, an electric field is applied to move the complex, and when the complex comes into contact with a second binding substance labeled with peroxidase present in the flow channel, a complex (peroxidase-labeled complex) is formed between the first binding substance, the antigen, and the second binding substance labeled with peroxidase. The electric field further moves the peroxidase-labeled complex through the flow channel, and the peroxidase-labeled complex undergoes B / F separation before reaching a detection unit in the flow channel. In the detection section, the amount of peroxidase in the peroxidase-labeled complex is measured, and the amount of antigen in the sample is determined based on the amount of peroxidase in the peroxidase-labeled complex.

[0045] In the measurement method of the present invention, the antigen in the sample and the first binding substance that recognizes the antigen immobilized on the surface of the solid phase carrier can be contacted by a conventional treatment such as stirring, mixing, etc. The reaction time can be appropriately set depending on the antigen and the first binding substance, but is usually 1 minute to 24 hours, preferably 1 minute to 1 hour, more preferably 1 to 10 minutes, and particularly preferably 1 to 5 minutes.

[0046] B / F separation in the measurement method of the present invention can be achieved, for example, by utilizing the magnetic properties of the solid carriers, collecting the solid carriers from the outside of the reaction vessel using a magnet or the like, discharging the reaction solution, adding a washing solution, removing the magnet, mixing and dispersing the solid carriers, and washing them. The above procedure may be repeated 1 to 3 times. The washing solution is not particularly limited as long as it is one commonly used in this field.

[0047] As the peroxidase, horseradish peroxidase (HRP) or microperoxidase can be used.

[0048] To bind peroxidase to the second or third binding substance, a method commonly used in this field, for example, a known labeling method commonly used in known EIA [e.g., Medical Chemistry Experiment Lectures, Vol. 8, edited by Yamamura Yuichi, 1st Edition, Nakayama Shoten, 1971; Illustrated Fluorescent Antibodies, by Kawao Akira, 1st Edition, Soft Science Co., Ltd., 1983; Enzyme Immunoassay, edited by Ishikawa Eiji, Kawai Tadashi, and Miyai Kiyoshi, 2nd Edition, Igaku Shoin, 1982, etc.] may be used.

[0049] The amount of peroxidase used is preferably such that the second or third binding substance and peroxidase are used in a molar ratio of, for example, usually 1:0.5 to 20, preferably 1:1 to 15, and more preferably 1:1 to 10. The binding substance that recognizes the antigen labeled with peroxidase may be used by being contained in a buffer solution commonly used in this field, such as Tris buffer, phosphate buffer, veronal buffer, borate buffer, or Good's buffer (e.g., MES (2-morpholinoethanesulfonic acid) buffer). The concentration of the peroxidase bound to the second binding substance or the third binding substance is not particularly limited, but is preferably 0.1 nmol / L or more and 5000 nmol / L or less, more preferably 1 nmol / L or more and 500 nmol / L or less, even more preferably 3 nmol / L or more and 300 nmol / L or less, and particularly preferably 5 nmol / L or more and 150 nmol / L or less.

[0050] When an antibody is used as the binding substance, the pH of the buffer solution may be within a range that does not inhibit the antigen-antibody reaction, and is typically between pH 5 and 9. The buffer solution may also contain stabilizers such as albumin, globulin, water-soluble gelatin, and polyethylene glycol, surfactants, and sugars, as long as they do not inhibit the antigen-antibody reaction. In this specification, pH refers to the value measured in accordance with JIS K0400-12-10:2000 (measurement temperature: 25°C).

[0051] <Kit for measuring human chorionic gonadotropin or estradiol> A second aspect of the present invention relates to a kit for measuring human chorionic gonadotropin or estradiol, comprising a peroxidase bound to a binding substance that recognizes an antigen, a chemiluminescent substance, an oxidizing agent, and one or more compounds represented by the following formula (1): A third aspect of the present invention relates to a kit for measuring human chorionic gonadotropin or estradiol, comprising a peroxidase bound to a binding substance that recognizes a complex of an antigen and the binding substance that recognizes the antigen, a chemiluminescent substance, an oxidizing agent, and one or more compounds represented by the following formula (1):

[0052] In the formula, X represents an unsubstituted or substituted phenyl group, an unsubstituted or substituted pyridyl group, an unsubstituted or substituted oxazolyl group, an unsubstituted or substituted thiophenyl group, or an unsubstituted or substituted pyrrolyl group, and the substituent is a carboxyl group, an amide group, a halogen atom, an alkoxy group having 1 to 5 carbon atoms, or an alkyl group having 1 to 5 carbon atoms.

[0053] Preferred embodiments and specific examples of the peroxidase bound to a binding substance that recognizes an antigen, the chemiluminescent substance, the oxidizing agent, and the one or more compounds represented by formula (1) are as described above in this specification. Furthermore, the peroxidase bound to a binding substance that recognizes an antigen, the peroxidase bound to a binding substance that recognizes a complex of an antigen and a binding substance that recognizes the antigen, the chemiluminescent substance, the oxidizing agent, and the one or more compounds represented by formula (1) in this reagent kit may be contained in the reagent at the concentrations described above in this specification during the reaction.

[0054] In the kit of the present invention, the concentration of the chemiluminescent substance (concentration as a reagent) is not particularly limited, but is preferably 0.2 mmol / L to 20 mmol / L, more preferably 0.4 mmol / L to 15.0 mmol / L, even more preferably 0.5 mmol / L to 10.0 mmol / L, and particularly preferably 0.5 mmol / L to 5.0 mmol / L. In the kit of the present invention, the concentration of the oxidizing agent (concentration as a reagent) is not particularly limited, but is preferably 1.0 mmol / L to 40 mmol / L, more preferably 2 mmol / L to 20 mmol / L, and even more preferably 2.8 mmol / L to 7.0 mmol / L. In the kit of the present invention, the concentration (reagent concentration) of one or more compounds represented by formula (1) is not particularly limited, but is preferably 20 μmol / L to 1000 μmol / L, more preferably 100 μmol / L to 1000 μmol / L, even more preferably 150 μmol / L to 800 μmol / L, and particularly preferably 200 μmol / L to 600 μmol / L. In the kit of the present invention, the concentration (reagent concentration) of the peroxidase bound to the second binding substance or the third binding substance is not particularly limited, but is preferably 0.1 nmol / L to 5000 nmol / L, more preferably 1 nmol / L to 500 nmol / L, even more preferably 3 nmol / L to 300 nmol / L, and particularly preferably 5 nmol / L to 150 nmol / L.

[0055] The assay kit of the present invention may further comprise a different binding substance that recognizes the antigen to be assayed, different from the above-mentioned binding substance bound to peroxidase, or a solid phase carrier to which the above-mentioned different binding substance is immobilized. The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0056] <Synthesis Examples> Synthesis of Various Compounds Purification by silica gel column chromatography used an automatic purification apparatus ISOLERA (manufactured by Biotage). The carrier used in normal phase chromatography was CHROMATOREX Q-PACK (manufactured by Fuji Silysia Chemical Ltd.). The carrier used in reverse phase chromatography was Sfar C18 (manufactured by Biotage). Mass (MS) spectra were measured using an ACQUITY SQD LC / MS System (manufactured by Waters) with an ionization method of ESI (ElectroSpray Ionization). Retention times (RT) were measured using an SQD (Waters) and are shown in minutes (min). Column: Waters BEH C18 1.7 μm, 2.1 × 30 mm Solvent: Solution A: 0.1% formic acid - water Solution B: 0.1% formic acid - acetonitrile Gradient cycle: 0.00 min (solution A / solution B = 95 / 5), 2.00 min (solution A / solution B = 5 / 95), 3.00 min (solution A / solution B = 95 / 5) Flow rate: 0.5 mL / min Column temperature: 40 ° C Detection wavelength: 254 nm

[0057] Synthesis of 3'-fluoro-[1,1'-biphenyl]-4-ol

[0058] To 4-hydroxyphenylboronic acid (100 mg), a solution of 1-bromo-3-fluorobenzene (93.0 mg) in N,N-dimethylformamide (1.0 mL), sodium carbonate (154 mg), water (100 μL), and tetrakis(triphenylphosphine)palladium (168 mg) were added, and the initiator ＴＭ The mixture was stirred at 120°C under a nitrogen atmosphere for 1 hour using a chromatograph (Biotage). The reaction mixture was purified by normal phase column chromatography (ethyl acetate / hexane = 0 / 100 → 100 / 0) and reverse phase column chromatography (0.1% formic acid-acetonitrile / 0.1% formic acid-water = 0 / 100 → 100 / 0) to obtain 3'-fluoro-[1,1'-biphenyl]-4-ol as a white solid (31.9 mg). MS (ESI m / z): 187.3 (M-H) RT (min): 1.37

[0059] The compounds shown in Table 1 below were synthesized in the same manner as in the synthesis of 3'-fluoro-[1,1'-biphenyl]-4-ol.

[0060]

[0061] Example 1 Evaluation of BiPCA in hCG Measurement 1. Preparation of Luminescent Reagent (hCG) The constituent reagents required for measurement were prepared using the following raw reagent materials: MES (2-morpholinoethanesulfonic acid monohydrate) (manufactured by Dojindo Laboratories), sodium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), BCN300S (manufactured by Nitta Gelatin Co., Ltd.), Block Ace (manufactured by KAC Corporation), BSA (bovine serum albumin) (manufactured by Sigma-Aldrich Japan Co., Ltd.), sodium orthovanadate (manufactured by Sigma-Aldrich Japan Co., Ltd.), luminol sodium salt (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), phosphoric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), EDTA-2Na (manufactured by Dojindo Laboratories), hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and dipotassium hydrogen phosphate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0062] (i) "First Reagent" Magnetic Particle-Solidified Antibody-Containing Reagent Maglapid MGP-010T (Sanyo Chemical Industries, Ltd.) was reacted with 3-Aminopropyltriethoxysilane (Tokyo Chemical Industry Co., Ltd.) and succinic anhydride (Fujifilm Wako Pure Chemical Industries, Ltd.), followed by magnetization with a neodymium magnet and removal of the supernatant to obtain magnetic particles having carboxyl groups. Next, the carboxyl groups were activated with commercially available N-hydroxysuccinimide (Fujifilm Wako Pure Chemical Industries, Ltd.) and WSC (water-soluble carbodiimide) (Dojindo Laboratories), and then the anti-hCG antibody Mab to hCG beta (Meridian Japan Co., Ltd.) was reacted at 26°C for 12 to 16 hours, followed by magnetization with a neodymium magnet and removal of the supernatant to prepare magnetic particles with immobilized anti-hCG antibodies, thereby preparing a first reagent having the following composition: "First reagent": anti-hCG antibody-immobilized magnetic particles 0.5 mg / mL, 50 mM MES (pH 5.5), 500 mM sodium chloride, 3.0% BCN300S

[0063] (ii) "Second Reagent" Reaction Buffer: 50 mM MOPS (3-morpholinopropanesulfonic acid) (manufactured by Dojindo Laboratories) (pH 7.5), 500 mM sodium chloride, 0.4% Block Ace

[0064] (iii) "Third Reagent" Labeled Antibody-Containing Reagent MAB<HCG>M-INN22 IgG (Roche Diagnostics), an anti-hCG antibody, was digested with pepsin, and then F(ab')2 was separated using a column (diameter: 1.5 cm x length: approximately 95 cm) packed with Sephacryl S-200HR (Cytiva). The resulting F(ab')2 was reduced with cysteamine hydrochloride (Sigma-Aldrich Japan), and then Fab' was separated using a column (diameter: 1.5 cm x length: approximately 40 cm) packed with G-25 Superfine (Cytiva). On the other hand, peroxidase (POD) (Roche Diagnostics) was maleimidated using a maleimidation reagent, Sulfo-KMUS (Dojindo Laboratories), and the reaction mixture was passed through a Sephadex G-25 column to remove unreacted Sulfo-KMUS, yielding maleimidated POD. The prepared Fab' and maleimidated POD were mixed and separated on a Sephacryl S-100HR column to produce a POD-labeled anti-hCG antibody. This was used to prepare a third reagent consisting of the following composition. "Third reagent": 6 nmol / L POD-labeled anti-hCG antibody, 50 mM MES (pH 6.5), 150 mM sodium chloride, 2.0% BSA

[0065] (iv) "Fourth Reagent" 1.82 g (50 mM) of 2-amino-2-hydroxymethyl-1,3-propanediol Tris(hydroxymethyl)aminomethane (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), 0.690 mL (0.069%) of 5N-HCl (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), 0.877 g (150 mM) of sodium chloride (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), 8.57 mg (1 mM) of sodium orthovanadate (manufactured by Sigma-Aldrich Japan K.K.), 0.1 g (5 mM) of sodium luminol (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), and BiPCA (a product of Tokyo Chemical Industry Co., Ltd.) were dissolved in 100 mL of purified water to a concentration of 0.2 mM to prepare a fourth reagent.

[0066] (V) "Fifth Reagent" "Fifth Reagent": 68 μL / L phosphoric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 4mM EDTA-2Na (manufactured by Dojindo Laboratories), 335 μL / L hydrogen peroxide (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.)

[0067] 2. Preparation of Blank Sample A blank sample was prepared by dissolving 10.46 g (50 mM) of bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 1.7 mL (0.17%) of 5N-HCl (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 20 g (2.0%) of bovine serum albumin (manufactured by Sigma-Aldrich Japan K.K.) in 1,000 mL of purified water.

[0068] 3. Preparation of measurement samples (hCG) Human chorionic gonadotropin (manufactured by Meridian Japan Co., Ltd.) was dissolved in 10 mL of the blank sample prepared in "2. Preparation of blank sample" to prepare a 1,000 mIU / mL high-unit hCG solution. Using the blank sample and high-unit hCG solution prepared in "2. Preparation of blank sample", 0, 10, 20,000, and 30,000 mIU / mL hCG solutions were prepared as measurement samples.

[0069] 4. Evaluation of Luminescent Reagents Using the first to fifth reagents prepared in "1. Preparation of Luminescent Reagent (hCG)" and Accura Seed B / F Separation Solution (Fujifilm Wako Pure Chemical Corporation), the luminescence intensity of each solution was measured using an automated chemiluminescent enzyme immunoassay analyzer, Accura Seed (Fujifilm Wako Pure Chemical Corporation), according to the following procedure. 50 μL of the first reagent added to a reaction cuvette was heated at 67°C for 20 seconds while magnetically collected using a neodymium magnet, and the supernatant was removed. Subsequently, 140 μL of the second reagent and 10 μL of a measurement sample (hCG solution) with concentrations of 0, 10, 20,000, or 30,000 mIU / mL were added, stirred, and heated at 37°C for 3 minutes. After heating, the solution was magnetically collected using a neodymium magnet, and the reagents other than the magnetic particles were removed. The solution was then washed three times with washing solution. Next, 50 μL of the third reagent was added, and the mixture was heated at 37° C. for 3 minutes. After heating, the mixture was collected using a neodymium magnet to remove all reagents other than the magnetic particles, and then washed three times with a washing solution. After washing, 100 μL of the fourth reagent and 100 μL of the fifth reagent were added, and after incubation at 37° C. for 20 seconds, the luminescence intensity was measured.

[0070] The low and high sensitivity were calculated from the obtained luminescence amount. The low sensitivity was calculated as (luminescence amount of 10 mIU / mL hCG solution) / (luminescence amount of blank sample), and the high sensitivity was calculated as (luminescence amount of 30,000 mIU / mL hCG solution) / (luminescence amount of 20,000 mIU / mL hCG solution). The results are shown in Table 4.

[0071] Comparative Example 1: Evaluation of thiazolephenol (TP) in hCG measurement A luminescence reagent was prepared and evaluated in the same manner as in Example 1, except for the following: In place of BiPCA (a product of Tokyo Chemical Industry Co., Ltd.) in the "fourth reagent" of Example 1(iv), thiazolephenol (TP) (Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved to a concentration of 0.2 mM was used as the "fourth reagent." The results are shown in Table 4.

[0072] Example 2 Evaluation of BiPCA in E2 Measurement 1. Preparation of Luminescent Reagent (E2) A luminescent reagent was prepared in the same manner as in "1. Preparation of Luminescent Reagent (hCG)" in Example 1, except for the following: In the "first reagent," Sheep Monoclonal Antibodies (SMAs) to E2 5A11 (manufactured by BioVentix Inc.) was used instead of the anti-hCG antibody Mab to hCG beta (manufactured by Meridian Japan Co., Ltd.) to prepare anti-E2 antibody-immobilized magnetic particles. The obtained anti-E2 antibody-immobilized magnetic particles were used instead of the anti-hCG antibody-immobilized magnetic particles in the "first reagent." Furthermore, in the "third reagent", an anti-immune complex M19-1G antibody produced by a hybridoma prepared by the method described below was used in place of the anti-hCG antibody MAB<HCG>M-INN22 IgG (manufactured by Roche Diagnostics K.K.), to prepare a POD-labeled anti-immune complex M19-1G antibody. Furthermore, the obtained POD-labeled anti-immune complex M19-1G antibody was used in place of the POD-labeled anti-hCG antibody in the "third reagent".

[0073] Hybridomas were produced by the following method. A complex of the immunogen E2 and an anti-E2 antibody was injected into a mouse to immunize it, and lymphocytes were then collected. The resulting lymphocytes were fused with myeloma cells, and hybridoma cells producing the desired antibody were selected. After cloning, hybridomas producing the desired antibody were produced.

[0074] 2. Preparation of Blank Sample A blank sample was prepared in the same manner as in Example 1, "2. Preparation of Blank Sample."

[0075] 3. Preparation of Measurement Sample (E2) β-estradiol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 10 mL of the blank sample prepared in "2. Preparation of Blank Sample" to prepare a 10,000 pg / mL high-unit E2 solution. Using the blank sample prepared in "2. Preparation of Blank Sample" and the high-unit E2 solution, measurement samples were prepared at 0, 200, 2,000, and 3,000 pg / mL E2 solutions.

[0076] 4. Evaluation of the luminescence reagent Using the above luminescence reagent, the luminescence intensity was measured in the same manner as in Example 1, "4. Evaluation of the luminescence reagent," and the low and high sensitivity values ​​were calculated. The low sensitivity value was calculated as (luminescence intensity of 200 pg / mL E2 solution) / (blank luminescence intensity), and the high sensitivity value was calculated as (luminescence intensity of 3000 pg / mL E2 solution) / (luminescence intensity of 2000 pg / mL E2 solution). The results are shown in Table 4 and Figure 1.

[0077] (Examples 3 to 17) Evaluation of Various Compounds in E2 Measurement Luminescence reagents were prepared and evaluated in the same manner as in Example 2, with the following exceptions. Instead of BiPCA (a product of Tokyo Chemical Industry Co., Ltd.) in the fourth reagent of Example 2, 4-(pyridin-2-yl)phenol (manufactured by Combi-Blocks), 4-(oxazol-4-yl)phenol (manufactured by Combi-Blocks), 4-(1H-pyrrol-1-yl)phenol (manufactured by Enamine), and the enhancer produced in the synthesis example were dissolved to 0.2 mM (reaction concentration during measurement was 0.1 mM) and used as the "fourth reagent." The structures of the enhancers used in Examples 3 to 17 are shown below. The results are shown in Table 4.

[0078]

[0079] Comparative Example 2: Evaluation of thiazolephenol (TP) in E2 measurement A luminescence reagent was prepared and evaluated in the same manner as in Example 2, except for the following. Instead of BiPCA (a product of Tokyo Chemical Industry Co., Ltd.) in the "fourth reagent" of Example 2, thiazolephenol (TP) (Fujifilm Wako Pure Chemical Industries, Ltd.) dissolved to a concentration of 0.2 mM was used as the "fourth reagent." The results are shown in Table 4 and Figure 1.

[0080] (Comparative Examples 3-4) Evaluation of Various Compounds in E2 Measurement A luminescence reagent was prepared and evaluated in the same manner as in Example 2, except for the following. Instead of BiPCA (a product of Tokyo Chemical Industry Co., Ltd.) in the fourth reagent of Example 2, the compounds listed in Table 3 below (all products of Tokyo Chemical Industry Co., Ltd.) were dissolved to a concentration of 0.2 mM (reaction concentration during measurement: 0.1 mM) and used as the "fourth reagent." The results are shown in Table 4.

[0081]

[0082] Comparative Example 5-6: Evaluation of BiPCA in HBs antigen measurement 1. Preparation of luminescent reagent (HBs antigen) A luminescent reagent was prepared using the same method as in Example 1, "1. Preparation of luminescent reagent (hCG)," except for the following: In the "first reagent," anti-HBs antibody produced by a hybridoma prepared by the method described below was used instead of the anti-hCG antibody Mab to hCG beta (manufactured by Meridian Japan Co., Ltd.) in the "first reagent." Anti-HBs antibody-immobilized magnetic particles were prepared using the resulting anti-HBs antigen-antibody-immobilized magnetic particles instead of the anti-hCG antibody-immobilized magnetic particles in the "first reagent." Hybridomas were prepared using the following method: HBs antigen, an immunogen, was injected into a mouse as an immunized animal to immunize the animal, and lymphocytes were then recovered. The resulting lymphocytes were fused with a myeloma, and hybridoma cells producing the target antibody were selected. After cloning, hybridomas producing the target antibody were prepared. In the "third reagent", an anti-HBs antigen antibody produced by the hybridoma prepared by the above method was used in place of the anti-hCG antibody MAB<HCG>M-INN22 IgG (manufactured by Roche Diagnostics K.K.), to obtain a POD-labeled anti-HBs antibody. In place of the POD-labeled hCG antibody in the "third reagent", a POD-labeled anti-HBs antibody was used. In the "fourth reagent", L-012 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used in place of luminol sodium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0083] 2. Preparation of Blank Sample A blank sample was prepared in the same manner as in Example 1, "2. Preparation of Blank Sample."

[0084] 3. Sample Preparation (HBs Antigen) HBs antigen (ad) (manufactured by Trina Bioreactives, Inc.) was dissolved in 10 mL of the blank sample prepared in "2. Preparation of Blank Sample" to prepare a 2,000 IU / mL high-unit HBs antigen solution. Using the blank sample prepared in "2. Preparation of Blank Sample" and the high-unit HBs antigen solution, 0, 0.2, 50, and 300 IU / mL HBs antigen solutions were prepared as measurement samples.

[0085] 4. Evaluation of luminescent reagents Using the above luminescent reagents, the luminescence intensity when each luminescent reagent was measured using an automatic chemiluminescent enzyme immunoassay analyzer, Accura Seed (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), was measured, and the low and high sensitivity values ​​were calculated. The low sensitivity value was calculated as (HBs antigen 0.2 IU / mL luminescence intensity) / (blank luminescence intensity), and the high sensitivity value was calculated as (HBs antigen 300 IU / mL luminescence intensity) / (HBs antigen 50 IU / mL luminescence intensity). The results are shown in Table 4.

[0086] <Examples 18 and 19> Evaluation of BiPCA in E2 Measurement Luminescence reagents were prepared and evaluated in the same manner as in Example 2, except that instead of 0.2 mM BiPCA (product of Tokyo Chemical Industry Co., Ltd.) in the "fourth reagent," BiPCA was used at the following concentrations. The results are shown in Table 4. Example 18: 0.3 mM BiPCA in the "fourth reagent" (reaction concentration at the time of measurement: 0.15 mM) Example 19: 0.6 mM BiPCA in the "fourth reagent" (reaction concentration at the time of measurement: 0.30 mM)

[0087] <Examples 20 and 21> Evaluation of BiPCA in hCG Measurement Luminescent reagents were prepared and evaluated in the same manner as in Example 1, except that instead of 0.2 mM BiPCA (product of Tokyo Chemical Industry Co., Ltd.) in the "fourth reagent," BiPCA was used at the following concentrations. The results are shown in Table 4. Example 20: 0.3 mM BiPCA in the "fourth reagent" (reaction concentration at the time of measurement: 0.15 mM) Example 21: 0.6 mM BiPCA in the "fourth reagent" (reaction concentration at the time of measurement: 0.30 mM)

[0088]

[0089] It is desirable that the luminescence intensity increases with the concentration of the analyte, at least within the concentration range required for each analyte. However, as shown in Table 4 and Figure 1, when hCG (Comparative Example 1) or E2 (Comparative Example 2) was measured using TP, which is known as an enhancer that enhances chemiluminescence, the luminescence intensity did not reach the expected level when hCG or E2 was present at high concentrations within the concentration range required for hCG or E2 measurement, and no increase in luminescence was observed, making it impossible to accurately measure the amount of the analyte.

[0090] Therefore, using TP as the standard, compounds that significantly improved high-value sensitivity compared to TP and achieved low-value sensitivity equivalent to that of TP were investigated. As a result, the compounds used in Comparative Examples 3 and 4 had the effect of enhancing chemiluminescence as an enhancer, and improved high-value sensitivity compared to TP in Comparative Example 2, but significantly reduced low-value sensitivity. On the other hand, the compounds used in Examples 1 to 17 achieved the same low-value sensitivity as TP in Comparative Example 2, and significantly improved high-value sensitivity by 1.2 times or more. As a result of Examples 18 to 21, similar effects were observed even when the reaction concentration of BiPCA during measurement was changed from 0.1 mM to 0.15 mM or 0.3 mM.

[0091] Furthermore, the results of Comparative Examples 5 and 6 showed that when BiPCA was used in the measurement of HBs antigen, the high-value sensitivity was slightly improved compared to TP, but the low-value sensitivity was significantly reduced. That is, the effect of the compound according to the present invention in improving the high-value sensitivity while maintaining the low-value sensitivity (same as when TP is used) was confirmed only in the measurement of hCG and E2.

Claims

1. A method for measuring an antigen contained in a test sample, the antigen being human chorionic gonadotropin or estradiol, comprising reacting (1) an antigen bound to peroxidase via a binding substance that recognizes the antigen, or an antigen bound to peroxidase via a binding substance that recognizes a complex of the antigen and a binding substance that recognizes the antigen, (2) a chemiluminescent substance, and (3) an oxidizing agent, in the presence of one or more compounds represented by the following formula (1): In the formula, X is an unsubstituted or substituted phenyl group, an unsubstituted or substituted pyridyl group, an unsubstituted or substituted oxazolyl group, an unsubstituted or substituted thiophenyl group, or an unsubstituted or substituted pyrrolyl group, and the substituent is a carboxyl group, an amide group, a halogen atom, an alkoxy group having 1 to 5 carbon atoms, or an alkyl group having 1 to 5 carbon atoms.

2. The method according to claim 1, wherein X is a phenyl group having a carboxyl group, an amide group, a halogen atom or an alkoxy group having 1 to 5 carbon atoms; an unsubstituted pyridyl group or a pyridyl group having an alkyl group having 1 to 5 carbon atoms or an alkoxy group having 1 to 5 carbon atoms; an oxazolyl group or an alkoxy group having 1 to 5 carbon atoms or an oxazolyl group having an alkyl group having 1 to 5 carbon atoms or an alkoxy group having 1 to 5 carbon atoms; an unsubstituted thiophenyl group or a pyrrolyl group having an alkyl group having 1 to 5 carbon atoms or an alkoxy group having 1 to 5 carbon atoms; 3. The method according to claim 1, wherein X is a phenyl group having a carboxyl group, an amide group, a halogen atom or an alkoxy group having 1 to 5 carbon atoms.

4. The measurement method according to claim 1, wherein the antigen to which peroxidase is bound via a binding substance that recognizes the antigen is obtained by reacting the antigen with a first binding substance that recognizes the antigen to form a complex, and then reacting the complex with a second binding substance to which peroxidase is bound that recognizes the antigen.

5. The measurement method according to claim 1, wherein the antigen to which peroxidase is bound via a binding substance that recognizes a complex of the antigen and a binding substance that recognizes the antigen is obtained by reacting the antigen with a first binding substance that recognizes the antigen to form a complex, and then reacting the complex with a "third binding substance that recognizes the complex of the antigen to which peroxidase is bound and the first binding substance that recognizes the antigen." 6. The method according to any one of claims 1 to 5, wherein the concentration of the compound represented by formula (1) is 10 μmol / L or more and 500 μmol / L or less.

7. The method according to any one of claims 1 to 5, wherein the chemiluminescent substance is luminol, a luminol derivative, 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione sodium salt, or a salt thereof.

8. The measurement method according to claim 7, wherein the concentration of the chemiluminescent substance is 0.1 mmol / L or more and 10 mmol / L or less.

9. A method for measuring the concentration of iodine according to any one of claims 1 to 5, wherein the oxidizing agent is hydrogen peroxide.

10. A kit for measuring human chorionic gonadotropin or estradiol, comprising a peroxidase bound to a binding substance that recognizes an antigen, a chemiluminescent substance, an oxidizing agent, and one or more compounds represented by the following formula (1): In the formula, X is an unsubstituted or substituted phenyl group, an unsubstituted or substituted pyridyl group, an unsubstituted or substituted oxazolyl group, an unsubstituted or substituted thiophenyl group, or an unsubstituted or substituted pyrrolyl group, and the substituent is a carboxyl group, an amide group, a halogen atom, an alkoxy group having 1 to 5 carbon atoms, or an alkyl group having 1 to 5 carbon atoms.

11. The kit according to claim 10, further comprising another binding substance that recognizes the antigen to be measured, different from the binding substance bound to peroxidase, or a solid phase carrier to which the other binding substance is immobilized.

12. A kit for measuring human chorionic gonadotropin or estradiol, comprising a peroxidase bound to a binding substance that recognizes a complex between an antigen and a binding substance that recognizes the antigen, a chemiluminescent substance, an oxidizing agent, and one or more compounds represented by the following formula (1): In the formula, X is an unsubstituted or substituted phenyl group, an unsubstituted or substituted pyridyl group, an unsubstituted or substituted oxazolyl group, an unsubstituted or substituted thiophenyl group, or an unsubstituted or substituted pyrrolyl group, and the substituent is a carboxyl group, an amide group, a halogen atom, an alkoxy group having 1 to 5 carbon atoms, or an alkyl group having 1 to 5 carbon atoms.

13. The kit according to claim 12, further comprising another binding substance that recognizes the antigen to be measured, different from the binding substance bound to peroxidase, or a solid phase carrier to which the other binding substance is immobilized.

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