Compound or salt thereof, chemiluminescent reagent, measurement kit, and measurement method

Abstract

Provided is a compound represented by formula (1) or a salt thereof. In formula (1), R1 represents a hydrocarbon group or a heterocyclic group, R2 represents a hydrocarbon group, and R3 and R4 each independently represent a hydrogen atom or a hydrocarbon group.

Description

Compound or salt thereof, chemiluminescent reagent, measurement kit, and measurement method

[0001] This disclosure relates to compounds or salts thereof, chemiluminescent reagents, measurement kits, and measurement methods.

[0002] Two types of chemiluminescent substrates are most well known: luminol and L-012 (both known compounds). L-012 is known to exhibit higher signal intensity and sensitivity than luminol, but it has low photostability, and its signal intensity decreases when exposed to fluorescent light.

[0003] Patent Document 1 contains the formula

[0004]

[0005] [In the formula, R １ Each of these represents an optionally substituted hydrocarbon group or heterocyclic group, and R ２ R represents a hydroxyl group, thiol group, amino group, or monosubstituted amino group. ２ If it is a monosubstituted amino group, R ２ is R １ They may also form a ring together. R ３ R represents a hydrogen atom, an optionally substituted hydroxyl group, an optionally substituted amino group, an optionally substituted thiol group, a halogen atom, a heterocyclic group, a nitro group, a cyano group, an optionally esterified or amidated carboxyl group, an azide group, a sulfo group, or an organic sulfonyl group, provided that R １ If it is an aliphatic group, R ３ The following describes an assay method characterized by utilizing the chemiluminescence of a pyridopyridazine derivative or a salt thereof represented by [X is not a hydrogen atom. X represents an oxygen atom or a sulfur atom.]

[0006] Patent Document 1: Patent No. 3167762

[0007] An object of an embodiment of the present disclosure is to provide a compound or a salt thereof having excellent signal intensity and photostability, and a chemiluminescent reagent. An object of another embodiment of the present disclosure is to provide a measurement kit using the above compound or a salt thereof, and a measurement method using the above measurement kit.

[0008] Means for solving the above problems include the following aspects. <1> A compound represented by the following formula (1) or a salt thereof.

[0009] In formula (1), R １ represents a hydrocarbon group or a heterocyclic group, R ２ represents a hydrocarbon group, R ３ and R ４ each independently represent a hydrogen atom or a hydrocarbon group.

[0010] <2> R １ is a hydrocarbon group, the compound or a salt thereof according to <1>. <3> R １ is an alkyl group, and R ２ is an alkyl group, the compound or a salt thereof according to <1>. <4> R １ is an alkyl group having 2 or less carbon atoms, and R ２ is an alkyl group having 2 or less carbon atoms, the compound or a salt thereof according to <1>. <5> R ３ and R ４ are hydrogen atoms, the compound or a salt thereof according to any one of <1> to <4>. <6> A chemiluminescent reagent comprising a compound represented by the following formula (1) or a salt thereof.

[0011] In formula (1), R １ represents a hydrocarbon group or a heterocyclic group, R ２ represents a hydrocarbon group, R ３ and R ４ each independently represent a hydrogen atom or a hydrocarbon group.

[0012] <7> R １ is a hydrocarbon group, the chemiluminescent reagent according to <6>. <8> R １ is an alkyl group, and R ２However, the chemiluminescent reagent described in <6> is an alkyl group. <9> R １ However, it is an alkyl group having 2 or fewer carbon atoms, and R ２ However, the chemiluminescent reagent described in <6> is an alkyl group having 2 or fewer carbon atoms. <10> R ３ and R ４ A chemiluminescent reagent described in any one of <6> to <9>, which is a hydrogen atom. <11> A kit for measuring an antigen contained in a test sample, comprising a peroxidase to which an antigen-recognizing binding substance is bound, an oxidizing agent, an enhancer compound, and a compound described in any one of <1> to <5> or a salt thereof. <12> A measurement method using the measurement kit described in <11>.

[0013] According to one embodiment of the present disclosure, a compound or salt thereof exhibiting excellent signal intensity and photostability, and a chemiluminescent reagent are provided. According to another embodiment of the present disclosure, a measurement kit using the above compound or salt thereof, and a measurement method using the above measurement kit are provided.

[0014] The contents of this disclosure will be described in detail below. In this specification, when groups (atomic groups) are not specified as substituted or unsubstituted, the notation includes both those with and without substituents. For example, "alkyl group" includes not only unsubstituted alkyl groups but also substituted alkyl groups. In this specification, Me in chemical formulas represents a methyl group, Et represents an ethyl group, Pr represents a propyl group, Bu represents a butyl group, Ac represents an acetyl group, Bn represents a benzyl group, and Ph represents a phenyl group. In this disclosure, "mass%" and "weight%" are synonymous, and "parts by mass" and "parts by weight" are synonymous. Furthermore, in this disclosure, a combination of two or more preferred embodiments is a more preferred embodiment.

[0015] "Room temperature" refers to the ambient temperature that is not specifically controlled, and in this disclosure, unless otherwise specified, it refers to "25°C".

[0016] (Compound or Salt Thereof) The compound or salt thereof according to the present disclosure is a compound represented by the following formula (1) or a salt thereof.

[0017] In formula (1), R １ represents a hydrocarbon group or a heterocyclic group, and R ２ represents a hydrocarbon group, and R ３ and R ４ each independently represent a hydrogen atom or a hydrocarbon group.

[0018] It is known that L-012 described in Patent Document 1 exhibits higher signal intensity and sensitivity than luminol, but it has a problem of low photostability, and the signal intensity decreases upon exposure to a fluorescent lamp. In the compound or salt thereof according to the present disclosure, by allowing R １ to be a hydrocarbon group or a heterocyclic group, and R ２ to be a hydrocarbon group, photostability can be improved while increasing signal intensity.

[0019] In formula (1), R １ is preferably a hydrocarbon group, more preferably an alkyl group, still more preferably an alkyl group having 8 or less carbon atoms, particularly preferably an alkyl group having 2 or less carbon atoms, and most preferably a methyl group, from the viewpoints of signal intensity and photostability.

[0020] In formula (1), R ２ is preferably an alkyl group, more preferably an alkyl group having 8 or less carbon atoms, still more preferably an alkyl group having 2 or less carbon atoms, and particularly preferably a methyl group, from the viewpoints of signal intensity and photostability.

[0021] In formula (1), R ３ and R ４ each independently is preferably a hydrogen atom, an alkyl group or an aryl group, more preferably a hydrogen atom or an alkyl group, and particularly preferably a hydrogen atom, from the viewpoint of signal intensity.

[0022] As the alkyl group, an alkyl group having 1 to 5 carbon atoms is preferable. 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. As the alkyl group having 1 to 5 carbon atoms, a methyl group is preferable.

[0023] As the aryl group, an aryl group having 6 to 14 carbon atoms is preferable, and examples thereof include a phenyl group, a 1-naphthyl group, a 2-naphthyl group, a biphenylyl group, and a 2-anthryl group.

[0024] As the heterocyclic group, a monocyclic or polycyclic heterocyclic group containing one or more atoms selected from an oxygen atom, a nitrogen atom and a sulfur atom as hetero atoms can be mentioned, and examples thereof include 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.

[0025] R in formula (1) １ to R ４ The hydrocarbon group in may be an alkyl group or an aryl group. R in formula (1) １ The hydrocarbon group and heterocyclic group in, as well as R ２ to R ４ The hydrocarbon group in may have a substituent. Examples of the substituent include an alkyl group, an aryl group, a heterocyclic group, an alkoxy group, a hydroxy group, a mercapto group, a halogen atom, a cyano group, a sulfo group, a carboxyl group, a nitro group, a hydroxamic acid group, a sulfino group, an acyl group, an alkoxycarbonyl group, an acyloxy group, an acylamino group, an alkoxycarbonylamino group, a sulfonylamino group, a sulfamoyl group, a carbamoyl group, an alkylthio group, a sulfonyl group, a sulfinyl group, and a ureido group.

[0026] Further, the compound represented by formula (1) can be converted into a salt of the compound represented by formula (1) by a known salt formation method. Further, the compound represented by formula (1) can also be purified by known purification methods such as column chromatography, thin-layer chromatography, recrystallization, and reprecipitation.

[0027] The method for synthesizing the compound represented by formula (1) is not particularly limited and can be done by applying known methods.

[0028] Specific examples of compounds represented by formula (1) are shown below, but it goes without saying that compounds represented by formula (1) are not limited to the examples below.

[0029]

[0030] (Chemiluminescent Reagent) The chemiluminescent reagent according to this disclosure comprises a compound represented by formula (1) or a salt thereof. Preferred embodiments of the compound represented by formula (1) or a salt thereof in the chemiluminescent reagent according to this disclosure are the same as the preferred embodiments described above. The concentration of the compound represented by formula (1) is not particularly limited, but is preferably 0.1 mmol / L or more and 10 mmol / L or less, more preferably 0.1 mmol / L or more and 7.5 mmol / L or less, even more preferably 0.1 mmol / L or more and 5.0 mmol / L or less, and particularly preferably 0.1 mmol / L or more and 2.5 mmol / L or less. The concentration referred to here preferably is the concentration in the reaction solution when a substance to be measured to which peroxidase is bound via a binding substance that recognizes the substance to be measured, the compound represented by formula (1), and an oxidizing agent are reacted in the presence of an enhancer compound.

[0031] Examples of test samples for the chemiluminescent reagents relating to this disclosure include, but are 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.

[0032] In this disclosure, 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 also be an antigen.

[0033] In this disclosure, the substances to be measured are not particularly limited, and all substances that can be measured by conventional immunological assays, such as steroids, peptides, hormones, antibodies, proteins, drugs, metabolites, and vitamins contained in biological samples, can be listed. Specifically, endocrine function-related substances include, for example, 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 antibody, and anti-T3 Antibodies, anti-T4 antibodies, anti-TSH antibodies, calcitonin, catecholamines, dopamine, serotonin, aldosterone, renin, angiotensin, cortisol, deoxycortisol, cortisone, corticosterone, deoxycorticosterone, androsterone, progesterone, pregnenolone, estrogen, estrone, estriol, estradiol, testosterone, human chorionic gonadotropin, insulin, anti-insulin antibodies, C-peptide, glucagon, g Examples of tumor-related substances include stringin, secretin, cyclic AMP, cyclic GMP, prostaglandins, thromboxane, erythropoietin, histamine, etc. Tumor-related substances include, for example, CEA, ferritin, β2-microglobulin, elastase, α-fetoprotein, nerve-specific enolase, prostate-specific antigen, CA19-9, etc. Drug and vitamin-related substances include, for example, phenobarbital, phenytoin, carbamazepine, primidone, ethosuximide, valpro Acids, acetazole amide, sultium, glutetimide, 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 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 antibodies, and C-reactive proteins. Examples of virus and infection-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] <Enhancer Compounds> The chemiluminescent reagents according to this disclosure preferably contain enhancer compounds. Examples of enhancer compounds include PPP (4-phenylphenol), TP (4-(thiazole-4-yl)phenol), BiPCA (4-(4-hydroxyphenyl)benzoic acid), and compounds represented by the following formula (E).

[0035] In formula (E), X represents a pyridyl group which may be substituted with one or more groups selected from the group consisting of alkyl groups, alkenyl groups, cycloalkyl groups, aryl groups, heterocyclic groups, alkoxy groups, cycloalkoxy groups, aryloxy groups, alkoxycarbonyl groups, cycloalkoxycarbonyl groups, amino groups, acylamino groups, silyl groups, silyloxy groups, hydroxyl groups, cyano groups, nitro groups, halogen atoms, carboxyl groups, sulfo groups, phosphonyl groups, phosphoryl groups, boric acid groups, and sulfanyl groups.

[0036] A preferred example of a compound represented by formula (E) is any of the following compounds, but the compound represented by formula (1) is not limited to the following compounds.

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043] The enhancer concentration is preferably 10 μmol / L or more and 500 μmol / L or less, more preferably 50 μmol / L or more and 500 μmol / L or less, even more preferably 75 μmol / L or more and 400 μmol / L or less, and particularly preferably 100 μmol / L or more and 300 μmol / L or less.

[0044] The chemiluminescent reagents relating to this disclosure preferably contain an oxidizing agent. Examples of oxidizing agents include aqueous solutions of known oxidizing agents described in Japanese Patent Publication No. 8-261943 and Japanese Patent Publication No. 2000-279196, etc. [inorganic peroxides (hydrogen peroxide, sodium perborate, potassium perborate, etc.), organic peroxides (dialkyl peroxides, acyl peroxides, etc.), peroxoacid compounds (peroxosulfuric acid, peroxophosphate, etc.)]. Of these, hydrogen peroxide, sodium perborate, and potassium perborate are preferred from the viewpoint of storage stability, and hydrogen peroxide is more preferred.

[0045] The concentration of the oxidizing agent is set appropriately depending on its type, the measurement method and conditions applied, etc., but from the viewpoint of enhancing chemiluminescence, it is preferably 0.5 mmol / L or more and 20 mmol / L or less, more preferably 1 mmol / L or more and 10 mmol / L or less, and even more preferably 1.4 mmol / L or more and 3.5 mmol / L or less.

[0046] The chemiluminescent reagent relating to this disclosure may contain other compounds. There are no particular restrictions on the other compounds, and known additives can be used.

[0047] (Measurement Kit and Measurement Method) The measurement kit relating to this disclosure is a kit used for measuring an antigen contained in a test sample, and comprises a peroxidase to which a binding substance that recognizes the antigen is bound, an oxidizing agent, an enhancer compound, and the compound relating to this disclosure or a salt thereof (a compound represented by formula (1) or a salt thereof). The measurement method relating to this disclosure is a measurement method using the measurement kit relating to this disclosure. The measurement kit relating to this disclosure may also comprise a peroxidase to which a binding substance that recognizes a complex of the substance to be measured and the binding substance that recognizes the substance to be measured is bound, an oxidizing agent, an enhancer compound, and the compound relating to this disclosure or a salt thereof (a compound represented by formula (1) or a salt thereof).

[0048] Preferred embodiments and specific examples of the oxidizing agent, enhancer compound, and the compound or salt thereof relating to this disclosure are as described above.

[0049] In the measurement kit according to this disclosure, the concentration (reagent concentration) of the compound or salt thereof according to this disclosure is not particularly limited, but is preferably 0.2 mmol / L or more and 20 mmol / L or less, more preferably 0.4 mmol / L or more and 15.0 mmol / L or less, even more preferably 0.5 mmol / L or more and 10.0 mmol / L or less, and particularly preferably 0.5 mmol / L or more and 5.0 mmol / L or less. In the measurement kit according to this disclosure, the concentration (reagent concentration) of the oxidizing agent is not particularly limited, but is preferably 1.0 mmol / L or more and 40 mmol / L or less, more preferably 2 mmol / L or more and 20 mmol / L or less, and even more preferably 2.8 mmol / L or more and 7.0 mmol / L or less.

[0050] In the measurement kit according to this disclosure, the concentration of the enhancer compound (concentration as a reagent) 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.

[0051] In the measurement kit according to this disclosure, the concentration (reagent concentration) of peroxidase to which the second or third binding substance is bound 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.

[0052] The measurement kit relating to this disclosure may further include another binding substance that recognizes the target substance, which is different from the binding substance bound to the peroxidase, or a solid support on which the other binding substance is immobilized.

[0053] The measurement method relating to this disclosure preferably comprises reacting (1) a substance to be measured to which peroxidase is bound via a binding substance that recognizes the substance to be measured, or a substance to be measured to which peroxidase is bound via a binding substance that recognizes a complex of the substance to be measured and the binding substance that recognizes the substance to be measured, (2) a compound relating to this disclosure or a salt thereof, and (3) an oxidizing agent, in the presence of an enhancer compound.

[0054] 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 that recognizes the substance to be measured and to which peroxidase is bound.

[0055] In one example, the substance to which peroxidase is bound via a binding substance that recognizes a complex of the substance to be measured and a first binding substance that recognizes the substance to be measured may be obtained by reacting the substance to be measured with the first binding substance that recognizes the substance to be measured to form a complex, and then reacting the complex with a "third binding substance that recognizes a complex of the substance to be measured and the first binding substance that recognizes the substance to be measured," to which peroxidase is bound.

[0056] For example, a target substance in a sample can be measured using a solid-phase support, such as magnetic silica particles, on which a first binding substance that recognizes the target substance is immobilized on the surface of the solid-phase support.

[0057] Examples of binding substances that recognize the target substance (first binding substance that recognizes the target substance, or second binding substance that recognizes the target substance) include those that bind to the target substance through interactions such as antigen-antibody reactions, glycan-protein reactions, glycan-lectin reactions, protein-peptide chain reactions, protein-protein reactions, and protein-nucleotide chain reactions.

[0058] Examples of binding substances that recognize the above complex include those that bind to the complex through interactions such as antigen-antibody reactions, glycan-protein reactions, glycan-lectin reactions, protein-peptide chain reactions, protein-protein reactions, and protein-nucleotide chain reactions.

[0059] The measurement method should be carried out in accordance with the sandwich method described in commonly referenced literature in this field [for example, Enzyme Immunoassay, 2nd Edition (edited by Eiji Ishikawa et al., Igaku-Shoin), 1982].

[0060] In the sandwich method, for example, a first binding substance that recognizes the substance to be measured is immobilized on the surface of a solid support, such as magnetic silica particles. A sample containing the substance to be measured, the solid support, and a second binding substance that recognizes the substance to be measured and is labeled with peroxidase are mixed, and the immobilized first binding substance, the substance to be measured in the sample, and the second binding substance are brought into contact. This forms a peroxidase-labeled complex, which is a composite of the immobilized first binding substance, the substance to be measured in the sample, and the peroxidase-labeled second binding substance. Next, the solid support carrying the peroxidase-labeled complex is subjected to B / F separation, 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.

[0061] Alternatively, for example, a first binding substance that recognizes the substance to be measured is immobilized on the surface of a solid support such as magnetic silica particles. A sample containing the substance to be measured, the solid support, and a third binding substance labeled with peroxidase that recognizes a composite of the substance to be measured and the first binding substance are mixed, and the immobilized first binding substance, the substance to be measured in the sample, and the third binding substance are brought into contact. This forms a peroxidase-labeled composite, which is a composite of the immobilized first binding substance, the substance to be measured in the sample, and the peroxidase-labeled third binding substance. Next, the solid support carrying the peroxidase-labeled composite is subjected to B / F separation, the amount of peroxidase in the peroxidase-labeled composite is measured, and the amount of the substance to be measured in the sample is measured based on the amount of peroxidase in the peroxidase-labeled composite.

[0062] As the solid phase support, any support used in conventional immunoassays (especially insoluble supports) can be used. Examples include organic materials 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 materials such as glass, silicon dioxide, diatoms, porous glass, frosted glass, alumina, silica gel, and metal oxides; magnetic materials such as iron, cobalt, nickel, magnetite, and chromite; and alloys of these magnetic materials.

[0063] Solidification can be performed on any solid phase (e.g., any surface such as beads, magnetic beads, membranes, or plates). Various commercially available magnetic beads can be used. For example, the beads disclosed in WO2012 / 173002A may be used as magnetic beads.

[0064] Specifically, for example, the substance to be measured in the sample is brought into contact with a first binding substance that recognizes the substance to be measured, which is immobilized on the surface of a solid support such as magnetic silica particles, to form a composite between the first binding substance that recognizes the substance to be measured immobilized on the surface of the solid support and the substance to be measured in the sample. Next, a composite with a second binding substance labeled with peroxidase is brought into contact with the above composite to form a composite (peroxidase-labeled composite) between the first binding substance that recognizes the substance to be measured immobilized on the solid support, the substance to be measured in the sample, and the second binding substance labeled with peroxidase. The peroxidase-labeled composite can be separated by B / F separation to measure the amount of peroxidase in the peroxidase-labeled composite, and the amount of the substance to be measured in the sample can be measured based on the amount of peroxidase in the peroxidase-labeled composite.

[0065] 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 the second binding substance labeled with peroxidase is reacted. However, the second binding substance labeled with peroxidase may be reacted with the substance to be measured in the sample, and then the first binding substance that recognizes the immobilized substance to be measured may be reacted, or all three may be reacted simultaneously.

[0066] In the sandwich method described above, B / F separation (Bond / Free separation) refers to the separation of a substance supported on a solid phase support from other substances. Specifically, B / F separation in the present invention involves: (1) the separation of a "complex of a first binding substance that recognizes the target substance immobilized on the surface of the solid phase support and the target substance in the sample" from the target substance and other substances in the reaction system that did not participate in the formation of the complex; 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 did not participate in the formation of the peroxidase-labeled complex.

[0067] The sandwich method can also be performed using a microfluidic chip (micro-total analysis systems; μTAS). The microfluidic chip has channels, each having a sample inlet. For example, at the sample inlet, a first binding agent that recognizes the target substance, to which a polycation or polyanion (e.g., DNA) is bound, is brought into contact with the target substance in the sample to form a complex of the first binding agent and the target substance. Next, an electric field is applied to move the complex, and when it comes into contact with a second binding agent labeled with peroxidase, which is present in the middle of the channel, a complex of the first binding agent, the target substance, and the second binding agent labeled with peroxidase (peroxidase-labeled complex) is formed. Further electric field causes the peroxidase-labeled complex to move along the channel, where it undergoes B / F separation and reaches the detection unit on the channel. In the detection unit, the amount of peroxidase in the peroxidase-labeled complex is measured, and the amount of the target substance in the sample is determined based on the amount of peroxidase in the peroxidase-labeled complex.

[0068] In the measurement method according to this disclosure, the method for bringing the substance to be measured in the sample and the first binding substance that recognizes the substance to be measured, which is immobilized on the surface of the solid support, into contact can be done by a process such as stirring or mixing, which is normally performed. The reaction time can be set appropriately depending on the substance to be measured 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.

[0069] B / F separation in the measurement method according to this disclosure is performed, for example, by utilizing the magnetism of the solid phase support, collecting the solid phase support 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 phase support, and washing it. The above operation may be repeated one to three times. The washing solution is not particularly limited as long as it is commonly used in this field.

[0070] As the peroxidase, horseradish peroxidase (HRP) or microperoxidase can be used.

[0071] To bind peroxidase to a second or third binding substance, one can use methods commonly used in this field, such as known labeling methods generally used in known EIAs [e.g., Medical Chemistry Experiment Course, Vol. 8, supervised by Yuichi Yamamura, 1st edition, Nakayama Shoten, 1971; Illustrated Fluorescent Antibodies, by Akira Kawai, 1st edition, Soft Science Co., Ltd., 1983; Enzyme Immunoassay, edited by Eiji Ishikawa, Tadashi Kawai, and Kiyoshi Miyai, 2nd edition, Igaku Shoin, 1982, etc.].

[0072] The amount of peroxidase used is preferably such that the second or third binding substance and peroxidase are 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 target substance labeled with peroxidase may be contained in a buffer commonly used in this field, such as Tris buffer, phosphate buffer, Veronal buffer, borate buffer, or Good's buffer (for example, MES (2-morpholinoethanesulfonic acid) buffer). The concentration of the peroxidase to which the second or third binding substance is bound 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.

[0073] When an antibody is used as the binding agent, the pH of the buffer solution should be within a range that does not inhibit the antigen-antibody reaction, preferably pH 5 to 9. Furthermore, the buffer solution may contain stabilizers such as albumin, globulin, water-soluble gelatin, polyethylene glycol, surfactants, sugars, etc., as long as they do not inhibit the antigen-antibody reaction. In this specification, pH refers to a value measured in accordance with JIS K0400-12-10:2000 (measurement temperature 25°C).

[0074] The present disclosure will be described in detail below with reference to examples, but the disclosure is not limited to these examples. In these examples, "%" and "parts" mean "mass%" and "parts by mass," respectively, unless otherwise specified.

[0075] Purification by silica gel column chromatography was performed using the ISOLERA automated purification system (Biotage). For normal-phase chromatography, CHROMATOREX Q-PACK (Fuji Silicia Co., Ltd.) was used as the support. For reverse-phase chromatography, Sfar C18 (Biotage) was used as the support. Mass (MS) spectra were measured using the ACQUITY SQD LC / MS System (Waters Corporation) and the ionization method: ESI (ElectroSpray Ionization). Retention time (RT) was measured using SQD (Waters Corporation) and expressed in minutes (min). Column: Waters BEH C18 1.7 μm, 2.1 x 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

[0076] (Example 1) <Synthesis of diethyl 5-bromopyridine-3,4-dicarboxylate>

[0077]

[0078] In a 300 mL three-necked flask, 10 g of diethylpyridine-3,4-dicarboxylate, 100 mL of methanol, 478 μL of sulfuric acid, and 690 mg of ammonium acetate were added, and the mixture was heated to 68°C while stirring. 1.59 g of N-bromosuccinimide was added and the mixture was stirred for 15 minutes. 3.99 g of N-bromosuccinimide was added and the mixture was stirred for 15 minutes. 3.99 g of N-bromosuccinimide was added and the mixture was stirred for 15 minutes. 3.99 g of N-bromosuccinimide was added and the mixture was stirred for 15 minutes. Under ice cooling, potassium carbonate solution was added dropwise to adjust the pH to around 8. After adding ethyl acetate, the organic layer was removed by vacuum distillation, and the resulting residue was purified by silica gel column chromatography (hexane-ethyl acetate) to obtain diethyl 5-bromopyridine-3,4-dicarboxylate (12 g). Mass spectrometry (MS) (ESI m / z): 302.1 (M+H) Retention time (RT) (min): 1.46

[0079] <Synthesis of diethyl 5-[(2,4-dimethoxyphenyl)methylamino]pyridine-3,4-dicarboxylate>

[0080]

[0081] Diethyl 5-bromopyridine-3,4-dicarboxylate (6.65 g), toluene (70 mL), cesium carbonate (14.3 g), 2,4-dimethoxybenzylamine (7.36 g), and 4,5-bis(diphenylphosphin)-9,9-dimethylxanthene (3.82 g) were added to a 100 ml three-necked flask and heated under reflux under a nitrogen atmosphere for 3 hours. After returning to room temperature, water was added, and the organic layer was removed by distillation under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane-ethyl acetate) to obtain diethyl 5-[(2,4-dimethoxyphenyl)methylamino]pyridine-3,4-dicarboxylate (6.7 g) as an oily substance. MS (ESI m / z): 389.5 (M + H) RT (min): 1.55

[0082] <Synthesis of diethyl 5-aminopyridine-3,4-dicarboxylate>

[0083]

[0084] Diethyl 5-[(2,4-dimethoxyphenyl)methylamino]pyridine-3,4-dicarboxylate (6.7 g) was mixed with dichloromethane (40 mL), followed by the addition of trifluoroacetic acid (3.9 mL) under ice cooling. After raising the temperature to room temperature, the mixture was stirred for 4 hours. The reaction mixture was cooled on ice, and sodium bicarbonate solution was added dropwise to adjust the pH to 8. The organic layer was then removed by reduced pressure distillation. The residue was purified by silica gel column chromatography (hexane-ethyl acetate) to obtain diethyl 5-aminopyridine-3,4-dicarboxylate (4.1 g). MS (ESI m / z): 239.4 (M + H) RT (min): 1.00

[0085] <Synthesis of diethyl 5-amino-2,6-dichloropyridine-3,4-dicarboxylate>

[0086]

[0087] Diethyl 5-aminopyridine-3,4-dicarboxylate (1.45 g), acetonitrile (8.0 mL), and N-chlorosuccinimide (2.03 g) were added to a 20 mL microwave reaction vial, and the mixture was irradiated with microwaves and stirred at 50°C for 1 hour. This reaction was repeated four times on the same scale. Ethyl acetate and sodium bicarbonate solution were added to the reaction mixture, and the organic layer was removed by vacuum distillation. The resulting residue was purified by silica gel column chromatography to obtain diethyl 5-amino-2,6-dichloropyridine-3,4-dicarboxylate (4.4 g). MS (ESI m / z): 307.3 (M + H) RT (min): 1.51

[0088] <Synthesis of diethyl 5-amino-2,6-dimethylpyridine-3,4-dicarboxylate>

[0089]

[0090] Diethyl 5-amino-2,6-dichloropyridine-3,4-dicarboxylate (786 mg), potassium carbonate (1.41 g), 1,1-bis(diphenylphosphino)ferrocenedichloropalladium(II) (374 mg), methylboronic acid (613 mg), tetrahydrofuran (7.86 mL), and water (786 μL) were added to a 20 mL microwave reaction vial. The mixture was then irradiated with microwaves under a nitrogen atmosphere and stirred at 120°C for 4 hours. The reaction solution was treated with ISOLUTE Si-Thiol to remove residual palladium, and then purified by silica gel column chromatography to obtain diethyl 5-amino-2,6-dimethylpyridine-3,4-dicarboxylate (205 mg). MS (ESI m / z): 267.4 (M+H) RT (min): 0.90

[0091] <Synthesis of 8-amino-5,7-dimethyl-2,3-dihydropyrido[3,4-d]pyridazine-1,4-dione>

[0092]

[0093] Diethyl 5-amino-2,6-dimethylpyridine-3,4-dicarboxylate (153 mg), methanol (765 μL), and hydrazine (306 μL) were added to a 20 mL microwave reaction vial. The mixture was then microwaved and stirred at 100°C for 4 hours. Acetic acid (612 μL) was added, and the mixture was microwaved again and stirred at 100°C for 1 hour. The precipitated solid was filtered through a Kiriyama funnel, washed twice with a 5% methanol acetate solution, and then washed once with methanol. The resulting solid was dried under reduced pressure to obtain 8-amino-5,7-dimethyl-2,3-dihydropyrido[3,4-d]pyridazine-1,4-dione (105 mg). MS (ESI m / z): 207.3 (M+H) RT (min): 0.30

[0094] (Example 2) The following compounds were synthesized in the same manner as the synthesis of diethyl 5-amino-2,6-dimethylpyridine-3,4-dicarboxylate.

[0095]

[0096] The following compounds were synthesized in the same manner as the synthesis of 8-amino-5,7-dimethyl-2,3-dihydropyrido[3,4-d]pyridazine-1,4-dione.

[0097]

[0098] <Evaluation> The following evaluations were performed using each compound. The evaluation results are summarized in Table 3. Note that L-012 was used as Comparative Example 1. (Example 101) 1. Preparation of chemiluminescent reagents The constituent reagents necessary for measurement were prepared using the following reagent raw materials: MES (2-morpholinoethanesulfonic acid monohydrate) (manufactured by Dojin Chemical Laboratories), sodium chloride (manufactured by Fujifilm Wako Pure Chemical Corporation), BCN300S (manufactured by Nitta Gelatin Co., Ltd.), Block Ace (manufactured by K.A.C. Co., Ltd.), BSA (bovine serum albumin) (manufactured by Sigma-Aldrich Japan Co., Ltd.), sodium orthovanadate (manufactured by Sigma-Aldrich Japan Co., Ltd.), phosphoric acid (manufactured by Fujifilm Wako Pure Chemical Corporation), EDTA-2Na (manufactured by Dojin Chemical Laboratories), hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Corporation), dipotassium hydrogen phosphate (manufactured by Fujifilm Wako Pure Chemical Corporation).

[0099] (i) The first reagent, Magrapid MGP-010T (manufactured by Sanyo Chemical Industries, Ltd.), containing a solid-phase antibody for magnetic particles, was reacted successively with 3-aminopropyltriethoxysilane (manufactured by Tokyo Chemical Industries, Ltd.) and succinic anhydride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). The mixture was then magnetized using a neodymium magnet, and the supernatant was removed to obtain magnetic particles having carboxyl groups. Next, the carboxyl groups were activated using commercially available N-hydroxysuccinimide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and WSC (water-soluble carbodiimide) (manufactured by Dojin Chemical Laboratories). Then, the anti-E2 antibody Sheep Monoclonal Antibodies (SMAs) to E2 5A11 (manufactured by BioVentix Inc.) was reacted at 26°C for 12 to 16 hours. Magnetic particles containing the anti-E2 antibody were collected with a neodymium magnet, and the supernatant was removed to prepare the first reagent, which consisted of the following composition: "First Reagent": Anti-E2 antibody-containing magnetic particles 0.25 mg / mL, 50 mM (= 50 mM / L) MES (pH 5.5), 500 mM Sodium chloride, 3.0% BCN300S

[0100] (ii) "Second Reagent" Reaction Buffer: 50 mM MOPS (3-morpholinopropanesulfonic acid) (manufactured by Dojin Chemical Laboratories) (pH 7.5), 500 mM sodium chloride, 0.4% Block Ace

[0101] (iii) "Third Reagent" A reagent containing labeled antibody: A complex of E2, an immunogen, and an anti-E2 antibody was injected into mice, which were immunized animals, and lymphocytes were collected. The obtained lymphocytes were fused with myeloma cells to select hybridoma cells that produced the target antibody. After cloning, hybridomas producing the target antibody were prepared. The anti-immune complex M19-1G antibody produced by the hybridomas prepared by the above method was digested with pepsin, and F(ab')2 was separated using a column packed with cefacrill S-200HR (Cytiva) (diameter: 1.5 cm x length: approximately 95 cm). The obtained F(ab')2 was reduced with cysteamine hydrochloride (Sigma-Aldrich Japan), and Fab' was separated using a column packed with G-25 Superfine (Cytiva) (diameter: 1.5 cm x length: approximately 40 cm). Meanwhile, peroxidase (POD) (Roche Diagnostics K.K.) was maleimidized using the maleimidization reagent Sulfo-KMUS (Dojin Chemical Laboratories), and the reaction mixture was removed using a Sephadex G-25 column to obtain maleimidized POD. The prepared Fab' and maleimidized POD were mixed and separated using a Cefacryl S-100HR column to produce the POD-labeled anti-immune complex M19-1G antibody. A third reagent consisting of the following composition was prepared using this antibody. "Third Reagent": 17.5 nmol / L POD-labeled anti-immune complex M19-1G antibody, 50 mM MES (pH 6.5), 150 mM sodium chloride, 2.0% BSA

[0102] (iv) "Fourth Reagent" 100 mL of purified water, 1.82 g (50 mM) of 2-amino-2-hydroxymethyl-1,3-propanediol Tris(hydroxymethyl)aminomethylane (manufactured by Fujifilm Wako Pure Chemical Corporation), 0.690 mL (0.069%) of 5N-HCl (manufactured by Fujifilm Wako Pure Chemical Corporation), 0.877 g (150 mM) of sodium chloride, 8.57 mg (1 mM) of sodium orthovanadate (manufactured by Sigma-Aldrich Japan Co., Ltd.), 8-amino-5 11.4 g (0.5 mM) of ,7-dimethyl-2,3-dihydropyrido[3,4-d]pyridazine-1,4-dione and 4-(4-thiazolyl)phenol (manufactured by Enamine Co., Ltd.) were dissolved to a concentration of 0.2 mM. The solution was then divided in half, one half was stored in the dark, and the other half was exposed to a 100 lux fluorescent lamp for 30 minutes, then stored in the dark. Fourth reagents with and without light exposure were prepared.

[0103] (v) "Fifth Reagent" "Fifth Reagent": 68 μL / L phosphoric acid, 4mM EDTA-2Na (manufactured by Dojindo Laboratories), 335 μL / L hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

[0104] 2. Preparation of the measurement sample: 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.), 20 g (2.0%) of bovine serum albumin (manufactured by Sigma-Aldrich Japan Ltd.), and 20 mg of β-estradiol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were dissolved in 1000 mL of purified water to prepare a 20 pg / mL E2 solution.

[0105] 3. Evaluation of Luminescent Reagents Using the first to fifth reagents prepared in "1. Preparation of Luminescent Reagents" and AccuraSeed B / F separation solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), the luminescence of the E2 solution was measured using the fourth reagent, with and without fluorescent lamp exposure, in an automated chemiluminescent enzyme immunoassay analyzer (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) according to the following procedure. 50 μL of the first reagent was added to the reaction cuvette and heated at 67°C for 20 seconds while being magnetized with a neodymium magnet, and the supernatant was removed. Subsequently, 140 μL of the second reagent and 10 μL of the sample to be measured were added and stirred, and the mixture was heated at 37°C for 3 minutes. After heating, the mixture was magnetized with a neodymium magnet to remove reagents other than magnetic particles, and washed three times with washing solution. Subsequently, 50 μL of the third reagent was added and heated at 37°C for 3 minutes. After heating, the mixture was magnetized with a neodymium magnet to remove reagents other than magnetic particles, and washed three times with washing solution. After washing was complete, 100 μL each of the fourth reagent and the fifth reagent were added, and after reacting at 37°C for 20 seconds, the amount of luminescence was measured for each reagent. The signal change rate was calculated from the obtained luminescence amounts as (luminescence amount when using the fourth reagent with fluorescent lamp exposure) / (luminescence amount when using the fourth reagent without fluorescent lamp exposure).

[0106] (Example 102) The chemiluminescent reagent was prepared and evaluated in the same manner as in Example 101, except as described below. Instead of 8-amino-5,7-dimethyl-2,3-dihydropyrido[3,4-d]pyridazine-1,4-dione in Example 1(iv) "Fourth Reagent", 12.8 mg (0.5 mM) of 8-amino-5,7-diethyl-2,3-diethyl-2,3-diethyl-2,3-dihydropyrido[3,4-d]pyridazine-1,4-dione was dissolved and used as "Fourth Reagent".

[0107] (Comparative Example 101) The chemiluminescent reagent was prepared and evaluated in the same manner as in Example 101, except as described below. In Comparative Example 1(iv) "Fourth Reagent", 15.5 mg (0.5 mM) of L-012 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in the solution of 8-amino-5,7-dimethyl-2,3-dihydropyrido[3,4-d]pyridazine-1,4-dione and used as "Fourth Reagent".

[0108] - Signal values ​​without fluorescent light exposure - The evaluation criteria are as follows: A: Over 100,000 cps B: Over 80,000 cps and 100,000 cps or less C: Over 70,000 cps and 80,000 cps or less D: 60,000 cps or more and 70,000 cps or less E: Less than 60,000 cps

[0109] -Changes in signal intensity after 30 minutes of exposure to fluorescent light- The signal value was measured after 30 minutes of exposure to fluorescent light. Subsequently, the rate of change in signal was calculated based on the following formula.

[0110]

[0111] The evaluation criteria are as follows: A: Signal change rate less than 10% B: Signal change rate 10% or more but less than 20% C: Signal change rate 20% or more but less than 30% D: Signal change rate 30% or more but 40% or less E: Signal change rate greater than 40%

[0112]

[0113] (Examples 3-5)

[0114]

[0115] Diethyl 5-amino-2,6-dichloropyridine-3,4-dicarboxylate (4.0 g), tripotassium phosphate (11.1 g), Sphos Pd G3 (4.0 g), 2,6-dimethoxyphenylboronic acid (2.61 g), and toluene (56 mL) were added to a 300 mL three-necked flask, and the mixture was stirred at 130 °C under a nitrogen atmosphere for 3 hours. The reaction mixture was treated with ISOLUTE Si-Thiol to remove residual palladium, and the insoluble matter was filtered off. The solvent was removed under reduced pressure, and the resulting residue was purified by silica gel column chromatography to obtain diethyl 5-amino-2-chloro-6-(2,6-dimethoxyphenyl)pyridine-3,4-dicarboxylate (2.0 g). MS (ESI m / z): 409.1 (M + H) RT (min): 1.54

[0116] The following compounds were synthesized in the same manner as the synthesis of diethyl 5-amino-2-chloro-6-(2,6-dimethoxyphenyl)pyridine-3,4-dicarboxylate.

[0117]

[0118]

[0119] Diethyl 5-amino-2-chloro-6-(2,6-dimethoxyphenyl)pyridine-3,4-dicarboxylate (2.0 g), tripotassium phosphate (4.1 g), Sphos Pd G3 (0.76 g), methylboronic acid (0.88 g), and toluene (28 mL) were added to a 300 mL three-necked flask, and the mixture was stirred at 115°C for 1 hour under a nitrogen atmosphere. The reaction mixture was treated with ISOLUTE Si-Thiol to remove residual palladium, and the insoluble matter was filtered off. The resulting residue was purified by silica gel column chromatography to obtain diethyl 5-amino-6-(2,6-dimethoxyphenyl)-2-methylpyridine-3,4-dicarboxylate (1.74 g). MS (ESI m / z): 389.4 (M + H) RT (min): 1.31

[0120] The following compounds were synthesized in the same manner as the synthesis of diethyl 5-amino-6-(2,6-dimethoxyphenyl)-2-methylpyridine-3,4-dicarboxylate.

[0121]

[0122] The following compounds were synthesized in the same manner as the synthesis of 8-amino-5,7-dimethyl-2,3-dihydropyrido[3,4-d]pyridazine-1,4-dione.

[0123]

[0124] <Evaluation> The following evaluations were performed using each compound. The evaluation results are summarized in Table 7. For Comparative Example 1, L-012 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used.

[0125] (Example 103) 1. Preparation of chemiluminescent reagents The first to fifth reagents necessary for measurement were prepared using the following reagent raw materials. MES (2-morpholinoethanesulfonic acid, manufactured by Dojin Chemical Laboratories), TAPSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid, manufactured by Dojin Chemical Laboratories), sodium chloride (manufactured by Fujifilm Wako Pure Chemical Corporation), BCN300S (manufactured by Nitta Gelatin Co., Ltd.), BSA (manufactured by Sigma-Aldrich Japan Co., Ltd.), boric acid (manufactured by Fujifilm Wako Pure Chemical Corporation), 4-(4-thiazolyl)phenol (manufactured by Enamine Co., Ltd.), phosphoric acid (manufactured by Fujifilm Wako Pure Chemical Corporation), hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Corporation), capture antibody: anti-amyloid β monoclonal antibody (manufactured by Fujifilm Wako Pure Chemical Corporation, recognizes the N-terminus), detection antibody: anti-amyloid β42 monoclonal antibody (both manufactured by Fujifilm Wako Pure Chemical Corporation)

[0126] (i) First Reagent (Antibody-Solid Magnetic Particle Reagent) Magrapid MGP-010T (manufactured by Sanyo Chemical Industries, Ltd.) was successively reacted with 3-aminopropyltriethoxysilane (manufactured by Tokyo Chemical Industries, Ltd.) and succinic anhydride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and the mixture was magnetized with a neodymium magnet, and the supernatant was removed to obtain magnetic particles having carboxyl groups. Next, the carboxyl groups were activated with commercially available N-hydroxysuccinimide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and WSC (water-soluble carbodiimide) (manufactured by Dojin Chemical Laboratories). Then, an anti-amyloid β monoclonal antibody was reacted at 25°C for 12 to 16 hours, and the supernatant was removed while the magnetic particles were magnetized with a neodymium magnet to prepare anti-amyloid β monoclonal antibody-solid magnetic particles, and the first reagent consisting of the following composition was prepared. • 0.50 mg / mL anti-amyloid-beta monoclonal antibody immobilized magnetic particles • 50 mM MES (pH 5.5) • 500 mM sodium chloride • 3.0% BCN300S

[0127] (ii) Second reagent (reaction buffer) A second reagent was prepared with the following composition: • 50 mM TAPSO (pH = 7.5) • 150 mM sodium chloride

[0128] (iii) Third Reagent (Reagent Containing Labeled Antibody) A third reagent containing a peroxidase-labeled anti-amyloid-β monoclonal antibody, which is a labeled antibody for measuring the amount of antigen in the sample, was prepared with the following composition: • 50 mM MES (pH = 6.5) • 150 mM sodium chloride • 2.0% BSA • 7.5 nM peroxidase-labeled anti-amyloid-β42 monoclonal antibody

[0129] (iv) Fourth reagent A fourth reagent consisting of the following composition was prepared: 50 mM TAPSO4, 0.9% boric acid, 0.50 mM 8-amino-7-(2,6-dimethoxyphenyl)-5-methyl-2,3-dihydropyrido[3,4-d]pyridazine-1,4-dione, 0.2 mM 4-(4-thiazolyl)phenol. The prepared fourth reagent was divided in half, one half was stored in the dark, and the other half was exposed to a 100 lux fluorescent lamp for 30 minutes and then stored in the dark to prepare fourth reagents with and without light exposure.

[0130] (v) A fifth reagent was prepared, consisting of the following components: 0.0068% phosphoric acid and 0.0201% hydrogen peroxide (30%)

[0131] 2. Preparation of Measurement Samples The measurement samples were prepared by dissolving amyloid-β1-42 peptide (manufactured by Peptide Laboratories, Inc.) in EDTA-2K plasma samples at a concentration of 1 pg / mL. A specified amount of whole blood sample was collected in a blood collection container containing EDTA-2K, and the whole blood sample containing EDTA-2K was subjected to centrifugation. The supernatant obtained by separating and removing the settled blood cell components was used as the EDTA-2K plasma sample.

[0132] 3. Evaluation of luminescent reagents Using the first to fifth reagents prepared in "1. Preparation of chemiluminescent reagents" and AccuraSeed B / F separation solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), the luminescence of the amyloid β1-42 peptide solution was measured using the fourth reagent, with and without fluorescent lamp exposure, in an automated chemiluminescent enzyme immunoassay analyzer (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) according to the following procedure. 50 μL of the first reagent was added to the reaction cuvette and heated at 67°C for 20 seconds while being magnetized with a neodymium magnet, and the supernatant was removed. Subsequently, 50 μL of the second reagent and 25 μL of the sample to be measured were added and stirred, and heated at 37°C for 3 minutes. After heating, the mixture was magnetized with a neodymium magnet to remove reagents other than magnetic particles, and washed three times with washing solution. Next, 50 μL of the third reagent was added and heated at 37°C for 3 minutes. After heating, the mixture was magnetized using a neodymium magnet to remove reagents other than magnetic particles, and then washed three times with the washing solution. After washing, 100 μL of the fourth reagent and 100 μL of the fifth reagent were added and reacted at 37°C for 20 seconds. The amount of luminescence was measured for each reagent, and the signal change rate was calculated from the obtained luminescence amounts as (luminescence amount when using the fourth reagent with fluorescent lamp exposure) / (luminescence amount when using the fourth reagent without fluorescent lamp exposure).

[0133] (Example 104) The chemiluminescent reagent was prepared and evaluated in the same manner as in Example 103, except as follows: (iv) Instead of 8-amino-7-(2,6-dimethoxyphenyl)-5-methyl-2,3-dihydropyrido[3,4-d]pyridazin-1,4-dione in "Reagent Four," 8-amino-7-(2,4-dimethylthiophen-3-yl)-5-methyl-2,3-dihydropyrido[3,4-d]pyridazin-1,4-dione was dissolved in 0.5 mM solution and used as "Reagent Four."

[0134] (Example 105) The chemiluminescent reagent was prepared and evaluated in the same manner as in Example 103, except as described below. (iv) Instead of 8-amino-7-(2,6-dimethoxyphenyl)-5-methyl-2,3-dihydropyrido[3,4-d]pyridazine-1,4-dione in "Reagent Fourth", 8-amino-5-methyl-7-(2,4,6-trimethoxyphenyl)-2,3-dihydropyrido[3,4-d]pyridazine-1,4-dione dissolved in 0.5 mM was used as "Reagent Fourth".

[0135] (Comparative Example 102) The chemiluminescent reagent was prepared and evaluated in the same manner as in Example 103, except as follows: (iv) Instead of 8-amino-7-(2,6-dimethoxyphenyl)-5-methyl-2,3-dihydropyrido[3,4-d]pyridazine-1,4-dione in "Reagent Fourth", a solution of L-012 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) in 0.5 mM was used as "Reagent Fourth".

[0136] - Signal values ​​without fluorescent light exposure - The evaluation criteria are as follows: A: Over 16,000 cps B: Over 14,000 cps and 16,000 cps or less C: Over 12,000 cps and 14,000 cps or less D: 10,000 cps or more and 12,000 cps or less E: Less than 10,000 cps

[0137] -Changes in signal intensity after 30 minutes of exposure to fluorescent light- The signal value was measured after 30 minutes of exposure to fluorescent light. Subsequently, the rate of change in signal was calculated based on the following formula.

[0138]

[0139] The evaluation criteria are as follows: A: Signal change rate less than 10% B: Signal change rate 10% or more but less than 20% C: Signal change rate 20% or more but less than 30% D: Signal change rate 30% or more but 40% or less E: Signal change rate greater than 40%

[0140]

[0141] The disclosure of Japanese Patent Application No. 2025-050775, filed on 25 March 2025, is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually noted to be incorporated by reference.

Claims

1. A compound represented by the following formula (1) or a salt thereof. In formula (1), R １ R represents a hydrocarbon group or a heterocyclic group. ２ represents a hydrocarbon group, R ３ and R ４ Each of these independently represents either a hydrogen atom or a hydrocarbon group.

2. R １ The compound or salt thereof according to claim 1, wherein the compound is a hydrocarbon group.

3. R １ However, it is an alkyl group and R ２ The compound or salt thereof according to claim 1, wherein the compound is an alkyl group.

4. R １ is an alkyl group having 2 or less carbon atoms, and R ２ is an alkyl group having 2 or less carbon atoms, the compound according to claim 1 or a salt thereof.

5. R ３ and R ４ The compound or salt thereof according to any one of claims 1 to 4, wherein the compound is a hydrogen atom.

6. A chemiluminescent reagent containing a compound represented by the following formula (1) or a salt thereof. In formula (1), R １ R represents a hydrocarbon group or a heterocyclic group. ２ represents a hydrocarbon group, R ３ and R ４ Each of these independently represents either a hydrogen atom or a hydrocarbon group.

7. R １ The chemiluminescent reagent according to claim 6, wherein the hydrocarbon group is...

8. R １ However, it is an alkyl group and R ２ However, the chemiluminescent reagent according to claim 6 is an alkyl group.

9. R １ However, it is an alkyl group having 2 or fewer carbon atoms, and R ２ The chemiluminescent reagent according to claim 6, wherein the alkyl group has two or fewer carbon atoms.

10. R ３ and R ４ However, the chemiluminescent reagent according to claim 6 is a hydrogen atom.

11. A kit for measuring antigens contained in a test sample, comprising a peroxidase to which an antigen-recognizing binding substance is bound, an oxidizing agent, an enhancer compound, and a compound or salt thereof as described in any one of claims 1 to 5.

12. A measurement method using the measurement kit described in claim 11.

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