Method for producing particle for immunoassay for detecting Anti-hbs antibody, and particle obtained by the production method

Abstract

To provide a method for producing a particle for immunoassay for detecting an anti-HBs antibody, which improves the detection sensitivity of the anti-HBs antibody immunoassay by removing impurities contained in the HBs antigen.SOLUTION: A method for producing a particle for immunoassay for detecting an anti-HBs antibody, comprising a first step of covalently binding an HBs antigen to a particle to obtain an HBs antigen-sensitized particle, and a second step of washing the HBs antigen-sensitized particle with a nonionic surfactant or an amphoteric surfactant to obtain a washed HBs antigen-sensitized particle. Also, a method for producing a particle for immunoassay for detecting an anti-HBs antibody, comprising a first step of covalently binding an HBs antigen to a particle to obtain an HBs antigen-sensitized particle, and a third step of washing the HBs antigen-sensitized particle with an acidic aqueous solution to obtain a washed HBs antigen-sensitized particle.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing particles for use in immunoassays for detecting anti-HBs antibodies, which are used in latex immunoagglutination, and to particles obtained by the method. [Background technology]

[0002] It is estimated that there are approximately 400 million people worldwide who are chronically infected with hepatitis B virus (HBV). HBV-infected individuals can progress from asymptomatic carriers to chronic hepatitis and then to cirrhosis. On the other hand, inactive carriers are known to have a low risk of progression of the disease or cancer, and a good long-term prognosis.

[0003] The re-proliferation of HBV in HBV-infected patients due to immunosuppression, chemotherapy, etc. is called HBV reactivation. HBV reactivation can lead to hepatitis B, and in some cases, it can become fulminant, so caution is required. Therefore, when administering immunosuppression and chemotherapy, it is necessary to screen for HBV infection by measuring hepatitis B surface antigen (HBs antigen), hepatitis B core antigen (HBc) antibody, and HBs antibody before starting treatment in all patients.

[0004] One method for measuring infectious disease-related antigens and antibodies is the latex immunoagglutination method. In this method, a dispersion of particles sensitized (bound) to antibodies or antigens as ligands is mixed with a sample that may contain the target substance (antigen or antibody). If the target substance (antigen or antibody) is present in the sample, the ligand-sensitized particles will undergo an agglutination reaction. This agglutination reaction can be measured as changes in scattered light intensity, transmitted light intensity, absorbance, etc., allowing the presence or absence of the target substance to be identified and quantified.

[0005] Latex agglutination test reagents for HBs antibodies measure HBs antibodies using particles sensitized with HBs antigen as a ligand. HBs antigen is an envelope protein that covers the surface of HBV, and many variations are known, including subtype antigen groups d and y, and w and r. HBs antigen used in latex agglutination test reagents can be either purified from natural HBV or recombinant protein antigen produced using E. coli or cells. For HBVs that contain many mutations, using HBs antigen derived from natural HBV allows for the reflection of HBs antigen mutation variations.

[0006] HBs antigen is known to exist in a particulate state, with antigen protein displayed on a lipid membrane. Patent Document 1 describes a process in which particulate HBs antigen is reduced in size using a surfactant or the like, and then reacted with a molecular chaperone. It reports that the resulting treated HBs antigen can then be sensitized to latex particles, thereby increasing detection sensitivity. Patent Document 2 also reports an example in which detection sensitivity is increased by denaturing HBs antigen using a denaturant or the like, exposing internal epitopes, and then detecting the antigen using an antibody that binds to the exposed epitope. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2000-105233 [Patent Document 2] Patent No. 04430677 Summary of the Invention [Problem to be solved by the invention]

[0008] HBs antigen derived from natural HBV is extracted from the living body and purified. Therefore, natural HBs antigen derived from HBV contains impurities such as lipids and unnecessary proteins. The presence of these impurities inhibits the binding of HBs antigen to anti-HBs antibodies, which is one of the reasons for the reduced sensitivity of the latex agglutination test. As described in Patent Documents 1 and 2, methods have been proposed for reducing particle size and removing impurities by pre-treating HBs antigen with a surfactant or the like. However, there is a problem that HBs antigen pre-mixed with a surfactant reduces the sensitization efficiency of latex particles having functional groups (e.g., carboxy groups). In addition, some of the surfactants and reducing agents disclosed in Patent Document 2 destroy the structure surrounding antigenic determinant a, the main conformational epitope of HBs antigen, and therefore cannot be used as a treatment reagent for HBs antigen in anti-HBs antibody tests. [Means for solving the problem]

[0009] The method for producing particles for anti-HBs antibody detection immunoassays according to the present invention is characterized by comprising a first step of covalently binding HBs antigen to particles to obtain HBs antigen-sensitized particles, and a second step of washing the HBs antigen-sensitized particles with a nonionic surfactant or an amphoteric surfactant to obtain washed HBs antigen-sensitized particles.

[0010] The method for producing particles for immunoassays for detecting anti-HBs antibodies according to the present invention is characterized by comprising a first step of covalently binding the HBs antigen to particles to obtain HBs antigen-sensitized particles, and a third step of washing the HBs antigen-sensitized particles with an acidic aqueous solution to obtain washed HBs antigen-sensitized particles.

[0011] The particles for immunoassays for detecting anti-HBs antibodies according to the present invention are characterized by being produced by comprising a first step of covalently binding HBs antigen to particles to obtain HBs antigen-sensitized particles, and a second step of washing the HBs antigen-sensitized particles with a nonionic surfactant or an amphoteric surfactant to obtain washed HBs antigen-sensitized particles.

[0012] The particles for anti-HBs antibody detection immunoassays according to the present invention are characterized in that they are produced by comprising a first step of covalently binding HBs antigen to particles to obtain HBs antigen-sensitized particles, and a third step of washing the HBs antigen-sensitized particles with an acidic aqueous solution to obtain washed HBs antigen-sensitized particles. [Effects of the Invention]

[0013] According to the present invention, impurities from HBs antigen derived from natural HBV covalently bound to particles can be removed, thereby improving the detection sensitivity of anti-HBs antibodies in immunoassays using the particles. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a graph showing the results of measuring the anti-HBs detection sensitivity of particles for immunoassay for detecting anti-HBs antibodies, which particles are produced by the production method according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, embodiments of the present invention will be described in detail, but the technical scope of the present invention is not limited to these embodiments.

[0016] (Manufacturing method) A method for producing particles for an immunoassay for detecting anti-HBs antibodies according to an embodiment of the present invention will now be described. The method for producing particles for an immunoassay for detecting anti-HBs antibodies according to the present invention is characterized by comprising a first step of covalently binding HBs antigen to particles to obtain HBs antigen-sensitized particles, and a second step of washing the HBs antigen-sensitized particles with a nonionic surfactant or an amphoteric surfactant to obtain washed HBs antigen-sensitized particles. Any bond can be used as the covalent bond in the first step as long as it can bind HBs antigen to the functional group of the particle. Since HBs antigen contains amino groups, carboxyl groups, thiol groups, etc., any functional group that can react with these can be used as the functional group of the particle.

[0017] Examples of functional groups that react with the amino group of HBs antigen to form a covalent bond include N-hydroxysuccinimide ester, sulfo-N-hydroxysuccinimide ester, isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imide ester, carbodiimide, anhydride, and fluoroester. Among these, N-hydroxysuccinimide ester and sulfo-N-hydroxysuccinimide ester are preferred, as they react efficiently even at near-neutral pH. Particles having a carboxy group as a functional group are preferred because they allow the introduction of N-hydroxysuccinimide ester or sulfo-N-hydroxysuccinimide ester via carbodiimide activation.

[0018] Examples of functional groups that react with the carboxyl groups of HBs antigen to form covalent bonds include amino groups, etc. In this case, the carboxyl groups of HBs antigen can be converted to N-hydroxysuccinimide esters or sulfo-N-hydroxysuccinimide esters via carbodiimide activation, and then bonded to the amino groups of the particles.

[0019] Examples of functional groups that react with the thiol group of HBs antigen to form a covalent bond include a maleimide group, a sulfide group, etc. In the present invention, particles obtained by covalently bonding HBs antigen to particles are called HBs antigen-sensitized particles.

[0020] Next, as the nonionic surfactant or amphoteric surfactant in the second step, any bond can be used as long as a suitable cleaning effect can be obtained. As will be shown in the examples below, positive surfactants are not preferred because the negatively charged particle surface interacts with the positively charged positive surfactant, making the particles hydrophobic, resulting in aggregation due to hydrophobic interactions, making them difficult to use as test reagents. Furthermore, as will be shown in the examples below, negative surfactants are not preferred because the particle surfaces are negatively charged, making it difficult for the surfactant to approach the surface and making it difficult for the reaction to remove impurities to proceed.

[0021] Biological components contained in patient samples, etc., are often negatively charged at near-neutral pH. To suppress nonspecific reactions with such biological components, the majority of particles used in specimen testing have a negatively charged particle surface. In other words, positive and negative surfactants are not preferred surfactants for use in the particle manufacturing method according to an embodiment of the present invention. On the other hand, nonionic surfactants and amphoteric surfactants are less susceptible to the charge on the particle surface and therefore provide a high cleaning effect. In the present invention, HBs antigen-sensitized particles washed with a nonionic surfactant or amphoteric surfactant are referred to as washed HBs antigen-sensitized particles.

[0022] The washing and replacement procedure here refers to mixing a nonionic surfactant or amphoteric surfactant with HBs antigen-sensitized particles and, after a certain period of time, replacing the mixture with a surfactant-free buffer solution. The mixing time, temperature, and whether or not shaking is performed can be set as desired as long as a cleaning effect can be achieved. For example, a high cleaning effect can be achieved by performing the mixing procedure for 22 hours at room temperature (20°C to 25°C) with shaking (microtube mixer MT-400 (Tomy Seiko Co., Ltd.), scale 5). The replacement with a surfactant-free buffer solution can be performed by dialysis using a dialysis membrane, crossflow using an ultrafiltration membrane, or centrifugation. In the present invention, the step of replacing with a buffer solution is referred to as the fourth step.

[0023] In the particle production method according to an embodiment of the present invention, it is necessary to wash the HBs antigen-sensitized particles obtained in the first step with a nonionic surfactant or an amphoteric surfactant in the second step, or to wash the HBs antigen-sensitized particles obtained in the first step with an acidic aqueous solution in the third step. Here, the second and third steps may be performed after the first step. As shown in the examples below, when the second step was performed first, the amount of HBs antigen sensitization was significantly reduced, and the detection sensitivity of anti-HBs antibodies was also significantly reduced. This is thought to be because the small amount of surfactant remaining even after thorough dialysis and purification in the second step inhibits the sensitization reaction with the particles.

[0024] Another method for producing particles for immunoassays for detecting anti-HBs antibodies according to the present invention comprises a first step of covalently binding HBs antigen to particles, and a third step of washing with an acidic aqueous solution. As shown in the examples below, alkaline aqueous solutions are undesirable because they cause partial dissociation or denaturation of the HBs antigen in the HBs antigen-sensitized particles, resulting in a decrease in the sensitization amount and a decrease in the sensitivity of anti-HBs antibody detection. Furthermore, reducing agents are undesirable because they destroy the structure around antigenic determinant a, the main conformational epitope of HBs antigen in the HBs antigen-sensitized particles, resulting in a decrease in the sensitivity of anti-HBs antibody detection. On the other hand, acidic aqueous solutions can achieve a high cleaning effect without significantly denaturing the HBs antigen. In the present invention, HBs antigen-sensitized particles washed with an acidic aqueous solution are also referred to as washed HBs antigen-sensitized particles.

[0025] (surfactant) The surfactant used in the production method according to this embodiment may be a nonionic surfactant or an amphoteric surfactant. Examples of nonionic surfactants include polyoxyethylene sorbitan fatty acid esters (e.g., compounds represented by formula (1)), Brij® 35, Brij® 58, Brij® 76, Brij® 98, Triton® X-100, Triton® X-114, Triton® X-305, Triton® N-101, Nonidet® P-40, IGEPAL® CO530, IGEPAL® CO630, IGEPAL® CO720, and IGEPAL® CO730. [ka] (In formula (1), R 21 ~R 24 are each independently selected from -H and -OCR'. R' is a saturated or unsaturated alkyl group having 1 to 18 carbon atoms. In addition, in formula (1), w1, x1, y1, and z1 are integers such that the sum of w1, x1, y1, and z1 is 10 to 30.

[0026] Examples of the polyoxyethylene sorbitan fatty acid ester represented by formula (1) include Tween (registered trademark) 20, Tween (registered trademark) 40, Tween (registered trademark) 60, Tween (registered trademark) 80, and Tween (registered trademark) 85. Examples of amphoteric surfactants used in the production method according to this embodiment include betaine type, alkylbetaine type, sulfobetaine type, etc. Here, the amphoteric surfactant is a surfactant in which the hydrophilic group portion becomes positively or negatively charged depending on the pH.

[0027] (acidic aqueous solution) The acidic aqueous solution used in the production method according to this embodiment may be an aqueous hydrochloric acid solution, an aqueous acetic acid solution, or an aqueous glycine hydrochloride solution. The concentration of the acidic aqueous solution is preferably in the range of 0.01 to 2 M. If the acid is too strong, the HBs antigen will be acid-denatured, reducing its binding to anti-HBs antibodies, which is not preferred. On the other hand, if the acid is too weak, a sufficient washing effect cannot be obtained.

[0028] (particle) The particles used in the manufacturing method of this embodiment are particles used in ordinary immunoassays, and any particles can be used as long as they have a functional group capable of covalently binding to HBs. Examples of particle materials include polymers such as polystyrene, styrene-methacrylic acid copolymer, styrene-glycidyl (meth)acrylate copolymer, acrylic acid polymer, agarose, and dextran; inorganic materials such as silica and alumina; and metals such as gold colloid, iron oxide particles, and magnetic particles. The particles can be synthesized and prepared according to known methods.

[0029] An example of a suitable particle is latex particles. Here, latex particles refer to polymer particles capable of immobilizing antigens or antibodies against target substances, and are used in latex agglutination reactions. Preferred examples of latex particles include polystyrene particles, polystyrene particles containing siloxane, and polystyrene particles containing polyglycidyl (meth)acrylate. These particles have advantages such as the relative ease of obtaining nano-sized particles and the ability to chemically modify the particle surface depending on the purpose, making them suitable for use with the latex agglutination reagent of this embodiment.

[0030] Among these particles, as will be described later in the Examples, particles that are styrene-glycidyl (meth)acrylate copolymers in which one or more glycidyl groups have been ring-opened with mercaptosuccinic acid and 3-mercapto-1,2-propanediol have chemically hydrophilized particle surfaces, and can highly suppress nonspecific adsorption. Furthermore, among the polystyrene particles containing the siloxane, as will be described later in the Examples, polystyrene-silica hybrid particles using 3-methacryloxypropyltrimethoxysilane as a monomer also have hydrophilic particle surfaces, which can highly suppress nonspecific adsorption.

[0031] The particle size, in terms of major axis diameter, is preferably 0.01 μm or more and 10 μm or less, and more preferably 0.05 μm or more and 1 μm or less. By setting the particle size to 0.01 μm or more, the absorbance in the visible range observed in latex agglutination measurement becomes within an appropriate range. By setting the particle size to 10 μm or less, the dispersion stability in solution is improved and the absorbance in the visible range becomes within an appropriate range. From the viewpoints of reactivity and dispersion stability, it is preferable to use particles of 0.05 μm or more and 0.50 μm or less. One suitable example of the size can be expressed as the average particle diameter. The average particle size may be a volume average particle size measured by scattering particle size distribution measurement. The preferred average particle size of the particles is 0.05 μm or more and 1 μm or less.

[0032] (HBs antigen) The HBs antigen used in the production method of this embodiment can be inactivated HBV (or Dane particles) collected from the blood of a patient with hepatitis B. For example, the virus can be inactivated by liquid heat treatment at 60°C for 10 hours, and then HBs antigen can be obtained by concentration gradient centrifugation purification using sucrose or potassium bromide or gel filtration purification. Furthermore, HBs antigen produced by genetic recombination can also be used. It is also possible to artificially produce HBs by introducing the gene encoding HBs into E. coli or mammalian cells. HBs produced by genetic recombination may have fewer mutation variations because the amino groups are determined based on genetic information. Therefore, it is necessary to devise a method such as using multiple genetic information.

[0033] (HBs antigen binding to particles) In this embodiment, the carboxyl group of the particle according to this embodiment reacts with the amino group of the HBs antigen to form an amide bond, thereby chemically immobilizing HBs. Any conventionally known method can be used as long as it is possible to achieve the object of the present invention. For example, carbodiimide-mediated reaction and NHS ester activation reaction are commonly used chemical reactions. However, the method of chemical reaction for chemically fixing the carboxy group and HBs in the present invention is not limited to these.

[0034] In the HBs-sensitized particles of this embodiment, hydrophilic molecules may be bound to the remaining active esterified carboxyl groups that have not bound HBs. This is generally referred to as active ester inactivation, carboxyl group blocking, or masking, and is performed to reduce nonspecific protein adsorption to carboxyl groups and improve particle dispersion stability. In this embodiment, the hydrophilic molecule is preferably polyethylene glycol (PEG), trishydroxymethylaminomethane (Tris), or ethanolamine. PEG can significantly reduce protein adsorption to particles. When active ester inactivation is performed using PEG, the molecular weight of the PEG is important; a large molecular weight may inhibit antigen-antibody reactions. Therefore, the molecular weight of the PEG is preferably 350 to 5000, and particularly preferably 1000 to 2000. In this embodiment, PEG has a functional group reactive to carboxyl groups or active esters, such as PEG with an amino group, and polyethylene glycol with a primary amine is particularly preferred. Polyethylene glycol may be a linear or branched polymer. Tris is represented by the following formula (2), and an example of PEG is represented by the following formulas (3) and (4): In formulas (3) and (4), n is an integer of 1 or more and indicates the number of oxyethylene units. [ka] CH3O-(CH2CH2O)n-CH2CH2NH2(3) CH3O-(CH2CH2O)n-CH2CH2CH2NH2(4)

[0035] The amount of HBs antigen bound is also an important factor; if the amount of HBs antigen bound (which can also be called the immobilization amount) is low, the antigen-antibody reactivity decreases, while antibody binding that crosslinks particles becomes more likely to occur. Conversely, if the amount of HBs antigen bound is high, the antigen-antibody reactivity improves, but the two antigen-antibody binding sites on one particle tend to bind more easily, making it difficult for crosslinking reactions to occur between particles. Depending on the particle size, if the average particle size is approximately 200 nm, the amount of HBs antigen bound per 1 mg of particles is preferably 1 μg to 500 μg, and particularly preferably 10 μg to 200 μg.

[0036] (Testing reagents) The in vitro diagnostic test reagent according to this embodiment contains particles for immunoassays for detecting anti-HBs antibodies according to this embodiment and a dispersion medium. The amount of particles for immunoassays for detecting anti-HBs antibodies contained in the test reagent according to this embodiment is preferably 0.001% to 20% by mass, more preferably 0.01% to 10% by mass. The reagent of the present invention may contain a third substance, such as a solvent or a blocking agent, in addition to the particles for immunoassays for detecting anti-HBs antibodies according to this embodiment, within a range that allows the object of the present invention to be achieved. Two or more types of third substances, such as solvents or blocking agents, may be used in combination. Examples of solvents used in the present invention include various buffer solutions, such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, but the solvents contained in the reagent according to this embodiment are not limited to these.

[0037] The in vitro diagnostic test kit of this embodiment includes the test reagent of this embodiment and a housing containing the test reagent. The test kit of this embodiment preferably further includes a reaction buffer solution containing albumin (hereinafter, Reagent 2) in addition to the test reagent of this embodiment (hereinafter, Reagent 1). Examples of the albumin include serum albumin, which may be protease-treated. The amount of albumin contained in Reagent 2 is approximately 0.001% to 5% by mass, although the test kit of this embodiment is not limited to this amount. A sensitizer for latex immunoagglutination assays may be contained in either or both of Reagent 1 and Reagent 2. Examples of sensitizers for latex immunoagglutination assays include, but are not limited to, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyalginic acid, etc. A surfactant may be contained in either or both of Reagent 1 and Reagent 2. Surfactants have the effect of stabilizing particles and proteins; for example, polyoxyethylene sorbitan monolaurate or poly(oxyethylene) octylphenyl ether is preferably used. Furthermore, the test kit in this embodiment may include a positive control, a negative control, a serum dilution solution, etc. in addition to Reagent 1 and Reagent 2. As the medium for the positive control and the negative control, serum not containing a measurable target substance, physiological saline, or a solvent may be used.

[0038] (Detection method) The method for detecting a target substance in a specimen by latex immunoagglutination of this embodiment is characterized by mixing the particles for anti-HBs antibody detection immunoassay of this embodiment with a specimen that may contain the target substance. Furthermore, mixing of the particles for anti-HBs antibody detection immunoassay of this embodiment with the specimen is preferably performed at a pH range of 3.0 to 11.0. The mixing temperature is in the range of 20°C to 50°C, and the mixing time is in the range of 10 seconds to 30 minutes. In this detection method of this embodiment, the concentration of the particles for anti-HBs antibody detection immunoassay of this embodiment in the reaction system is preferably 0.001% to 5% by mass, more preferably 0.01% to 1% by mass. The detection method of this embodiment optically detects an agglutination reaction that occurs as a result of mixing the particles for anti-HBs antibody detection immunoassay of this embodiment with the specimen. Specifically, optical detection of the agglutination reaction allows the target substance in the specimen to be detected and the concentration of the target substance to be measured. The agglutination reaction can be optically detected by measuring the changes in scattered light intensity, transmitted light intensity, absorbance, etc. using an optical device capable of detecting these values.

[0039] (Other immunoassay reagents) The particles produced by the manufacturing method according to the embodiment of the present invention can also be used in immunoassay reagents other than latex immunoagglutination assays, including, but not limited to, immunochromatography, chemiluminescent enzyme immunoassay, electrochemiluminescent immunoassay, and enzyme immunoassay (including competitive assays). [Example]

[0040] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0041] Example 1: Synthesis of particles A 2L four-neck separable flask was charged with 12.0 g of styrene (St: Kishida Chemical Industry Co., Ltd.), 17.9 g of glycidyl methacrylate (GMA: Tokyo Chemical Industry Co., Ltd.), 0.45 g of divinylbenzene (DVB: Kishida Chemical Industry Co., Ltd.), and 2168.6 g of ion-exchanged water to prepare a mixture. The mixture was stirred at 200 rpm and maintained at 70 °C. A nitrogen flow was introduced at a flow rate of 200 ml / min to deoxygenate the four-neck separable flask. Next, a separately prepared solution of 1.13 g of V-50 (Fujifilm Wako Pure Chemical Industries Co., Ltd.) dissolved in 30 g of ion-exchanged water was added to the mixture to initiate soap-free emulsion polymerization. Two hours after the start of polymerization, 3.1 g of GMA was added to the four-neck separable flask, and the mixture was maintained at 70 °C with stirring at 200 rpm for an additional 22 hours, yielding an aqueous dispersion containing granular copolymer A. After the dispersion was slowly cooled to room temperature, a portion was sampled and the polymerization conversion was evaluated using proton NMR, gas chromatography, and gel permeation chromatography, confirming that it was essentially 100%. The dry particle size of granular copolymer A was 151.4 nm, and the particle size in water was 160.2 nm. Granular copolymer A was concentrated by ultrafiltration or diluted with ion-exchanged water to give a 2.5 w / v% aqueous dispersion, and stored at 4°C under light-shielded conditions. Next, 24 g of a 2.5 w / v% aqueous dispersion of granular copolymer A, 3.3 g of ion-exchanged water, 40 mg (0.26 mmol) of mercaptosuccinic acid (Fujifilm Wako Pure Chemical Corporation), and 0.214 mL (2.34 mmol) of 3-mercapto-1,2-propanediol (Fujifilm Wako Pure Chemical Corporation) were weighed into a 100 mL round-bottom flask, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. The contents of the round-bottom flask were then heated to 70 °C while stirring at 200 rpm and held at this temperature for an additional 18 hours to obtain a particle dispersion. The particles were separated from the dispersion using a centrifuge and redispersed in ion-exchanged water eight times to purify the particles. The aqueous dispersion was then adjusted to a particle concentration of 1.0 w / v% and stored at 4 °C in the dark.

[0042] (Example 2: HBs antigen sensitization) 0.1 mL (1 mg of particles) of the particle dispersion liquid (1.0% by mass solution, 10 mg / mL) prepared in Example 1 was transferred to each microtube (1.5 mL capacity). 0.12 mL of activation buffer (25 mM MES buffer, pH 6.0) was added to each tube, and the mixture was centrifuged at 4°C and 15,000 rpm (20,400 g) for 5 minutes. After centrifugation, the supernatant was discarded using a pipettor. Next, 0.12 mL of activation buffer was added, and the mixture was dispersed ultrasonically using an ultrasonic cleaner (product name: MODEL VS-100III AS ONE 3-frequency ultrasonic cleaner, AS ONE Corporation, 28 kHz). Next, the mixture was centrifuged at 4°C and 15,000 rpm (20,400 g) for 5 minutes. The supernatant was discarded using a pipettor, and 0.12 mL of activation buffer was added, followed by ultrasonic dispersion. The mixture was then centrifuged at 15,000 rpm (20,400 g) for 5 minutes at 4°C, and the supernatant was discarded using a pipettor. Next, 60 μL each of WSC solution (50 mg of WSC dissolved in 1 mL of activation buffer) and N-hydroxysulfosuccinimide (Sulfo NHS) solution (50 mg of Sulfo NHS dissolved in 1 mL of activation buffer) was added. After addition, the mixture was dispersed ultrasonically. The particles were further stirred at room temperature for 30 minutes to convert the carboxyl groups to active esters.

[0043] Next, the dispersion was centrifuged at 15,000 rpm (20,400 g) for 5 minutes at 4°C, and the supernatant was discarded using a pipettor. 0.2 mL of immobilization buffer (25 mM MES buffer, pH 4.0) was added, and the mixture was dispersed by ultrasonication. The mixture was centrifuged at 15,000 rpm (20,400 g) for 5 minutes at 4°C, and the supernatant was discarded using a pipettor. 50 μL of immobilization buffer was added per 1 mg of particles, and the carboxyl-activated particles were dispersed by ultrasonication. HBs antigen (manufactured by Specialized Immunology Research Institute) was diluted with immobilization buffer to 100 μg / 50 μL (hereinafter referred to as the HBs antigen solution). 50 μL of the HBs antigen solution was added to 50 μL of the carboxyl-activated particle solution (containing 1 mg of particles), and the particles were dispersed using ultrasound. The amount of HBs antigen added was 100 μg per 1 mg of particles (100 μg / mg). The tube was stirred at 23°C for 210 minutes to immobilize the antigen to the carboxyl groups of the particles. The mixture was then centrifuged at 4°C for 5 minutes at 15,000 rpm (20,400 g), and the supernatant was discarded using a pipettor. 0.24 mL of an active ester inactivation buffer containing tris(hydroxymethyl)aminomethane (Tris) (1 M Tris buffer, pH 8.0 containing 0.1% Tween (registered trademark) 20) was added, and the mixture was dispersed by ultrasonic waves. After stirring at room temperature for 1 hour to allow Tris to bind to the remaining activated esters, the mixture was left standing at 4°C overnight. The mixture was then centrifuged at 15,000 rpm (20,400 g) for 5 minutes at 4°C, and the supernatant was discarded using a pipettor. 0.2 mL of washing and storage buffer (10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer, pH 7.9) was added, and the mixture was dispersed by ultrasonication. After repeating the washing procedure twice with 0.2 mL of washing and storage buffer, 1.0 mL of washing and storage buffer was added, and the mixture was dispersed by ultrasonication. Since there was almost no particle loss during the above process, the final concentration of HBs antigen-sensitized particles was 0.6% by mass (6 mg / mL). The mixture was stored in a refrigerator, and redispersed by ultrasonication before use.

[0044] (Example 3: Treatment with surfactant, etc.) 0.166 mL (1 mg) of the HBs antigen-sensitized particles prepared in Example 2 was mixed with 0.05 mL of 10% dodecyl-β-D-maltoside aqueous solution, or 0.05 mL of 10% Triton (registered trademark) X-100 aqueous solution, or 0.05 mL of 10% 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) aqueous solution, and 0.034 mL of 10 mM HEPES buffer (pH 7.9), and the mixture was shaken for 22 hours at room temperature (20°C to 25°C) using a microtube mixer MT-400 (manufactured by Tomy Seiko) at scale 5 to obtain a solution containing washed HBs antigen-sensitized particles. After the reaction, the particle solution was washed three times with 0.25 mL of 10 mM HEPES buffer (pH 7.9) and finally redispersed in 0.33 mL of 10 mM HEPES buffer (pH 7.9) containing 0.01% Tween (registered trademark) 20 to obtain a solution containing particles for immunoassays for detecting anti-HBs antibodies.

[0045] (Example 4: Treatment with an acidic aqueous solution) 0.166 mL (1 mg) of the HBs antigen-sensitized particles prepared in Example 2 was mixed with 0.084 mL of 1 M hydrochloric acid aqueous solution, and the mixture was shaken for 22 hours at room temperature (20°C to 25°C) using a microtube mixer MT-400 (Tomy Seiko Co., Ltd.) at scale 5 to obtain a solution containing washed HBs antigen-sensitized particles. After the reaction, the particle solution was washed three times with 0.25 mL of 10 mM HEPES buffer (pH 7.9) and finally redispersed in 0.33 mL of 10 mM HEPES buffer (pH 7.9) containing 0.01% Tween (registered trademark) 20 to obtain a solution containing particles for immunoassays for detecting anti-HBs antibodies.

[0046] (Comparative Example 1) Particles for immunoassays for detecting anti-HBs antibodies were prepared by surfactant treatment in the same manner as in Example 3, except that 0.05 mL of a 10% aqueous solution of sodium N-dodecanoyl sarcosinate or 0.05 mL of a 10% aqueous solution of dodecyltrimethylammonium chloride was used instead of the various surfactants used in Example 3.

[0047] (Comparative Example 2) Particles for immunoassays for detecting anti-HBs antibodies were prepared in the same manner as in Example 4, except that 0.084 mL of 1 M sodium hydroxide aqueous solution, 0.084 mL of 0.1 M dithiothreitol aqueous solution, or 0.84 mL of 10 mM HEPES buffer (pH 7.9) was used instead of the hydrochloric acid aqueous solution used in Example 4. The particle size of the obtained particles for immunoassays to detect anti-HBs antibodies was evaluated by dynamic light scattering (DLS). A Zetasizer Nano ZS (Malvern Instruments) was used for the measurement. The measured volume-average particle size and polydispersity index are summarized in Table 1. Protein quantification confirmed that HBs antigen had been sensitized (immobilized) to the particles. Specifically, this method involves reacting particles for anti-HBs antibody detection immunoassays with BCA reagents. First, 25 μL (25 μg particle amount) of a dispersion solution (0.1% solution) of particles for anti-HBs antibody detection immunoassays was taken. 7 mL of solution A and 140 μL of solution B from the Protein Assay BCA Kit (Wako Pure Chemical Industries) were mixed to make solution AB. 200 μL of solution AB was added to the particle solution (25 μL) and incubated at 60°C for 30 minutes. The solution was centrifuged at 15,000 rpm (20,400 g) for 5 min at 4°C, and 200 μL of the supernatant was collected using a pipettor. The absorbance at 562 nm was measured using a multimode microplate reader (SynergyMX, BioTek) along with standard samples (HBs antigen in 10 mM HEPES at several concentrations ranging from 0 to 200 μg / mL). The amount of HBs antigen was calculated from the standard curve. The amount of HBs antigen sensitized to the particles (HBs antigen binding amount per particle weight (HBs antigen immobilization amount) (μg / mg)) was calculated by dividing the calculated amount of HBs antigen by the particle weight (0.025 mg in this case). The measured amounts of HBs antigen sensitized are summarized in Table 1.

[0048] [Table 1]

[0049] (Example 5) Evaluation of sensitivity to anti-HBs antibodies The sensitivity of the particles for use in immunoassays for detecting anti-HBs antibodies according to the present invention was evaluated by a latex immunoagglutination method. Specifically, the particles for use in immunoassays for detecting anti-HBs antibodies were reacted with anti-HBs antibodies to form aggregates of immune complexes. The aggregates were then irradiated with light, and the attenuation of the irradiated light due to scattering (absorbance) was measured using an absorption spectrometer. The proportion of aggregates increases depending on the amount of antigen contained in the sample, resulting in an increase in absorbance. In evaluating sensitivity, a large increase in absorbance (expressed as ΔODx10,000) at a given anti-HBs antibody concentration is desirable. Absorbance was measured using a UV-visible spectrophotometer (Biospetrometer Kinetic (Eppendorf)), with the sample poured into a plastic cuvette and measured at an optical path length of 2 mm. The specific measurement method is described below.

[0050] Specifically, 1 μL of anti-HBs mouse antibody solution (anti-HBs mouse antibody concentration: 0 mg / mL or 0.1 mg / mL) was used as a sample. This sample was mixed with 60 μL of dilution buffer (PBS, 0.01% Tween® 20) in a plastic cuvette and heated at 37°C for 5 minutes. 30 μL of a dispersion solution of particles for anti-HBs antibody detection immunoassay (particle concentration: 0.3 wt%, 10 mM HEPES, pH 7.9, 0.01 wt% Tween® 20) was added to 61 μL of dilution buffer containing anti-HBs mouse antibody and quickly pipetted, taking care to avoid introducing air bubbles, to prepare a sample. The absorbance of the sample at 572 nm was measured and designated Abs1. After heating the sample at 37°C for 5 minutes, the absorbance of the sample at 572 nm was measured and designated Abs2. The value obtained by subtracting Abs1 from Abs2 was multiplied by 10,000 to obtain the ΔOD x 10,000 value.

[0051] The results are shown in Table 2. The particles for anti-HBs antibody detection immunoassays of this example showed an increase in ΔODx10,000 in the presence of anti-HBs mouse antibodies. This was the result of the particles for anti-HBs antibody detection immunoassays binding to the anti-HBs mouse antibodies to form particle aggregates, demonstrating their function as particles for use in latex immunoagglutination. Furthermore, the particles for anti-HBs antibody detection immunoassays produced in Examples 3 and 4 were found to have a larger ΔODx10,000 value than the particles for anti-HBs antibody detection immunoassays produced in Comparative Examples 1 and 2, demonstrating the effectiveness of the production method of the present invention.

[0052] [Table 2]

[0053] As can be seen from Table 1, the particle size of the particles for anti-HBs antibody detection immunoassays treated with a positive surfactant (dodecyltrimethylammonium chloride) was larger. This is thought to be because the negatively charged particle surface interacted with the positively charged positive surfactant, making the particles hydrophobic, resulting in aggregation due to hydrophobic interactions. The particle size and ΔODx10,000 value of particles for anti-HBs antibody detection immunoassays treated with a negative surfactant (sodium N-dodecanoyl sarcosinate) were not different from those of the reference (treated with 10 mM HEPES buffer (pH 7.9)). This is thought to be because the particle surface is negatively charged, making it difficult for the surfactant to approach, and the reaction to remove impurities did not proceed.

[0054] For the above reasons, it is believed that the effects of the present invention could not be obtained when the positive surfactant and negative surfactant were mixed with the HBs antigen-sensitized particles. On the other hand, the nonionic surfactants and amphoteric surfactants resulted in slightly smaller particle sizes compared to the reference (Table 1) and larger ΔODx10,000 values (Table 2). These surfactants are thought to have improved sensitivity by reacting favorably with the HBs antigen-sensitized particles sensitized on the particle surface and removing impurities.

[0055] Table 1 shows that the amount of sensitization of particles for immunoassays to detect anti-HBs antibodies treated with an alkaline aqueous solution (sodium hydroxide) was reduced. This is thought to be because a portion of the HBs antigen bound to the particles was dissociated by prolonged reaction with a high concentration of alkaline aqueous solution. As a result, the ΔODx10,000 value dropped significantly, making it difficult to detect the presence of anti-HBs mouse antibodies. One possible reason for this is that the HBs antigen was denatured by the alkaline aqueous solution.

[0056] As can be seen from Table 1, particles for immunoassays for detecting anti-HBs antibodies treated with a reducing agent (dithiothreitol) showed a slight increase in particle size and a significant decrease in the ΔODx10,000 value, making it difficult to detect the presence of anti-HBs mouse antibodies. This is thought to be because the reducing agent destroyed the structure around antigenic determinant a, the main conformational epitope of HBs antigen, preventing the binding of anti-HBs mouse antibodies. On the other hand, particles for anti-HBs antibody detection immunoassays treated with an acidic aqueous solution (hydrochloric acid) had a slightly smaller particle size than the reference (Table 1) and a larger ΔODx10,000 value (Table 2). Unlike alkaline aqueous solutions or reducing agents, the acidic aqueous solution reacted favorably with the HBs antigen sensitized on the particle surface and had the effect of removing impurities, which is thought to have improved sensitivity.

[0057] (Example 6) Evaluation of sensitivity to anti-HBs antibodies using a practical device Next, the sensitivity of the particles for immunoassays for detecting anti-HBs antibodies that had been reacted with 10% CHAPSO aqueous solution prepared in Example 3, and the particles for immunoassays for detecting anti-HBs antibodies (reference) that had been mixed with 10 mM HEPES buffer (pH 7.9) prepared in Comparative Example 2, was evaluated by latex immunoagglutination using a clinical testing device, TBA-120FR (Canon Medical Corporation). The anti-HBs antibody concentration in the serum of a human anti-HBs antibody-positive patient was measured in advance, and then the serum was diluted with human anti-HBs antibody-negative serum to achieve anti-HBs antibody concentrations of 0, 200, and 1000 mIU / mL. The solution mixing conditions for measurement were 35 μL of each of the aforementioned serum samples, 25 μL of a sample dilution buffer solution (PBS, 0.01% Tween® 20, 0.5% PEG500K), and 20 μL of a dispersion solution of particles for immunoassays for detecting anti-HBs antibodies (particle concentration 0.3 wt%, 10 mM HEPES, pH 7.9, 0.01 wt% Tween® 20). The absorbance at 572 nm was measured according to a standard measurement protocol. Specifically, the mixture of serum and sample dilution buffer solution was heated at 37°C for 5 minutes, after which the dispersion solution of particles for immunoassays for detecting anti-HBs antibodies was added, and the absorbance at 572 nm was measured immediately after the addition and 5 minutes after the addition. The absorbance immediately after addition was subtracted from the absorbance after 5 minutes, and the resulting value was multiplied by 10,000 to obtain the ΔOD x 10,000 value.

[0058] The results are shown in Figure 1. The anti-HBs antibody detection immunoassay particles reacted with CHAPSO aqueous solution had a higher ΔODx10,000 value than the anti-HBs antibody detection immunoassay particles (reference) mixed with 10 mM HEPES buffer. Furthermore, there was no significant difference in the ΔODx10,000 value between the anti-HBs antigen antibody concentrations of 0 mIU / mL and 200 mIU / mL for the reference anti-HBs antibody detection immunoassay particles. On the other hand, the anti-HBs antibody detection immunoassay particles reacted with CHAPSO aqueous solution had a significantly higher ΔODx10,000 value at the anti-HBs antibody concentration of 200 mIU / mL compared to 0 mIU / mL, demonstrating the ability to distinguish between 0 mIU / mL and 200 mIU / mL. In other words, the manufacturing method of the present invention enables the measurement of lower concentrations of anti-HBs antibody. This is thought to be due to the CHAPSO aqueous solution reacting favorably with the HBs antigen-sensitized particles on the particle surface, thereby removing impurities, resulting in improved sensitivity.

[0059] (Comparative Example 3) 0.125 mL of a 1 mg / mL aqueous solution of HBs antigen, 0.1 mL of a 10% aqueous solution of dodecyl-β-D-maltoside, and 0.275 mL of PBS were mixed and shaken at 25°C for 24 hours. The solution was then purified and concentrated by dialysis and an Amicon Ultra (Merck) centrifugal ultrafiltration filter. The resulting HBs antigen solution was used to sensitize particles in the same manner as in Example 2. The sensitization level was only 33 μg / mg, indicating a low sensitization efficiency. This is thought to be due to residual surfactant in the HBs antigen solution. Even after thorough dialysis and purification, the small amount of surfactant remaining is thought to inhibit the sensitization reaction (binding reaction) with the particles. Furthermore, when these particles for immunoassay for detecting anti-HBs antibodies were used to evaluate their sensitivity to anti-HBs antibodies by the method described in Example 5, the ΔODx10000 value was 60 even at an anti-HBs mouse antibody concentration of 1 mg / mL. From the above, it can be said that treatment with a surfactant or the like before covalent bonding to the particles does not contribute to improving the sensitivity of anti-HBs antibody detection.

[0060] Example 7: Synthesis of polystyrene particles having siloxane A 200 mL flask was charged with 90 g of phosphate buffer solution (Kishida Chemical Co., Ltd., pH 7.4), and 0.9 g of polyvinylpyrrolidone K-30 (Kishida Chemical Co., Ltd., molecular weight 40,000) was dissolved therein. Next, 3.0 g of 3-methacryloxypropyltrimethoxysilane (product name: LS-3380, Shin-Etsu Chemical Co., Ltd.) and 9.0 g of styrene (Kishida Chemical Co., Ltd.) were added, and the mixture was stirred at room temperature for 10 minutes while blowing in nitrogen. The emulsion in the flask was then heated to 70°C in an oil bath. A solution of 0.3 g of potassium peroxodisulfate (Wako Pure Chemical Industries, Ltd.) in 15 mL of phosphate buffer solution (Kishida Chemical Co., Ltd., pH 7.4) was added to the emulsion heated to 70°C. The mixture was stirred at 70°C for 7 hours and then returned to room temperature, yielding a dispersion of polystyrene-silica hybrid microparticles. To the resulting dispersion of polystyrene-silica hybrid microparticles, 2 g of Tween 20 (Tokyo Chemical Industry Co., Ltd.) was added as a surfactant, and 0.1 mL of X-12-1135 (Shin-Etsu Chemical Co., Ltd.), a silane coupling agent with a carboxyl group, was added and stirred at room temperature for 14 hours to obtain carboxylated polystyrene-silica hybrid microparticles.

[0061] (Example 8: Sensitization with HBs antigen and treatment with surfactant, etc.) In the same manner as in Examples 2 to 4, the carboxylated polystyrene-silica hybrid microparticles were sensitized with HBs antigen and treated with a surfactant or the like.

[0062] (Comparative Example 3) Carboxylated polystyrene-silica hybrid microparticles sensitized with HBs antigen were treated in the same manner as in Example 8, and particles for immunoassays for detecting anti-HBs antibodies were prepared in the same manner as in Example 8, except that 0.84 mL of 10 mM HEPES buffer (pH 7.9) was used instead of the surfactants and the like used in Example 8.

[0063] The physical properties of the particles for immunoassay for detecting anti-HBs antibodies prepared in Example 8 and Comparative Example 3 are summarized in Table 3.

[0064] [Table 3]

[0065] Example 9: Evaluation of sensitivity to anti-HBs antibodies The sensitivity to anti-HBs antibodies was evaluated using the same method as described in Example 5. The results are summarized in Table 4.

[0066] [Table 4]

[0067] Particles for anti-HBs antibody detection immunoassays treated with a nonionic surfactant, an amphoteric surfactant, or an acidic aqueous solution (hydrochloric acid) had a larger ΔODx10,000 value compared to the reference (Table 4). These surfactants and acidic aqueous solutions are thought to have improved sensitivity by reacting favorably with the HBs antigen-sensitized particles on the particle surface and removing impurities.

[0068] The disclosure of this embodiment includes the following methods and configurations. [Method 1] A method for producing particles for immunoassays to detect anti-HBs antibodies, comprising: a first step of covalently binding HBs antigen to particles to obtain HBs antigen-sensitized particles; and a second step of washing the HBs antigen-sensitized particles with a nonionic surfactant or an amphoteric surfactant to obtain washed HBs antigen-sensitized particles. [Method 2] A method for producing particles for immunoassays for detecting anti-HBs antibodies, comprising: a first step of covalently binding HBs antigen to particles to obtain HBs antigen-sensitized particles; and a third step of washing the HBs antigen-sensitized particles with an acidic aqueous solution to obtain washed HBs antigen-sensitized particles. [Method 3] The production method according to Method 1 or 2, further comprising a fourth step of replacing the dispersion of the washed HBs antigen-sensitized particles with a buffer solution. [Method 4] 4. The method according to any one of methods 1 to 3, wherein the covalent bond is an amide bond between an amino group of the HBs antigen and a carboxy group of the particle. [Method 5] 5. The method of claim 4, wherein the particles are styrene-glycidyl (meth)acrylate copolymers in which one or more glycidyl groups are ring-opened with mercaptosuccinic acid and 3-mercapto-1,2-propanediol. [Method 6] 5. The method of claim 4, wherein the particles are styrene-3-methacryloxypropyltrimethoxysilane copolymers and the particles contain polyvinylpyrrolidone. [Method 7] The method according to any one of methods 1 and 3 to 5, wherein the nonionic surfactant is one or more selected from the group consisting of dodecyl-β-D-maltoside and Triton X-100, or the amphoteric surfactant is selected from CHAPSO. [Method 8] 7. The production method according to any one of Methods 2 to 6, wherein the acidic aqueous solution is one or more selected from the group consisting of an aqueous hydrochloric acid solution, an aqueous acetic acid solution, and an aqueous glycine hydrochloride solution. [Configuration 1] Particles for immunoassays for detecting anti-HBs antibodies, which are produced by a manufacturing method comprising the steps of: a first step of covalently binding HBs antigen to particles to obtain antigen-sensitized particles; and a second step of washing the antigen-sensitized particles with a nonionic surfactant or an amphoteric surfactant to obtain washed HBs antigen-sensitized particles. [Configuration 2] Particles for immunoassays for detecting anti-HBs antibodies, which are produced by a manufacturing method comprising the steps of: a first step of covalently bonding HBs antigen to particles to obtain antigen-sensitized particles; and a third step of washing the antigen-sensitized particles with an acidic aqueous solution to obtain washed HBs antigen-sensitized particles. [Configuration 3] 3. Particles for immunoassay for detecting anti-HBs antibody according to configuration 1 or 2, wherein the production method further comprises a fourth step of replacing the dispersion of the washed HBs antigen-sensitized particles with a buffer solution. [Configuration 4] 4. The particles for immunoassay for detecting anti-HBs antibody according to any one of configurations 1 to 3, wherein the covalent bond is an amide bond between an amino group of the HBs antigen and a carboxy group of the particle. [Configuration 5] 5. Particles for immunoassays for detecting anti-HBs antibodies according to claim 4, wherein the particles are styrene-glycidyl (meth)acrylate copolymers in which one or more glycidyl groups are ring-opened with mercaptosuccinic acid and 3-mercapto-1,2-propanediol. [Configuration 6] 5. Particles for immunoassay for detecting anti-HBs antibodies according to configuration 4, wherein the particles are styrene-3-methacryloxypropyltrimethoxysilane copolymers and contain polyvinylpyrrolidone. [Configuration 7] 6. The particles for immunoassay for detecting anti-HBs antibodies according to any one of configurations 1 and 3 to 5, wherein the nonionic surfactant is one or more selected from the group consisting of dodecyl-β-D-maltoside and Triton X-100, or the amphoteric surfactant is selected from CHAPSO. [Configuration 8] 7. Particles for an immunoassay for detecting anti-HBs antibodies according to any one of configurations 2 to 6, wherein the acidic aqueous solution is at least one selected from the group consisting of an aqueous hydrochloric acid solution, an aqueous acetic acid solution, and an aqueous glycine hydrochloride solution.

Claims

1. A method for producing particles for immunoassays for detecting anti-HBs antibodies, comprising: a first step of covalently bonding HBs antigen to particles to obtain HBs antigen-sensitized particles; and a second step of washing the HBs antigen-sensitized particles with a nonionic surfactant or an amphoteric surfactant to obtain washed HBs antigen-sensitized particles.

2. A method for producing particles for immunoassays for detecting anti-HBs antibodies, comprising: a first step of covalently bonding HBs antigen to particles to obtain HBs antigen-sensitized particles; and a third step of washing the HBs antigen-sensitized particles with an acidic aqueous solution to obtain washed HBs antigen-sensitized particles.

3. The method according to claim 1, further comprising a fourth step of replacing the dispersion of the washed HBs antigen-sensitized particles with a buffer solution.

4. The method according to claim 2, further comprising a fourth step of replacing the dispersion of the washed HBs antigen-sensitized particles with a buffer solution.

5. The method of claim 1 , wherein the covalent bond is an amide bond between an amino group of the HBs antigen and a carboxy group of the particle.

6. The method of claim 2, wherein the covalent bond is an amide bond between an amino group of the HBs antigen and a carboxy group of the particle.

7. The method of claim 5, wherein the particles are styrene-glycidyl (meth)acrylate copolymers in which one or more glycidyl groups are ring-opened with mercaptosuccinic acid and 3-mercapto-1,2-propanediol.

8. The method of claim 6, wherein the particles are styrene-glycidyl (meth)acrylate copolymers in which one or more glycidyl groups are ring-opened with mercaptosuccinic acid and 3-mercapto-1,2-propanediol.

9. The method of claim 5, wherein the particles are styrene-3-methacryloxypropyltrimethoxysilane copolymer and the particles contain polyvinylpyrrolidone.

10. 7. The method of claim 6, wherein the particles are styrene-3-methacryloxypropyltrimethoxysilane copolymer and the particles contain polyvinylpyrrolidone.

11. The method according to any one of claims 1, 3, 5, 7, and 9, wherein the nonionic surfactant is one or more selected from the group consisting of dodecyl-β-D-maltoside and Triton X-100, or the amphoteric surfactant is selected from CHAPSO.

12. 11. The method according to claim 2, wherein the acidic aqueous solution is at least one selected from the group consisting of an aqueous hydrochloric acid solution, an aqueous acetic acid solution, and an aqueous glycine hydrochloride solution.

13. Particles for immunoassays for detecting HBs antibodies, which are produced by a manufacturing method comprising: a first step of covalently bonding HBs antigen to particles to obtain antigen-sensitized particles; and a second step of washing the antigen-sensitized particles with a nonionic surfactant or an amphoteric surfactant to obtain washed HBs antigen-sensitized particles.

14. Particles for immunoassays for detecting HBs antibodies, which are produced by a manufacturing method comprising: a first step of covalently bonding HBs antigen to particles to obtain antigen-sensitized particles; and a third step of washing the antigen-sensitized particles with an acidic aqueous solution to obtain washed HBs antigen-sensitized particles.

15. 14. The particles for immunoassay for detecting anti-HBs antibodies according to claim 13, wherein the production method further comprises a fourth step of replacing the dispersion of the washed HBs antigen-sensitized particles with a buffer solution.

16. 15. The particles for immunoassay for detecting anti-HBs antibody according to claim 14, wherein the production method further comprises a fourth step of replacing the dispersion of the washed HBs antigen-sensitized particles with a buffer solution.

17. 14. The particle for an immunoassay for detecting anti-HBs antibody according to claim 13, wherein the covalent bond is an amide bond between an amino group of the HBs antigen and a carboxy group of the particle.

18. 15. The particle for immunoassay for detecting anti-HBs antibody according to claim 14, wherein the covalent bond is an amide bond between an amino group of the HBs antigen and a carboxy group of the particle.

19. 18. The particle for an immunoassay for detecting anti-HBs antibodies according to claim 17, wherein the particle is a styrene-glycidyl (meth)acrylate copolymer in which one or more glycidyl groups are ring-opened with mercaptosuccinic acid and 3-mercapto-1,2-propanediol.

20. 19. The particles for an immunoassay for detecting anti-HBs antibodies according to claim 18, wherein the particles are styrene-glycidyl (meth)acrylate copolymers in which one or more glycidyl groups are ring-opened with mercaptosuccinic acid and 3-mercapto-1,2-propanediol.

21. 18. Particles for an immunoassay for detecting anti-HBs antibodies according to claim 17, wherein the particles are styrene-3-methacryloxypropyltrimethoxysilane copolymers and contain polyvinylpyrrolidone.

22. 19. Particles for an immunoassay for detecting anti-HBs antibodies according to claim 18, wherein the particles are styrene-3-methacryloxypropyltrimethoxysilane copolymers and contain polyvinylpyrrolidone.

23. 22. The particle for immunoassay for detecting anti-HBs antibodies according to claim 13, 15, 17, 19, or 21, wherein the nonionic surfactant is one or more selected from the group consisting of dodecyl-β-D-maltoside and Triton X-100, or the amphoteric surfactant is selected from CHAPSO.

24. 23. The particles for immunoassay for detecting anti-HBs antibodies according to claim 14, 16, 18, 20, or 22, wherein the acidic aqueous solution is at least one selected from the group consisting of an aqueous hydrochloric acid solution, an aqueous acetic acid solution, and an aqueous glycine hydrochloride solution.

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