Particles, reagents, test kits, and methods for detecting target substances for immunoturbidimetry.

Abstract

This invention provides particles, reagents, and test kits for immunoturbidimetry that can detect trace components in low-concentration ranges and improve storage stability, as well as a method for detecting target substances. [Solution] A particle for immunoturbidimetry containing a first resin and titanium dioxide, comprising a first layer having the first resin and a second layer having the titanium dioxide, wherein the second layer is located outside the first layer and the density of the titanium dioxide is 3.40 g / cm³. 3 Particles for immunoturbidimetry, characterized in that the titanium dioxide content in the particles is 10% by mass or more and 80% by mass or less.

Description

[Technical Field]

[0001] The present invention relates to particles for immunoturbidimetry, reagents, test kits, and methods for detecting target substances. [Background technology]

[0002] In recent years, immunoturbidimetric testing has attracted attention as a simple and rapid immunoassay method. Immunoturbidimetric testing involves mixing a dispersion of particles having antibodies or antigens on their surface as ligands with a sample that may contain a target substance (antigen or antibody). If the sample contains the target substance (antibody or antigen), the particles will agglutinate. This agglutination reaction can be optically detected as changes in scattered light intensity, transmitted light intensity, absorbance, etc., to identify the presence or absence of disease.

[0003] Traditionally, polystyrene-based latex particles, primarily composed of polystyrene, have been used as particles for immunoturbidimetric methods because they are easy to sensitize (immobilize) with antigens or antibodies, are relatively inexpensive, and their polymerization reactions are easy to control. However, immunoturbidimetric methods using polystyrene latex particles sometimes had the problem of being unable to detect trace components in the low-concentration range. Therefore, there was a need to develop particles that were more sensitive than polystyrene latex particles.

[0004] To solve the above problem, it is important to increase the absorbance change due to particle aggregation associated with immune complex formation. To improve the absorbance change, particle development using high refractive index materials is being pursued. For example, particle development is underway using titanium dioxide, which is well known as a high refractive index material. Patent document 1 proposes particles for immunoturbidimetry in which titanium dioxide nanoparticles are coated onto carboxylic acid-modified polystyrene particles, and a ligand is attached to these particles. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2008-241357 [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] In the method described in Patent Document 1, many particles containing titanium oxide have a high density of titanium oxide and a large particle size from the beginning. Therefore, when used as an inspection reagent, the sedimentation rate is high, and there is room for improvement in the storage stability of the reagent. The present disclosure has been made in view of these background arts and problems. Specifically, an object of the present disclosure is to provide particles for immunoturbidimetry, a reagent, an inspection kit, and a method for detecting a target substance that can detect trace components in a low concentration region and improve storage stability in immunoturbidimetry. [Means for Solving the Problems]

[0007] The particles for immunoturbidimetry according to the present disclosure are particles for immunoturbidimetry containing a first resin and titanium oxide, having a first layer having the first resin and a second layer having the titanium oxide, where the second layer is outside the first layer, where the density of the titanium oxide is 3.40 g / cm 3 or less, and the content rate of the titanium oxide in the particles is 10 mass% or more and 80 mass% or less. The particles for immunoturbidimetry are characterized by this.

[0008] In addition, the reagent according to the present disclosure is a reagent characterized in that the above particles for immunoturbidimetry are dispersed in an aqueous solution. Further, the inspection kit according to the present disclosure is an inspection kit characterized by having the above reagent and a container containing the above reagent. Also, the method for detecting a target substance according to the present disclosure is a method for detecting a target substance in a specimen by in vitro diagnosis, and is characterized in that the reagent and a specimen that may contain the target substance are mixed. Also, the method for detecting a target substance according to the present disclosure is a method for detecting a target substance in a specimen by in vitro diagnosis, and includes a step of mixing a specimen that may contain the target substance with the reagent to obtain a mixed solution, a step of irradiating the mixed solution with light, and a step of detecting at least one of transmitted light and scattered light from the light irradiated on the mixed solution.

Advantages of the Invention

[0009] According to the present disclosure, it is possible to provide particles for immunoturbidimetry, a reagent, a test kit, and a method for detecting a target substance that can detect trace components in a low-concentration region of the target substance and improve storage stability.

Embodiments for Carrying Out the Invention

[0010] Hereinafter, embodiments of the present disclosure will be described in detail, but the technical scope of the present invention is not limited to these embodiments. The particles for immunoturbidimetry according to the present disclosure are particles for immunoturbidimetry containing a first resin and titanium oxide, having a first layer having the first resin and a second layer having titanium oxide, the second layer being outside the first layer, and the density of titanium oxide being 3.40 g / cm 3 Hereinafter, and the content rate of the titanium oxide in the particles is 10 mass% or more and 80 mass% or less.

[0011] The inventors of the present invention contain a first resin as the first layer at the center of the particle, and a layer containing titanium oxide having a density of 3.40 g / cm as the second layer outside thereof. 3 By forming a layer containing titanium oxide hereinafter, it has been found that trace components in a low-concentration region of the target substance can be detected and storage stability is improved. The reason is considered as follows. A resin layer as the first layer and a density of 3.40 g / cm as the second layer. 3By providing a layer containing titanium oxide as described below, it is possible to realize a system in which, even though the particle density is lower than in the case of particles containing only titanium oxide, the turbidity change during particle aggregation is large. Thus, it is thought that it has become possible to detect trace components in a low concentration region of a target substance and to improve storage stability.

[0012] The titanium oxide contained in the particles for immunoturbidimetry of the present disclosure has a density of 3.40 g / cm 3 It is characterized as follows. Generally, titanium oxide distributed on the market is produced by, for example, the sulfuric acid method, the chlorine method, etc., and has a high crystallinity of crystal structures such as the anatase type, the brookite type, the rutile type, etc. Titanium oxide having a high crystallinity has a high density, which is different from the titanium oxide used in the present disclosure. The inventors of the present invention have conceived that by containing low-density, i.e., lightweight, titanium oxide within a certain range, it is possible to detect trace components in a low concentration region of a target substance and to improve storage stability, leading to the present disclosure.

[0013] In order to enable detection of trace components in a low concentration region of a target substance, the density of the titanium oxide of the present disclosure is preferably 1.50 g / cm 3 or more and 3.40 g / cm 3 or less, and more preferably 2.50 g / cm 3 or more and 3.40 g / cm 3 or less.

[0014] The titanium oxide used in the present disclosure is not particularly limited as long as its density is within the above range, but can be obtained, for example, by a method of hydrolyzing a metal oxide precursor containing titanium called the sol-gel method. That is, it can be obtained by coexisting a metal oxide precursor, an oxygen-containing organic solvent, and water and performing a hydrolysis reaction.

[0015] Examples of metal oxide precursors include metal chlorides, metal acetates, metal alkoxides, metal hydroxides, etc. Among these, from the viewpoint of by-produced impurities (e.g., chlorides, etc.), metal alkoxides, metal acetates, and metal hydroxides are preferably used. Among these, particularly, the chemical formula Mx (OR) y (Here, M represents a metal element, R represents an alkyl group, and x and y each independently represent integers between 1 and 4.) A metal alkoxide represented by the chemical formula M x’ (OH) y’ Preferred are metal hydroxides represented by nH2O (where M represents a metal element, x' and y' each independently represent integers between 1 and 4, and n represents an integer of 1 or greater), and compounds containing the above-mentioned metal alkoxides and / or metal hydroxides. Specifically, examples include titanium methoxide, titanium ethoxide, titanium diisopropoxide bis(2,4-pentanedione), titanium diisopropoxide bis(ethyl acetate), titanium-n-butoxide, titanium isopropoxide, titanium methoxypropoxide, titanium-n-nonyloxide, titanium-n-propoxide, titanium stearyl oxide, titanium triisostearyl isopropoxide, titanium trimethylsiloxide, and the like.

[0016] Examples of oxygen-containing organic solvents include alcohols, ketones, aldehydes, ethers, esters, and siloxanes.

[0017] The immunoturbidimetric particles according to this disclosure are characterized by having a first layer having a first resin and a second layer having titanium dioxide, wherein the second layer is located outside the first layer. By containing the first resin as the center of the particle, i.e., the first layer, and containing titanium dioxide as the second layer outside thereof, it is possible to include a sufficient amount of resin and titanium dioxide to detect trace components in the low-concentration range and to improve storage stability.

[0018] The immunoturbidimetric particles according to this disclosure are not particularly limited as long as there is a second layer outside the first layer, but can be obtained, for example, by further coexisting a first resin with the hydrolysis reaction described above. That is, they can be obtained by coexisting a metal oxide precursor, an oxygen-containing organic solvent, water, and a first resin and carrying out a hydrolysis reaction. The first resin can be obtained by general polymer particle manufacturing methods such as emulsion polymerization, soap-free polymerization, dispersion polymerization, suspension polymerization, phase inversion emulsion, wet grinding, and dry grinding, and is not particularly limited. The first resin obtained by emulsion polymerization and soap-free polymerization is preferred because it provides a desired volume-average particle size and a uniform particle size distribution for use as immunoturbidimetric particles.

[0019] The preferred range for volume-average particle size for immunoturbidimetry particles is 150 nm to 1000 nm, and more preferably 180 nm to 800 nm. Furthermore, the preferred range for particle size distribution for immunoturbidimetry particles is a value of 1.00 to 1.25 for the ratio of volume-average particle size to number-average particle size.

[0020] The first resin of this disclosure is not particularly limited, but preferably has a high refractive index. Preferred specific structures for this purpose include a styrene structure, a fluorene structure, a dinaphthothiophene structure, a naphthalene structure, an anthracene structure, and a phenanthrene structure.

[0021] The titanium dioxide content in the immunoturbidimetric particles of this disclosure is 10% by mass or more and 80% by mass or less, preferably 21% by mass or more and 60% by mass or less. If the titanium dioxide content is less than 10% by mass, it may be difficult to detect trace components in the low-concentration range of the target substance, and if it is more than 80% by mass, the sedimentation rate increases, which may cause problems with storage stability.

[0022] The density of the particles used for immunoturbidimetry in this disclosure is 1.20 g / cm³. 3 More than 1.54g / cm 3 The following is preferable: The titanium dioxide disclosed herein has a density of 3.40 g / cm³. 3By incorporating a sufficient amount of the following titanium dioxide into the particles and keeping the particle density within the aforementioned range, it is possible to achieve both improved sensitivity and suppression of sedimentation.

[0023] The circularity of the particles for immunoturbidimetry according to this disclosure is preferably 0.85 to 1.00. By keeping the circularity of the particles within the above range, the particles approach each other smoothly in the antigen-antibody reaction, thereby achieving a higher level of sensitivity.

[0024] Furthermore, the immunoturbidimetric particles according to this disclosure preferably have a third layer containing a second resin on the outside of a second layer containing titanium dioxide. The presence of the third layer improves the adsorption rate of antibodies and antigens that serve as biosensors, thereby achieving higher sensitivity. The second resin contained in the third layer preferably has the structure shown in formula (1) below. This can further improve the adsorption rate of antibodies and antigens that serve as biosensors. [ka] (In formula (1), R 1 This represents a hydrogen atom or a methyl group. R 2 (This indicates a structure having an optionally substituted phenyl group, or a structure having an ester group.)

[0025] Furthermore, the second resin contained in the third layer more preferably has at least one of the structures shown in formula (1-1) and formula (1-2) below, as represented by formula (1). [ka] (In formula (1-1), R 3 This represents a hydrogen atom or a methyl group. R 4 (This indicates a structure having a hydroxyl group or a carboxyl group.) [ka] (In formula (1-2), R 5 This represents a hydrogen atom or a methyl group. R 6 (This indicates a structure having a hydroxyl group or a carboxyl group.)

[0026] By including at least one of the structures represented by formula (1-1) and formula (1-2), the coating resin can interact well with titanium dioxide and provide uniform coating without exposure of titanium dioxide. This suppresses the desorption of the resin. Specific examples of the structures represented by formula (1-1) or formula (1-2) are shown in formulas (1-A-1) to (1-A-12) below, but are not limited to these. [ka]

[0027] Furthermore, the second resin preferably has at least one of the structures represented by formula (2) and formula (3) below. [ka] (In formula (2), R 7 This represents a hydrogen atom or a methyl group. n1 is an integer between 1 and 3 (inclusive), m1 is an integer between 0 and 2 (inclusive), and n1 + m1 is 3. *1 independently indicates bonding to a titanium atom or a silicon atom, or to a hydrogen atom, a methyl group, or an ethyl group. 8 Each of these independently represents either a methyl group or an ethyl group. In other words, the structure shown in formula (2) may be bonded to the titanium atoms of titanium oxide via an oxygen atom, or it may be bonded to the silicon atoms of another structure shown in formula (2) or formula (3) via an oxygen atom. [ka] (In formula (3), R 9 This represents a hydrogen atom or a methyl group. R10 This represents a single bond, a phenylene group, or an alkylene group having 3 or fewer carbon atoms. n2 represents an integer between 1 and 3 (inclusive), m2 represents an integer between 0 and 2 (inclusive), and n2 + m2 is 3. *2 independently indicates bonding to a titanium atom or a silicon atom, or to a hydrogen atom, a methyl group, or an ethyl group. 11 Each of these independently represents either a methyl group or an ethyl group. In other words, the structure shown in formula (3) may be bonded to the titanium atoms of titanium oxide via an oxygen atom, or it may be bonded to the silicon atoms of another structure shown in formula (3) or formula (2) via an oxygen atom.

[0028] A resin containing the structures shown in formulas (1-1) and (1-2), as well as the structures shown in formulas (2) and (3), is a vinyl polymer and also contains an alkoxysilane. Through interaction with titanium dioxide, the exposure of titanium dioxide is further suppressed, resulting in a uniform coating. Particles having the structure shown in formula (1-1) or formula (1-2) can be obtained, for example, by performing a polymerization reaction by coexisting titanium dioxide-containing particles and monomers in an aqueous medium and adding a polymerization initiator, i.e., by seed polymerization. The monomer is not particularly limited, but for example, it can be obtained by performing seed polymerization using glycidyl (meth)acrylate as the monomer, followed by a ring-opening reaction of the glycidyl group. In addition, the monomer may be used alone or as a mixture of two or more. The monomers to be mixed are not particularly limited, but if they contain the structure shown in formula (2) or formula (3), they can be obtained by mixing the monomers from which the structure shown in formula (2) or formula (3) is derived, and performing seed polymerization by copolymerization of two or more monomers. The monomers from which the structure shown in formula (2) or formula (3) is derived are not particularly limited as long as they have the structure from which the structure shown in formula (2) or formula (3) is derived. Examples include vinyltrimethoxysilane, vinyltriethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, and 3-acryloxypropyltrimethoxysilane. These can be used individually or in combination of two or more. Particles having a second resin can also be obtained, for example, by coexisting titanium dioxide-containing particles and the second resin in an aqueous medium and allowing them to adsorb. In this case, the second resin is not particularly limited, but it can be any material that dissolves in an aqueous medium and can adsorb to the second layer containing titanium dioxide. For example, water-soluble polymers such as poly(sodium acrylate), polysaccharides such as carboxymethyl dextran sodium, and proteins such as bovine serum albumin can be used.

[0029] The immunoturbidimetric particles according to this disclosure may be pre-sensitized particles without ligands attached, or they may be affinity particles having ligands further attached to their surface. The affinity particles have a selective or specific high affinity for the target substance due to the ligand attached to the particle surface. In particular, it is preferable that the ligand is attached to the surface of the particle by chemical bonding. In this disclosure, a ligand is a compound that specifically binds to a receptor on a particular target substance. The binding site of the ligand to the target substance is fixed, and it has a selective or specific high affinity. Examples include antigens and antibodies, enzyme proteins and their substrates, signaling substances such as hormones and neurotransmitters and their receptors, and nucleic acids, but ligands in this disclosure are not limited to these. Examples of nucleic acids include deoxyribonucleic acid. In this disclosure, affinity particles have a selective or specific high affinity for the target substance. It is preferable that the ligand in this disclosure is an antibody, an antigen, or a nucleic acid.

[0030] Furthermore, the reagents relating to this disclosure are reagents in which the immunoturbidimetric particles relating to this disclosure are dispersed in an aqueous solution. The reagent is not particularly limited in its target of testing. Examples of reagents include in vitro diagnostic medical devices, such as antigen detection reagents for testing antigens and antibody detection reagents for testing antibodies. The reagent may consist of pre-sensitized particles without ligands dispersed in an aqueous solution, with the expectation that the user will attach a desired ligand, such as an antibody, to the particles. Alternatively, it may consist of affinity particles with a ligand already attached, dispersed in an aqueous solution, with a specific target substance in mind.

[0031] The reagents relating to this disclosure comprise immunoturbidimetric particles (pre-sensitization particles or affinity particles) relating to this disclosure and a dispersion medium for dispersing the immunoturbidimetric particles. To the extent that the objectives of this disclosure can be achieved, the reagents relating to this disclosure may also include third substances such as solvents and blocking agents in addition to the immunoturbidimetric particles relating to this disclosure. Two or more types of third substances such as solvents and blocking agents may be included in combination. Examples of dispersion media used include various buffers such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, but the dispersion media included in the reagents are not limited to these.

[0032] The test kit according to this disclosure comprises the reagent according to this disclosure and a container containing the reagent. In addition to the reagent described above (hereinafter referred to as Reagent 1), the test kit may also include a reaction buffer (hereinafter referred to as Reagent 2). Reagents 1 and 2, or either one of them, may contain a sensitizer. Furthermore, the test kit according to this disclosure may also include, in addition to Reagent 1 and Reagent 2, a positive control, a negative control, a serum diluent, a primary antibody, and a secondary antibody. As the medium for the positive control and negative control, serum, physiological saline, or a solvent may be used, provided that serum does not contain the target substance that can be measured.

[0033] The method for detecting a target substance according to this disclosure is a method for detecting a target substance in a sample by in vitro diagnostics, and may involve mixing the reagent according to this disclosure with a sample that may contain the target substance. Alternatively, the method for detecting a target substance according to this disclosure is a method for detecting a target substance in a sample by in vitro diagnostics, and may include the steps of: obtaining a mixture by mixing the reagent according to this disclosure with a sample that may contain the target substance; irradiating the mixture with light; and detecting at least one of the transmitted light and scattered light from the light irradiated onto the mixture.

[0034] (Method for measuring the titanium dioxide content in particles used for immunoturbidimetry) An example of a method for measuring the titanium dioxide content in particles for immunoturbidimetry according to this disclosure is described below. The titanium dioxide content in the particles is measured using a thermomass spectrometer. For example, STA200 (manufactured by Hitachi High-Tech Science) is used. 5 to 10 mg of the cured material is weighed into an aluminum pan and measured under a nitrogen atmosphere. The temperature conditions are to hold at 30°C for 30 minutes, then raise the temperature from 30°C to 500°C at a rate of 10°C / min, and then hold at 500°C for 10 minutes. For evaluation purposes, the titanium dioxide content in the particles can be measured from the mass loss of the resin component at 300°C.

[0035] (Method for measuring the density of titanium dioxide in particles used for immunoturbidimetry) An example of a method for measuring the density of titanium dioxide in this disclosure is described below. The density of titanium dioxide can be measured by separating titanium dioxide from immunoturbidimetric particles and performing dry density measurement of the separated titanium dioxide using the constant volume expansion method. For example, the measurement is performed using an AccuPic II (manufactured by Shimadzu Corporation) and averaged 10 times at 25°C. The method for separating titanium dioxide from particles is not particularly limited, but for example, titanium dioxide can be extracted from particles by dissolving and washing the resin in the particles with a solvent. Furthermore, the density of titanium dioxide can be calculated using the following formula. D P =W T ×D T +(1-W T )×D R D P : Particle density measured by the method illustrated herein D T Titanium oxide density D R : Resin density W T : Titanium dioxide content in particles measured by the method exemplified herein However, the resin density is 1.10 g / cm³. 3 It is calculated as follows.

[0036] (Method for measuring the density of particles used in immunoturbidimetry) An example of a method for measuring the density of particles for immunoturbidimetry in this disclosure is described. The particle density is measured, for example, by dry density measurement using the constant volume expansion method. For example, the measurement is performed using an Accupic II (manufactured by Shimadzu Corporation) and averaged 10 times at 25°C.

[0037] (Method for measuring volume-average particle size and particle size distribution in an aqueous dispersion of particles for immunoturbidimetry) This disclosure describes a method for measuring the volume-average particle size (Dv) of particles used in immunoturbidimetry. The Dv of particles present in an aqueous dispersion is measured by dynamic light scattering. For example, a zetasizer (zetasizer ultra: Malvern Panalytical) is used, and the measurement is performed at 25°C. Furthermore, the particle size distribution in this disclosure is calculated by measuring the number-average particle size (Dn) using the dynamic light scattering method described above, and then using the ratio of Dv to Dn (Dv / Dn).

[0038] (Method for measuring the circularity of particles used in immunoturbidimetry) This disclosure describes a method for measuring the circularity of particles used in immunoturbidimetry. 500 particles are extracted from a scanning electron microscope (SEM) image, and the circularity of each particle is calculated using image analysis software. The average value of the 500 particles is then used as the circularity. For example, a Hitachi High-Tech S4800 scanning electron microscope is used, and for example, Image-J is used as the image analysis software. [Examples]

[0039] The present disclosure will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0040] [Examples of particle production] (Preparation of particle 1) (Resin-containing first layer formation process) A mixture was prepared by weighing 12.68 g of styrene (St: Kishida Chemical Co., Ltd.), 0.23 g of divinylbenzene (DVB: Kishida Chemical Co., Ltd.), and 1512.02 g of deionized water into a 2 L four-neck separable flask. This mixture was maintained at 70°C while stirring at 140 rpm, and the inside of the four-neck separable flask was deoxygenated by flowing nitrogen at a flow rate of 200 ml / min. Next, a solution prepared separately by dissolving 0.55 g of V-50 (Fujifilm Wako Pure Chemical Corporation) in 20 g of deionized water was added to the mixture to initiate soap-free polymerization. After 23 hours of reaction from the start of polymerization, a dispersion of first-layer forming particles 1, consisting of a copolymer of styrene and divinylbenzene, was obtained. A portion of this dispersion was taken and evaluated using dynamic light scattering (Zetasizer Ultra: Malvern Panalytical), and the volume-average particle size was found to be 190 nm.

[0041] (Titanium dioxide-containing second layer formation process) For the first layer forming particle 1, a solution was prepared with deionized water to a solid content concentration of 0.6%. 20 g of this dispersion was mixed with 404.50 g of ethanol (Kishida Chemical Co., Ltd.) containing 0.2% polyvinylpyrrolidone K-30 (PVP K-30: Kishida Chemical Co., Ltd.), and the mixture was maintained at 70°C while stirring at 140 rpm. Next, a solution prepared by mixing 5.0 ml of titanium(IV)n-butoxide monomer (TBOT: Kishida Chemical Co., Ltd.), which had been prepared separately, with 197.50 g of ethanol was added to the above mixture to initiate the sol-gel reaction. The reaction was allowed to proceed for 24 hours from the start of the sol-gel reaction, and the particles dispersed in the above mixture were separated by centrifuge and redispersed in ethanol. Further separation of the particles from the above dispersion by centrifuge and redispersion of the particles in deionized water was repeated twice to purify the mixture and obtain the second layer forming particle 1. The second layer-forming particles 1 were stored in an aqueous dispersion adjusted to a final concentration of 5.0% by mass. A portion of this dispersion was taken, and the dynamic scattering of the second layer-forming particles 1 was evaluated, revealing a volume-average particle size of 200 nm. Furthermore, the metal oxide content was evaluated using differential thermal-thermogravimetric analysis (NEXTA STA200RV: Hitachi High-Tech Corporation), which showed it to be 45% of the particle weight.

[0042] (Resin-containing third layer formation process) A dispersion of second-layer-forming particles 1 with a solid content of 0.2% was prepared using deionized water to a volume of 149.55 g. 0.135 g of glycidyl methacrylate (GMA: Kishida Chemical Co., Ltd.) and 0.015 g of 3-methacryloxypropyltrimethoxysilane (MPS: Shin-Etsu Chemical Co., Ltd.) were added, and the mixture was maintained at 70°C while stirring at 100 rpm. The four-necked separable flask was deoxygenated by flowing nitrogen at a flow rate of 200 ml / min. Then, a solution of 0.03 g of V-50 dissolved in 0.3 g of deionized water, which had been prepared separately, was added to the above mixture to initiate shell formation. After stirring continued for 18 hours after the start of the reaction, a dispersion containing third-layer-forming particles 1 was obtained.

[0043] (Process for imparting reactive functional groups) An aqueous solution of mercaptosuccinic acid (MSA: Wako Pure Chemical Industries, Ltd.), which had been prepared in advance, was added to a dispersion containing the third layer-forming particle 1 (the total number of moles of MSA was equal to the number of moles of the glycidyl methacrylate). Triethylamine (Kishida Chemical Co., Ltd.) was then added to adjust the pH to 10. Next, the mixture was heated to 70°C while stirring at 800 rpm, and maintained in this state for 18 hours to obtain a dispersion containing particle 1.

[0044] (Particle washing process) Particle 1 was separated from the above dispersion using a centrifuge, and then redispersed in ion-exchanged water. This process was repeated eight times, and the particle concentration was finally adjusted to 5.0% by mass to obtain a dispersion of particle 1.

[0045] (Preparation of particles 2 to 6) In the resin-containing third layer formation step, instead of adding 0.135 g of glycidyl methacrylate and 0.015 g of 3-methacryloxypropyltrimethoxysilane, 0.15 g of glycidyl methacrylate was added. Otherwise, the dispersion of particle 2 was obtained using the same procedure as for particle 1.

[0046] In the resin-containing third layer formation step, instead of adding 0.135 g of glycidyl methacrylate and 0.015 g of 3-methacryloxypropyltrimethoxysilane, 0.135 g of styrene and 0.015 g of methacrylic acid (MAA: Kishida Chemical Co., Ltd.) were added. Furthermore, the reactive functional group imparting step was omitted. Otherwise, a dispersion of particle 3 was obtained using the same procedure as for particle 1.

[0047] In the titanium dioxide-containing second layer formation step, a dispersion of particle 4 was obtained using the same procedure as for particle 2, except that the amount of titanium(IV)n-butoxide monomer was changed from 5.0 ml to 3.0 ml.

[0048] In the titanium dioxide-containing second layer formation step, a dispersion of particle 5 was obtained using the same procedure as for particle 2, except that the amount of titanium(IV)n-butoxide monomer was changed from 5.0 ml to 7.5 ml.

[0049] In the titanium dioxide-containing second layer formation step, a dispersion of particle 6 was obtained using the same procedure as for particle 2, except that the amount of titanium(IV)n-butoxide monomer was changed from 5.0 ml to 2.0 ml.

[0050] (Preparation of particle 7) Bovine serum albumin (hereinafter sometimes abbreviated as BSA) was used as the resin-containing third layer. First, 30 mg of BSA was added to 1 mL of deionized water to prepare an aqueous BSA solution. 0.5 mL of the aqueous BSA solution was placed in a 1.5 mL tube, and then 0.5 mL of a 2.0 mass% solution of the second layer-forming particle 1 was added. After mixing well, it was left at room temperature overnight. Next, the particles were separated from the dispersion using a centrifuge, and the process of redispersing them in deionized water was repeated three times, and the particle concentration was finally adjusted to 5.0 mass%, thereby obtaining a dispersion of particle 7 having a third layer containing BSA.

[0051] (Preparation of particle 8) Poly(sodium acrylate) (hereinafter sometimes abbreviated as PAANa) was used as the resin-containing third layer. The molecular weight of PAANa ranged from 22,000 to 66,000. First, 10 mL of deionized water was added to 10 mg of PAANa to prepare an aqueous PAANa solution. 0.5 mL of the aqueous PAANa solution was placed in a 1.5 mL tube, and then 0.5 mL of a 2.0 mass% solution of the second layer-forming particle 1 was added. After thorough mixing, the mixture was left at room temperature overnight. Next, the particles were separated from the dispersion using a centrifuge, and the mixture was redispersed in deionized water. This process was repeated three times, and the particle concentration was finally adjusted to 5.0 mass%, thereby obtaining a dispersion of particle 8 having a third layer containing PAANa.

[0052] (Preparation of particle 9) As the resin-containing third layer, carboxymethyl dextran sodium (hereinafter sometimes abbreviated as Dex) was used. The molecular weight of Dex was 40,000. First, 20 mg of Dex was added to 1 mL of deionized water to prepare an aqueous solution of Dex. 0.5 mL of the aqueous Dex solution was placed in a 1.5 mL tube, and then 0.5 mL of a 2.0 mass% solution of the second layer-forming particle 1 was added. After mixing well, it was left at room temperature overnight. Next, the particles were separated from the dispersion using a centrifuge, and the process of redispersing them in deionized water was repeated three times, and finally the particle concentration was adjusted to 5.0 mass%, thereby obtaining a dispersion of particle 9 having a third layer containing Dex.

[0053] (Preparation of particle 10 and particle 11) A dispersion of particle 10 was obtained using the same procedure as for particle 2, except that the amount of styrene was changed from 12.68 g to 101.44 g and the amount of divinylbenzene was changed from 0.23 g to 1.84 g in the resin-containing first layer formation process.

[0054] In the resin-containing first layer formation process, a dispersion of particle 11 was obtained using the same procedure as for particle 2, except that divinylbenzene was not added and styrene was replaced with methyl methacrylate (MMA: Kishida Chemical Co., Ltd.).

[0055] [Examples of comparative particle production] 50 μL of titanium dioxide microparticle dispersion and 950 μL of γ-aminopropyltriethoxysilane aqueous solution (pure water containing 1% by mass of γ-aminopropyltriethoxysilane) were added to a centrifuge tube and stirred at 100 rpm using a tube roller for 1 hour at room temperature (20°C to 30°C). Next, the centrifuge tube was centrifuged at 15,000 rpm (approximately 20,000 G) for 15 minutes, and the supernatant was removed by aspirator. 1,500 μL of acetate buffer (0.01 M, pH 5.0) was added to the precipitate, and it was redispersed using a vortex mixer. The mixture was centrifuged again at 15,000 rpm (approximately 20,000 G) for 15 minutes, and the supernatant was removed by aspirator. Furthermore, this process (adding acetate buffer, centrifuging, and removing the supernatant) was repeated three times. 500 μL of phosphate buffer (0.01 M, pH 6.0) was added to the obtained precipitate, and the mixture was dispersed using an ultrasonic disruptor for 2 minutes to obtain a dispersion of amino group-introduced titanium dioxide microparticles.

[0056] The following materials were added to a centrifuge tube and stirred at 100 rpm using a tube roller for 1 hour at room temperature (20°C to 30°C). • Carboxylated polystyrene microparticle dispersion [Product name "Imtex", manufactured by JSR Corporation, carboxylated latex, volume average particle size of contained microparticles 200 nm, microparticle content 10%] (B-1) 100 μL ·900μL of HEPES buffer containing 1% bovine serum albumin • Soluble carbodiimide solution [1M aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, the same applies hereafter] 500 μL Next, the centrifuge tube was centrifuged at 15,000 rpm (approximately 20,000 G) for 15 minutes. The supernatant was then removed by aspirator, and 1,500 μL of pure water was added to the precipitate. The mixture was then redispersed using a vortex mixer. The mixture was again centrifuged at 15,000 rpm (approximately 20,000 G) for 15 minutes, the supernatant was removed by aspirator, and 500 μL of 1% bovine serum albumin-containing HEPES buffer was added to this precipitate. The mixture was then redispersed using an ultrasonic disruptor for 2 minutes to obtain a dispersion of carboxyl-activated polystyrene microparticles.

[0057] 88 μL of amino group-introduced titanium dioxide microparticle dispersion and 500 μL of carboxyl group-activated polystyrene microparticle dispersion were placed in a centrifuge tube and stirred at 100 rpm using a tube roller for 2 hours at room temperature (20-30°C). Next, the centrifuge tube was centrifuged at 15,000 rpm (approximately 20,000 G) for 15 minutes, and the supernatant was removed by aspirator. 1,500 μL of phosphate buffer (0.1 M, pH 7.1) was added to the precipitate and redispersed using a vortex mixer to obtain a titanium dioxide-polystyrene composite microparticle dispersion.

[0058] A titanium dioxide-polystyrene composite fine particle dispersion was centrifuged using density gradient centrifugation to obtain a fraction with a density of 1.60 (20% titanium dioxide content in the composite particles) as comparison particle 1. Similarly, a fraction with a density of 1.20 (5% titanium dioxide content in the composite particles) was obtained as comparison particle 2. The physical properties of the obtained particles 1-11 and comparison particles 1 and 2 are summarized in Table 1.

[0059] [Table 1]

[0060] (Production of affinity particles) For the dispersion of particle 1, 300 μL of the dispersion (3 mg of particle solids) was diluted with deionized water to a solid content concentration of 1.0% by mass, and this was placed in a 1.5 mL microtube. To this, 90 μL of a 5.0% aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (manufactured by Tokyo Chemical Industry Co., Ltd.) and 90 μL of a 5.0% aqueous solution of N-hydroxysulfosuccinimide sodium (manufactured by Tokyo Chemical Industry Co., Ltd.) were added. By stirring at room temperature for 30 minutes, an activated particle dispersion containing carboxyl groups was obtained.

[0061] After centrifugal washing, 270 μL of pH 5.5 phosphate buffer-physiological saline (PBS) was added, and the particles with activated carboxyl groups were dispersed using ultrasound. To this, 24 μL of a 5.0 mg / mL dispersion of mouse monoclonal anti-ferritin antibody (isoelectric point 7.1) (0.12 mg of antibody) was added, and the mixture was stirred at room temperature for 3 hours to obtain affinity particle 1, in which particle 1 was sensitized with the antibody. In the preparation of affinity particle 1, affinity particles 2-9 and comparative affinity particles 1 and 2 were prepared using the same experimental procedure as in the preparation of affinity particle 1, except that the types of particles were changed to particles 2-9 and comparative particles 1 and 2, respectively.

[0062] (Preparation of the first reagent) The first reagent was prepared by dissolving 50 mM HEPES, 0.05% by mass TritonX-100, and 1.0% by mass sodium chloride (manufactured by Kishida Chemical Co., Ltd.) in deionized water.

[0063] (Preparation of the second reagent) Affinity particles 1 were centrifuged and washed, then redispersed in 500 μL of buffer (HEPES buffer) containing 10 mM HEPES, 0.01% by mass polyoxyethylene nonylphenyl ether (Triton X-100, Kishida Chemical Co., Ltd.), and 10% by mass sucrose (viscosity modifier) ​​dissolved in deionized water. Subsequently, the mixture was mixed and diluted with HEPES buffer until affinity particles 1 comprised 0.1% by mass to obtain the second reagent 1. In the preparation of the second reagent 1, the second reagents 2 to 9 and the comparative second reagents 1 and 2 were prepared using the same experimental procedure as in the preparation of the second reagent 1, except that the types of particles were changed to affinity particles 2 to 9 and comparative affinity particles 1 and 2, respectively.

[0064] (Measurement of change in absorbance) A mixture was prepared by mixing 15 μL each of samples with ferritin concentrations of 0.0 ng / mL (physiological saline) and 100 ng / mL with 60 μL of the first reagent 1, and incubating the mixture at 37°C for 290 seconds. Next, 30 μL each of the second reagents 1-9 and comparative second reagents 1 and 2 were mixed into these mixtures, and the absorbance was measured after 42 seconds of stirring. Furthermore, this mixture was allowed to stand at 37°C for 253 seconds, and the absorbance was measured again. The difference from the absorbance measured after 42 seconds of stirring was defined as the absorbance change ΔABS. Absorbance measurements were performed using an Eppendorf BIOSPECTROMETER spectrophotometer at a wavelength of 572 nm.

[0065] (Calculation of storage stability index) The change in absorbance at a ferritin concentration of 100 ng / mL was measured. Second reagents 1-9 and comparative second reagents 1 and 2 were left to stand at 4°C for a certain period, and the upper half of the container was sampled. For these samples, the change in absorbance at a ferritin concentration of 100 ng / mL was measured again under the same conditions, and the change after a certain period was calculated. A smaller change is expected to indicate better storage stability. The storage stability was evaluated as follows based on the values ​​of the storage stability index shown below. A: The change after 14 days was within 10%. B: The change after 7 days was within 10%, but the change after 14 days was greater than 10%. C: The change after 7 days was greater than 10%. The results are shown in Table 2.

[0066] (Calculation of sensitivity index) For the second reagent, which had storage stability indices of A and B, the sensitivity index was calculated as follows: The change in absorbance when the ferritin concentration was 0.0 ng / mL (physiological saline) was defined as ΔABS(0), and the change in absorbance when it was 100 ng / mL was defined as ΔABS(100). The value of ΔABS(100) × 10000 / ΔABS(0) × 10000 was calculated and used as the sensitivity index value. A higher sensitivity index value is expected to indicate that the target substance can be detected with higher sensitivity. The evaluation was based on the sensitivity index values ​​as follows. A: The sensitivity index value was greater than 500. B: The sensitivity index value was greater than 100 and less than or equal to 500. C: The sensitivity index value was 100 or less. The results are shown in Table 2.

[0067] [Table 2] As described above, the immunoturbidimetric particles 1-9 of this disclosure exhibit high sensitivity and excellent storage stability, achieving both improved sensitivity and storage stability. On the other hand, Comparative Example 1 had poor storage stability. Comparative Example 2 had excellent storage stability but low sensitivity. In other words, Comparative Example 1 and Comparative Example 2 were unable to achieve both improved sensitivity and storage stability.

[0068] This embodiment includes the following configurations and methods. (Composition 1) A particle for immunoturbidimetry containing a first resin and titanium dioxide, The first layer has the first resin, and the second layer has the titanium oxide, The aforementioned second layer is located outside the aforementioned first layer. The density of the titanium dioxide is 3.40 g / cm³. 3 The following: Particles for immunoturbidimetry, characterized in that the titanium dioxide content in the particles is 10% by mass or more and 80% by mass or less. (Configuration 2) The immunoturbidimetric particles according to configuration 1, characterized in that the titanium dioxide content in the immunoturbidimetric particles is 21% by mass or more and 60% by mass or less. (Composition 3) A particle for immunoturbidimetry according to configuration 1 or 2, characterized in that its circularity is 0.85 or more and 1.00 or less. (Composition 4) A particle for immunoturbidimetry according to any one of configurations 1 to 3, characterized in that it has a third layer containing a second resin on the outside of the second layer having titanium dioxide. (Composition 5) Density is 1.20 g / cm³ 3 More than 1.54g / cm 3 A particle for immunoturbidimetry according to any one of configurations 1 to 4, characterized in that it is as follows. (Composition 6) The immunoturbidimetric particle according to configuration 4, characterized in that the second resin contains a structure represented by the following formula (1). [ka] (In formula (1), R 1 This represents a hydrogen atom or a methyl group. R 2 (This indicates a structure having an optionally substituted phenyl group, or a structure having an ester group.) (Composition 7) The density of the titanium dioxide is 2.50 g / cm³. 3 More than 3.40g / cm 3 A particle for immunoturbidimetry according to any one of configurations 1 to 6, characterized in that it is as follows. (Composition 8) A particle for immunoturbidimetry according to any one of configurations 1 to 7, characterized in that it further has a ligand on its surface. (Composition 9) A reagent characterized in that the immunoturbidimetry particles described in claim 1 or 8 are dispersed in an aqueous solution. (Composition 10) A test kit characterized by comprising the reagent described in configuration 9 and a container containing the reagent. (Method 11) A method for detecting a target substance in a specimen by in vitro diagnostics, characterized by mixing the reagent described in configuration 9 with a specimen that may contain the target substance. (Method 12) A method for detecting a target substance in a specimen by in vitro diagnostics, comprising the steps of: mixing a specimen potentially containing the target substance with the reagent described in configuration 9 to obtain a mixture; irradiating the mixture with light; and detecting at least one of transmitted light and scattered light from the light irradiated onto the mixture.

Claims

1. A particle for immunoturbidimetry containing a first resin and titanium dioxide, The first layer has the first resin, and the second layer has the titanium oxide, The aforementioned second layer is located outside the aforementioned first layer. The density of the titanium dioxide is 3.40 g / cm³. ３ The following: Particles for immunoturbidimetry, characterized in that the titanium dioxide content in the particles is 10% by mass or more and 80% by mass or less.

2. The immunoturbidimetric particles according to claim 1, characterized in that the titanium dioxide content in the immunoturbidimetric particles is 21% by mass or more and 60% by mass or less.

3. Particles for immunoturbidimetry according to claim 1, characterized in that the circularity is 0.85 or more and 1.00 or less.

4. The immunoturbidimetric particle according to claim 1, characterized in that it has a third layer containing a second resin on the outside of the second layer having titanium dioxide.

5. Density is 1.20 g / cm³ ３ 1.54g / cm or more ３ The immunoturbidimetric particle according to claim 1, characterized in that it is as follows.

6. The immunoturbidimetric particle according to claim 4, characterized in that the second resin contains a structure represented by the following formula (1). 【Chemistry 1】 (In formula (1), R １ This represents a hydrogen atom or a methyl group. R ２ (This indicates a structure having an optionally substituted phenyl group, or a structure having an ester group.)

7. The density of the titanium oxide is 2.50 g / cm³. ３ 3.40g / cm or more ３ The immunoturbidimetric particle according to claim 1, characterized in that it is as follows.

8. Particles for immunoturbidimetry according to claim 1, characterized in that they further have a ligand on their surface.

9. A reagent characterized in that the immunoturbidimetry particles described in claim 1 or 8 are dispersed in an aqueous solution.

10. A test kit comprising the reagent described in claim 9 and a container containing the reagent.

11. A method for detecting a target substance in a specimen by in vitro diagnostics, characterized by mixing the reagent described in claim 9 with a specimen that may contain the target substance.

12. A method for detecting a target substance in a specimen by in vitro diagnostics, comprising the steps of: mixing a specimen potentially containing the target substance with the reagent described in claim 9 to obtain a mixture; irradiating the mixture with light; and detecting at least one of transmitted light and scattered light from the light irradiated onto the mixture.

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