Condition change method for detection method of target substance using sensitization particles, and detection method for detecting target substance

Abstract

To provide a condition change method for a detection method of a target substance using sensitization particles capable of relatively reducing an amount of antibody to be used during measurement.SOLUTION: A condition change method for a detection method of target substance using sensitization particles includes: a prozone detection step which detects that a prozone phenomenon has occurred in which an increase in optical density is suppressed after the optical density increases in accordance with an increase in density of the target substance when optical density of sensitization particles X where blocking protein is physically combined with and a ligand is chemically combined with the surface of core particles is measured by a coagulation method using density x; a condition changing step changing conditions of the following (a) and (b) after the prozone detection step; where (a) changing sensitization particles X into sensitization particles Y in which at least blocking protein and the ligand are chemically combined with the surface of core particles and (b) changing density y of the sensitization particles Y into one higher than the density x of the sensitization particles X.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for changing conditions for a method for detecting a target substance using sensitized particles, and a method for detecting a target substance. [Background technology]

[0002] A latex immunoagglutination method is known as a method for measuring a target substance in a sample. The latex immunoagglutination method is a measurement method in which, for example, latex particles carrying antibodies against a target substance are used, and the degree of agglutination (turbidity) of the latex particles caused by binding of the target substance, an antigen, to the antibody-carrying latex particles is detected by optical means or the like. Known examples of this type of technology include the technology described in Patent Document 1. Patent Document 1 describes a reagent for measuring a target substance in a sample using a latex immunoagglutination method, which includes latex particles carrying an antibody that specifically recognizes the target substance in the sample (claim 7 of Patent Document 1). Furthermore, Patent Document 1 describes that after reacting an antibody with latex particles, a blocking treatment is carried out using bovine serum albumin (BSA) (paragraph 0029 of Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2021-162593 Summary of the Invention [Problem to be solved by the invention]

[0004] It is known that in the blocking treatment and washing treatment described in Patent Document 1, BSA is physically adsorbed onto the surface of latex particles. In the latex immunoagglutination method, there is a prozone phenomenon in which the antigen-antibody reaction is inhibited by an excess of either the antigen or the antibody. When using latex particles with physically adsorbed BSA, one possible solution to the prozone phenomenon in high antigen concentration regions is to increase the number of latex particles, which is presumably because the total surface area of ​​the particles increases, increasing the area where the antigen-antibody reaction occurs. However, as the number of latex particles increases, the amount of antibody used during measurement also increases in order to cover the entire surface of the increased number of particles. [Means for solving the problem]

[0005] After further investigation, the inventors found that when the prozone phenomenon occurs in a high concentration region of a target substance such as an antigen, even if conditions are changed to increase the number of sensitized particles contained in the reagent, the amount of antibody used during measurement can be relatively reduced by using sensitized particles to which a blocking protein is chemically bonded, compared to when sensitized particles to which a blocking protein is not chemically bonded are used, and thus completed the present invention.

[0006] According to one aspect of the present invention, there are provided a method for changing conditions for a method for detecting a target substance using sensitized particles, and a method for detecting a target substance, as follows. 1. A method for changing conditions for a method for detecting a target substance using sensitized particles, comprising: a prozone detection step of detecting the occurrence of a prozone phenomenon in which, when absorbance is measured by an agglutination method using sensitized particles X, each of which has a blocking protein physically bound to the surface of a core particle and a ligand chemically bound to the surface of the core particle at a concentration x, the absorbance increases in response to an increase in the concentration of the target substance, and then the increase in absorbance is suppressed; a condition changing step of changing the conditions as follows (a) and (b) after the prozone detection step; (a) changing the sensitized particle X to a sensitized particle Y having at least a blocking protein and a ligand chemically bonded to the surface of a core particle; (b) changing the concentration y of the sensitized particles Y to be higher than the concentration x of the sensitized particles X; and a method for changing conditions. 2. The method for changing conditions described in 1., A condition-changing method, wherein the concentration y / the concentration x is 1.1 or more. 3. The method for changing conditions according to 1. or 2., A condition-changing method, wherein the maximum concentration of the target substance in the prozone detection step is 25 ng / mL or more and 9000 ng / mL or less. 4. The condition change method according to any one of 1. to 3., the amount of ligand used in producing the sensitized particles Y at the concentration y is changed to 0.9 or less when the amount of ligand used in producing the sensitized particles X at the concentration x is taken as 1 in the condition changing step. 5. The condition change method according to any one of 1. to 4., The method for changing conditions, wherein the average particle diameter of the core particles is 80 nm or more and 700 nm or less. 6. The condition change method according to any one of 1. to 5., The method for changing conditions, wherein the CV value of the average particle diameter of the core particles is 15% or less. 7. The condition change method according to any one of 1. to 6., The condition-changing method, wherein the core particles are polystyrene-based latex particles. 8. The condition change method according to any one of 1. to 7., The method for changing conditions, wherein the ligand is any one of an antibody, an antigen, a protein, and a nucleic acid. 9. A detection method for detecting a target substance by an agglutination method using sensitized particles, comprising: a contacting step of contacting the target substance with the sensitized particles to obtain a mixture; a detection step of detecting the agglutination of the sensitized particles by optical means after the contact step and measuring the concentration of the target substance; Including, the sensitized particle is a sensitized particle Y in which at least a blocking protein and a ligand are chemically bonded to the surface of a core particle, the measured concentration of the target substance in the mixture in the detection step of measuring the concentration of the target substance is 0.5 ng / mL or more and 9000 ng / mL or less; A detection method for detecting a target substance. 10. A detection method for detecting a target substance according to 9., comprising: The average particle diameter of the core particles is 200 nm or more and 700 nm or less. A detection method for detecting a target substance, wherein the CV value of the average particle diameter of the core particles is 15% or less. [Effects of the Invention]

[0007] According to the present invention, there are provided a method for changing the conditions of a method for detecting a target substance using sensitized particles, which allows a relative reduction in the amount of antibody used during measurement, and a method for detecting a target substance. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is a cross-sectional view schematically illustrating an example of the configuration of a sensitized particle according to the present embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all drawings, similar components are designated by similar reference numerals, and descriptions thereof will be omitted as appropriate. Furthermore, the drawings are schematic diagrams and do not correspond to actual dimensional proportions.

[0010] An outline of a method for changing conditions for the target substance detection method of this embodiment will be described.

[0011] The method for changing the conditions of the method for detecting a target substance using sensitized particles of this embodiment is as follows: a prozone detection step of detecting the occurrence of a prozone phenomenon in which, when absorbance is measured by an agglutination method using sensitized particles X, each of which has a blocking protein physically bound to the surface of a core particle and a ligand chemically bound to the surface of the core particle at a concentration x, the absorbance increases in response to an increase in the concentration of the target substance, and then the increase in absorbance is suppressed; After the prozone detection step, a condition change step is performed to change the following conditions (a) and (b): (a) Changing the sensitizing particle X to a sensitizing particle Y formed by chemically bonding at least a blocking protein and a ligand to the surface of the core particle (b) Changing the concentration y of the sensitizing particle Y to be higher than the concentration x of the sensitizing particle X This is a condition change method including the above.

[0012] According to the findings of the present inventors, when a prozone phenomenon occurs in a high-concentration region of a target substance such as an antigen, even if a condition change is carried out to increase the number of sensitizing particles contained in the reagent, that is, to increase the concentration y compared to the concentration x before the condition change, by using the sensitizing particle Y to which a blocking protein is chemically bonded, it has been found that the amount of antibody used at the time of measurement can be relatively reduced compared to before the condition change using the sensitizing particle X to which no blocking protein is chemically bonded. For example, when the concentration y of the sensitizing particle X is increased to 1.5 times, the amount of antibody used at the time of sensitization also needs to be increased to 1.5 times. In contrast, even when the concentration y of the sensitizing particle Y is increased to 1.5 times, it is possible to make the amount of antibody used at the time of sensitization less than 1.5 times. That is, when the concentration y of the sensitizing particle Y is increased to y1 times (where 1 < y1 < 4), it is possible to make the amount of antibody used at the time of sensitization less than y1 times, preferably 1 time or less, more preferably 4 / 5 times or less, and even more preferably 3 / 5 times or less.

[0013] Although the detailed mechanism is not clear, on the surface of the sensitizing particle, in addition to physical adsorption, by introducing a blocking protein (BSA) by chemical bonding, the blocking protein (BSA) enters the gaps of the antibody (ligand) and is stably fixed, so that the surface unevenness of the sensitizing particle becomes more uniform (the surface becomes smooth), and the reaction efficiency between the antigen (target substance) and the antibody (ligand) is increased. Thus, it is推测 that even if the number of sensitizing particles Y, that is, the concentration y in the reagent, is increased, and even if the amount of antibody used at the time of sensitization is the same as or reduced compared to before the condition change, the decrease in sensitivity in the high-concentration region of the target substance can be suppressed.

[0014] Here, the concentration of the target substance is defined as a low concentration range of 0.5 ng / mL or more but less than 25 ng, and a high concentration range of 25 ng / mL or more but less than 9000 ng / mL. The upper limit of the concentration in the high concentration range is not limited to this and may be even higher. The prozone phenomenon that occurs in latex immunoagglutination is a phenomenon in which an antigen-antibody reaction is inhibited by an excess of either the antigen or the antibody. This can be explained as follows using the ratio of the amount of antibody to the amount of antigen depending on the concentration range of the target substance. When the concentration of the target substance (antigen) is in the low concentration range, it is considered measurable when the mass ratio of antibody amount / antigen amount is within a range of, for example, 20 to 400,000; when it is outside this range, it is considered a detection condition in which the prozone phenomenon may occur. When the concentration of the target substance (antigen) is in the high concentration range, it is considered measurable when the mass ratio of antibody amount / antigen amount is within the range of, for example, 2 to 10,000, and when it is outside this range, it becomes a detection condition where the prozone phenomenon may occur.

[0015] The lower limit of the maximum concentration of the target substance in the prozone detection step is, for example, 25 ng / mL or more, preferably 30 ng / mL or more, and more preferably 35 ng / mL or more. The region above this concentration lower limit may be defined as the high concentration region of the target substance. On the other hand, the upper limit of the maximum concentration of the target substance in the prozone detection step is, for example, 9000 ng / mL or less, preferably 8500 ng / mL or less, and more preferably 8000 ng / mL or less.

[0016] The lower limit of concentration y / concentration x is, for example, 1.1 or more, preferably 1.15 or more, and more preferably 1.2 or more. On the other hand, the upper limit of concentration y / concentration x is, for example, 10 or less, preferably 9 or less, and more preferably 8 or less.

[0017] In the condition changing step, when the amount of ligand used in producing sensitized particles X at a concentration x is taken as 1, the upper limit of the amount of ligand used in producing sensitized particles Y at a concentration y may be changed to, for example, 0.9 or less, preferably 0.8 or less, and more preferably 0.7 or less. On the other hand, the lower limit of the amount of ligand used when producing sensitized particles Y with a concentration y is, for example, 0.01 or more, preferably 0.02 or more, and more preferably 0.03 or more.

[0018] The lower limit of the average particle size of the core particles is, for example, 80 nm or more, preferably 90 nm or more, and more preferably 100 nm or more. If the solution to the prozone phenomenon in high target substance concentration regions were to reduce the particle size of sensitized particle X, the surface area of ​​the entire particle would increase, which would likely increase the amount of antibody used during sensitization. In contrast, by increasing the particle size of the core particle of sensitized particle Y relatively, the increase in the amount of antibody used can be suppressed. On the other hand, the upper limit of the average particle size of the core particles is, for example, 700 nm or less, preferably 650 nm or less, and more preferably 600 nm or less.

[0019] The upper limit of the CV value of the average particle diameter of the core particles is, for example, 15% or less, preferably 10% or less, and more preferably 5% or less. One possible solution to the prozone phenomenon in high target substance concentration areas is to use both large and small particle sizes. However, by using sensitized particles Y, which have a small variation in particle size and are relatively uniform, the quality can be stabilized. On the other hand, the lower limit of the CV value of the average particle diameter of the core particles is, for example, 0.01% or more, preferably 0.02% or more, and more preferably 0.03% or more.

[0020] In the condition changing method of this embodiment, it is possible to suppress a decrease in sensitivity in a high concentration region of the target substance. Furthermore, the target substance detection method of this embodiment can be applied to a wide range of target substance concentrations, from low to high, i.e., it can be used in the low concentration range of 0.5 ng / mL or more and less than 25 ng, and it can also be used in the high concentration range of 25 ng / mL or more and 9000 ng / mL or less.

[0021] The target substance detection method of this embodiment includes: A detection method for detecting a target substance by an agglutination method using sensitized particles, comprising: a contacting step of contacting a target substance with sensitized particles to obtain a mixture; a detection step of detecting the agglutination of the sensitized particles by optical means after the contact step and measuring the concentration of the target substance; Including, the sensitized particle is a sensitized particle Y having at least a blocking protein and a ligand chemically bonded to the surface of a core particle; The measured concentration of the target substance in the mixture in the detection step of measuring the concentration of the target substance is 0.5 ng / mL or more and 9000 ng / mL or less.

[0022] In the target substance detection method, the average particle diameter of the core particles is, for example, 200 nm to 700 nm, and the CV value of the average particle diameter of the core particles is, for example, 15% or less. By using sensitized particles Y having such a relatively large particle size and a relatively uniform particle size distribution, it is possible to widen the range of detectable target substance concentrations.

[0023] The configuration of this embodiment will be described in detail below.

[0024] FIG. 1 is a cross-sectional view schematically showing an example of the configuration of a sensitized particle 20 according to this embodiment. An example of a sensitized particle 20 has a core particle 1 and, on at least a portion of the surface of the core particle 1, a ligand 3 immobilized by a chemical bond, a blocking protein 5 immobilized by a chemical bond, and another blocking protein 5 immobilized by a physical bond.

[0025] The sensitized particles 20 of this embodiment can be used as particles for agglutination methods used to detect target substances by agglutination methods.

[0026] The agglutination method is a method in which a sample containing a target substance (e.g., an antigen) is contacted in a dispersion medium with sensitized particles 20 carrying a ligand 3 (e.g., an antibody) that specifically recognizes (binds to) the target substance, and the ligand 3 binds to the target substance, forming a complex between the target substance, the ligand 3, and the sensitized particles 20, thereby selectively agglutinating the sensitized particles 20. The immunoagglutination method (also called immunoturbidimetry), which is one of the agglutination methods, utilizes an antigen-antibody reaction to cause an antigen to react with an antibody provided on the surface of the sensitized particles 20, thereby agglutinating the sensitized particles 20. In a method for detecting a target substance using the agglutination method, the degree of agglutination of the sensitized particles 20 can be characterized and quantified by optical measurement methods such as absorbance, scattered light intensity, and transmitted light intensity.

[0027] According to this embodiment, the dispersibility of the sensitized particles 20 in the dispersion medium can be increased, which prevents the sensitized particles 20 from aggregating with each other in the dispersion medium before reacting with the target substance, i.e., prevents a nonspecific reaction.

[0028] Furthermore, according to this embodiment, the dispersibility of the sensitized particles 20 can be improved, thereby increasing the change in the degree of aggregation of the sensitized particles 20 before and after reacting with the target substance. In other words, when comparing the presence and absence of the target substance, the difference in optical measurement values ​​such as absorbance in a dispersion medium containing the sensitized particles 20 increases, making it possible to improve the sensitivity of the sensitized particles 20, i.e., the detection sensitivity of the target substance.

[0029] An example of the target substance detection reagent of this embodiment includes the particle group of the sensitized particles 20 and a dispersion medium. The concentration (wt %) of the sensitized particles 20 in the dispersion medium can be set appropriately. As the dispersion medium, for example, the above-mentioned aqueous solvents are preferably used. The aqueous solvent may contain, within the scope that does not impair the effects of the invention, a third substance such as a buffering agent, a preservative (such as sodium azide), a blocking agent such as a protein (albumin), a sensitizer such as a water-soluble polymer (such as a sugar, polyethylene glycol, or dextran), a salt (such as sodium chloride or an amino acid), or a surfactant. These may be used alone or in combination of two or more.

[0030] Examples of the sensitizer include polyethylene glycol, dextran, and a sensitizer having a phosphorylcholine group. Preferably, the sensitizer may include a sensitizer having a phosphorylcholine group. This can further improve the dispersibility of the sensitized particles 20.

[0031] Examples of sensitizers having a phosphorylcholine group include polymers having a phosphorylcholine group. These may be used alone or in combination of two or more. The polymer having a phosphorylcholine group has at least one or two or more structural units M having a phosphorylcholine group in the side chain of the polymer. The phosphorylcholine group may be partially modified as long as it can be used as a polar group of phospholipids, which are components of biological membranes. The polymer having a phosphorylcholine group may be a homopolymer having only the structural unit M, or may be a copolymer having the structural unit M and another structural unit N. The structural unit N may be a structural unit having, for example, any one of a hydrophobic group, an anionic group, a cationic group, and a hydrogen-bonding group on the side chain of the polymer. The main chain of the polymer having a phosphorylcholine group may have a structure in which polymerizable groups in the raw material monomers such as 2-methacryloyloxyethyl phosphorylcholine (MPC) are linked to each other.

[0032] The target substance detection reagent of this embodiment can also be in the form of a kit from the viewpoints of storage, transportation, distribution, etc. The form of the kit is not particularly limited as long as it is a form that enables the measurement method of the present invention. An example of the target substance detection kit of this embodiment includes a reagent container containing the above-described target substance detection reagent.

[0033] The kit can take various forms, such as the following: The kit of the first type comprises a first reagent container containing a particle group of sensitized particles 20 and a dispersion medium. The kit of the second embodiment includes a second reagent container A containing a particle group of sensitized particles 20, and a second reagent container B containing a dispersion medium. The first reagent container of the kit of the first form and the second reagent containers A and B of the kit of the second form may each contain at least one or more of the third substances exemplified above. Furthermore, each of the kits of the first and second forms may further include a third reagent container containing at least one of the third substances. Furthermore, each of the kits of the first and second forms may include, in addition to the above-mentioned reagent container, a fourth reagent container containing one or more of a positive control, a negative control, a diluent, a washing solution, instructions for use, etc. As the media for the positive and negative controls, serum, physiological saline, solvents, etc. that do not contain the target substance to be measured are used.

[0034] An example of the method for detecting a target substance according to this embodiment includes a contact step of mixing a target substance with a target substance detection reagent. In the contact step, a sample containing a target substance and sensitized particles 20 having ligands 3 are added to the same reaction vessel and allowed to coexist, allowing the ligands 3 bound to the sensitized particles 20 to come into contact with the target substance. The sample containing the standard substance and the target substance reagent containing the sensitized particles 20 may be added in any order. Furthermore, the target substance and the sensitized particles 20 may be mixed at a pH of about 3.0 or more and about 11.0 or less, and the temperature of the mixture may be about 20°C or more and about 50°C or less.

[0035] The target substance is brought into contact with the ligand 3 and reacted with each other, and the substance to be measured in the sample reacts with the antibody carried by the core particle, forming a complex between the target substance, the ligand 3, and the sensitized particle 20, causing the sensitized particle 20 to aggregate as a result of the complex formation.

[0036] The method for detecting a target substance may include, after the contact step, a detection step of optically detecting the agglutination of the sensitized particles 20. Examples of a method for optically measuring agglutination include a method of measuring absorbance, scattered light intensity, or transmitted light intensity using an optical device. The wavelength for measuring absorbance is usually 340 nm to 1000 nm, preferably 500 nm to 900 nm. The time for measuring an agglutination reaction can be measured by measuring the rate of change per unit time or the amount of change per unit time. For example, when measuring absorbance, the rate of change per unit time from 30 seconds to 5 minutes after the start of the agglutination reaction can be measured by measuring the amount of change in absorbance per unit time or the amount of change in absorbance per unit time.

[0037] The sample is not particularly limited as long as it has the potential to contain the target substance, and examples thereof include one or more biological samples selected from the group consisting of whole blood, plasma, serum, urine, cerebrospinal fluid, saliva, amniotic fluid, urine, sweat, and pancreatic juice. The sample is preferably whole blood, plasma, serum, or urine. The sample may be an aqueous solvent containing the target substance, for example, the target substance diluted with phosphate buffered saline or the like.

[0038] The aqueous solvent is not particularly limited as long as it allows the aggregation method, and examples thereof include deionized water, distilled water, buffer solutions, etc., with buffer solutions being preferred. Examples of buffering agents contained in buffer solutions include phosphate-based, acetate-based, Tris-based, borate-based, carbonate-based, glycine-based, and Gut-based buffer solutions. Suitable Good's buffers include, for example, HEPES, PIPES, and MOPS. The buffering agent may be used alone or in combination of two or more kinds. Acids that can be used to adjust the pH of a buffer solution containing a buffer component include ordinary hydrochloric acid, sulfuric acid, nitric acid, and organic acids such as acetic acid. Alkalis that can be used include sodium hydroxide, potassium hydroxide, lithium hydroxide, and ammonium hydroxide.

[0039] The target substance is not particularly limited as long as it is a target substance that allows for the agglutination method, and examples thereof include CRP (C-reactive protein); prostate-specific antigen (PSA); ferritin; α2-macroglobulin; β2-microglobulin; myoglobin; fibrin; fibrinogen degradation products; D-dimer; thrombin-antithrombin III complex (TAT); soluble fibrin (SF); soluble interleukin-2 receptor (sIL-2R); and sodium-reducing peptides such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). Examples include urinary peptides; N-terminal pro-brain natriuretic peptide (NT-proBNP); antistreptolysin O; rheumatoid factor; transferrin; haptoglobin; α1-antitrypsin; α1-acidoglycoprotein; hemopexin; antithrombin-III; α-fetoprotein; CEA (carcinoembrionic antigen); HBs-Ag (hepatitis B envelope antigen); Anti-HBs (anti-hepatitis B envelope antibody); HBe-Ag (hepatitis B e antigen); Anti-HBe (anti-hepatitis B e antibody); Anti-HBc (anti-hepatitis B core antibody); IgG; IgA; IgM, etc. The target substance is not particularly limited as long as it is in a form that allows it to bind to an antibody, and may be, for example, in a free form as a simple substance, or in a complex form with another substance (for example, a protein, etc.).

[0040] Next, each component of the sensitized particle of this embodiment will be described.

[0041] An example of a sensitized particle 20 of this embodiment has a core particle 1 having multiple reactive functional groups (A1) on its surface, a ligand 3 chemically bonded to some of the reactive functional groups (A1), and a blocking protein 5 chemically bonded to other parts of the reactive functional groups (A1).

[0042] When the core particle 1 has a reactive functional group (A1) on its surface, the ligand 3 can be chemically bound to the surface of the core particle 1 via the reactive functional group (A1). Furthermore, when the core particle 1 has a reactive functional group (A1) on its surface, the blocking protein 5 can be chemically bound to the surface of the core particle 1 via the reactive functional group (A1).

[0043] The reactive functional group (A1) may contain one or more groups selected from the group consisting of nucleophilic groups such as carboxyl groups, amino groups, and thiol groups, and electrophilic groups such as epoxy groups, maleimide groups, acrylic groups, methacrylic groups, and acid anhydride groups. Examples of acid anhydride groups include maleic anhydride groups and succinic anhydride groups. Among these, the reactive functional group (A1) may contain a carboxyl group.

[0044] (core particle) The core particle 1 is a particle that serves as the core of the sensitized particle 20, and may be composed of either an organic particle or an inorganic particle.

[0045] The organic particles may be particles made of an organic polymer. Examples of organic polymers include synthetic resins such as polystyrene, polyvinyl chloride, polypropylene, (meth)acrylic resin, polymethyl methacrylate, and silicone rubber; cellulose derivatives such as nitrocellulose, cellulose, and methylcellulose; phenolic resins, polylactic acid, polyethylene, and melamine phenol; copolymers containing one of these as the main component (e.g., copolymers containing styrene as the main component); and copolymers containing two or more of these. These organic polymers may be used alone or in combination. The organic polymers constituting the organic particles are not particularly limited as long as they are composed of a polymer different from the surface-modifying polymer.

[0046] As the organic particles, known latex particles can be used, for example, aromatic latex particles such as styrene-based latex particles, acrylic acid-based latex particles, various modified latex particles (for example, carboxylic acid-modified latex particles in which a carboxyl group is introduced into the above-mentioned polystyrene), colored latex particles, fluorescent latex particles, etc. The latex particles can be produced by a known method, or commercially available particles may be used. Two or more types of the latex particles may be used in combination.

[0047] Examples of methods for producing organic particles (resin particles) include a method in which raw material monomers (styrene, methacrylic acid, etc.) are polymerized with a polymerization initiator (radical polymerization initiator, etc.) to form a granular organic polymer in a solvent (water, etc.). Methods for forming the granular organic polymer include radical polymerization methods such as emulsion polymerization, soap-free emulsion polymerization, and suspension polymerization, but are not limited to radical polymerization.

[0048] Examples of methods for producing aromatic latex particles include polymerizing one or more of the following raw material components using a polymerization initiator (such as a radical polymerization initiator) to form a granular organic polymer in a solvent (such as water). When forming the granular organic polymer, a crosslinked structure may be formed by a coupling reaction, if necessary.

[0049] Examples of raw material components include main skeleton monomers (monofunctional polymerizable compounds having aromatic groups, polyfunctional polymerizable compounds having aromatic groups), polymerizable silane coupling agents, dispersants, crosslinking agents, etc. These may be used alone or in combination of two or more. When the raw material components include a main skeleton monomer having an aromatic group, a polymerizable silane coupling agent, and a dispersant, each of these components preferably has a polymerizable functional group, more preferably has a functional group having an unsaturated double bond capable of radical polymerization.In addition, when the raw material components further include a crosslinking agent, the polymerizable silane coupling agent preferably has a hydrolyzable silyl group, and the crosslinking agent preferably has two or more functional groups in the molecule that react with the hydrolyzable silyl group. The main skeleton monomer, or the polymerizable compound, may be a monomer or a polymer such as an oligomer or a prepolymer.

[0050] Examples of monofunctional polymerizable compounds having an aromatic group include vinyl compounds such as styrene-based monomers, α-methylstyrene, and chlorostyrene; naphthalene compounds such as 1-vinylnaphthalene and 2-vinylnaphthalene; anthracenyl compounds such as 2-vinylanthracene and 9-vinylanthracene; phenanthrene compounds such as 3-vinylphenanthrene and 9-vinylphenanthrene; dinaphthothiophene compounds such as 6-vinyldinaphthothiophene and 6-vinyletherdinaphthothiophene; carbazole compounds such as 9-vinylcarbazole; and (meth)acrylic compounds such as aromatic (meth)acrylate compounds, such as benzyl (meth)acrylate; and polycyclic aromatic (meth)acrylate compounds, such as 2-(1-naphthyl)methyl (meth)acrylate, phenantyl (meth)acrylate, 6-(meth)acryloyloxymethyldinaphthothiophene, and 6-(meth)acryloyloxyethyldinaphthothiophene.

[0051] Examples of polyfunctional polymerizable compounds having an aromatic group include vinyl compounds such as divinylbenzene, 2,12-divinyldinaphthothiophene, 3,11-divinyldinaphthothiophene, 5,9-divinyldinaphthothiophene, 2,12-divinyloxymethyldinaphthothiophene, 3,11-divinyloxydinaphthothiophene, 1,4-divinyloxybutane, and divinyl sulfone; and (meth)acrylic compounds such as 2,12-di(meth)acrylonitrile, ... Examples of the acryloyloxymethyl dinaphthothiophene include 3,11-di(meth)acryloyloxymethyl dinaphthothiophene, 9,9-bis(4-(meth)acryloyloxyphenyl)fluorene, 9,9-bis(4-(meth)acryloyloxymethoxyphenyl)fluorene, and 9,9-bis(4-(2-(meth)acryloyloxyethoxy)phenyl)fluorene; and examples of allyl compounds include 9,9'-bis(4-allyloxyphenyl)fluorene.

[0052] As the polymerizable silane coupling agent, a silane coupling agent having a polymerizable functional group and a hydrolyzable silyl group can be used, and examples thereof include 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, and 3-acryloxypropyltrimethoxysilane, which enables orientation control.

[0053] As the dispersant, a dispersant having a polymerizable group can be used, but an anionic dispersant is preferred. Examples of anionic dispersants having a polymerizable group include sodium methacrylate, sodium parastyrene, etc. These may be contained alone or in any combination of two or more.

[0054] As the crosslinking agent, a compound having two or more functional groups in the molecule that react with the hydrolyzable silyl group in the polymerizable silane coupling agent can be used, for example, a silane coupling agent having a carboxylic acid group. The functional group that reacts with the hydrolyzable silyl group is not limited to a carboxylic acid group (carboxyl group), but may include a hydroxyl group, an amino group, a thiol group, etc. These may be used alone or in combination of two or more. The crosslinking mechanism of the crosslinking agent is thought to be that a polymerizable silane coupling agent such as 3-methacryloxypropyltrimethoxysilane undergoes radical polymerization with a polymerizable group contained in a monofunctional polymerizable compound having an aromatic group such as styrene or a dispersant such as sodium methacrylate, is incorporated into the main skeleton, and the alkoxysilyl group in the side chain undergoes a coupling reaction with a crosslinking agent such as a silane coupling agent having a carboxylic acid group.

[0055] The aromatic latex particles may also include a silane coupling agent having a carboxy group. In this case, the content of the silane coupling agent having a carboxy group relative to the structural unit A having an aromatic group is, in mass terms, for example, 0.1 to 20 mass%, preferably 0.3 to 17.5 mass%, and more preferably 0.5 to 15 mass%.

[0056] The aromatic latex particles may comprise a structural unit A having an aromatic group and a structural unit B having an ionic group. In this case, the ratio of the structural unit B to the structural unit A is, for example, 0.01 to 15%, preferably 0.03 to 12.5%, and more preferably 0.05 to 10%, on a molar basis. The ionic group refers to a sulfonic acid group and / or a carboxy group.

[0057] The aromatic latex particles may have a structural unit A having an aromatic group and a structural unit C having a Si element. In this case, the ratio of the structural unit C to the structural unit A is, for example, 0.01 to 40% by mass, preferably 0.3 to 30% by mass, and more preferably 0.05 to 20% by mass.

[0058] The aromatic latex particles may include, in addition to the structural unit A having an aromatic group, a structural unit B having an ionic group, a structural unit C having an Si element, and a silane coupling agent having a polycarboxy group.

[0059] The inorganic particles may be particles made of an inorganic substance. Examples of inorganic substances include inorganic materials such as metals, ceramics, and glass. Specific examples of inorganic substances include silica. These may be used alone or in combination of two or more. Magnetic inorganic materials may also be used as inorganic substances. In this way, non-magnetic particles or magnetic particles may be used as the inorganic particles.

[0060] The shape of the core particle 1 is not particularly limited, but examples thereof include a spherical shape and an elliptical shape.

[0061] (ligand) Ligand 3 is a compound that specifically binds to a receptor possessed by the target substance described above. The site at which the ligand 3 binds to the target substance is fixed, and the ligand 3 has high affinity selectively or specifically. Examples of the ligand 3 include, but are not limited to, antigens and antibodies, enzyme proteins and their substrates, signal substances such as hormones and neurotransmitters and their receptors, and nucleic acids. Examples of the ligand 3 include antibodies such as full-length antibodies and antigen-binding fragments (e.g., antibody fragments such as Fab, F(ab')2, F(ab'), Fv, and scFv); antigens; nucleic acids such as naturally occurring nucleic acids and artificial nucleic acids; proteins such as aptamers, peptide aptamers, oligopeptides, enzymes, and coenzymes; etc. Commercially available antibodies can be used as the antibodies that specifically recognize the target substance.

[0062] (Blocking protein) Examples of blocking proteins 5 include animal-derived albumins such as bovine serum albumin, casein, gelatin, ovalbumin, and gamma-globulin. Among these, naturally occurring blocking proteins are preferred from the viewpoint of dispersibility, and naturally occurring blocking proteins are preferred over synthetic polymers from the viewpoint of sensitivity. These may be used alone or in combination of two or more.

[0063] Here, one example of the method for producing sensitized particles of this embodiment includes a step of obtaining sensitized particles 20 in which ligands 3 and blocking proteins 5 are immobilized by chemical bonds to at least a portion of the surface of core particles 1 . Specifically, the process of obtaining such sensitized particles 20 includes a reaction treatment in which some of the reactive functional groups (A1) present on the surface of the core particle 1 are chemically bonded to the blocking protein 5. An example of this reaction treatment is to add a condensing agent that chemically bonds the other part of the reactive functional group (A1) with the blocking protein 5 and the blocking protein 5. The addition of the blocking protein 5 and the condensing agent (reaction treatment) is not particularly limited, as long as it is performed during a series of treatments including the sensitization treatment, washing treatment, and blocking treatment described below, or before or after each treatment in the series of treatments, which are carried out during the process of obtaining the sensitized particles 20. Specifically, the addition of the blocking protein 5 and the condensing agent may be performed, for example, simultaneously with the blocking treatment, before the blocking treatment, simultaneously with any of a series of treatments other than the blocking treatment, or before or after any of the series of treatments. It is preferable that the addition of the blocking protein 5 and the condensing agent includes at least one of the following: adding the condensing agent after the blocking protein 5; adding the condensing agent and then adding the blocking protein 5; or adding a mixture of the blocking protein 5 and the condensing agent. Furthermore, the number of times that the blocking protein 5 and the condensing agent are each added may be one or more than one. When adding them multiple times, one or more of the above-mentioned treatments may be performed between additions.

[0064] As the condensing agent, those used in the immobilization method utilizing a carboxy group or an amino group, which are known as methods for immobilizing antibodies, can be used. As the condensation agent, for example, a carbodiimide-based crosslinking agent or an amine-reactive crosslinking agent is used. Carboxy groups and amino groups can be reacted using a carbodiimide crosslinking agent such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, also known as EDAC or WSC). Amine-reactive crosslinkers can also be used to react primary or secondary amino groups with synthetic chemical groups such as isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluoroesters.

[0065] In the above-mentioned sensitization treatment, the ligand 3 can be chemically reacted with at least a part of the surface of the core particle 1 . Sensitization treatment for binding (carrying) ligand 3 to core particle 1 can be performed, for example, by suspending ligand 3 (antibody) and core particle 1 in a solvent such as a buffer solution and reacting them at approximately 20 to 37°C for a predetermined period of time.

[0066] The buffer solution may have a pH of, for example, 5.0 to 10.0. The buffer solution may contain, for example, an aqueous solvent, a buffering agent, a preservative (such as sodium azide), a blocking agent such as a protein (albumin), a sensitizer such as a water-soluble polymer (such as a sugar, polyethylene glycol, or dextran), a salt (such as sodium chloride or an amino acid), a surfactant, etc. These may be used alone or in combination of two or more.

[0067] In the sensitization treatment, a conventionally known method can be used as a chemical reaction method for chemically immobilizing the reactive functional group (A1) present on the surface of the core particle 1 and the ligand 3. Examples of chemical reaction methods that can be used include, but are not limited to, a carbodiimide-mediated reaction and an NHS ester activation reaction. If necessary, the above-mentioned condensing agent may also be used.

[0068] The process for obtaining the sensitized particles 20 may include, in addition to the above-mentioned sensitization treatment, one or more treatments commonly used in the technical field of sensitized particles, such as a washing treatment and a blocking treatment. If necessary, other treatments such as centrifugation, ultrasonic treatment, heating (aging) treatment, etc. may be carried out at appropriate times. In the washing treatment, for example, the sensitized product obtained in the sensitization treatment can be washed by a known method. A washing solution or the like can be used for washing. The washing treatment may be carried out once or twice or more times. In the blocking treatment, another blocking protein that physically binds to the sensitized product obtained by the sensitization treatment, for example, by physical adsorption, can be added. At this time, a blocking solution containing the other blocking protein may be added.

[0069] In another embodiment, other blocking proteins may be present on at least a portion of the surface of the sensitized particle 20 and / or the surface of the blocking protein 5, by physical adsorption or the like. At least one or more of the washing solution that can be used in the washing process, the blocking solution that can be used in the blocking process, and the dispersion medium may contain other blocking proteins that physically bind by physical adsorption or the like, such as those exemplified for the above-mentioned blocking protein 5.

[0070] Furthermore, one example of the method for producing the sensitized particle dispersion of this embodiment includes a dispersing step of dispersing the obtained sensitized particles 20 in a dispersion medium. In this dispersing step, the sensitized particles 20 produced in the step of obtaining the sensitized particles 20 are dispersed in the above-mentioned dispersion medium, thereby producing a target substance detection reagent.

[0071] In another embodiment, the method may include an introduction step of introducing a surface-modifying polymer having the above-mentioned reactive functional group (A1) or a coupling agent having the above-mentioned reactive functional group (A1) onto at least a portion of the surface of the core particle. The introduction step can be performed at any timing during the process of obtaining the sensitized particles 20, but is preferably performed before the sensitization treatment or the binding treatment. As a result, the reactive functional group (A1) is formed on the surface of the core particle 1 via the surface-modifying polymer and / or the coupling agent. Sensitized particles 20 of this type may include a ligand 3 and / or a blocking protein 5 bound to at least some of the reactive functional groups (A1) of the surface-modifying polymer and / or coupling agent.

[0072] The surface-modifying polymer may be formed on at least a part of the surface of the core particle 1, and may form a polymer coating layer that covers a part or the entire surface of the core particle 1. The surface-modified polymer is physically bonded to the surface of the core particle 1, and / or a specific functional group contained in the surface-modified polymer is chemically bonded to a specific functional group present on the surface of the core particle 1.

[0073] The surface-modifying polymer may be composed of a (meth)acrylic polymer, a maleic acid polymer, a methylene malonate polymer, a phenolic resin, an epoxy resin, or the like. These may be used alone or in combination of two or more. However, the surface-modifying polymer may not include a copolymer having a unit derived from a styrene-based monomer and a unit derived from a glycidyl group-containing monomer. According to this embodiment, by using a surface-modified polymer in addition to the core particle 1, a wide variety of molecular designs become possible for the surface-modified polymer, and the degree of freedom in molecular design increases.

[0074] Examples of the (meth)acrylic polymer include methacrylic polymers and acrylic polymers. Examples of maleic acid-based polymers include maleic anhydride-norbornene copolymers, styrene-maleic anhydride copolymers, maleic anhydride homopolymers, methyl vinyl ether-maleic anhydride copolymers, and olefin-maleic anhydride copolymers.

[0075] The surface-modifying polymer may be any of a homopolymer, a random copolymer, an alternating copolymer, a block copolymer, a periodic copolymer, and the like.

[0076] The weight average molecular weight of the surface modifying polymer is, for example, 1,000 or more and 10,000,000 or less, preferably 2,000 or more and 5,000,000 or less, and more preferably 3,000 or more and 1,000,000 or less. The weight average molecular weight can be determined from a polystyrene equivalent value obtained from a calibration curve of standard polystyrene (PS) obtained by gel permeation chromatography measurement.

[0077] The surface modifying polymer may comprise a structural unit A1 that comprises a reactive functional group (A1) capable of binding to a ligand 3.

[0078] The method for forming the surface-modifying polymer on the surface of the core particle 1 uses, for example, a polymer introduction step in which a surface-modifying polymer is introduced onto the surface of the core particle 1 by a chemical reaction. For the chemical reaction, an immobilization method using a carboxy group or an immobilization method using an amino group, which are known as methods for immobilizing antibodies, can be used. These chemical reactions may involve the use of condensing agents, such as carbodiimide-based crosslinkers and amine-reactive crosslinkers, if desired. Carboxy groups and amino groups can be reacted using a carbodiimide crosslinking agent such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, also known as EDAC or WSC). Amine-reactive crosslinkers can also be used to react primary or secondary amino groups with synthetic chemical groups such as isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluoroesters.

[0079] When the core particle 1 is an organic particle (resin particle), a method is used in which a polymer reactive group (such as a carboxy group or an amino group) contained in the raw material monomer of the organic particle reacts with a predetermined reactive group contained in the surface-modifying polymer. For example, in the manufacturing process of the organic particle, it is possible to incorporate a polymer reactive group into the organic polymer during the polymerization of the raw material monomer. Commercially available polystyrene particles containing a carboxy group may also be used as the core particle 1. As a specific example, when a surface-modifying polymer containing a carboxy group is immobilized on a core particle 1 containing a carboxy group, primary amino groups (-NH2) may be introduced by amino treatment to some of the carboxy groups in the core particle 1 (amino treatment). Then, the primary amino groups (polymer reactive groups) in the core particle 1 after the amino treatment and the predetermined reactive groups in the surface-modifying polymer are chemically reacted, if necessary, by adding the above-mentioned condensing agent. When the surface-modifying polymer contains an acrylic group / methacrylic group, it is possible to react the primary amino group (polymer reactive group) with the carboxy group (predetermined reactive group) contained in the acrylic group / methacrylic group via the above-mentioned condensing agent. In another embodiment, when the surface-modifying polymer contains an anhydride group, it is possible to react a primary amino group (polymer reactive group) with an anhydride group (predetermined reactive group) such as a maleic anhydride group without using the above-mentioned condensing agent. On the other hand, when the core particle 1 is an inorganic particle, a method is used in which a polymer reactive group is introduced onto the surface of the inorganic particle using a coupling agent or the like as needed, and then the polymer reactive group is reacted with a predetermined reactive group contained in the surface-modifying polymer.

[0080] Here, an example of a method for synthesizing a surface-modifying polymer will be described. The raw material monomers used in the polymer precursor production process can be one or more of raw material monomer A containing a diene compound and a diene parent compound, and raw material monomer B containing a functional group and an unsaturated group such as a double bond.

[0081] For the polymer of a diene compound and a diene parent compound, (i) if an anhydride group is contained, the reactive functional group (A1), the acidic functional group (A2), or other functional groups (functional functional groups) may be introduced into the side chain by ring-opening the anhydride group; (ii) if a carboxyl group is contained, various functional groups may be introduced into the side chain by a condensation reaction or the like; and (iii) if an unsaturated group such as a double bond is contained, the various functional groups described above may be introduced into the side chain by an addition reaction or the like. Examples of the other functional groups mentioned above include known functional groups such as various reactive groups, hydrophobic groups, hydrophilic groups (water-soluble groups), and acidic groups. Furthermore, (i) when an anhydride group is contained, the anhydride group may be ring-opened with water, alcohol, etc., or may be ring-opened with an organic base. Furthermore, the functional group described above may be introduced into the side chain of the polymer precursor by a condensation reaction or the like with respect to a functional group such as a carboxyl group formed by ring-opening of the anhydride group, as in (ii) above.

[0082] For the synthesis of a polymer precursor of a maleic anhydride-based polymer, for example, a norbornene-based monomer such as norbornene can be used as the diene compound, and an unsaturated carboxylic acid anhydride having a cyclic structure in the molecule such as maleic anhydride can be used as the new diene compound.

[0083] On the other hand, the raw material monomer B may be a raw material monomer B1 having a reactive functional group (A1) and an acidic functional group (A2), or a combination of a raw material monomer B2 having a reactive functional group (A1) and a raw material monomer B2 having an acidic functional group (A2). If necessary, in addition to these, a raw material monomer B3 having a functional group other than the reactive functional group (A1) and the acidic functional group (A2) may be used.

[0084] To synthesize a polymer precursor of a (meth)acrylic polymer, for example, (meth)acrylic acid such as acrylic acid, methacrylic acid, (meth)acrylic acid esters such as methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, butyl (meth)acrylate, isobutyl (meth)acrylate, hexyl (meth)acrylate, and ethylhexyl (meth)acrylate can be used. One or more of these (meth)acrylic monomers can be used.

[0085] The polymerization may be, but is not limited to, addition polymerization and / or condensation polymerization. For example, addition polymerization may be carried out by radical polymerization. Alternatively, the reactive functional group (A1), the acidic functional group (A2), or other functional groups (functional functional groups) may be introduced into the side chains of the resulting polymer precursor by ring-opening reaction, condensation reaction, addition reaction, or the like. A specific example will be described. First, one or more raw material monomers and a polymerization initiator are dissolved in a solvent, and then the solution is heated for a predetermined period of time to carry out solution polymerization. The heating temperature can be, for example, 50°C to 80°C. The heating time can be, for example, 1 hour to 20 hours. It is more preferable to carry out solution polymerization after removing dissolved oxygen in the solvent by nitrogen bubbling.

[0086] As the polymerization initiator, for example, an azo compound, an organic peroxide, or the like can be used. Specific examples of the azo compound include azobisisobutyronitrile (AIBN), dimethyl 2,2'-azobis(2-methylpropionate), and 1,1'-azobis(cyclohexanecarbonitrile) (ABCN). Examples of organic peroxides include hydrogen peroxide, ditertiary butyl peroxide (DTBP), benzoyl peroxide (benzoyl peroxide, BPO), and methyl ethyl ketone peroxide (MEKP). As for the polymerization initiator, one type may be used alone, or two or more types may be used in combination.

[0087] Furthermore, a molecular weight modifier or a chain transfer agent can be used as needed. Examples of the chain transfer agent include thiol compounds such as dodecyl mercaptan, mercaptoethanol, and 4,4-bis(trifluoromethyl)-4-hydroxy-1-mercaptobutane. These chain transfer agents can be used alone or in combination of two or more.

[0088] Examples of solvents used in the polymerization reaction include one or more esters such as methyl ethyl ketone (MEK), propylene glycol monomethyl ether, diethyl ether, tetrahydrofuran (THF), toluene, ethyl acetate, and butyl acetate. Examples of the polymerization initiator include one or more azo compounds and organic peroxides. Examples of azo compounds include azobisisobutyronitrile (AIBN), dimethyl 2,2'-azobis(2-methylpropionate), and 1,1'-azobis(cyclohexanecarbonitrile) (ABCN). Examples of organic peroxides include hydrogen peroxide, ditertiarybutyl peroxide (DTBP), benzoyl peroxide (benzoyl peroxide (BPO)), and methyl ethyl ketone peroxide (MEKP).

[0089] The reaction solution containing the (co)polymer thus obtained is added to an alcohol or ether, such as hexane or methanol, to precipitate the polymer. The polymer is then filtered, washed with an alcohol, such as hexane or methanol, and then dried. In this manner, the polymer can be synthesized. This allows the removal of low-molecular-weight components, such as residual monomers, oligomers, and polymerization initiators.

[0090] Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various other configurations may be adopted. Furthermore, the present invention is not limited to the above-described embodiments, and modifications and improvements within the scope of achieving the object of the present invention are included in the present invention. [Example]

[0091] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the descriptions of these examples.

[0092] <Production of Sensitized Particle Dispersion> [Example 1] (sensitization treatment) 940 μL of latex solution A (0.4% w / vol; the number of latex particles and total particle surface area are shown in Table 1) containing the following latex particles to which the following condensing agent was further added was mixed with 940 μL of the following antibody A solution (0.07 mg / mL), and the mixture was shaken and stirred at 20°C for 180 minutes to obtain a mixed solution. Condensing agent: Carbodiimide crosslinking agent (EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, also known as WSC) Latex solution A: Carboxyl group-modified polystyrene latex particles (manufactured by JSR, product name: IMMUNTEX, average particle diameter d: 400 × 10 -7 cm, solid content: 10%) Antibody A solution: Ferritin antibody (Mikuri Immuno Research Institute, Ferritin clone No. 14), 10 mM MES (pH 6.5) The particle number and total surface area of ​​the core particles (latex particles) in Table 1 were calculated as follows. First, the weight and density of the whole particles were measured, and the total volume of the particles was calculated from the weight / density. The volume per particle is 4 / 3×π×(d / 2) 3 Calculated from. From the above, the number of latex particles was calculated by dividing the total volume of particles by the volume per particle. Also, the surface area per particle is 4×π×(d / 2) 2 Calculated from. From the above, the total particle surface area of ​​the latex particles was calculated by multiplying the number of particles by the surface area per particle.

[0093] (Cleaning process) The resulting mixture was centrifuged at 10,000 rpm for 15 minutes at 10° C. using a centrifuge (rotor: R15A (HITACHI)). The supernatant was decanted, and 5 mL of the following HEPES buffer solution was added to the resulting sediment, which was then dispersed by sonication. Subsequently, the mixture was centrifuged at 10,000 rpm for 15 minutes at 10° C. using the above centrifuge. The supernatant was decanted, and 5 mL of the following HEPES buffer solution was added to the resulting sediment, which was then dispersed by sonication to obtain a suspension. HEPES buffer: 0.1% BSA, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.2), 0.1% Tween 20, 0.05% NaN

[0094] (Blocking process) To the resulting suspension, add 5 mL of the following blocking solution A, and then add 30 μL of additional blocking solution B (amount added in terms of coverage: 50%, amount of BP (blocking protein) added per total particle surface area: 113 ng / cm). 2The ratio of the amount of BP added (in blocking solution B) during the reaction treatment to the total amount of BP added (in blocking solution A and blocking solution B) during the reaction treatment, sensitization treatment, washing treatment, and blocking treatment (ratio of amount added to the total amount): 0.10%), and 19 μL of the above condensing agent (5 mg / mL WSC solution) were added, thoroughly dispersed, and then stirred at 37°C for 1 hour (reaction treatment). After this, the mixture was centrifuged at 10,000 rpm at 10°C for 15 minutes using the above centrifuge to obtain ligand-sensitized particles. Blocking solution A: Bovine serum albumin (BSA) solution (1% BSA, 20 mM MES buffer (pH 6.0), 0.05% NaN3) Blocking solution B: Bovine serum albumin (BSA) solution (the molecular weight M of BSA is approximately 66,500, the Stokes radius r of BSA is 3.5 nm, 0.2% BSA, 20 mM MES buffer (pH 6.0), 0.05% NaN3)

[0095] (reagent processing) The following components were mixed in a predetermined mixing ratio and uniformly dispersed by ultrasonic treatment (dispersion treatment) to prepare a reagent (sensitized particle dispersion) with a solid content of 0.135%. The ligand-sensitized particles obtained above HEPES buffer containing 0.5% bovine serum albumin (BSA) 100mM NaCl ·0.09%NaN3 ·100mM HEPES buffer (pH7.0)

[0096] [Example 2] (Production of aromatic latex particles) A reaction vessel was charged with 30 mL of ion-exchanged water, styrene (main backbone monomer), 3-methacryloxypropyltrimethoxysilane (polymerizable silane coupling agent), sodium methacrylate (dispersant 1), and sodium parastyrene (dispersant 2) in molar ratios of 1.00, 0.02, 0.032, and 0.002. A carboxylic acid-containing oligomeric silane coupling agent (crosslinker, Shin-Etsu Chemical Co., Ltd., X-12-1135) was then added at a ratio of 2.2 wt% per 100 parts by weight of styrene. The mixture was stirred, and the atmosphere in the reaction vessel was then purged with nitrogen. After the temperature in the reaction vessel reached 70°C, 0.75 mL of a 1.0 wt% aqueous potassium persulfate solution was added dropwise. The reaction was terminated 24 hours after the addition of the potassium persulfate solution. The particle suspension was recovered by filtration. The suspension was centrifuged and washed with methanol and ion-exchanged water to obtain a dispersion of aromatic latex particles (polystyrene latex particles). A reagent (sensitized particle dispersion) with a solid content of 0.18% was prepared in the same manner as in Example 1, except that in the above (sensitization treatment), the obtained dispersion of aromatic latex particles was used instead of the above latex liquid A.

[0097] [Example 3] (Preparation of polymer-attached particles) Diamine was mixed with the above latex solution A, and the diamine was reacted with the carboxyl groups of the polystyrene latex particles to introduce primary amino groups, followed by purification (amino treatment). Next, the above-mentioned maleic anhydride polymer was mixed in a predetermined mixing ratio with a solution containing polystyrene latex particles having primary amino groups, to react the primary amino groups with the maleic anhydride groups, and the mixture was purified to obtain a dispersion containing core particles with a surface-modifying polymer introduced onto their surfaces (polymer introduction treatment). A reagent (sensitized particle dispersion) with a solid concentration of 0.135% was prepared in the same manner as in Example 1, except that in the above (sensitization treatment), the dispersion containing the obtained core particles into which the surface-modified polymer had been introduced was used instead of the above latex liquid A.

[0098] [Comparative Example 1] A reagent (sensitized particle dispersion) with a solids concentration of 0.1% was prepared in the same manner as in Example 1, except that in the above (blocking treatment), the amount of blocking solution A added was changed and blocking solution B and the condensing agent were not added.

[0099] Comparative Example 2 A reagent (sensitized particle dispersion) with a solids concentration of 0.15% was prepared in the same manner as in Example 1, except that in the above (blocking treatment), the amount of blocking solution A added was changed and blocking solution B and the condensing agent were not added.

[0100] <Absorbance measurement> Using an automatic analyzer TBA-120FR (manufactured by Canon Medical Systems Inc.), the absorbance at a wavelength of 700 nm was measured for each of the reagents of each example and comparative example, the measurement reagents listed below, and the target substance (ferritin antigen solution), all kept at 10°C. Next, 20 μL of each reagent from each Example and Comparative Example was mixed with 80 μL of the following measurement reagent and 4 μL of the target substance (antigen amount: 0 ng / mL, or antigen amount: any of the ranges from 10 ng / mL to 2000 ng / mL), and the mixture was stirred uniformly and maintained at 37°C. The absorbance at a wavelength of 700 nm was measured immediately after adding the measurement reagent and after 5 minutes had elapsed. The absorbance of each reagent was calculated by subtracting the absorbance immediately after adding the measurement reagent from the absorbance after 5 minutes.

[0101] (measurement reagents) The following components were mixed in a predetermined ratio to prepare a measuring reagent. 0.1% Tween 20 Polyethylene glycol (PEG) (5000 to 50000) However, the concentration used in each example and comparative example was set so that the absorbance value was less than 0.01 at an antigen concentration of 0 ng / mL. 1% bovine serum albumin (BSA) in HEPES buffer 150mM NaCl ·0.09%NaN3 ·100mM HEPES buffer (pH7.6)

[0102] In the reagent (sensitized particle dispersion) obtained in the above <Production of Sensitized Particle Dispersion>, when the particle concentration and the amount of antibody at time of measurement in Comparative Example 1 were each normalized to 1, the particle concentration in Comparative Example 2 was 1.5 and the amount of antibody at time of measurement was 1.5, the particle concentration in Examples 1 and 3 was 1.35 and the amount of antibody at time of measurement was 0.45, and the particle concentration in Example 2 was 1.8 and the amount of antibody at time of measurement was 0.6. That is, the detection methods in Examples 1 to 3 were modified from those in Comparative Example 1 by (a) changing to sensitized particles Y to which a blocking protein and an antibody were chemically bonded, and (b) changing the concentration of sensitized particles Y to be higher than the concentration x of sensitized particles X in Comparative Example 1. As a result of the above <Absorbance Measurement>, when the reagent of Comparative Example 1 was used, the prozone phenomenon occurred in the antigen concentration range of about 1000 ng / mL or more. In Examples 1 to 3 and Comparative Example 2, it was found that the prozone phenomenon did not occur even in the antigen concentration range of about 1000 ng / mL or more. Furthermore, when Examples 1 to 3 and Comparative Example 2, in which the particle concentration was higher than that of Comparative Example 1, were compared, it was found that Examples 1 to 3 could significantly reduce the amount of antibody during measurement compared to Comparative Example 2. It was also found that the detection methods of Examples 1 to 3 could measure antigen concentrations in the range of about 10 to 2000 ng / mL. [Explanation of symbols]

[0103] 1. Core particle 3. Ligand 5. Blocking Proteins 20 Sensitizing particles

Claims

1. A method for changing conditions of a method for detecting a target substance using sensitized particles, comprising: a prozone detection step of detecting the occurrence of a prozone phenomenon in which, when absorbance is measured by an agglutination method using sensitized particles X, each of which has a blocking protein physically bound to the surface of a core particle and a ligand chemically bound to the surface of the core particle at a concentration x, the absorbance increases in response to an increase in the concentration of the target substance, and then the increase in absorbance is suppressed; a condition changing step of changing the conditions as follows (a) and (b) after the prozone detection step: (a) changing the sensitized particle X to a sensitized particle Y having at least a blocking protein and a ligand chemically bonded to the surface of a core particle; (b) changing the concentration y of the sensitized particles Y to be higher than the concentration x of the sensitized particles X; and a method for changing conditions.

2. 2. The condition changing method according to claim 1, The condition changing method, wherein the concentration y / the concentration x is 1.1 or more.

3. 3. The condition change method according to claim 1 or 2, A condition-changing method, wherein the maximum concentration of the target substance in the prozone detection step is 25 ng / mL or more and 9000 ng / mL or less.

4. 3. The condition change method according to claim 1 or 2, a condition changing step of changing the amount of ligand used in producing the sensitized particles Y at the concentration y to 0.9 or less, where the amount of ligand used in producing the sensitized particles X at the concentration x is 1.

5. 3. The condition changing method according to claim 1 or 2, The method for changing conditions, wherein the average particle diameter of the core particles is 80 nm or more and 700 nm or less.

6. The polystyrene-based latex particles according to claim 1 or 2, The method for changing conditions, wherein the CV value of the average particle diameter of the core particles is 15% or less.

7. 3. The condition changing method according to claim 1 or 2, The condition-changing method, wherein the core particles are polystyrene-based latex particles.

8. 3. The condition changing method according to claim 1 or 2, The method for changing conditions, wherein the ligand is any one of an antibody, an antigen, a protein, and a nucleic acid.

9. A detection method for detecting a target substance by an agglutination method using sensitized particles, comprising: a contacting step of contacting the target substance with the sensitized particles to obtain a mixture; a detection step of detecting the agglutination of the sensitized particles by optical means after the contact step and measuring the concentration of the target substance; Including, the sensitized particle is a sensitized particle Y in which at least a blocking protein and a ligand are chemically bonded to the surface of a core particle, the measured concentration of the target substance in the mixture in the detection step of measuring the concentration of the target substance is 0.5 ng / mL or more and 9000 ng / mL or less; A detection method for detecting a target substance.

10. A method for detecting a target substance according to claim 9, comprising: The average particle diameter of the core particles is 200 nm or more and 700 nm or less. A method for detecting a target substance, wherein the CV value of the average particle diameter of the core particles is 15% or less.

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