Enhancer reagent for chemiluminescent reaction, and compound

Abstract

The present invention addresses the problem of providing: an enhancer reagent that increases the amount of chemiluminescence, the enhancer regent being capable of, in measuring a measurement substance, extending the measurement concentration upper limit while allowing for luminescent measurement at the measurement concentration lower limit; and a compound that is capable of increasing the amount of chemiluminescence. The present invention provides an enhancer reagent for a chemiluminescent reaction, the enhancer reagent containing a compound represented by formula (1). In the formula, X represents a pyridyl group that may be substituted by one or more groups.

Description

Chemiluminescence reaction enhancer reagents and compounds

[0001] The present invention relates to a chemiluminescent reaction enhancer reagent that can enhance the amount of chemiluminescence when measuring a substance to be measured using chemiluminescence. The present invention further relates to a compound useful as a chemiluminescent reaction enhancer reagent.

[0002] In a chemiluminescence measurement system using luminol or the like, it is known that PPP (4-phenylphenol) and TP (thiazolephenol) are used as enhancers to increase the amount of chemiluminescence.

[0003] Patent Document 1 describes TP (thiazole phenol) as an enhancer, and Patent Document 2 describes PPP, but neither mentions the measurement range on the high concentration side. As an effect, they describe an improvement in sensitivity in the low concentration range.

[0004] Patent No. 03030043 Publication Special Publication No. 03-005539

[0005] For example, in the measurement of human chorionic gonadotropin (hCG) or estradiol (E2), it is necessary to measure samples with a wide range of antigen concentrations, from low to high. However, through studies by the present inventors, it has been found that measurements using TP or PPP have the problem of being unable to measure samples with a wide range of antigen concentrations, i.e., unable to extend the upper limit of measurement while maintaining the lower limit of measurement, and unable to maintain linearity over a wide range.

[0006] An object of the present invention is to provide an enhancer reagent for enhancing the amount of chemiluminescence, which can achieve excellent linearity in the high concentration range (hereinafter referred to as high-value slope) in the measurement of a substance to be measured. Another object of the present invention is to provide a compound that can enhance the amount of chemiluminescence.

[0007] The present inventors have conducted extensive research to solve the above problems and, as a result of screening various compounds, have found that enhancer reagents having a pyridine skeleton can achieve an excellent high slope. The present invention was completed based on the above findings. Specifically, the present invention provides the following:

[0008] <1> A chemiluminescent reaction enhancer reagent comprising a compound represented by formula (1). In the formula, X represents a pyridyl group optionally substituted with one or more groups selected from the group consisting of an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, a heterocyclic group, an alkoxy group, a cycloalkoxy group, an aryloxy group, an alkoxycarbonyl group, a cycloalkoxycarbonyl group, an amino group, an acylamino group, a silyl group, a silyloxy group, a hydroxyl group, a cyano group, a nitro group, a halogen atom, a carboxy group, a sulfo group, a phosphonyl group, a phosphoryl group, a boric acid group, and a sulfanyl group. <2> The enhancer reagent according to <1>, wherein X is a 3-pyridyl group optionally substituted with one or more groups selected from the group consisting of an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, a heterocyclic group, an alkoxy group, a cycloalkoxy group, an aryloxy group, an alkoxycarbonyl group, a cycloalkoxycarbonyl group, an amino group, an acylamino group, a silyl group, a silyloxy group, a hydroxyl group, a cyano group, a nitro group, a halogen atom, a carboxy group, a sulfo group, a phosphonyl group, a phosphoryl group, a boric acid group, and a sulfanyl group. <3> The enhancer reagent according to <1>, wherein X is a 3-pyridyl group substituted with one or more groups selected from the group consisting of an alkyl group, an alkoxy group, a halogen atom, and an amino group. <4> The enhancer reagent according to <1>, wherein X is a 3-pyridyl group substituted with one or more groups selected from the group consisting of an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, and a fluorine atom. <5> The enhancer reagent according to <1>, wherein the compound represented by formula (1) is any one of the following compounds: <6> The compound shown below.

[0009] The chemiluminescent reaction enhancer reagent and compound of the present invention can achieve an excellent high value gradient in the measurement of a substance to be measured.

[0010] The present invention will be described in detail below. In this specification, mM represents mmol / L.

[0011] <Chemiluminescent Reaction Enhancer Reagent> The chemiluminescent reaction enhancer reagent of the present invention comprises a compound represented by formula (1). The chemiluminescent reaction enhancer reagent is a reagent that can be used to enhance the amount of chemiluminescence, increase the luminescence rate, and extend the luminescence time when measuring a substance to be measured using chemiluminescence. In the formula, X represents a pyridyl group optionally substituted with one or more groups selected from the group consisting of an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, a heterocyclic group, an alkoxy group, a cycloalkoxy group, an aryloxy group, an alkoxycarbonyl group, a cycloalkoxycarbonyl group, an amino group, an acylamino group, a silyl group, a silyloxy group, a hydroxyl group, a cyano group, a nitro group, a halogen atom, a carboxy group, a sulfo group, a phosphonyl group, a phosphoryl group, a boric acid group, and a sulfanyl group.

[0012] Preferably, X is a 3-pyridyl group optionally substituted with one or more groups selected from the group consisting of an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, a heterocyclic group, an alkoxy group, a cycloalkoxy group, an aryloxy group, an alkoxycarbonyl group, a cycloalkoxycarbonyl group, an amino group, an acylamino group, a silyl group, a silyloxy group, a hydroxyl group, a cyano group, a nitro group, a halogen atom, a carboxy group, a sulfo group, a phosphonyl group, a phosphoryl group, a boric acid group, and a sulfanyl group.

[0013] More preferably, X is a 3-pyridyl group substituted with one or more groups selected from the group consisting of an alkyl group, an alkoxy group, a halogen atom, and an amino group.

[0014] More preferably, X is a 3-pyridyl group substituted with one or more groups selected from the group consisting of an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, and a fluorine atom.

[0015] The alkyl group is preferably an alkyl group having 1 to 5 carbon atoms. 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. The alkyl group having 1 to 5 carbon atoms is preferably a methyl group. The alkenyl group is preferably an alkyl group having 2 to 5 carbon atoms. Examples of the alkyl group having 2 to 5 carbon atoms include an ethenyl group, a propenyl group, a butenyl group, and a pentenyl group, which may be linear or branched.

[0016] The cycloalkyl group is preferably a cycloalkyl group having 3 to 6 carbon atoms. Examples of the cycloalkyl group having 3 to 6 carbon atoms include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group.

[0017] The aryl group is preferably an aryl group having 6 to 14 carbon atoms, and examples thereof include a phenyl group, a 1-naphthyl group, a 2-naphthyl group, a biphenylyl group, and a 2-anthryl group.

[0018] Examples of the heterocyclic group include monocyclic or bicyclic or higher heterocyclic groups containing one or more heteroatoms selected from oxygen, nitrogen, and sulfur atoms, such as a pyridyl group, a pyrazolyl group, an imidazolyl group, a benzimidazolyl group, a thiazolyl group, an oxazolyl group, a pyrimidyl group, an isoxazolyl group, a triazolyl group, a furanyl group, a thiophenyl group, and a pyrrole group.

[0019] The alkoxy group is preferably an alkoxy group having 1 to 5 carbon atoms. 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, and these may be linear or branched. The alkoxy group having 1 to 5 carbon atoms is preferably a methoxy group. The cycloalkoxy group is preferably a cycloalkoxy group having 3 to 6 carbon atoms. Examples of the cycloalkoxy group having 3 to 6 carbon atoms include a cyclopentyloxy group and a cyclohexyloxy group. The aryloxy group is preferably an aryloxy group having 6 to 20 carbon atoms. Examples of the aryloxy group having 6 to 20 carbon atoms include a phenoxy group.

[0020] The alkoxycarbonyl group is preferably an alkoxycarbonyl group having 2 to 6 carbon atoms. Examples of the alkoxycarbonyl group having 2 to 6 carbon atoms include a methoxycarbonyl group, an ethoxycarbonyl group, a propoxycarbonyl group, a butoxycarbonyl group, and a pentyloxycarbonyl group. The cycloalkoxycarbonyl group is preferably a cycloalkoxycarbonyl group having 4 to 7 carbon atoms. Examples of the cycloalkoxycarbonyl group having 4 to 7 carbon atoms include a cyclopentyloxycarbonyl group and a cyclohexyloxycarbonyl group.

[0021] Examples of the acylamino group include a formylamino group, an alkylcarbonylamino group having 2 to 30 carbon atoms, and an arylcarbonylamino group having 6 to 30 carbon atoms. Specific examples of the acylamino group include a formylamino group, an acetylamino group, a pivaloylamino group, a lauroylamino group, and a benzoylamino group.

[0022] Examples of the silyl group include —Si(R １ ) (R ２ ) (R ３ ) groups represented by R １ , R ２ and R ３ each independently represents a hydrogen atom or a substituent, and the substituent is preferably an alkyl group, an alkoxy group, an aryl group or a heterocyclic group.

[0023] The silyloxy group is —O—Si(R １ ) (R ２ ) (R ３ ) groups represented by R １ , R ２ and R ３ each independently represents a hydrogen atom or a substituent, and the substituent is preferably an alkyl group, an alkoxy group, an aryl group or a heterocyclic group.

[0024] The halogen atom includes fluorine, chlorine, bromine, or iodine, and is preferably fluorine or chlorine.

[0025] Preferred examples of the compound represented by formula (1) include any of the following compounds, but the compound represented by formula (1) is not limited to the following compounds.

[0026]

[0027] <Compounds> According to the present invention, the following compounds are provided: The compounds shown below are useful as enhancer reagents for chemiluminescent reactions.

[0028] The compound of the present invention can be synthesized in accordance with the method described in "Synthesis of 4-(5-fluoro-6-methoxypyridin-3-yl)phenol" or "Synthesis of 4-(6-methoxypyridin-2-yl)phenol" in the Examples below.

[0029] <Method for measuring a target substance> The chemiluminescent reaction enhancer reagent of the present invention can be used for measuring a target substance. The method for measuring a target substance using the chemiluminescent reaction enhancer reagent of the present invention may be a method for measuring a target substance contained in a test sample.

[0030] The method for measuring a substance to be measured using the chemiluminescent reaction enhancer reagent of the present invention comprises reacting (1) the substance to be measured to which peroxidase is bound via a binding substance that recognizes the substance to be measured, or the substance to be measured to which peroxidase is bound via a binding substance that recognizes a complex of the substance to be measured and a binding substance that recognizes the substance to be measured, (2) a chemiluminescent substance, and (3) an oxidizing agent, in the presence of one or more compounds represented by formula (1).

[0031] Examples of test samples include, but are not limited to, biological fluids such as serum, blood, plasma, and urine, and biological samples such as lymph, blood cells, and various types of cells.

[0032] In the present invention, the substance to be measured contained in the test sample is not particularly limited, and may be any substance to be measured. The substance to be measured may be an antigen.

[0033] In the present invention, the substance to be measured is not particularly limited, and examples thereof include all substances that are considered to be measurable by ordinary immunological assays, such as steroids, peptides, hormones, antibodies, proteins, drugs, metabolites, vitamins, etc. contained in biological samples. Specific examples of substances related to endocrine function include thyroid-stimulating hormone (TSH), parathyroid hormone (iPTH), growth hormone (GH), somatomedin C (IGF-1), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), adrenocorticotropic hormone (ACTH), vasopressin, oxytocin, somatostatin, enkephalin, β-endorphin, thyroxine, triiodothyronine, thyroglobulin, anti-thyroglobulin antibodies, and anti-T3 Antibody, anti-T4 antibody, anti-TSH antibody, calcitonin, catecholamine, dopamine, serotonin, aldosterone, renin, angiotensin, cortisol, deoxycortisol, cortisone, corticosterone, deoxycorticosterone, androsterone, progesterone, pregnenolone, estrogen, estrone, estriol, estradiol, testosterone, human chorionic gonadotropin, insulin, anti-insulin antibody, C-peptide, glucagon, galectin Examples of substances related to tumors include CEA, ferritin, β2-microglobulin, elastase, α-fetoprotein, nerve-specific enolase, prostate-specific antigen, CA19-9, etc. Examples of drugs and vitamin-related substances include phenobarbital, phenytoin, carbamazepine, primidone, ethosuximide, valproate, etc. Acid, acetazolamide, sulthiame, glutethimide, clonazepam, nitrazepam, diazepam, pentobarbital, secobarbital, bupivacaine, mepivacaine, lidocaine, procainamide, quinidine, digoxin, digitoxin, theophylline, amitriptyline, imipramine, amikacin, gentamicin, tobramycin, cephalexin, sulfamethoxazole, methotrexate, cyclosporine, methylprednisolone, salicylic acid, acetaminophen,Examples of such substances include indomethacin, allopurinol, vitamin A, carotene, vitamin B1, vitamin B2, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin D, and vitamin E. Examples of serum or plasma protein-related substances include albumin, α1-microglobulin, α1-antitrypsin, α2-macroglobulin, haptoglobulin, hemopexin, transferrin, myoglobin, IgG, IgM, IgA, IgD, IgE, fibrinogen, antithrombin, plasminogen, antiplasmin, protein C, rheumatoid factor, anti-DNA antibody, and C-reactive protein. Examples of virus and infectious disease-related substances include, but are not limited to, HBs antigen, HBs antibody, HBc antibody, HTLV-I antibody, HTLV-III antibody, TPHA, various viral antigens, and various viral antibodies.

[0034] The concentration of the compound represented by formula (1) is preferably 10 μmol / L to 500 μmol / L, more preferably 50 μmol / L to 500 μmol / L, even more preferably 75 μmol / L to 400 μmol / L, and particularly preferably 100 μmol / L to 300 μmol / L. The concentration referred to here is the concentration in a reaction solution when a analyte bound to peroxidase via a binding substance that recognizes the analyte, a chemiluminescent substance, and an oxidizing agent are reacted in the presence of one or more compounds represented by formula (1).

[0035] The substance to be measured, to which peroxidase is bound via a binding substance that recognizes the substance to be measured, a chemiluminescent substance, and an oxidizing agent are reacted in the presence of one or more compounds represented by the above formula (1).

[0036] 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.

[0037] The structure of L-012 is shown below.

[0038] 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 (e.g., alkali metal salts) are preferred, luminol and its metal salts are more preferred, and luminol sodium salt is particularly preferred.

[0039] 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 a analyte to which peroxidase is bound via a binding substance that recognizes the analyte, a chemiluminescent substance, and an oxidizing agent are reacted in the presence of one or more compounds represented by formula (1).

[0040] 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, hydrogen peroxide, sodium perborate, and potassium perborate are preferred, and hydrogen peroxide is more preferred.

[0041] The concentration of the oxidizing agent is set appropriately 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 chemiluminescence enhancement effect, etc. The concentration referred to here is the concentration in a reaction solution when a analyte bound to peroxidase via a binding substance that recognizes the analyte, a chemiluminescent substance, and an oxidizing agent are reacted in the presence of one or more compounds represented by formula (1).

[0042] In one example, the substance to be measured to which peroxidase is bound via a binding substance that recognizes the substance to be measured may be obtained by reacting the substance to be measured with a first binding substance that recognizes the substance to be measured to form a complex, and then reacting the complex with a second binding substance to which peroxidase is bound that recognizes the substance to be measured.

[0043] In one example, the analyte to which peroxidase is bound via a binding substance that recognizes a complex of the analyte and a first binding substance that recognizes the analyte may be obtained by reacting the analyte with the first binding substance that recognizes the analyte to form a complex, and then reacting the complex with a "third binding substance that recognizes the complex of the analyte to which peroxidase is bound and the first binding substance that recognizes the analyte."

[0044] For example, a substance to be measured 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 substance to be measured is immobilized.

[0045] Examples of binding substances that recognize the analyte (first binding substances that recognize the analyte, or second binding substances that recognize the analyte) include substances that bind to the analyte 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.

[0046] 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.

[0047] The measurement may be carried out in accordance with the sandwich method described in literature commonly referred to in the art [for example, Enzyme Immunoassay, 2nd Edition (edited by Ishikawa Eiji et al., Igaku Shoin), 1982].

[0048] In the sandwich method, a first binding substance that recognizes the analyte is immobilized on the surface of a solid-phase carrier, such as magnetic silica particles. A sample containing the analyte is mixed with the solid-phase carrier and a second binding substance labeled with peroxidase that recognizes the analyte. The immobilized first binding substance, the analyte in the sample, and the second binding substance are brought into contact with each other. This results in the formation of a peroxidase-labeled complex, which is a complex of the immobilized first binding substance, the analyte 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 to measure the amount of peroxidase in the peroxidase-labeled complex, and the amount of the analyte in the sample is determined based on the amount of peroxidase in the peroxidase-labeled complex.

[0049] Alternatively, for example, a first binding substance that recognizes the analyte is immobilized on the surface of a solid-phase carrier such as magnetic silica particles. A sample containing the analyte is mixed with the solid-phase carrier and a peroxidase-labeled "third binding substance that recognizes the complex between the analyte and the first binding substance that recognizes the analyte." The immobilized first binding substance is then contacted with the analyte in the sample and the third binding substance. This results in the formation of a peroxidase-labeled complex, which is a complex between the immobilized first binding substance, the analyte 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 to measure the amount of peroxidase in the peroxidase-labeled complex, and the amount of the analyte in the sample is measured based on the amount of peroxidase in the peroxidase-labeled complex.

[0050] 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, diatomaceous earth, 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.

[0051] 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.

[0052] Specifically, for example, a target substance in a sample is contacted with a first binding substance immobilized on the surface of a solid-phase carrier such as magnetic silica particles, which recognizes the target substance, to form a complex between the target substance in the sample and the first binding substance immobilized on the surface of the solid-phase carrier, which recognizes the target substance. Next, the complex is contacted with a complex of a second binding substance labeled with peroxidase, to form a complex (peroxidase-labeled complex) between the first binding substance immobilized on the solid-phase carrier, which recognizes the target substance in the sample, and the second binding substance labeled with peroxidase. 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 the target substance in the sample can be determined based on the amount of peroxidase in the peroxidase-labeled complex.

[0053] In the above method, the substance to be measured in the sample is reacted with a first binding substance that recognizes the immobilized substance to be measured, and then a second binding substance labeled with peroxidase is reacted. However, it is also possible to react the second binding substance labeled with peroxidase with the substance to be measured in the sample, and then react with the first binding substance that recognizes the immobilized substance to be measured, or these three reactions may be performed simultaneously.

[0054] 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 a substance to be measured immobilized on the surface of a solid phase carrier and the substance to be measured in a sample" from the substance to be measured or other substances in the reaction system that were not involved in the formation of the complex; and (2) the separation of a peroxidase-labeled complex from a second binding substance labeled with peroxidase or other substances in the reaction system that were not involved in the formation of the peroxidase-labeled complex.

[0055] 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, a first binding substance bound to a polycation or polyanion (e.g., DNA) that recognizes the analyte in the sample is contacted at the sample inlet to form a complex between the first binding substance that recognizes the analyte and the analyte. 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 analyte, and the second binding substance labeled with peroxidase. The electric field further moves the peroxidase-labeled complex through the flow channel, where it undergoes B / F separation and reaches 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 the substance to be measured in the sample is determined based on the amount of peroxidase in the peroxidase-labeled complex.

[0056] In the measurement method of the present invention, the method for contacting the analyte in the sample with the first binding substance that recognizes the analyte immobilized on the surface of the solid phase carrier may be carried out by a conventional treatment such as stirring, mixing, etc. The reaction time may be appropriately set depending on the analyte 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.

[0057] 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.

[0058] As the peroxidase, horseradish peroxidase (HRP) or microperoxidase can be used.

[0059] 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.

[0060] 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, typically 1:0.5 to 20, preferably 1:1 to 15, and more preferably 1:1 to 10. The binding substance that recognizes the peroxidase-labeled analyte may be 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.

[0061] 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).

[0062] <Measurement Kit> The present invention can provide a measurement kit comprising a peroxidase bound to a binding substance that recognizes a substance to be measured, a chemiluminescent substance, an oxidizing agent, and the chemiluminescent reaction enhancer reagent of the present invention. The present invention further provides a measurement kit comprising a peroxidase bound to a binding substance that recognizes a complex of the substance to be measured and the binding substance that recognizes the substance to be measured, a chemiluminescent substance, an oxidizing agent, and the chemiluminescent reaction enhancer reagent of the present invention.

[0063] Preferred embodiments and specific examples of the peroxidase bound to a binding substance that recognizes the analyte, the peroxidase bound to a binding substance that recognizes a complex between the analyte and a binding substance that recognizes the analyte, the chemiluminescent substance, the oxidizing agent, and the chemiluminescent reaction enhancer reagent of the present invention are as described above in this specification. Furthermore, in this measurement kit, the peroxidase bound to a binding substance that recognizes the analyte, the peroxidase bound to a binding substance that recognizes a complex between the analyte and a binding substance that recognizes the analyte, the chemiluminescent substance, the oxidizing agent, and the chemiluminescent reaction enhancer reagent of the present invention may be contained in the reagent so as to achieve the concentrations described above in this specification during the reaction.

[0064] In the measurement 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 measurement 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.

[0065] In the measurement kit of the present invention, the concentration (concentration as a reagent) of one or more compounds represented by formula (1) is not particularly limited, but is preferably 20 μmol / L or more and 1000 μmol / L or less, more preferably 100 μmol / L or more and 1000 μmol / L or less, even more preferably 150 μmol / L or more and 800 μmol / L or less, and particularly preferably 200 μmol / L or more and 600 μmol / L or less.

[0066] In the measurement kit of the present invention, the concentration (concentration as a reagent) 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.

[0067] The measurement kit of the present invention may further include a different binding substance that recognizes the substance to be measured, 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.

[0068] 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.

[0069] <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

[0070] Synthesis of 4-(5-fluoro-6-methoxypyridin-3-yl)phenol

[0071] 5-Bromo-3-fluoro-2-methoxypyridine (239 mg), potassium carbonate (321 mg), water (160 μL), 1,1-bis(diphenylphosphino)ferrocene dichloropalladium(II) (170 mg), tetrahydrofuran (1.6 mL), and water (160 μL) were added to 4-hydroxyphenylboronic acid (160 mg), and the mixture was stirred at 47°C for 1 hour. ＴＭ The mixture was stirred at 120°C under a nitrogen atmosphere for 1 hour using a solvent-free solvent system (Biotage). The reaction mixture was purified by normal phase column chromatography (ethyl acetate / hexane = 0 / 100 → 70 / 30) and reverse phase column chromatography (acetonitrile / water = 0 / 100 → 100 / 0) to obtain 4-(5-fluoro-6-methoxypyridin-3-yl)phenol as a white solid (48.0 mg). MS (ESI m / z): (M+H) 220.4 RT (min): 1.26

[0072] The compounds shown in Table 1 below were synthesized in the same manner as in the synthesis of 4-(5-fluoro-6-methoxypyridin-3-yl)phenol, except that the raw materials shown in Table 1 were used instead of 5-bromo-3-fluoro-2-methoxypyridine.

[0073]

[0074] Synthesis of 4-(6-methoxypyridin-2-yl)phenol

[0075] To 4-hydroxyphenylboronic acid (100 mg), a solution of 2-bromo-6-methoxypyridine (136 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 solvent-free solvent system (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 4-(6-methoxypyridin-2-yl)phenol as a white solid (31.6 mg). MS (ESI m / z): 202.4 (M-H) RT (min): 0.48

[0076] The compounds shown in Table 2 below were synthesized in the same manner as in the synthesis of 4-(6-methoxypyridin-2-yl)phenol, except that the raw materials listed in Table 1 were used instead of 2-bromo-6-methoxypyridine.

[0077]

[0078] Examples 1 to 29 Evaluation of Each Enhancer in the Measurement of Human Chorionic Gonadotropin (hCG) 1. Preparation of Luminescent Reagent (hCG) The constituent reagents necessary for the 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.).

[0079] (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 reacted with Mab to hCG beta (Meridian Japan Co., Ltd.), an anti-hCG antibody, at 26°C for 12 to 16 hours. The particles were then magnetized with a neodymium magnet and the supernatant was removed to prepare magnetic particles with immobilized anti-hCG antibodies, and a first reagent having the following composition was prepared. "First reagent": anti-hCG antibody-immobilized magnetic particles 0.75 mg / mL, 50 mM MES (pH 5.5), 500 mM sodium chloride, 3.0% BCN300S

[0080] (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

[0081] (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": 16 nmol / L POD-labeled anti-hCG antibody, 50 mM MES (pH 6.5), 150 mM sodium chloride, 2.0% BSA

[0082] (iv) “Fourth Reagent” A fourth reagent was prepared by dissolving 1.295 g (50 mM) of TAPSO (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), 0.09 g (0.09%) of boric acid (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), 0.28 mL (0.280%) of 5 mol / L NaOH (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), and 0.1555 g (0.5 mM) of L-012 (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) in 100 mL of purified water to give a concentration of 0.2 mM for each of the synthesized compounds.

[0083] (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.)

[0084] 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.

[0085] 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 20,000 mIU / mL high-unit hCG solution. Using the blank sample and high-unit hCG solution prepared in "2. Preparation of blank sample", 0, 1, 3, 7000, and 10,000 mIU / mL hCG solutions were prepared as measurement samples.

[0086] 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 measurement samples (hCG solutions) with concentrations of 0, 1, 3, 7000, and 10,000 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 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, and all reagents except the magnetic particles were removed. The mixture was then washed three times with washing solution. After washing, 100 μL of the fourth reagent and 100 μL of the fifth reagent were added, and the mixture was incubated at 37°C for 20 seconds, after which the luminescence intensity was measured. The sensitivity and high-value slope were calculated from the obtained luminescence intensity. The sensitivity was calculated as (luminescence intensity of 3 mIU / mL hCG solution) / (luminescence intensity of blank sample), and the high-value slope was calculated as (luminescence intensity of 10,000 mIU / mL hCG solution) / (luminescence intensity of 7,000 mIU / mL hCG solution).

[0087] Comparative Example 1 Evaluation of PPP in hCG Measurement Luminescent reagents were prepared and evaluated in the same manner as in Examples 1 to 29, except for the following: Instead of each synthesized compound in Example 1 (iv) "fourth reagent," PPP was dissolved to a concentration of 0.2 mM and used as the "fourth reagent."

[0088]

[0089] Examples 30 to 35 Evaluation of Enhancers in Estradiol (E2) Measurement 1. Preparation of Luminescence Reagent (E2) The constituent reagents required for measurement were prepared using the following raw reagent materials: MES (2-morpholinoethanesulfonic acid monohydrate) (Dojindo Laboratories), sodium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.), BCN300S (Nitta Gelatin Co., Ltd.), Block Ace (KAC Corporation), BSA (bovine serum albumin) (Sigma-Aldrich Japan Co., Ltd.), sodium orthovanadate (Sigma-Aldrich Japan Co., Ltd.), luminol sodium salt (Fujifilm Wako Pure Chemical Industries, Ltd.), phosphoric acid (Fujifilm Wako Pure Chemical Industries, Ltd.), EDTA-2Na (Dojindo Laboratories), hydrogen peroxide (Fujifilm Wako Pure Chemical Industries, Ltd.), and dipotassium hydrogen phosphate (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0090] (i) "First Reagent": Magnetic particle-immobilized antibody-containing reagent Maglapid MGP-010T (manufactured by Sanyo Chemical Industries, Ltd.) was reacted with 3-aminopropyltriethoxysilane (manufactured by Tokyo Chemical Industry Co., Ltd.) and succinic anhydride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) in turn, and the mixture was collected with a neodymium magnet, followed by removal of the supernatant, to obtain magnetic particles having carboxyl groups. Next, the carboxyl groups were activated using commercially available N-hydroxysuccinimide (Fujifilm Wako Pure Chemical Industries, Ltd.) and WSC (water-soluble carbodiimide) (Dojindo Laboratories), and then the anti-E2 antibody, Sheep Monoclonal Antibodies (SMAs) to E2 5A11 (BioVentix Co., Ltd.), was reacted at 26°C for 12 to 16 hours. Magnetic particles bearing the immobilized anti-E2 antibody were prepared by magnetic collection with a neodymium magnet and removal of the supernatant, and a first reagent consisting of the following composition was prepared: "First reagent": 0.5 mg / mL anti-E2 antibody-immobilized magnetic particles, 50 mM MES (pH 5.5), 500 mM sodium chloride, 3.0% BCN300S

[0091] (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

[0092] (iii) "Third Reagent" - Labeled Antibody-Containing Reagent A complex of E2, an immunogen, and an anti-E2 antibody was injected into a mouse as an immunized animal for immunization, 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 prepared. The anti-immunoconjugate M19-1G antibody produced by the hybridoma prepared by the above method was digested with pepsin, and F(ab')2 was separated using a column (diameter: 1.5 cm x length: approximately 95 cm) packed with Sephacryl S-200HR (manufactured by 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 × length: approximately 40 cm) packed with G-25 Superfine (Cytiva). Meanwhile, peroxidase (POD) (Roche Diagnostics) was maleimidated using the 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 POD-labeled anti-immune complex M19-1G antibody. This was used to prepare a third reagent having the following composition: "Third reagent": 6 nmol / L POD-labeled anti-immunocomplex M19-1G antibody, 50 mM MES (pH 6.5), 150 mM sodium chloride, 2.0% BSA

[0093] (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 each compound listed in the table were dissolved in 100 mL of purified water to a concentration of 0.2 mM to prepare a fourth reagent.

[0094] (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.)

[0095] 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.

[0096] 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.

[0097] 4. Evaluation of luminescence reagents The first to fifth reagents and Accura Seed B / F separation solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were used to measure the luminescence intensity, and the sensitivity and high-value slope were calculated using the same method as in Example 1. The sensitivity was calculated from (luminescence intensity of 200 pg / mL E2 solution) / (blank luminescence intensity), and the high-value slope was calculated from (luminescence intensity of 3000 pg / mL E2 solution) / (luminescence intensity of 2000 pg / mL E2 solution).

[0098] Comparative Example 2: Evaluation of TP in E2 Measurement Luminescence reagents were prepared and evaluated in the same manner as in Examples 30 to 35, except for the following: Example 30-35(iv) "Fourth Reagent" Instead of each compound described in Examples 30 to 35, TP was dissolved to a concentration of 0.2 mM and used as the "fourth reagent."

[0099]

Claims

1. A chemiluminescence reaction enhancer reagent comprising a compound represented by formula (1). In the formula, X represents a pyridyl group which may be substituted with one or more groups selected from the group consisting of an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, a heterocyclic group, an alkoxy group, a cycloalkoxy group, an aryloxy group, an alkoxycarbonyl group, a cycloalkoxycarbonyl group, an amino group, an acylamino group, a silyl group, a silyloxy group, a hydroxyl group, a cyano group, a nitro group, a halogen atom, a carboxy group, a sulfo group, a phosphonyl group, a phosphoryl group, a boronic acid group, and a sulfanyl group.

2. The enhancer reagent according to claim 1, wherein X is a 3-pyridyl group optionally substituted with one or more groups selected from the group consisting of an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, a heterocyclic group, an alkoxy group, a cycloalkoxy group, an aryloxy group, an alkoxycarbonyl group, a cycloalkoxycarbonyl group, an amino group, an acylamino group, a silyl group, a silyloxy group, a hydroxyl group, a cyano group, a nitro group, a halogen atom, a carboxy group, a sulfo group, a phosphonyl group, a phosphoryl group, a boric acid group, and a sulfanyl group.

3. The enhancer reagent according to claim 1, wherein X is a 3-pyridyl group substituted with one or more groups selected from the group consisting of an alkyl group, an alkoxy group, a halogen atom and an amino group.

4. The enhancer reagent according to claim 1, wherein X is a 3-pyridyl group substituted with one or more groups selected from the group consisting of an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, and a fluorine atom.

5. The enhancer reagent according to claim 1, wherein the compound represented by formula (1) is any of the following compounds:

6. The compound shown below.

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