Immuno-nephelometry particle, reagent, test kit and method for detecting target substance

Abstract

Provided are a particle for immunoturbidimetry, a reagent, a test kit, and a method for detecting a target substance, each of which can detect a trace component in a low concentration region and improve storage stability. The particle for immunoturbidimetry is a particle for immunoturbidimetry including a first resin and titanium oxide, wherein the particle for immunoturbidimetry includes a first layer containing the first resin and a second layer containing the titanium oxide, wherein the second layer is arranged on the outside of the first layer, and wherein the density of the titanium oxide is 3.40 g / cm 3 Hereinafter, and wherein the content of the titanium oxide in the particle for immunoturbidimetry is 10 mass% or more and 80 mass% or less.

Description

Technical Field

[0001] This disclosure relates to a method for detecting a target substance using an immunoturbidimetric assay, comprising particles, reagents, a detection kit, and a method thereof. Background Technology

[0002] In recent years, immunoturbidimetry has gained attention as a simple and rapid immunological detection method. Immunoturbidimetry involves mixing a dispersion of particles with antibodies or antigens on their surface as ligands with a sample that may contain the target substance (antigen or antibody). When the sample contains the target substance (antibody or antigen), the particles induce an agglutination reaction, and the presence or absence of a disease can therefore be determined by optically detecting changes in the intensity of scattered light, transmitted light, or absorbance.

[0003] Polystyrene-based latex particles, with polystyrene as the main component, have been used for immunoturbidimetric assays because they are easy to sensitize (immobilize) antigens or antibodies, relatively inexpensive, and their polymerization reaction is easy to control.

[0004] For example, particles using titanium dioxide have been developed, which is a well-known high refractive index material. In Japanese Patent Publication No. 2008-241357, a particle for immunoturbidimetry is proposed, wherein a ligand is bound to particles obtained by coating carboxylic acid-modified polystyrene particles with titanium dioxide microparticles. Summary of the Invention

[0005] In immunoturbidimetric assays using polystyrene latex particles, a problem arises in some cases where trace components in low-concentration regions cannot be detected. Therefore, there is a need to develop particles with higher sensitivity than polystyrene latex particles.

[0006] To address these issues, it is important to increase the absorbance variation caused by particle aggregation and the formation of immune complexes. To improve absorbance variation, particles using high-refractive-index materials have been developed.

[0007] In the method described in Japanese Patent Publication No. 2008-241357, the particles containing a large amount of titanium oxide have high-density titanium oxide and large particle size. Therefore, when these particles are used as a detection reagent, the sedimentation rate is fast, and there is potential for improving the storage stability of the reagent.

[0008] This disclosure is made in view of the aforementioned background technology and problems. Specifically, this disclosure relates to providing particles, reagents, detection kits, and methods for detecting target substances in immunoturbidimetry, which can each detect trace components in low concentration regions and improve storage stability in immunoturbidimetry.

[0009] The immunoturbidimetric particles according to this disclosure are immunoturbidimetric particles comprising a first resin and titanium dioxide, wherein the immunoturbidimetric particles comprise a first layer containing the first resin and a second layer containing the titanium dioxide, wherein the second layer is disposed outside the first layer, and wherein the titanium dioxide has a concentration of 3.40 g / cm³. 3 The following density, and the content of titanium dioxide in the particles used in the immunoturbidimetric assay is more than 10% by mass and less than 80% by mass.

[0010] Furthermore, the reagent according to this disclosure comprises the above-described immunoturbidimetric particles and an aqueous solution, wherein the immunoturbidimetric particles are dispersed in the aqueous solution. Furthermore, the detection kit according to this disclosure comprises the above-described reagent and a container configured to contain the above-described reagent.

[0011] Furthermore, the method for detecting a target substance according to this disclosure is a method for detecting a target substance in a sample via in vitro diagnostics, the method comprising mixing the above-described reagents and a sample that may contain the target substance. Additionally, the method for detecting a target substance according to this disclosure is a method for detecting a target substance in a sample via in vitro diagnostics, the method comprising: mixing the above-described reagents and a sample that may contain the target substance to provide a mixed solution; irradiating the mixed solution with light; and detecting at least one of transmitted light and scattered light from the light that has irradiated the mixed solution.

[0012] The features of this disclosure will become apparent from the following description of the implementation schemes. The following description of the implementation schemes is illustrated by way of example. Detailed Implementation

[0013] The embodiments of this disclosure are described in detail below. However, the technical scope of this disclosure is not limited to the described embodiments.

[0014] The immunoturbidimetric particles according to this disclosure are immunoturbidimetric particles comprising a first resin and titanium dioxide, wherein the immunoturbidimetric particles comprise a first layer containing the first resin and a second layer containing the titanium dioxide, wherein the second layer is disposed outside the first layer, and wherein the titanium dioxide has a concentration of 3.40 g / cm³. 3 The following density, and the content of titanium dioxide in the particles used in the immunoturbidimetric assay is more than 10% by mass and less than 80% by mass.

[0015] The inventors have discovered that when the particles include a first resin as its core (i.e., the first layer) and contain a density of 3.40 g / cm³, 3When the following titanium oxide-containing layer is formed as a second layer on its outer side, trace components in low-concentration regions of the target substance can be detected, and storage stability can be improved. The reason is believed to be as follows. The resin layer as the first layer and the titanium oxide-containing layer as the second layer have a density of 3.40 g / cm³. 3 The arrangement of the titanium oxide layers below enables a system with significant turbidity changes during particle agglomeration, but with a lower particle density than in the case of particles consisting solely of titanium oxide. This system is believed to be capable of detecting trace components in low-concentration regions of the target substance and improving storage stability.

[0016] The immunoturbidimetric method disclosed herein uses titanium dioxide particles with a concentration of 3.40 g / cm³. 3 The following density. Typically, commercially available titanium dioxide is produced, for example, by the sulfate or chloride process, and has a highly crystalline crystal structure, such as anatase, brookite, or rutile. Highly crystalline titanium dioxide has a high density and differs from the titanium dioxide used in this disclosure. The inventors conceived of introducing a range of low-density, i.e., lightweight titanium dioxide, to enable the detection of trace components in low-concentration regions of the target substance and to improve storage stability, thus realizing the present invention.

[0017] The density of the titanium oxide disclosed herein is preferably 1.50 g / cm³. 3 Above and 3.40 g / cm 3 The following is more preferably 2.50 g / cm³. 3 Above and 3.40 g / cm 3 The following is to enable the detection of trace components in low-concentration regions of the target substance.

[0018] The titanium oxide used in this disclosure is not particularly limited, as long as its density is within the aforementioned range. However, it can be obtained, for example, by hydrolyzing a titanium-containing metal oxide precursor, a method known as the sol-gel method. In other words, the titanium oxide used in this disclosure can be obtained by coexisting a metal oxide precursor, an oxygen-containing organic solvent, and water, and then carrying out a hydrolysis reaction.

[0019] Examples of metal oxide precursors include metal chlorides, metal acetates, metal alkoxides, and metal hydroxides. Among these, metal alkoxides, metal acetates, and metal hydroxides are preferred from the viewpoint of impurities (e.g., chlorides) generated as byproducts. Of these, those with the chemical formula M are particularly preferred. x (OR) y The metal alkoxide (where M represents a metal element, R represents an alkyl group, and "x" and "y" each independently represent an integer greater than 1 and less than 4), is represented by the chemical formula M x′ (OH) y′•nH₂O represents metal hydroxides (where M represents a metal element, x′ and y′ each independently represent an integer greater than or less than 1 and less than 4, and “n” represents an integer greater than or equal to 1), and compounds containing the above-mentioned metal alkoxides and / or metal hydroxides. Specific examples include titanium methoxide, titanium ethanol, titanium diisopropoxydi(2,4-pentanedione), titanium diisopropoxydi(ethyl acetoacetate), titanium n-butoxide, titanium isopropoxide, titanium methoxypropoxide, titanium n-nonoxide, titanium n-propoxide, titanium stearate, titanium triisostearylpropoxide, and titanium trimethylsilyl alcohol.

[0020] Examples of oxygen-containing organic solvents include alcohols, ketones, aldehydes, ethers, esters, and siloxanes.

[0021] The turbidimetric particles according to this disclosure include a first layer containing a first resin and a second layer containing titanium dioxide, with the second layer disposed outside the first layer. When the particles include the first resin as its core (i.e., the first layer) and include titanium dioxide disposed outside it as the second layer, sufficient amounts of resin and titanium dioxide can be included to detect trace components in low concentration regions and improve storage stability.

[0022] The particles used in the immunoturbidimetric assay according to this disclosure are not particularly limited, as long as the particles comprise a second layer outside the first layer. However, they can be obtained, for example, by coexisting the first resin in the aforementioned hydrolysis reaction. That is, the particles can be obtained by coexisting a metal oxide precursor, an oxygen-containing organic solvent, water, and the first resin and carrying out a hydrolysis reaction. The first resin can be obtained by general polymer particle production methods, such as emulsion polymerization, soap-free polymerization, dispersion polymerization, suspension polymerization, phase inversion emulsification, wet milling, or dry milling, and there are no particular limitations. The first resin obtained by each of the emulsion polymerization and soap-free polymerization methods is preferred because it achieves the volume-average particle size required for the immunoturbidimetric particles and obtains a uniform particle size distribution.

[0023] For particles used in immunoturbidimetry, the preferred range for volume average particle size is 150 nm or more and 1000 nm or less, more preferably 180 nm or more and 800 nm or less. Furthermore, the preferred range for particle size distribution for particles used in immunoturbidimetry is a ratio of volume average particle size to number average particle size of 1.00 or more and 1.25 or less.

[0024] The first resin disclosed herein is not particularly limited, but preferably has a high refractive index. Preferred specific structures for this purpose may include styrene structures, fluorene structures, dinaphthothiophene structures, naphthalene structures, anthracene structures, and phenanthrene structures.

[0025] The immunoturbidimetric method disclosed herein uses particles containing 10% by mass or more and 80% by mass or less, preferably 21% by mass or more and 60% by mass or less. When the titanium oxide content is less than 10% by mass, it may be difficult to detect trace components in low concentration regions of the target substance. When the titanium oxide content is greater than 80% by mass, the sedimentation rate may increase, thereby causing problems related to storage stability.

[0026] The preferred particle density for the immunoturbidimetric method according to this disclosure is 1.20 g / cm³. 3 Above and 1.54 g / cm 3 the following.

[0027] For the titanium oxide of this disclosure, when a sufficient amount of 3.40 g / cm³ is incorporated into the particles... 3 When the titanium dioxide content is set within the above range and the particle density is within the range specified, both improved sensitivity and suppression of sedimentation can be achieved simultaneously.

[0028] According to the immunoturbidimetric method of this disclosure, the circularity of the particles is preferably 0.85 or higher and 1.00 or lower. When the circularity of the particles is set within the above range, the particles smoothly approach each other during the antigen-antibody reaction, thus achieving higher sensitivity.

[0029] Furthermore, the particles used in the immunoturbidimetric assay according to this disclosure preferably comprise a third layer containing a second resin outside the second layer containing titanium dioxide. When the particles include this third layer, the adsorption rate of antibodies or antigens acting as biosensors is improved, and higher sensitivity can be achieved.

[0030] The second resin in the third layer preferably has a structure represented by the following formula (1). This can further improve the adsorption rate of antibodies or antigens that act as biosensors.

[0031]

[0032] In equation (1), R 1 R represents a hydrogen atom or a methyl group. 2 This indicates a structure with a substituted phenyl group or a structure with an ester group.

[0033] Furthermore, the second resin in the third layer more preferably has at least one of the structures represented by formula (1-1) and formula (1-2) as the structure represented by formula (1):

[0034]

[0035] In equation (1-1), R 3 R represents a hydrogen atom or a methyl group. 4This indicates a structure having a hydroxyl group or a structure having a carboxyl group; and

[0036]

[0037] In equation (1-2), R 5 R represents a hydrogen atom or a methyl group. 6 This indicates a structure containing a hydroxyl group or a carboxyl group.

[0038] When the coating resin has at least one of the structures represented by formula (1-1) and formula (1-2), the coating resin interacts satisfactorily with titanium oxide and the titanium oxide can be uniformly coated without exposing it. This can suppress the desorption of the resin. Specific examples of the structures shown in formula (1-1) or (1-2) are shown in formulas (1-A-1) to (1-A-12) below, but this disclosure is not limited thereto.

[0039]

[0040] Furthermore, the second resin preferably has at least one of the structures shown in formula (2) and formula (3).

[0041]

[0042] In equation (2), R 7 Represents a hydrogen atom or a methyl group; n1 represents an integer greater than or equal to 1 and less than or equal to 3; m1 represents an integer greater than or equal to 0 and less than or equal to 2; n1+m1 equals 3; *1 independently represents a bond with a titanium or silicon atom, or represents a hydrogen atom, a methyl group, or an ethyl group; R 8 Each can be represented independently as methyl or ethyl. That is, the structure shown in formula (2) can be bonded by an oxygen atom to a titanium atom of titanium oxide, or by an oxygen atom to a silicon atom of another structure shown in formula (2) or formula (3).

[0043]

[0044] In equation (3), R 9 R represents a hydrogen atom or a methyl group. 10 The symbol represents a single bond, a phenylene group, or an alkylene group having three or fewer carbon atoms; n2 represents an integer greater than or equal to 1 and less than 3; m2 represents an integer greater than or equal to 0 and less than 2; n2+m2 equals 3; *2 independently represents a bond with a titanium or silicon atom, or represents a hydrogen atom, a methyl group, or an ethyl group; and R 11 Each can be represented independently as methyl or ethyl. That is, the structure shown in formula (3) can be bonded by oxygen atoms to titanium atoms of titanium oxide, or by oxygen atoms to silicon atoms of another structure shown in formula (3) or formula (2).

[0045] In addition to the structures represented by formula (1-1) or formula (1-2), the resins containing the structures represented by formula (2) or formula (3) are vinyl polymers and have alkoxysilanes. Uniform coating is achieved by further suppressing titanium oxide exposure through interaction with titanium oxide.

[0046] Particles having the structure shown in Formula (1-1) or Formula (1-2) can be obtained, for example, by coexisting titanium oxide-containing particles and monomers in an aqueous medium and adding a polymerization initiator thereto to carry out a polymerization reaction, i.e., by seed polymerization. The monomer is not particularly limited, but for example, the particles can be obtained by seed polymerization using glycidyl methacrylate as the monomer followed by a ring-opening reaction of the glycidyl group. Furthermore, the monomer can be used alone or as a mixture thereof. The monomers to be mixed are not particularly limited, but when the monomers contain the structure shown in Formula (2) or Formula (3), the particles can be obtained by seed polymerization of monomers deriving the structure shown in Formula (2) or Formula (3) through copolymerization of two or more monomers. The monomers deriving the structure shown in Formula (2) or Formula (3) are not particularly limited, as long as the monomer has a structure deriving the structure shown in Formula (2) or Formula (3). Examples include vinyltrimethoxysilane, vinyltriethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, and 3-acryloxypropyltrimethoxysilane. These monomers can be used alone or in combination.

[0047] The particles including the second resin can also be obtained, for example, by coexisting the titanium oxide-containing particles and the second resin in an aqueous medium to allow an adsorption reaction to occur between them. In this case, the second resin is not particularly limited, as long as it is soluble in the aqueous medium and can be adsorbed onto the titanium oxide-containing second layer. For example, water-soluble polymers such as sodium poly(acrylate), polysaccharides such as sodium carboxymethyl dextran, and proteins such as bovine serum albumin can be used.

[0048] The particles used in the immunoturbidimetric assay according to this disclosure can be pre-sensitized particles without ligands, or affinity particles whose surfaces further include ligands. Due to the ligands added to the particle surface, affinity particles exhibit selective or specific high affinity for the target substance. In particular, it is preferable to add the ligands to the particle surface via chemical bonds.

[0049] The ligands in this disclosure are compounds that specifically bind to a receptor of a particular target substance. The binding site of the ligand to the target substance is fixed and exhibits selective or specific high affinity. Examples include antigens and antibodies, enzyme proteins and their substrates, signaling molecules (such as hormones or neurotransmitters) and their receptors, and nucleic acids, but the ligands in this disclosure are not limited to these. An example of a nucleic acid is deoxyribonucleic acid (DNA). The affinity particles in this disclosure exhibit selective or specific high affinity for the target substance. Preferably, the ligands in this disclosure are any one of antibodies, antigens, and nucleic acids.

[0050] Furthermore, the reagent according to this disclosure is a reagent in which particles are dispersed in an aqueous solution according to the immunoturbidimetric method of this disclosure.

[0051] There are no particular limitations on the target substances that the reagent can detect. Examples of reagents are in vitro diagnostic drugs, and examples include antigen detection reagents that use antigens as the target substances and antibody detection reagents that use antibodies as the target substances. A reagent can be one in which ligand-free pre-sensitized particles are dispersed in an aqueous solution, assuming the user adds the desired ligand, such as an antibody, to the particles. Alternatively, assuming a specific target substance, the reagent can be one in which affinity particles with added ligands are pre-dispersed in an aqueous solution.

[0052] The reagents according to this disclosure include immunoturbidimetric particles (pre-sensitized particles or affinity particles) according to this disclosure and a dispersion medium for dispersing said immunoturbidimetric particles. In addition to the immunoturbidimetric particles according to this disclosure, the reagents according to this disclosure may also include a third substance, such as a solvent or blocking agent, to the extent that the objectives of this disclosure can be achieved. Two or more third substances (such as solvents and blocking agents) may be added 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 in the reagents are not limited to these.

[0053] The test kit according to this disclosure includes reagents according to this disclosure and a container for containing said reagents. In addition to the reagents described above (hereinafter referred to as "Reagent 1"), the test kit may include a reaction buffer (hereinafter referred to as "Reagent 2"). A sensitizer may be incorporated into each of Reagent 1 and Reagent 2, or either of said reagents. Furthermore, in addition to Reagent 1 and Reagent 2, the test kit of this disclosure may also include a positive control, a negative control, a serum diluent, a primary antibody, a secondary antibody, etc. The medium used as a positive or negative control may be a solvent, in addition to serum and physiological saline, each of which does not contain the measurable target substance.

[0054] The method for detecting a target substance according to this disclosure can be a method for detecting a target substance in a sample via in vitro diagnostics, the method comprising mixing a reagent according to this disclosure with a sample that may contain the target substance. Furthermore, the method for detecting a target substance according to this disclosure can be a method for detecting a target substance in a sample via in vitro diagnostics, the method comprising: mixing a reagent according to this disclosure with a sample that may contain the target substance to provide a mixed solution; irradiating the mixed solution with light; and detecting at least one of transmitted light and scattered light from the light that has irradiated the mixed solution.

[0055] (Immunoturbidimetric method for measuring titanium dioxide content in particles)

[0056] An example of a method for measuring the content of titanium dioxide in particles using immunoturbidimetry according to this disclosure is described. The content of titanium dioxide in the particles is measured using a thermogravimetric analyzer. For example, a STA200 (manufactured by Hitachi High-Tech Science Corporation) is used. 5 to 10 mg of the cured product is weighed into an aluminum pan and measured under a nitrogen atmosphere. The temperature conditions are as follows: the temperature is maintained at 30°C for 30 minutes, then the temperature is increased from 30°C to 500°C at a rate of 10°C / min, and then maintained at 500°C for 10 minutes.

[0057] As an evaluation, the titanium dioxide content in the particles can be measured by the mass loss of the resin components at 300°C.

[0058] (Immunoturbidimetric method for measuring the density of titanium dioxide in particles)

[0059] Examples of methods for measuring the density of titanium oxide as described in this disclosure are presented. The density of titanium oxide can be measured by separating the titanium oxide with an immunoturbidimetric assay using particles and then performing a dry density measurement on the separated titanium oxide using a constant volume expansion method. For example, the measurement was performed using an AccuPyc II (manufactured by Shimadzu Corporation) at 25°C, and the average of 10 measurements was used.

[0060] There are no particular limitations on the methods for separating titanium dioxide from particles, but titanium dioxide can be removed from particles, for example, by dissolving and washing the resin in the particles with a solvent.

[0061] Alternatively, the density of titanium oxide can be calculated using the following formula.

[0062]

[0063] D P The density of particles measured using the method presented in this article as an example.

[0064] DT Titanium oxide density

[0065] D R Resin density

[0066] W T The content of titanium dioxide in particles was measured using the method presented in this article as an example.

[0067] The calculated resin density is 1.10 g / cm³. 3 .

[0068] (Immunoturbidimetric assay is a method for measuring particle density)

[0069] Examples of methods for measuring particle density using immunoturbidimetry as described in this disclosure are presented. The particle density is measured, for example, by dry density measurement using a constant volume expansion method. For example, an AccuPyc II (manufactured by Shimadzu Corporation) is used, and measurements are performed at 25°C, using the average of 10 measurements.

[0070] (Immunoturbidimetric assay for aqueous dispersions is a method for measuring the volume-average particle size and particle size distribution of particles.)

[0071] This disclosure describes a method for measuring the volume-average particle size (Dv) of particles using immunoturbidimetry. The Dv of particles present in an aqueous dispersion is measured by a dynamic light scattering method. For example, a Zetasizer (Zetasizer ultra: manufactured by Malvern Panalysis Ltd.) is used and the measurement is performed at 25°C.

[0072] Furthermore, the particle size distribution of the particles in this disclosure is calculated by measuring the number-average particle size (Dn) using the dynamic light scattering method described above, in order to determine the ratio of Dv to Dn (Dv / Dn).

[0073] (Immunoturbidimetric method for determining the roundness of particles)

[0074] This disclosure describes a method for measuring particle roundness using immunoturbidimetry. Five hundred particles are extracted from a scanning electron microscope (SEM) image, and the roundness of each particle is calculated using image analysis software. The average of the five hundred particles is taken as the roundness. Roundness is calculated using, for example, an S4800 manufactured by Hitachi High-Tech Corporation as the scanning electron microscope and, for example, Image-J as the image analysis software.

[0075] [Example]

[0076] The present disclosure is described in detail below with reference to embodiments, but the present disclosure is not limited to these embodiments.

[0077] [Example of Particle Preparation]

[0078] (Preparation of Particle 1)

[0079] (The steps for forming the first layer containing resin)

[0080] 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 were weighed and added to a 2 L four-necked separable flask to prepare a mixed solution. Oxygen was removed from the inside of the flask by maintaining the mixed solution at 70 °C, stirring at 140 rpm, and applying a nitrogen flow at a rate of 200 ml / min. Next, a solution of 0.55 g of V-50 (FUJIFILM Wako Pure Chemical Corporation) dissolved in 20 g of separately prepared deionized water was added to the mixed solution to initiate soap-free polymerization. A dispersion containing the copolymer of styrene and divinylbenzene, known as particle 1, was obtained after reacting for 23 hours from the start of polymerization. A portion of the dispersion was collected and evaluated using dynamic light scattering (ZetasizerUltra: Malvern Panalysis Ltd.), resulting in a volume-average particle size of 190 nm.

[0081] (The steps for forming the second layer containing titanium oxide)

[0082] The first layer of forming particles 1 was adjusted to a solids concentration of 0.6% using deionized water. 20 g of dispersion was mixed into 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 of 5.0 ml of titanium butoxide (IV) monomer (TBOT: Kishida Chemical Co., Ltd.) mixed with 197.50 g of separately prepared ethanol was added to the mixture to initiate the sol-gel reaction. The reaction was carried out for 24 hours from the start of the sol-gel reaction. The dispersed particles in the mixture were separated by centrifugation and redispersed in ethanol. Furthermore, the process of separating the particles from the dispersion by centrifugation and redispersing them in deionized water was repeated twice for purification, thereby providing the second layer of forming particles 1. The second-layer forming particles 1 were stored as an aqueous dispersion, ultimately adjusted to 5.0% by mass. A portion of the dispersion was collected, and the dynamic scattering of the second-layer forming particles 1 was evaluated. As a result, the volume-average particle size was 200 nm. Furthermore, when the metal oxide content was evaluated using differential thermal-thermogravimetric analysis (NEXTA STA200RV: Hitachi High-Tech Corporation), the content was 45% of the particle weight.

[0083] (The step of forming the third layer containing resin)

[0084] The mixture was adjusted with deionized water to provide a dispersion of 149.55 g of second-layer forming particles 1 with a solids concentration of 0.2%. 0.135 g of glycidyl methacrylate (GMA: Kishida Chemical Co., Ltd.) and 0.015 g of 3-methacryloyloxypropyltrimethoxysilane (MPS: Shin-Etsu Chemical Co., Ltd.) were added, and the mixture was maintained at 70 °C while stirring at 100 rpm. Oxygen was removed from the inside of the four-necked separable flask by a nitrogen flow at a rate of 200 ml / min. Then, a separately prepared solution of 0.03 g of V-50 dissolved in 0.3 g of deionized water was added to the mixture to initiate shell formation. A dispersion containing third-layer forming particles 1 was obtained by continuing stirring for 18 hours after the reaction began.

[0085] (The steps of assigning reactive functional groups)

[0086] A pre-prepared aqueous solution containing dissolved mercaptosuccinic acid (MSA: Wako Pure Chemical Industries, Ltd.) (the total moles of MSA equal to the moles of glycidyl methacrylate) was added to the dispersion containing the third layer forming particles 1. Triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. Next, the mixture was heated to 70°C while being stirred at 800 rpm. The mixture was maintained at this temperature for another 18 hours to provide a dispersion containing particles 1.

[0087] (Particle cleaning steps)

[0088] The process of separating particle 1 from the dispersion using a centrifuge and redispersing particle 1 in ion-exchanged water was repeated eight times, and finally the particle concentration was adjusted to 5.0% by mass. Thus, a dispersion of particle 1 was obtained.

[0089] (Preparation of particles 2 to 6)

[0090] In the step of forming the resin-containing third layer, 0.15 g of glycidyl methacrylate is added in place of 0.135 g of glycidyl methacrylate and 0.015 g of 3-methacryloyloxypropyltrimethoxysilane. Apart from the foregoing, a dispersion of particles 2 is obtained by the same procedure as for particles 1.

[0091] In the step of forming the third layer containing the resin, 0.135 g of styrene and 0.015 g of methacrylic acid (MAA: Kishida Chemical Co., Ltd.) were added in place of 0.135 g of glycidyl methacrylate and 0.015 g of 3-methacryloyloxypropyltrimethoxysilane. Furthermore, the step of imparting reactive functional groups was not performed. Apart from the foregoing, the dispersion of particles 3 was obtained by the same operation as for particles 1.

[0092] The dispersion of particles 4 was obtained by the same operation as that of particles 2, except that the amount of titanium butoxide (IV) monomer was changed from 5.0 ml to 3.0 ml in the step of forming the second layer containing titanium oxide.

[0093] The dispersion of particles 5 was obtained by the same operation as that for particles 2, except that the amount of titanium butoxide (IV) monomer was changed from 5.0 ml to 7.5 ml in the step of forming the second layer containing titanium oxide.

[0094] The dispersion of particle 6 was obtained by the same operation as particle 2, except that the amount of titanium butoxide (IV) monomer was changed from 5.0 ml to 2.0 ml in the step of forming the second layer containing titanium oxide.

[0095] (Preparation of Particle 7)

[0096] Bovine serum albumin (hereinafter sometimes simply referred to as "BSA") was used in the resin-containing third layer. First, 1 mL of deionized water was added to 30 mg of BSA to prepare an aqueous BSA solution. 0.5 mL of the BSA solution was placed in a 1.5 mL tube, and then 0.5 mL of a 2.0% by mass solution of the second layer forming particles 1 was added. After thorough mixing, the dispersion was allowed to stand overnight at room temperature. Next, the process of separating the particles from the dispersion using a centrifuge and redispersing the particles in deionized water was repeated three times, finally adjusting the particle concentration to 5.0% by mass. This yielded a dispersion of particles 7 comprising the BSA-containing third layer.

[0097] (Preparation of Particle 8)

[0098] Sodium poly(acrylate) (hereinafter sometimes referred to as "PAANa") is used for the third layer containing the resin. PAANa has a molecular weight of 22,000 to 66,000. First, 10 mL of deionized water is added to 10 mg of PAANa to prepare an aqueous solution of PAANa. 0.5 mL of the PAANa aqueous solution is placed in a 1.5 mL tube, and then 0.5 mL of a 2.0% by mass solution of the second layer forming particles 1 is added. After thorough mixing, the dispersion is allowed to stand overnight at room temperature. Next, the process of separating the particles from the dispersion using a centrifuge and redispersing the particles in deionized water is repeated three times, finally adjusting the particle concentration to 5.0% by mass. Thus, a dispersion of particles 8 comprising a third layer containing PAANa is obtained.

[0099] (Preparation of Particle 9)

[0100] Sodium carboxymethyl dextran (hereinafter sometimes simply referred to as "Dex") was used for the third layer containing the resin. Dex has a molecular weight of 40,000. First, 1 mL of deionized water was added to 20 mg of Dex to prepare an aqueous solution of Dex. 0.5 mL of the Dex aqueous solution was placed in a 1.5 mL tube, and then 0.5 mL of a 2.0% by mass solution of the second layer forming particles 1 was added. After thorough mixing, the dispersion was allowed to stand overnight at room temperature. Next, the process of separating the particles from the dispersion using a centrifuge and redispersing the particles in deionized water was repeated three times, finally adjusting the particle concentration to 5.0% by mass. Thus, a dispersion of particles 9, comprising the third layer containing Dex, was obtained.

[0101] (Preparation of particles 10 and 11)

[0102] The dispersion of particles 10 is obtained by the same operation as particles 2, except that in the step of forming the first layer containing resin, the amount of styrene is changed from 12.68 g to 101.44 g and the amount of divinylbenzene is changed from 0.23 g to 1.84 g.

[0103] The dispersion of particles 11 is obtained by the same operation as particles 2, except that divinylbenzene is not added in the step of forming the first layer containing resin and styrene is converted into methyl methacrylate (MMA: Kishida Chemical Co., Ltd.).

[0104] [Comparative Particle Preparation Examples]

[0105] 50 μL of titanium dioxide fine particle dispersion and 950 μL of γ-aminopropyltriethoxysilane aqueous solution (pure water containing 1% γ-aminopropyltriethoxysilane by mass) were placed in centrifuge tubes and stirred at 100 rpm for 1 hour at room temperature (20°C to 30°C) using a roller. Next, the tubes were centrifuged at 15,000 rpm (approximately 20,000 G) for 15 minutes, and the supernatant was removed by aspiration. 1500 μL of acetate buffer (0.01 M, pH: 5.0) was added to the precipitate, and the mixture was redispersed using a vortex mixer and centrifuged again at 15,000 rpm (approximately 20,000 G) for 15 minutes, with the supernatant removed by aspiration. This process (addition of acetate buffer, centrifugation, and removal of supernatant) was repeated three times. Add 500 μL of phosphate buffer (0.01 M, pH: 6.0) to the resulting precipitate, disperse it using an ultrasonic homogenizer for 2 minutes, and then redisperse it to provide a dispersion of amino-introduced titanium dioxide microparticles.

[0106] Add the following materials to a centrifuge tube and stir the mixture for 1 hour at 100 rpm using a tube roller at room temperature (20°C to 30°C).

[0107] • Carboxyl-modified polystyrene microparticle dispersion [trade name: "IMMUTEX", manufactured by JSR Corporation, carboxyl-modified latex, containing microparticles with a volume average particle size of 200 nm and a microparticle content of 10%] (B-1) 100 μL

[0108] • 900 μL of HEPES buffer containing 1% bovine serum albumin

[0109] • Soluble carbodiimide solution [1M aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; hereinafter the same] 500 μL

[0110] Next, centrifuge the tubes at 15,000 rpm (approximately 20,000 G) for 15 minutes, then remove the supernatant by aspiration. Add 1500 μL of pure water to the precipitate and redisperse it using a vortex mixer. Centrifuge the result again at 15,000 rpm (approximately 20,000 G) for 15 minutes, remove the supernatant by aspiration, and add 500 μL of HEPES buffer containing 1% bovine serum albumin to the precipitate. Disperse the result using an ultrasonic homogenizer for 2 minutes and redisperse to provide a dispersion of carboxyl-activated polystyrene microparticles.

[0111] 88 μL of amino-introduced titanium dioxide microparticle dispersion and 500 μL of carboxyl-activated polystyrene microparticle dispersion were loaded into centrifuge tubes and stirred at 100 rpm for 2 hours at room temperature (20°C to 30°C) using a roller. The mixture was then centrifuged at 15,000 rpm (approximately 20,000 G) for 15 minutes, and the supernatant was removed by aspiration. 1,500 μL of phosphate buffer (0.1 M, pH 7.1) was added to the precipitate, and the mixture was redispersed using a vortex mixer to provide a titanium dioxide-polystyrene composite microparticle dispersion.

[0112] The titanium dioxide-polystyrene composite fine particle dispersion was centrifuged using density gradient centrifugation to obtain a fraction with a density of 1.60 (titanium dioxide content in the composite particles: 20%), which was used as comparative particle 1. Similarly, a fraction with a density of 1.20 (titanium dioxide content in the composite particles: 5%) was obtained as comparative particle 2.

[0113] The physical properties of the obtained particles 1 to 11, as well as the comparative particles 1 and 2, are shown in Tables 1-1 and 1-2.

[0114] Table 1-1

[0115]

[0116] Table 1-2

[0117]

[0118] (Preparation of affinity particles)

[0119] 300 μL (3 mg based on particulate solids) of particle 1 dispersion diluted with deionized water to a solids concentration of 1.0% by mass was aliquoted into 1.5 mL microtubes. 90 μL of a 5.0% aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (Tokyo Chemical Industry Co., Ltd.) and 90 μL of a 5.0% aqueous solution of N-hydroxysulfosuccinimide sodium salt (Tokyo Chemical Industry Co., Ltd.) were added. The mixture was stirred at room temperature for 30 minutes to provide a dispersion of particles with activated carboxyl groups (activated particle dispersion).

[0120] After centrifugation and washing, 270 μL of phosphate-buffered saline (PBS) with a pH of 5.5 was added, and the particles with activated carboxyl groups were dispersed by ultrasonication.

[0121] 24 μL (0.12 mg based on antibody amount) of a 5.0 mg / mL dispersion of mouse monoclonal antiferritin antibody (isoelectric point: 7.1) was added to the mixture, and the mixture was stirred at room temperature for 3 hours to provide affinity particle 1, wherein particle 1 was sensitized by the antibody.

[0122] Affinity particles 2 to 9 and comparative affinity particles 1 and 2 were prepared using the same experimental procedures as those used for preparing affinity particles 1, except that the particle types used in preparing affinity particles 1 were changed to particles 2 to 9 and comparative particles 1 and 2, respectively.

[0123] (Preparation of the first reagent)

[0124] The first reagent was obtained by dissolving 50 mM HEPES, 0.05% by mass Triton X-100 and 1.0% by mass sodium chloride (Kishida Chemical Co., Ltd.) in ion-exchanged water.

[0125] (Preparation of the second reagent)

[0126] After centrifuging and washing the affinity particles 1, the resulting product was redispersed in 500 μL of buffer (HEPES buffer) containing 10 mM HEPES, 0.01 wt% polyoxyethylene nonylphenyl ether (Triton X-100, Kishida Chemical Co., Ltd.), and 10 wt% sucrose (viscosity modifier) ​​dissolved in deionized water. The contents were then mixed and diluted with HEPES buffer to a concentration of 0.1 wt% for providing the second reagent 1.

[0127] The second reagents 2 to 9 and the comparative second reagents 1 and 2 were prepared by the same experimental procedures as those used in the preparation of the second reagent 1, except that the particle types used in the preparation of the second reagent 1 were changed to affinity particles 2 to 9 and comparative affinity particles 1 and 2, respectively.

[0128] (Measurement of the change in absorbance)

[0129] A mixed solution was prepared by mixing 15 μL of each sample prepared at 0.0 ng / mL (physiological saline) and 100 ng / mL ferritin concentration with 60 μL of reagent 1 (first reagent), and incubating at 37°C for 290 seconds. Next, 30 μL each of reagents 1 to 9 (second reagents) and comparative reagents 1 and 2 (comparative reagents) were added to the mixed solution, the mixture was stirred, and absorbance was measured after 42 seconds. The mixed solution was then allowed to stand at 37°C for 253 seconds, and absorbance was measured again. The difference in absorbance after 42 seconds of stirring was taken as the absorbance change ΔABS. Absorbance was measured at a measurement wavelength of 572 nm using a BIOSPECTROMETER spectrophotometer manufactured by Eppendorf Co.

[0130] (Calculation of storage stability index)

[0131] The change in absorbance at a ferritin concentration of 100 ng / mL was measured. Reagents 1 to 9 and Comparative Reagents 1 and 2 were allowed to stand at 4°C for a certain period, and then samples were taken from the upper half of the container. Under the same conditions, the change in absorbance at a ferritin concentration of 100 ng / mL was measured again, and the change after a certain period was calculated. It is expected that a smaller change indicates greater improvement in storage stability. Storage stability was evaluated based on the values ​​of the storage stability index shown below.

[0132] A: The change after 14 days is within 10%.

[0133] B: The change after 7 days is less than 10%, but the change after 14 days is greater than 10%.

[0134] C: The change after 7 days is greater than 10%.

[0135] The results are shown in Table 2.

[0136] (Calculation of the sensitivity index)

[0137] For a second reagent with a storage stability index of A or B, the sensitivity index is calculated as follows. The change in absorbance at a ferritin concentration of 0.0 ng / mL (physiological saline) is defined as ΔABS(0), and the change in absorbance at a ferritin concentration of 100 ng / mL is defined as ΔABS(100). The value of ΔABS(100) × 10,000 / ΔABS(0) × 10,000 is calculated and used as the sensitivity index. It is expected that as the sensitivity index increases, the sensitivity for detecting the target substance becomes higher.

[0138] The evaluation is based on the value of the sensitivity index, as described below.

[0139] A: The sensitivity index value is greater than 500.

[0140] B: The sensitivity index value is greater than 100 and less than 500.

[0141] C: The sensitivity index value is below 100.

[0142] The results are shown in Tables 2-1 and 2-2.

[0143] Table 2-1

[0144]

[0145] Table 2-2

[0146]

[0147] As described above, the immunoturbidimetric assay according to this disclosure uses particles 1 to 9, which exhibit high sensitivity and excellent storage stability, and are able to achieve a balance between improved sensitivity and improved storage stability. Meanwhile, the particles of Comparative Example 1 exhibit low storage stability. Furthermore, the particles of Comparative Example 2 exhibit excellent storage stability but low sensitivity. In other words, neither Comparative Example 1 nor Comparative Example 2 can simultaneously achieve an improvement in both sensitivity and improved storage stability.

[0148] According to this disclosure, an immunoturbidimetric assay can be provided using particles, reagents, a detection kit, and a method for detecting a target substance, which can detect trace components in low concentration regions of the target substance and improve storage stability.

[0149] Although this disclosure has been described with reference to embodiments, it should be understood that this disclosure is not limited to the disclosed embodiments. The scope of the following claims should be given the broadest interpretation in order to cover all such modifications and equivalent structures and functions.

Claims

1. A particle for immunoturbidimetry, comprising a first resin and titanium dioxide, in, The particles used in the immunoturbidimetric assay comprise a first layer containing the first resin and a second layer containing the titanium dioxide. The second layer is arranged outside the first layer. The titanium oxide has a content of 3.40 g / cm³. 3 The following densities, and The titanium dioxide content in the particles used in the immunoturbidimetric assay is 10% by mass or more and 80% by mass or less.

2. The granules for immunoturbidimetry according to claim 1, wherein, The content of titanium dioxide in the particles used in the immunoturbidimetric assay is 21% by mass or more and 60% by mass or less.

3. The granules for immunoturbidimetry according to claim 1, wherein, The particles used in the immunoturbidimetric assay have a sphericity of 0.85 or higher and 1.00 or lower.

4. The granules for immunoturbidimetry according to claim 1, wherein, The particles used in the immunoturbidimetric assay are comprised of a third layer containing a second resin outside the second layer containing the titanium dioxide.

5. The granules for immunoturbidimetry according to claim 1, wherein, The immunoturbidimetric assay uses particles with a particle size of 1.20 g / cm³. 3 Above and 1.54 g / cm 3 The following densities.

6. The granules for immunoturbidimetry according to claim 4, wherein, The second resin has a structure represented by the following formula (1): In equation (1), R 1 R represents a hydrogen atom or a methyl group. 2 This indicates a structure with a substituted phenyl group or a structure with an ester group.

7. The granules for immunoturbidimetry according to claim 1, wherein, The density of the titanium oxide is 2.50 g / cm³. 3 Above and 3.40 g / cm 3 the following.

8. The granules for immunoturbidimetry according to claim 1, wherein, Its surface further contains ligands.

9. A reagent comprising the granules for immunoturbidimetry as described in any one of claims 1 to 8 and an aqueous solution. in, The immunoturbidimetric method uses particles dispersed in the aqueous solution.

10. A test kit comprising the reagent of claim 9 and a container configured to contain the reagent.

11. A method for detecting a target substance in a sample by in vitro diagnostics, the method comprising mixing the reagent of claim 9 with a sample that may contain the target substance.

12. A method for detecting a target substance in a sample via in vitro diagnostics, the method comprising: The reagent of claim 9 is mixed with a sample that may contain the target substance to provide a mixed solution; The mixed solution was irradiated with light; as well as Detect at least one of the transmitted light and the scattered light from the light that has irradiated the mixed solution.

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