Immunoassay method and cleaning agent

Abstract

To provide a thyroglobulin immunoassay measurement method and cleansing agent that reduce an impact of interference of an anti-thyroglobulin antibody, and can measure an accurate amount of thyroglobulin by a simple operation.SOLUTION: The present invention is an immunoassay measurement method that measures thyroglobulin in a sample, and the immunoassay measurement method includes the steps and the like of mixing the sample with a solid-phase carrier body (a), obtaining a mixture containing a composite body (J1) of the solid-phase carrier body (a) and the thyroglobulin, and thereafter cleaning the composite body (J1) by a cleansing agent (C). The solid-phase carrier body (a) is a magnetic particle that contains superparamagnetic metal oxide of a specific particle size at 60 to 95 wet.%, and the cleansing agent (C) contains a halide of an alkaline metal and / or a halide of an alkaline earth metal, as well as a nonionic surfactant at specific concentration, in which ph of the cleansing agent is 6.5 to 8.0.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to an immunoassay method and a cleaning agent. [Background technology]

[0002] Thyroglobulin (hereinafter sometimes abbreviated as "Tg") is a glycoprotein with a molecular weight of 660,000 that is produced only by thyroid follicular cells. Biosynthesized Tg is released into the follicular lumen. During this process, iodine molecules bind to the tyrosine group in the Tg molecule under the action of peroxidase, leading to the synthesis of thyroid hormones. The Tg in the follicular lumen is taken up again by follicular cells, where it is broken down and thyroid hormones are released. This process is also activated by the action of thyroid-stimulating hormone (TSH). Therefore, under normal conditions, only a very small amount of Tg itself is released into the bloodstream, and it is known that Tg release into the bloodstream indicates some kind of thyroid abnormality. Transgenic thyroid gland (Tg) is known as a highly organ-specific and useful marker for various thyroid diseases. In particular, serum Tg is used as a postoperative evaluation of differentiated thyroid carcinoma and as a marker to determine the presence or absence of postoperative recurrence and metastasis. It is also useful as an indicator of treatment effectiveness and remission in Graves' disease, and for determining the subtype of congenital hypothyroidism. Furthermore, when combined with imaging studies, it has been suggested that Tg may be useful for the preoperative diagnosis of nodular goiter and for differentiating between benign thyroid diseases and malignant tumors.

[0003] However, if a subject tests positive for anti-thyroglobulin antibodies (hereinafter sometimes abbreviated as "TgAb"), the Tg concentration may actually be high, but the measurement may show a low value due to measurement errors. For example, in samples from patients with thyroid cancer, Hashimoto's disease, and other autoimmune diseases (such as Graves' disease) who may be TgAb positive, there was a problem in accurately measuring the amount of Tg. To solve the above problems, a technique for pre-treating the measurement sample is known (Patent Document 1). On the other hand, there was a need for a measurement method that did not require a pretreatment step in order to perform measurements quickly and easily. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Patent No. 7007278 [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention aims to provide a thyroglobulin immunoassay method and a washing agent that reduce the effects of interference from anti-thyroglobulin antibodies and enable accurate measurement of thyroglobulin levels with simple procedures. [Means for solving the problem]

[0006] The inventors of this invention have diligently studied and developed the present invention in order to achieve the above objective. That is, the present invention is An immunoassay method for measuring the concentration of thyroglobulin in a sample, The aforementioned immunoassay method, The steps include: mixing the sample with a solid support (a) to obtain a mixture containing a composite (J1) of the solid support (a) and thyroglobulin, and then washing the composite (J1) with a cleaning agent (C); The process includes, in this order, mixing the aforementioned complex (J1) with a substance labeled with a labeling substance (b) and a substance (F) that specifically binds to thyroglobulin, to obtain a complex (J2) of a solid support (a), thyroglobulin, and substance (F). The solid support (a) is a magnetic particle containing a superparamagnetic metal oxide with a volume-average particle diameter of 1 to 20 nm. The weight percentage of the superparamagnetic metal oxide is 60 to 95% by weight, based on the weight of the solid support (a). The cleaning agent (C) contains an alkali metal halide and / or an alkaline earth metal halide, and a nonionic surfactant. The total weight ratio of the alkali metal halides and alkaline earth metal halides is 0.010 to 0.50% by weight, based on the weight of the cleaning agent (C). The weight percentage of the nonionic surfactant is 0.001 to 1% by weight, based on the weight of the detergent (C). An immunoassay method wherein the pH of the detergent (C) is 6.5 to 8.0; a detergent used in the immunoassay method. [Effects of the Invention]

[0007] The immunoassay method using the cleaning agent of the present invention has the advantage of enabling accurate measurement of thyroglobulin levels with simple operation, even when the substance to be measured is a sample containing anti-thyroglobulin antibodies. [Modes for carrying out the invention]

[0008] The present invention relates to an immunoassay method for measuring the concentration of thyroglobulin in a sample, The aforementioned immunoassay method is, The steps include: mixing the sample with the solid support to obtain a mixture containing a composite (J1) of the solid support (a) and thyroglobulin, and then washing the composite (J1) with a cleaning agent (C); The process includes, in this order, mixing the aforementioned complex (J1) with a substance (F) labeled with a labeling substance (b) that specifically binds to thyroglobulin, to obtain a complex (J2) of a solid support (a), thyroglobulin, and substance (F).

[0009] The immunoassay method of the present invention is The steps include: mixing the sample with a solid support (a) to obtain a mixture containing a composite (J1) of the solid support (a) and thyroglobulin; The method includes, in this order, mixing the aforementioned complex (J1) with a substance labeled with a labeling substance (b) and a substance (F) that specifically binds to thyroglobulin, in order to obtain a complex (J2) of a solid support (a), thyroglobulin, and substance (F). It can be used in methods commonly practiced in the field of immunoassay as an immunoassay method for quantifying thyroglobulin as a substance to be measured in a sample. Specifically, it can be used in sandwich methods and the like described in literature [for example, Enzyme Immunoassay, 2nd Edition (edited by Eiji Ishikawa et al., Medical View Co., Ltd.) 1982].

[0010] [[ID=⑥]]Among the immunoassay methods of the present invention, as a method using a solid-phase carrier reagent (A) containing a solid-phase carrier (a) and using, as the solid-phase carrier (a), magnetic particles (H) having a substance (D) that specifically binds to the substance to be measured (thyroglobulin), specifically, the following sandwich method is included.

[0011] <Sandwich method> Specific examples of applying the immunoassay method of the present invention to the sandwich method include the following methods. That is, a sample containing thyroglobulin as a substance to be measured is brought into contact with magnetic particles (H) {solid-phase carrier (a) in the solid-phase carrier reagent (A)} having a substance (D) [such as an antibody against thyroglobulin] that specifically binds to thyroglobulin, and a complex of the substance (D) that specifically binds to thyroglobulin and thyroglobulin [that is, a complex (J1) of the solid-phase carrier (a) and thyroglobulin] is formed on the surface of the magnetic particles (H). Thereafter, the complex (J1) is washed with a cleaning agent (C) described in detail later. During washing, it is preferable to hold the complex (J1) by magnetic force or the like to prevent loss of the complex (J1). Thereafter, the complex (J1) is contacted with a substance (F) {in the labeling reagent (B)} that is labeled with a labeling substance (b) and specifically binds to thyroglobulin, to form a labeled complex of a substance (D) that specifically binds to thyroglobulin immobilized on magnetic particles (H), thyroglobulin, and a labeled substance (F) that specifically binds to thyroglobulin [i.e., a complex (J2) of the solid-phase carrier (a), thyroglobulin, and substance (F)]. The labeled complex (J2) is subjected to B / F separation, the amount of the labeling substance (b) in the complex (J2) is measured, and based on the result, the amount of the substance to be measured (thyroglobulin) in the sample is measured. Note that the B / F separation in the above sandwich method means separation of the complex (J2) from the substance (F) not involved in the formation of the complex (J2). Specifically, it means separation of the complex (J2), the complex (J1), and the magnetic particles (H) immobilized with the substance (D) from other components [components other than thyroglobulin in the sample, the substance (F) not involved in the formation of the complex (J2), etc.].

[0012] The cleaning agent (C) in the present invention contains a halide of an alkali metal and / or a halide of an alkaline earth metal and a nonionic surfactant.

[0013] Preferred examples of the halide of an alkali metal include sodium chloride, potassium chloride, sodium iodide, and the like. Preferred examples of the halide of an alkaline earth metal include magnesium chloride, calcium chloride, and the like.

[0014] As the nonionic surfactant contained in the cleaning agent (C), a water-soluble nonionic surfactant is preferred. Note that water-soluble means dissolving 10 g in 100 g of water at 25°C. As the water-soluble nonionic surfactant, specifically, polyoxyethylene nonylphenyl ether with an HLB of 12 or more (preferably an HLB of 12 to 16), polyoxyethylene alkyl ether (such as polyoxyethylene octyl ether), polyoxyethylene sorbitan aliphatic ester, and the like can be mentioned. In the present invention, the HLB (Hydrophile-Lipophile Balance) value means the HLB value that can be measured by the Griffin method [the Griffin method described on page 142 of "Introduction to Surfactants" (published by Sanyo Chemical Industries, Ltd. in 2007, written by Takehiko Fujimoto). In the calculation of HLB in the present application, the oxypropylene group is treated as a hydrophilic group and calculated.] From the viewpoint of reducing the influence of the anti-thyroglobulin antibody, polyoxyalkylene alkyl ether represented by the following general formula (1) is preferable. R 1 O-[(A 1 O) x / (A 2 O) y -H (1)

[0015] In the general formula (1), A 1 is an ethylene group. In the general formula (1), A 2 is a propylene group. In the general formula (1), R 1 is an alkyl group having 8 to 30 carbon atoms. In the general formula (1), x is an integer of 1 to 50. In the general formula (1), y is an integer of 0 to 10. Also, in the general formula (1), [(A 1 O) x / (A 2 O) y represents that the bonding order of x divalent groups (A 1 O) units and y divalent groups (A 2 O) units is arbitrary.

[0016] The proportion of the total weight of alkali metal halides and alkaline earth metal halides contained in the aforementioned cleaning agent (C) is 0.010 to 0.50% by weight, based on the weight of the cleaning agent (C). If the percentage of the total weight mentioned above exceeds 0.50% by weight, there is a problem in that the effect of anti-thyroglobulin antibodies in the sample cannot be reduced.

[0017] The cleaning agent (C) is preferably a buffer solution, and preferably contains a buffering component. Examples of buffer solutions commonly used in immunoassays include Tris buffer, phosphate buffer, Veronal buffer, borate buffer, and Good's buffer. For example, when using a phosphate buffer, one example is a buffer that uses potassium dihydrogen phosphate and dipotassium hydrogen phosphate as buffering components. The total molar concentration of the buffering component in the detergent (C) is preferably 0.1 to 20 mM, and more preferably 0.1 to 5 mM.

[0018] The cleaning agent (C) may also contain preservatives and water in addition to the above-mentioned ingredients. Examples of preservatives include methylisothiazolinone, sodium azide, 5-bromo-5-nitro-1,3-dioxane, and imidazolidinylurea. The weight percentage of the preservative is preferably 0.05 to 0.5% by weight, based on the weight of the cleaning agent (C).

[0019] The weight percentage of the nonionic surfactant contained in the aforementioned detergent (C) is 0.001 to 1% by weight, based on the weight of the detergent (C), from the viewpoint of reducing the influence of anti-thyroglobulin antibodies and improving operability due to foaming during washing.

[0020] The pH of the aforementioned cleaning agent (C) is 6.5 to 8.0, from the viewpoint of the stability of the substance being measured. In this application, pH can be measured using a pH meter at 25°C.

[0021] The cleaning operation of the composite (J1) using the cleaning agent (C) preferably involves holding the composite (J1) by magnetic force or the like, as described above, and removing components other than thyroglobulin from the sample. Specifically, this involves dispersing a composite (J1) in a cleaning agent (C) [the composite (J1) may be recovered before cleaning by magnetic B / F separation], and then using magnetism to collect the composite (J1) or the like to remove any unwanted components other than the composite (J1). If necessary, the above-mentioned unwanted components may be removed using an aspirator or similar device.

[0022] In the step of washing the complex (J1) with the aforementioned detergent (C), the temperature during washing [when dispersing the complex (J1) in the detergent (C), etc.] [the temperature of the detergent (C), the temperature of the mixture containing the complex (J1), etc.] is preferably 30 to 45°C from the viewpoint of reducing the influence of anti-thyroglobulin antibodies. Furthermore, the weight percentage of the cleaning agent (C) used [as described later, if the washing operation is performed two or more times, the weight percentage of the cleaning agent (C) used per wash] is preferably 1,000 to 20,000% by weight, based on the weight of the solid support (a) used, from the viewpoint of reducing the influence of anti-thyroglobulin antibodies. The time for washing the complex (J1) with the aforementioned washing agent (C) [such as the time for dispersing the complex (J1) in the washing agent (C)] is preferably 1 second or more, and more preferably 2 seconds or more, from the viewpoint of reducing the influence of anti-thyroglobulin antibodies. Furthermore, from the viewpoint of shortening the measurement time, the time for washing the composite (J1) with the cleaning agent (C) is preferably 100 seconds or less, more preferably 50 seconds or less, and particularly preferably 10 seconds or less. It is preferable to perform the above cleaning operation two or more times, and from the viewpoint of shortening the measurement time, it is preferable to perform it 10 times or less.

[0023] The immunoassay method of the present invention measures thyroglobulin in the sample. The effects of the present invention are particularly effective when the thyroglobulin to be measured is thyroglobulin contained in serum or plasma.

[0024] In the immunoassay method of the present invention, it is preferable to use a solid-phase carrier reagent (A) containing a solid-phase carrier (a) on which a substance (D) that specifically binds to thyroglobulin is immobilized. The solid phase carrier (a) may be used alone or in combination of two or more types.

[0025] In the present invention, the solid phase support (a) is a magnetic particle containing 60 to 95% by weight [weight ratio based on the weight of the solid phase support (a)] of a superparamagnetic metal oxide with a volume-average particle diameter of 1 to 20 nm. Of these, from the viewpoint of shortening the measurement time and accuracy in immunoassays, it is preferable to use silica particles containing the metal oxide described in Japanese Patent Application Publication No. 2014-210680 and Japanese Patent Application Publication No. 2013-019889. Furthermore, from the viewpoint of measurement sensitivity and reduction of measurement time, the magnetic particles are preferably magnetic particles (H) having antibodies against thyroglobulin [such as magnetic particles with antibodies against thyroglobulin immobilized as a substance (D) that specifically binds to thyroglobulin].

[0026] Preferably, silica particles containing metal oxides have a volume-average particle diameter of 1 to 20 nm and exhibit superparamagnetism, in which the metal oxide is dispersed in a silica matrix. Superparamagnetism refers to the phenomenon where, in the presence of an external magnetic field, the individual atomic magnetic moments of a substance align and induced a temporary magnetic field, and when the external magnetic field is removed, the partial alignment is lost and the magnetic field is no longer observed. Furthermore, the volume-average particle diameter of the metal oxide and the silica particles containing the metal oxide in this invention is the average value of the particle diameters measured by observing any 200 particles with a scanning electron microscope (JEOL Ltd. "JSM-7000F").

[0027] Examples of superparamagnetic metal oxides exhibiting superparamagnetism with a volume-average particle size of 1 to 20 nm include oxides of iron, cobalt, nickel, and their alloys, but iron oxide is particularly preferred due to its excellent sensitivity to magnetic fields. One type of superparamagnetic metal oxide may be used alone, or two or more types may be used in combination.

[0028] Various known iron oxides can be used as the iron oxide. Among iron oxides, at least one selected from the group consisting of magnetite, γ-hematite, magnetite-α-hematite intermediate iron oxide, and γ-hematite-α-hematite intermediate iron oxide is preferred due to its excellent chemical stability, and magnetite is even more preferred because it has a large saturation magnetization and excellent sensitivity to external magnetic fields.

[0029] The lower limit of the superparamagnetic metal oxide content in silica particles containing metal oxides is 60% by weight, preferably 65% ​​by weight, based on the weight of the silica particles containing metal oxides [i.e., the weight of the solid support (a)]. Furthermore, the upper limit of the superparamagnetic metal oxide content is preferably 95% by weight, and more preferably 90% by weight, based on the weight of silica particles containing the metal oxide [i.e., the weight of the solid support (a)], from the viewpoint of enhancing the effect of reducing the influence of anti-thyroglobulin antibodies by the cleaning agent (C). A superparamagnetic metal oxide content of 60% by weight or more is preferable because it results in sufficient magnetism of the resulting silica particles containing the metal oxide, allowing for quick separation operations in practical applications, and also tends to improve the reduction of the effect of anti-thyroglobulin antibodies by the cleaning agent (C). Furthermore, a content of 95% by weight or less facilitates synthesis.

[0030] The method for producing superparamagnetic metal oxides is not particularly limited, but they can be synthesized by a coprecipitation method using a water-soluble iron salt and ammonia, based on the method reported by Massart (R. Massart, IEEE Trans.Magn. 1981, 17, 1247), or by a method using the oxidation reaction of a water-soluble iron salt in an aqueous solution.

[0031] The volume-average particle size of the silica particles containing metal oxides is preferably 1 to 5 μm, and more preferably 1 to 3 μm. When the volume-average particle diameter is 1 μm or larger, separation and recovery tend to be possible in a short time. When it is 5 μm or smaller, the surface area is appropriate, allowing for an appropriate amount of immobilized substance [such as a substance that specifically binds to thyroglobulin (D)] to be bound, resulting in good binding efficiency.

[0032] The volume-average particle size of silica particles containing metal oxides can be controlled by adjusting the mixing conditions (shear force, etc.) when preparing the oil-in-water emulsion, as described later, to adjust the particle size of the emulsion. Furthermore, the volume-average particle size can also be set to a desired value by changing the conditions of the water washing process during the production of silica particles containing metal oxides, or by using methods such as conventional classification.

[0033] The silica particles containing metal oxides in the present invention are obtained by mixing a dispersion containing, for example, superparamagnetic metal oxide particles with a volume average particle diameter of 1 to 20 nm, 30 to 500% by weight of (alkyl)alkoxysilane based on the weight of the superparamagnetic metal oxide particles, and optionally a dispersant, with a solution containing water, a water-soluble organic solvent, a nonionic surfactant, and a catalyst for hydrolysis of (alkyl)alkoxysilane to form an oil-in-water emulsion, then carrying out hydrolysis and condensation reactions of (alkyl)alkoxysilane to obtain an aqueous dispersion of magnetic particles in which the superparamagnetic metal oxide is encapsulated in silica, and finally separating the aqueous dispersion of magnetic particles by centrifugation and / or magnetic collection, and washing with water or methanol, etc. Furthermore, if necessary, the magnetic particles obtained in the above operation, (alkyl)alkoxysilane, water, water-soluble organic solvent, nonionic surfactant, and a catalyst for hydrolysis of (alkyl)alkoxysilane may be mixed, and hydrolysis and condensation reactions of (alkyl)alkoxysilane may be carried out to obtain silica particles having a core-shell structure. In the above and below, (alkyl)alkoxysilane means alkylalkoxysilane or alkoxysilane.

[0034] Silica particles containing metal oxides allow for the immobilization of substances (D), which specifically bind to many thyroglobulins, on their surface, because the superparamagnetic metal oxides are embedded in the silica and present in relatively small amounts on the particle surface.

[0035] In the present invention, substance (D) can be an antibody against thyroglobulin (anti-thyroglobulin antibody), etc. Antibodies against thyroglobulin are not particularly limited as long as they are commonly measured in this field. Furthermore, the antibodies used in this invention also include proteolytic enzymes such as papain and pepsin, as well as degradation products such as Fab and F(ab')2 fragments produced by chemical degradation.

[0036] In the present invention, one method for immobilizing an antibody against thyroglobulin on magnetic particles [immobilized as a substance (D) that specifically binds to thyroglobulin] to form magnetic particles (H) is to physically adsorb the antibody against thyroglobulin onto the magnetic particles as described above. However, from the viewpoint of more efficiently immobilizing the substance to be measured, it is preferable to bind at least one organic compound selected from the group consisting of glutaraldehyde, albumin, carbodiimide, streptavidin, biotin, and alkylalkoxysilane having a functional group to the surface of the magnetic particles, and to immobilize the antibody against thyroglobulin on the magnetic particles via these compounds. More preferably, the immobilization is carried out via an alkylalkoxysilane having a functional group (such as an ethylenically unsaturated group, epoxy group, amino group, mercapto group, and isocyanate group). Alkylalkoxysilanes having such functional groups include vinyltrimethoxysilane, vinyltriethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyl Examples include trimethoxysilane, N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, N-2-(aminoethyl)-3-aminopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-triethoxysilyl-N-(1,3-dimethylbutylidene)propylamine, N-phenyl-3-aminopropyltrimethoxysilane, tris-(trimethoxysilylpropyl)isocyanurate, 3-ureidopropyltrialkoxysilane, 3-mercaptopropylmethyldimethoxysilane, 3-mercaptopropyltrimethoxysilane, and 3-isocyanatetopropyltriethoxysilane.

[0037] The content of solid support (a) in solid support reagent (A) is preferably 0.001 to 10% by weight, and more preferably 0.01 to 1% by weight, from the viewpoint of the washability of the solid support.

[0038] In addition to the solid support (a), the solid support reagent (A) may also contain gelatin, proteins other than gelatin, serum (such as mouse serum), sugars, surfactants, inorganic salts, ethylenediaminetetraacetic acid, and water.

[0039] The gelatin may include any known gelatin, and is not limited in terms of molecular weight or properties; it may be obtained from any animal (mammals, birds, fish, etc.). Examples of gelatin include acid-treated gelatin and alkali-treated gelatin, which are produced by chemically treating collagen with an acid or alkali followed by heat treatment. Furthermore, gelatin derivatives can also be used, which are chemically modified by introducing functional groups such as amino groups, imino groups, carboxyl groups, mercapto groups, and hydroxyl groups using well-known methods. From the viewpoint of the storage stability of the solid-phase support reagent (A), the gelatin content is preferably 1 to 8% by weight, and more preferably 2 to 5% by weight, based on the weight of the solid-phase support reagent (A).

[0040] Other proteins besides gelatin are not particularly limited as long as they are commonly used in the field of immunoassays, and examples include bovine serum albumin (BSA), casein, and skim milk. One protein may be used alone, or two or more may be used in combination. From the viewpoint of the storage stability of the solid support reagent (A), the protein content is preferably 0 to 15% by weight, and more preferably 0.1 to 15% by weight, based on the weight of the solid support reagent (A). Furthermore, the serum content is preferably 0 to 15% by weight, and more preferably 0.1 to 10% by weight, based on the weight of the solid-phase carrier reagent (A).

[0041] Sugars include monosaccharides, disaccharides, and polysaccharides. Examples of monosaccharides include trioses (ketotriose, etc.), tetroses (ketotetrose, etc.), pentoses (ketopentoses, aldopentoses, and deoxy sugars, etc.), hexoses [ketohexoses (psicose, fructose, sorbose, and tagatose, etc.), aldohexoses (allose, altrose, glucose, mannose, gross, idose, galactose, and talose, etc.), and deoxy sugars (fucose, fuculose, and rhamnose, etc.)] and heptoses (sedoheptulose, etc.). Disaccharides include those formed by the dehydration condensation of two molecules of the above-mentioned monosaccharides to form a glycosidic bond. Specifically, examples include sucrose, lactose, maltose, and cellobiose. Polysaccharides include those formed by the dehydration condensation of three or more molecules of the above-mentioned monosaccharides to form glycosidic bonds. Specifically, examples include amylose, amylopectin, glycogen, cellulose, hyaluronic acid, chondroitin sulfate, and heparin. Sugars may be used individually or in combination of two or more types. As for the sugars, disaccharides are preferred from the viewpoint of storage stability of the solid-phase support reagent (A), and sucrose and lactose are more preferred. From the viewpoint of the storage stability of the solid-phase support reagent (A), the sugar content is preferably 5 to 40% by weight, and more preferably 10 to 20% by weight, based on the weight of the solid-phase support reagent (A).

[0042] Examples of surfactants include those exemplified in the explanation in (B), which will be described in detail later, and the same applies to preferred surfactants. Furthermore, the surfactant content is preferably 0 to 2% by weight, and more preferably 0.01 to 1% by weight, based on the weight of the solid support reagent (A).

[0043] Examples of inorganic salts include alkali metal salts [halides (sodium chloride, potassium chloride, sodium bromide, and sodium fluoride, etc.), sulfates (sodium sulfate and potassium sulfate, etc.), nitrates (sodium nitrate and potassium nitrate, etc.), and phosphates (sodium phosphate and potassium phosphate, etc.)], and alkaline earth metal salts [halides (calcium chloride and magnesium chloride, etc.) and sulfates (magnesium sulfate, etc.)]. Inorganic salts may be used individually or in combination of two or more. Of these, at least one selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, sodium phosphate, sodium sulfate, potassium sulfate, sodium nitrate, and potassium nitrate is preferred from the viewpoint of storage stability of the solid-phase support reagent (A). From the viewpoint of the storage stability of the solid-phase support reagent (A), the inorganic salt content is preferably 0.1 to 2% by weight, and more preferably 0.5 to 1% by weight, based on the weight of the solid-phase support reagent (A). Furthermore, the ethylenediaminetetraacetic acid content is preferably 0 to 2% by weight, and more preferably 0.01 to 1% by weight, based on the weight of the solid support reagent (A).

[0044] As described in the description of specific immunoassay methods (sandwich method, competitive method, etc.) of the present invention, it is preferable to use a labeling reagent (B) that contains a substance (F) labeled with a labeling substance (b) and specifically binds to thyroglobulin.

[0045] The substance labeled with labeling substance (b) and used in substance (F) which specifically binds to thyroglobulin, is the same as the substance that specifically binds to thyroglobulin (D) described above, and the preferred substances are also the same.

[0046] The labeling substance (b) used for labeling is: For example, enzymes used in enzyme-mediated immunoassay (EIA), such as alkaline phosphatase, β-galactosidase, peroxidase, microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, luciferase, tyrosinase, and acid phosphatase; For example, radioisotopes such as 99mTc, 131I, 125I, 14C, 3H, and 32P used in radioimmunoassay (RIA); For example, fluorescent substances such as fluorescein, dansyl, fluorescein, coumarin, naphthylamine or their derivatives, and green fluorescent protein (GFP) used in immunofluorescence assays (FIA); For example, luminescent substances such as luciferin, isoluminol, luminol, and bis(2,4,6-trifluorophenyl)oxalate; For example, substances that absorb ultraviolet light, such as phenol, naphthol, anthracene, or derivatives thereof; Examples include substances that have properties as spin labeling agents, such as compounds having an oxyl group, like 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-cyclohexadiene-1-ylidene)-p-trioxyl. Of these, enzymes and fluorescent substances are preferred from the viewpoint of sensitivity, and alkaline phosphatase, peroxidase, and glucose oxidase are more preferred, with peroxidase being particularly preferred.

[0047] To bind the labeled substance (b) to a substance that specifically binds to thyroglobulin, one can use methods commonly employed in the field of immunoassays, such as known labeling methods commonly used in EIA, RIA, and FIA [e.g., Medical Chemistry Experiment Course, Vol. 8, supervised by Yuichi Yamamura, 1st edition, Nakayama Shoten, 1971; Illustrated Fluorescent Antibodies, by Akira Kawai, 1st edition, Soft Science Co., Ltd., 1983; Enzyme Immunoassay, edited by Eiji Ishikawa, Tadashi Kawai, and Kiyoshi Muroi, 2nd edition, Igaku Shoin, 1982, etc.].

[0048] The amount of labeling substance (b) used varies depending on the type of labeling substance (b) used and cannot be stated in general terms. However, for example, when using peroxidase (hereinafter abbreviated as POD) as labeling substance (b), the substance that specifically binds to thyroglobulin and the labeling substance (b) should be included in the buffer solution in a molar ratio of, for example, preferably 1:1 to 20 (more preferably 1:1 to 10, and particularly preferably 1:1 to 2). Examples of buffer solutions commonly used in immunoassays include Tris buffer, phosphate buffer, Veronal buffer, borate buffer, and Good's buffer. The pH should be within a range that does not suppress the antigen-antibody reaction, and is preferably between 5 and 9. Furthermore, such buffer solutions may contain stabilizers such as albumin, globulin, water-soluble gelatin, and polyethylene glycol, as well as surfactants and sugars, provided they do not inhibit the desired antigen-antibody reaction.

[0049] From the viewpoint of sensitivity, the content of the substance (F) labeled with the labeling substance (b) in the labeling reagent (B), which specifically binds to thyroglobulin, is preferably 0.01 to 40 μg / mL, and more preferably 0.1 to 20 μg / mL.

[0050] Labeling reagent (B) may also contain proteins, surfactants, and polymer compounds in addition to those mentioned above. The protein used is not particularly limited as long as it is commonly measured in the field of immunoassays, and examples include bovine serum albumin (BSA), casein, and skim milk. One protein may be used alone, or two or more proteins may be used in combination. From the viewpoint of sensitivity and storage stability of the reagent, the protein content is preferably 0.001 to 8% by weight, based on the weight of the labeled reagent (B).

[0051] Examples of surfactants include well-known nonionic surfactants, amphoteric surfactants, and anionic surfactants, but from the viewpoint of reducing nonspecific adsorption, water-soluble nonionic surfactants are preferred. Furthermore, "water-soluble" means that 10g dissolves in 100g of water at 25°C. Examples of water-soluble nonionic surfactants include polyoxyethylene nonylphenyl ethers, polyoxyethylene alkyl ethers (such as polyoxyethylene octyl ether), and polyoxyethylene sorbitan aliphatic esters, all of which have an HLB of 12 or higher. The surfactant content is 0.001 to 4% by weight, based on the weight of the labeled reagent (B), from the viewpoint of particle cleaning performance.

[0052] The polymer compounds are not particularly limited as long as they are commonly used in the field of immunoassays, and examples include polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, Blockmaster (manufactured by JSR Corporation), and Lipidure (manufactured by NOF Corporation). Polymer compounds may be used individually or in combination of two or more types. From the viewpoint of suppressing nonspecific adsorption, the content of the polymer compound is preferably 0.001 to 3% by weight, and more preferably 0.5 to 1% by weight, based on the weight of the labeled reagent (B).

[0053] Methods for measuring the amount of labeled substance (b) include radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), and chemiluminescence immunoassay (CLIA and CLEIA). From the viewpoint of sensitivity in rapid immunoassay, EIA, CLIA, and CLEIA are preferred, with CLEIA being the most preferred.

[0054] For example, when measuring the amount of labeled substance using the chemiluminescence method, chemiluminescent reagent (E) is used. The chemiluminescent reagent (E) is selected based on the labeling substance (b) described above. For example, if the labeling substance (b) is a peroxidase, it comprises a first chemiluminescent reagent solution containing a 2,3-dihydro-1,4-phthalazinedione compound and a chemiluminescence enhancer as essential components, and a second chemiluminescent reagent solution containing an oxidizing agent and water as essential components.

[0055] As the 2,3-dihydro-1,4-phthalazinedione compound, for example, known 2,3-dihydro-1,4-phthalazinedione compounds and mixtures thereof described in Japanese Patent Publication No. 2-291299, Japanese Patent Publication No. 10-319015, and Japanese Patent Publication No. 2000-279196 can be used. Of these, luminol, isoluminol, N-aminohexyl-N-ethylisoluminol (AHEI), N-aminobutyl-N-ethylisoluminol (ABEI), and their metal salts (alkali metal salts, etc.) are preferred, more preferably luminol and its metal salts, and particularly preferably sodium salt of luminol.

[0056] As chemiluminescence enhancers, for example, known chemiluminescence enhancers described in Japanese Patent Publication No. 59-500252, Japanese Patent Publication No. 59-171839, and Japanese Patent Publication No. 2-291299, and mixtures thereof can be used. Of these, phenol is preferred from the viewpoint of chemiluminescence enhancing effect, more preferably p-iodophenol, 4-(cyanomethylthio)phenol, and 4-cyanomethylthio-2-chlorophenol, and particularly preferably 4-(cyanomethylthio)phenol.

[0057] The first chemiluminescent reagent is preferably alkaline from the viewpoint of the fluorescence intensity of the enzyme, and the pH of the first reagent is preferably 7 to 11, and more preferably 8 to 10. Furthermore, pH is measured at a measurement temperature of 25°C in accordance with JIS K0400-12-10:2000.

[0058] Examples of oxidizing agents contained in the second chemiluminescent reagent include known oxidizing agents described in Japanese Patent Publication No. 8-261943 and Japanese Patent Publication No. 2000-279196, etc. [inorganic peroxides (hydrogen peroxide, sodium perborate, potassium perborate, etc.), organic peroxides (dialkyl peroxides, acyl peroxides, etc.), peroxoacid compounds (peroxosulfuric acid, peroxophosphate, etc.)]. Of these, hydrogen peroxide, sodium perborate, and potassium perborate are preferred from the viewpoint of storage stability, and hydrogen peroxide is even more preferred.

[0059] The immunoassay kit of the present invention comprises a solid-phase support reagent (A), a labeling reagent (B), an immunoassay buffer (W), and a washing agent (C). In the immunoassay kit of the present invention, the solid-phase support reagent (A) can be the solid-phase support reagent (A) described above, the labeling reagent (B) can be the labeling reagent (B) described above, and the washing agent (C) can be the washing agent (C) described above.

[0060] The immunoassay buffer (W) used in the immunoassay kit of the present invention may contain buffers commonly used in the field of immunoassay, such as Tris buffer, phosphate buffer, Veronal buffer, borate buffer, and Good's buffer. Its pH may be within a range that does not inhibit the effects of the present invention, and is preferably 5 to 9. Furthermore, such buffer solutions may contain salts (such as sodium chloride and ethylenediaminetetraacetic acid), albumin (such as bovine serum albumin), globulin, protein (casein hydrolysate), water-soluble gelatin, stabilizers such as polyethylene glycol, surfactants [such as the surfactants exemplified in the description of the labeling reagent (B) above], and sugars [such as the sugars exemplified in the description of the solid support reagent (A) above], as long as they do not inhibit the effects of the present invention.

[0061] The immunoassay kit of the present invention preferably further includes a chemiluminescent reagent (E). Furthermore, it is preferable that the immunoassay kit of the present invention includes a chemiluminescent reagent (E) as a component, comprising a luminol luminescence reagent (E1) [a chemiluminescent reagent solution 1 in which the 2,3-dihydro-1,4-phthalazinedione compound is luminol and / or its metal salt] and a hydrogen peroxide solution (E2) [a chemiluminescent reagent solution 2 in which the oxidizing agent is hydrogen peroxide], and the labeling substance (b) in the labeling reagent (B) is a peroxidase.

[0062] The composition, content, and preferred ranges of each component of the solid-phase support reagent (A), labeling reagent (B), washing agent (C), and chemiluminescent reagent (E) in the immunoassay kit of the present invention are the same as those described in the immunoassay method described above. [Examples]

[0063] The present invention will be further described below with reference to examples, but the present invention is not limited thereto. In the following, "parts" refers to parts by weight.

[0064] <Example 1> An immunoassay kit (S-1) of the present invention, consisting of a solid-phase support reagent (A-1), a labeling reagent (B-1), an immunoassay buffer (W), a luminol luminescence reagent (E1), and a hydrogen peroxide solution (E2), was obtained by the method described below.

[0065] Fabrication of magnetic particles (PH-1): <Fabrication of magnetic metal oxide particles> 186 parts of iron(III) chloride hexahydrate, 68 parts of iron(II) chloride tetrahydrate, and 1288 parts of water were charged into a reaction vessel and dissolved. The mixture was heated to 50°C, and while stirring, the temperature was maintained at 50-55°C. 280 parts of 25% by weight aqueous ammonia were added dropwise over 1 hour to obtain magnetite particles in water. 64 parts of oleic acid, a dispersant, were added to the obtained magnetite particles, and stirring was continued for 2 hours. After cooling to room temperature, solid-liquid separation was performed by decantation, and the magnetite particles with adsorbed oleic acid were washed three times with 1000 parts of water, followed by two washes with 1000 parts of acetone. The mixture was then dried at 40°C for 2 days to obtain superparamagnetic metal oxide particles with a volume-average particle size of 15 nm.

[0066] <Core layer fabrication> 80 parts of superparamagnetic metal oxide particles were added to 240 parts of tetraethoxysilane and dispersed to prepare dispersion (1). Next, 5050 parts of water, 3500 parts of 25% by weight aqueous ammonia solution, and 400 parts of nonionic surfactant ("NSA-17", manufactured by Sanyo Chemical Industries, Ltd.) were added to the reaction vessel and mixed using Clear Mix (manufactured by M-Technique Co., Ltd.) to obtain solution (2). After raising the temperature to 50°C, the dispersion (1) was added dropwise to solution (2) over 1 hour while stirring with Clear Mix at a rotation speed of 6,000 rpm, and the mixture was reacted at 50°C for 1 hour. After the reaction, the mixture was centrifuged at 2,000 rpm for 20 minutes to remove the supernatant containing fine particles and obtain the core layer.

[0067] <Fabrication of magnetic particles> 80 parts of core layer, 2500 parts of deionized water, 260 parts of 25% ammonia aqueous solution, 2500 parts of ethanol, and 1200 parts of tetraethoxysilane were added to a reaction vessel and mixed using Clear Mix (manufactured by M-Technique Co., Ltd.). The mixture was reacted for 2 hours while stirring at a rotation speed of 6,000 rpm with the Clear Mix. After the reaction, the supernatant containing fine particles was removed by centrifugation at 2,000 rpm for 20 minutes. 4000 parts of deionized water were added to the precipitated particles after centrifugation to redisperse them, and the dispersed particles were collected using a magnet, with the supernatant removed. This process was repeated 10 times. Next, 5,000 parts of water were added to the obtained solid phase to disperse the particles, and the mixture was centrifuged at 600 rpm for 10 minutes. This process of removing the supernatant containing fine particles was repeated 20 times. Subsequently, 5,000 parts of water were added to the obtained solid phase to disperse the particles, and the mixture was centrifuged at 300 rpm for 10 minutes to settle and remove the larger particle sizes, thereby performing classification. Furthermore, the particles were magnetized using a magnet and the supernatant was removed. Then, 5000 parts of water were added to disperse the core-shell particles, and the process of magnetizing the particles and removing the supernatant was repeated 10 times to obtain magnetic particles (PH-1) with the target volume-average particle diameter of 2.0 μm. The content of superparamagnetic metal oxide particles in the obtained magnetic particles (PH-1) was measured and found to be 81% by weight.

[0068] <Method for measuring the volume-average particle diameter of particles (superparamagnetic metal oxide particles and magnetic particles)> Using a scanning electron microscope (model: JSM-7000F, manufacturer: JEOL Ltd.), we observed 200 arbitrary superparamagnetic metal oxide particles, measured their particle sizes, and determined the volume-average particle size. The volume-average particle diameter was determined for magnetic particles using a similar method.

[0069] <Method for measuring the content of superparamagnetic metal oxide particles> For 20 arbitrary magnetic particles, observation was performed using a scanning electron microscope (model JSM-7000F, manufacturer JEOL Ltd.), and the content of superparamagnetic metal oxide particles was measured using an energy-dispersive X-ray spectrometer (model INCA Wave / Energy, manufacturer Oxford). The average value was defined as content S. The silica content was also measured using the same method, and the average value was defined as content T. The content of superparamagnetic metal oxide particles was calculated using the following formula (1). Content of superparamagnetic metal oxide particles (wt%)=(S) / (S+T)×100...(1)

[0070] Preparation of magnetic particles (H1-1) and solid-phase support reagent (PA-1): 40 mg of the prepared magnetic particles (PH-1) were added to a lidded polyethylene bottle containing 40 mL of an acetone solution containing 1 wt% γ-aminopropyltriethoxysilane. The mixture was reacted at 25°C for 1 hour. The magnetic particles were then magnetized with a neodymium magnet, and the liquid was removed by aspirator. Next, 40 mL of deionized water was added, the bottle was sealed, and the polystyrene bottle was slowly inverted and stirred twice. The magnetic particles were then magnetized with a neodymium magnet, and the liquid was removed by aspirator to wash them. This washing operation was repeated 5 times. Next, the washed magnetic particles were added to a lidded polyethylene bottle containing 40 mL of an aqueous solution containing 2 wt% glutaraldehyde, and the mixture was reacted at 25°C for 1 hour. Then, 40 mL of deionized water was added, the bottle was sealed, and the polystyrene bottle was slowly inverted and stirred twice. The magnetic particles were then magnetized with a neodymium magnet, and the liquid was removed by aspirator to wash them. This washing operation was repeated 10 times. Furthermore, the washed magnetic particles were added to a lidded polyethylene bottle containing 120 mL of 0.02 M phosphate buffer (pH 8.7) with 10 μg / mL of anti-Tg monoclonal antibody (mouse) (Monoclonal mouse anti-human thyroglobulin 5F9cc, Hytest), and reacted at 25°C for 1 hour. After the reaction, the magnetic particles were magnetized with a neodymium magnet, and the phosphate buffer containing the anti-Tg monoclonal antibody was removed to obtain magnetic particles (H1-1) to which the anti-Tg monoclonal antibody was bound. [Note that the weight of magnetic particles (H1-1) was equivalent to that of magnetic particles (PH-1), and the content of superparamagnetic metal oxide particles in magnetic particles (H1-1) was 81% by weight.] Next, the magnetic particles (H1-1) were diluted with the diluent described later to a concentration of 0.01% by weight, and a solid-phase support reagent (PA-1) containing magnetic particles (H1-1) was prepared.

[0071] Preparation of magnetic particles (H1-2) and solid-phase support reagent (PA-2): In the "Preparation of Magnetic Particles (H1-1) and Solid-Phase Support Reagent (PA-1)" described above, a polyethylene bottle with a lid containing 120 mL of 0.02 M phosphate buffer (pH 8.7) containing 10 μg / mL of anti-Tg monoclonal antibody (mouse) (Monoclonal mouse anti-human thyroglobulin 5F9cc, Hytest) was replaced with a polyethylene bottle with a lid containing 120 mL of 0.02 M phosphate buffer (pH 8.7) containing 10 μg / mL of anti-Tg monoclonal antibody (mouse) (Monoclonal Antibody to Human Thyrogloblin, E01326M, meridian)). The procedure was carried out in the same manner as in the "Preparation of Magnetic Particles (H1-1) and Solid-Phase Support Reagent (PA-1)," except that the "polyethylene bottle with a lid containing 120 mL of 0.02 M phosphate buffer (pH 8.7) containing 10 μg / mL of anti-Tg monoclonal antibody (mouse) (Monoclonal mouse anti-human thyroglobulin 5F9cc, Hytest)" was replaced with a polyethylene bottle with a lid containing 120 mL of anti-Tg monoclonal antibody (mouse) (Monoclonal Antibody to Human Thyrogloblin, E01326M, meridian) was used. A solid-phase support reagent (PA-2) containing magnetic particles (H1-2) to which anti-Tg monoclonal antibody was bound was prepared. [Note that the weight of magnetic particles (H1-2) is equivalent to that of magnetic particles (PH-1), and the content of superparamagnetic metal oxide particles in magnetic particles (H1-2) is 81% by weight.]

[0072] Preparation of solid-phase support reagent (A-1): Solid-phase support reagent (PA-1) and solid-phase support reagent (PA-2) were mixed in a weight ratio of 1:1 to prepare solid-phase support reagent (A-1) containing magnetic particles (H1-1) and magnetic particles (H1-2).

[0073] Preparation of the diluent: A diluent containing 10% by weight BSA, 0.1% by weight Naroacty CL-100 [polyoxyalkylene alkyl ether, manufactured by Sanyo Chemical Industries, Ltd.], 0.1% by weight EDTA (ethylenediamine-N,N,N',N'-tetraacetate disodium salt dihydrate, manufactured by Dojin Chemical Laboratories Co., Ltd.), 5% by weight mouse serum [manufactured by Cosmo Bio Co., Ltd.], and 0.02M sodium phosphate (pH 7.0) was prepared and stored under refrigeration (2-10°C).

[0074] Preparation of immunoassay buffer (W): A 0.02 M phosphate buffer (pH 7.0) containing 0.1% by weight of bovine serum albumin (manufactured by Boval Company), 1% by weight of Emulmin L-90-S (manufactured by Sanyo Chemical Industries, Ltd.), and 0.85% by weight of sodium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was prepared and stored under refrigeration (2-10°C).

[0075] Preparation of labeling reagent (B-1): Using anti-Tg monoclonal antibody (mouse) (Monoclonal mouse anti-human thyroglobulin 5F6cc, Hytest), POD-labeled anti-Tg monoclonal antibody (mouse) (F) was prepared according to the method described in the literature (S. Yoshitake, M. Imagawa, E. Ishikawa, Etol; J. Biochem, Vol. 92, 1982, 1413-1424). This was diluted to a concentration of 100 nM as POD-labeled anti-Tg monoclonal antibody (mouse) (F) in 0.02 M phosphate buffer (pH 7.0) containing 0.5 wt% bovine serum albumin and 1 wt% Naroacty CL-100 as a surfactant, to prepare labeling reagent (B-1), which was stored under refrigeration (2-10°C).

[0076] Preparation of luminol luminescence reagent (E1): 0.7 g of sodium luminol [manufactured by Sigma-Aldrich Japan Co., Ltd.] and 0.1 g of 4-(cyanomethylthio)phenol were placed in a 1,000 mL volumetric flask. 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid / sodium hydroxide buffer (10 mM, pH=8.6) was added to the flask to a total volume of 1,000 mL, and the mixture was homogenized at 25°C to prepare the luminol luminescence reagent (E1). The solution was stored refrigerated (2-10°C) until use in measurement.

[0077] Preparation of hydrogen peroxide solution (E2): 6.6 g of hydrogen peroxide [manufactured by Wako Pure Chemical Industries, Ltd., reagent grade, concentration 30% by weight] was placed in a 1,000 mL volumetric flask. Deionized water was added to bring the total volume of the solution to 1,000 mL, and the mixture was homogenized at 25°C to prepare hydrogen peroxide solution (E2). The solution was stored refrigerated (2-10°C) until use in the measurement.

[0078] Preparation of cleaning agent (C-1): The nonionic surfactant, buffering component, alkali metal halide, alkaline earth metal halide, and ion-exchanged water listed in Table 1 were mixed in the weight portions listed in Table 1 to obtain detergent (C-1).

[0079] <Examples 2-13 and Comparative Examples 1-2> In the production of the immunoassay kit (S-1) in Example 1, the immunoassay kits (S-2) to (S-13) and (S'-1) to (S'-2) were obtained in the same manner as in Example 1, except that the washing agents (C-2) to (C-13) and (C'-1) to (C'-2), obtained by the following method, were used instead of the washing agent (C-1) used in Example 1. <Preparation of cleaning agents (C-2) to (C-13) and (C'-1) to (C'-2)> Each component shown in Table 1 or Table 2 was mixed in the weight amounts listed in Table 1 or Table 2 to obtain immunoassay buffers (C-2) to (C-13) and (C'-1) to (C'-2).

[0080] The following nonionic surfactants were used as listed in Table 1 or Table 2. • Naroacty CL-100 [Polyoxyalkylene alkyl ether, HLB=13.3, manufactured by Sanyo Chemical Industries, Ltd.] • Naroacty CL-200 [Polyoxyethylene alkyl ether, HLB=16.0, manufactured by Sanyo Chemical Industries, Ltd.] • Naroacty CL-85 [Polyoxyalkylene alkyl ether, HLB=12.6, manufactured by Sanyo Chemical Industries, Ltd.] • Emulmin LS-90 [Polyoxyethylene lauryl ether, HLB=13.6, manufactured by Sanyo Chemical Industries, Ltd.]

[0081] <Example 14> In the <core layer preparation> during the preparation of magnetic particles (PH-1) in Example 1, the amount of superparamagnetic metal oxide particles added was changed from 80 parts to 63 parts. The procedure was carried out in the same manner as in Example 1 for the preparation of magnetic particles (PH-1), and magnetic particles (PH-2) with a volume-average particle diameter of 2.0 μm were obtained. The content of superparamagnetic metal oxide particles in the obtained magnetic particles (PH-2) was measured and found to be 61% by weight. The subsequent steps in Example 1 were carried out in the same manner as in Example 1, except that magnetic particles (PH-2) were used instead of magnetic particles (PH-1). The solid support reagent (A-2) containing magnetic particles (H2-1) instead of magnetic particles (H1-1) and magnetic particles (H2-2) instead of magnetic particles (H1-2) was prepared. The immunoassay kit (S-14) of the present invention was obtained in the same manner as in Example 1. Furthermore, the weight of magnetic particles (H2-1) is equivalent to that of magnetic particles (PH-2), and the content of superparamagnetic metal oxide particles in magnetic particles (H2-1) is 61% by weight. Similarly, the weight of magnetic particles (H2-2) is equivalent to that of magnetic particles (PH-2), and the content of superparamagnetic metal oxide particles in magnetic particles (H2-2) is 61% by weight.

[0082] <Example 15> In the <core layer preparation> during the preparation of magnetic particles (PH-1) in Example 1, the amount of superparamagnetic metal oxide particles added was changed from 80 parts to 92 parts. Otherwise, the procedure was carried out in the same manner as in Example 1, and magnetic particles (PH-3) with a volume-average particle diameter of 2.0 μm were obtained. The content of superparamagnetic metal oxide particles in the obtained magnetic particles (PH-3) was measured to be 90% by weight. The subsequent steps in Example 1 were carried out in the same manner as in Example 1, except that magnetic particles (PH-3) were used instead of magnetic particles (PH-1). The solid support reagent (A-3) containing magnetic particles (H3-1) instead of magnetic particles (H1-1) and magnetic particles (H3-2) instead of magnetic particles (H1-2) was prepared. The immunoassay kit (S-15) of the present invention was obtained in the same manner as in Example 1. Furthermore, the weight of magnetic particle (H3-1) is equivalent to that of magnetic particle (PH-3), and the content of superparamagnetic metal oxide particles in magnetic particle (H3-1) is 90% by weight. Similarly, the weight of magnetic particle (H3-2) is equivalent to that of magnetic particle (PH-3), and the content of superparamagnetic metal oxide particles in magnetic particle (H3-2) is 90% by weight.

[0083] <Examples 16-36 and Comparative Examples 3-4> Immunoassays were performed using the immunoassay kits (S-1) to (S-15) or (S'-1) to (S'-2) listed in Table 1, Table 2, or Table 3, and the thyroglobulin concentration in the sample was measured using the following method (sandwich method).

[0084] <Sensitivity Evaluation> 0.5 mL of each solid-phase support reagent (A) from the immunoassay kit was placed in a test tube. Magnetic particles were collected from the outside of the test tube using a neodymium magnet for 10 seconds. The liquid inside the test tube was removed with an aspirator, and the neodymium magnet was moved sufficiently away from the side. Next, 0.2 mL of immunoassay buffer (W) and 0.05 mL of serum samples [TgAb-positive samples 1-5 and TgAb-negative samples 6-10, whose TgAb concentrations were previously measured using AccuraSeed TgAb] were injected into a test tube, and the mixture was allowed to react in the test tube at 37°C for 3 minutes to form a complex [magnetic particles immobilized with anti-Tg monoclonal antibody / Tg complex]. After the reaction, magnetic particles were collected from the outside of the test tube using a neodymium magnet for 10 seconds, the liquid in the test tube was removed with an aspirator, and the neodymium magnet was moved sufficiently away from the side. Then, at the temperatures specified in Table 1, Table 2, or Table 3, the cleaning agent (C) was added in the amount specified in Table 1, Table 2, or Table 3, and the magnetic particles were dispersed and collected for the time specified in Table 1, Table 2, or Table 3. This cleaning operation, in which the liquid was removed with an aspirator, was repeated twice.

[0085] Next, 0.05 mL of the labeling reagent (B) corresponding to each immunoassay kit was injected into a test tube, and the mixture was reacted in the test tube at 37°C for 3 minutes to form a complex [magnetic particle immobilized with anti-Tg monoclonal antibody / Tg / POD-labeled anti-Tg mouse monoclonal antibody complex]. After the reaction, magnetic particles were collected from the outside of the test tube using a neodymium magnet for 10 seconds, the liquid in the test tube was removed with an aspirator, and the neodymium magnet was moved sufficiently away from the side. Then, 0.5 mL of physiological saline was added to disperse the magnetic particles, and the washing procedure of removing the liquid with an aspirator was repeated twice after magnetization. Finally, 0.07 mL of chemiluminescent reagent solution 1 (E1) and 0.07 mL of chemiluminescent reagent solution 2 (E2) were added simultaneously, and the luminescence reaction was allowed to proceed at 37°C for 45 seconds. The average luminescence amount from 43 to 45 seconds after the addition of the chemiluminescent reagents was measured using a luminometer [Lumat LB9507, manufactured by Berthold Japan]. This average luminescence amount was defined as the average luminescence amount (Z).

[0086] Furthermore, in the same procedure as described above, except that the standard solutions listed below were used instead of serum samples, the average luminescence was measured using a luminometer, and a calibration curve showing the relationship between luminescence and Tg concentration was created. From the obtained calibration curve, the measured concentration (ng / mL) in the above immunoassay method was determined. The results are shown in Tables 1, 2, and 3. Standard solution: Pooled serum with Tg concentrations adjusted to 0, 5, 30, 50, 100, 500, or 1000 ng / mL.

[0087] Furthermore, comparative experiments were conducted in the same manner as described above, except that physiological saline solution was used instead of the washing agent (C), and the measured concentrations in the immunoassay method described above were determined. The results are shown in Tables 1, 2, and 3.

[0088] In Examples 1-36, even when the measurement samples were TgAb-positive, the measured concentration of thyroglobulin was higher compared to each comparative experiment. Examples 1-36 demonstrate that washing with detergent (C) reduces the influence of TgAb contained in the samples. On the other hand, in Comparative Examples 1 and 2, where the total concentration of alkali metal halides and alkaline earth metal halides in the detergent was outside the range of the present invention, the measured concentration of thyroglobulin was low, similar to that in each comparative experiment, when the measurement sample was a TgAb-positive sample. This indicates that the influence of TgAb contained in the sample could not be reduced in Comparative Examples 1 and 2.

[0089] [Table 1]

[0090] [Table 2]

[0091] [Table 3] [Industrial applicability]

[0092] The immunoassay method using the cleaning agent of the present invention is easy to operate and highly accurate, making it widely applicable to clinical tests such as radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, and chemiluminescence immunoassay.

Claims

1. An immunoassay method for measuring the concentration of thyroglobulin in a sample, The aforementioned immunoassay method, The process includes, in this order, mixing the sample with a solid support (a) to obtain a mixture containing a complex (J1) of the solid support (a) and thyroglobulin, then washing the complex (J1) with a cleaning agent (C), and mixing the complex (J1) with a substance (F) labeled with a labeling substance (b) that specifically binds to thyroglobulin to obtain a complex (J2) of the solid support (a), thyroglobulin, and substance (F). The solid-phase support (a) is a magnetic particle containing a superparamagnetic metal oxide with a volume-average particle diameter of 1 to 20 nm. The weight percentage of the superparamagnetic metal oxide is 60 to 95% by weight, based on the weight of the solid support (a). The cleaning agent (C) contains alkali metal halides and / or alkaline earth metal halides and polyoxyalkylene alkyl ethers. The total weight ratio of the alkali metal halides and alkaline earth metal halides is 0.010 to 0.50% by weight, based on the weight of the cleaning agent (C). The weight percentage of the polyoxyalkylene alkyl ether is 0.001 to 1% by weight, based on the weight of the detergent (C). An immunoassay method wherein the pH of the cleaning agent (C) is 6.5 to 8.

0.

2. A thyroglobulin measurement kit used in the immunoassay method described in claim 1, comprising: a solid-phase support reagent (A) containing the solid-phase support (a); a labeling reagent (B) containing a substance (F) labeled with the labeling substance (b); and a washing agent (C), wherein the solid-phase support (a) is a magnetic particle containing a superparamagnetic metal oxide having a volume-average particle diameter of 1 to 20 nm, The weight percentage of the superparamagnetic metal oxide is 60 to 95% by weight, based on the weight of the solid support (a). The cleaning agent (C) contains alkali metal halides and / or alkaline earth metal halides and polyoxyalkylene alkyl ethers. The total weight ratio of the alkali metal halides and alkaline earth metal halides is 0.010 to 0.50% by weight, based on the weight of the cleaning agent (C). The weight percentage of the polyoxyalkylene alkyl ether is 0.001 to 1% by weight, based on the weight of the detergent (C). A thyroglobulin measurement kit wherein the pH of the cleaning agent (C) is 6.5 to 8.0.

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