Magnetic-responsive particles, immunoassay method using the same, and reagent for immunoassay

Abstract

Sensitized magnetically responsive particles according to the present invention comprise: core particles; magnetically responsive particles having at least one magnetic layer comprising microparticles of a magnetic metal and / or an oxide thereof disposed upon the core particles; and a substance that specifically acts upon a target material supported on the magnetically responsive particles. Defining vc and wc as the volume and weight of the core particles and ve and we as the volume and weight of the magnetically responsive particles, the magnetic body density [(we–wc) / (ve-vc)] of the sensitized magnetically responsive particles satisfies formula 1: 2.0 ≤ (we–wc) / (ve–vc). The magnetically responsive particles have high magnetism-collecting properties despite having a small particle size. Provided is an immunoassay reagent that has superior magnetic separation ability and is capable of realizing high sensitivity as a result of these magnetically responsive particles being used.

Description

Technical Field

[0001] The present invention relates to magnetic-responsive particles used in immunodetection reagents, an immunodetection method using the same, and an immunodetection reagent.

Background Art

[0002] Conventionally, as a method for measuring or purifying a target protein or the like from a sample containing a biological substance, a substance that specifically acts on the target is immobilized on a solid-phase carrier, bound to the target in a biological sample, and unbound substances other than the target are removed by washing, and the amount of the target bound to the solid-phase carrier is measured.

[0003] When removing unbound substances, magnetic-responsive particles are used as the solid-phase carrier because they are easy to separate and recover. As such particles, for example, Patent Document 1 describes particles for clinical test drugs in which a magnetic layer is formed on the surface of a core particle and a polymer layer is formed thereon. However, only particles with a broad particle diameter could be obtained for the disclosed particles. Regarding magnetic-responsive particles, in addition to the above problems, further improvement in magnetic responsiveness has been demanded.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] As a means for enhancing magnetic responsiveness, there is a method of increasing the magnetic substance content. However, increasing the magnetic substance content has a problem that the average particle diameter increases and the surface area of the particle with respect to the weight decreases, leading to a decrease in the binding volume with biochemical substances. Therefore, there has been a demand for particles that exhibit high magnetic aggregability without reducing the surface area of the particles. An object of the present invention is to provide a magnetoresponsive particle having a high magnetic aggregating property despite a small average particle diameter, and a reagent for immunoassay using the same, which can achieve excellent magnetic separability and high sensitivity.

Means for Solving the Problems

[0006] As a result of intensive studies to solve the above problems, the present inventors have found that magnetoresponsive particles that can realize a highly sensitive immunoassay reagent with good magnetic separability can be obtained by setting the magnetic substance density of the magnetoresponsive particles to a certain level or higher. That is, the present invention relates to the following contents. [1] Magnetoresponsive particles having at least one magnetic layer composed of core particles and fine particles of a magnetic metal and / or its oxide disposed on the core particles, and a substance that specifically acts on an object carried on the magnetoresponsive particles, When the volume and weight of the core particles are v c , w c , and the volume and weight of the magnetoresponsive particles are v e , w e , the magnetic substance density [(w e - w c ) / (v e - v c )] satisfies the following formula 1 2.0 ≦ (w e - w c ) / (v e - v c ) Formula 1 The sensitized magnetoresponsive particles satisfying the above. [2] The sensitized magnetoresponsive particles according to [1], having a magnetic separability of 40% or more. [3] The sensitized magnetoresponsive particles according to [1] or [2], further having a non-magnetic layer composed of a non-magnetic metal oxide and / or an organometallic compound between the magnetic layer and the substance that specifically acts on the object. [4] The sensitized magnetoresponsive particles according to any one of [1] to [3], wherein the substance that specifically acts on the object is chemically bonded onto the magnetic layer by one-step or multi-step reactions. [5] The sensitized magnetic-responsive particles according to [3], wherein the substance that specifically acts on the target is bonded to the non-magnetic layer via one or more stages of chemical bonds. [6] The sensitized magnetic-responsive particles according to any one of [1] to [5], wherein the coefficient of variation of the weight average particle diameter of the magnetic-responsive particles is 15% or less. [7] The sensitized magnetic-responsive particles according to [6], wherein the coefficient of variation of the volume average particle diameter of the magnetic-responsive particles is 20% or less. [8] A heterogeneous immunoassay method using the sensitized magnetic-responsive particles according to any one of [1] to [7]. [9] An immunoassay reagent using the sensitized magnetic-responsive particles according to any one of [1] to [7]. [Advantages of the Invention]

[0007] According to the present invention, there are provided magnetic-responsive particles having high magnetic aggregability despite their small particle diameters, and an immunoassay reagent having excellent magnetic separability and capable of achieving high sensitivity using the same. [Brief Description of the Drawings]

[0008]

Figure 1

Figure 2

[0009] Hereinafter, the present invention will be described with reference to embodiments, but the present invention is not limited to the following embodiments.

[0010] 1. Magnetic-responsive particles and method for producing the same As a result of intensive research, the inventor has found that when the volume and weight of the core particles are v c , w c , and the volume and weight of the magnetic-responsive particles are v e , w e , the magnetic density [(w e - w c ) / (v e - v c )] satisfies the following formula 1 2.0 ≦ (w e - w c ) / (v e - v c ) Formula 1 When this condition is satisfied, magnetic-responsive particles having a high magnetic separability of 40% or more can be obtained despite the small particle size. There has been no report on the relationship between the magnetic density and the magnetic separability. Hereinafter, each element and the like will be described in detail.

[0011] 1.1 Core particles The magnetic-responsive particles of the present invention have a magnetic layer composed of fine particles of at least one or more magnetic metals and / or their oxides disposed on core particles (core particles). The core particles of the present invention may be inorganic materials or organic materials and are not particularly limited. However, when used in immunoassay reagents, resin particles composed of resin are preferred because they have better dispersibility when the specific gravity is small. Resin particles are basically non-magnetic substances, and for example, organic substances such as polymers can be used.

[0012] The material constituting the resin particles is not particularly limited. For example, polyolefins such as polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinylidene chloride, polytetrafluoroethylene, polyisobutylene, and polybutadiene; acrylic resins such as polymethyl methacrylate and polymethyl acrylate; copolymer resins of acrylate and divinylbenzene, polyalkylene terephthalate, polysulfone, polycarbonate, polyamide, phenol formaldehyde resin, melamine formaldehyde resin, benzoguanamine formaldehyde resin, urea formaldehyde resin, etc. may be mentioned. These materials constituting the resin fine particles may be used alone or in combination of two or more.

[0013] In the present invention, the average particle diameter of the core particles is preferably 0.5 to 10 μm, more preferably 1 to 5 μm, and most preferably 2.5 to 4 μm. When the average particle diameter of the core particles is less than 0.5 μm, the area to which the magnetic substance can adhere per particle is small, and sufficient magnetic separability may not be obtained. Further, when the average particle diameter of the core particles exceeds 10 μm, when used as a biochemical carrier after adhering the magnetic substance, the surface area as a reaction field may be small.

[0014] Magnetic separability is an index indicating the responsiveness of magnetic-responsive particles to a magnet. A magnet is applied to a magnetic-responsive particle aqueous dispersion, and for example, the attenuation rate of the absorbance over time can be calculated and evaluated with a spectrophotometer (U-3900H manufactured by Hitachi, Ltd.). The larger the attenuation rate, the better the responsiveness to the magnet. When used as a reagent for immunoassay, it can be said to be a magnetic-responsive particle that can efficiently separate the target substance in a short time.

[0015] In addition, the coefficient of variation (CV value) of the volume average particle diameter of the core particles is 20% or less, preferably 15% or less, more preferably 10% or less. When using particles with a large CV value for the core, the surface area per particle varies, and when forming the magnetic layer, there may be variations in the amount of magnetic substance coating. Variations in the coating amount lead to variations in magnetic separability and may lead to deterioration of measurement reproducibility when used as an immunoassay reagent, which is not desirable. Regarding the control of the particle diameter, either a method of controlling the particle diameter in the manufacturing process of the core particles or a method of controlling the particle diameter by classification after producing the core particles can be applied, and these methods can also be used in combination.

[0016] The average particle diameter in the present invention can be determined, for example, by observing the particles with a scanning electron microscope ("S-4800" manufactured by Hitachi High-Technologies Corporation) and calculating the arithmetic mean of the maximum diameters of 50 randomly selected particles in the observed image.

[0017] The volume average particle diameter in the present invention is the volume average particle diameter obtained by measurement with, for example, a laser diffraction / scattering particle size distribution measuring device ("LS 13 320" manufactured by Beckman Coulter, Inc.).

[0018] The above core particles may have reactive functional groups on the particle surface. This can be used, for example, as a bonding site when coating the magnetic substance.

[0019] As the above core particles, particles that have absorbed or adsorbed fine powders of a liquid substance or a solid substance can also be used. Thereby, magnetic-coated particles containing the above liquid substance or solid substance inside and / or on the surface can be obtained. Note that the absorption or adsorption of the above substance means absorption or adsorption, or adhesion, etc. on the particle surface and inside the pores, and this absorption and adsorption can be carried out by a conventionally known method, such as impregnation.

[0020] 1.2 Magnetic Substance In the present invention, the magnetic metal and / or magnetic metal oxide that coats the surface of the core particles may use only a single species, or may be used in combination of two or more species. Further, the metal and / or metal oxide may be provided with reactive functional groups on the particle surface. This can be used, for example, as a bonding site when coating the core particles.

[0021] From the viewpoint of magnetic separability, the magnetic metal and / or magnetic metal oxide preferably contains at least one selected from the metals described in Groups 8 to 10 of the 4th to 6th periods of the periodic table and lanthanoids. Alternatively, iron oxide-based substances, specifically, ferrites represented by MFe2O4 (M = Co, Ni, Mn, Zn, Mg, Cu, Li 0.5 Fe 0.5 etc.), magnetite represented by Fe3O4, or γFe2O3 are preferred. In particular, Fe3O4 and γFe2O3 are most preferred as magnetic materials having a strong saturation magnetization and a small residual magnetization.

[0022] 1.3 Magnetic layer In the magnetic-coated particles (hereinafter referred to as "magnetic-responsive particles" in this specification) in the present invention, a magnetic layer is formed by adsorbing a magnetic metal and / or metal oxide on the surface of the core particles. Here, the metal and / or metal oxide can be coated on the surface of the resin particles by physical adsorption or chemical bonding. The physical adsorption of the metal and / or metal oxide in the present invention refers to adsorption and bonding without a chemical reaction. Examples include fusion bonding or adsorption, sintering bonding or adsorption, hydrogen bonding, van der Waals bonding, electrostatic interaction, heteroaggregation, etc. Coating by chemical bonding is that functional groups provided on the surface of the resin particles and the surface of the metal and / or metal oxide are bonded by a chemical reaction, and the metal and / or metal oxide is supported on the surface of the core particles. Among these, coating by physical adsorption is more preferred from the viewpoint of simplicity of preparation.

[0023] For the same core particles, magnetic composite particles may be produced by performing multiple complexation steps using a magnetic metal and / or metal oxide. In each step, the metal and / or metal oxide used for complexation is not particularly limited, and only a single species may be used, or two or more species may be used in combination. Also, the method of adding the metal and / or metal oxide is not particularly limited, and any of a batch method, a divided method, or a continuous addition method may be used. Further, when two or more species are used in any ratio in combination, the order of addition is not particularly limited, and all may be added after mixing, or all may be added separately, or some may be added after mixing and some may be added separately, etc., and they may be added in any order and combination. The number of addition times is also not particularly limited.

[0024] Also, if necessary, as a substance for forming a magnetic layer, a functional substance other than a magnetic substance may be added during complexation. There are no particular restrictions on the type of such functional substance, and it can be appropriately selected from organic substances or inorganic substances according to the purpose of complexation, and it is not limited to only a single species, and two or more species can also be used in combination. The purpose here refers to adding functions other than imparting magnetic separability, such as imparting electrical properties or coloring.

[0025] For the same core particles, complexation may be performed multiple times using a magnetic substance and / or a functional substance. In each step, the magnetic substance and / or functional substance used for complexation is not particularly limited, and only a magnetic substance may be used, only a functional substance may be used, or both a magnetic substance and a functional substance may be used. Also, for both the magnetic substance and the functional substance, only a single species may be used, or two or more species may be used in combination. Further, the addition method for each is not particularly limited, and any of a batch method, a divided method, or a continuous addition method may be used. When two or more species are used in any ratio in combination, the order of addition is not particularly limited, and all may be added after mixing, or all may be added separately, or some may be added after mixing and some may be added separately, etc., and they may be added in any order and combination. The number of addition times is also not particularly limited.

[0026] When performing complexation multiple times using a metal and / or metal oxide having magnetism on the same core particle, after forming a non-magnetic layer on the magnetic layer, a magnetic layer may be further formed, or a plurality of magnetic layers and non-magnetic layers may be alternately formed.

[0027] The magnetic substance content in the magnetic-responsive particles is preferably 10% by weight or more and 50% by weight or less. When the content of the magnetic substance exceeds 50% by weight, there is a concern that the specific gravity of the magnetic-responsive particles as the final product is also large, the sedimentation property increases, and the dispersibility of the particles deteriorates. In addition, when the magnetic substance content increases, the coefficient of variation (CV value) of the weight-average particle diameter increases accordingly, which may have an adverse effect on the reproducibility when used as an immunoassay reagent. If it is less than 10% by weight, sufficient magnetic separability cannot be obtained, and when used as an immunoassay reagent, separation and recovery become difficult.

[0028] The dispersibility of the magnetic-responsive particles can be evaluated by the dispersion rate, and the change rate of the absorbance can be calculated from the absorbance before magnetic collection of the magnetic-responsive particles and the absorbance after magnetic collection and dispersion. The dispersion rate is 85% or more, preferably 90% or more, and more preferably 95% or more. If the dispersion rate is less than 85%, the magnetic particles may not be sufficiently redispersed after magnetic separation, and the measurement accuracy, measurement sensitivity, and measurement reproducibility may decrease.

[0029] The coefficient of variation (CV value) of the weight-average particle diameter of the magnetic-responsive particles is preferably 15% or less, and more preferably 10% or less. The CV value of the weight-average particle diameter indicates the variation in particle diameter and the variation in density. A low CV value indicates that the particle diameter and density are uniform, while a high CV value indicates that the particle diameter or density or both are non-uniform. The CV value of the weight-average particle diameter of the present invention is a value obtained by, for example, a disk centrifuge particle size distribution measuring device ("DC24000UHR" manufactured by CPS Instruments). The particles of the present invention having a low CV value of the weight-average particle diameter of 10% or less have a uniform magnetic substance content (amount) among the particles. Therefore, the magnetic separability is better than that of conventional magnetic-responsive particles.

[0030] The average particle diameter of the magnetic-responsive particles is preferably 0.5 to 10 μm, more preferably 1 to 8 μm, and most preferably 2 to 5 μm. The average particle diameter of the present invention is a value obtained by, for example, a scanning electron microscope ("S-4800" manufactured by Hitachi High-Technologies Corporation).

[0031] The CV value of the volume average particle diameter of the magnetic-responsive particles is 20% or less, preferably 15% or less, and more preferably 10% or less. The CV value of the volume average particle diameter is a value reflecting the variation in particle diameter, indicating that the particle diameter is uniform when low and non-uniform when high.

[0032] The magnetic separability of the magnetic-responsive particles is preferably 40% or more, more preferably 50% or more, and most preferably 60% or more.

[0033] Note that the magnetic layer made of metal and / or metal oxide is about 10 to 200 nm, and the non-magnetic layer made of metal oxide and / or organometallic compound, which will be described later, is about 10 to 500 nm, and the average particle diameter of the finally obtained magnetic-responsive particles is about 0.5 to 10 μm.

[0034] 1.4 Non-magnetic layer The magnetic-responsive particles of the present invention may have a layer of non-magnetic metal oxide and / or non-magnetic organometallic compound on the surface of the magnetic layer. This non-magnetic layer is formed for coating the magnetic layer and / or imparting functionality to the particles themselves.

[0035] In applications as a biochemical carrier such as an immunoassay reagent, it can be selected according to the purpose of the surface characteristics of the non-magnetic layer. By forming the non-magnetic layer by the method described below, elution of impurities from the particles, elution of the magnetic substance itself, or elution of impurities from the magnetic layer can be firmly prevented, and in particular, a more suitable state as carrier particles for immunoassay reagents can be realized.

[0036] The non-magnetic layer can be formed by reacting a non-magnetic metal oxide and / or a non-magnetic organometallic compound as the main raw materials, and optionally adding other auxiliary raw materials in the presence of particles in a liquid phase. The non-magnetic metal oxide and / or the non-magnetic organometallic compound used at this time preferably has a functional group capable of reacting with the magnetic body surface. By using a metal oxide and / or an organometallic compound that directly reacts with the magnetic body surface, the magnetic layer and the non-magnetic layer are firmly bonded, maintaining high adhesion, and showing good effects in preventing leakage and immobilizing the components of the magnetic layer. In contrast, when using a radically polymerizable monomer typified by a vinyl-based monomer, since it does not directly bond to the magnetic body, the adhesion between the magnetic layer and the polymer layer is poor, the immobilization of the components of the magnetic layer is insufficient, and leakage of the components and instability of the shape and magnetic separability may occur.

[0037] The method of reacting the magnetic surface with the non-magnetic layer is not particularly limited, and examples include covalent bonds and coordination bonds.

[0038] Hereinafter, the main raw materials of the non-magnetic layer will be described. For convenience, only single-molecule compounds are exemplified, but it is sufficient to include one or more functional groups capable of reacting with the magnetic body surface in one molecule, and it may be a dimer to a polymer in which a plurality of single molecules are polycondensed. Also, the number of functional groups capable of reacting with the magnetic body is not particularly limited.

[0039] The non-magnetic metal oxide and / or non-magnetic organometallic compound contains at least one selected from Si, Ge, Ti, and Zr, and preferably has a functional group capable of reacting with the surface of the magnetic layer as described above. Specifically, silane compounds typified by tetraethyl orthosilicate and its hydrolyzates, germanium compounds typified by germanium tetraethoxide and its hydrolyzates, titanium compounds typified by titanium tetraethoxide and its hydrolyzates, zirconium compounds typified by zirconium tetrabutoxide and its hydrolyzates, etc. can be exemplified. Here, considering the maintenance of the dispersibility of the particles, the specific gravity of the non-magnetic layer is preferably as small as possible, and among the above examples, the silane compound is most preferable.

[0040] Furthermore, in the metal oxide and / or organometallic compound used as the main raw material of the non-magnetic layer, by using a metal oxide and / or organometallic compound having a portion with a separate function in addition to the functional group that reacts with the surface of the magnetic layer, the functions derived from the metal oxide and / or organometallic compound can also be provided to the magnetic responsive particles.

[0041] Regarding the metal oxide and / or organometallic compound having a portion with a separate function, specific examples will be given in detail taking silane compounds as an example, but the metal oxide and / or organometallic compound to be used is not limited to these.

[0042] For example, vinyl group-containing compounds such as vinyltrimethoxysilane, vinyltriethoxysilane, 7-octenyltrimethoxysilane; epoxy group-containing compounds such as 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, 8-glycidoxyoctyltrimethoxysilane; styryl group-containing compounds such as p-styryltrimethoxysilane; methacryl group-containing compounds such as 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 8-methacryloxyoctyltrimethoxysilane; acrylic group-containing compounds such as 3-acryloxypropyltrimethoxysilane; amino group-containing compounds such as N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, N-2-(aminoethyl)-3-aminopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-triethoxysilyl-N-(1,3-dimethyl-butylidene)propylamine, N-phenyl-3-aminopropyltrimethoxysilane, N-(vinylbenzyl)-2-aminoethyl-3-aminopropyltrimethoxysilane hydrochloride, N-2-(aminoethyl)-8-aminooctyltrimethoxysilane; isocyanurate group-containing compounds such as tris-(trimethoxysilylpropyl)isocyanurate; ureido group-containing compounds such as 3-ureidopropyltrialkoxysilane; mercapto group-containing compounds such as 3-mercaptopropylmethyldimethoxysilane, 3-mercaptopropyltrimethoxysilane; isocyanate group-containing compounds such as 3-isocyanatopropyltriethoxysilane; carboxylic anhydride-containing compounds such as 3-trimethoxysilylpropylsuccinic anhydride; carboxylic acid-containing compounds such as the hydrolysis product of 3-trimethoxysilylpropylsuccinic anhydride, and the like can be mentioned.

[0043] Among these, when used as a carrier for biochemistry, an epoxy group-containing compound, an amino group-containing compound, a mercapto group-containing compound, a carboxylic anhydride-containing compound, and a carboxylic acid-containing compound are preferable. Further, for example, those obtained by epoxidizing the double bond portions of vinyl group-containing compounds and styryl group-containing compounds, and those obtained by introducing vinyl group-containing compounds and styryl group-containing compounds into particles and then oxidizing the double bond portions to form epoxy groups, etc., which have undergone functional group conversion before and after introduction into the particles to improve the reactivity with biorelated substances, are similarly preferable.

[0044] The metal oxides and / or organometallic compounds used as the main raw materials for the nonmagnetic layer described above may be used alone or in any combination of two or more. When using two or more of these metal oxides and / or organometallic compounds, the combination of two or more of these metal oxides and / or organometallic compounds may be a combination of two or more metal oxides and / or organometallic compounds having separately functional parts, a combination of two or more metal oxides and / or organometallic compounds having no separately functional parts, or a combination of one or more metal oxides and / or organometallic compounds having separately functional parts and one or more metal oxides and / or organometallic compounds having no separately functional parts.

[0045] The method for adding the metal oxides and / or organometallic compounds in the formation of the nonmagnetic layer is not particularly limited, and may be any of a batch method, a divided method, or a continuous addition method. Further, when using two or more in any combination, the addition order is not particularly limited, and all may be mixed and added, or all may be added separately, or some may be mixed and some may be added separately, etc., and they can be added in any order and combination. The number of addition times is also not particularly limited.

[0046] It is also possible to repeatedly perform coating with a non-magnetic layer and coating with a magnetic layer on core particles whose surface is coated with a magnetic layer. For example, for core particles whose surface is coated with a magnetic layer, coating with a non-magnetic layer and then coating this surface layer with a magnetic layer and a non-magnetic layer in this order can be considered. At this time, as long as it contains at least one magnetic layer and the outermost layer is a non-magnetic layer, the number of coatings (number of layers), as well as the number (breakdown) and types of magnetic layers / non-magnetic layers in the entire coating layer with respect to the core particles are not limited. In addition, when two or more magnetic layers are included, compared with the case where only one magnetic layer is included, the magnetic substance content rate of the finished particles can be improved, and the magnetic responsiveness can be further enhanced.

[0047] When forming the non-magnetic layer, in addition to the main raw material metal oxide and / or organometallic compound, auxiliary raw materials can be used as necessary. The auxiliary raw materials are not particularly limited, but in accordance with the present invention, for example, when reacting the magnetic body surface with a metal oxide and / or an organometallic compound, generally an acid or a base is added to promote the reaction.

[0048] The coefficient of variation (CV value) of the weight average particle diameter of the magnetic responsive particles having a non-magnetic layer formed on the magnetic layer surface is preferably 15% or less, more preferably 10% or less. Also, the average particle diameter is preferably 0.5 to 10 μm, more preferably 1 to 8 μm, and most preferably 2 to 5 μm. The CV value of the volume average particle diameter is 20% or less, preferably 15% or less, more preferably 10% or less.

[0049] The volume and weight of the core particles are v c , w c , and the volume and weight of the magnetic responsive particles are v e , w e . When the magnetic substance density [(w e - w c ) / (v e - v c )] is given by the following formula 1 2.0 ≦ (w e - w c ) / (v e - v c ) Formula 1 It is preferable to satisfy this. This is because magnetic-responsive particles having a high magnetic separability of 40% or more can be obtained despite the small particle size. In this case, the magnetic density is preferably 2.0 to 5.0, and more preferably 2.0 to 3.5.

[0050] When the particle size of the core particles is R c When the thickness of the magnetic layer is De, it is preferably 0 nm < De ≤ 200 nm, more preferably 30 nm ≤ De ≤ 150 nm, further preferably 40 nm < De ≤ 120 nm, and even more preferably 50 nm < De ≤ 100 nm.

[0051] 1.5 Loading of the loading substance and the biologically related substance <Target substance> The target substance in the present invention means a substance to be measured and captured, for example, a substance present in a living body or a biological sample such as blood (whole blood), red blood cells, serum, plasma, urine, saliva, or sputum, such as a diseased tissue, a diseased cell, an inflammation-related marker such as CRP (C-reactive protein), IgA, IgG, IgM, a fibrin degradation product (for example, D-dimer), soluble fibrin, a coagulation / fibrinolysis marker such as TAT (thrombin-antithrombin complex), PIC (plasmin-plasmin inhibitor complex), oxidized LDL, BNP (brain natriuretic peptide), H-FABP (heart-type fatty acid-binding protein), a circulation-related marker such as cardiac troponin I (cTnI), a metabolism-related marker such as adiponectin, a tumor marker such as CEA (carcinoembryonic antigen), AFP (α-fetoprotein), PIVKA-II, CA19-9, CA125, PSA (prostate-specific antigen), an infectious disease-related marker such as HBV (hepatitis B virus), HCV (hepatitis C virus), Chlamydia trachomatis, Neisseria gonorrhoeae, a respiratory-related marker such as KL-6, an allergen-specific IgE (immunoglobulin E), a hormone, a drug, etc. are exemplified.

[0052] <Substance that acts> In the present invention, substances that specifically act on the target substance include proteins, peptides, amino acids, lipids, carbohydrates, DNA, RNA, receptors, haptens, biotin, avidin, etc. There is no particular limitation on the molecular weight or origin such as natural or synthetic. For example, antibodies or antigens that can be used in immunological assays utilizing immune reactions are included. Further, "act" means reaction, binding, etc.

[0053] The main use of the above magnetic-responsive particles is as a carrier for biochemistry such as immunological assay reagents. By immobilizing a target substance, a substance similar to the target substance, or a substance that specifically acts on the target substance (hereinafter, these may be collectively abbreviated as "substances for loading") on the particles as a carrier, magnetic-responsive particles serving as a carrier for biochemistry (hereinafter, referred to as "sensitized magnetic-responsive particles") can be produced.

[0054] The method for immobilizing the substance for loading on the magnetic-responsive particles of the present invention to produce sensitized magnetic-responsive particles is not particularly limited. It can be immobilized by conventionally known physical and / or chemical bonds. When immobilizing by chemical bonds, a non-magnetic layer is formed using a metal oxide and / or an organometallic compound having a functional group capable of binding to a biologically related substance as exemplified in paragraph 0042, and the functional group present on the surface of the magnetic-responsive particles is used as a scaffold for binding the substance for loading, whereby the substance for loading can be immobilized.

[0055] By the above method, it is preferable to provide an epoxy group, an amino group, a mercapto group, a carboxy group, or a carboxylic anhydride structure on the surface of the magnetic-responsive particles, and to support the substance for loading on the particle surface through these structures.

[0056] Alternatively, a biological substance that specifically binds to the above-mentioned supporting substance can be supported on magnetic-responsive particles, and the supporting substance can be bound to the magnetic-responsive particles through the biological substance in the reaction system. Examples of the biological substance that can be used for this purpose include avidin and streptavidin. Such magnetic-responsive particles carrying a biological substance for binding to the supporting substance can also be treated as sensitized magnetic-responsive particles.

[0057] The obtained sensitized magnetic-responsive particles are, if necessary, coated (blocked) with various polymer compounds and proteins (e.g., bovine serum albumin, etc.), and dispersed in an appropriate buffer solution to be used as a sensitized magnetic particle dispersion. The sensitized particle dispersion can be used as a reagent for immunoassay. Furthermore, by combining a diluent (buffer solution) and a standard substance used for measurement, etc., it can be used as a measurement reagent kit.

[0058] The coefficient of variation (CV value) of the weight-average particle diameter of the sensitized magnetic-responsive particles is preferably 15% or less, more preferably 10% or less. Also, the average particle diameter is preferably 0.5 to 10 μm, more preferably 1 to 8 μm, and most preferably 2 to 5 μm. The CV value of the volume-average particle diameter is 20% or less, preferably 15% or less, and more preferably 10% or less.

[0059] The above-mentioned reagent for immunoassay and diluent may contain various sensitizers for the purpose of improving the measurement sensitivity and promoting the specific reaction between the target substance and the supporting substance. Also, the above-mentioned reagent for immunoassay and diluent may contain various polymer compounds, proteins, and their degradation products, amino acids, surfactants, etc. for the purpose of suppressing non-specific reactions caused by substances other than the measurement target substance present in the measurement sample and improving the stability of the measurement reagent.

[0060] The method for measuring a substance to be measured in the immunoassay reagent of the present invention is not particularly limited, except that the method is carried out using the magnetically responsive particles of the present invention described above. For example, the method may be carried out in accordance with the sandwich method, competitive method, or the like, which are commonly used in this field and described in the literature (for example, "Enzyme Immunoassay Methods 2nd Edition," edited by Ishikawa Eiji et al., Igaku-Shoin, 1982).

[0061] The measurement of the target substance includes the steps of contacting the sample, sensitized magnetically responsive particles, labeled target-binding substance, labeled measurement target substance or its analogue, and B / F separation (separation of bound labeled antibody and unbound labeled antibody). The former step may be performed by dispersing the magnetic particles by a usual process such as stirring or mixing. The latter step may be performed, for example, by utilizing the magnetism of the magnetic particles, collecting the magnetic particles from the outside of the reaction vessel with a magnet or the like, discharging the reaction liquid, adding a washing liquid, removing the magnet, mixing and dispersing the magnetic particles, and washing them. The above operation may be repeated 1 to 3 times. The washing liquid is not particularly limited as long as it is one usually used in this field.

[0062] The measurement result of the target substance is calculated from the measured amount or activity of the target substance or its analogues, which are labeled with a labeling substance. The method for measuring the labeling substance or its activity can be any commonly used method without any particular limitation. Specific examples include radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA), electrochemiluminescence assay (ECLIA), chemiluminescence immunoassay (CLIA and CLEIA), absorbance measurement, surface plasmon resonance, etc. The optical equipment used for the measurement is also not particularly limited, and typically any biochemical automatic analyzer widely used in clinical testing can be used.

[0063] When using magnetic-responsive particles as a carrier for biochemistry such as immunoassay reagents as described above, high magnetic separability is very important. By using particles with excellent magnetic separability, the washing and purification efficiency can be improved, and the amount of particle loss can be reduced. Therefore, high performance can be exhibited in immunoassay reagents. The magnetic-responsive particles of the present invention can achieve high magnetic separability, so they are suitable as a carrier for biochemistry such as immunoassay reagents.

Example

[0064] 2. Example Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[0065] The average particle diameter, CV value of the CV value of the average density, magnetic separability, and magnetic substance content of the particles obtained in the examples and comparative examples were measured by the following methods.

[0066] 2.1 Physical property measurement method 2.1.1 Average particle diameter The particles were observed with a scanning electron microscope (Hitachi High-Technologies Corporation S-4800), and the arithmetic mean of the maximum diameters of 50 randomly selected particles in the observed image was obtained.

[0067] 2.1.2 Measurement of CV value of volume average particle diameter The volume average particle diameter distribution was measured with a laser diffraction / scattering particle size distribution measuring device (Beckman Coulter "LS 13 320"), and the CV value of the volume average particle diameter was calculated.

[0068] 2.1.3 Measurement of CV value of weight average particle diameter The weight average particle diameter distribution was measured with a disk centrifuge particle size distribution measuring device (CPS Instruments "DC24000UHR"), and the CV value of the weight average particle diameter was calculated. Specifically, an 8% and 24% sucrose solution was mixed, and the solution with a concentration gradient was rotated at 5000 rpm, and a particle aqueous dispersion (0.1 mL) adjusted to an absorbance of 1.0 was added and measured.

[0069] 2.1.4 Evaluation of Magnetic Separability Using a spectrophotometer (U-3900H manufactured by Hitachi, Ltd.), the absorbance at 550 nm was measured and used for evaluation. With a magnet (2800 G, W10 mm × D10 mm × H1 mm) applied to the bottom of the quartz cell installed in the spectrophotometer, a particle aqueous dispersion (1.3 mL) adjusted to an absorbance of 1.0 was introduced, and the absorbances 5 seconds and 125 seconds after sample introduction were measured. The absorbance decay rate during these 120 seconds was calculated and used as an index of magnetic separability.

[0070] 2.1.5 Measurement of Magnetic Substance Content The magnetic substance content of the magnetic-responsive particles is determined from the residue obtained by decomposing the resin portion when the particles are heated to 1000 °C under air. That is, the dry weight (A) of the magnetic-responsive particles is accurately weighed, and the temperature is raised from 35 °C to 1000 °C at a rate of 5 °C / min using a differential thermal thermogravimetric simultaneous measurement device (TG-DTA6300 manufactured by Hitachi High-Tech Science Corporation), and after maintaining at 1000 °C for 5 minutes, the weight (B) of the obtained residue is measured. The ratio of B to A is obtained as a percentage and used as the magnetic substance content.

[0071] 2.1.6. Evaluation of Dispersibility As a sample solution, a liquid in which magnetic particles adjusted to an absorbance of 1.0 at a wavelength of 550 nm were dispersed in water was prepared. 1.3 mL of the sample solution was introduced into a quartz cell installed in a spectrophotometer (U-3900H manufactured by Hitachi, Ltd.), and the absorbance at a wavelength of 550 nm was measured. Next, using a magnet (28000 G, W40 mm × D40 mm × H10 mm), magnetic collection was performed until the absorbance of the supernatant became 0. Thereafter, the magnetic particles were dispersed by vortexing at 2000 rpm for 5 seconds, and the absorbance at a wavelength of 550 nm was measured. From the absorbance before magnetic collection and the absorbance after magnetic collection and dispersion, the change rate of the absorbance was calculated by the following formula and used as the dispersibility. Dispersibility (%) = {(Absorbance after magnetic collection and dispersion) / (Absorbance before magnetic collection)} × 100

[0072] 2.2.1 Example 1-1 2.0 g of Micropearl EX-003 (particle size 3.01 μm, CV 3.1%, manufactured by Sekisui Chemical Co., Ltd.) as resin particles was ultrasonically dispersed in 40.0 g of ion-exchanged water to obtain a core particle dispersion liquid. Subsequently, while stirring under ultrasonic irradiation, 4.0 mL of magnetic fluid EMG707 (manufactured by Ferrotec Corporation) was added, and ultrasonic dispersion was further performed for 30 minutes. The obtained dispersion liquid was filtered, washed with ion-exchanged water, and excess magnetic fluid was removed to obtain magnetic-responsive particles [1].

[0073] 2.2.2 Example 1-2 1.0 g of the magnetic-responsive particles [1] obtained in Example 1-1 was ultrasonically dispersed in 400 g of ethanol. Thereafter, 10 mL of 28% aqueous ammonia solution (manufactured by Nacalai Tesque, Inc.), 1.0 g of tetraethyl orthosilicate, and 3.0 g of 8-glycidoxy octyltrimethoxysilane were added, and ultrasonic dispersion was performed for 3 hours. After the obtained dispersion liquid was filtered, dispersion in ion-exchanged water and centrifugation were repeated three times to obtain magnetic-responsive particles EP[1] having epoxy groups on the surface.

[0074] After ultrasonically dispersing the above magnetic-responsive particles EP[1] in PBS to a concentration of 3.0% by weight, 0.5 mL was aliquoted into a test tube. After collecting the magnetic-responsive particles EP[1] on the wall surface of the test tube with a magnet, the dispersion medium was removed, 0.5 mL of a PBS solution (0.75 mg / mL) of anti-KL-6 antibody was added, and sensitization was performed by stirring overnight at 25°C to obtain anti-KL-6 antibody-sensitized magnetic-responsive particles [1]. Thereafter, 1.5 mL of 1.0% by weight BSA solution was added, and stirring was performed at 25°C for 4 hours. After collecting the sensitized magnetic-responsive particles on the wall surface of the test tube with a magnet, the dispersion medium was removed, and 1.5 mL of 1.0% by weight BSA solution was newly added and dispersed. This operation was repeated three times to obtain a dispersion liquid of anti-KL-6 antibody-sensitized magnetic-responsive particles [1].

[0075] 2.2.3 Example 1-3 Streptavidin was dissolved in 0.1 M boric acid aqueous solution to prepare a streptavidin solution of 0.1 μg / mL. After ultrasonic dispersion of the magnetic-responsive particles EP[1] obtained in Example 1-2 to a concentration of 3.0 wt% in a 0.1 M boric acid aqueous solution, 0.5 mL was aliquoted into a test tube. After collecting the magnetic-responsive particles EP[1] on the wall surface of the test tube with a magnet, the dispersion medium was removed, 0.5 mL of the above streptavidin solution was added, and the mixture was stirred at 37 °C for 18 hours to obtain streptavidin-sensitized magnetic-responsive particles [1]. Thereafter, 0.5 mL of a 1.0 wt% BSA solution was added, and the mixture was stirred at a reaction temperature of 37 °C for 4 hours. After collecting the sensitized magnetic-responsive particles on the wall surface of the test tube with a magnet, the dispersion medium was removed, and newly, 1.5 mL of a 1.0 wt% BSA solution was added and dispersed. This operation was repeated 3 times to obtain a dispersion of streptavidin-sensitized magnetic-responsive particles [1].

[0076] 2.2.4 Example 2-1 As resin particles, 2.0 g of Micropearl SP-203 (particle size 3.02 μm, CV 4.9%, manufactured by Sekisui Chemical Co., Ltd.) was ultrasonically dispersed in 40.0 g of ion-exchanged water to obtain a core particle dispersion liquid. Subsequently, while stirring under ultrasonic irradiation, 4.0 mL of magnetic fluid EMG707 (manufactured by Ferrotec Corporation) was added, and further ultrasonic dispersion was performed for 30 minutes. The obtained dispersion liquid was filtered, washed with ion-exchanged water, and excess magnetic fluid was removed to obtain magnetic-responsive particles [2].

[0077] 2.2.5 Example 2-2 The same operations as in Example 1-2 were performed except that the magnetic-responsive particles [2] prepared in Example 2-1 were used, and anti-KL-6 antibody-sensitized magnetic-responsive particles [2] and a dispersion of anti-KL-6 antibody-sensitized magnetic-responsive particles [2] were obtained.

[0078] 2.2.6 Example 3-1 As resin particles, 2.0 g of Micropearl SP-203 (particle size 3.02 μm, CV 4.9%, manufactured by Sekisui Chemical Co., Ltd.) was ultrasonically dispersed in 40.0 g of ion-exchanged water to obtain a core particle dispersion liquid. Subsequently, while stirring under ultrasonic irradiation, 5.0 mL of magnetic fluid EMG707 (manufactured by Ferrotec Corporation) was added, and ultrasonic dispersion was further performed for 30 minutes. The obtained dispersion was filtered, washed with ion-exchanged water, and excess magnetic fluid was removed to obtain magnetic-responsive particles [3].

[0079] 2.2.7 Example 3-2 The same operations as in Example 1-2 were performed except that the magnetic-responsive particles [3] prepared in Example 3-1 were used, and anti-KL-6 antibody-sensitized magnetic-responsive particles [3] and an anti-KL-6 antibody-sensitized magnetic-responsive particles [3] dispersion were obtained.

[0080] 2.3.1 Comparative Example 1-1 As the magnetic-responsive particles, Magnosphere MS300 Tosyl (manufactured by JSR Life Sciences Co., Ltd.) was used as magnetic-responsive particles [4].

[0081] 2.3.2 Comparative Example 1-2 After ultrasonically dispersing the above magnetic-responsive particles [4] in PBS to a concentration of 3.0% by weight, 0.5 mL was aliquoted into a test tube. After collecting the magnetic-responsive particles [4] on the wall surface of the test tube with a magnet, the dispersion medium was removed, 0.5 mL of a PBS solution (0.75 mg / mL) of anti-KL-6 antibody was added, and sensitization was carried out by stirring overnight at 25°C to obtain anti-KL-6 antibody-sensitized magnetic-responsive particles [4]. Then, 1.5 mL of a 1.0% by weight BSA solution was added and stirred at 25°C for 4 hours. After collecting the sensitized magnetic-responsive particles on the wall surface of the test tube with a magnet, the dispersion medium was removed, and 1.5 mL of a new 1.0% by weight BSA solution was added and dispersed. This operation was repeated 3 times to obtain an anti-KL-6-sensitized magnetic-responsive particles [4] dispersion.

[0082] 2.3.3 Comparative Example 2 As the resin particles, 2.0 g of Micropearl SP-203 (particle size 3.02 μm, CV 4.9%, manufactured by Sekisui Chemical Co., Ltd.) was ultrasonically dispersed in 40.0 g of a 10 mM NaCl aqueous solution to obtain a core particle dispersion. Subsequently, while stirring under ultrasonic irradiation, 8.0 mL of magnetic fluid EMG707 (manufactured by Ferrotec Corporation) was added, and ultrasonic dispersion was further carried out for 30 minutes. The obtained dispersion was allowed to stand for 3 minutes. After removing the supernatant, it was redispersed in ion-exchanged water, filtered, and washed with ion-exchanged water to obtain magnetic-responsive particles [5].

[0083] 2.3.4 Reference Example Magnetic-responsive particles [6] were prepared in the same manner as in Example 2-1, except that 2.0 mL of magnetic fluid EMG707 (manufactured by Ferrotec Corporation) was used.

[0084] Table 1 shows the measurement results of the CV values of the volume-average particle diameter, the CV values of the weight-average particle diameter, the magnetic substance content, the magnetic separability, and the dispersibility for the particles of Examples 1-1 to 3-2, Comparative Examples 1-1 to 2, and the reference example. [Table 1]

[0085] For the magnetic-responsive particles of Examples 1 to 3, regardless of before and after sensitization, the CV value of the weight-average particle diameter was 15% or less, and the CV of the volume-average particle diameter was 20% or less. They had a similar particle diameter and showed better magnetic separability than the magnetic-responsive particles of Comparative Example 1, where the CV value of the weight-average particle diameter was greater than 15% and the CV of the volume-average particle diameter was greater than 20%. The magnetic-responsive particles of the present invention have a CV value of the weight-average particle diameter of 15% or less, and since the magnetic substance contained in each magnetic-responsive particle is uniform, it has been shown that they exhibit excellent magnetic separability. In addition, the magnetic-responsive particles of Comparative Example 2 had a CV of the weight-average particle diameter adjusted to 15% or more by magnetic purification. Although they exhibited high magnetic separability due to a high magnetic substance content, they showed low dispersibility due to the large specific gravity of the particles.

[0086] In addition, since the CV value of the volume average particle diameter of the magnetic responsive particles and the sensitized magnetic responsive particles of Example 3 is 15% or more, although they have practical magnetic separability, compared with the magnetic responsive particles and the sensitized magnetic responsive particles of Examples 1 and 2, the magnetic separability showed a low value. This suggests that when the variation in the volume average particle diameter increases, the magnetic separation also becomes non-uniform.

[0087] Reference Example 2 Magnetic responsive particles were obtained in the same manner as in Example 1-1 except that ion-exchanged water was replaced with a 0.1% aqueous sodium chloride solution to obtain a core particle dispersion liquid, and the magnetic substance content was improved compared to Example 1-1.

[0088] For some of the particles obtained in the above Examples and Comparative Examples, practicality evaluation was carried out by the following method.

[0089] 2.4.1 Reagent Evaluation Using the sensitized magnetic responsive particles obtained in Example 1-2 with anti-KL-6 antibody immobilized on the particle surface and the sensitized magnetic responsive particles of Comparative Example 1-2, the following immunoassay was performed, and the difference in luminescence amount was determined when immunoassay was performed using 0 U / mL of KL-6 concentration and when immunoassay was performed using a standard solution of 5000 U / mL and each antigen solution obtained by diluting the standard solution to 10, 50, 100, 500, 1000, 2500 U / mL.

[0090] Preparation of ruthenium complex-labeled anti-KL-6 antibody: 0.5 mL of a PBS solution (2.0 mg / mL) of anti-KL-6 antibody was added to a polypropylene tube, and then 13 μL of Ru-NHS (1 mg / mL) was added. After shaking and stirring at 25 °C, purification was performed using a Sephadex G25 column and used for evaluation.

[0091] KL-6 Immunoassay Method: Using an automatic analyzer (Picolumi III manufactured by Sekisui Medical Co., Ltd.) based on the principle of electrochemiluminescence immunoassay, the luminescence intensity was measured. After adding 20 μL of the sample to 200 μL of the reaction solution, 25 μL of anti-KL-6 antibody-conjugated magnetic particles was added. After reacting at 30 °C for 9 minutes, 350 μL of 10 mM Tris buffer was added, and the particles were washed 3 times while trapping the particles with a magnet. Next, 200 μL of a ruthenium-labeled antibody solution containing 1.0 μg / mL of ruthenium complex-labeled anti-KL-6 antibody was added, and after reacting at 30 °C for 9 minutes, 350 μL of 10 mM Tris buffer was added, and the particles were washed 3 times while trapping the particles with a magnet. 300 μL of a luminescence electrolyte solution containing 0.1 M tripropylamine was added, fed to the electrode surface, and the luminescence intensity of the ruthenium complex bound to the particles was measured.

[0092] The practicality evaluation results of the sensitized magnetic-responsive particles of Examples 1-2 and Comparative Examples 1-2 are shown below according to the above method.

Table 2

[0093] When the particles of Examples 1-2 were reacted with the same concentration of antigen compared to the particles of Comparative Examples 1-2, the difference in luminescence intensity from 0 U / mL was large and the sensitivity was high, that is, they showed good reagent performance. Compared with the particles of Comparative Examples 1-2, the CV values of the weight-average particle diameter and the volume-average particle diameter of the particles of Examples 1-2 were both low, and the magnetic separability was excellent. The movement of the particles in the liquid after antibody sensitization was uniform, and when the particles were trapped with a magnet, most of the particles did not vary and were efficiently captured. Therefore, it was considered that they showed good reagent performance in the reagent evaluation.

[0094] Example 4-1 2.0 g of Micropearl EX-003 (particle size 3.01 μm, CV 3.1%, manufactured by Sekisui Chemical Co., Ltd.) as resin particles (core particles) was ultrasonically dispersed in 40.0 g of a 0.1% sodium chloride aqueous solution to obtain a core particle dispersion. Subsequently, while stirring under ultrasonic irradiation, 4.0 mL of magnetic fluid EMG707 (manufactured by Ferrotec Corporation) was added, and ultrasonic dispersion was further performed for 30 minutes. The obtained dispersion was filtered, washed with ion-exchanged water, and excess magnetic fluid was removed to obtain magnetic-responsive particles [4-1].

[0095] Example 4-2, Example 4-3, Example 4-4, Example 4-5, Example 4-6 Magnetic-responsive particles [4-3] to [4-6] were obtained in the same manner as in Example 4-2, except that the addition amounts of magnetic fluid EMG707 were 2.0 mL (Example 4-2), 3.5 mL (Example 4-3), 2.5 mL (Example 4-4), 3.0 mL (Example 4-5), and 1.0 mL (Example 4-6), respectively.

[0096] Comparative Example 4-1, Comparative Example 4-2 As the magnetic-responsive particles, the following magnetic-responsive particles were obtained, respectively. Comparative Example 4-1: Magnosphere MS300 COOH (particle size 2.80 μm, CV value of weight-average particle size: 19.1%, CV value of volume-average particle size: 30.4%, manufactured by JSR Life Sciences Co., Ltd.) Comparative Example 4-2: M280 (particle size 2.8 μm, CV value of weight-average particle size: 6.3%, CV value of volume-average particle size: 8.3%, manufactured by Dynabeads Corporation). Note that the particles of Comparative Example 4-1 are core-shell type particles, and the particles of Comparative Example 4-2 are particles in which iron is distributed throughout the inside of the mother particles.

[0097] The physical properties of each of the obtained magnetic-responsive particles were measured. At that time, the average particle size, magnetic substance content ratio, and magnetic substance content were measured based on the descriptions of "average particle size" and "measurement of magnetic substance content ratio" in the column of the above physical property measurement method. The magnetic separability was measured based on the description of "evaluation of magnetic separability" in the column of the above physical property measurement method. The thickness of the magnetic substance layer and the magnetic substance density were calculated based on the following formula. Note that the physical property measurement and calculation were also performed in the same manner for Examples 5-1 to 5-5 and Comparative Examples 5-1 to 5-2 described later. · Thickness of magnetic substance layer = (average particle size - particle size of mother particle) / 2 ·Magnetic substance density = {(weight after magnetic substance adsorption) - (weight before magnetic substance adsorption)} / {(volume after magnetic substance adsorption) - (volume before magnetic substance adsorption)}

[0098] The physical properties (average particle diameter, magnetic substance density, magnetic substance content, magnetic separability, thickness of magnetic substance layer, magnetic substance content rate) of the obtained magnetoresponsive particles are collectively shown in Table 3.

Table 3

[0099] Figure 1A shows the relationship between the magnetic substance content and the magnetic separability when the particle diameter of the core particles is fixed and the magnetic substance content (thickness of the magnetic layer) is changed. In Figure 1A, the circles indicate examples and the triangles indicate comparative examples (the same notation will be used in the following figures). From Figure 1A, it was confirmed that as the magnetic substance content increases, the magnetic separability improves. Figure 1B shows the relationship between the magnetic separability and the magnetic substance density when the particle diameter of the core particles is fixed and the magnetic substance content (thickness of the magnetic layer) is changed. From the comparison between the example group (4-1 to 4-6) and the comparative example group (4-1 to 4-2) in Figure 1B, it was confirmed that when the magnetic substance density is 2.00 or more, the magnetic separability is 40% or more. Also, in Examples 4-1 to 4-6, it was confirmed that the magnetic separability can be controlled by controlling the magnetic substance density.

[0100] Example 5-1 2.0 g of Micropearl EX-002 (particle diameter 2 μm, CV 3.1%, manufactured by Sekisui Chemical Co., Ltd.) as resin particles (core particles) was ultrasonically dispersed in 40.0 g of 0.1% aqueous sodium chloride solution to obtain a core particle dispersion liquid. Subsequently, while stirring under ultrasonic irradiation, 6.0 mL of magnetic fluid EMG707 (manufactured by Ferrotec Corporation) was added, and further ultrasonically dispersed for 30 minutes. The obtained dispersion liquid was filtered, washed with ion-exchanged water, and excess magnetic fluid was removed to obtain magnetoresponsive particles [5-1].

[0101] Examples 5-2, 5-3, 5-4, 5-5 The particle sizes of the resin particles were 2.5 μm (Example 5-2), 3.0 μm (Example 5-3), 3.5 μm (Example 5-4), and 4 μm (Example 5-5), and the magnetic fluid EMG707 (manufactured by Ferrotec Corporation) added was 4.8 mL (Example 5-2), 4.0 mL (Example 5-3), 3.4 mL (Example 5-4), and 3.4 mL (Example 5-5). Magnetic-responsive particles [5-2], [5-3], [5-4], and [5-5] were obtained in the same manner as in Example 5-1, except for the above. Note that the magnetic-responsive particles of Example 5-3 were prepared under the same conditions as those of Example 4-4 described above. However, for ease of comparative study, they are referred to as Example 5-3 here.

[0102] The physical properties (average particle diameter, magnetic substance density, magnetic substance content, magnetic separability, thickness of the magnetic substance layer, magnetic substance content ratio) of the obtained magnetic-responsive particles [5-1] to [5-5] are summarized in Table 4. The relationship between the magnetic substance content and the magnetic separability is shown in Fig. 2A, and the relationship between the magnetic separability and the magnetic substance density is shown in Fig. 2B.

Table 4

[0103] Fig. 2A shows the relationship between the magnetic substance content and the magnetic separability when the particle diameter of the core particles is changed. From Fig. 2A, it was confirmed that as the magnetic substance content increases, the magnetic separability improves. Fig. 2B shows the relationship between the magnetic substance density and the magnetic separability when the particle diameter of the core particles is changed. From the comparison between the example group (5-1 to 5-5) and the comparative example group (5-1 to 5-2) in Fig. 2B, it was confirmed that when the magnetic substance density is 2.00 or more, the magnetic separability is 40% or more. Also, in Examples 5-1 to 5-5, it was confirmed that the magnetic separability can be controlled by controlling the magnetic substance density.

[0104] Example 5-1-2 1.0 g of the magnetic-responsive particles [5-1] obtained in Example 5-1 was ultrasonically dispersed in 400 g of ethanol. Then, 10 mL of 28% aqueous ammonia solution (manufactured by Nacalai Tesque, Inc.), 1.0 g of tetraethyl orthosilicate, and 3.0 g of 8-glycidoxy octyltrimethoxysilane were added, and ultrasonic dispersion was performed for 3 hours. After filtering the obtained dispersion, dispersion in ion-exchanged water and centrifugation were repeated three times to obtain magnetic-responsive particles [5-1-1] having epoxy groups on the surface.

[0105] After ultrasonically dispersing the above magnetic-responsive particles [5-1-1] in PBS to a concentration of 3.0 wt%, 0.5 mL was aliquoted into a test tube. Using a magnet, the magnetic-responsive particles [5-1-1] were collected on the wall surface of the test tube, and then the dispersion medium was removed. 0.5 mL of a PBS solution (0.75 mg / mL) of anti-KL-6 antibody was added, and sensitization was carried out by stirring overnight at 25 °C to obtain anti-KL-6 antibody-sensitized magnetic-responsive particles [5-1-2]. Then, 1.5 mL of 1.0 wt% BSA solution was added, and stirring was carried out at 25 °C for 4 hours. After collecting the sensitized magnetic-responsive particles on the wall surface of the test tube with a magnet, the dispersion medium was removed, and 1.5 mL of 1.0 wt% BSA solution was newly added and dispersed. This operation was repeated three times to obtain a dispersion of anti-KL-6 antibody-sensitized magnetic-responsive particles [5-1-2].

[0106] Example 5-2-2, Example 5-3-2, Example 5-5-2 Anti-KL-6 antibody-sensitized magnetic-responsive particles [5-2-2], [5-3-2], and [5-5-2] were obtained in the same manner as in Example 5-1-2, except that the magnetic-responsive particles used in the reaction were [5-2] (Example 5-2-1), [5-3] (Example 5-3-1), and [5-5] (Example 5-5-1).

[0107] Comparative Example 4-1-2 After ultrasonically dispersing the above Comparative Example 4-1 particles in PBS to a concentration of 3.0% by weight, 0.5 mL was aliquoted into a test tube. After collecting the magnetic-responsive particles on the wall surface of the test tube using a magnet, the dispersion medium was removed. Then, 0.5 mL of a PBS solution of anti-KL-6 antibody (0.75 mg / mL) and 0.1 mL of an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride at 10 mg / mL were added, and the mixture was sensitized by stirring overnight at 25°C to obtain anti-KL-6 antibody-sensitized magnetic-responsive particles. Thereafter, 1.5 mL of a 1.0% by weight BSA solution was added, and the mixture was stirred at 25°C for 4 hours. After collecting the sensitized magnetic-responsive particles on the wall surface of the test tube using a magnet, the dispersion medium was removed, and 1.5 mL of a fresh 1.0% by weight BSA solution was added and dispersed. This operation was repeated three times to obtain a dispersion of anti-KL-6-sensitized magnetic-responsive particles [4-1-2]. According to the above KL-6 immunoassay method, practicality evaluations were performed on the anti-KL-6 antibody-sensitized magnetic-responsive particles of Example 4-1-2, Example 5-1-2, 5-2-2, 5-3-2, Example 5-5-2, and Comparative Example 4-1-2. The obtained results are shown below.

Table 5

[0108] The particles of Example 5-1-2, Example 5-2-2, Example 5-3-2, and Example 5-5-2 showed a large difference in luminescence intensity from 0 U / mL and high sensitivity, that is, good reagent performance. The particles of Example 5-1-2, 5-2-2, 5-3-2, and 5-5-2 had a low particle CV and excellent magnetic separability of 40% or more. They were particles with a narrow particle size distribution and high magnetic separability, and since the movement of the particles in the liquid after antibody sensitization was uniform, when the particles were trapped with a magnet, most of the particles were not scattered and were efficiently captured, showing good reagent performance. Also, by reducing the particle diameter, the surface area per unit weight could be increased. As a result, an increase in luminescence intensity for each antigen concentration, that is, high-sensitivity reagent performance with a large difference in luminescence intensity from 0 U / mL was shown.

Industrial Applicability

[0109] According to the present invention, by using magnetic-responsive particles with a CV value of the weight-average particle diameter of 15% or less, a highly sensitive immunoassay reagent that can easily carry biological-related substances and has high separation and purification efficiency due to excellent magnetic separability is provided. According to the present invention, magnetic-responsive particles having high magnetic aggregability despite their small particle diameter, and a reagent for immunoassay having excellent magnetic separability using the same and capable of realizing high sensitivity are provided.

Claims

1. Magnetoresponsive particles having at least one magnetic layer composed of core particles and fine particles of a magnetic metal and / or its oxide disposed on the core particles, and a substance that specifically acts on the target carried on the magnetoresponsive particles, The volume and weight of the core particles are v ｃ , w ｃ , the volume and weight of the magnetic-responsive particles are v ｅ , w ｅ When the magnetic density [(w ｅ - w ｃ ) / (v ｅ - v ｃ )] satisfies the following formula 1 2.0 ≤ (w ｅ - w ｃ ) / (v ｅ - v ｃ ) Equation 1 The sensitized magnetoresponsive particles satisfying the above.

2. The sensitized magnetoresponsive particles according to Claim 1, having a magnetic separability of 40% or more.

3. The sensitized magnetoresponsive particles according to Claim 1 or 2, further having a nonmagnetic layer composed of a nonmagnetic metal oxide and / or an organometallic compound between the magnetic layer and the substance that specifically acts on the target.

4. The sensitized magnetoresponsive particles according to any one of Claims 1 to 3, wherein the substance that specifically acts on the target is chemically bonded onto the magnetic layer by one or multiple steps of reaction.

5. The sensitized magnetoresponsive particles according to Claim 3, wherein the substance that specifically acts on the target is bonded via one or multiple steps of chemical bonding onto the nonmagnetic layer.

6. The sensitized magnetoresponsive particles according to any one of Claims 1 to 5, wherein the coefficient of variation of the weight average particle diameter of the magnetoresponsive particles is 15% or less.

7. The sensitized magnetoresponsive particles according to Claim 6, wherein the coefficient of variation of the volume average particle diameter of the magnetoresponsive particles is 20% or less.

8. A heterogeneous immunoassay method using the sensitized magnetoresponsive particles according to any one of Claims 1 to 7.

9. An immunoassay reagent using the sensitized magnetoresponsive particles according to any one of Claims 1 to 7.

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