Magnetic-responsive particles and immunoassay methods and reagents using them

Abstract

This invention discloses a sensitized magnetically responsive particle, comprising: a magnetically responsive particle having a nucleus and at least one magnetic layer disposed on the nucleus, the magnetic layer containing particles of a magnetic metal and / or its oxide; and a substance that specifically interacts with an analyte, the substance being loaded onto the magnetically responsive particle, wherein it is assumed that the volume and weight of the nucleus are v0 and v1, respectively. c and w c Furthermore, the volume and weight of the magnetically responsive particles are v0 and v1, respectively. e and w e Then the density of the magnetic material [(w e -w c ) / (v e -v c The expression 1 satisfies the following condition: 2.0 ≤ (w) e -w c ) / (v e -v c Expression 1. Magnetic-responsive particles have a small particle size but high magnetic collection ability. When magnetic-responsive particles are used, reagents for immunoassays with excellent magnetic separation rates and high sensitivity can be provided.

Description

Technical Field

[0001] This invention relates to magnetically responsive particles used in immunoassay reagents, as well as immunoassay methods and reagents using said particles. Background Technology

[0002] As a procedure for measuring and / or purifying a target protein from a sample containing biological material, known assays include: immobilizing a substance that specifically interacts with the analyte onto a solid support; binding the substance to the analyte in the biological sample; washing away unbound material other than the analyte; and measuring the amount of analyte bound to the solid support.

[0003] Magnetically responsive particles are used as solid loads because unbound material is easily separated and collected upon removal. For example, Patent Document 1 discloses clinical test reagent particles as such particles, which have a magnetic layer formed on the surface of a nuclear particle and a polymer layer formed thereon. However, regarding the disclosed particles, only particles with a wide range of particle sizes are obtained.

[0004] In addition to the problems mentioned above, further improvements are needed to enhance the magnetic responsiveness of magnetically responsive particles.

[0005] List of cited references

[0006] Patent documents

[0007] Patent Document 1: Japanese Patent Publication No. 2004-205481 Summary of the Invention

[0008] Technical issues

[0009] One way to improve magnetic responsiveness is to increase the content of magnetic material. However, as the content of magnetic material increases, the average particle size increases and the particle surface area decreases relative to weight, which leads to a decrease in the binding volume with biochemical substances.

[0010] Therefore, there is a need for a particle that can achieve high magnetic collection properties without reducing the particle surface area.

[0011] The purpose of this invention is to provide a magnetically responsive particle with small particle size but high magnetic collection ability, and an immunoassay reagent using said magnetically responsive particle that can achieve excellent magnetic separation rate and high sensitivity.

[0012] Solution to the problem

[0013] To address the aforementioned problems, the inventors conducted in-depth research and discovered that magnetically responsive particles with a magnetic material density not less than a specific value can produce magnetically responsive particles with good magnetic separation efficiency and capable of realizing highly sensitive reagents for immunoassays. Specifically, this invention relates to the following items:

[0014] [1] A sensitized magnetically responsive particle, the sensitized magnetically responsive particle comprising:

[0015] Magnetic-responsive particles, the magnetic-responsive particles having a nucleus and at least one magnetic layer disposed on the nucleus, the magnetic layer comprising particles of a magnetic metal and / or its oxide; and

[0016] A substance that specifically interacts with the analyte, said substance being loaded onto magnetically responsive particles.

[0017] Here, it is assumed that the volume and weight of the nuclear particle are v, respectively. c and w c Furthermore, the volume and weight of the magnetically responsive particles are v0 and v1, respectively. e and w e Then the density of the magnetic material [(w e -w c ) / (v e -v c The following expression 1 is satisfied:

[0018] 2.0≤(w e -w c ) / (v e -v c Expression 1.

[0019] [2] The sensitized magnetic responsive particles according to [1], wherein the magnetic separation rate is 40% or more.

[0020] [3] According to [1] or [2], the sensitized magnetic responsive particles further comprise a non-magnetic layer between the magnetic layer and the substance that specifically interacts with the analyte, the non-magnetic layer comprising a non-magnetic metal oxide and / or organometallic compound.

[0021] [4] Sensitized magnetic responsive particles according to any one of [1] to [3], wherein the substance that specifically interacts with the analyte is chemically bonded to the magnetic layer through one or more steps of reaction.

[0022] [5] According to [3], the sensitized magnetic responsive particles, wherein the substance that specifically interacts with the analyte is bonded to a nonmagnetic layer via one or more chemical bonds.

[0023] [6] The sensitized magnetically responsive particles according to any one of [1] to [5], wherein the coefficient of variation of the weight-average particle size of the magnetically responsive particles is less than 15%.

[0024] [7] The sensitized magnetically responsive particles according to [6], wherein the coefficient of variation of the volume average particle size of the magnetically responsive particles is less than 20%.

[0025] [8] A heterogeneous immunoassay method, the method using sensitized magnetically responsive particles according to any one of [1] to [7].

[0026] [9] An immunoassay reagent comprising sensitized magnetically responsive particles according to any one of [1] to [7].

[0027] Beneficial effects of the invention

[0028] The present invention can provide a magnetically responsive particle with small particle size but high magnetic collection ability, and an immunoassay reagent using the magnetically responsive particle with excellent magnetic separation rate and high sensitivity. Attached Figure Description

[0029] [Figure 1] Figure 1A This is a graph showing the relationship between magnetic material content and magnetic separation rate when the magnetic material content (magnetic layer thickness) is varied while keeping the nuclear particle size constant. Figure 1B This is a graph showing the relationship between magnetic separation rate and magnetic material density when the magnetic material content (magnetic layer thickness) is varied while keeping the nuclear particle size constant.

[0030] [Figure 2] Figure 2A This is a graph showing the relationship between the magnetic material content and magnetic separation rate obtained when the nuclear particle size is changed. Figure 2B This is a graph showing the relationship between the density and magnetic separation rate of the magnetic material obtained when the particle size of the nuclear particles is changed. Detailed Implementation

[0031] The invention will be described below with reference to embodiments. However, the invention is not limited to the following embodiments.

[0032] 1. Magnetic-responsive particles and their preparation methods

[0033] As a result of in-depth research, the inventors discovered that: when assuming the volume and weight of the nuclear particle are respectively v c and w c Furthermore, the volume and weight of the magnetically responsive particles are v e and w e Magnetic material density [(w e -w c) / (v e -v c When the following expression 1 is satisfied:

[0034] 2.0≤(w e -w c ) / (v e -v c Expression 1

[0035] It is possible to obtain magnetically responsive particles with small particle size but a high magnetic separation rate of over 40%.

[0036] The relationship between the density of magnetic materials and the magnetic separation rate has not been reported before. The various factors will be described in detail below.

[0037] 1.1 Nuclear particles

[0038] The magnetically responsive particles of the present invention have at least one magnetic layer disposed on a particle (nuclear particle) serving as a nucleus and comprising a magnetic metal and / or its oxide.

[0039] The nuclear particles of the present invention can be made of inorganic or organic materials and are not particularly limited thereto. However, when used as an immunoassay reagent, resin particles made of resin are preferred because their lower specific gravity provides better dispersibility. The resin particles are essentially non-magnetic materials and, for example, organic materials (such as polymers) can be used.

[0040] Examples of materials constituting the aforementioned resin particles include, but are not particularly limited to: polyolefins, such as polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinylidene chloride, polytetrafluoroethylene, polyisobutylene, and polybutadiene; acrylic resins, such as polymethyl methacrylate and polymethyl acrylate; acrylate / divinylbenzene copolymer resins, polyalkylene terephthalate, polysulfone, polycarbonate, polyamide, phenolic resins, melamine-formaldehyde resins, benzoguanamine-formaldehyde resins, and urea-formaldehyde resins. These materials constituting the resin particles may be used alone or in combination of two or more.

[0041] The average particle size of the nuclear particles in this invention is preferably 0.5 to 10 μm, more preferably 1 to 5 μm, and most preferably 2.5 to 4 μm. If the average particle size is less than 0.5 μm, the area on which the magnetic material can adhere to each particle is small. This may result in insufficient magnetic separation. Furthermore, if the average particle size exceeds 10 μm, the surface area serving as reaction sites may become smaller when the nuclear particles are used as carriers for biochemical applications after being attached to magnetic materials.

[0042] Magnetic separation efficiency is an indicator of the response of magnetically responsive particles to a magnet. It can be evaluated by applying a magnet to an aqueous dispersion of the magnetically responsive particles and calculating the decrease in absorbance over time using, for example, a spectrophotometer (U-3900H, manufactured by Hitachi, Ltd.). A greater decrease indicates a better response to the magnet. When used as a reagent in immunoassays, this means that magnetically responsive particles can separate analytes more effectively in a shorter time.

[0043] Furthermore, the coefficient of variation (CV value) of the volume average particle size of the nuclear particles is 20% or less, preferably 15% or less, and more preferably 10% or less. Using particles with large CV values ​​as the nucleus leads to variations in the surface area of ​​each particle. This can result in variations in the amount of magnetic material coating during the formation of the magnetic layer. Variations in coating are undesirable because they lead to changes in magnetic separation efficiency, and therefore, in the case of use as an immunoassay reagent, assay reproducibility may deteriorate. To control particle size, any method for controlling particle size can be applied: a method of performing a particle size control process during the step of manufacturing the nuclear particles; or a method of performing a particle size control process by fractionation after the manufacturing of the nuclear particles. In addition, these methods can be used in combination.

[0044] The average particle size in this invention can be determined by observing the particles, for example, under a scanning electron microscope (“S-4800”, manufactured by Hitachi High-Technologies Corporation), and calculating the average of the maximum diameters of 50 randomly selected particles in the observed image.

[0045] The volume average particle size in this invention is obtained by measuring, for example, a laser diffraction and scattering particle size distribution analyzer (“LS13 320”, manufactured by Beckman Coulter Inc.).

[0046] The aforementioned nuclear particles can have reactive functional groups on their surface. For example, this can be used as a binding site when coating magnetic materials.

[0047] The aforementioned nuclear particles can be particles that absorb or adsorb fine powders of liquid or solid materials. This can produce particles coated with magnetic materials containing the aforementioned liquid or solid materials internally and / or on their surface. Note that the aforementioned material absorption / adsorption means absorption / adsorption or adhesion, etc., through the particle surface and inside the pores / on the particle surface and inside the pores. This absorption and adsorption can be carried out by conventionally known procedures, such as impregnation.

[0048] 1.2 Magnetic Materials

[0049] The magnetic metal and / or magnetic metal oxide used in this invention for coating the surface of nuclei can be used alone or in combination of two or more. Furthermore, the metal and / or metal oxide can have reactive functional groups on the surface of the particle. For example, this can be used as a binding site when coating nuclei.

[0050] From the perspective of magnetic separation rate, the magnetic metal and / or magnetic metal oxide preferably contains at least one metal selected from Groups 8 to 10 of Periods 4 to 6 of the periodic table or a lanthanide element. Alternatively, iron oxide-based materials are preferred. Specific examples include MFe₂O₄ (where M = Co, Ni, Mn, Zn, Mg, Cu, Li). 0.5 Fe 0.5 Ferrites such as Fe3O4, Fe3O4, or γFe2O3 are preferred. Fe3O4 or γFe2O3 are particularly preferred as magnetic materials with strong saturation magnetization and low remanent magnetization.

[0051] 1.3 Magnetic Layer

[0052] The magnetic metal-coated particles of this invention (hereinafter referred to as "magnetically responsive particles") have a magnetic layer formed by adsorbing magnetic metals and / or metal oxides onto the surface of the core particle. Here, the metals and / or metal oxides can be coated by physical adsorption or via one or more chemical bonds on the surface of the resin particles. Physical adsorption of metals and / or metal oxides in this invention refers to adsorption / bonding without any chemical reaction. Examples include melt bonding or adsorption, fusion bonding or adsorption, hydrogen bonding, van der Waals bonding, electrostatic interaction, or heterogeneous aggregation. Coating via one or more chemical bonds means that functional groups provided on the surface of the resin particles bond with functional groups provided on the metals and / or metal oxides through a chemical reaction, such that the surface of the core particle is loaded with the metals and / or metal oxides on the surface of the core particle. Among these coating methods, coating by physical adsorption is preferred because it is convenient to prepare.

[0053] Using magnetic metals and / or metal oxides, the formation of the composite can be repeated multiple times on the same nucleus to prepare magnetically responsive particles. In each step, there are no particular restrictions on the metals and / or metal oxides used to form the composite, and they can be used alone or in combination of two or more. Furthermore, there are no particular restrictions on the method of adding the metals and / or metal oxides, and any of batch, partial, or continuous addition processes is permitted. Moreover, when two or more metals and / or metal oxides are used in combination at a given ratio, there are no particular restrictions on the order of addition. They can be all mixed and added, each can be added individually, some can be mixed and others added individually, and so on. They can be added in any given order and combination. There are also no particular restrictions on the number of times they are added.

[0054] Furthermore, if desired, functional materials other than magnetic materials can be added as magnetic layer forming materials during the formation of the composite. There are no particular limitations on the type of such functional materials, and they can be selected from organic or inorganic materials, depending on the purpose of composite formation, if appropriate. The type is not limited to just one, and two or more can be used in combination. The purpose here refers, for example, to adding one or more functions besides imparting magnetic separation, such as imparting electrical properties, coloring, etc.

[0055] The formation of the complex can be repeated multiple times on the same nucleoparticle using magnetic and / or functional materials. There are no particular restrictions on the magnetic and / or functional materials used to form the complex in each step. Only magnetic materials or only functional materials may be used. Alternatively, both magnetic and functional materials may be used. Furthermore, either magnetic or functional materials may be used alone, or in combination of two or more. Moreover, there are no particular restrictions on each process of adding materials. Batch, intermittent, or continuous addition processes are permitted. When two or more are used in combination at a given ratio, there are no particular restrictions on the order of addition. They can be all mixed and added, each can be added individually, some can be mixed and others added individually, and so on. They can be added in any given order and combination. There are also no particular restrictions on the number of times they are added.

[0056] When a complex is repeatedly formed on the same nucleus particle using magnetic metals and / or metal oxides, a non-magnetic layer can be formed on top of a magnetic layer, followed by the formation of a further magnetic layer. Magnetic and non-magnetic layers can be formed alternately to create multiple layers.

[0057] The magnetic material content of the magnetically responsive particles is preferably between 10% and 50% by weight. When the magnetic material content exceeds 50% by weight, the specific gravity of the magnetically responsive particles in the final product is relatively high, and the sedimentation rate increases. This raises concerns about poor particle dispersibility. Furthermore, the higher the magnetic material content, the larger the coefficient of variation (CV) of the weight-average particle size, which may adversely affect reproducibility when used as an immunoassay reagent. If the content is less than 10% by weight, sufficient magnetic separation cannot be obtained, and therefore separation and collection become difficult when used as an immunoassay reagent.

[0058] The dispersibility of magnetically responsive particles can be evaluated by the dispersion rate. The dispersion rate can be calculated by the rate of change of absorbance from the magnetically responsive particles before magnetic collection and after magnetic collection and dispersion. The dispersion rate is 85% or more, preferably 90% or more, and even more preferably 95% or more. If the dispersion rate is less than 85%, the magnetic particles after magnetic separation are not sufficiently redispersed, which may lead to a decrease in measurement precision, measurement sensitivity, and measurement reproducibility.

[0059] The coefficient of variation (CV) of the weight-average particle size of the magnetically responsive particles is preferably less than 15%, and more preferably less than 10%. The CV value of the weight-average particle size indicates how the particle size varies and how the density varies. A lower CV value indicates uniform particle size and uniform density. A higher CV value indicates non-uniform particle size or density, or both. The CV value of the weight-average particle size in this invention is obtained using, for example, a disk centrifugal particle size distribution analyzer (“DC24000UHR”, manufactured by CPS Instruments, Inc.). The CV value of the weight-average particle size of the particles in this invention is as low as 10%, and the particles in this invention each have similar amounts of magnetic material. Therefore, the magnetic separation rate is superior to that of conventional magnetically responsive particles.

[0060] The average particle size of the magnetically responsive particles is preferably 0.5 to 10 μm, more preferably 1 to 8 μm, and even more preferably 2 to 5 μm. The average particle size in this invention is a value obtained using, for example, a scanning electron microscope (“S-4800”, manufactured by Hitachi High-Technologies Corporation).

[0061] The volume-average particle size (CV) value of the magnetically responsive particles is 20% or less, preferably 15% or less, and more preferably 10% or less. The CV value of the volume-average particle size reflects the variation in particle size. This indicates that if the value is low, the particle size is uniform, and if the value is high, the particle size is non-uniform.

[0062] The magnetic separation rate of the magnetically responsive particles is preferably 40% or more, more preferably 50% or more, and most preferably 60% or more.

[0063] Please note that the magnetic layer, composed of metals and / or metal oxides, has a thickness of about 10 to 200 nm; the non-magnetic layer, composed of metal oxides and / or organometallic compounds, has a thickness of about 10 to 500 nm; and the resulting magnetically responsive particles have an average particle size of about 0.5 to 10 μm.

[0064] 1.4 Non-magnetic layer

[0065] The magnetically responsive particles of this invention may have a non-magnetic metal oxide layer and / or a non-magnetic organometallic compound layer on the surface of the magnetic layer. This non-magnetic layer is formed to encapsulate the magnetic layer and / or to impart functionality to the particles.

[0066] Regarding its use as a carrier for biochemical applications (such as immunoassay reagents), the properties of the surface of the non-magnetic layer can be selected according to the purpose. The following procedure can be used to form a non-magnetic layer to strongly prevent the eluting of impurities from the particles, the eluting of the magnetic material itself, or the eluting of impurities from the magnetic layer. This can achieve a more preferred state, particularly for carrier particles used as immunoassay reagents.

[0067] Non-magnetic layers can be formed by adding non-magnetic metal oxides and / or non-magnetic organometallic compounds, as main raw materials, and optionally other auxiliary materials in the presence of particles, and reacting them in a liquid phase. The non-magnetic metal oxides and / or non-magnetic organometallic compounds used here preferably have functional groups capable of reacting with the surface of magnetic materials. Using metal oxides and / or organometallic compounds that can directly react with the surface of magnetic materials allows the magnetic and non-magnetic layers to be firmly bonded together and maintain a high degree of adhesion, thus producing excellent effects in preventing leakage of magnetic layer components and fixing the components. Conversely, when using free-radical polymerizable monomers, such as vinyl monomers, the monomers do not directly bond with the magnetic material. Therefore, the adhesion between the magnetic layer and the polymer layer is poor, and the magnetic layer components are not sufficiently fixed. This can lead to component leakage and instability in shape / magnetic separation rate.

[0068] There are no particular restrictions on the procedure for reacting magnetic surfaces and non-magnetic layers. Examples include covalent bonding or coordination bonding.

[0069] The main raw materials for the non-magnetic layer will be described below. For simplicity, only monomolecular compounds will be used as examples. However, compounds containing one or more functional groups that can react with the surface of magnetic materials are acceptable. The compound can be a dimer or polymer formed by the condensation of multiple monomolecules. Furthermore, there is no particular limitation on the number of functional groups that can react with magnetic materials.

[0070] The nonmagnetic metal oxide and / or nonmagnetic organometallic compound preferably contains at least one selected from Si, Ge, Ti, or Zr. As mentioned above, it is preferred to have one or more functional groups that can react with the surface of the magnetic layer. Specific examples include: silane compounds, represented by alkoxysilanes, such as tetraethyl orthosilicate and its hydrolysis products; germanium compounds, represented by alkoxygermanium, such as tetraethanolgermanium and its hydrolysis products; titanium compounds, represented by alkoxytitanium, such as tetraethanoltitanium and its hydrolysis products; or zirconium compounds, represented by alkoxyzirconium, such as tetrabutoxidezirconium and its hydrolysis products. Here, the specific gravity of the nonmagnetic layer should be as small as possible in order to maintain particle dispersion. In the above examples, silane compounds are most preferred.

[0071] Furthermore, in the metal oxides and / or organometallic compounds used as the main raw materials for the non-magnetic layer, metal oxides and / or organometallic compounds having other functional groups besides those that react with the surface of the magnetic layer can be used. In this case, the magnetically responsive particles can also possess other functions derived from the metal oxides and / or organometallic compounds.

[0072] Metal oxides and / or organometallic compounds having other functional moieties will be specifically exemplified with reference to silane compounds. However, the metal oxides and / or organometallic compounds used are not limited to them.

[0073] Examples include: compounds containing vinyl groups, such as vinyltrimethoxysilane, vinyltriethoxysilane, and 7-octenyltrimethoxysilane; compounds containing epoxy groups, such as 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, and 8-glycidoxyoctyltrimethoxysilane; and compounds containing styrene groups. Compounds, such as p-styryltrimethoxysilane; compounds containing a methacryl group, such as 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 8-methacryloxyoctyltrimethoxysilane; compounds containing an acryl group, such as 3-acryloxypropyltrimethoxysilane; compounds containing an amino group, such as N-2-(aminoethyl)- 3-Aminopropylmethyldimethoxysilane, N-2-(aminoethyl)-3-aminopropyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-triethoxysilyl-N-(1,3-dimethylbutylene)propylamine, N-phenyl-3-aminopropyltrimethoxysilane, N-(vinylbenzyl)-2-aminoethyl-3-aminopropyltrimethoxysilane hydrochloride, N-2-(aminoethyl)-8-aminooctyltrimethoxysilane; compounds containing isocyanurates Compounds, such as tri-(trimethoxysilylpropyl)isocyanurate; compounds containing a urea group, such as 3-ureopropyltrialkoxysilane; compounds containing a mercapto group, such as 3-mercaptopropylmethyldimethoxysilane and 3-mercaptopropyltrimethoxysilane; compounds containing isocyanates, such as propyltriethoxysilane-3-isocyanate; compounds containing carboxylic anhydrides, such as 3-trimethoxysilylpropylsuccinic anhydride; and compounds containing carboxylic acids, such as hydrolyzed 3-trimethoxysilylpropylsuccinic anhydride.

[0074] When used as a carrier for biochemical applications, compounds containing epoxy groups, amino groups, mercapto groups, carboxylic anhydrides, or carboxylic acids are preferred. Similarly, preferred examples include those in which one or more double bond portions of vinyl-containing / styrene-containing compounds are oxidized to form an epoxidized compound; or those in which vinyl-containing / styrene-containing compounds are introduced onto particles and then their double bond portions are oxidized to obtain epoxy groups, thereby achieving enhanced reactivity with biorelevant materials by transforming one or more functional groups before or after introduction onto the particles.

[0075] As described above, the metal oxides and / or organometallic compounds used as the main raw materials for the non-magnetic layer can be of a single type, or two or more can be used in combination in a given ratio. When using two or more metal oxides and / or organometallic compounds, the combination of the two or more metal oxides and / or organometallic compounds is any of the following combinations: a combination of two or more metal oxides and / or organometallic compounds having another functional portion; a combination of two or more metal oxides and / or organometallic compounds not having another functional portion; or a combination of metal oxides and / or organometallic compounds having one or more other functional portions with metal oxides and / or organometallic compounds not having one or more other functions.

[0076] There are no particular restrictions on the procedure for adding metal oxides and / or organometallic compounds during the formation of the nonmagnetic layer, and any of the batch, stepwise, or continuous addition processes are permitted. Furthermore, there are no particular restrictions on the order of addition when two or more compounds are used in a given ratio. They can be mixed and added together, each can be added individually, some can be mixed and others added individually, and so on. They can be added in any given order and combination. There are also no particular restrictions on the number of times they can be added.

[0077] The coating of non-magnetic layers and magnetic layers can be repeated on nuclei whose surfaces are already coated with magnetic layers. For example, a nucleus whose surface is already coated with a magnetic layer is then coated with a non-magnetic layer. This surface layer can then be further coated with magnetic and non-magnetic layers in this sequential manner. There are no restrictions on the number of coatings (layers) or the number and type of magnetic / non-magnetic layers (inner layers) on the nucleus, as long as at least one magnetic layer is included and the outermost surface layer is a non-magnetic layer. Note that when more than two magnetic layers are included, the magnetic material content of the higher-order particles is improved compared to particles with only one magnetic layer, resulting in a higher magnetic response.

[0078] In forming the non-magnetic layer, auxiliary materials may optionally be used in addition to the metal oxides and / or organometallic compounds that serve as the main raw materials. There are no particular limitations on the auxiliary materials. For example, in cases where the surface of a magnetic material is reacted with the metal oxides and / or organometallic compounds of this invention, an acid or base may often be added to facilitate the reaction.

[0079] The coefficient of variation (CV value) of the weight-average particle size of the magnetic-responsive particles having a non-magnetic layer formed on the surface of the magnetic layer is preferably 15% or less, and more preferably 10% or less. Further, the average particle size is preferably 0.5 to 10 μm, more preferably 1 to 8 μm, and still more preferably 2 to 5 μm. The CV value of the volume-average particle size is 20% or less, preferably 15% or less, and more preferably 10% or less.

[0080] Assume that the volume and weight of the core particles are v c and w c , and the volume and weight of the magnetic-responsive particles are v e and w e , then the magnetic material density [(w e -w c ) / (v e -v c )] preferably satisfies the following Expression 1:

[0081] 2.0 ≤ (w e -w c ) / (v e -v c ) Expression 1

[0082] Therefore, even though the particle size is small, magnetic-responsive particles having a high magnetic separation rate of 40% or more can be obtained. In this case, the magnetic material density is preferably 2.0 to 5.0, and more preferably 2.0 to 3.5.

[0083] Assume that the particle size of the core particles is R c and the thickness of the magnetic material layer is De, which preferably satisfies 0 nm < De ≤ 200 nm, and more preferably satisfies 30 nm ≤ De ≤ 150 nm, further preferably satisfies 四十 nm < De ≤ 120 nm, and still further preferably satisfies 五十 nm < De ≤ 100 nm.

[0084] 1.5 Load Substances and How to Load Bio-Related Materials

[0085] <Analyte>

[0086] 请注意,原文中“四十nm”和“五十nm”应是“40nm”和“50nm”的错误表述,我在翻译中已修正。如果这并非错误,请告知我,我会按照你的要求进行翻译。In this invention, analyte refers to a substance to be measured / captured. For example, such a substance is present in the body or in biological samples, such as blood (whole blood), red blood cells, serum, plasma, urine, saliva, or sputum. Examples include: inflammation-related markers such as lesion tissue, lesion cells, CRP (C-reactive protein), IgA, IgG, and IgM; coagulation and fibrinolysis markers such as fibrin degradation products (e.g., D-dimer), soluble fibrin, TAT (thrombin-antithrombin complex), and PIC (plasmin-plasmin inhibitor complex); circulation-related markers such as oxidized LDL, BNP (brain natriuretic peptide), H-FABP (cardiac fatty acid-binding protein), and cardiac troponin I (cTnI); metabolism-related markers such as adiponectin; tumor markers such as CEA (carcinoembryonic antigen), AFP (alpha-fetoglobulin), PIVKA-II, CA19-9, CA125, and PSA (prostate-specific antigen); and infection-related markers such as HBV (hepatitis B virus), HCV (hepatitis C virus), Chlamydia trachomatis, and Neisseria gonorrhoeae. (gonorrhoeae); respiratory markers such as KL-6; allergen-specific IgE (immunoglobulin E); hormones; and drugs.

[0087] <Interacting Matter>

[0088] Examples of substances that specifically interact with the analyte in this invention include: proteins, peptides, amino acids, lipids, carbohydrates, DNA, RNA, receptors, haptens, biotin, and avidin. There are no particular limitations on the molecular weight or whether the interacting substance is naturally occurring or synthetic. Examples include one or more antibodies or one or more antigens that can be used in immunoassays utilizing immune responses. The term "interaction" refers to a reaction or binding.

[0089] The primary use of the aforementioned magnetically responsive particles is as carriers for biochemical applications (such as immunoassay reagents). Magnetically responsive particles (hereinafter referred to as "sensitized magnetically responsive particles") used as carriers for biochemical applications can be prepared by using the particles as carriers and immobilizing analytes, analyte analogs, or substances that specifically interact with the analytes (hereinafter sometimes simply referred to as "loaded substances").

[0090] There are no particular limitations to the procedure for immobilizing the load material onto the magnetically responsive particles to prepare the sensitized magnetically responsive particles of the present invention. Conventionally known physical and / or chemical bonds can be used for immobilization. When immobilized via chemical bonds, functional groups present on the surface of the magnetically responsive particles can be immobilized as a scaffold for binding the load material by forming a nonmagnetic layer using metal oxides and / or organometallic compounds having bio-related material binding functional groups (as illustrated in paragraph 0042).

[0091] The above-mentioned technology is preferably used to provide epoxy groups, amino groups, mercapto groups, carboxylic acid groups or carboxylic anhydride structures on the surface of magnetically responsive particles and to load the loading material onto the particle surface via each structure.

[0092] Furthermore, bio-related materials that can specifically bind to the aforementioned loading substances can be loaded onto magnetically responsive particles, and in the reaction system, the loading substances can bind to the magnetically responsive particles via the bio-related materials. Examples of bio-related materials that can be used for this purpose include avidin and streptavidin. Magnetically responsive particles on which bio-related materials are loaded to mediate binding with the loading substances can also be considered sensitized magnetically responsive particles.

[0093] The resulting sensitized magnetic responsive particles can optionally be coated (blocked) with various polymer compounds or proteins (e.g., bovine serum albumin) and dispersed in a suitable buffer solution, then used as a sensitized magnetic particle dispersion. The sensitized particle dispersion can be used as a reagent for immunoassays. Furthermore, diluents (buffer solutions) and / or standards for the assay can be combined to provide a kit containing the assay reagents.

[0094] The coefficient of variation (CV) of the weight-average particle size of the sensitized magnetically responsive particles is preferably 15% or less, and more preferably 10% or less. Furthermore, the average particle size 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 size is 20% or less, preferably 15% or less, and more preferably 10% or less.

[0095] Reagents and diluents used in immunoassays may contain various sensitizers to increase assay sensitivity and promote specific reactions between the analyte and the loading substance. Additionally, reagents and diluents used in immunoassays may contain, for example, various polymer compounds / proteins and their degradation products, amino acids, and / or surfactants to inhibit non-specific reactions caused by one or more substances present in the test sample other than the target analyte and to improve the stability of the assay reagents.

[0096] There are no particular limitations on the method for measuring the target substance using the immunoassay reagent of the present invention, as long as the magnetically responsive particles of the present invention are used. For example, sandwich assays, competitive assays, etc., which are commonly performed in the art, can be performed according to the description in the literature (e.g., "Enzymatic Immunoassay, 2nd Edition", edited by Eiji Ishikawa et al., Igaku-Shoin Ltd., 1982).

[0097] The analyte determination includes the following steps: contacting the sample, sensitized magnetically responsive particles, labeled analyte-binding material, labeled target analyte or its analogue, etc.; and performing B / F separation (separating the bound labeled antibody from the free labeled antibody). In the former step, the magnetic particles can be dispersed by conventional treatments (such as stirring or mixing). The latter step is performed, for example, by using the magnetism of the magnetic particles to collect them by using a magnet applied from outside the reaction vessel, etc.; draining the reaction solution; adding a washing solution; removing the magnet; and mixing, dispersing, and then washing the magnetic particles. The above operations can be repeated one to three times. There are no particular limitations on the washing solution, as long as it is a solution conventionally used in the art.

[0098] The results of analyte measurement can be calculated from values ​​obtained by labeling the analyte or its analogues with a labeled substance and measuring its amount or activity. Conventional protocols can be used as procedures for measuring labeled substances or their activity, with no particular limitations. Specific examples include radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), electrochemiluminescence immunoassay (ECLIA), chemiluminescence immunoassay (CLIA and CLEIA), absorbance measurement, or surface plasmon resonance. There are no particular limitations on the optical instruments used in the measurement. As a representative example, any automated biochemical analyzer widely used in clinical testing can be used.

[0099] As described above, improving magnetic separation efficiency is crucial when using magnetically responsive particles as carriers for biochemical applications (such as immunoassay reagents). Using particles with excellent magnetic separation efficiency allows for superior washing / purification efficiency and reduces particle loss. This results in high performance, particularly for immunoassay reagents. The magnetically responsive particles of the present invention achieve high magnetic separation efficiency and are therefore suitable for use as carriers for biochemical applications (such as immunoassay reagents).

[0100] Example

[0101] 2. Example

[0102] The invention will be described in more detail below with reference to embodiments. However, the invention is not limited to these embodiments.

[0103] The following methods are used to measure the average particle size and its CV value, the CV value of the average density, the magnetic separation rate, and the content of magnetic material of the particles obtained in each embodiment or comparative example.

[0104] 2.1 Scheme for measuring physical properties

[0105] 2.1.1 Average particle size

[0106] The average particle size can be determined by observing the particles under a scanning electron microscope (“S-4800”, manufactured by Hitachi High-Technologies Corporation) and calculating the average of the maximum diameters of 50 randomly selected particles from the observed image.

[0107] 2.1.2 Measurement of CV value of volume average particle size

[0108] The volume average particle size CV value is calculated by measuring the volume average particle size distribution using a laser diffraction and scattering particle size distribution analyzer (“LS 13320”, manufactured by Beckman Coulter Inc.).

[0109] 2.1.3 Measurement of CV value of weight-average particle size

[0110] The CV value of weight-average particle size was calculated by measuring the weight-average particle size distribution using a disk centrifugal particle size distribution analyzer (“DC24000UHR”, manufactured by CPS Instruments, Inc.). Specifically, a solution with a density gradient obtained by mixing 8% and 24% sucrose solutions was centrifuged at 5000 rpm while a 0.1 mL aqueous dispersion of particles with the absorbance adjusted to 1.0 was placed on it for measurement.

[0111] 2.1.4 Evaluation of Magnetic Separation Rate

[0112] The absorbance at 550 nm was measured using a spectrophotometer (U-3900H, manufactured by Hitachi, Ltd.) for evaluation. A magnet (2800G, W10 mm × D10 mm × H1 mm) was attached to the bottom of a quartz cell in the spectrophotometer, and 1.3 mL of an aqueous dispersion of particles with the absorbance adjusted to 1.0 was placed in it. The absorbance was read at 5 or 125 seconds after sample placement. The absorbance decay during this 120-second period was calculated to give an index of magnetic separation efficiency.

[0113] 2.1.5 Measurement of Magnetic Material Content

[0114] The magnetic material content of the magnetically responsive particles can be calculated from the residue obtained by decomposing the resin portion of the particles in air at temperatures up to 1000°C. Specifically, the dry weight (A) of the magnetically responsive particles is accurately weighed; the temperature is increased from 35°C to 1000°C using a simultaneous thermogravimetric analyzer (TG-DTA6300, manufactured by Hitachi High-Tech Science Corporation) at a heating rate of 5°C / min; the temperature is held at 1000°C for 5 minutes; then the weight (B) of the resulting residue is measured; and the ratio of B to A is calculated as a percentage to determine the magnetic material content.

[0115] 2.1.6 Evaluation of Dispersion

[0116] The sample solutions used were aqueous dispersions of magnetic particles in which the absorbance at 550 nm was adjusted to 1.0. Here, 1.3 mL of each sample solution was dispensed into a quartz cell in a spectrophotometer (“U-3900H”, manufactured by Hitachi, Ltd.), and the absorbance at 550 nm was read. Next, the particles in the solution were magnetically collected using a magnet (28000G, W40 mm × D40 mm × H10 mm) until the absorbance of the supernatant became 0. Then, the solution was vortexed at 2000 rpm for 5 seconds to disperse the magnetic particles, and the absorbance at 550 nm was measured. The change in absorbance before magnetic collection and after magnetic collection and dispersion was used to calculate the dispersion using the following equation, thus obtaining the dispersion rate.

[0117] Dispersion rate (%) = [(absorbance after magnetic collection and dispersion) / (absorbance before magnetic collection)] × 100.

[0118] 2.2.1 Example 1-1

[0119] First, 2.0 g of Micropearl EX-003 (particle size of 3.01 μm and CV of 3.1%, manufactured by SEKISUI CHEMICAL CO.,LTD.) as resin particles was ultrasonically dispersed into 40.0 g of ion-exchanged water to obtain a nuclear particle dispersion.

[0120] Subsequently, 4.0 mL of magnetic fluid EMG707 (manufactured by Ferrotec Corporation) was added while stirring under ultrasonic irradiation for ultrasonic dispersion for an additional 30 minutes. The resulting dispersion was filtered and washed with deionized water, and then excess magnetic fluid was removed to prepare magnetically responsive particles [1].

[0121] 2.2.2 Examples 1-2

[0122] First, 1.0 g of the magnetically responsive particles [1] obtained in Example 1-1 were ultrasonically dispersed into 400 g of ethanol.

[0123] Then, 10 mL of 28% ammonia solution (manufactured by NACALAI TESQUE, INC.), 1.0 g of tetraethyl orthosilicate and 3.0 g of 8-glycidoxyoctyltrimethoxysilane were added and dispersed by ultrasonication for 3 hours. After filtering the resulting dispersion, the dispersion and centrifugation in ion-exchanged water were repeated three times to obtain magnetically responsive particles EP with epoxy groups on their surface [1].

[0124] The magnetically responsive particles EP[1] were dispersed in PBS at 3.0 wt% by sonication, and 0.5 mL of the dispersion was transferred to a test tube. After collecting the magnetically responsive particles EP[1] onto the test tube wall using a magnet, the dispersion medium was removed. Then, 0.5 mL of PBS solution containing anti-KL-6 antibody (0.75 mg / mL) was added. The mixture was stirred overnight at 25°C to sensitize the particles, thereby preparing anti-KL-6 antibody-sensitized magnetically responsive particles[1]. Subsequently, 1.5 mL of 1.0 wt% BSA solution was added, and the mixture was stirred at 25°C for 4 hours. The sensitized magnetically responsive particles were collected onto the test tube wall using a magnet. Afterward, the dispersion medium was removed, and 1.5 mL of 1.0 wt% BSA solution was added and dispersed. This operation was repeated three times to prepare a dispersion of anti-KL-6 antibody-sensitized magnetically responsive particles[1].

[0125] 2.2.3 Examples 1-3

[0126] Streptomycin was dissolved in 0.1M boric acid aqueous solution to prepare a 0.1 μg / mL streptomycin solution.

[0127] The magnetically responsive particles EP[1] obtained in Examples 1-2 were ultrasonically dispersed at 3.0 wt% in a 0.1 M boric acid aqueous solution, and 0.5 mL of the dispersion was transferred to a test tube. After collecting the magnetically responsive particles EP[1] onto the test tube wall using a magnet, the dispersion medium was removed. Next, 0.5 mL of the above-mentioned streptavidin solution was added, and the mixture was stirred at 37°C for 18 hours to prepare streptavidin-sensitized magnetically responsive particles[1]. Then, 0.5 mL of 1.0 wt% BSA solution was added, and the mixture was stirred at a reaction temperature of 37°C for 4 hours. The sensitized magnetically responsive particles were collected onto the test tube wall using a magnet. Afterward, the dispersion medium was removed, and 1.5 mL of 1.0 wt% BSA solution was added and dispersed. This operation was repeated three times to prepare a dispersion of streptavidin-sensitized magnetically responsive particles[1].

[0128] 2.2.4 Example 2-1

[0129] First, 2.0 g of Micropearl SP-203 (particle size 3.02 μm and CV 4.9%, manufactured by SEKISUI CHEMICAL CO.,LTD.) as resin particles was ultrasonically dispersed into 40.0 g of ion-exchanged water to prepare a nuclear particle dispersion.

[0130] Subsequently, 4.0 mL of magnetic fluid EMG707 (manufactured by Ferrotec Corporation) was added while stirring under ultrasonic irradiation for ultrasonic dispersion for an additional 30 minutes. The resulting dispersion was filtered and washed with deionized water. Excess magnetic fluid was then removed to prepare magnetically responsive particles [2].

[0131] 2.2.5 Example 2-2

[0132] Except for using the magnetically responsive particles [2] prepared in Example 2-1, the same operations as in Example 1-2 were performed to prepare magnetically responsive particles [2] sensitized with anti-KL-6 antibody and dispersions of magnetically responsive particles [2] sensitized with anti-KL-6 antibody.

[0133] 2.2.6 Example 3-1

[0134] First, 2.0 g of Micropearl SP-203 (particle size 3.02 μm and CV 4.9%, manufactured by SEKISUI CHEMICAL CO.,LTD.) as resin particles was ultrasonically dispersed into 40.0 g of ion-exchanged water to prepare a nuclear particle dispersion.

[0135] Subsequently, 5.0 mL of magnetic fluid EMG707 (manufactured by Ferrotec Corporation) was added while stirring under ultrasonic irradiation for ultrasonic dispersion for an additional 30 minutes. The resulting dispersion was filtered and washed with deionized water. Excess magnetic fluid was then removed to prepare magnetically responsive particles [3].

[0136] 2.2.7 Example 3-2

[0137] Except for using the magnetically responsive particles[3] prepared in Example 3-1, the same operations as in Example 1-2 were performed to prepare magnetically responsive particles[3] sensitized with anti-KL-6 antibody and dispersions of magnetically responsive particles[3] sensitized with anti-KL-6 antibody.

[0138] 2.3.1 Comparative Example 1-1

[0139] Magnosphere MS300 Tosyl (manufactured by JSR Life Sciences Corporation) was used as the magnetically responsive particle[4].

[0140] 2.3.2 Comparative Examples 1-2

[0141] The above magnetically responsive particles [4] were dispersed in PBS at 3.0 wt% by sonication, and 0.5 mL of the dispersion was transferred to a test tube. After collecting the magnetically responsive particles [4] onto the test tube wall using a magnet, the dispersion medium was removed. Then, 0.5 mL of PBS solution containing anti-KL-6 antibody (0.75 mg / mL) was added. The mixture was stirred overnight at 25°C to sensitize the particles, thereby preparing anti-KL-6 antibody-sensitized magnetically responsive particles [4]. Subsequently, 1.5 mL of 1.0 wt% BSA solution was added, and the mixture was stirred at 25°C for 4 hours. The sensitized magnetically responsive particles were collected onto the test tube wall using a magnet. Afterward, the dispersion medium was removed, and 1.5 mL of 1.0 wt% BSA solution was added and dispersed. This operation was repeated three times to prepare a dispersion of anti-KL-6 antibody-sensitized magnetically responsive particles [4].

[0142] 2.3.3 Comparative Example 2

[0143] First, 2.0 g of Micropearl SP-203 (particle size of 3.02 μm and CV of 4.9%, manufactured by SEKISUI CHEMICAL CO.,LTD.) as resin particles was ultrasonically dispersed into 40.0 g of 10 mM NaCl solution to prepare a nuclear particle dispersion.

[0144] Subsequently, 8.0 mL of magnetic fluid EMG707 (manufactured by Ferrotec Corporation) was added while stirring under ultrasonic irradiation for ultrasonic dispersion for another 30 minutes. The resulting dispersion was allowed to stand for 3 minutes and the supernatant was removed. The particles were then redispersed in ion-exchanged water, filtered, and washed with ion-exchanged water to obtain magnetically responsive particles [5].

[0145] 2.3.4 Reference Example

[0146] Except for changing the volume of the magnetic fluid EMG707 (manufactured by Ferrotec Corporation) to 2.0 mL, the same procedures as in Example 2-1 were performed to prepare magnetically responsive particles [6].

[0147] Table 1 shows the results of measuring the volume average particle size (CV), weight average particle size (CV), magnetic material content, magnetic separation rate, and dispersion rate of the particles obtained in Examples 1-1 to 3-2, Comparative Examples 1-1 to 2, or Reference Examples.

[0148] [Table 1]

[0149]

[0150]

[0151] Regardless of whether before or after sensitization, the magnetically responsive particles in Examples 1 to 3 have a weight-average particle size CV value of 15% or less and a volume-average particle size CV value of 20% or less, exhibiting better magnetic separation efficiency compared to the magnetically responsive particles in Comparative Example 1, which have a similar particle size but a weight-average particle size CV value greater than 15% and a volume-average particle size CV value greater than 20%. This demonstrates that the magnetically responsive particles of the present invention have a weight-average particle size CV value of 15% or less and contain uniform magnetic material in each magnetically responsive particle, indicating excellent magnetic separation efficiency.

[0152] On the other hand, magnetically responsive particles, as in Comparative Example 2, were prepared such that the CV of the weight-average particle size was set to 15% or more through magnetic purification. Although a high magnetic separation rate was exhibited due to the increased content of magnetic material, low dispersibility resulted from the high particle specific gravity.

[0153] The volume-average particle size (CV) values ​​of the magnetically responsive particles and sensitized magnetically responsive particles in Example 3 are greater than 15% and do indeed exhibit practical magnetic separation rates; however, their magnetic separation rates are lower than those of the magnetically responsive particles and sensitized magnetically responsive particles in Examples 1 or 2. This indicates that magnetic separation becomes more non-uniform as the volume-average particle size varies further.

[0154] See Example 2

[0155] Except for replacing the ion-exchanged water with a 0.1% sodium chloride aqueous solution to obtain a nuclear particle dispersion, the same operation as in Example 1-1 was performed, and it was found that the magnetic material content was thus improved compared to Example 1-1.

[0156] The following procedure is used to evaluate the utility of some particles obtained in the above embodiments or comparative examples.

[0157] 2.4.1 Reagent Evaluation

[0158] The following immunoassays were performed using the sensitized magnetic responsive particles obtained in Examples 1-2 or Comparative Examples 1-2, wherein the particle surface had immobilized anti-KL-6 antibody. The differences in luminescence levels were measured between immunoassays using a KL-6 concentration of 0 U / mL and immunoassays using a standard solution of 5000 U / mL or standard solutions wherein the antigen had been diluted to 10, 50, 100, 500, 1000, or 2500 U / mL.

[0159] Preparation of ruthenium complex-labeled anti-KL-6 antibody

[0160] First, 0.5 mL of PBS solution containing anti-KL-6 antibody (2.0 mg / mL) was added to a polypropylene tube. Next, 13 μL of Ru-NHS (1 mg / mL) was added. The mixture was stirred with shaking at 25 °C, purified by Sephadex G25 column, and then evaluated.

[0161] KL-6 Immunoassay:

[0162] The luminescence level was measured using an automated analyzer (“Picolumi III”, manufactured by SEKISUI MEDICAL CO.,LTD.), with electrochemiluminescence assay used as the measurement principle. After adding 20 μL of sample to 200 μL of reaction solution, 25 μL of anti-KL-6 antibody-conjugated magnetic particles were added. The mixture was reacted at 30 °C for 9 min; 350 μL of 10 mM Tris buffer was added; and the particles were washed three times while being captured by the magnet. Next, 200 μL of ruthenium-labeled antibody solution containing 1.0 μg / mL of ruthenium complex-labeled anti-KL-6 antibody was added. After reacting at 30 °C for 9 min, 350 μL of 10 mM Tris buffer was added. The particles were washed three times while being captured by the magnet. Then, 300 μL of luminescent electrolyte containing 0.1 M tripropylamine was added, and the solution was supplied to the electrode surface. Finally, the luminescence level of the ruthenium complex bound to the particles was measured.

[0163] The following shows the results of the practicality evaluation of the sensitized magnetic responsive particles of Examples 1-2 or Comparative Examples 1-2 evaluated according to the above procedure.

[0164] [Table 2]

[0165]

[0166] Light emission level [count]

[0167] When reacting with the same concentration of antigen, the particles in Examples 1-2 exhibited a greater difference in luminescence levels from 0 U / mL compared to the particles in Comparative Examples 1-2, indicating higher sensitivity and superior reagent performance. Compared to the particles in Comparative Examples 1-2, the particles in Examples 1-2 had lower CV values ​​for both weight-average particle size and volume-average particle size, indicating excellent magnetic separation. The superior reagent performance obtained in the reagent evaluation is likely due to the uniform movement of particles in the liquid after antibody sensitization, and the fact that most particles did not disperse upon magnetic capture and were effectively trapped.

[0168] Example 4-1

[0169] First, 2.0 g of Micropearl EX-003 (with a particle size of 3.01 μm and a CV of 3.1%, manufactured by SEKISUI CHEMICAL CO.,LTD.) as resin particles (nuclear particles) was ultrasonically dispersed into 40.0 g of 0.1% sodium chloride aqueous solution to obtain a nucleus particle dispersion.

[0170] Subsequently, while stirring under ultrasonic irradiation, 4.0 mL of magnetic fluid EMG707 (manufactured by Ferrotec Corporation) was added for ultrasonic dispersion for an additional 30 minutes. The resulting dispersion was filtered and washed with deionized water, and then excess magnetic fluid was removed to prepare magnetically responsive particles [4-1].

[0171] Examples 4-2, 4-3, 4-4, 4-5, and 4-6

[0172] Except that the amount of magnetic fluid EMG707 added was changed to 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, the same operation as in Example 4-2 was performed to prepare magnetically responsive particles [4-3] to [4-6].

[0173] Comparative Example 4-1 and Comparative Example 4-2

[0174] As magnetically responsive particles, the following magnetically responsive particles were obtained respectively:

[0175] Comparative Example 4-1: Magnosphere MS300 COOH (particle size 2.80 μm, CV value of weight average particle size 19.1%, and CV value of volume average particle size 30.4%, manufactured by JSR Life Sciences)

[0176] Comparative Example 4-2: M280 (particle size 2.8 μm, CV value of weight-average particle size 6.3%, and CV value of volume-average particle size 8.3%, manufactured by Dynabeads)

[0177] The particle in Comparative Example 4-1 is a core-shell particle, and the particle in Comparative Example 4-2 is a particle in which iron is distributed throughout the parent particle.

[0178] The physical properties of the individual magnetically responsive particles obtained therefrom were measured. In the measurements, the average particle size, magnetic material content, and magnetic material concentration were measured based on the descriptions given above in the entries for "Average Particle Size" and "Measurement of Magnetic Material Content." The magnetic separation rate was measured based on the description given above in the entry for "Evaluation of Magnetic Separation Rate." The thickness of the magnetic material layer and the magnetic material density were calculated based on the following equations. In Examples 5-1 to 5-5 and Comparative Examples 5-1 to 5-2 described below, the material properties were measured and calculated in the same manner.

[0179] -Thickness of the magnetic material layer = (Average particle size - Particle size of the parent particle) / 2

[0180] - Magnetic material density = {(weight after magnetic material adsorption) - (weight before magnetic material adsorption)} / {(volume after magnetic material adsorption) - (volume before magnetic material adsorption)}

[0181] The physical properties of the magnetically responsive particles obtained therefrom (average particle size, magnetic material density, magnetic material content, magnetic separation rate, magnetic material layer thickness, and magnetic material content) are shown in Table 3.

[0182] [Table 3]

[0183]

[0184] Figure 1A This illustrates the relationship between magnetic material content and magnetic separation rate when the magnetic material content (magnetic layer thickness) is varied while keeping the nuclear particle size constant. Figure 1A In the figures, circles represent the results of the embodiments, and triangles represent the results of the comparative examples (this also applies to the other figures mentioned below). Figure 1A This confirms that the magnetic separation rate improves with increasing magnetic material content.

[0185] Figure 1B This illustrates the relationship between magnetic separation rate and magnetic material density when the magnetic material content (magnetic layer thickness) is varied while keeping the nuclear particle size constant. According to... Figure 1BA comparison of Examples 4-1 to 4-6 and Comparative Examples 4-1 to 4-2 confirmed that when the magnetic material density is 2.00 or higher, the magnetic separation rate is 40% or higher. Examples 4-1 to 4-6 also demonstrated that the magnetic separation rate can be controlled by controlling the magnetic material density.

[0186] Example 5-1

[0187] First, 2.0 g of Micropearl EX-002 (with a particle size of 2 μm and a CV of 3.1%, manufactured by SEKISUI CHEMICAL CO.,LTD.) as resin particles (nuclear particles) was ultrasonically dispersed into 40.0 g of 0.1% sodium chloride aqueous solution to obtain a nuclear particle dispersion.

[0188] Subsequently, 6.0 mL of magnetic fluid EMG707 (manufactured by Ferrotec Corporation) was added to the mixture while stirring under ultrasonic irradiation for ultrasonic dispersion for an additional 30 minutes. The resulting dispersion was filtered and washed with deionized water, and then excess magnetic fluid was removed to prepare magnetically responsive particles [5-1].

[0189] Examples 5-2, 5-3, 5-4 and 5-5

[0190] Except that the particle size of the resin particles was changed to 2.5 μm (Example 5-2), 3.0 μm (Example 5-3), 3.5 μm (Example 5-4) and 4 μm (Example 5-5) respectively, and the amount of magnetic fluid EMG707 added was changed to 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) respectively, the same operation as in Example 5-1 was performed to prepare magnetically responsive particles [5-2], [5-3], [5-4] and [5-5].

[0191] The magnetically responsive particles in Examples 5-3 were prepared under the same conditions as those in Examples 4-4 above, but are shown as Examples 5-3 for ease of comparison and discussion.

[0192] The physical properties (average particle size, magnetic material density, magnetic material content, magnetic separation rate, magnetic material layer thickness, and magnetic material content) of the magnetically responsive particles [5-1] to [5-5] obtained therefrom are shown in Table 4.

[0193] The relationship between magnetic material content and magnetic separation rate is as follows: Figure 2A The relationship between magnetic separation rate and magnetic material density is shown in [the figure]. Figure 2B As shown in the image.

[0194]

[0195] Figure 2A The relationship between the magnetic material content and magnetic separation rate obtained by changing the nuclear particle size is shown. According to... Figure 2A This confirms that the magnetic separation rate improves with increasing magnetic material content.

[0196] Figure 2B The relationship between the density and magnetic separation rate of magnetic materials obtained by changing the particle size of the nuclei is shown. According to... Figure 2B A comparison of Examples 5-1 to 5-5 and Comparative Examples 5-1 to 5-2 confirmed that when the magnetic material density is 2.00 or higher, the magnetic separation rate is 40% or higher. Examples 5-1 to 5-5 also demonstrated that the magnetic separation rate can be controlled by controlling the magnetic material density.

[0197] Example 5-1-2

[0198] First, 1.0 g of the magnetically responsive particles obtained in Example 5-1 [5-1] were ultrasonically dispersed in 400 g of ethanol. Next, 10 mL of 28% ammonia solution (manufactured by NACALAI TESQUE, INC.), 1.0 g of tetraethyl orthosilicate, and 3.0 g of 8-glycidoxyoctyltrimethoxysilane were added, and the mixture was ultrasonically dispersed for 3 hours. After filtering the resulting dispersion, the dispersion and centrifugation in ion-exchanged water were repeated three times to obtain magnetically responsive particles with epoxy groups on their surface [5-1-1].

[0199] The magnetically responsive particles [5-1-1] were dispersed in PBS at 3.0 wt% by sonication, and 0.5 mL of this dispersion was transferred to a test tube. After collecting the magnetically responsive particles [5-1-1] onto the test tube wall using a magnet, the dispersion medium was removed. Then, 0.5 mL of PBS solution containing anti-KL-6 antibody (0.75 mg / mL) was added. The mixture was stirred overnight at 25°C to sensitize, thereby preparing anti-KL-6 antibody-sensitized magnetically responsive particles [5-1-2]. Subsequently, 1.5 mL of 1.0 wt% BSA solution was added, and the mixture was stirred at 25°C for 4 hours. The sensitized magnetically responsive particles were collected onto the test tube wall using a magnet. Afterward, the dispersion medium was removed, and 1.5 mL of 1.0 wt% BSA solution was added and dispersed. This operation was repeated three times to prepare a dispersion of anti-KL-6 antibody-sensitized magnetically responsive particles [5-1-2].

[0200] Examples 5-2-2, 5-3-2, and 5-5-2

[0201] Except for replacing the magnetically responsive particles used in the reaction with magnetically responsive particles [5-2] (Example 5-2-1), [5-3] (Example 5-3-1), and [5-5] (Example 5-5-1), the same operations as in Example 5-1-2 were performed to prepare the anti-KL-6 antibody-sensitized magnetically responsive particles [5-2-2], [5-3-2], and [5-5-2].

[0202] Comparative Example 4-1-2

[0203] The particles described in Comparative Example 4-1 were dispersed in PBS at 3.0 wt% by sonication, and 0.5 mL of this concentration was transferred to a test tube. After collecting the magnetically responsive particles onto the test tube wall using a magnet, the dispersion medium was removed. Then, 0.5 mL of PBS solution containing anti-KL-6 antibody (0.75 mg / mL) and 0.1 mL of 10 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride aqueous solution were added. The mixture was stirred overnight at 25°C for sensitization, thereby preparing anti-KL-6 antibody-sensitized magnetically responsive particles. Subsequently, 1.5 mL of 1.0 wt% BSA solution was added, and the mixture was stirred at 25°C for 4 hours. The sensitized magnetically responsive particles were collected onto the test tube wall using a magnet. Afterward, the dispersion medium was removed, and 1.5 mL of fresh 1.0 wt% BSA solution was added and dispersed. This operation was repeated three times to prepare a dispersion of magnetically responsive particles [4-1-2] sensitized with anti-KL-6 antibody.

[0204] Based on the above KL-6 immunoassay, the practicality of the anti-KL-6 antibody-sensitized magnetically responsive particles of Examples 4-1-2, 5-1-2, 5-2-2, and 5-3-2, Example 5-5-2, and Comparative Example 4-1-2 was evaluated. The results obtained are as follows:

[0205]

[0206] The particles in Examples 5-1-2, 5-2-2, 5-3-2 and 5-5-2 exhibited luminescence levels that differed significantly from 0 U / mL and demonstrated high sensitivity, i.e., good reagent performance.

[0207] The particles in Examples 5-1-2, 5-2-2, 5-3-2, and 5-5-2 exhibit low particle volume coefficients (CVs) and excellent magnetic separation rates exceeding 40%. This superior reagent performance is likely due to the narrow particle distribution and high magnetic separation rate of these particles, the uniform particle movement in the liquid after antibody sensitization, and the fact that most particles remain intact and are effectively captured by the magnet.

[0208] Furthermore, due to the reduced particle size, the surface area per unit weight can be increased. Therefore, it exhibits high-sensitivity reagent performance with a greater difference in luminescence levels compared to different antigen concentrations (i.e., a larger difference in luminescence levels compared to 0 U / mL).

[0209] Industrial applicability

[0210] This invention provides highly sensitive immunoassay reagents by using magnetically responsive particles with a weight-average particle size (CV) of less than 15%. These reagents are readily loaded with biorelated materials and exhibit improved separation / purification efficiency due to their excellent magnetic separation rate. This invention provides magnetically responsive particles with small particle size but high magnetic collection properties, and immunoassay reagents using these particles that achieve excellent magnetic separation and high sensitivity.

Claims

1. A sensitized magnetically responsive particle, comprising: a magnetically responsive particle having a core particle and at least one magnetic layer disposed on the core particle, the magnetic layer comprising microparticles of a magnetic metal and / or an oxide thereof; and a substance that specifically interacts with an analyte, the substance being loaded on the magnetically responsive particle. wherein Assuming that the volume and weight of the core particle are v c and w c , respectively, and the volume and weight of the magnetically responsive particle are v e and w e , respectively, the magnetic layer density [(w e - w c ) / (v e - v c )] satisfies the following expression 1: 2.0 ≤ (w e - w c ) / (v e - v c Expression 1.

2. The sensitized magnetically responsive particle according to claim 1, wherein a magnetic separation rate is 40% or more.

3. The sensitized magnetically responsive particle according to claim 1 or 2, further comprising a non-magnetic layer between the magnetic layer and the substance that specifically interacts with an analyte, the non-magnetic layer comprising a non-magnetic metal oxide and / or an organometallic compound.

4. The sensitized magnetically responsive particle according to claim 1 or 2, wherein the substance that specifically interacts with an analyte is chemically bonded to the magnetic layer through one-step or multi-step reaction.

5. The sensitized magnetically responsive particle according to claim 3, wherein the substance that specifically interacts with an analyte is bonded to the non-magnetic layer via one or more chemical bonds.

6. The sensitized magnetically responsive particle according to claim 1 or 2, wherein a coefficient of variation of a weight average particle size of the magnetically responsive particle is 15% or less.

7. The sensitized magnetically responsive particle according to claim 6, wherein a coefficient of variation of a volume average particle size of the magnetically responsive particle is 20% or less.

8. A heterogeneous immunoassay method using the sensitized magnetically responsive particle according to any one of claims 1 to 7.

9. An immunoassay reagent comprising the sensitized magnetically responsive particle according to any one of claims 1 to 7.

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