Method for detecting substance to be measured, method for reducing influence of hemolysis, composition, and kit for measuring substance to be measured

Abstract

The present invention addresses the problem of providing: a method for detecting a substance to be measured and a kit for detecting a substance to be measured, the method and kit making it possible to reduce the influence of hemolytic interference in a sample and then carry out measurement; a method for reducing the influence of hemolysis when detecting a substance to be measured in a hemolytic sample; and a composition that could be used in the aforementioned detection method and detection kit. The present invention provides a method for detecting a substance to be measured, the method comprising bringing an iron-binding protein and a hemolytic sample into contact with each other and detecting a substance to be measured in the hemolytic sample.

Description

Method for detecting a substance to be measured, method for reducing the influence of hemolysis, composition, and kit for measuring a substance to be measured

[0001] The present invention relates to a method for detecting a substance to be measured and a method for reducing the influence of hemolysis, and further relates to a composition and a kit for measuring a substance to be measured that can be used in the above method.

[0002] BACKGROUND ART In the fields of clinical testing and biochemistry, blood samples such as serum or plasma are collected and a substance to be measured in the blood sample is measured.

[0003] Patent Document 1 describes an incubation medium for solid-phase immunoassays containing lactoferrin as an inhibitor of nonspecific binding of accompanying substances in a sample to the solid phase. Patent Document 1 uses serum as a sample, but does not describe hemolyzed samples. Patent Document 2 describes a measurement method for hemolyzed samples, characterized in that ethacrynic acid or its salt is added to a measurement solution containing the sample.

[0004] Japanese Patent No. 2714572 Japanese Patent Laid-Open No. 2001-231596

[0005] When measuring a substance to be measured in a sample, if the sample is a hemolyzed sample, there is a problem in that the measurement of the substance to be measured is interfered with by hemolyzed components.

[0006] An object of the present invention is to provide a method for detecting a substance to be measured and a kit for measuring a substance to be measured that can reduce the influence of hemolysis interference in a sample.A further object of the present invention is to provide a method for reducing the influence of hemolysis when detecting a substance to be measured in a hemolyzed sample.A further object of the present invention is to provide a composition that can be used in the above-mentioned detection method and detection kit.

[0007] As a result of extensive research aimed at solving the above problems, the present inventors have found that by contacting an iron-binding protein with a hemolyzed sample, the influence of hemolyzed components on the measurement of a target substance can be reduced, thereby enabling more accurate measurement of the target substance. The present invention was completed based on the above findings.

[0008] That is, the present invention provides the following: <1> A method for detecting a target substance, comprising contacting an iron-binding protein with a hemolyzed sample and detecting the target substance in the hemolyzed sample. <2> The method according to <1>, in which the iron-binding protein is one or more selected from the group consisting of lactoferrin, transferrin, and ferritin. <3> The method according to <1> or <2>, in which the concentration of the iron-binding protein when contacting the hemolyzed sample with the iron-binding protein is 0.1% (w / v) or more. <4> The method according to any one of <1> to <3>, in which the detection is performed using iron ions as a catalyst. <5> The method according to any one of <1> to <4>, in which the detection is performed using a substance that binds to the target substance bound to a labeling substance, or a substance that competes with the target substance bound to a labeling substance. <6> The method according to any one of <1> to <5>, wherein the detection comprises contacting the hemolyzed sample with heparin or a heparin salt. <7> The method according to <6>, wherein the concentration of heparin or heparin salt when the hemolyzed sample is contacted with the heparin or heparin salt is 0.1% (w / v) or more and 3% (w / v) or less. <8> The method according to any one of <1> to <7>, wherein the detection comprises contacting the hemolyzed sample with an anionic surfactant. <9> A method for reducing the effects of hemolysis, comprising contacting an iron-binding protein with the hemolyzed sample and detecting a substance to be measured in the hemolyzed sample. <10> A composition for detecting a substance to be measured in a hemolyzed sample, comprising an iron-binding protein. <11> The composition according to <10>, wherein the iron-binding protein is one or more selected from the group consisting of lactoferrin, transferrin, and ferritin. <12> The composition according to <10>, for use in the method according to any one of <1> to <9>. <13> A kit for measuring a substance to be measured, comprising an iron-binding protein, an immobilized substance to be measured, and a labeled substance that binds to the substance to be measured. <14> A kit for measuring a substance to be measured, comprising an iron-binding protein, an immobilized substance that binds to the substance to be measured, and a labeled substance to be measured.<15> A kit for measuring a substance to be measured, comprising an iron-binding protein, an analyte-binding substance, and a labeled binding substance capable of binding to a complex of the analyte and the analyte-binding substance. <16> The kit for measuring a substance to be measured according to <15>, wherein the analyte-binding substance is immobilized. <17> The kit for measuring a substance to be measured according to any one of <13> to <16>, further comprising heparin or a heparin salt. <18> The kit for measuring a substance to be measured according to any one of <13> to <17>, further comprising an anionic surfactant.

[0009] According to the present invention, the influence of hemolytic components interfering with the measurement of a substance to be measured can be reduced, and the substance to be measured can be measured more accurately.

[0010] Figure 1 shows the relationship between hemolyzed hemoglobin concentration and the measured value (measurement ratio) when the measured value without hemolyzed hemoglobin is set to 100%. Figure 2 shows the relationship between lactoferrin concentration and the measured value (measurement ratio) when the measured value without hemolyzed hemoglobin is set to 100%. Figure 3 shows the relationship between transferrin concentration and the measured value (measurement ratio) when the measured value without hemolyzed hemoglobin is set to 100%. Figure 4 shows the relationship between lactoferrin concentration and Neoperex concentration and the measured value (measurement ratio) when the measured value without hemolyzed hemoglobin is set to 100% under each condition. Figure 5 shows the relationship between heparin concentration and the measured value (measurement ratio) when the measured value without hemolyzed hemoglobin is set to 100%. Figure 6 shows the relationship between lactoferrin concentration and the measured value (measurement ratio) when the measured value without hemolyzed hemoglobin is set to 100%. FIG. 7 shows the relationship (measurement value ratio) between the presence or absence of lactoferrin and various surfactants, with the measurement value without hemolyzed hemoglobin being taken as 100%.

[0011] The present invention will be described in detail below. In this specification, the term "to" is used to mean that the numerical values ​​before and after it are included as the lower and upper limits. In the present invention, measurement may be either qualitative or quantitative.

[0012] <Method for detecting a substance to be measured and method for reducing the influence of hemolysis> The present invention provides a method for detecting a substance to be measured, which comprises contacting an iron-binding protein with a hemolyzed sample and detecting the substance to be measured in the hemolyzed sample. The present invention also provides a method for reducing the influence of hemolysis, which comprises contacting an iron-binding protein with a hemolyzed sample and detecting the substance to be measured in the hemolyzed sample.

[0013] It was previously unknown and completely unexpected that contacting an iron-binding protein with a hemolyzed sample reduces the effect of hemolyzed components interfering with the measurement of the target substance, enabling more accurate measurement of the target substance.

[0014] In the present invention, the substance to be measured is not particularly limited, and examples thereof include all substances that are considered to be measurable by ordinary immunological assays, such as steroids, peptides, hormones, antibodies, proteins, drugs, metabolites, vitamins, etc. contained in biological samples. Specific examples of substances related to endocrine function include thyroid stimulating hormone (TSH), parathyroid hormone (iPTH), growth hormone (GH), somatomedin C (IGF-1), luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), adrenocorticotropic hormone (ACTH), vasopressin, oxytocin, somatostatin, enkephalin, β-endorphin, thyroxine, triiodothyronine, thyroglobulin, antithyroglobulin antibody, anti-T3 antibody, and the like. Body, anti-T4 antibody, anti-TSH antibody, calcitonin, catecholamine, dopamine, serotonin, aldosterone, renin, angiotensin, cortisol, deoxycortisol, cortisone, corticosterone, deoxycorticosterone, androsterone, progesterone, pregnenolone, estrogen, estrone, estriol, estradiol, testosterone, gonadotropins, insulin, anti-insulin antibody, C-peptide, glucagon, gastrin, sex Examples of the anti-inflammatory drugs include retin, cyclic AMP, cyclic GMP, prostaglandins, thromboxane, erythropoietin, and histamine. Examples of tumor-related substances include CEA, ferritin, β2-microglobulin, elastase, α-fetoprotein, nerve-specific enolase, prostate-specific antigen, and CA19-9. Examples of drugs and vitamin-related substances include phenobarbital, phenytoin, carbamazepine, primidone, ethosuximide, valproic acid, and acetazole. Amides, sulthiame, glutethimide, clonazepam, nitrazepam, diazepam, pentobarbital, secobarbital, bupivacaine, mepivacaine, lidocaine, procainamide, quinidine, digoxin, digitoxin, theophylline, amitriptyline, imipramine, amikacin, gentamicin, tobramycin, cephalexin, sulfamethoxazole, methotrexate, cyclosporine, methylprednisolone, salicylic acid, acetaminophen, indomethacin,Examples of serum or plasma protein-related substances include albumin, α1-microglobulin, α1-antitrypsin, α2-macroglobulin, haptoglobulin, hemopexin, transferrin, myoglobin, IgG, IgM, IgA, IgD, IgE, fibrinogen, antithrombin, plasminogen, antiplasmin, protein C, rheumatoid factor, anti-DNA antibody, and C-reactive protein. Examples of virus and infectious disease-related substances include HBs antigen, HBs antibody, HBc antibody, HTLV-I antibody, HTLV-III antibody, TPHA, various viral antigens, and various viral antibodies. An example of a substance to be measured is testosterone, as measured in the experiments described below, but is not limited to this.

[0015] The iron-binding protein is preferably one or more selected from the group consisting of lactoferrin, transferrin, and ferritin, more preferably lactoferrin or transferrin.When the iron-binding protein is brought into contact with the hemolyzed sample, the concentration of the iron-binding protein is preferably 0.1% (w / v) or more, more preferably 0.2% (w / v) to 5.0% (w / v), even more preferably 0.5% (w / v) to 5.0% (w / v), preferably 1.0% (w / v) to 5.0% (w / v), and particularly preferably 1.5% (w / v) to 5.0% (w / v).When the hemolyzed sample is diluted by pretreatment, the concentration of the iron-binding protein when the hemolyzed sample is brought into contact with the iron-binding protein may be adjusted according to the dilution ratio, etc.

[0016] In the present invention, even when a hemolyzed sample is used as the sample, the influence of hemolyzed components interfering with the measurement of the target substance can be reduced, allowing for more accurate measurement of the target substance. The hemolyzed sample is not particularly limited as long as it is a sample that may contain hemoglobin due to hemolysis (a phenomenon in which hemoglobin and the like are released from red blood cells). For example, compared to a biological sample that does not contain hemoglobin or contains hemoglobin in an amount that does not affect the measurement of the target substance, a sample that may contain hemoglobin in an amount that affects the measurement of the target substance due to hemolysis is preferred.

[0017] The biological sample is not particularly limited, but animal-derived samples are preferred, and human-derived samples are more preferred. Examples of biological samples include, but are not limited to, blood-derived samples (serum, plasma, whole blood, etc.), feces, urine, saliva, lymph, cerebrospinal fluid, pleural effusion, ascites, tears, semen, bladder washings, tissue extracts, tissue sections, tissue biopsy samples, and samples prepared from these. Blood-derived samples are preferred, with serum, plasma, or whole blood being particularly preferred. Biological samples may be directly collected or may be pretreated, for example, by collection, concentration, purification, isolation, dilution with a buffer solution, or filtration sterilization. These pretreatments may be performed appropriately according to conventional methods. Hemolysis can occur in the biological sample due to physical stimuli such as suction during blood collection, freezing or heating of biological samples such as blood, or contact with compounds that cause hemolysis, such as anionic surfactants. Examples of hemolyzed samples include serum containing hemoglobin and plasma containing hemoglobin.

[0018] The detection of the analyte is not particularly limited, and can be carried out, for example, by an immunological measurement method. The detection of the analyte is preferably detection using iron ions as a catalyst. Examples of detection using iron ions as a catalyst include detection using luciferin or L-012 as a luminescent substance (substrate) and peroxidase or horseradish peroxidase (HPR) as an enzyme. The detection of the analyte is preferably carried out by an immune reaction using a "substance that binds to the analyte" (also referred to as an analyte-binding substance) bound to a labeled substance, or an immune reaction using a "substance that competes with the analyte" bound to a labeled substance.

[0019] The substance that binds to the analyte (analyte-binding substance) can be an antibody that binds to the analyte, a peptide such as a cyclic peptide, an aptamer, etc. Antibodies that bind to the analyte include Fab, Fab', F(ab')2, Fv, Fd, single-chain Fv (scFv), disulfide-bonded Fv (sdFv), V L , V H , Fv-clasp, diabody ((V L -V H )2 or (V H -V L The antibody may be a portion or a modified form of an antibody such as a triabody (trivalent antibody), a tetrabody (tetravalent antibody), a minibody ((scFV-CH3)2), an IgG-delta-CH2, an scFv-Fc, or an (scFv)2-Fc fragment. The substance that binds to the analyte may be labeled or immobilized. Details of labeling and immobilization will be described later.

[0020] The detection of the analyte preferably includes contacting the hemolyzed sample with heparin or a heparin salt. Examples of heparin salts include sodium heparin and potassium heparin. The use of heparin or a heparin salt further reduces the influence of hemolysis components that interfere with the measurement of the analyte, enabling more accurate measurement of the analyte. The concentration of the heparin or heparin salt when contacting the hemolyzed sample with the heparin or heparin salt is preferably 0.1% (w / v) to 3% (w / v), more preferably 0.1% (w / v) to 2% (w / v), and even more preferably 0.2% (w / v) to 0.8% (w / v).

[0021] The detection of the target substance preferably includes contacting the hemolyzed sample with an anionic surfactant. The iron-binding protein can reduce the influence of hemolysis components on the measurement of the target substance, even when the hemolysis is caused by the anionic surfactant, thereby enabling more accurate measurement of the target substance.

[0022] The anionic surfactant is not particularly limited, but from the viewpoint of easily causing hemolysis even at low concentrations, examples thereof include anionic surfactants having an alkylbenzenesulfonic acid skeleton, alkanesulfonic acids or alkanesulfonate salts (sodium alkanesulfonate, potassium alkanesulfonate, etc.), naphthalenesulfonic acids or naphthalenesulfonate salts (sodium alkanesulfonate, potassium alkanesulfonate, etc.), alkylnaphthalenesulfonic acids or alkylnaphthalenesulfonate salts (sodium alkylnaphthalenesulfonate, potassium alkylnaphthalenesulfonate, etc.), alkyldiphenyletherdisulfonic acids or alkyldiphenyletherdisulfonate salts (sodium alkyldiphenyletherdisulfonate, potassium alkyldiphenyletherdisulfonate, etc.), polyoxyethylene alkylethersulfonic acids or polyoxyethylene alkylethersulfonate salts (sodium polyoxyethylene alkylethersulfonate, potassium polyoxyethylene alkylethersulfonate, etc.), polyoxyethylene alkylethersulfonic acids or polyoxyethylene alkylethersulfonate salts (sodium polyoxyethylene alkylethersulfonate, potassium polyoxyethylene alkylethersulfonate, etc.), polyoxyethylene alkylethersulfonic acids or polyoxyethylene alkylethersulfonates ... sulfonic acid-based anionic surfactants such as polyoxyethylene fatty acid amide ether sulfate or polyoxyethylene fatty acid amide ether sulfate salts (sodium polyoxyethylene fatty acid amide ether sulfate, potassium polyoxyethylene fatty acid amide ether sulfate, etc.), sodium lauroyl methyl taurate, sodium myristoyl methyl taurate, sodium palmitoyl methyl taurate, sodium stearoyl methyl taurate, sodium cocoyl methyl taurate, and sodium laurate taurate; amino acid-based anionic surfactants such as acyl methyl alanine or acyl methyl alanine salts (sodium acyl methyl alanine, potassium acyl methyl alanine, etc.), acyl sarcosine or acyl sarcosine salts (sodium acyl sarcosine, potassium acyl sarcosine, etc.); carboxylic acid-based anionic surfactants such as polyoxyethylene alkyl ether acetate or polyoxyethylene alkyl ether acetate salts (sodium polyoxyethylene alkyl ether acetate, potassium polyoxyethylene alkyl ether acetate, etc.), and polycarboxylic acid-type polymer surfactants;Phosphates such as lauryl phosphate, oleyl phosphate, polyoxyethylene lauryl ether phosphate, polyoxyethylene alkyl (C12-15) ether phosphate, polyoxyethylene cetyl ether phosphate, polyoxyethylene stearyl ether phosphate, dipolyoxyethylene alkyl (C12-15) ether phosphate, tripolyoxyethylene alkyl (C12-15) ether phosphate, polyoxyethylene oleyl ether phosphate, and polyoxyethylene coconut oil fatty acid monoethanolamide phosphate, which are salts of sodium, potassium, ammonium, triethanolamine, etc.; deoxycholic acid and its salts (sodium deoxycholate, etc.), cholic acid and its salts (sodium cholate, etc.), etc.;

[0023] Examples of the anionic surfactant having an alkylbenzenesulfonic acid skeleton include anionic surfactants having an alkylbenzenesulfonic acid skeleton represented by the following formula [1]: (In the above general formula [1], X represents a hydrogen atom or an alkali metal, and R １ , R ２ , R ３ , R ４ and R ５ is an optional substituent, and R １ , R ２、 R ３ , R ４ and R ５ At least one of R represents a linear or branched alkyl group having 1 to 30 carbon atoms. １ , R ２ , R ４ and R ５ are each independently a hydrogen atom or a linear or branched alkyl group having 4 to 20 carbon atoms, and more preferably a hydrogen atom. ３ is preferably a linear or branched alkyl group having 1 to 30 carbon atoms, and more preferably a linear or branched alkyl group having 4 to 20 carbon atoms. １ , R ２ , R ４ and R ５ is a hydrogen atom, and R３ is a linear or branched alkyl group having 1 to 30 carbon atoms. １ , R ２ , R ４ and R ５ is a hydrogen atom, and R ３ is a straight or branched alkyl group having 4 to 20 carbon atoms.

[0024] Examples of the alkanesulfonic acid or alkanesulfonate include alkanesulfonic acids represented by the following formula [2] and formula [3]. Formula [2] In the formula, X represents a hydrogen atom or an alkali metal; １１ represents a linear, branched, or cyclic alkyl or alkenyl group having 4 to 30 carbon atoms, preferably a linear or branched alkyl or alkenyl group having 8 to 20 carbon atoms, and more preferably a linear or branched alkyl group having 10 to 18 carbon atoms. In the formula, X represents a hydrogen atom or an alkali metal; １２ represents a linear, branched, or cyclic alkyl or alkenyl group having 4 to 30 carbon atoms, preferably a linear or branched alkyl or alkenyl group having 8 to 20 carbon atoms, and more preferably a linear or branched alkyl group having 10 to 18 carbon atoms.

[0025] Examples of the naphthalenesulfonic acid or naphthalenesulfonate salt, or the alkylnaphthalenesulfonic acid or alkylnaphthalenesulfonate salt include (alkyl)naphthalenesulfonic acids represented by the following formula [4]: In the formula, X represents a hydrogen atom or an alkali metal; ２１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 20 carbon atoms, preferably a hydrogen atom or a linear or branched alkyl group having 1 to 10 carbon atoms, and more preferably a linear or branched alkyl group having 1 to 8 carbon atoms.

[0026] Examples of alkyl diphenyl ether disulfonic acids or alkyl diphenyl ether disulfonate salts include alkyl diphenyl ether disulfonic acids represented by the following formula [5]: In the formula, X represents a hydrogen atom or an alkali metal; ３１ and R ３２ each independently represents a hydrogen atom or a linear or branched alkyl or alkenyl group having 1 to 30 carbon atoms, preferably a hydrogen atom or a linear or branched alkyl or alkenyl group having 1 to 20 carbon atoms, more preferably R ３１ and R ３２ is a hydrogen atom, and the other is a linear or branched alkyl group having 6 to 20 carbon atoms.

[0027] Examples of polyoxyethylene alkyl ether sulfonic acids or polyoxyethylene alkyl ether sulfonate salts include polyoxyethylene alkyl ether sulfonic acids represented by the following formulas [6], [7], and [8]. Formula [6] In the formula, X represents a hydrogen atom or an alkali metal; ４１ represents a linear or branched alkyl group having 6 to 20 carbon atoms, preferably a linear or branched alkyl group having 8 to 15 carbon atoms, and n1 represents an integer of 1 to 12. In the formula, X represents a hydrogen atom or an alkali metal; ４２ represents a linear or branched alkyl group having 4 to 20 carbon atoms, preferably a linear or branched alkyl group having 6 to 15 carbon atoms, and n2 represents an integer of 1 to 10. In the formula, X represents a hydrogen atom or an alkali metal; ４３ represents a linear or branched alkyl group having 4 to 20 carbon atoms, preferably a linear or branched alkyl group having 6 to 15 carbon atoms; n3 represents an integer of 1 to 10;

[0028] Examples of acyl sarcosine or acyl sarcosine salts, or acyl methyl alanine or acyl methyl alanine salts include acyl sarcosines or acyl methyl alanines represented by the following formula [9]: Formula [9] In the formula, X represents a hydrogen atom or an alkali metal; ５１ represents a linear or branched alkyl or alkenyl group having 1 to 30 carbon atoms, preferably a linear or branched alkyl or alkenyl group having 5 to 30 carbon atoms, more preferably a linear or branched alkyl or alkenyl group having 5 to 20 carbon atoms, and m represents an integer of 1 to 3. In the above formulas [1] and [3] to [9], examples of the alkali metal represented by X include sodium, potassium, and lithium, with sodium being preferred.

[0029] Specific examples of anionic surfactants include sodium dodecylbenzenesulfonate, SDS (sodium lauryl sulfate), STS (sodium tridecyl sulfate), NLS (sodium N-lauroylsarcosinate), lithium dodecyl sulfate, and sodium deoxycholate.

[0030] The concentration of the anionic surfactant when the hemolyzed sample is brought into contact with the anionic surfactant is preferably 0.01% (w / v) to 2.0% (w / v), more preferably 0.02% (w / v) to 1.0% (w / v), and even more preferably 0.05% (w / v) to 0.5% (w / v).

[0031] 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.

[0032] In a first aspect of the method for detecting a substance to be measured of the present invention, a hemolyzed sample containing the substance to be measured, a solid phase carrier (e.g., magnetic particles) to which the substance to be measured is bound, and an iron-binding protein are brought into contact and reacted, and then a labeled "substance that binds to the substance to be measured" is reacted, and B / F separation (Bound / Free separation) is performed to detect the label bound to the solid phase.

[0033] 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 substance to be measured is bound. The substance to be measured can be bound to the particles 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.

[0034] The second reagent is a reaction buffer containing an iron-binding protein. 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.

[0035] The third reagent is a reagent containing a labeled "substance that binds to the analyte." The third reagent preferably contains a labeled "substance that binds to the analyte," a buffer, and the like. Examples of the buffer include those described below in the section "Composition of the Present Invention."

[0036] 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>.

[0037] 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.

[0038] 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.

[0039] (2) Detection Method The second reagent and the specimen (i.e., hemolyzed sample containing the substance to be measured) 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 fifth reagent are added, and after the reaction, the amount of luminescence is measured.

[0040] In a second aspect of the method for detecting a substance to be measured of the present invention, a hemolyzed sample containing the substance to be measured, a solid phase carrier (e.g., magnetic particles) to which the substance to be measured is bound, an iron-binding protein, and a labeled "substance that binds to the substance to be measured" are reacted, and B / F separation (Bound / Free separation) is performed to detect the label bound to the solid phase (the label that binds to the complex produced by the reaction).

[0041] In a third aspect of the method for detecting a substance to be measured of the present invention, a hemolyzed sample containing the substance to be measured is contacted with a solid phase carrier (e.g., magnetic particles) to which the substance to be measured is bound. Then, a labeled "substance that binds to the substance to be measured" and an iron-binding protein are added to the mixture and reacted, followed by B / F separation (bound / free separation) to detect the label bound to the solid phase (the label that binds to the complex produced by the reaction).

[0042] In a fourth aspect of the method for detecting a substance to be measured of the present invention, a hemolyzed sample containing the substance to be measured is contacted with a solid phase carrier (e.g., magnetic particles) to which a substance that binds to the substance to be measured is bound, and an iron-binding protein to cause a reaction, and then a labeled substance to be measured 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).

[0043] In a fifth aspect of the method for detecting a substance to be measured of the present invention, a hemolyzed sample containing the substance to be measured, a solid phase carrier (e.g., magnetic particles) to which a substance that binds to the substance to be measured is bound, an iron-binding protein, and a labeled substance to be measured 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).

[0044] In a sixth aspect of the method for detecting a substance to be measured of the present invention, a hemolyzed sample containing the substance to be measured is brought into contact with a solid phase carrier (e.g., magnetic particles) to which a substance that binds to the substance to be measured is bound, and a reaction is then allowed to occur. Thereafter, a labeled substance to be measured and an iron-binding protein 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 that binds to the complex produced by the reaction).

[0045] A seventh aspect of the method for detecting a substance to be measured of the present invention is a case in which, in the fourth to sixth aspects of the method for detecting a substance to be measured of the present invention, the solid phase carrier to which the substance that binds to the substance to be measured is bound is indirectly immobilized to the solid phase carrier via an anti-IgG antibody.

[0046] In an eighth aspect of the method for detecting a substance to be measured of the present invention, a hemolyzed sample containing the substance to be measured is contacted with a substance that binds to the substance to be measured and an iron-binding protein to cause a reaction, followed by reaction with a labeled antibody that recognizes a complex between the "substance to be measured" and the "substance that binds to the substance to be measured," followed by B / F separation (Bound / Free separation) to detect the label bound to the solid phase.

[0047] In the eighth aspect, the "substance that binds to the analyte" may or may not be immobilized. When a non-immobilized "substance that binds to the analyte" is used, the "substance that binds to the analyte" may be labeled with a polyanionic substance, if desired. Examples of methods for B / F separation (bound / free separation) include electrophoresis 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.

[0048] Of the above-mentioned embodiments, from the viewpoint of the dissociation reaction of the steroid hormone by the hydrogen halide salt or 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.

[0049] <Composition> According to the present invention, a composition for detecting a substance to be measured in a hemolyzed sample is provided, which contains an iron-binding protein. The iron-binding protein is preferably one or more selected from the group consisting of lactoferrin, transferrin, and ferritin, more preferably lactoferrin or transferrin, and particularly preferably lactoferrin. The composition of the present invention is preferably used in the method for detecting a substance to be measured of the present invention and the method for reducing the influence of hemolysis of the present invention.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] Examples of the stabilizer include known chelating agents and protease inhibitors.

[0055] 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.

[0056] Examples of the adsorption inhibitor include surfactants and inhibitors of peptide adsorption such as albumin (e.g., bovine serum albumin).

[0057] Examples of preservatives include salicylic acid and benzoic acid.

[0058] Examples of the solvent include water and water-soluble organic solvents.

[0059] <Measurand substance detection kit> A first aspect of the analyte substance detection kit is an analyte substance measurement kit comprising: an iron-binding protein; a solid-phase analyte substance; and a labeled analyte substance-binding substance.

[0060] A second embodiment of the analyte detection kit is an analyte measurement kit comprising an iron-binding protein, an immobilized analyte-binding substance, and a labeled analyte.

[0061] A third aspect of the analyte detection kit is an analyte measurement kit comprising: an iron-binding protein; an analyte-binding substance; and a labeled binding substance capable of binding to a complex of the analyte and the analyte-binding substance. In the third aspect of the analyte detection kit described above, the analyte-binding substance may or may not be immobilized.

[0062] The above-mentioned kit for measuring a substance to be measured of the present invention may further contain heparin or a heparin salt, such as sodium heparin or potassium heparin.

[0063] The above-mentioned kit for measuring a substance to be measured of the present invention may further contain an anionic surfactant.

[0064] The anionic surfactant is not particularly limited, but from the viewpoint of easily causing hemolysis even at low concentrations, examples thereof include anionic surfactants having an alkylbenzenesulfonic acid skeleton, alkanesulfonic acids or alkanesulfonate salts (sodium alkanesulfonate, potassium alkanesulfonate, etc.), naphthalenesulfonic acids or naphthalenesulfonate salts (sodium alkanesulfonate, potassium alkanesulfonate, etc.), alkylnaphthalenesulfonic acids or alkylnaphthalenesulfonate salts (sodium alkylnaphthalenesulfonate, potassium alkylnaphthalenesulfonate, etc.), alkyldiphenyletherdisulfonic acids or alkyldiphenyletherdisulfonate salts (sodium alkyldiphenyletherdisulfonate, potassium alkyldiphenyletherdisulfonate, etc.), polyoxyethylene alkylethersulfonic acids or polyoxyethylene alkylethersulfonate salts (sodium polyoxyethylene alkylethersulfonate, potassium polyoxyethylene alkylethersulfonate, etc.), polyoxyethylene alkylethersulfonic acids or polyoxyethylene alkylethersulfonate salts (sodium polyoxyethylene alkylethersulfonate, potassium polyoxyethylene alkylethersulfonate, etc.), polyoxyethylene alkylethersulfonic acids or polyoxyethylene alkylethersulfonates ... sulfonic acid-based anionic surfactants such as polyoxyethylene fatty acid amide ether sulfate or polyoxyethylene fatty acid amide ether sulfate salts (sodium polyoxyethylene fatty acid amide ether sulfate, potassium polyoxyethylene fatty acid amide ether sulfate, etc.), sodium lauroyl methyl taurate, sodium myristoyl methyl taurate, sodium palmitoyl methyl taurate, sodium stearoyl methyl taurate, sodium cocoyl methyl taurate, and sodium laurate taurate; amino acid-based anionic surfactants such as acyl methyl alanine or acyl methyl alanine salts (sodium acyl methyl alanine, potassium acyl methyl alanine, etc.), acyl sarcosine or acyl sarcosine salts (sodium acyl sarcosine, potassium acyl sarcosine, etc.); carboxylic acid-based anionic surfactants such as polyoxyethylene alkyl ether acetate or polyoxyethylene alkyl ether acetate salts (sodium polyoxyethylene alkyl ether acetate, potassium polyoxyethylene alkyl ether acetate, etc.), and polycarboxylic acid-type polymer surfactants;Phosphates such as lauryl phosphate, oleyl phosphate, polyoxyethylene lauryl ether phosphate, polyoxyethylene alkyl (C12-15) ether phosphate, polyoxyethylene cetyl ether phosphate, polyoxyethylene stearyl ether phosphate, dipolyoxyethylene alkyl (C12-15) ether phosphate, tripolyoxyethylene alkyl (C12-15) ether phosphate, polyoxyethylene oleyl ether phosphate, and polyoxyethylene coconut oil fatty acid monoethanolamide phosphate, which are salts of sodium, potassium, ammonium, triethanolamine, etc.; deoxycholic acid and its salts (sodium deoxycholate, etc.), cholic acid and its salts (sodium cholate, etc.), etc.;

[0065] Examples of the anionic surfactant having an alkylbenzenesulfonic acid skeleton include anionic surfactants having an alkylbenzenesulfonic acid skeleton represented by the following formula [1]: (In the above general formula [1], X represents a hydrogen atom or an alkali metal, and R １ , R ２ , R ３ , R ４ and R ５ is an optional substituent, and R １ , R ２ , R ３ , R ４ and R ５ At least one of R represents a linear or branched alkyl group having 1 to 30 carbon atoms. １ , R ２ , R ４ and R ５ are each independently a hydrogen atom or a linear or branched alkyl group having 4 to 20 carbon atoms, and more preferably a hydrogen atom. ３ is preferably a linear or branched alkyl group having 1 to 30 carbon atoms, and more preferably a linear or branched alkyl group having 4 to 20 carbon atoms. １ , R ２ , R ４ and R ５is a hydrogen atom, and R ３ is a linear or branched alkyl group having 1 to 30 carbon atoms. １ , R ２、 R ４ and R ５ is a hydrogen atom, and R ３ is a straight or branched alkyl group having 4 to 20 carbon atoms.

[0066] Examples of the alkanesulfonic acid or alkanesulfonate include alkanesulfonic acids represented by the following formula [2] and formula [3]. Formula [2] In the formula, X represents a hydrogen atom or an alkali metal; １１ represents a linear, branched, or cyclic alkyl or alkenyl group having 4 to 30 carbon atoms, preferably a linear or branched alkyl or alkenyl group having 8 to 20 carbon atoms, and more preferably a linear or branched alkyl group having 10 to 18 carbon atoms. In the formula, X represents a hydrogen atom or an alkali metal; １２ represents a linear, branched, or cyclic alkyl or alkenyl group having 4 to 30 carbon atoms, preferably a linear or branched alkyl or alkenyl group having 8 to 20 carbon atoms, and more preferably a linear or branched alkyl group having 10 to 18 carbon atoms.

[0067] Examples of the naphthalenesulfonic acid or naphthalenesulfonate salt, or the alkylnaphthalenesulfonic acid or alkylnaphthalenesulfonate salt include (alkyl)naphthalenesulfonic acids represented by the following formula [4]: In the formula, X represents a hydrogen atom or an alkali metal; ２１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 20 carbon atoms, preferably a hydrogen atom or a linear or branched alkyl group having 1 to 10 carbon atoms, and more preferably a linear or branched alkyl group having 1 to 8 carbon atoms.

[0068] Examples of alkyl diphenyl ether disulfonic acids or alkyl diphenyl ether disulfonate salts include alkyl diphenyl ether disulfonic acids represented by the following formula [5]: In the formula, X represents a hydrogen atom or an alkali metal; ３１ and R ３２ each independently represents a hydrogen atom or a linear or branched alkyl or alkenyl group having 1 to 30 carbon atoms, preferably a hydrogen atom or a linear or branched alkyl or alkenyl group having 1 to 20 carbon atoms, more preferably R ３１ and R ３２ is a hydrogen atom, and the other is a linear or branched alkyl group having 6 to 20 carbon atoms.

[0069] Examples of polyoxyethylene alkyl ether sulfonic acids or polyoxyethylene alkyl ether sulfonate salts include polyoxyethylene alkyl ether sulfonic acids represented by the following formulas [6], [7], and [8]. Formula [6] In the formula, X represents a hydrogen atom or an alkali metal; ４１ represents a linear or branched alkyl group having 6 to 20 carbon atoms, preferably a linear or branched alkyl group having 8 to 15 carbon atoms, and n1 represents an integer of 1 to 12. In the formula, X represents a hydrogen atom or an alkali metal; ４２ represents a linear or branched alkyl group having 4 to 20 carbon atoms, preferably a linear or branched alkyl group having 6 to 15 carbon atoms, and n2 represents an integer of 1 to 10. In the formula, X represents a hydrogen atom or an alkali metal; ４３ represents a linear or branched alkyl group having 4 to 20 carbon atoms, preferably a linear or branched alkyl group having 6 to 15 carbon atoms; n3 represents an integer of 1 to 10;

[0070] Examples of acyl sarcosine or acyl sarcosine salts, or acyl methyl alanine or acyl methyl alanine salts include acyl sarcosines or acyl methyl alanines represented by the following formula [9]: Formula [9] In the formula, X represents a hydrogen atom or an alkali metal; ５１ represents a linear or branched alkyl or alkenyl group having 1 to 30 carbon atoms, preferably a linear or branched alkyl or alkenyl group having 5 to 30 carbon atoms, more preferably a linear or branched alkyl or alkenyl group having 5 to 20 carbon atoms, and m represents an integer of 1 to 3. In the above formulas [1] and [3] to [9], examples of the alkali metal represented by X include sodium, potassium, and lithium, with sodium being preferred.

[0071] Specific examples of anionic surfactants include sodium dodecylbenzenesulfonate, SDS (sodium lauryl sulfate), STS (sodium tridecyl sulfate), NLS (sodium N-lauroylsarcosinate), lithium dodecyl sulfate, and sodium deoxycholate.

[0072] The substance that binds to the analyte may be an antibody that binds to the analyte, a peptide such as a cyclic peptide, an aptamer, etc. Antibodies that bind to the analyte may include Fab, Fab', F(ab')2, Fv, Fd, single-chain Fv (scFv), disulfide-bonded Fv (sdFv), V L , V H , Fv-clasp, diabody ((V L -V H )2 or (V H -V L )2), triabody (trivalent antibody), tetrabody (tetravalent antibody), minibody ((scFV-CH3)2), IgG-delta-CH2, scFv-Fc, (scFv)2-Fc fragment, or a modified form of an antibody.

[0073] Examples of solid-phase carriers for immobilizing the analyte or the analyte-binding substance include insoluble solid-phase carriers used in conventional immunoassays. Specifically, substrates used in conventional protein immobilization methods, such as slide glasses, ELISA plates, microtiter plates, beads (magnetic beads, etc.), microparticles (latex particles, etc.), filters, tubes, films, and membranes, can be used. Materials for the substrate include synthetic polymer compounds 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 (porous glass, etc.), ground glass, silicon, ceramics, alumina, silica gel, activated carbon, and metal oxides.

[0074] The method for immobilizing the substance to be measured or the substance binding to the substance to be measured on the solid phase carrier is not particularly limited, and known methods such as chemical bonding methods (methods of immobilization by covalent bonding) and physical adsorption methods can be applied.

[0075] As the labeling substance used to label the substance to be measured or the substance to be measured, 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.

[0076] 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;

[0077] Fluorescent substances such as HiLyte 647 (manufactured by AhaSpec), 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, and L-012;

[0078] 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, and 2,6-di-t-butyl-α-(3,5-di-t-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxyl;

[0079] 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);

[0080] 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

[0081] The labeling substance used to bind to the analyte or label the analyte is preferably one used for detection using iron ions as a catalyst, and is preferably an enzyme such as peroxidase or HPR, or a luminescent substance (substrate) such as luciferin or L-012, with enzymes being more preferred.

[0082] The method for binding a labeled substance to an analyte-binding substance or an analyte can be carried out by appropriately utilizing a labeling method used in conventional immunoassays, etc., and examples thereof include those described in "Medical Chemistry Experiment Lectures," Vol. 8, edited by Yamamura Yuichi, 1st Edition, Nakayama Shoten, 1971; "Illustrated Fluorescent Antibodies," by Kawao Akira, 1st Edition, Soft Sciences Co., Ltd., 1983; and "Enzyme Immunoassay," edited by Ishikawa Eiji, Kawai Tadashi, and Muroi Kiyoshi, 2nd Edition, Igaku Shoin, 1982. The labeled substance may be directly bound to the analyte-binding substance or the analyte, or may be indirectly bound to the analyte-binding substance or the analyte via a suitable spacer (e.g., via a combination of one or several amino acids, one or several amino acids and a linker, or avidins (e.g., streptavidin, tamavidin) and a substance having affinity for biotin).

[0083] 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.

[0084] The kit of the present invention may further include a standard sample. The standard sample is preferably a sample containing a known concentration of the substance to be measured. Standard samples are used as calibrators or controls for creating calibration curves showing the correlation between a known amount (concentration) of the substance to be measured contained in the standard sample and measurements obtained by various analytical methods, for confirming the effectiveness of accuracy control and calibration of analytical instruments, and for investigating the stability of quantitative analysis over time. Examples of the measured values ​​include absorbance, change in absorbance, amount of transmitted light, change in transmitted light, luminescence, and change in luminescence. Furthermore, conversion values ​​such as the amount and concentration of the substance to be measured in a sample calculated from the measured value may also be referred to as measured values ​​in this specification.

[0085] The kit of the present invention may further include an instruction manual, etc. The instruction manual may describe the components of the reagent of the present invention, the operating procedure and principles of the method of detecting a substance to be measured of the present invention, etc.

[0086] 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.

[0087] 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 (Fujifilm Wako Pure Chemical Industries, Ltd.) and 4 mL of acetic acid were added, and 9 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-phenyl-1H-indol-3-yl)methanamine (Compound 2) (yield: 11.7 g, 90%).

[0088] 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%).

[0089]

[0090] <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)

[0091]

[0092] Example 1 Evaluation of Lactoferrin's Reduction of Hemolytic Effect (1) Preparation of Reagents The constituent reagents required for the measurement were prepared using the following raw reagent materials: MES (2-morpholinoethanesulfonic acid monohydrate) (manufactured by Dojindo Laboratories, Inc.), 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, Inc.), boric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), L-012 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). (Fujifilm Wako Pure Chemical Industries, Ltd.), phosphoric acid (Fujifilm Wako Pure Chemical Industries, Ltd.), EDTA-2Na (Dojindo Laboratories, Ltd.), hydrogen peroxide (Fujifilm Wako Pure Chemical Industries, Ltd.), heparin sodium (Fujifilm Wako Pure Chemical Industries, Ltd.), lactoferrin (Fujifilm Wako Pure Chemical Industries, Ltd.), transferrin (Fujifilm Wako Pure Chemical Industries, Ltd.), Neopelex G-65 (Kao Corporation).

[0093] (i) "First Reagent": Antigen-Solidified Magnetic Particle-Containing Reagent. The carboxyl groups of modified testosterone, TES-19CME (Shinsei Chemical Co., Ltd.), were activated with commercially available N-hydroxysuccinimide (Fujifilm Wako Pure Chemical Industries, Ltd.) and WSC (water-soluble carbodiimide) (Dojindo Laboratories, Inc.). The resulting mixture was then reacted with a BSA (bovine serum albumin) solution at 4°C for 18-24 hours and purified using a PD-10 column (Cytiva) to prepare BSA-bound testosterone. 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 using a neodymium magnet and removal of the supernatant, yielding magnetic particles bearing carboxyl groups. Next, the carboxyl groups were activated with commercially available N-hydroxysuccinimide (WSC), and then BSA-bound testosterone was reacted at 26°C for 12 to 16 hours. The particles were collected with a neodymium magnet, and the supernatant was removed to prepare testosterone-immobilized magnetic particles. The first reagent was composed of the following: "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.

[0094] (ii) "Second Reagent" Reaction Buffer: 40 mM MES (pH 5.5), 150 mM sodium chloride, 2.0% (w / v) BSA, 0.25% (w / v) heparin sodium, 0.62% (w / v) Neoperex (equivalent to 0.4% sodium dodecylbenzenesulfonate as the main component), 0% or 3.0% (w / v) lactoferrin, 0.5 mM 3-((1H-imidazol-1-yl)methyl)-2-phenyl-1H-indole hydrochloride

[0095] (iii) "Third Reagent" Labeled Antibody-Containing Reagent Anti-testosterone antibodies were produced by the following method. Testosterone, an immunogen, was injected into a rabbit as an immunization animal to immunize the animal, and lymphocytes were then recovered. Antibody gene cassettes were obtained from the resulting lymphocytes by single PCR, and these gene cassettes were each 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 to produce anti-testosterone antibodies. The produced anti-testosterone antibodies were reduced with cysteamine hydrochloride (Sigma-Aldrich Japan), and IgG was then separated using a column (diameter: 1.5 cm x length: approximately 40 cm) packed with G-25 Superfine (Cytiva). On the other hand, peroxidase (POD) (Roche Diagnostics) was maleimidated using the maleimidation reagent Sulfo-KMUS (Dojindo Laboratories, Ltd.), 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 on a Sephacryl S-100HR column to produce a POD-labeled anti-testosterone antibody. This was used to prepare a third reagent consisting of the following composition: "Third Reagent": 20 pmol / L POD-labeled anti-testosterone antibody, 50 mM MES (pH 5.5), 150 mM sodium chloride, 2.0% BSA

[0096] (iv) "Fourth Reagent" Luminescence Reagent: 50 mM TAPSO, 0.9% (w / v) boric acid, 0.50 mM L-012, 200 mM thiazolephenol

[0097] (v) “Fifth Reagent” “Fifth Reagent”: 68 μL / L phosphoric acid, 4mM EDTA-2Na, 335 μL / L hydrogen peroxide

[0098] (vi) Other Reagents As samples for the calibration curve, diluted samples of testosterone (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were prepared at 0.07 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.

[0099] (2) Preparation of hemolyzed hemoglobin samples Hemolyzed hemoglobin and blank solutions included with Interference Check A Plus (Sysmex Corporation) were prepared and added to serum samples to prepare 0, 100, 200, 300, 400, and 500 mg / dL hemolyzed hemoglobin samples.

[0100] (3) 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 Accura Seed automated chemiluminescent enzyme immunoassay system (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 collected using a neodymium magnet, and the supernatant was removed. Subsequently, 50 μL of the second reagent and 25 μL of hemolyzed hemoglobin samples with concentrations of 0, 100, 200, 300, 400, and 500 mg / dL were added, stirred, and heated at 37°C for 3 minutes. After heating, 50 μL of the third reagent was added and heated at 37°C for 3 minutes. After heating, the solution was magnetically collected using a neodymium magnet, and the reagents other than the magnetic particles were removed. The solution was then washed three times with washing solution. After washing, 100 μL of the fourth reagent and 100 μL of the fifth reagent were added, and the luminescence intensity was measured after 20 seconds of reaction at 37° C. A calibration curve showing the relationship between the amount of testosterone and the amount of luminescence was created from the luminescence intensity when each calibration curve sample was measured, and the amount of testosterone in the hemolyzed sample was calculated.

[0101] Figure 1 shows the relationship between the hemolyzed hemoglobin concentration in the second reagent and the measured value (the ratio of the measured value when the measured value without hemolyzed hemoglobin is set to 100%). When lactoferrin is 0%, the measured value decreases according to the hemolyzed hemoglobin concentration, confirming that hemolysis affects the measured value. On the other hand, when lactoferrin is 3% (w / v), the decrease in the measured value is suppressed, indicating that lactoferrin reduces the influence of hemolysis.

[0102] Example 2: Evaluation of the Effect of Lactoferrin on Concentration-Dependent Reduction of Hemolysis A luminescent reagent was prepared and evaluated in the same manner as in Example 1, except as follows. Example 1 (ii) The lactoferrin concentration in the "second reagent" was adjusted to 1%, 2%, 3%, 4%, or 5.0% (w / v), and measurements were performed using 0 and 500 mg / dL hemolyzed hemoglobin samples, and the amount of testosterone in the sample was calculated. Figure 2 shows the relationship between the lactoferrin concentration in the second reagent and the measured value (measurement ratio when the measured value without hemolyzed hemoglobin is set to 100%). The decrease in the measured value was suppressed in a lactoferrin concentration-dependent manner, approaching the measured value (100%) of the 0 mg / dL hemolyzed hemoglobin sample (no added sample). In other words, it was found that the hemolysis effect was reduced in a lactoferrin concentration-dependent manner.

[0103] Example 3: Evaluation of the Effect of Transferrin on Concentration-Dependent Reduction of Hemolytic Influence A luminescent reagent was prepared and evaluated in the same manner as in Example 2, with the following exceptions. Measurements were conducted by changing the lactoferrin in the "second reagent" of Example 2 to 0%, 1%, or 5% (w / v) transferrin, and the amount of testosterone in the sample was calculated. Figure 3 shows the relationship between the transferrin concentration in the second reagent and the measured value (measurement value ratio when the measurement value without hemolyzed hemoglobin was set to 100%). The reduction in the measured value was suppressed in a transferrin concentration-dependent manner, approaching the measurement value of a sample with 0 mg / dL hemolyzed hemoglobin. In other words, it was found that transferrin also reduces the hemolytic influence in a concentration-dependent manner.

[0104] Example 4: Evaluation of the Effect of Neopelex on Hemolysis Reduction. A luminescent reagent was prepared and evaluated using the same method as in Example 1, except for the following: Measurements were performed using the "second reagent" of Example 1, with the amount of Neopelex changed to 0% or 0.62% (w / v), and the amount of testosterone in the sample was calculated. Figure 4 shows the relationship between the lactoferrin concentration and the Neopelex concentration in the second reagent and the measured values ​​(the ratio of the measured values ​​under each condition, with the measured value without hemolyzed hemoglobin set to 100%). The reduction rate of the 500 mg / dL hemolyzed hemoglobin sample measured value relative to the 0 mg / dL hemolyzed hemoglobin sample measured value (the measured value without hemolyzed hemoglobin) was found to be greater with 0.62% (w / v) Neopelex compared to 0% Neopelex (no Neopelex added). This indicates that hemolysis occurs due to the anionic surfactant Neopelex (sodium dodecylbenzenesulfonate). Lactoferrin was also found to reduce the hemolytic effects caused by anionic surfactants.

[0105] Example 5: Evaluation of Reduction of Hemolytic Effects by Heparin Sodium A luminescent reagent was prepared and evaluated in the same manner as in Example 1, except as noted below. Measurements were conducted by changing the heparin sodium in the "second reagent" of Example 1 to 0.25%, 0.50%, 0.75%, and 1.00% (w / v), and testosterone in the sample was calculated. Figure 5 shows the relationship between the heparin concentration in the second reagent and the measured value (measurement value ratio when the measurement value without hemolyzed hemoglobin was set to 100%). The decrease in the measured value was suppressed in a heparin sodium concentration-dependent manner, approaching the measured value of a 0 mg / dL hemolyzed hemoglobin sample. In other words, it was confirmed that the use of heparin sodium further reduced the hemolytic effects compared to lactoferrin alone.

[0106] Example 6: Evaluation of the Effect of Lactoferrin Concentration-Dependent Reduction of Hemolysis in the Absence of Added Heparin Sodium. A luminescent reagent was prepared and evaluated in the same manner as in Example 2, except for the following: Example 2 (ii) The amount of testosterone in the sample was calculated, including the point where the heparin sodium concentration in the "second reagent" was 0% (w / v) and the lactoferrin concentration was 0% (w / v). Figure 6 shows the relationship between the lactoferrin concentration in the second reagent and the measured value (measurement value ratio when the measured value without added hemolyzed hemoglobin is taken as 100%). It was confirmed that the decrease in the measured value was suppressed in a lactoferrin concentration-dependent manner, even when heparin sodium was not added.

[0107] Example 7: Evaluation of the effect of anionic surfactants on reducing the hemolytic effect of lactoferrin. A luminescent reagent was prepared and evaluated in the same manner as in Example 1, with the following exceptions: Neopelex in the "second reagent" of Example 1 was replaced with various anionic surfactants at 0.50% (w / v), and the amount of testosterone in the sample was calculated at lactoferrin concentrations of 0% and 3% (w / v). The anionic surfactants used were as follows: Softanol MES-5 (polyoxyethylene alkyl ether sulfosuccinic acid half ester, manufactured by Nippon Shokubai Co., Ltd.), Sannol LMT-1430 (polyoxyethylene alkyl ether sulfate sodium salt, manufactured by Lion Corporation), Enerdicol L-30AN (N-lauroyl-N-methyl β-alanine sodium salt, manufactured by Lion Corporation), Pelex NBL (sodium alkylnaphthalenesulfonate, manufactured by Kao Corporation), Pelex SS-H (sodium alkyldiphenyletherdisulfonate, manufactured by Kao Corporation), Latemul PS (sodium alkanesulfonate, manufactured by Kao Corporation), SDS (sodium lauryl sulfate, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

[0108] Figure 7 shows the relationship between the anionic surfactant type and lactoferrin concentration in the second reagent and the measured values ​​(the ratio of the measured values ​​under each condition to the measured value without hemolyzed hemoglobin, taken as 100%). As with Neopelex, Figure 7 shows that the measured values ​​decreased with each anionic surfactant. This indicates that the hemolytic effect is exacerbated by the anionic surfactant. Furthermore, the hemolytic effect was suppressed by the addition of lactoferrin, regardless of the anionic surfactant type. This indicates that lactoferrin suppresses the hemolytic effect.

Claims

1. A method for detecting a substance to be measured, comprising contacting an iron-binding protein with a hemolyzed sample and detecting the substance to be measured in the hemolyzed sample.

2. The method of claim 1, wherein the iron-binding protein is one or more selected from the group consisting of lactoferrin, transferrin, and ferritin.

3. The method according to claim 1, wherein the concentration of the iron-binding protein when contacting the iron-binding protein with the hemolyzed sample is 0.1% (w / v) or more.

4. The method of claim 1, wherein the detection is an iron ion-catalyzed detection.

5. The method according to claim 1, wherein the detection is carried out by using a substance that binds to the analyte bound to a label, or by using a substance that competes with the analyte bound to a label.

6. The method of claim 1, wherein said detecting comprises contacting said hemolyzed sample with heparin or a heparin salt.

7. The method according to claim 6, wherein the concentration of heparin or heparin salt when the hemolyzed sample is brought into contact with the heparin or heparin salt is 0.1% (w / v) or more and 3% (w / v) or less.

8. The method of claim 1, wherein said detecting comprises contacting said hemolyzed sample with an anionic surfactant.

9. A method for reducing the effects of hemolysis, comprising contacting an iron-binding protein with a hemolyzed sample and detecting a substance to be measured in the hemolyzed sample.

10. A composition for detecting a substance to be measured in a hemolyzed sample, comprising an iron-binding protein.

11. The composition according to claim 10, wherein the iron-binding protein is one or more selected from the group consisting of lactoferrin, transferrin, and ferritin.

12. A composition according to claim 10 for use in a method according to any one of claims 1 to 9.

13. A kit for measuring a substance to be measured, comprising an iron-binding protein, a solid-phased substance to be measured, and a labeled substance that binds to the substance to be measured.

14. A kit for measuring a substance to be measured, comprising an iron-binding protein, a solid-phase substance to be measured that binds to the substance to be measured, and a labeled substance to be measured.

15. A kit for measuring a substance to be measured, comprising: an iron-binding protein; a substance to be measured that binds to the substance to be measured; and a labeled binding substance that can bind to a complex of the substance to be measured and the substance to be measured that binds to the substance to be measured.

16. The kit for measuring a substance to be measured according to claim 15, wherein the substance to be measured that binds to the substance is immobilized.

17. The kit for measuring a substance to be measured according to any one of claims 13 to 16, further comprising heparin or a heparin salt.

18. The kit for measuring a substance to be measured according to any one of claims 13 to 16, further comprising an anionic surfactant.

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