Steroid hormone detection method, compound, inclusion complex, composition, and kit

Abstract

The present invention addresses the problem of providing: a steroid hormone detection method and steroid hormone detection kit that are capable of avoiding the influence of steroid hormone-binding proteins during the measurement of steroid hormones in a biological sample; a compound and inclusion complex that can be used in the abovementioned detection method and detection kit; and a composition containing the abovementioned compound or inclusion complex. The present invention provides a steroid hormone detection method involving: bringing a hydrogen halide salt or cyclodextrin inclusion complex of a compound represented by formula [1] into contact with a complex of a steroid hormone-binding protein and steroid hormones in a biological sample; and detecting steroid hormones. In the formula, R1, R2, and R3 are each independently a hydrogen atom or 1-3C alkyl group. R4 represents a halogen atom, a 1-3C alkyl group, a phenyl group, a methoxy group, or an ethoxy group.

Description

Method for detecting steroid hormones, compounds, inclusion complexes, compositions and kits

[0001] The present invention relates to a method for detecting steroid hormones and a kit for detecting steroid hormones. The present invention further relates to compounds and inclusion complexes that can be used in the above-mentioned detection methods and kits, and compositions containing the compounds or inclusion complexes.

[0002] Steroid hormones are a general term for hormones with a steroid skeleton and are synthesized in the adrenal cortex and gonads using cholesterol as a raw material. Examples of steroid hormones include adrenal cortical hormones such as glucocorticoids (cortisol) and mineralocorticoids (aldosterone), as well as sex hormones such as male hormones (testosterone) and female hormones (progesterone, estrone, and estradiol). Steroid hormones are involved in the regulation of various physiological phenomena, including metabolism, neurotransmission, gene expression, reproduction, blood pressure, and vascular permeability. Therefore, measuring the amount of steroid hormones in vivo is used for diagnostic purposes. For example, testosterone measurements are used to diagnose infertility treatments in both women and men. Meanwhile, steroid hormone-binding proteins (e.g., sex hormone-binding globulin (SHBG)) are known to exist in the body. Most testosterone is bound to albumin or SHBG in the blood.

[0003] Non-Patent Document 1 describes that 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole (hereinafter also referred to as IPI) binds to SHBG with an affinity similar to that of testosterone and estradiol.

[0004] Journal of Biological Chemistry(2020), 295(5), 1202-1211

[0005] When measuring steroid hormones in biological samples, there is a problem that the steroid hormones cannot be accurately quantified due to the influence of steroid hormone-binding proteins (such as sex hormone-binding globulin (SHBG)). Furthermore, it was found that the IPI described in Non-Patent Document 1 has low water solubility and is not suitable for use as a reagent.

[0006] An object of the present invention is to provide a method for detecting steroid hormones and a steroid hormone detection kit that can avoid the influence of steroid hormone-binding proteins (e.g., sex hormone-binding globulin (SHBG)) when measuring steroid hormones in biological samples.A further object of the present invention is to provide a compound and an inclusion complex that can be used in the above-mentioned detection method and detection kit, as well as a composition containing the compound or the inclusion complex.

[0007] As a result of intensive research aimed at solving the above problems, the present inventors have found that, in the measurement of steroid hormones in biological samples, a highly sensitive measurement system can be realized by using a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1] as a dissociating agent, thereby avoiding the influence of steroid hormone-binding proteins. The present invention was completed based on the above findings.

[0008] That is, the present invention provides the following inventions: <1> A method for detecting a steroid hormone, which comprises contacting a hydrogen halide salt or a cyclodextrin inclusion complex of a compound represented by formula [1] with a complex of a steroid hormone binding protein and a steroid hormone in a biological sample, and detecting the steroid hormone; In the formula, R １ , R ２ and R3 each independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ４ represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, or a phenyl group. １ and R ２is a hydrogen atom. <3> The method according to <1> or <2>, wherein the steroid hormone is one or more selected from the group consisting of testosterone, dihydrotestosterone, progesterone, estradiol, and cortisol. <4> The method according to any one of <1> to <3>, wherein the steroid hormone-binding protein is one or more selected from the group consisting of sex hormone-binding globulin, albumin, and cortisol-binding protein. <5> The method according to any one of <1> to <4>, wherein the concentration of the hydrogen halide salt or cyclodextrin inclusion complex of the compound represented by formula [1] upon contact with the complex is 0.1 to 10 mmol / L. <6> The method according to any one of <1> to <5>, wherein the sample is serum, plasma, or whole blood. <7> The method according to any one of <1> to <6>, wherein the detection is carried out by an immune reaction using a substance that binds to the steroid hormone, or an immune reaction using a substance that competes with the steroid hormone. <8> A steroid hormone detection kit comprising: a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1]; an immobilized steroid hormone; and a labeled steroid hormone binding substance. In the formula, R １ , R ２ , and R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ４ represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, or a phenyl group. <9> A steroid hormone detection kit comprising a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1], a solid-phase steroid hormone binding substance, and a labeled steroid hormone. In the formula, R １ , R ２ , and R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ４represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, or a phenyl group. <10> A steroid hormone detection kit comprising a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1], a steroid hormone binding substance, and a binding substance capable of binding to a complex of a steroid hormone and the steroid hormone binding substance, the binding substance being labeled. In the formula, R １ , R ２ , and R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ４ represents a hydrogen atom, a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, or a phenyl group. <11> The kit according to <10>, wherein the steroid hormone binding substance is immobilized. <12> A compound represented by formula [2]: In the formula, R １ , R ２ , and R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ４ represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, or a phenyl group, and X represents a chlorine group, a bromine group, or an iodo group. <13> A cyclodextrin inclusion complex of a compound represented by formula [1]: In the formula, R １ , R ２ , and R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ４represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, or a phenyl group. <14> The cyclodextrin inclusion complex according to <13>, wherein the cyclodextrin is γ-cyclodextrin, α-cyclodextrin, or β-cyclodextrin. <15> A composition for dissociating a steroid hormone binding protein and a steroid hormone in a biological sample, comprising the compound according to <12> or the cyclodextrin inclusion complex according to <13> or <14>. <16> The composition according to <15>, which is used in the method according to any one of <1> to <7>. <17> A composition for detecting a steroid hormone in a biological sample, which comprises the compound according to <12> or the cyclodextrin inclusion complex according to <13> or <14>. <18> The composition according to <17>, which is used in the method according to any one of <1> to <7>.

[0009] According to the present invention, the influence of steroid hormone binding proteins can be avoided when measuring steroid hormones in biological samples.

[0010] Figure 1 shows the results of an evaluation of the relationship between the type and concentration of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole salt and sensitivity. Figure 2 shows the results of an evaluation of the effect of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole salt on the measurement values ​​of two types of testosterone-containing specimens. Figure 3 shows the results of an evaluation of the relationship between the type and concentration of α-, β-, and γ-cyclodextrin inclusion complexes and sensitivity. Figure 4 shows the results of an evaluation of the effect of α-, β-, and γ-cyclodextrin inclusion complexes on the measurement values ​​of two types of testosterone-containing specimens.

[0011] The present invention will be described in detail below. In this specification, the word "to" is used to mean that the numerical values ​​before and after it are included as the lower limit and upper limit.

[0012] <Method for detecting steroid hormones> According to the present invention, there is provided a method for detecting steroid hormones, which comprises contacting a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1] with a complex of a steroid hormone binding protein and a steroid hormone in a biological sample, and detecting the steroid hormone. In the formula, R １ , R ２ , and R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ４ represents a hydrogen atom, a halogen atom (fluorine atom, chlorine atom, bromine atom, or iodine atom), an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, or a phenyl group.

[0013] As mentioned above, the IPI described in Non-Patent Document 1 had low water solubility and was not suitable for use as a reagent. As a result of evaluating various salts and inclusion complexes to increase the water solubility of IPI, it was found that the performance of a hydrogen halide salt or a cyclodextrin inclusion complex was superior. The above findings were unexpected.

[0014] Specific examples and preferred embodiments of the hydrogen halide salt or cyclodextrin clathrate of the compound represented by formula [1] will be described later in this specification. １ and R ２ is a hydrogen atom.

[0015] The steroid hormone is preferably one or more selected from the group consisting of testosterone, dihydrotestosterone, progesterone, estradiol, androstenediol, estrone, dehydroepiandrosterone, and cortisol, more preferably testosterone, dihydrotestosterone, and estradiol, and particularly preferably testosterone.

[0016] The steroid hormone binding protein is preferably one or more members selected from the group consisting of sex hormone binding globulin, albumin and cortisol binding protein, and particularly preferably sex hormone binding globulin.

[0017] When a complex of a steroid hormone binding protein and a steroid hormone in a biological sample is contacted, the concentration of the hydrogen halide salt or cyclodextrin inclusion complex of the compound represented by formula [1] at the time of contact with the complex is preferably 0.1 to 10 mmol / L, more preferably 0.1 to 5.0 mmol / L, and even more preferably 0.1 to 2.0 mmol / L.

[0018] The biological sample in the present invention is not particularly limited, but is preferably an animal-derived sample, and more preferably a human-derived sample. Examples of biological samples include, but are not limited to, blood-derived samples (serum, plasma, whole blood, etc.), urine, saliva, lymph, cerebrospinal fluid, pleural effusion, ascites, tears, semen, bladder washings, tissue extracts, tissue sections, tissue biopsy samples, and samples prepared therefrom. Blood-derived samples are preferred, with serum, plasma, or whole blood being particularly preferred. The biological sample may be directly collected or may be pretreated, for example, by collection, concentration, purification, isolation, dilution with a buffer solution, filtration sterilization, or the like. These pretreatments may be carried out as appropriate according to conventional methods.

[0019] The detection of steroid hormones is not particularly limited, and can be carried out, for example, by immunological assay. Preferably, the detection of steroid hormones can be carried out by an immune reaction using a substance that binds to steroid hormones (also referred to as a steroid hormone-binding substance), or an immune reaction using a substance that competes with steroid hormones. Examples of substances that bind to steroid hormones (steroid hormone-binding substances) that can be used include antibodies that bind to steroid hormones (anti-steroid hormone antibodies), peptides such as cyclic peptides, and aptamers. Anti-steroid hormone antibodies include Fab, Fab', F(ab')2, Fv, Fd, single-chain Fv (scFv), disulfide-linked Fv (sdFv), and V L , V H , Fv-clasp, diabody ((V L -V H )2 or (V H -V LThe steroid hormone-binding substance may be a portion or modified form of an antibody such as a triabody (trivalent antibody), a tetrabody (tetravalent antibody), a minibody ((scFV-CH3)2), an IgG-delta-CH2, scFv-Fc, or an (scFv)2-Fc fragment. The substance that binds to a steroid hormone may be labeled or immobilized. Details of labeling and immobilization will be described later.

[0020] The substance that competes with the steroid hormone may be any substance that competes with the steroid hormone to be detected, and for example, the same steroid hormone as the steroid hormone to be detected, a steroid hormone analog, or a steroid hormone derivative may be used. The steroid hormone used as the competing substance may be labeled or immobilized. Details of labeling and immobilization will be described later.

[0021] The measurement principle of immunoassay is not particularly limited, and examples thereof include competitive assay, sandwich assay, anti-immune complex antibody assay, immunochromatography, capillary electrophoresis, Western blotting, and surface plasmon resonance (SPR) assay. Furthermore, the measurement process is not particularly limited, and examples thereof include one-step assay, two-step assay, and one-step delay reaction assay. The measurement method in immunoassay is also not particularly limited, and examples thereof include enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), and the like.

[0022] In a first embodiment of the steroid hormone detection method of the present invention, a biological sample containing a complex of a steroid hormone binding protein and a steroid hormone is contacted with a solid phase carrier (e.g., magnetic particles) to which a steroid hormone is bound, and a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1] to cause a reaction, and then a labeled anti-steroid hormone antibody is reacted, and B / F separation (Bound / Free separation) is performed to detect the label bound to the solid phase.

[0023] Specific examples of the first aspect described above are shown below. (1) Preparation of Reagent The first reagent is a reagent containing particles to which a steroid hormone is bound. The steroid hormone can be bound to the particles directly or indirectly by a known method. It is preferable to use magnetic particles as the particles. The first reagent may contain a dispersion medium such as water, or may be used in the form of particles without containing a dispersion medium.

[0024] The second reagent is a reaction buffer solution containing a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1]. The second reagent preferably contains a buffering agent. Examples of the buffering agent include those described in the "Composition of the Present Invention" section below.

[0025] The third reagent is a reagent containing a labeled anti-steroid hormone antibody. The third reagent preferably contains a labeled anti-steroid hormone antibody, a buffer, and the like. Examples of the buffer include those described in the "Composition of the Present Invention" section below.

[0026] The fourth reagent is a reagent containing a luminescent substance. Examples of the luminescent substance include known luminescent substances such as luciferin and L-012 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). It is preferable that the fourth reagent further contains a buffer and the like. Examples of the buffer include those described below in the section <Composition of the Present Invention>.

[0027] The fifth reagent is a reagent containing hydrogen peroxide. Preferably, the fifth reagent further contains a buffering agent. Examples of the buffering agent include those described in the "Composition of the Present Invention" section below.

[0028] As the washing liquid, known washing liquids such as water, buffer solution, etc. may be used. Alternatively, commercially available washing liquids such as Accura Seed B / F Separation Liquid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) may be used.

[0029] (2) Detection Method The second reagent and the specimen (i.e., a biological sample containing a complex of a steroid hormone binding protein and a steroid hormone) are added to the first reagent, stirred, and heated, for example, at 37°C for 1 to 10 minutes. After heating, the third reagent is added, and heated, for example, at 37°C for 1 to 10 minutes. After heating, for example, if the particles in the first reagent are magnetic particles, reagents other than the magnetic particles are removed by magnetic collection using a magnet, and washed with a washing solution. After washing, the fourth reagent and the fifth reagent are added, and after the reaction, the amount of luminescence is measured.

[0030] In a second embodiment of the steroid hormone detection method of the present invention, a biological sample containing a complex of a steroid hormone binding protein and a steroid hormone, a solid phase carrier (e.g., magnetic particles) to which a steroid hormone is bound, a hydrogen halide salt or cyclodextrin inclusion complex of the compound represented by formula [1], and a labeled anti-steroid hormone antibody are reacted, and B / F separation (Bound / Free separation) is performed to detect the label bound to the solid phase (the label bound to the complex produced by the reaction).

[0031] In a third embodiment of the steroid hormone detection method of the present invention, a biological sample containing a complex between a steroid hormone-binding protein and a steroid hormone is contacted with a solid support (e.g., magnetic particles) to which a steroid hormone is bound. Then, a labeled anti-steroid hormone antibody and a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1] are added to the mixture, and the reaction is allowed to proceed. Bound / Free separation (B / F separation) is performed to detect the label bound to the solid support (the label bound to the complex produced by the reaction).

[0032] In a fourth aspect of the steroid hormone detection method of the present invention, a biological sample containing a complex of a steroid hormone binding protein and a steroid hormone is contacted with a solid phase carrier (e.g., magnetic particles) to which an anti-steroid hormone antibody is bound, and a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1] to cause a reaction, and then a labeled steroid hormone is reacted, and B / F separation (Bound / Free separation) is performed to detect the label bound to the solid phase (the label bound to the complex produced by the reaction).

[0033] In a fifth aspect of the steroid hormone detection method of the present invention, a biological sample containing a complex of a steroid hormone binding protein and a steroid hormone, a solid phase carrier (e.g., magnetic particles) to which an anti-steroid hormone antibody is bound, a hydrogen halide salt or cyclodextrin inclusion complex of the compound represented by formula [1], and a labeled steroid hormone are reacted, and B / F separation (Bound / Free separation) is performed to detect the label bound to the solid phase (the label bound to the complex produced by the reaction).

[0034] In a sixth aspect of the steroid hormone detection method of the present invention, a biological sample containing a complex of a steroid hormone binding protein and a steroid hormone is contacted with a solid phase carrier (e.g., magnetic particles) to which an anti-steroid hormone antibody is bound, and a reaction is allowed to occur. Thereafter, a labeled steroid hormone and a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1] are added and allowed to react, and B / F separation (Bound / Free separation) is performed to detect the label bound to the solid phase (the label bound to the complex produced by the reaction).

[0035] A seventh aspect of the steroid hormone detection method of the present invention is a case in which, in the fourth to sixth aspects of the steroid hormone detection method of the present invention, the solid phase carrier to which the anti-steroid hormone antibody is bound is indirectly immobilized to the solid phase carrier via an anti-IgG antibody.

[0036] In an eighth embodiment of the steroid hormone detection method of the present invention, a biological sample containing a complex of a steroid hormone-binding protein and a steroid hormone is contacted with an anti-steroid hormone antibody and a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1] to cause a reaction, followed by reaction with a labeled antibody that recognizes the complex of the steroid hormone and the anti-steroid hormone antibody, followed by B / F separation (Bound / Free separation) to detect the label bound to the solid phase (the label bound to the complex produced by the reaction).

[0037] In the eighth aspect, the anti-steroid hormone antibody may or may not be immobilized. When a non-immobilized anti-steroid hormone antibody is used, the anti-steroid hormone antibody may be labeled with a polyanionic substance, if desired. Examples of methods for B / F separation (bound / free separation) include electrophoretic methods such as chromatography, high-performance liquid chromatography, capillary electrophoresis, capillary tip electrophoresis, LBA (liquid-phase binding assay), and LBA-EATA (liquid-phase binding assay and electrokinetic analyte transport assay). An immunoassay method based on the LBA-EATA method is described in Japanese Patent No. 4862093. The entire contents of Japanese Patent No. 4862093 are incorporated herein by reference.

[0038] Of the above-mentioned embodiments, from the viewpoint of the dissociation reaction of a steroid hormone by a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1], the first, second, fourth, fifth, seventh, and eighth embodiments are preferred, the first, second, fourth, seventh, and eighth embodiments are more preferred, the first and second embodiments are even more preferred, and the first embodiment is particularly preferred.

[0039] <Steroid hormone detection kit> A first embodiment of the steroid hormone detection kit is a steroid hormone detection kit comprising: a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by the above formula [1]; an immobilized steroid hormone; and a labeled steroid hormone-binding substance.

[0040] A second embodiment of the steroid hormone detection kit is a steroid hormone detection kit comprising a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by the above formula [1], an immobilized steroid hormone binding substance, and a labeled steroid hormone.

[0041] A third embodiment of the steroid hormone detection kit is a steroid hormone detection kit comprising: a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by the above formula [1]; a steroid hormone binding substance; and a binding substance capable of binding to a complex of a steroid hormone and the steroid hormone binding substance, the binding substance being labeled. In the third embodiment of the steroid hormone detection kit, the steroid hormone binding substance may or may not be immobilized.

[0042] As the steroid hormone binding substance, an anti-steroid hormone antibody can be used.

[0043] Examples of solid-phase supports for immobilizing steroid hormones or steroid hormone-binding substances include insoluble solid-phase supports used in conventional immunoassays. Specific examples include substrates used in conventional protein immobilization methods, such as slide glasses, ELISA plates, microtiter plates, beads (e.g., magnetic beads), microparticles (e.g., latex particles), filters, tubes, films, and membranes. Examples of substrate materials include synthetic polymers such as polycarbonate, polystyrene, polyurethane, polypropylene, polyacrylic acid, polymethacrylic acid, polyacrylamide, polyglycidyl methacrylate, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, and silicone rubber, as well as inorganic materials such as glass (e.g., porous glass), ground glass, silicon, ceramics, alumina, silica gel, activated carbon, and metal oxides.

[0044] The method for immobilizing the steroid hormone or steroid hormone-binding substance on the solid phase carrier is not particularly limited, and known methods such as chemical binding methods (methods for immobilizing by covalent bonding) and physical adsorption methods can be applied.

[0045] As labeling substances used to label steroid hormone binding substances or steroid hormones, for example, the following substances can be used, but there is no particular limitation, and any labeling substance normally used in this field can be used.

[0046] Enzymes such as peroxidase, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, acetylcholinesterase, malate dehydrogenase, and luciferase; ９９ｍ Tc, １３１ I, １２５ I, １４ C. ３ H. ３２ P, ３５ Radioactive isotopes such as S;

[0047] Fluorescent substances such as HiLyte 647 (manufactured by Anaspec), fluorescein, dansyl, fluorescamine, coumarin, naphthylamine, fluorescein isothiocyanate (FITC), rhodamine, rhodamine X isothiocyanate, sulforhodamine 101, Lucifer Yellow, acridine, acridine isothiocyanate, riboflavin, or derivatives thereof; luminescent substances such as luciferin, isoluminol, luminol, L-012, etc.;

[0048] Substances that absorb in the ultraviolet region, such as phenol, naphthol, anthracene, or derivatives thereof; substances that have properties as spin labeling agents, such as compounds having an oxyl group, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, 2,6-di-t-butyl-α-(3,5-di-t-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxyl;

[0049] HiLyte dyes such as HiLyte Fluor 647, HiLyte Fluor 488, HiLyte Fluor 555, HiLyte Fluor 680, and HiLyte Fluor 750 (all trade names of HiLyte Bioscience, Inc.); Alexa Fluor Dye 350, Alexa Fluor Dye 430, Alexa Fluor Dye 488, Alexa Fluor Dye 532, Alexa Fluor Dye 546, Alexa Fluor Dye 555, and Alexa Fluor Dye Alexa dyes such as Alexa Fluor Dye 568, Alexa Fluor Dye 594, Alexa Fluor Dye 633, Alexa Fluor Dye 647, Alexa Fluor Dye 660, Alexa Fluor Dye 680, Alexa Fluor Dye 700, and Alexa Fluor Dye 750 (all trade names of Molecular Probes);

[0050] CyDye dyes such as Cy3, Cy3.5, Cy5, Cy5.5, and Cy7 (all trade names of Amersham Biosciences); dyes such as Coomassie Brilliant Blue R250 and Methyl Orange

[0051] The method for binding a labeling substance to a steroid hormone-binding substance or a steroid hormone can be carried out by appropriately utilizing a labeling method used in conventional immunoassays and the like, such as those described in "Medical Chemistry Experiment Lectures," Vol. 8, edited by Yamamura Yuichi, 1st ed., Nakayama Shoten, 1971; "Illustrated Fluorescent Antibodies," by Kawao Akira, 1st ed., Soft Science Co., Ltd., 1983; and "Enzyme Immunoassay," edited by Ishikawa Eiji, Kawai Tadashi, and Muroi Kiyoshi, 2nd ed., Igaku-Shoin, 1982. The labeling substance may be directly bound to the steroid hormone-binding substance or the steroid hormone, or may be indirectly bound to the steroid hormone-binding substance or the steroid hormone via a suitable spacer (e.g., a combination of one or several amino acids, one or several amino acids and a linker, or a substance having affinity, such as an avidin (e.g., streptavidin, tamavidin) and a biotin).

[0052] An example of the kit of the present invention may include the first reagent, the second reagent, the third reagent, the fourth reagent, and the fifth reagent. The kit of the present invention may further include the washing solution.

[0053] The kit of the present invention may further include a standard sample. The standard sample is preferably a sample containing a known concentration of a steroid hormone. The standard sample is used as a calibrator or control for creating a calibration curve showing the correlation between a known amount (concentration) of steroid hormone contained in the standard sample and the measured value obtained by various analytical methods, for confirming the effectiveness of quality control and calibration of analytical instruments, and for investigating the stability of quantitative analysis over time. Examples of the measured value include absorbance, change in absorbance, amount of transmitted light, change in transmitted light, luminescence, and change in luminescence. Furthermore, the equivalent value of the amount, concentration, etc. of a steroid hormone in a sample calculated from the measured value may also be referred to as a measured value in this specification.

[0054] The kit of the present invention may further include instructions for use (package insert), etc. The instructions may describe the components of the reagent of the present invention, the operating procedures and principles of the method for detecting steroid hormones of the present invention, etc.

[0055] <Compound and Inclusion Complex> According to the present invention, there is provided a compound represented by formula [2]. In the formula, R １ , R ２ , and R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ４ represents a hydrogen atom, a halogen atom (a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, or a phenyl group, and X represents a chlorine group, a bromo group, or an iodo group.

[0056] In formula [2], R １ , R ２ , R ３ , and R ４ In formula [2], the alkyl group having 1 to 3 carbon atoms is preferably an alkyl group having 1 to 2 carbon atoms, and more preferably an alkyl group having 1 carbon atom. ４ As the halogen atom in formula [2], a fluorine atom and a chlorine atom are preferred. ４As the alkoxy group having 1 to 3 carbon atoms, an alkoxy group having 1 to 2 carbon atoms is preferred, and an alkoxy group having 1 carbon atom is more preferred.

[0057] In formula [2], R １ , R ２ , R ３ , and R ４ is preferably a hydrogen atom. In formula [2], X is preferably a chlorine group.

[0058] The compound represented by formula [2] can be produced according to the description in the Examples below. IPI is dissolved in a suitable solvent (e.g., ethyl acetate, etc.), and a hydrohalic acid (hydrogen chloride (hydrochloric acid), hydrogen bromide, or hydrogen iodide) is added thereto and reacted to produce the compound represented by formula [2]. The reaction temperature is not particularly limited, but is generally 0°C to 25°C. The reaction time is also not particularly limited, but is generally 1 hour to 24 hours.

[0059] According to the present invention, there is provided a cyclodextrin inclusion complex of the compound represented by formula [1]. In the formula, R １ , R ２ , and R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ４ represents a hydrogen atom, a halogen atom (fluorine atom, chlorine atom, bromine atom, or iodine atom), an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, or a phenyl group.

[0060] In formula [1], R １ , R ２ , R ３ , and R ４ In formula [1], the alkyl group having 1 to 3 carbon atoms is preferably an alkyl group having 1 to 2 carbon atoms, and more preferably an alkyl group having 1 carbon atom. ４ As the halogen atom in formula [1], a fluorine atom and a chlorine atom are preferred. ４ As the alkoxy group having 1 to 3 carbon atoms, an alkoxy group having 1 to 2 carbon atoms is preferred, and an alkoxy group having 1 carbon atom is more preferred.

[0061] In formula [1], R １ , R ２ , R ３ , and R ４ is preferably a hydrogen atom. The cyclodextrin is preferably γ-cyclodextrin, α-cyclodextrin, or β-cyclodextrin, more preferably α-cyclodextrin.

[0062] The cyclodextrin inclusion complex of the compound represented by formula [1] can be produced according to the description in the Examples below. IPI is dissolved in a suitable solvent (e.g., methanol, dichloromethane, and water), and cyclodextrin is added thereto for reaction to produce the cyclodextrin inclusion complex of the compound represented by formula [1]. The reaction temperature is not particularly limited, but is generally 0°C to 40°C. The reaction time is also not particularly limited, but is generally 6 to 120 hours.

[0063] <Composition> The present invention provides a composition for dissociating a steroid hormone from a steroid hormone-binding protein in a biological sample, which comprises the compound of the present invention or the cyclodextrin inclusion complex of the present invention. The composition of the present invention is preferably used in the method for detecting a steroid hormone of the present invention.

[0064] The present invention further provides a composition for detecting a steroid hormone in a biological sample, which comprises the compound of the present invention or the cyclodextrin inclusion complex of the present invention. The composition of the present invention is preferably used in the method for detecting a steroid hormone of the present invention.

[0065] The composition of the present invention may further contain other components, including, but not limited to, buffers, inorganic salts, inorganic acids or inorganic bases, stabilizers, non-specific reaction inhibitors, adsorption inhibitors, preservatives, solvents, etc.

[0066] Examples of the buffer include N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N-(2-acetamido)iminodiacetic acid (ADA), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N,N-bis(2-hydroxyethyl)glycine (Bicine), bis(2-hydroxyethyl)iminotris(hydroxyethyl)methane (Bis-Tris), N-cyclohexyl-3-aminopropanesulfonic acid (CA PS), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) , 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPSO), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), 2-hydroxy-3-(N-morpholino)propanesulfonic acid (MOPSO), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) (P OPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), 2-hydroxy-N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSO), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), N-[tris(hydroxymethyl)methyl]glycine (Tricine), phosphate, acetate, citrate, tris(hydroxymethyl)aminomethane, and the like.

[0067] Examples of inorganic salts include those known in the art, such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, sodium bicarbonate, and sodium dihydrogen phosphate.

[0068] Examples of inorganic acids or inorganic bases include those known in the art, such as hydrochloric acid, sulfuric acid, boric acid, phosphoric acid, sodium hydroxide, and potassium hydroxide.

[0069] Examples of the stabilizer include known chelating agents and protease inhibitors.

[0070] As the non-specific reaction inhibitor, any of those known in the art can be used. For example, milk proteins such as skim milk, egg albumin, commercially available blocking agents (e.g., Block Ace (manufactured by Sumitomo Dainippon Pharma Co., Ltd.), Blockmaster TM PA series (manufactured by JSR Corporation), RNase, etc.

[0071] Examples of the adsorption inhibitor include surfactants and inhibitors of peptide adsorption such as albumin (e.g., bovine serum albumin).

[0072] Examples of preservatives include salicylic acid and benzoic acid.

[0073] Examples of the solvent include water and water-soluble organic solvents.

[0074] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In this specification, mM means mmol / L.

[0075] Synthesis Example 1 Synthesis of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole (IPI) (Compound 3) 10 g (0.052 mol) of 2-phenylindole (Compound 1) was dissolved in 60 mL of ethanol. To the resulting solution, 9 mL of dimethylamine (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) and 4 mL of acetic acid were added, and 9 mL of formaldehyde (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) was added dropwise, followed by stirring and reaction at 0°C for 2 hours. Next, ice water was added to the reaction solution, and the mixture was neutralized with potassium carbonate. The reaction solution was evaporated under reduced pressure, extracted with ether, and dried over sodium sulfate to obtain N,N-dimethyl-1-(2-phenyl-1H-indol-3-yl)methanamine (Compound 2) (yield: 11.7 g, 90%).

[0076] 11.6 g (0.046 mol) of compound 2 was dissolved in xylene, and 3.82 g (0.056 mol) of imidazole (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the resulting solution, followed by heating under reflux at 140° C. for 2 hours. The reaction solution was then evaporated under reduced pressure, and the residue was dissolved in 50 mL of methanol by heating, allowed to stand overnight at −20° C., and the precipitated crystals were collected by filtration to obtain 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole (IPI) (compound 3) (yield: 5.3 g, 42%).

[0077]

[0078] Example 1 Synthesis of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole hydrochloride (IPI-HCl) (Compound 4) 4.65 g of IPI (Compound 3) obtained in Synthesis Example 1 was dissolved in 50 mL of ethyl acetate, and 25 mL of 4 mol / L hydrochloric acid / ethyl acetate was added thereto, followed by stirring at 0°C for 4 hours. After completion of the reaction, the precipitated crystals were collected by filtration and washed with ethyl acetate to obtain 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole hydrochloride (Compound 4) (yield: 5.2 g, 99%). MS: 274.1 (posi) 1H-NMR (CD3OD); 5.72 (2H, d), 7.08-7.13 (1H, m), 7.19-7.24 (1H, m), 7.42-7.59 (9H, m), 8.82 (1H, s)

[0079]

[0080] For the synthesis of IPI-HBr and IPI-HI, the acid reagent was changed to hydrobromic acid or hydroiodic acid, but the synthesis was carried out in the same manner as for IPI-HCl, and the target products were obtained.

[0081] Example 2 Synthesis of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole·γ-cyclodextrin (IPI-γCD) (Compound 5) 25 mg of IPI (Compound 3) obtained in Synthesis Example 1 was dissolved in 1 mL of methanol, 1 mL of dichloromethane, and 1 mL of purified water, and 119 mg of γ-cyclodextrin was added thereto, followed by stirring at room temperature for 72 hours. After completion of the reaction, the reaction solution was evaporated under reduced pressure and dried under reduced pressure to obtain 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole·γ-cyclodextrin (Compound 5) (yield: 62 mg, 43%).

[0082]

[0083] IPI-αCD and IPI-βCD were synthesized by the same procedure as for IPI-γCD, except that γ-cyclodextrin was replaced with α-cyclodextrin or β-cyclodextrin, to obtain the target products. IPI-α-cyclodextrin and IPI-β-cyclodextrin were synthesized by the same procedure as for IPI-γ-cyclodextrin, except that γ-cyclodextrin was replaced with α-cyclodextrin or β-cyclodextrin, to obtain the target products.

[0084] Synthesis Example 2: Synthesis of 3-((1H-imidazol-1-yl)methyl)-2-(pyridin-2-yl)-1H-indole (IPYI) (Compound 8) 1 g (0.005 mol) of 2-pyridinindole (Compound 6) was dissolved in 4 mL of ethanol. To the resulting solution, 1.35 mL of dimethylamine (FUJIFILM Wako Pure Chemical Industries, Ltd.) and 0.6 mL of acetic acid were added, and 1.35 mL of formaldehyde (FUJIFILM Wako Pure Chemical Industries, Ltd.) was added dropwise, followed by stirring at 0°C for 2 hours. Next, ice water was added to the reaction solution, and the mixture was neutralized with potassium carbonate. The reaction solution was evaporated under reduced pressure, extracted with ether, and dried over sodium sulfate to obtain N,N-dimethyl-1-(2-pyridin-1H-indol-3-yl)methanamine (Compound 7) (yield: 0.65 g, 51%).

[0085] 0.65 g (0.0025 mol) of compound 7 was dissolved in xylene, and 0.19 g (0.0028 mol) of imidazole (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the resulting solution, followed by heating under reflux at 140° C. for 2 hours. The reaction solution was then evaporated under reduced pressure, and the residue was dissolved in 5 mL of methanol by heating. The mixture was allowed to stand overnight at −20° C., and the precipitated crystals were collected by filtration to give 3-((1H-imidazol-1-yl)methyl)-2-pyridine-1H-indole (IPYI) (compound 8) (yield: 0.31 g, 44%).

[0086]

[0087] Example 3 The relationship between the type and concentration of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole salt and sensitivity was evaluated for two samples containing testosterone. (1) Preparation of Reagents The constituent reagents required for measurement were prepared using the following raw reagent materials. MES (2-morpholinoethanesulfonic acid monohydrate) (manufactured by Dojindo Laboratories, Ltd.), sodium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), BCN300S (manufactured by Nitta Gelatin Co., Ltd.), BSA (bovine serum albumin) (manufactured by Sigma-Aldrich Japan K.K.), TAPSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid) (manufactured by Dojindo Laboratories, Ltd.), boric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), L-012 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), phosphoric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), EDTA-2Na (manufactured by Dojindo Laboratories, Ltd.), and hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0088] (i) "First Reagent" Testosterone-Solidified Magnetic Particle-Containing Reagent The carboxyl group of modified testosterone, TES-19CME (Shinsei Chemical Co., Ltd.), was activated with commercially available N-hydroxysuccinimide (Fujifilm Wako Pure Chemical Industries, Ltd.) and WSC (water-soluble carbodiimide) (Dojindo Laboratories, Ltd.). The resulting mixture was then reacted with a BSA (bovine serum albumin) solution at 4°C for 18 to 24 hours, and purified using a PD-10 column (Cytiva) to prepare BSA-bound testosterone.

[0089] 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 magnetic collection with a neodymium magnet and removal of the supernatant, yielding magnetic particles bearing carboxyl groups. The carboxyl groups were then activated with commercially available N-hydroxysuccinimide (WSC), followed by reaction with BSA-conjugated testosterone at 26°C for 12 to 16 hours. The particles were then magnetically collected with a neodymium magnet and the supernatant was removed to yield testosterone-immobilized magnetic particles. This gave a first reagent consisting of the following composition: "First Reagent": 0.25 mg / mL testosterone-immobilized magnetic particles, 50 mM MES (pH 5.5), 500 mM sodium chloride, 3.0% (w / v) BCN300S.

[0090] (ii) "Second Reagent" Reaction Buffer: 40 mM MES (pH 5.5), 150 mM sodium chloride, 2.0% (w / v) BSA, 0.0, 0.25, 0.5, or 1.0 mM of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole hydrochloride (Compound 4), bromide, or iodide.

[0091] (iii) "Third Reagent" - Labeled Antibody-Containing Reagent An anti-testosterone antibody was produced by the following method. Testosterone, an immunogen, was injected into a rabbit, which was an animal to be immunized, and lymphocytes were then collected. Antibody gene cassettes were obtained from the obtained lymphocytes by single PCR, and each gene cassette was introduced into HEK293 cells, and genes producing the target antibodies were selected. The genes producing the target antibodies were then transfected into CHO-Spica cells, and anti-testosterone antibodies were produced.

[0092] The prepared anti-testosterone antibody was reduced with cysteamine hydrochloride (Sigma-Aldrich Japan), and the IgG 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, Inc.), and the reaction mixture was passed through a Sephadex G-25 column to remove unreacted Sulfo-KMUS, yielding maleimidated POD. The prepared IgG and maleimidated POD were mixed and separated using a Sephacryl S-100HR column to produce a POD-labeled anti-testosterone antibody. Using this, a third reagent consisting of the following composition was prepared. "Third reagent": 20 pmol / L POD-labeled anti-testosterone antibody, 50 mM MES (pH 5.5), 150 mM sodium chloride, 2.0% BSA

[0093] (iv) "Fourth Reagent" Luminescence Reagent: 50 mM TAPSO, 0.9% (w / v) boric acid, 0.50 mM L-012, 200 mM thiazolephenol

[0094] (v) “Fifth Reagent” “Fifth Reagent”: 68 μL / L phosphoric acid, 4mM EDTA-2Na, 335 μL / L hydrogen peroxide

[0095] (vi) Other Reagents As samples for the calibration curve, diluted samples of testosterone (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were prepared at 0.00 ng / mL, 0.07 ng / mL, 0.20 ng / mL, 0.50 ng / mL, 1.00 ng / mL, 2.00 ng / mL, 4.00 ng / mL, 8.00 ng / mL, 12.00 ng / mL, and 20.00 ng / mL.

[0096] (2) Evaluation of Luminescent Reagents Using the first to fifth reagents prepared in "(1) Preparation of Reagents" and Accura Seed B / F Separation Solution (Fujifilm Wako Pure Chemical Industries, Ltd.), the luminescence intensity of each solution was measured using an automated chemiluminescent enzyme immunoassay analyzer, Accura Seed (Fujifilm Wako Pure Chemical Industries, Ltd.), 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 attracted using a neodymium magnet, and the supernatant was removed. Subsequently, 50 μL of the second reagent and 25 μL of the sample were added, stirred, and heated at 37°C for 3 minutes. After heating, 50 μL of the third reagent was added, and the mixture was heated at 37°C for 3 minutes. After heating, magnetically attracted using a neodymium magnet, and reagents other than 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. A calibration curve showing the relationship between the amount of testosterone and the amount of luminescence was created from the amount of luminescence measured for each calibration sample, and the amount of testosterone in the sample was determined.

[0097] Figure 1 shows the relationship between the type and concentration of salt in the second reagent and sensitivity. Generally accurate measurements are possible if the ratio of the luminescence intensity when measuring 4.00 ng / mL of testosterone to the luminescence intensity when measuring 0.00 ng / mL of the calibration curve sample is 25% or less. Figure 1 shows that the hydrochloride, bromide, and iodide salts of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole all met the above criteria. In particular, it was found that the hydrochloride salt of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole had the highest sensitivity.

[0098] Example 4 Evaluation of the effect of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole salt on the measured values ​​of samples The same procedure as in Example 3 was carried out to evaluate the effect of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole salt on the measured values ​​of two types of testosterone-containing samples.

[0099] The results are shown in Figure 2. The sample measurement value when no 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole salt was added was defined as 100%, and the measurement values ​​when 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole hydrochloride (Compound 4), bromide, or iodide salt was added were defined as the "testosterone measurement ratio relative to the testosterone measurement value without 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole salt added." When any of the 3-((13H-imidazol-1-yl)methyl)-2-phenyl-1H-indole salts was used, the measurement value increased compared to the testosterone measurement value without addition. This is thought to be because the 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole salt dissociated testosterone from the testosterone complex in the sample, avoiding the influence of steroid hormone-binding proteins, resulting in an increase in the measurement value.

[0100] Comparative Example 1 Evaluation of the effect of 3-((1H-imidazol-1-yl)methyl)-2-(pyridin-2-yl)-1H-indole (IPYI) on the measured value of a specimen The effect of 3-((1H-imidazol-1-yl)methyl)-2-(pyridin-2-yl)-1H-indole (IPYI) (compound 8) was used instead of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole salt, and the same method as in Example 4 was used to evaluate the effect of 3-((1H-imidazol-1-yl)methyl)-2-(pyridin-2-yl)-1H-indole (IPYI) (compound 8). As a result, no dissociation effect was obtained with 3-((1H-imidazol-1-yl)methyl)-2-(pyridin-2-yl)-1H-indole (IPYI) (compound 8).

[0101] Example 5 Accelerated Stability of Reagent Containing 3-((1H-Imidazol-1-yl)methyl)-2-phenyl-1H-indole Salt In order to evaluate the effect of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole salt on the stability of the reagent, a reagent having the same composition as in Example 3 was prepared and subjected to refrigerated storage or an accelerated test (37°C for 1 week, equivalent to refrigerated storage for 1 year). Measurements were performed in the same manner as in Example 3, except that the reagents stored in the refrigerator or after the accelerated test were used, and the fluctuations in the luminescence intensity of the calibration curve samples were evaluated. The results are shown in Table 1. The ratio of the luminescence intensity when the reagent after the accelerated test was used to the luminescence intensity when the reagent stored in the refrigerator was used decreased by an average of about 40 to 50% in all cases when 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole hydrochloride (Compound 4), bromide, or iodide was added to the reagent. For accurate measurements, it is desirable that the luminescence intensity fluctuates at a similar rate for all concentrations of calibration curve samples. From the viewpoint of the effect on reagent stability, 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole hydrochloride (Compound 4) was found to be the most excellent.

[0102] Example 6 Relationship between Sensitivity and Type and Concentration of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole Cyclodextrin Inclusion Complex A luminescent reagent was prepared and evaluated in the same manner as in Example 3, with the exception of the following: The amount of testosterone in a sample was calculated using α-, β-, or γ-cyclodextrin inclusion complexes instead of the 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole salt in the "second reagent" of Example 3.

[0103] Figure 3 shows the relationship between the type and concentration of cyclodextrin inclusion complex in the second reagent and sensitivity. The α-, β-, and γ-cyclodextrin inclusion complexes of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole all achieved a luminescence intensity ratio of 25% or less when measuring 4.00 ng / mL of testosterone compared to 0.00 ng / mL of the calibration curve sample. In particular, the α-cyclodextrin inclusion complex of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole was found to have the highest sensitivity.

[0104] Example 7 Evaluation of the Effect of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole Cyclodextrin Inclusion Complex on the Measurement Values ​​of Samples The same procedures as in Example 4 were carried out, except that an α-cyclodextrin inclusion complex, a β-cyclodextrin inclusion complex, or a γ-cyclodextrin inclusion complex of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole (Compound 5) was used instead of the 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole salt, and the effect of the 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole cyclodextrin inclusion complex on the measurement values ​​of two testosterone-containing samples was evaluated.

[0105] The results are shown in Figure 4. The sample measurement value without the addition of any 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole cyclodextrin inclusion complex was defined as 100%, and the measurement values ​​obtained when the α-cyclodextrin inclusion complex, β-cyclodextrin inclusion complex, or γ-cyclodextrin inclusion complex of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole (Compound 5) was added were defined as the "testosterone measurement ratio relative to the testosterone measurement value without the addition of any 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole cyclodextrin inclusion complex." When any of the 3-((13H-imidazol-1-yl)methyl)-2-phenyl-1H-indole cyclodextrin inclusion complexes was used, the measurement value increased compared to the testosterone measurement value without the addition of any of the 3-((13H-imidazol-1-yl)methyl)-2-phenyl-1H-indole cyclodextrin inclusion complexes. This is thought to be because the 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole cyclodextrin inclusion complex dissociated testosterone from the testosterone complex in the sample, avoiding the influence of steroid hormone-binding proteins, resulting in an increase in the measured value.

[0106] Example 8 Accelerated Stability of Reagents Containing 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole Cyclodextrin Inclusion Complex Measurements were conducted in the same manner as in Example 5, except that α-, β-, and γ-cyclodextrin inclusion complexes were used instead of the 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole salt in the "second reagent" of Example 3, and the fluctuations in the luminescence intensity of the calibration curve samples were evaluated. The results are shown in Table 2. The ratios of the luminescence intensity obtained using the reagent after the accelerated test to the luminescence intensity obtained using the reagent stored in a refrigerator decreased to an average of approximately 63%, 70%, and 96%, respectively, by adding the α-cyclodextrin inclusion complex, β-cyclodextrin inclusion complex, and γ-cyclodextrin inclusion complex of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole (Compound 5) to the reagent. For accurate measurements, it is desirable that the luminescence intensity fluctuate at a similar rate for all concentrations of calibration curve samples. The α-cyclodextrin and β-cyclodextrin inclusion complexes of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole were found to be more stable than the γ-cyclodextrin inclusion complex, and the α-cyclodextrin inclusion complex of 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole was found to be the most stable reagent.

[0107]

[0108]

Claims

1. A method for detecting a steroid hormone, comprising contacting a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1] with a complex of a steroid hormone binding protein and a steroid hormone in a biological sample, and detecting the steroid hormone; In the formula, R １ , R ２ , and R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ４ represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, or a phenyl group.

2. In formula [1], R １ , R ２ , R ３ , and R ４ The method of claim 1 , wherein is a hydrogen atom.

3. The method of claim 1, wherein the steroid hormone is one or more selected from the group consisting of testosterone, dihydrotestosterone, progesterone, estradiol and cortisol.

4. The method of claim 1, wherein the steroid hormone binding protein is one or more selected from the group consisting of sex hormone binding globulin, albumin, and cortisol binding protein.

5. The method according to claim 1, wherein the concentration of the hydrogen halide salt or cyclodextrin inclusion complex of the compound represented by formula [1] when contacted with the complex is 0.1 to 10 mmol / L.

6. The method of claim 1, wherein the sample is serum, plasma, or whole blood.

7. The method of claim 1, wherein the detection is carried out by an immunological reaction using a substance that binds to the steroid hormone or a substance that competes with the steroid hormone.

8. A steroid hormone detection kit comprising a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1], an immobilized steroid hormone, and a labeled steroid hormone binding substance. In the formula, R １ , R ２ , and R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ４ represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, or a phenyl group.

9. A steroid hormone detection kit comprising a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1], an immobilized steroid hormone binding substance, and a labeled steroid hormone. In the formula, R １ , R ２ , and R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ４ represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, or a phenyl group.

10. A steroid hormone detection kit comprising a hydrogen halide salt or a cyclodextrin inclusion complex of the compound represented by formula [1], a steroid hormone binding substance, and a labeled binding substance capable of binding to a complex of a steroid hormone and the steroid hormone binding substance. In the formula, R １ , R ２ , and R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ４ represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, or a phenyl group.

11. The kit according to claim 10, wherein the steroid hormone binding substance is immobilized.

12. A compound represented by formula [2]: In the formula, R １ , R ２ , and R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ４ represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, or a phenyl group, and X represents a chlorine group, a bromine group, or an iodo group.

13. A cyclodextrin inclusion complex of the compound represented by formula [1]: In the formula, R １ , R ２ , and R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms; R ４ represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, or a phenyl group.

14. The cyclodextrin inclusion complex of claim 13, wherein the cyclodextrin is γ-cyclodextrin, α-cyclodextrin, or β-cyclodextrin.

15. A composition for dissociating a steroid hormone from a steroid hormone binding protein in a biological sample, comprising the compound of claim 12 or the cyclodextrin inclusion complex of claim 13 or 14.

16. The composition of claim 15 used in the method of claim 1.

17. A composition for detecting a steroid hormone in a biological sample, comprising the compound of claim 12 or the cyclodextrin inclusion complex of claim 13 or 14.

18. The composition of claim 17 used in the method of claim 1.

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