Manufacturing method of sensitized particle dispersion, target substance detection reagent, target substance detection kit, and target substance detection method

Abstract

To provide a manufacturing method of a sensitized particle dispersion including a ligand sensitized particle excellent in dispersibility.SOLUTION: A manufacturing method of a sensitized particle dispersion includes a step of obtaining a ligand sensitized particle including: a core particle having a plurality of reactive functional groups (A1) on a surface thereof; a ligand having a specific bonding property to a target substance chemically bonded to some of the reactive functional groups (A1); and a blocking protein chemically bonded to the other of the reactive functional groups (A1).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a sensitized particle dispersion, a target substance detection reagent, a target substance detection kit, and a method for detecting a target substance. [Background technology]

[0002] The 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 between the target substance, an antigen, and 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, the reagent including latex particles carrying an antibody that specifically recognizes the target substance in the sample (claim 7, etc.). 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, etc.).

[0003] Furthermore, Patent Document 2 describes a method of blocking antigen-sensitized latex using a BSA solution containing BSA, phosphate buffer, and NaN3 (preservative), and then washing the latex three times with the BSA solution (paragraph 0030, etc.). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2021-162593 A [Patent Document 2] JP 2004-151000 A Summary of the Invention [Problem to be solved by the invention]

[0005] In the blocking and washing treatments described in the above Patent Documents 1 and 2, it is known that blocking proteins such as BSA are physically adsorbed onto the surface of latex particles. However, as a result of investigations conducted by the present inventors, it was found that the ligand-sensitized latex particles after the blocking treatment described in Patent Documents 1 and 2 above have room for improvement in terms of dispersibility. [Means for solving the problem]

[0006] After further investigation, the inventors found that by introducing a blocking protein that chemically bonds to the surface of core particles such as latex particles, the dispersibility of ligand-sensitized particles to which ligands are chemically bonded in a dispersion medium before the ligand and target substance bind to each other can be improved, thereby completing the present invention.

[0007] According to one aspect of the present invention, there are provided the following method for producing a sensitized particle dispersion, a target substance detection reagent, a target substance detection kit, and a target substance detection method.

[0008] 1. A method for producing a sensitized particle dispersion, comprising a step of obtaining ligand-sensitized particles having core particles having a plurality of reactive functional groups (A1) on the surface thereof, a ligand having specific binding ability to a target substance and chemically bonded to a portion of the reactive functional groups (A1), and a blocking protein and chemically bonded to another portion of the reactive functional groups (A1). 2. A method for producing the sensitized particle dispersion according to 1., comprising the steps of: The step of obtaining the ligand-sensitized particles includes a reaction treatment of bonding another part of the reactive functional groups (A1) with the blocking protein via a condensing agent. 3. A method for producing the sensitized particle dispersion according to 2., comprising the steps of: The step of obtaining the ligand-sensitized particles includes the reaction treatment, a sensitization treatment for reacting a part of the reactive functional groups (A1) with the ligand, a washing treatment, and a blocking treatment. 4. A method for producing the sensitized particle dispersion according to 2. or 3., comprising the steps of: a method for producing a sensitized particle dispersion, wherein, in the reaction treatment, an amount of the blocking protein added during the reaction treatment is X (ng), and a total amount of the blocking protein added in the reaction treatment, the sensitization treatment, the washing treatment, and the blocking treatment is Y (ng), and X / Y×100 is 0.01% or more and 5% or less. 5. A method for producing a sensitized particle dispersion according to any one of 2. to 4., comprising the steps of: a method for producing a sensitized particle dispersion, wherein, in the reaction treatment, an amount of the blocking protein added during the reaction treatment is X (ng), and an amount of the blocking protein added in terms of a 1% coverage of the core particles calculated by the following procedure is Z (ng), the blocking protein is added together with the condensing agent in an amount calculated as a coverage conversion from X / Z of 5% or more. (procedure) When the Stokes radius of the blocking protein is r (nm) and the molecular weight is M, 1cm 2 Mass of the blocking protein per unit volume (ng / cm 2 ) to [M / (24×r 2 )]. When the average particle diameter (cm) of the core particles is d and the total number of the core particles is Pn, The total surface area (cm) of all the core particles 2 ) to [π×d 2 ×Pn]. In this case, the amount of the blocking protein added, Z (ng), converted into a 1% coverage of the core particles is expressed as [π×d 2 ×Pn]×[M / (24×r 2 ) / 100]. 6. A method for producing a sensitized particle dispersion according to any one of 1. to 5., comprising the steps of: A method for producing a sensitized particle dispersion, wherein the reactive functional group (A1) contains one or more groups selected from the group consisting of a carboxy group, an amino group, a thiol group, an epoxy group, a maleimide group, an acryl group, a methacryl group, and an acid anhydride group. 7. A method for producing a sensitized particle dispersion according to any one of 1. to 6., comprising the steps of: The method for producing a sensitized particle dispersion, wherein the core particles are organic particles or inorganic particles. 8. A method for producing a sensitized particle dispersion according to any one of 1. to 7., comprising the steps of: The method for producing a sensitized particle dispersion, wherein the ligand is any one of an antibody, an antigen, a protein, and a nucleic acid. 9. A method for producing a sensitized particle dispersion according to any one of 1. to 8., comprising the steps of: A method for producing a sensitized particle dispersion, comprising the step of introducing a surface-modifying polymer having a reactive functional group (A1) or a coupling agent having a reactive functional group (A1) to at least a portion of the surface of the core particle in the step of obtaining the ligand-sensitized particle. 10. A target substance detection reagent comprising a sensitized particle dispersion obtained by the method for producing a sensitized particle dispersion according to any one of 1. to 9. 11. The target substance detection reagent according to 10., A target substance detection reagent comprising a sensitizer having a phosphorylcholine group. 12. A target substance detection kit comprising a reagent container containing the target substance detection reagent described in 10. or 11. 13. A method for detecting a target substance, comprising a step of mixing a target substance with the target substance detection reagent described in any one of 10. to 12. Effect of the Invention

[0009] According to the present invention, there are provided a method for producing a sensitized particle dispersion containing ligand-sensitized particles with excellent dispersibility, and a target substance detection reagent, a target substance detection kit, and a target substance detection method using the same. [Brief description of the drawings]

[0010] [Figure 1]FIG. 1 is a cross-sectional view illustrating a schematic example of the configuration of a ligand-sensitized particle according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. In all drawings, similar components are given similar reference numerals and their explanations will be omitted as appropriate. Also, the drawings are schematic diagrams and do not correspond to the actual dimensional ratios.

[0012] The method for producing the sensitized particle dispersion of this embodiment will be outlined below.

[0013] The method for producing the sensitized particle dispersion of this embodiment includes a step of obtaining ligand-sensitized particles having a core particle having a plurality of reactive functional groups (A1) on its surface, a ligand having specific binding ability to a target substance and chemically bonded to a portion of the reactive functional group (A1), and a blocking protein and chemically bonded to another portion of the reactive functional group (A1).

[0014] According to the findings of the present inventors, it has been found that in ligand-sensitized particles in which a ligand is formed on the surface of a core particle such as a latex particle (organic particle) or an inorganic particle, the dispersibility of the ligand-sensitized particles in a dispersion medium before the ligand and target substance bind to each other can be improved by introducing a blocking protein that chemically bonds to the surface of the core particle.

[0015] Although the detailed mechanism is unclear, it is believed that the blocking protein is introduced to the surface of the core particle by chemical bonding in addition to physical adsorption, which stably suppresses self-aggregation of the ligand-sensitized particles in a dispersion medium that does not contain the target substance, thereby improving dispersibility as described above.

[0016] FIG. 1 is a cross-sectional view that illustrates an example of the configuration of a ligand-sensitized particle 20 according to this embodiment. An example of the ligand-sensitized particle 20 has at least a core particle 1 , and a ligand 3 and a blocking protein 5 chemically immobilized to at least a portion of the surface of the core particle 1 .

[0017] The ligand-sensitized particles 20 of this embodiment can be used as particles for an agglutination method used in detecting a target substance by an agglutination method.

[0018] 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 ligand-sensitized particles 20 carrying a ligand 3 (e.g., an antibody) that specifically recognizes (binds to) the target substance, causing the ligand 3 to bind to the target substance, forming a complex between the target substance, the ligand 3, and the ligand-sensitized particles 20, and selectively agglutinating the ligand-sensitized particles 20. The immunoagglutination method (also called immunoturbidimetry), which is one of the agglutination methods, utilizes an antigen-antibody reaction to react an antigen with an antibody provided on the surface of the ligand-sensitized particles 20, thereby agglutinating the ligand-sensitized particles 20. In a method for detecting a target substance using an agglutination method, the degree of agglutination of the ligand-sensitized particles 20 can be characterized and quantified by optical measurement such as absorbance, scattered light intensity, or transmitted light intensity.

[0019] According to the present embodiment, the dispersibility of the ligand-sensitized particles 20 in the dispersion medium can be increased. This makes it possible to suppress the ligand-sensitized particles 20 from aggregating with each other in the dispersion medium before reacting with the target substance, i.e., to suppress nonspecific reactions.

[0020] Furthermore, according to this embodiment, the dispersibility of the ligand-sensitized particles 20 can be improved, so that the change in the degree of aggregation of the ligand-sensitized particles 20 before and after the reaction with the target substance can be increased. 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 ligand-sensitized particles 20 becomes large, so that it is possible to improve the sensitivity of the ligand-sensitized particles 20, i.e., the detection sensitivity of the target substance.

[0021] The target substance detection reagent of this embodiment contains the above-mentioned particle group of the ligand-sensitized particles 20 and a dispersion medium. The concentration (wt %) of the ligand-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 of the invention, a third substance such as a buffer, a preservative (e.g., sodium azide), a blocking agent such as a protein (albumin), a sensitizer such as a water-soluble polymer (e.g., sugar, polyethylene glycol, dextran), a salt (e.g., sodium chloride, amino acid), a surfactant, etc. These may be used alone or in combination of two or more kinds.

[0022] 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 ligand-sensitized particles 20. 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 phospholipid polar group, which is a component of biomembranes. The polymer having a phosphorylcholine group may be a homopolymer having only the structural unit M, or 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 a side chain of the polymer. The main chain of the polymer having a phosphorylcholine group may have a structure in which polymerizable groups in raw material monomers such as 2-methacryloyloxyethyl phosphorylcholine (MPC) are linked to each other.

[0023] The target substance detection reagent of this embodiment may 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. The target substance detection kit of the present embodiment includes a reagent container containing the above-mentioned target substance detection reagent.

[0024] The kit can take various forms, such as the following: The kit of the first type includes a first reagent container containing a particle group of the ligand-sensitized particles 20 and a dispersion medium. The kit of the second embodiment includes a second reagent container A containing a particle group of the ligand-sensitized particles 20, and a second reagent container B containing a dispersion medium. The first reagent container of the kit of the first embodiment and the second reagent container A and second reagent container B of the kit of the second embodiment may each contain at least one or more of the third substances exemplified above. Furthermore, each of the kits of the first and second embodiments may further include a third reagent container containing at least one of the third substances. In addition, 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 a measurable target substance are used.

[0025] The method for detecting a target substance of the present embodiment includes a contacting step of mixing a target substance with a target substance detection reagent. In the contact step, a sample containing a target substance and ligand-sensitized particles 20 having ligands 3 are added to the same reaction vessel and allowed to coexist, allowing the ligands 3 bound to the ligand-sensitized particles 20 to come into contact with the target substance. The sample containing the standard substance and the target substance reagent containing the ligand-sensitized particles 20 may be added in any order. Furthermore, the target substance and the ligand-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 50° C. or less.

[0026] The target substance is brought into contact with the ligand 3 and reacted with each other, so that the substance to be measured in the sample reacts with the antibody supported by the latex particles, forming a complex between the target substance, the ligand 3, and the ligand-sensitized particles 20, and causing aggregation of the ligand-sensitized particles 20 due to the formation of the complex.

[0027] The method for detecting a target substance may include, after the contact step, a detection step of optically measuring the agglutination of the ligand-sensitized particles 20. Examples of the method for optically measuring the agglutination include a method of measuring absorbance, scattered light intensity, or transmitted light intensity with an optical device. The wavelength for measuring absorbance is usually 340 nm to 1000 nm, and preferably 500 nm to 900 nm. The time for measuring the agglutination reaction can be measured based on the rate of change per unit time during which the agglutination reaction is occurring, or the amount of change per unit time. For example, when measuring absorbance, the time can be measured based on the rate of change in absorbance per unit time from 30 seconds to 5 minutes after the start of the agglutination reaction, or the amount of change in absorbance per unit time.

[0028] The sample is not particularly limited as long as it may contain the target substance, and examples thereof include any 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 medium containing the target substance, for example the target substance diluted in phosphate buffered saline or the like.

[0029] The aqueous solvent is not particularly limited as long as it is an aqueous solvent that allows the aggregation method, and examples thereof include deionized water, distilled water, and buffer solutions, with buffer solutions being preferred. Examples of the buffering agent contained in the buffer solution include phosphate-based, acetate-based, Tris-based, borate-based, carbonate-based, glycine-based, and Gut-based buffer solutions. As the Good's buffer, for example, HEPES, PIPES, MOPS, etc. are preferably used. The buffering agent may be used alone or in combination of two or more kinds. In addition, 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, and alkalis that can be used include sodium hydroxide, potassium hydroxide, lithium hydroxide, ammonium hydroxide, and the like.

[0030] 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 diuretic peptides such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). These 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 binding to an antibody, and may be, for example, in a free form as a simple substance, or in a complexed form with another substance (such as a protein).

[0031] The steps of the method for producing the sensitized particle dispersion of this embodiment will be described in detail below.

[0032] An example of a method for producing the sensitized particle dispersion of this embodiment includes at least a step of obtaining ligand-sensitized particles 20, which includes a sensitization treatment for chemically reacting ligands 3 with core particles 1, and a dispersion step of dispersing the ligand-sensitized particles 20 obtained by the step of obtaining the ligand-sensitized particles in a dispersion medium.

[0033] The process for obtaining the ligand-sensitized particles 20 may include, in addition to the sensitization treatment, one or more treatments that are commonly used in the technical field of sensitized particles, such as a washing treatment and a blocking treatment. 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. In the blocking treatment, another blocking protein that physically binds to the sensitized product obtained by the sensitization treatment by physical adsorption or the like can be added. In this case, a blocking solution containing the other blocking protein may be added. In the dispersion step, the ligand-sensitized particles 20 produced in the step of obtaining ligand-sensitized particles after, for example, a blocking treatment are dispersed in the above-mentioned dispersion medium, thereby producing a target substance detection reagent.

[0034] In the above sensitization treatment, a conventionally known method can be applied as a chemical reaction method for chemically fixing the reactive functional group (A1) present on the surface of the core particle 1 to the ligand 3. As the chemical reaction method, a carbodiimide-mediated reaction, an NHS ester activation reaction, etc. can be used, but are not limited thereto. If necessary, a condensation agent may be used.

[0035] For example, to bind (support) the ligand 3 to the core particle 1, the ligand 3 (antibody) and the core particle 1 may be suspended in a solvent such as a buffer solution and reacted for a predetermined time at about 20 to 37°C for sensitization, followed by washing and blocking treatments, etc. If necessary, other operational treatments such as centrifugation, ultrasonic treatment, and heating (aging) treatment may be performed at appropriate times. The washing treatment may be performed once or more than twice.

[0036] 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 sugars, polyethylene glycol, dextran), a salt (such as sodium chloride, amino acid), a surfactant, etc. These may be used alone or in combination of two or more kinds.

[0037] The core particle 1 is a particle that serves as the nucleus of the ligand-sensitized particle 20, and may be composed of either an organic particle or an inorganic particle.

[0038] As the organic particles, particles made of an organic polymer can be used. Examples of organic polymers include synthetic resins such as polystyrene, polyvinyl chloride, polypropylene, (meth)acrylic resins, 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 a main component (for example, copolymers containing styrene as a main component); and copolymers containing two or more of these. The organic polymer may be used alone or in combination of two or more. The organic polymer constituting the organic particles is not particularly limited as long as it is composed of a polymer different from the surface-modifying polymer.

[0039] As the organic particles, known latex particles can be used, for example, styrene-based latex particles such as polystyrene latex, 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, and the like. The latex particles can be produced by a known method, or may be commercially available. Two or more kinds of the latex particles may be used in combination.

[0040] 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.). The method for forming the granular organic polymer can be a radical polymerization method such as emulsion polymerization, soap-free emulsion polymerization, or suspension polymerization, but is not limited to radical polymerization.

[0041] As the inorganic particles, particles made of an inorganic substance can be used. 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. In addition, inorganic materials having magnetic properties may be used as inorganic substances. In this way, the inorganic particles may be non-magnetic particles or magnetic particles.

[0042] The shape of the core particle 1 is not particularly limited, but examples thereof include a spherical shape, an elliptical shape, and an irregular shape.

[0043] The average particle diameter (D50) of the core particles 1 may be, for example, 0.01 μm to 1.0 μm, 0.02 μm to 0.8 μm, or 0.03 μm to 0.6 μm. The particle size of the core particle 1 can be measured, for example, by a laser diffraction particle size distribution analyzer. The average particle size (D50) is defined as the particle size at which the integrated value from the small particle side in the volume-based particle size distribution is 50%.

[0044] The reactive functional group (A1) may contain one or more 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 the acid anhydride groups include maleic anhydride groups and succinic anhydride groups. Among these, the reactive functional group (A1) may contain a carboxyl group.

[0045] The ligand 3 is a compound that specifically binds to a receptor of a specific target substance. The site at which a ligand binds to a target substance is fixed, and the ligand has a high affinity selectively or specifically. Examples of ligands 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 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.

[0046] The step of obtaining the ligand-sensitized particles 20 also includes a reaction treatment for chemically bonding a part of the reactive functional groups (A1) present on the surface of the core particles 1 with the blocking protein 5. In one example of the reaction treatment, for example, addition of a condensing agent that chemically bonds the other portion of the reactive functional groups (A1) to the blocking protein 5 and the blocking protein 5 can be carried out. The addition (reaction treatment) of the blocking protein 5 and the condensing agent is not particularly limited as long as it is during a series of treatments including the above-mentioned sensitization treatment, washing treatment, blocking treatment, etc., or before or after each treatment in the series of treatments, which are carried out during the process of obtaining the ligand-sensitized particles 20. Specifically, the addition of the blocking protein 5 and the condensing agent may be, for example, simultaneous with the blocking treatment, before the blocking treatment, simultaneous with any of a series of treatments other than the blocking treatment, or before or after any of the series of treatments. In addition, the addition of the blocking protein 5 and the condensing agent preferably includes at least one embodiment, such as 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 blocking protein 5 and the condensing agent may each be added once or twice or more. When they are added multiple times, one or more of the above-mentioned treatments may be performed between the additions.

[0047] The amount of blocking protein added during the reaction treatment is designated as X (ng), and the total amount of blocking protein added in the reaction treatment, sensitization treatment, washing treatment, and blocking treatment is designated as Y (ng). In this case, in one example of the above reaction process, the lower limit of X / Y×100 is, for example, 0.01% or more, preferably 0.03% or more, and more preferably 0.05% or more, thereby making it possible to further improve the detection sensitivity of the target substance. On the other hand, the upper limit of X / Y×100 is, for example, 5% or less, preferably 4% or less, and more preferably 3% or less, which makes it possible to suppress a decrease in dispersibility.

[0048] In addition, in one example of the above reaction process, the amount of blocking protein added during the reaction process is X (ng), and the amount of blocking protein added when converted into 1% coverage of core particles calculated by the following procedure is Z (ng). At this time, in one example of the above-mentioned reaction treatment, a blocking protein may be added together with the condensing agent such that the amount added in terms of coverage calculated from X / Z is 5% or more. The lower limit of the amount added in terms of coverage is, for example, 5% or more, preferably 7% or more, and more preferably 9% or more, which makes it possible to increase the sensitivity while suppressing the decrease in dispersibility in the above reaction treatment. The upper limit of the amount of addition in terms of coverage is not particularly limited, and may be set according to the molecular weight and type of the blocking protein. For example, in the case of bovine serum albumin (BSA), the upper limit of the amount of addition in terms of coverage may be set to 300% or less. (procedure) When the Stokes radius of the blocking protein is r (nm) and the molecular weight is M, 1cm 2 Mass of blocking protein per unit mass (ng / cm 2 ) to [M / (24×r 2 )]. When the average particle diameter (cm) of the core particles is d and the total number of core particles is Pn, Total surface area of ​​all core particles (cm 2 ) to [π×d 2 ×Pn]. In this case, the amount of blocking protein added when converting to a 1% coverage of the core particles, Z (ng), is expressed as [π×d 2 ×Pn]×[M / (24×r 2 ) / 100].

[0049] Examples of blocking proteins 5 include animal-derived albumins such as bovine serum albumin, casein, gelatin, ovalbumin, and gamma-globulin. Among these, from the viewpoint of dispersibility, it is preferable to use blocking proteins derived from natural products, and from the viewpoint of sensitivity, blocking proteins derived from natural products are preferable to synthetic polymers. These may be used alone or in combination of two or more kinds. 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 a blocking protein that physically binds by physical adsorption or the like, such as the same type as exemplified above as blocking protein 5.

[0050] As the condensation agent, those which are known to be used in the immobilization method utilizing a carboxy group or an amino group for immobilization of an antibody 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 with each other by 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, fluoroesters, and the like.

[0051] In another embodiment, another blocking protein may be present on at least a portion of the surface of the ligand-sensitized particle 20 and / or the surface of the blocking protein 5, the blocking protein being physically bound, for example, by physical adsorption.

[0052] 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) to at least a part of the surface of the core particle. The introduction step can be performed at any timing in the process of obtaining the ligand-sensitized particle 20, but is preferably performed before the sensitization treatment or the binding treatment. As a result, the surface of the core particle 1 is in a state in which the reactive functional group (A1) is formed via the surface-modifying polymer and / or the coupling agent. This form of ligand-sensitized particle 20 may include a ligand 3 and / or a blocking protein 5 bound to at least a portion of the multiple reactive functional groups (A1) of the surface-modifying polymer and / or coupling agent.

[0053] The surface-modifying polymer may be formed on at least a part of the surface of the core particle 1, and a polymer coating layer that covers a part or the entire surface of the core particle 1 may be formed. The surface-modifying polymer is physically bonded to the surface of the core particle 1, and / or a specific functional group contained in the surface-modifying polymer is chemically bonded to a specific functional group present on the surface of the core particle 1.

[0054] 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 monomer and a unit derived from a glycidyl group-containing monomer. According to this embodiment, by using a surface-modifying polymer in addition to the core particle 1, a wide variety of molecular designs become possible for the surface-modifying polymer, and the degree of freedom in molecular design becomes high.

[0055] 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.

[0056] 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.

[0057] 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 using a polystyrene equivalent value from a calibration curve of standard polystyrene (PS) obtained by gel permeation chromatography measurement.

[0058] The surface modifying polymer may include a structural unit A1 that includes a reactive functional group (A1) capable of binding to a ligand 3.

[0059] A method for forming a surface-modifying polymer on the surface of the core particle 1 may, for example, be a polymer introduction step in which a surface-modifying polymer is introduced by chemical reaction onto the surface of the core particle 1. 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, may be used. These chemical reactions may involve the use of condensing agents, if desired, such as carbodiimide-based crosslinkers and amine-reactive crosslinkers. Carboxy groups and amino groups can be reacted with each other by 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, fluoroesters, and the like.

[0060] 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 is reacted with a predetermined reactive group contained in the surface-modifying polymer. For example, in the manufacturing process of the organic particle, it becomes possible to include a polymer reactive group in the organic polymer during polymerization of the raw material monomer. In addition, commercially available polystyrene particles containing a carboxy group may be used as the core particle 1. As a specific example, when a surface-modifying polymer containing a carboxy group is fixed to a core particle 1 containing a carboxy group, a primary amino group (-NH2) may be introduced by amino treatment to some of the carboxy groups in the core particle 1 (amino treatment). Then, the primary amino group (polymer reactive group) in the core particle 1 after the amino treatment and a predetermined reactive group in the surface-modifying polymer are chemically reacted with each other by adding the above-mentioned condensing agent if necessary. When the surface-modifying polymer contains an acrylic group / methacrylic group, it is possible to react a primary amino group (polymer reactive group) with a 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 necessary, and then the polymer reactive group is reacted with a specific reactive group contained in the surface-modifying polymer.

[0061] 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.

[0062] For the polymer of a diene compound and a parent diene compound, (i) if an anhydride group is contained, the anhydride group may be ring-opened to introduce a reactive functional group (A1), an acidic functional group (A2), or other functional groups (functional functional groups) into the side chain, (ii) if a carboxy 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 above-mentioned various functional groups 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. In addition, (i) when an anhydride group is contained, the anhydride group may be ring-opened with water, an alcohol, etc., or may be ring-opened with an organic base. In addition, 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 the above (ii).

[0063] In synthesizing 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.

[0064] 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), etc. 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.

[0065] For the synthesis of the polymer precursor of the (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 are used. One or more of these (meth)acrylic monomers can be used.

[0066] 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. In addition, 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 obtained 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 polymerized by heating for a predetermined time. 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 remove dissolved oxygen in the solvent by nitrogen bubbling before carrying out the solution polymerization.

[0067] As the polymerization initiator, for example, an azo compound, an organic peroxide, etc. 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, di-tertiary butyl peroxide (DTBP), benzoyl peroxide (benzoyl peroxide, BPO), and methyl ethyl ketone peroxide (MEKP). As for the polymerization initiator, only one type may be used, or two or more types may be used in combination.

[0068] In addition, a molecular weight regulator or a chain transfer agent can be used as necessary. 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 may be used alone or in combination of two or more.

[0069] As the solvent used in the polymerization reaction, for example, one or more of esters such as methyl ethyl ketone (MEK), propylene glycol monomethyl ether, diethyl ether, tetrahydrofuran (THF), toluene, ethyl acetate, and butyl acetate can be used. As the polymerization initiator, one or more of azo compounds and organic peroxides can be used. As the azo compound, for example, azobisisobutyronitrile (AIBN), dimethyl 2,2'-azobis(2-methylpropionate), and 1,1'-azobis(cyclohexanecarbonitrile) (ABCN) can be used. As the organic peroxide, for example, hydrogen peroxide, ditertiary butyl peroxide (DTBP), benzoyl peroxide (benzoyl peroxide (BPO)), and methyl ethyl ketone peroxide (MEKP) can be used.

[0070] The reaction liquid 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 makes it possible to remove low molecular weight components such as residual monomers, oligomers, and polymerization initiators.

[0071] Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various configurations other than those described above can be adopted. Furthermore, the present invention is not limited to the above-described embodiments, and modifications and improvements within the scope of the present invention are included in the present invention. EXAMPLES

[0072] 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.

[0073] <Production of Sensitized Particle Dispersion> [Example 1] (Sensitization treatment) 940 μL of latex liquid A (0.4% w / vol, the number of particles and the total particle surface area of ​​the latex particles are shown in Table 1) containing the following latex particles to which the following condensation 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 liquid. Condensation agent: Carbodiimide crosslinking agent (EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, also known as WSC) Latex solution A: Carboxyl-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 entire particles were measured, and the total volume of the particles was calculated from the weight / density. The volume of one 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. In addition, the surface area of ​​each 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.

[0074] (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 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 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% NaN3

[0075] (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 reaction treatment to the total amount of BP added (in blocking solution A and blocking solution B) added during 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 condensation 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°C for 15 minutes at 10,000 rpm 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)

[0076] (Reagent processing) The following components were mixed in a given mixing ratio and uniformly dispersed by ultrasonic treatment (dispersion treatment) to prepare a reagent (sensitized particle dispersion) with a solid content of 0.1%. 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)

[0077] [Example 2] In the blocking treatment, 5.015 mL of blocking solution A was added, and 15 μL of additional blocking solution B was added (coverage conversion amount: 25%, BP addition amount per total particle surface area: 56.5 ng / cm 2 A reagent (sensitized particle dispersion) was prepared in the same manner as in Example 1, except that the ratio of the amount added to the total amount was changed to 0.05%.

[0078] [Example 3] In the blocking treatment, 4.97 mL of blocking solution A was added, and 60 μL of additional blocking solution B was added (coverage conversion amount: 100%, BP addition amount per total particle surface area: 226 ng / cm 2 A reagent (sensitized particle dispersion) was prepared in the same manner as in Example 1, except that the ratio of the amount added to the total amount was changed to 0.20%).

[0079] [Example 4] In the blocking treatment, 4.91 mL of blocking solution A was added, and 120 μL of additional blocking solution B was added (coverage conversion amount: 200%, BP addition amount per total particle surface area: 452 ng / cm 2 A reagent (sensitized particle dispersion) was prepared in the same manner as in Example 1, except that the ratio of the amount added to the total amount was changed to 0.40%).

[0080] [Example 5] A reagent (sensitized particle dispersion) was prepared in the same manner as in Example 1, except that in the preparation of the reagent, polyethylene glycol was changed to 2-methacryloyloxyethyl phosphorylcholine (MPC).

[0081] [Example 6] <Synthesis of surface-modifying polymer> (Maleic anhydride polymer) Norbornene and maleic anhydride were reacted by radical polymerization at a predetermined ratio using an azo compound (polymerization initiator) to obtain polymer precursor A. Subsequently, various functional monomers having the following functional groups 1 to 2 were subjected to a ring-opening reaction in the presence of an organic base (triethylamine) at a predetermined ratio to introduce them into the side chains (ring-opened maleic anhydride groups) of each polymer precursor A, and then reprecipitation was performed with hexane or diethyl ether to obtain maleic anhydride-based polymers.

[0082] [ka]

[0083] [ka]

[0084] The weight average molecular weight (Mw) of each of the maleic anhydride polymers obtained above was calculated based on a calibration curve of a standard PEG sample using GPC (gel permeation chromatography) under the following measurement conditions. The Mw of the maleic anhydride polymer obtained was 13×10 3 It was. (GPC measurement conditions) For the GPC measurement, one Tosoh TSKgel SuperAWM-H, one TSKgel SuperAW2500, and one TSKgel guardcolumn SuperAW-H were used, and the developing solvent was 70% methanol / 30% HO (with 10 mM LiBr added), with a column temperature of 40°C and a flow rate of 0.6 ml / min.

[0085] The maleic anhydride polymer obtained above was subjected to the following measurement conditions: 13 The C-NMR spectrum was measured. It was found that the maleic anhydride polymer contains structural units having each functional group represented by the above general formula (A). R in general formula (A) represents functional group 1 or functional group 2 exemplified above. Based on the results of the NMR spectrum, the composition ratio (content of structural units having functional groups) in the maleic anhydride polymer was calculated. The maleic anhydride polymer had 17% dicarboxylic acid structure, 36% maleic anhydride structure, 20% functional group 1 structure, and 27% functional group 2 structure. The ring-opening rate (%) of the maleic anhydride group was 64%. (NMR measurement conditions) NMR measurements were performed using a JEOL ECA500 with deuterated dimethyl sulfoxide or deuterated ethanol as the solvent.

[0086] <Preparation of particles with polymer> Diamine was mixed into the above-mentioned latex liquid 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 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).

[0087] In the subsequent <Preparation of Sensitized Particle Dispersion>, a reagent (sensitized particle dispersion) was prepared in the same manner as in Example 1, except that a dispersion containing core particles to which a surface-modifying polymer had been introduced was used instead of latex liquid A.

[0088] [Comparative Example 1] A reagent (sensitized particle dispersion) was prepared in the same manner as in Example 1, except that in the blocking treatment, the amount of blocking solution A added was 5.03 mL, and blocking solution B and a condensing agent were not added.

[0089] The production conditions for the above Examples 1 to 6 and Comparative Example 1 are summarized in the following Table 1. Note that in Table 1, the amount of blocking protein added during the blocking treatment includes the amount added during the reaction treatment.

[0090] [Table 1]

[0091] <Absorbance> 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) held at 10°C. Next, 20 μL of each of the reagents from each Example and Comparative Example was mixed with 80 μL of the measurement reagent described below and 4 μL of the target substance (antigen amount 0 ng / mL or 300 ng / mL), and the mixture was stirred uniformly and kept 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 when the antigen amount was 0 ng / mL and 300 ng / mL was calculated by subtracting the absorbance immediately after adding the measurement reagent from the absorbance after 5 minutes. As an index of sensitivity, the difference in absorbance (ΔAbs) between the presence and absence of added target substance was calculated by subtracting the absorbance at a wavelength of 700 nm when the antigen amount was 0 ng / mL from the absorbance at a wavelength of 700 nm when the antigen amount was 300 ng / mL.

[0092] (Measuring Reagents) The following components were mixed in a given ratio to prepare a measuring reagent. 0.1% Tween20 Polyethylene glycol (PEG) (5000~50000) ·1% bovine serum albumin (BSA) in HEPES buffer 150mM NaCl ·0.09%NaN3 ·100mM HEPES buffer (pH7.6)

[0093] The sensitized particle dispersions obtained in Examples 1 to 6 were found to have excellent dispersibility, since they showed lower absorbance at a wavelength of 700 nm when the antigen amount was 0 ng / mL, i.e., lower absorbance when no target substance (antigen) was added, compared to Comparative Example 1. Furthermore, in Example 1, bovine serum albumin (BSA) was compared with porcine gelatin (Example 1a) and bovine casein (BlockAce: casein and buffer salt, Example 1b), which are classified as blocking proteins derived from the same natural products. As a result, both Examples 1a and 1b showed low absorbance at a wavelength of 700 nm when the antigen amount was 0 ng / mL, and excellent dispersibility, similar to Example 1.

[0094] Furthermore, the above <absorbance> was also examined in the low antigen concentration range of 1 to 10 ng / mL. As a result, the sensitized particle dispersions of Examples 1 to 6 showed higher absorbance differences (ΔAbs) between the presence and absence of target substance addition in the low antigen concentration range of 1 to 10 ng / mL compared to Comparative Example 1, and showed superior sensitivity.

[0095] It was found that the sensitized particle dispersions obtained in Examples 1 to 6 can be suitably used as sensitized particle dispersions (reagents) for use in the immune agglutination method. [Explanation of symbols]

[0096] 1. Core Particle 3. Ligand 5. Blocking Proteins 20 Ligand-sensitized particles

Claims

1. A method for producing a sensitized particle dispersion, comprising: obtaining ligand-sensitized particles having core particles having a plurality of reactive functional groups (A1) on the surface thereof; a ligand having specific binding ability to a target substance and chemically bonded to a portion of the reactive functional groups (A1); and a blocking protein chemically bonded to another portion of the reactive functional groups (A1).

2. A method for producing the sensitized particle dispersion according to claim 1, comprising the steps of: The step of obtaining the ligand-sensitized particles includes a reaction treatment of bonding another part of the reactive functional groups (A1) to the blocking protein via a condensing agent.

3. A method for producing the sensitized particle dispersion according to claim 2, comprising the steps of: The step of obtaining the ligand-sensitized particles includes the reaction treatment, a sensitization treatment of reacting a part of the reactive functional groups (A1) with the ligand, a washing treatment, and a blocking treatment.

4. A method for producing the sensitized particle dispersion according to claim 2, comprising the steps of: a blocking protein added during the reaction treatment, the blocking protein being added to the reaction treatment, the sensitization treatment, the washing treatment, and the blocking treatment, the blocking protein being added in a total amount of the blocking protein added during ...

5. A method for producing the sensitized particle dispersion according to claim 2, comprising the steps of: a method for producing a sensitized particle dispersion, wherein, in the reaction treatment, an amount of the blocking protein added during the reaction treatment is X (ng), and an amount of the blocking protein added in terms of a 1% coverage of the core particles calculated by the following procedure is Z (ng), the blocking protein is added together with the condensing agent in an amount calculated as a coverage conversion from X / Z of 5% or more. (procedure) When the Stokes radius of the blocking protein is r (nm) and the molecular weight is M, 1 cm ２ The mass of the blocking protein per unit area (ng / cm ２ ) to [M / (24×r ２ )]. When the average particle diameter (cm) of the core particles is d and the total number of the core particles is Pn, The total surface area (cm) of the total number of core particles ２ ) to [π × d ２ ×Pn]. At this time, the amount of the blocking protein added, Z (ng), converted into a 1% coverage of the core particles, is expressed as [π×d ２ ×Pn]×[M / (24×r ２ ) / 100].

6. A method for producing the sensitized particle dispersion according to claim 1, comprising the steps of: The reactive functional group (A1) contains one or more groups selected from the group consisting of a carboxy group, an amino group, a thiol group, an epoxy group, a maleimide group, an acryl group, a methacryl group, and an acid anhydride group.

7. A method for producing the sensitized particle dispersion according to claim 1, comprising the steps of: The method for producing a sensitized particle dispersion, wherein the core particles are organic particles or inorganic particles.

8. A method for producing the sensitized particle dispersion according to claim 1, comprising the steps of: The method for producing a sensitized particle dispersion, wherein the ligand is any one of an antibody, an antigen, a protein, and a nucleic acid.

9. A method for producing the sensitized particle dispersion according to claim 1, comprising the steps of: A method for producing a sensitized particle dispersion, comprising the step of introducing a surface-modifying polymer having a reactive functional group (A1) or a coupling agent having a reactive functional group (A1) to at least a portion of the surface of the core particle in the step of obtaining the ligand-sensitized particle.

10. A target substance detection reagent comprising a sensitized particle dispersion obtained by the method for producing a sensitized particle dispersion according to any one of claims 1 to 9.

11. The target substance detection reagent according to claim 10, A target substance detection reagent comprising a sensitizer having a phosphorylcholine group.

12. A target substance detection kit comprising a reagent container containing the target substance detection reagent according to claim 10.

13. A method for detecting a target substance, comprising the step of mixing a target substance with the target substance detection reagent according to claim 10.

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