JP2021032702A - Particles for inspecting sample and method for manufacturing the same - Google Patents

Abstract

To provide particles with a high efficiency of sensibilization of a ligand, the particles having a small nonspecific adsorption property, being used for a method for condensation, and having a reactive functional group for fixing a ligand, to provide ligand sensibilization particles used for a method for condensation formed by a chemical bonding of an antigen or an antibody as a ligand, and to provide a method for detecting a target material.SOLUTION: The present invention relates to particles for a method for condensation including a copolymer having a unit derived from a styrene monomer and a unit derived from a glycidyl group-containing monomer. The copolymer is particles having a partial structure shown by the following formula (1). In the formula (1), the counter ions R+ of a carboxyl group are organic amine and * denotes a part of the copolymer that combines with the unit derived from the glycidyl group-containing monomer.SELECTED DRAWING: None

Description

The present invention relates to particles for sample testing for the agglutination method and a method for producing the same.

Among the agglutination methods, the latex immunoagglutination method is used as a method for measuring a substance to be measured in a biological sample (hereinafter referred to as "specimen") in the clinical laboratory field. In this method, a dispersion of particles obtained by chemically immobilizing an antibody or an antigen as a ligand (hereinafter referred to as "ligand-sensitized particles", "antibody-sensitized particles", or "antigen-sensitized particles") and a measurement target. Mix with a sample that may contain the substance (antigen or antibody). At that time, if the substance to be measured (antibody or antigen) is contained in the sample, the sensitized particles cause an agglutination reaction, and this agglutination reaction is used as the amount of change in scattered light intensity, transmitted light intensity, absorbance, etc. The presence or absence of the substance to be measured can be specified or quantified by optically detecting it.

For particles used in the latex immunoagglutination method, improvement of detection sensitivity and suppression of non-specific reaction are issues. To improve the detection sensitivity, it has been proposed to increase the amount of ligand sensitization of the particles (ligand sensitization means immobilization of the ligand on the particles. Therefore, the ligand-sensitized particles immobilized the ligand. Means particles). For example, in Patent Document 1, the amount of antibody sensitization is increased by increasing the specific surface area of the particles. As the amount of antibody sensitization increases, the amount of antigen that can be detected increases. To suppress non-specific reactions, Patent Document 2 discloses a method of post-coating antibody-sensitized particles with albumin or a hydrophilic polymer in order to avoid adsorption of contaminants in a sample to particles. Particles with an increased antibody sensitization amount in Patent Document 1 are also post-coated with albumin to suppress non-specific reactions.

By the way, in recent years, studies have been widely conducted to purify or quantify a target substance by using an affinity particle in which a ligand having an affinity for the target substance and the particle are chemically fixed. As the particles used for such a purpose, for example, in Patent Document 3, resin particles whose surfaces are coated with polyglycidyl methacrylate by emulsion polymerization using both styrene and glycidyl methacrylate monomers (hereinafter referred to as “SG particles”). .) Is disclosed. In the SG particles, an epoxy group derived from polyglycidyl methacrylate is partially ring-opened to form a glycol, and due to the hydrophilicity of the glycol, non-specific adsorption to particles such as proteins is suppressed. When the ligand is chemically immobilized on the surface of SG particles, the epoxy group derived from polyglycidyl methacrylate can be used as it is. However, usually, a method of chemically reacting this reactive functional group with a ligand after undergoing a step of converting an epoxy group into another reactive functional group such as a carboxy group, an amino group, or a thiol group is common. .. Among them, particles obtained by converting an epoxy group of SG particles into a carboxy group via an appropriate spacer are the most versatile and preferable forms for chemically immobilizing a ligand on the particle surface.

Japanese Unexamined Patent Publication No. 2006-266970 International Publication No. 2017/138608 Japanese Unexamined Patent Publication No. 2003-26698

However, with the particles described in these documents, it has been difficult to achieve both improvement in detection sensitivity and suppression of non-specific reactions. That is, although the sensitivity can be improved by increasing the amount of ligand sensitized to the particles, post-coating is required to suppress the non-specific reaction. However, the amount of ligand sensitization to the particles is limited in order to secure the post-coat area. Further, although postcoating is effective for hydrophilization of the particle surface, since it is a temporary surface modification based on physical adsorption, particles that do not require postcoating and suppress non-specific reactions are desired.

On the other hand, Patent Document 3 discloses a spacer on the surface of the particle for fixing the ligand to the particle. However, since Patent Document 3 mainly aims to isolate and purify the target protein by immobilizing a ligand of a low molecular weight compound on the particles, the spacer on the particle surface requires a length of a certain size, and the spacer itself is also hydrophilic. Since it is essential to have sex, there is no specific description of the optimal spacer structure for the latex immunoaggregation method.

The problem to be solved by the present invention is to achieve both improvement of detection sensitivity and suppression of non-specific reaction of particles for agglutination method. Specifically, the present invention provides particles having a small non-specific adsorptivity and having a reactive functional group for immobilizing a ligand, and having a high sensitization efficiency of the ligand, and a method for producing the same. Another object of the present invention is to provide a method for detecting a sensitized particle for an agglutination method in which an antigen or a ligand is chemically immobilized as a ligand, and a target substance.

As a result of searching for particles having a small non-specific adsorptivity and having a reactive functional group for immobilizing a ligand as particles for the aggregation method, the present inventors have found SG particles having a short spacer containing a carboxy group. It was clarified that it shows high ligand sensitization efficiency and can suppress non-specific reactions.

That is, the present invention is particles for the aggregation method containing a copolymer having a unit derived from a styrene-based monomer and a unit derived from a glycidyl group-containing monomer, and the copolymer is represented by the following formula (1). It is a particle having the partial structure shown. In the formula (1), the counter ion R ⁺ of the carboxy group is an organic amine, and * indicates a portion of the copolymer that binds to a unit derived from the glycidyl group-containing monomer.

式（１）中、カルボキシ基のカウンターイオンＲ^＋は水素イオン、または有機アミンであり、式（１）および式（２）中、＊は前記共重合体のうち、前記グリシジル基含有モノマーに由来するユニットに結合する部分を示す。 Further, the present invention is particles for the aggregation method containing a copolymer having a unit derived from a styrene-based monomer and a unit derived from a glycidyl group-containing monomer, and the copolymer is represented by the following formula (1). It is a particle having a partial structure and a partial structure represented by the following formula (2).

In the formula (1), the counter ion R ⁺ of the carboxy group is a hydrogen ion or an organic amine, and in the formulas (1) and (2), * is derived from the glycidyl group-containing monomer among the copolymers. The part to be connected to the unit to be used is shown.

According to the present invention, a particle having a small non-specific adsorptivity and having a reactive functional group for immobilizing a ligand, which exhibits high sensitization efficiency of the ligand, and is optimal for a ligand-immobilized method for an aggregation method. Can be provided.

It is a schematic diagram of the ligand sensitized particle of this invention. It is a schematic diagram of the ligand sensitized particle which has a hydrophilic chain of this invention. It is a schematic diagram of the ligand sensitized particle which has polyethylene glycol of this invention.

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.

The particles of the present invention will be described.
The particles of the present invention are particles for the aggregation method containing a copolymer having a unit derived from a styrene-based monomer and a unit derived from a glycidyl group-containing monomer, and the copolymer is represented by the following formula (1). It is a particle having the partial structure shown. In the formula (1), the counter ion R ⁺ of the carboxy group is an organic amine, and * indicates a portion of the copolymer that binds to a unit derived from the glycidyl group-containing monomer. The partial structure represented by the following formula (1) preferably exists on the particle surface.

The particles of the present invention are particles for the agglutination method, and can be used, for example, as particles for the latex immunoagglutination method. The particles can immobilize the ligand. Since the obtained ligand-sensitized particles bind to the target substance, the target substance can be measured by the latex immunoagglutination method.

The agglutination method particles according to the present invention have an antibody or a carboxy group capable of chemically immobilizing an antigen as a ligand on the particle surface.

The matrix of the particles of the present invention (1 in FIG. 1) contains a copolymer having a unit derived from a styrene-based monomer and a unit derived from a glycidyl group-containing monomer. In the present invention, the "unit" means a unit structure corresponding to one monomer.

The chemical structure of the unit derived from the styrene-based monomer is not limited as long as the object of the present invention can be achieved, but it is preferably at least one selected from the group derived from styrenes. Styrenes are styrene, α-methylstyrene, β-methylstyrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, pn-butylstyrene, p-tert- Butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn-dodecylstyrene, p-methoxystyrene, p-phenylstyrene, etc. Can be given.

The chemical structure of the unit derived from the glycidyl group-containing monomer is not limited as long as the object of the present invention can be achieved, but it is preferably at least one selected from glycidyl methacrylate and glycidyl acrylate.

For a copolymer having a unit derived from a styrene-based monomer and a unit derived from a glycidyl group-containing monomer, the composition ratio of the units is not limited as long as the object of the present invention can be achieved. Preferably, the "unit derived from a styrene-based monomer" / "unit derived from a glycidyl group-containing monomer" is 0.1 or more and 10 or less (mol fraction), preferably 0.2 or more and 5 or less (mol fraction). More preferably, it is 0.5 or more and 2 or less (mоl fraction). This preferable range is a numerical value determined by the relationship between the particle strength of the particles derived from the "styrene-based monomer", the ability to suppress non-specific adsorption derived from the "glycidyl group-containing monomer", and the reaction efficiency of the carboxy group and the ligand. Is. When the above relationship is satisfied, the particle strength, the ability to suppress non-specific adsorption, and the ligand reaction efficiency are well balanced.

In the particles of the present invention, the spacer (2 in FIG. 1) having a carboxy group capable of fixing the ligand (3 in FIG. 1) exists on the surface of the parent particle and is represented by the following formula (1). In the formula (1), the counter ion R ⁺ of the carboxy group is an organic amine, and * indicates a portion of the copolymer that binds to a unit derived from the glycidyl group-containing monomer.

A spacer having a carboxy group capable of immobilizing a ligand is a side chain portion of a glycidyl group-containing monomer and is bonded to a polymer having a unit derived from the glycidyl group-containing monomer. Since this spacer molecule has a hydrophilic carboxy group, it not only enhances the hydrophilicity of the particle surface and contributes to the dispersion stability of the particles, but also functions as a scaffold for fixing the ligand. The surface density of carboxy groups (number of moles of carboxy groups / surface area cm ² ) is 0.01 or more and 100 or less (nanomoles / cm ² ), preferably 0.1 or more and 10 or less (nanomoles / cm ² ). This preferable range is a numerical value determined by the relationship between the ability to suppress non-specific adsorption of particles and the reaction efficiency between the carboxy group and the ligand. When the above relationship is satisfied, the particles can be stably dispersed and the ligand can be efficiently immobilized while maintaining the nonspecific adsorption inhibitory ability. Therefore, when used in the latex immunoaggregation measurement method, the target substance is high. It is advantageous to detect with sensitivity. When it is less than 0.01 (nanomol / cm ² ), the amount of ligand sensitization is small and the sensitivity is lowered because there are few carboxy groups on the particle surface. In addition, the dispersion stability of the particles may be impaired. On the other hand, if ^{it exceeds 100 (nanomol / cm 2} ), the carboxy group on the particle surface becomes excessive, so that the unreacted carboxy group remaining after the ligand is chemically immobilized may be involved in the non-specific reaction, which is not preferable. .. When the surface density of the carboxy group of a particle having a particle size of 200 nm is 0.01 or more and 100 or less (nanomoles / cm ² ), the number of moles of the carboxy group is 2.9 or more and 28571 or less (nanomol / mg) per 1 mg of the particle. Corresponds to.

^{The counter ion R +} of the carboxy group in the formula (1) is an organic amine. Examples of the organic amine include trimethylamine, triethylamine, N, N-dimethylpropylamine, N-ethyl-N-methylbutylamine and the like, but triethylamine is preferable. The presence of the organic amine as a counter ion of a carboxy group improves the hydrophilicity of the particles and improves the dispersion stability in water. The dispersion stability of the particles in water also affects the sensitization efficiency of immobilizing the ligand. That is, when the dispersion stability is poor, the immobilization of the ligand becomes non-uniform, and the amount of immobilization may decrease. The particles of the present invention are suitable as particles used in the latex immunoagglutination measurement method by using an organic amine as a counter ion.

Therefore, an example of a preferable embodiment as the particles of the present invention is particles for the aggregation method containing a copolymer of styrene and glycidyl methacrylate, and a part of the side chain of glycidyl methacrylate is represented by the following formula (1). It is a particle that is a spacer having a carboxy group capable of immobilizing a ligand. In formula (1), the counter ion R ⁺ of the carboxy group is triethylamine. * Indicates a portion that binds to a unit derived from glycidyl methacrylate.

Another embodiment of the present invention is particles for the aggregation method containing a copolymer having a unit derived from a styrene-based monomer and a unit derived from a glycidyl group-containing monomer, wherein the copolymer is described below. It is a particle having a partial structure represented by the formula (1) and a partial structure represented by the following formula (2). In the formula (1), the counter ion R ⁺ of the carboxy group is a hydrogen ion or an organic amine. In formulas (1) and (2), * indicates a portion of the copolymer that binds to a unit derived from the glycidyl group-containing monomer. The partial structure represented by the following formula (1) and the partial structure represented by the following formula (2) are preferably present on the particle surface.

The hydrophilic chain (4 in FIG. 2) is a side chain portion of the glycidyl group-containing monomer and is bonded to the polymer skeleton of the glycidyl group-containing monomer. Here, the hydrophilic chain is a chain having a plurality of hydroxyl groups. It is obtained by reacting a glycidyl group with trishydroxymethylaminomethane (Tris). As shown in the formula (2), the presence of a plurality of hydroxyl groups makes it possible to make the particle surface hydrophilic, and it is possible to suppress the dispersion stability of the particles and the non-specific adsorption of proteins. When the counter ion of the carboxy group on the particle surface is a hydrogen ion, the dispersion of the particle in water may become unstable, but in the case of a particle having a hydrophilic chain, when the counter ion of the carboxy group on the particle surface is a hydrogen ion. However, the dispersibility becomes good, and the particles are suitable as particles used in the latex immunoaggregation measurement method.

Therefore, an example of a preferable embodiment as the particles of the present invention is particles for the aggregation method containing a copolymer of styrene and glycidyl methacrylate, and a part of the side chain of glycidyl methacrylate is a ligand represented by the following formula (1). These are spacers having a carboxy group capable of fixing the particles, and particles having a hydrophilic chain represented by the following formula (2).
In the formula (1), the counter ion R ⁺ of the carboxy group is a hydrogen ion or an organic amine, and in the formulas (1) and (2), * indicates a portion of glycidyl methacrylate that binds to the polymer skeleton.

The particles according to the present invention have a form in which non-specific adsorption is suppressed, the reaction efficiency with the ligand is excellent, and the dispersion stability is just right for the agglutination method. The copolymer of styrene and glycidyl methacrylate is physically strong and does not crack or chip even after repeated centrifugation operations.

The particles of the present invention may be crosslinked. Particles can be crosslinked by using a monomer such as divinylbenzene during particle synthesis. Cross-linking of particles improves the physical strength of the particles and is advantageous for particle handling (such as centrifugation during production and ligand immobilization). By using divinylbenzene, the solvent resistance of the particles is also improved.

The particle size of the particles of the present invention is 0.05 μm or more and 1 μm or less, preferably 0.1 μm or more and 0.5 μm or less, and more preferably 0.15 μm or more and 0.3 μm or less in terms of the number average particle size in water. When it is 0.15 μm or more and 0.3 μm or less, the handleability in centrifugal operation is excellent, and the size of the specific surface area, which is a characteristic of particles, stands out. The particle size of the particles of the present invention was evaluated by a dynamic light scattering method.

The present invention also relates to a ligand sensitized particle for an agglutination method in which a ligand is chemically fixed to the carboxy group of the particle of the present invention.

A ligand is a compound that specifically binds to a receptor possessed by a specific target substance. The site where the ligand binds to the target substance is fixed and has a high affinity selectively or specifically. For example, antigens and antibodies, enzyme proteins and their substrates, signaling substances such as hormones and neurotransmitters and their receptors, nucleic acids, etc. are exemplified, but ligands are not limited thereto. Examples of ligands include antigens, antibodies, antigen-binding fragments (eg, Fab, F (ab') 2, F (ab'), Fv, scFv, etc.), naturally occurring nucleic acids, artificial nucleic acids, aptamers, peptide aptamers, oligos. Examples include peptides, enzymes, coenzymes and the like. The sensitized particles for the agglutination method in the present invention mean the sensitized particles for the agglutination method which have a high affinity (affinity) selectively or specifically with respect to the target substance.

In the present invention, as a method of a chemical reaction for chemically fixing a carboxy group and a ligand of the particles of the present invention, conventionally known methods can be applied as long as the object of the present invention can be achieved. For example, carbodiimide-mediated reactions and NHS ester activation reactions are commonly used chemical reactions. However, the method of the chemical reaction for chemically fixing the carboxy group and the ligand in the present invention is not limited to these.

In the ligand-sensitized particles of the present invention, a hydrophilic molecule may be bound to the remaining carboxy group (active esterified one) to which the ligand is not fixed. This is generally referred to as active ester inactivation, carboxy group blocking treatment, masking treatment, etc., in order to suppress non-specific adsorption of proteins to carboxy groups and improve the dispersion stability of ligand-sensitized particles. Will be done. In the ligand-sensitized particles of the present invention, polyethylene glycol (PEG) or trishydroxymethylaminomethane (Tris) is preferable as the hydrophilic molecule to be bound. Polyethylene glycol is particularly preferable because it can significantly suppress the adsorption of proteins. As will be described later in Examples, when active ester inactivation is performed using polyethylene glycol, the molecular weight of polyethylene glycol is important, and a large molecular weight may inhibit the antigen-antibody reaction. The optimum molecular weight is 350 or more and 5000 or less, and particularly preferably 1000 or more and 2000 or less. As the polyethylene glycol, those having a functional group reactive with a carboxy group or an active ester, for example, polyethylene glycol having an amino group is preferable, and polyethylene glycol having a primary amine is particularly preferable. The polyethylene glycol may be a linear polymer or a branched polymer. The following formula (3) shows an example of trishydroxymethylaminomethane (Tris), and the following formulas (4) to (5) show an example of polyethylene glycol. In the formula, n is an integer of 1 or more indicating the number of oxyethylene units.

The amount of ligand immobilization is also an important factor, and if the amount of ligand immobilization is small, the reactivity of the antigen antibody decreases, which is not preferable. On the contrary, when the amount of ligand immobilization is large, it causes deterioration of the dispersibility of the ligand sensitized particles. Although it depends on the particle size, if the average particle size is about 200 nm, it is preferably 1 μg to 500 μg per 1 mg of particles. In particular, 10 to 200 μg is preferable.

The ligand-sensitized particles for the agglutination method of the present invention can be preferably applied to a latex immunoagglutination measurement method in which an antibody or an antigen is used as a ligand and is widely used in fields such as clinical examination and biochemical research. When general particles are applied to the latex immunoaggregation measurement method, target substances such as antigens (antibodies) and foreign substances in serum are non-specifically adsorbed on the particle surface, and unintended particle aggregation is detected due to this. The problem is that it hinders accurate measurement. Therefore, for the purpose of reducing deceptive noise, particles are usually coated with a biological substance such as albumin as a blocking agent to suppress non-specific adsorption on the particle surface. However, since the characteristics of such biological substances differ slightly depending on the lot, the particles coated by these have different ability to suppress non-specific adsorption depending on the coating treatment. Therefore, there is a problem in stably supplying particles having the same level of ability to suppress non-specific adsorption. In addition, the biological substance coated on the particle surface may exhibit hydrophobicity due to denaturation, and does not necessarily have an excellent ability to suppress non-specific adsorption. Biological pollution is also an issue. Patent Document 2 discloses post-coated ligand-sensitized particles for latex immunoagglutination in order to suppress non-specific reactions. However, since the postcoating agent is water-soluble and coats the particle surface by physical adsorption, there is a concern that it is essentially liberated by dilution. The ligand-sensitized particles of the present invention are highly hydrophilic particles and have enhanced ability to suppress the above-mentioned non-specific adsorption. The above problems can be solved without the need for a postcoat such as albumin.

The reagent for the agglutination method is characterized by containing the ligand-sensitized particles for the agglutination method of the present invention. The amount of the ligand sensitized particles for the agglutination method of the present invention contained in the reagent is preferably 0.001% by mass or more and 20% by mass or less, and more preferably 0.01% by mass or more and 10% by mass or less. .. The reagent may contain a third substance such as a solvent or a blocking agent in addition to the ligand-sensitized particles for the aggregation method of the present invention to the extent that the object of the present invention can be achieved. A combination of two or more third substances such as a solvent and a blocking agent may be contained. Examples of the solvent to be used include various buffer solutions such as phosphate buffer solution, glycine buffer solution, Good's buffer solution, Tris buffer solution, and ammonia buffer solution, but the solvent contained in the reagent is not limited thereto.

A kit for use in detecting a target substance in a sample by the agglutination method is characterized by containing at least the above reagents. The kit preferably includes a reaction buffer solution containing albumin (hereinafter, reagent 2) in addition to the above-mentioned reagent (hereinafter, reagent 1). Examples of the albumin include serum albumin and the like, and those treated with protease may be used. The amount of albumin contained in the reagent 2 is, as a guide, 0.001% by mass or more and 5% by mass or less, but the kit is not limited to this. Reagent 1 and / or one of Reagent 2 may contain a sensitizer for measuring latex immunoagglutination. Examples of the sensitizer for measuring latex immunoaggregation include polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyalginic acid, and the like, but the kit is not limited thereto. A surfactant may be contained in both or one of the reagent 1 and the reagent 2. Since the surfactant has an effect of stabilizing particles and proteins, for example, polyoxyethylene sorbitan monolaurate and poly (oxyethylene) octylphenyl ether are preferably used. Further, the kit may include a positive control, a negative control, a serum diluent and the like in addition to the reagents 1 and 2. As a medium for positive control and negative control, a solvent may be used in addition to serum and physiological saline containing no measurable target substance.

The method for detecting a target substance in a sample by the agglutination method of the present invention is characterized by mixing the ligand-sensitized particles for the agglutination method of the present invention with a sample that may contain the target substance. .. Further, the mixing of the ligand-sensitized particles for the agglutination method of the present invention with the sample is preferably performed at pH 3.0 or higher and pH 11.0 or lower. The mixing temperature is 20 ° C. or higher and 50 ° C. or lower, and the mixing time is 10 seconds or longer and 30 minutes or shorter. Further, the concentration of the ligand-sensitized particles for the aggregation method of the present invention in the detection method of the present invention is preferably 0.001% by mass or more and 5% by mass or less, more preferably 0.01% by mass or more 1 in the reaction system. It is mass% or less. The detection method of the present invention is characterized by optically detecting the agglutination reaction generated as a result of mixing the ligand-sensitized particles for the agglutination method of the present invention with a sample, and optically detecting the agglutination reaction. Therefore, the target substance in the sample is detected, and the concentration of the target substance can also be measured. As a method for optically detecting the aggregation reaction, the amount of change in these values may be measured using an optical device capable of detecting scattered light intensity, transmitted light intensity, absorbance and the like.

A preferred method for producing the particles of the present invention will be described.
The method for producing particles is to mix styrene as a monomer, glycidyl methacrylate as a monomer, water, and a radical polymerization initiator to form a granular copolymer, and obtain an aqueous dispersion of the granular copolymer (step). 1).
By mixing and heating the aqueous dispersion of the granular copolymer and aqueous ammonia, the epoxy group derived from glycidyl methacrylate of the granular copolymer is reacted with ammonia to react the epoxy group of the granular copolymer. Introduces an amino group into the epoxy (step 2).
The aqueous dispersion of the granular copolymer and succinic anhydride are mixed and reacted to react the amino group of the granular copolymer with succinic anhydride (step 3).
By mixing and reacting the aqueous dispersion of the granular copolymer with an organic amine, the proton of the carboxy group of the granular copolymer is replaced with the organic amine (step 4).

The radical polymerization initiator is at least 4,4'-azobis (4-cyanovalerian acid), 2,2'-azobis [2-methyl-N- (2-hydroxyethyl) propionamide], 2,2'-. Azobis [N- (2-carboxyethyl) -2-methylpropion amidine] tetrahydrate, 2,2'-azobis (2-methylpropion amidine) dihydrodochloride, 2,2'-azobis [2- (2) -Imidazoline-2-yl) propane], 2,2'-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride, 2,2'-azobis [2- (2-imidazolin-2-yl) propane] ) Propane] Disulfate Dihydrate.

Radical polymerization initiators are 2,2'-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride, 2,2'-azobis [2- (2-imidazolin-2-yl) propane] disulfide. Dihydrate, 2,2'-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride, 2,2'-azobis [2- (2-imidazolin-2-yl) propane]. Is preferable.

The method for forming the granular copolymer is not limited to radical polymerization as long as the object of the present invention can be achieved. Among the radical polymerizations, emulsion polymerization, soap-free emulsion polymerization, and suspension polymerization are preferably used, and emulsion polymerization or soap-free emulsion polymerization is more preferable. More preferably, soap-free emulsion polymerization is used. In general, emulsion polymerization and soap-free emulsion polymerization can obtain a granular copolymer having a sharp particle size distribution as compared with suspension polymerization. Further, when the particles are chemically immobilized with the ligand, there is a concern that the ligand will be denatured if an anionic surfactant generally used in emulsion polymerization is present. Therefore, soap-free emulsion polymerization is the most preferable method for forming the granular copolymer.

In step 1 of the method for producing particles, it is preferable to further contain a crosslinkable radical polymerization monomer in addition to styrene and glycidyl methacrylate. By including the crosslinkable radically polymerizable monomer, the obtained granular copolymer becomes physically strong, and there is no concern that particles will be cracked or chipped even if the centrifugation operation is repeated during purification.

Specific examples of the crosslinkable radically polymerizable monomer that can be used in the present invention are listed below, but the present invention is not limited thereto. Further, two or more kinds of oil-based radically polymerizable monomers may be used. When divinylbenzene is used in the exemplified radical polymerizable monomer, it is preferable because it is excellent in handleability during the radical polymerization reaction.

Crosslinkable radical polymerizable monomer: diethylene glycol diacrylate, triethylene glycol diacrylate, tetraethylene glycol diacrylate, polyethylene glycol diacrylate, 1,6-hexanediol diacrylate, neopentyl glycol diacrylate, tripropylene glycol diacrylate, polypropylene Glycol diacrylate, 2,2'-bis (4- (acryloxydiethoxy) phenyl) propane, trimethyl propan triacrylate, tetramethylol methanetetraacrylate, ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, Tetraethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, 1,3-butylene glycol dimethacrylate, 1,6-hexanediol dimethacrylate, neopentyl glycol dimethacrylate, polypropylene glycol dimethacrylate, 2,2'-bis (4-( Methacryloxydiethoxy) phenyl) propane, 2,2'-bis (4- (methacryloxypolyethoxy) phenyl) propane, trimethylolpropane trimethacrylate, tetramethylolmethanetetramethacrylate, divinylbenzene, divinylnaphthalin, and divinyl ether etc.

In step 1 of the method for producing particles, a step of further mixing the monomer glycidyl methacrylate and coating the surface of the granular copolymer with polyglycidyl methacrylate is further included in the step of forming the granular copolymer. Is preferable.

The radical polymerization initiator is at least 4,4'-azobis (4-cyanovalerian acid), 2,2'-azobis [2-methyl-N- (2-hydroxyethyl) propionamide], 2,2'-. Azobis [N- (2-carboxyethyl) -2-methylpropion amidine] tetrahydrate, 2,2'-azobis (2-methylpropion amidine) dihydrodochloride, 2,2'-azobis [2- (2) -Imidazolin-2-yl) propane], 2,2'-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride, 2,2'-azobis [2- (2-imidazolin-2-yl) propane] ) Propane] disulfate The reason why it is dihydrolate is that the epoxy group derived from glycidyl methacrylate is not opened in the step 1 of obtaining the aqueous dispersion of the granular copolymer. For example, when potassium persulfate is used as the radical polymerization initiator, the radical polymerization reaction field becomes acidic due to the influence of the initiator residue, and the epoxy group derived from glycidyl methacrylate reacts with water to open the ring and form glycol. It may form. Further, when ammonium persulfate is used as the radical polymerization initiator, the epoxy group derived from glycidyl methacrylate may react with ammonia. Further, when an anionic radical polymerization initiator having a carboxy group is used as the radical polymerization initiator, the epoxy group derived from glycidyl methacrylate reacts with the carboxy group derived from the polymerization initiator, and the granular copolymer agglomerates. ..

Step 2 is a step of introducing an amino group into the epoxy group derived from glycidyl methacrylate of the granular copolymer. In general, epoxy groups can easily react with ammonia to introduce amino groups. Since the reaction with ammonia is a strong base condition, the chemical reaction between the epoxy group and water is suppressed, and the amino group can be efficiently introduced into the epoxy group.

Step 2 may include a step of introducing a hydrophilic chain. Generally, it is a step of introducing trishydroxymethylaminomethane (Tris) into an epoxy group derived from glycidyl methacrylate of a granular copolymer. Since the reaction between the epoxy group and trishydroxymethylaminomethane (Tris) is a strong base condition, the chemical reaction between the epoxy group and water is suppressed, and trishydroxymethylaminomethane (Tris) is efficiently introduced into the epoxy group. can do. The introduction of trishydroxymethylaminomethane into the epoxy group may be performed before or after the reaction with ammonia, or may be reacted at the same time as ammonia.

Step 3 is a step of reacting the amino group of the granular copolymer with succinic anhydride to introduce a carboxy group. The succinic anhydride can easily react with a primary amine to form an amide bond and introduce a carboxy group.

Step 4 is a step of reacting the carboxy group of the granular copolymer with an organic amine to replace the proton of the carboxy group with the organic amine. By using the organic amine as a counter ion of a carboxy group, the water dispersibility of the granular copolymer is dramatically improved. In step 4, examples of the organic amine include trimethylamine, triethylamine, N, N-dimethylpropylamine, N-ethyl-N-methylbutylamine and the like, but triethylamine is preferable.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

(Example 1)
(Synthesis of SGNST particles)
1.2 g (1.32 mL) of styrene (manufactured by Kishida Chemical), 1.8 g (1.68 mL) of glycidyl methacrylate (manufactured by Kishida Chemical), 0.04 g (0.044 mL) of divinylbenzene (Kishida) in a 300 mL flask. (Chemical), 115 g (115 mL) of ion-exchanged water was weighed to obtain a mixed solution.
Then, the mixed solution was kept at 70 ° C. with stirring at 200 rpm, and nitrogen bubbling was performed for 30 minutes. Next, nitrogen bubbling was switched to nitrogen flow.
As a polymerization initiator, 0.06 g of V-50 (2,2'-azobis (2-methylpropionamidine) dihydrochloride, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 5 g (5 mL) of pure water to dissolve V-50. Obtained liquid.
Radical polymerization (soap-free emulsion polymerization) was started by adding this V-50 solution to the mixed solution (hereinafter referred to as "radical polymerization solution"). Two hours after the start of the polymerization, 0.3 g (0.28 mL) of glycidyl methacrylate was added to the radical polymerization solution, and the mixture was kept at 70 ° C. with stirring at 200 rpm for 7 hours and then slowly cooled to room temperature. At this point, the contents of the 300 ml flask were sampled, and the radical polymerization conversion rate was evaluated using proton NMR, gas chromatography, and gel permeation chromatography. As a result, it was confirmed that the content was substantially 100%.

ＳＧ粒子は、分散媒体をイオン交換水として保存した。ＳＧ粒子の水分散液をＳＧ粒子分散液とする。ＳＧ粒子分散液の固形分濃度は、１０．４ｗｔ％（１０４ｍｇ／ｍＬ）であった。動的光散乱（ゼータサイザー：マルバーン）を用いてＳＧ粒子を評価したところ、平均粒径が２２０ｎｍであった。 Next, the contents of the 300 mL flask (dispersion solution of the granular copolymer) were centrifuged (15000 rpm). The solids were washed with ion-exchanged water. Centrifugation was performed again, and this washing was performed twice. The obtained particles are referred to as "SG particles". The surface of the SG particles is coated with polyglycidyl methacrylate, and as shown in the formula (6), an epoxy group is present. In the formula, * indicates a portion that binds to the polymethacrylic acid skeleton.

For SG particles, the dispersion medium was stored as ion-exchanged water. The aqueous dispersion of SG particles is referred to as the SG particle dispersion. The solid content concentration of the SG particle dispersion was 10.4 wt% (104 mg / mL). When SG particles were evaluated using dynamic light scattering (zetasizer: Malvern), the average particle size was 220 nm.

ＳＧＮ粒子は、分散媒体をイオン交換水として保存した。ＳＧＮ粒子の水分散液をＳＧＮ粒子分散液とする。ＳＧＮ粒子分散液の固形分濃度は、７．５４ｗｔ％（７５．４ｍｇ／ｍＬ）であった。動的光散乱（ゼータサイザー：マルバーン）を用いてＳＧＮ粒子を評価したところ、平均粒径が２２６．３ｎｍであった。ゼータ電位（ゼータサイザー：マルバーン）の測定より、ＳＧＮ粒子のゼータ電位は５５．０ｍＶであった。 Next, 19 g (solid content 1.98 g) of the SG particle dispersion was transferred to a 200 mL flask. 28% ammonia water (55.4 g) was added dropwise with a dropping funnel over about 15 minutes while irradiating the SG particle dispersion with ultrasonic waves in ice water containing a salt. The obtained SG particle dispersion was transferred to an autoclave bezel containing a rotor, sealed tightly, and then stirred in an oil bath at 70 ° C. for 24 hours. A 50-fold molar amount of ammonia was reacted with respect to the molar amount of glycidyl methacrylate in the particles. After slowly cooling to room temperature, centrifugation (15000 rpm) and washing with ion-exchanged water were repeated three times. The obtained particles are particles in which an amino group is introduced on the surface of SG particles, and are called "SGN particles". The surface of the SGN particles is coated with polyglycidyl methacrylate, and as shown in the formula (7), an amino group is present. In the formula, * indicates a portion that binds to the polymethacrylic acid skeleton.

For SGN particles, the dispersion medium was stored as ion-exchanged water. The aqueous dispersion of SGN particles is referred to as the SGN particle dispersion. The solid content concentration of the SGN particle dispersion was 7.54 wt% (75.4 mg / mL). When the SGN particles were evaluated using dynamic light scattering (zetasizer: Malvern), the average particle size was 226.3 nm. From the measurement of the zeta potential (zetasizer: Malvern), the zeta potential of the SGN particles was 55.0 mV.

動的光散乱（ゼータサイザー：マルバーン）を用いてＳＧＮＳ粒子を評価したところ、平均粒径が２２１．２ｎｍであった。ゼータ電位（ゼータサイザー：マルバーン）の測定より、ＳＧＮＳ粒子のゼータ電位は−４１．１ｍＶであった。 Next, a carboxy group was introduced into the amino group on the particle surface. The solid content concentration of the SGN particle dispersion was adjusted to 0.75 wt% (7.5 mg / mL) using ion-exchanged water. 6 mL of this solution (0.045 g as SGN particles) was transferred to a 15 mL centrifuge tube. Centrifugation was performed, SGN particles were washed with methanol (manufactured by Kishida Chemical Co., Ltd.), and the dispersion medium was 6 mL of methanol. To this, 0.186 g of succinic anhydride (manufactured by Tokyo Chemical Industry Co., Ltd.) was added, and the mixture was stirred at 30 ° C. for 24 hours. The obtained particles are particles in which a carboxy group is introduced on the surface of SGN particles, and are called "SGNS particles". The surface of the SGNS particles is coated with polyglycidyl methacrylate, and as shown in the formula (8), a carboxy group is present. In the formula, * indicates a portion that binds to the polymethacrylic acid skeleton.

When SGNS particles were evaluated using dynamic light scattering (zetasizer: Malvern), the average particle size was 221.2 nm. From the measurement of the zeta potential (zetasizer: Malvern), the zeta potential of the SGNS particles was -41.1 mV.

動的光散乱（ゼータサイザー：マルバーン）を用いてＳＧＮＳＴ粒子を評価したところ、平均粒径が２２２．６ｎｍであった。ゼータ電位（ゼータサイザー：マルバーン）の測定より、ＳＧＮＳＴ粒子のゼータ電位は−４３．６ｍＶであった。 Next, the proton of the carboxy group on the particle surface was replaced with triethylamine.
A methanol solution (containing SGNS particles) reacted with succinic anhydride was centrifuged. The particles were redispersed in an aqueous triethylamine solution (3 wt%) with respect to the precipitate. Centrifugation and redispersion of the triethylamine aqueous solution were repeated twice. Next, the dispersion medium was replaced with ion-exchanged water, and washing was performed three times (centrifugation and redispersion using ion-exchanged water). The obtained particles are particles in which the proton of the carboxy group on the surface of the SGNS particles is replaced with triethylamine, and are called "SGNST particles". The surface of the SGNST particles according to this embodiment is coated with polyglycidyl methacrylate and has a spacer molecule represented by the formula (1). In the formula (1), the counter ion R ⁺ of the carboxy group is It is triethylamine. In the formula, * indicates a portion that binds to the polymethacrylic acid skeleton.

When SGNST particles were evaluated using dynamic light scattering (zetasizer: Malvern), the average particle size was 222.6 nm. From the measurement of the zeta potential (zetasizer: Malvern), the zeta potential of the SGNST particles was -43.6 mV.

(Comparative Example 1)
(Polystyrene particles)
As Comparative Example 1, polystyrene particles Imtex (manufactured by JSR, P0113, 188 nm) were used. Imtex diluted with ion-exchanged water in a 0.1 wt solution was used.

(Reference example 1)
(Post-coated polystyrene particles)
As Reference Example 1, particles coated with Imtex with albumin were prepared. First, 300 μL of a 0.1 wt% aqueous solution of Imtex (manufactured by JSR, P0113, 188 nm) was placed in an Eppen tube, and 80 μL of 1% BSA / PBS (10 mg / mL) was added to this solution. The mixture was stirred at room temperature for 30 minutes. Centrifugation was performed for 20 minutes, the supernatant was removed and the particles were washed twice with PBS.

(Example 2)
(Evaluation of non-specific reaction (non-specific aggregation) of particles)
The non-specific reaction (non-specific aggregation) of the particles was evaluated by the immunoturbidimetric method.

This is a method in which human serum and particles are brought into contact with each other, and nonspecific particle agglutination is measured with an absorbance meter using turbidity as an index. Absorbance increases when non-specific aggregation occurs. The absorbance was measured using an ultraviolet-visible spectrophotometer (trade name: GeneQuant 1300, GE Healthcare), and the sample was injected into a plastic cell (sample volume minimum 70 μL) and measured at an optical path length of 10 mm. The specific measurement method is shown below.

16 μL of human serum (Kishida Chemistry, Specimen No. 7) and 60 μL of R1 buffer were mixed in a plastic cell and heated at 37 ° C. for 5 minutes. 30 μL of a particle dispersion solution (particle concentration 0.1 wt%, ion-exchanged water) was added to R1 buffer solution (76 μL) containing human serum, and quick pipetting was performed carefully so as not to contain air bubbles to prepare a sample. The absorbance at 572 nm of the sample was read and used as Abs1. After heating the sample at 37 ° C. for 5 minutes, the absorbance at 572 nm of the sample was read and used as Abs2. The value obtained by subtracting Abs1 from Abs2 is obtained, and the value obtained by multiplying by 10000 is defined as the ΔODx10000 value. If the ΔODx10000 value is 1000 or more, it is determined that a non-specific reaction has occurred.

The results are shown in Table 1. The ΔODx10000 of the SGNST particles of this example was 1000 or less, and no non-specific reaction was observed. On the other hand, in the polystyrene particles Imtex, 1000 or more ΔODx10000 was observed. Since it can be considered that the increase in the absorbance of the dispersion liquid is caused by the occurrence of non-specific adsorption of the particles in the dispersion liquid, resulting in interparticle agglutination, it was found that Imtex was non-specifically aggregated by serum. Post-coating of Imtex no longer showed non-specific aggregation. It was confirmed that the SGNST particles of this example are superior in the ability to suppress non-specific adsorption as compared with commercially available polystyrene particles.

(Example 3)
(Preparation of antibody-sensitized SGNST particles)
Transfer 0.1 mL (1 mg of particles) of the dispersion liquid (concentration 1.0 wt% solution, 10 mg / mL) of SGNST particles of this example to an Eppen tube (volume 1.5 mL), and 0.12 mL of activation buffer solution. (25 mM MES, pH 6.0) was added and centrifuged at 15000 rpm (20400 g) for 5 minutes at 4 ° C. After centrifugation, the supernatant was discarded (decanted) with a pipetter. 0.12 mL of activation buffer (25 mM MES, pH 6.0) was added and dispersed by ultrasonic waves (AS ONE 3-frequency ultrasonic cleaner MODEL VS-100III, 28 kHz). Next, it was centrifuged at 15000 rpm (20400 g) at 4 ° C. for 5 minutes. The supernatant was discarded with a pipettor, 0.12 mL of activation buffer (25 mM MES, pH 6.0) was added and dispersed ultrasonically. Centrifugation was performed at 4 ° C. at 15,000 rpm (20400 g) for 5 minutes. Discard the supernatant with a pipettor and add 60 μL each of WSC solution (50 mg of WSC dissolved in 1 mL of activation buffer) and Sulfo NHS solution (50 mg of Sulfo NHS dissolved in 1 mL of activation buffer). , Dispersed by ultrasonic waves. The carboxy group of the particles was converted to an active ester by stirring at room temperature for 30 minutes. Centrifugation was carried out at 4 ° C. at 15000 rpm (20400 g) for 5 minutes, and the supernatant was discarded with a pipetter. 0.2 mL of immobilized buffer (25 mM MES, pH 5.0) was added and dispersed by ultrasonic waves. Centrifugation was carried out at 4 ° C. at 15000 rpm (20400 g) for 5 minutes, and the supernatant was discarded with a pipetter. 50 μL of immobilized buffer (per 1 mg of particles) was added to ultrasonically disperse the carboxy group activated particles.

The anti-PSA antibody (monoclonal antibody) was diluted with an immobilized buffer solution to 100 μg / 50 μL (described as an antibody solution). 50 μL of the antibody solution was added to 50 μL of the solution of the particles in which the carboxy group was activated (including 1 mg of the particles), and the particles were dispersed by ultrasonic waves. The amount of antibody charged is 100 μg per 1 mg of particles (100 μg / mg). The tube was stirred at room temperature for 60 minutes to immobilize the antibody on the carboxy group of the particles. Then, the mixture was centrifuged at 15000 rpm (20400 g) at 4 ° C. for 5 minutes, and the supernatant was discarded with a pipetter.
0.24 mL of an active ester inactivation buffer containing Tris (1 M Tris, pH 8.0 containing 0.1% Tween 20) was added and dispersed by ultrasonic waves. The mixture was stirred at room temperature for 2 hours, Tris was bound to the remaining activated ester, or the remaining activated ester was hydrolyzed (returned to a carboxy group), and then allowed to stand at 4 ° C. overnight.
Next, the mixture was centrifuged at 15000 rpm (20400 g) at 4 ° C. for 5 minutes, and the supernatant was discarded with a pipetter. 0.2 mL of wash / storage buffer (10 mM HEPES, pH 7.9) was added and dispersed by ultrasonic waves. After repeating the washing operation with 0.2 mL of the washing / storage buffer (10 mM HEPES, pH 7.9) twice, 0.5 mL of the washing / storage buffer was added and dispersed by ultrasonic waves. Since almost no particle loss was observed in the sensitization step, the antibody sensitized particle concentration was finally 0.2 wt% (2 mg / mL). It was stored in a refrigerator and redispersed by ultrasonic waves when used. The obtained antibody-sensitized particles are hereinafter referred to as "antibody-sensitized SGNST particles".
As shown in FIG. 3, in the antibody-sensitized SGNST particles, the antibody is immobilized on the surface of the particles via spacers, and trishydroxymethylaminomethane (Tris) is contained in some spacers on which the antibody is not immobilized. It is an antibody sensitized particle that is bound.

(Comparative Example 3)
(Antibody sensitization to polystyrene particles)
As a comparative example, an antibody was sensitized to polystyrene particles and Imtex manufactured by JSR.
Antibody-sensitized polystyrene particles as a comparative example were obtained by the same experimental operation as in Example 3 except that the particles of Example 3 were changed to Imtex and post-coating of albumin was performed after antibody fixation. The obtained particles are hereinafter referred to as "antibody-sensitized polystyrene particles".

(Example 4)
(Measurement of antibody sensitization efficiency)
It was confirmed by protein quantification that the antibody was sensitized (fixed) to the particles. Specifically, it is a method of reacting antibody-sensitized particles with a BCA reagent. First, 12.5 μL (25 μg of particle volume) of a dispersion of antibody-sensitized particles (0.2% solution) is separated, and 12.5 μL of 10 mM HEPES (pH 7.9) is added thereto. 7 mL of solution A and 140 μL of solution B of the protein assay BCA kit (Wako Pure Chemical Industries, Ltd.) were mixed to obtain solution AB. To the particle solution (25 μL), 200 μL of AB solution was added, and the mixture was incubated at 60 ° C. for 30 minutes. The solution was centrifuged at 4 ° C. at 15000 rpm (20400 g) for 5 minutes, and 200 μL of the supernatant was collected with a pipetter. Absorbance at 562 nm was measured with a multimode microplate reader (trade name: SynergyMX, BioTek) along with a standard sample (several points of antibody in the range of 0 to 200 μg / mL at 10 mM HEPES). The amount of antibody was calculated from the standard curve. The amount of antibody sensitized to the particles (antibody fixation amount per particle weight (μg / mg)) was determined by dividing the calculated antibody amount by the particle weight (here 0.025 mg). The sensitization efficiency was determined from the amount of antibody charged. The results are shown in Table 2. It was found that the antibody-sensitized particles of this example had higher sensitization efficiency than the comparative examples.

The high sensitization efficiency of the particles of this example will be described. In the particles of this example, the carboxy group is located near the surface, and almost no amino group, which is a scaffold for introducing the carboxy group, remains. That is, the surface charge is negative and uniform. On the other hand, the region other than the carboxy group is a hydroxyl group obtained by ring-opening of the glycidyl group and is extremely hydrophilic. The hydrophilic surface exhibits a high ability to suppress non-specific adsorption. Upon sensitization, the antibody is in a catalytic state and electrostatically attracts the negative surface of the carboxy group region of the particle. As a result, the antibody is concentrated on the particle surface, and the reaction between the antibody and the carboxy group on the particle surface is greatly promoted. As a result, the antibody sensitization rate to the particles is considered to be improved. Since the NHS-activated ester of the carboxy group is rapidly hydrolyzed in water, the action of concentrating the antibody on the particle surface is an important process. On the other hand, since the particle surface of commercially available polystyrene is hydrophobic, physical adsorption of the antibody to the hydrophobic portion other than the carboxy group region occurs. As a result of repeated desorption and adsorption of physical adsorption, it is considered that the sensitization efficiency of commercially available polystyrene particles is low.

(Example 5)
(Sensitivity of antibody-sensitized particles to human PSA antigen and evaluation of non-specific reaction)
The sensitivity of antibody-sensitized particles was evaluated by the latex immunoagglutination method. Specifically, antibody-sensitized particles are reacted with an antigen to form an aggregate of an immune complex, the aggregate is irradiated with light, and the attenuation (absorbance) of the irradiation light due to scattering is measured with an absorptiometer. The method. The proportion of aggregates increases depending on the amount of antigen contained in the sample, and the absorbance increases. In the evaluation of sensitivity, it is desirable that the amount of increase in absorbance (described as ΔODx10000) at a predetermined PSA concentration is large. The absorbance was measured using an ultraviolet-visible spectrophotometer (trade name: GeneQuant 1300, GE Healthcare), and the sample was injected into a plastic cell and measured at an optical path length of 10 mm. The measurement method is specifically shown below.

Using 16 μL of PSA solution (PSA concentration 91.7 ng / mL) as a sample, this sample and 60 μL of R1 buffer were mixed in a plastic cell and heated at 37 ° C. for 5 minutes. Add 30 μL of a dispersion solution of antibody-sensitized particles (particle concentration 0.2 wt%, 10 mM HEPES, pH 7.9) to R1 buffer (76 μL) containing PSA, and quickly pipette while being careful not to allow air bubbles to enter. It was used as a sample. The absorbance at 572 nm of the sample was read and used as Abs1. After heating the sample at 37 ° C. for 5 minutes, the absorbance at 572 nm of the sample was read and used as Abs2. The value obtained by subtracting Abs1 from Abs2 was obtained, and the value obtained by multiplying by 10000 was defined as the ΔODx10000 value.
The results are shown in Table 3.

In the antibody-sensitized SGNST particles of this example, an increase of ΔODx10000 was observed in the presence of PSA. This is a result of antibody-sensitized particles binding to PSA, which is an antigen, to form particle aggregates, and it was found that the particles function as particles for use in the latex immunoaggregation method.

When a PSA-free solution (PSA concentration 0 ng / mL) was used as a sample, the ΔODx10000 of the sensitized particle solution did not change, as shown in Table 3.

When human sera (sample numbers 3-2 and 3-5) containing no PSA were used as samples, the ΔODx10000 of the sensitized particle solution did not change, as shown in Table 3.

From the above results, the non-specific reactivity of the antibody-sensitized SGNST particles of this example was not observed. It was found that the particles of this example can suppress non-specific reactions even without postcoating with albumin.

(Example 6)
(Synthesis of SGNST-2 particles)
An SG particle dispersion was prepared in the same manner as in Example 1. Next, the SG particle dispersion (solid content 3.0 g) was transferred to a 200 mL flask. 28% ammonia water (40.8 g) was added with a dropping funnel while stirring and ultrasonically irradiating the SG particle dispersion in ice water. After the addition, stirring and ultrasound were continued for 15 minutes. The obtained SG particle dispersion was transferred to an autoclave bezel containing a rotor, sealed tightly, and then stirred in an oil bath at 70 ° C. for 24 hours. A 25-fold molar amount of ammonia was reacted with respect to the molar amount of glycidyl methacrylate in the particles. After slowly cooling to room temperature, centrifugation (15000 rpm) and washing with ion-exchanged water were repeated three times. The obtained particles are particles in which an amino group is introduced on the surface of SG particles, and are called "SGN-2 particles". In the same manner as in Example 1, a carboxy group was introduced into the SGN-2 particles, and the proton of the carboxy group on the particle surface was replaced with triethylamine. The obtained particles are called "SGNST-2 particles".

When SGNST-2 particles were evaluated using dynamic light scattering (trade name: Zetasizer, Malvern), the average particle size was 222.4 nm. From the measurement of the zeta potential (zetasizer: Malvern), the zeta potential of the SGNST-2 particles was −52.3 mV.

(Example 7)
(Preparation of antibody-sensitized SGNST-2 particles)
The antibody-sensitized SGNST-2 particles according to this example were obtained by the same experimental operation as in Example 3 except that the SGNST particles of Example 3 were changed to SGNST-2 particles. The obtained particles are hereinafter referred to as "antibody sensitized SGNST-2 particles".

(Example 8)
(Sensitivity of antibody-sensitized particles to human PSA antigen and evaluation of non-specific reaction)
The sensitivity and non-specific reaction of antibody-sensitized SGNST-2 particles were evaluated in the same manner as in Example 5. The results are shown in Table 4. The antibody-sensitized SGNST-2 particles showed an increase in ΔODx10000 in the presence of PSA. This is a result of antibody-sensitized particles binding to PSA, which is an antigen, to form particle aggregates, and it was found that the particles function as particles for use in the latex immunoaggregation method. As shown in Table 4, when a PSA-free solution (PSA concentration 0 ng / mL) was used as a sample and when PSA-free human serum (sample numbers 3-2 and 3-5) was used as a sample. , ΔODx10000 did not change. From the above results, the non-specific reactivity of the antibody-sensitized SGNST-2 particles of this example was not observed. It was found that the particles of this example can suppress non-specific reactions even without postcoating with albumin.

(Example 9)
(Preparation of antibody-sensitized SGNST particles modified with PEG having a molecular weight of 5000)
The same as in Example 4 except that the anti-PSA antibody (monoclonal antibody) of Example 3 was changed to an anti-CRP antibody (monoclonal antibody) and the active ester inactivation buffer was changed to a PEG modification buffer (PEG molecular weight 5000). By experimental operation, PEG-modified antibody-sensitized SGNST particles according to this example were obtained. The obtained particles are hereinafter referred to as "antibody sensitized SGNST particles (P5000)". As shown in FIG. 3, the antibody-sensitized SGNST particles (P5000) have antibody 3 immobilized on the surface of the particles 1 via spacers 2, and PEG5 (PEG5) on some spacers on which antibody 3 is not immobilized. It is an antibody sensitized particle to which PEG molecular weight 5000) is bound.

(Example 10)
(Preparation of antibody-sensitized SGNST particles modified with PEG having a molecular weight of 2000)
The same as in Example 3 except that the anti-PSA antibody (monoclonal antibody) of Example 3 was changed to an anti-CRP antibody (monoclonal antibody) and the active ester inactivation buffer was changed to a PEG modification buffer (PEG molecular weight 2000). By experimental operation, PEG-modified antibody-sensitized SGNST particles according to this example were obtained. The obtained particles are hereinafter referred to as "antibody sensitized SGNST particles (P2000)". As shown in FIG. 3, the antibody-sensitized SGNST particles (P2000) have an antibody immobilized on the particle surface via spacers, and PEG (PEG molecular weight 2000) is attached to some spacers on which the antibody is not immobilized. Is an antibody sensitized particle to which is bound.

(Example 11)
(Preparation of antibody-sensitized SGNST particles modified with PEG having a molecular weight of 1000)
The same as in Example 3 except that the anti-PSA antibody (monoclonal antibody) of Example 3 was changed to an anti-CRP antibody (monoclonal antibody) and the active ester inactivation buffer was changed to a PEG modification buffer (PEG molecular weight 1000). By experimental operation, PEG-modified antibody-sensitized SGNST particles according to this example were obtained. The obtained particles are hereinafter referred to as "antibody sensitized SGNST particles (P1000)". As shown in FIG. 3, the antibody-sensitized SGNST particles (P1000) have an antibody immobilized on the particle surface via spacers, and PEG (PEG molecular weight 1000) is attached to some spacers on which the antibody is not immobilized. Is an antibody sensitized particle to which is bound.

(Example 12)
(Preparation of antibody-sensitized SGNST particles modified with PEG having a molecular weight of 550)
The same as in Example 3 except that the anti-PSA antibody (monoclonal antibody) of Example 3 was changed to an anti-CRP antibody (monoclonal antibody) and the active ester inactivation buffer was changed to a PEG modification buffer (PEG molecular weight 550). By experimental operation, PEG-modified antibody-sensitized SGNST particles according to this example were obtained. The obtained particles are hereinafter referred to as "antibody sensitized SGNST particles (P550)". As shown in FIG. 3, the antibody-sensitized SGNST particles (P550) have an antibody immobilized on the particle surface via spacers, and PEG (PEG molecular weight 550) is attached to some spacers on which the antibody is not immobilized. Is an antibody sensitized particle to which is bound.

(Example 13)
(Preparation of antibody-sensitized SGNST particles modified with PEG having a molecular weight of 350)
The same as in Example 3 except that the anti-PSA antibody (monoclonal antibody) of Example 3 was changed to an anti-CRP antibody (monoclonal antibody) and the active ester inactivation buffer was changed to a PEG modification buffer (PEG molecular weight 350). By experimental operation, PEG-modified antibody-sensitized SGNST particles according to this example were obtained. The obtained particles are hereinafter referred to as "antibody sensitized SGNST particles (P350)". As shown in FIG. 3, the antibody-sensitized SGNST particles (P350) have an antibody immobilized on the particle surface via spacers, and PEG (PEG molecular weight 350) is attached to some spacers on which the antibody is not immobilized. Is an antibody sensitized particle to which is bound.

(Example 14)
(Preparation of antibody-sensitized SGNST particles without PEG modification)
The antibody-sensitized SGNST particles according to this example were obtained by the same experimental procedure as in Example 3 except that the anti-PSA antibody (monoclonal antibody) of Example 3 was changed to an anti-CRP antibody (monoclonal antibody). The obtained particles are hereinafter referred to as "antibody sensitized SGNST particles (N)". As shown in FIG. 1, the antibody-sensitized SGNST particles (N) are antibody-sensitized particles in which no hydrophilic chain is present on the particle surface.

(Example 15)
Sensitization efficiency, antibody immobilization amount, average particle size, and polydispersity index (PDI) were measured for the antibody-sensitized particles obtained in Examples 9 to 14. Here, the polydispersity index (PDI) is an index of the width of the particle size distribution in the dynamic light scattering (DLS) measurement, and is represented by a numerical value of 0 to 1. A distribution with a PDI value of 0.1 or less is called monodispersity. The smaller the value, the narrower the particle size distribution. Table 5 shows the measurement results. The antibody sensitization efficiency and the amount of immobilization are the same because all the examples use the same lot of particles (only the particles of Example 15 were measured). It was found that in the antibody-sensitized SGNST particles (P1000) and the antibody-sensitized SGNST particles (P2000), the polydispersity index (PDI) was small and the particle size distribution was narrow. It is considered that the dispersibility of the sensitized particles was improved by the PEG having an appropriate size.

(Example 16)
The sensitivity of the antibody-sensitized particles obtained in Examples 9 to 14 was evaluated by the latex immunoagglutination method in the same manner as in Example 5.
Unlike Example 5, CRP was used as the antigen instead of PSA. Specifically, 1 μL of CRP solution (CRP concentration 4 mg / dL) was used as a sample, and this sample and 50 μL of R1 buffer were mixed in a plastic cell and heated at 37 ° C. for 5 minutes. Add 50 μL of a dispersion solution of antibody-sensitized particles (particle concentration 0.05 wt%, 10 mM HEPES, pH 7.9) to R1 buffer (51 μL) containing CRP, and quickly pipette while being careful not to allow air bubbles to enter. It was used as a sample. The absorbance at 572 nm of the sample was read and used as Abs1. After heating the sample at 37 ° C. for 5 minutes, the absorbance at 572 nm of the sample was read and used as Abs2. The value obtained by subtracting Abs1 from Abs2 was obtained, and the value obtained by multiplying by 10000 was defined as the ΔODx10000 value. In order to evaluate the non-specific reaction, a serum solution containing no CRP (CRP concentration 0 mg / dL) was used as a sample.

The results are shown in Table 6. The antibody-sensitized particles obtained in Examples 9 to 14 showed an increase in ΔODx10000 in the presence of CRP (CRP concentration 4 mg / dL). This is a result of antibody-sensitized particles binding to CRP, which is an antigen, to form particle aggregates, and it was found that the particles function as particles for use in the latex immunoaggregation method.

It was found that the antibody-sensitized SGNST particles (P1000) and the antibody-sensitized SGNST particles (P2000) had a larger ΔODx10000 and higher sensitivity than the other antibody-sensitized particles. On the other hand, antibody-sensitized SGNST particles (P5000) tended to have lower sensitivity. If the PEG molecular weight is high, the PEG on the particle surface may inhibit the reaction between the antibody and the antigen. The sensitivity of antibody-sensitized particles can be controlled by introducing PEG into the particles. When a CRP-free solution (CRP concentration 0 mg / dL) was used as a sample, ΔODx10000 did not change. From the above results, it was found that the antibody-sensitized particles obtained in Examples 10 to 15 can suppress the non-specific reaction even without post-coating with albumin.

(Example 17)
(Synthesis of SGNST-3 particles with hydrophilic chains introduced on the surface)
In the same manner as in Example 1, SGN particles having an amino group introduced on the surface of the SG particles were synthesized. Next, the unmodified residue (residual epoxy group) of the epoxy group existing on the surface of the SGN particle can be modified with trishydroxymethylaminomethane (Tris) as follows to introduce a hydrophilic chain onto the particle surface. You can.

The SGN particle dispersion was adjusted with ultrapure water so that the solid content was 5 wt%, Tris (5 times the amount charged in GMA at the time of SGN particle synthesis) was added thereto, and then Tris was dissolved by stirring. .. Next, the pH was adjusted to 11 with triethylamine (TEA). Then, the mixture was stirred at 70 ° C. for 24 hours. Then, centrifugation was carried out at 4 ° C. and 20,500 G for 10 minutes, the supernatant was discarded, and purification for redispersing the precipitate with ultrapure water was carried out a total of 5 times.

Then, in the same manner as in Example 1, a carboxy group is introduced into the amino group on the particle surface, and finally the proton of the carboxy group on the particle surface is replaced with triethylamine. The obtained particles are referred to as "SGNST-3 particles". The SGNST-3 particles have a hydrophilic chain introduced on the surface and have high dispersion stability in water.

(Example 18)
(Synthesis of antibody-sensitized SGNST-3 particles)
The antibody-sensitized SGNST-3 particles according to this example can be obtained by the same experimental operation as in Example 14. As shown in FIG. 2, the antibody-sensitized SGNST-3 particles are antibody-sensitized particles in which hydrophilic chains are present on the particle surface. Since the antibody does not occupy the entire surface of the particle, non-specific adsorption of contaminating proteins on the surface of the particle may be a problem. In that case, it is possible to suppress non-specific adsorption of proteins by introducing a hydrophilic chain onto the particle surface. Antibody-sensitized SGNST-3 particles can suppress non-specific reactions without postcoating with albumin.

1 Particle 2 Spacer molecule 3 Ligand 4 Hydrophilic chain 5 Polyethylene glycol

Claims (13)

Particles for the agglutination method containing a copolymer having a unit derived from a styrene-based monomer and a unit derived from a glycidyl group-containing monomer.
Particles in which the copolymer has a partial structure represented by the following formula (1).

(In the formula (1), the counter ion R ⁺ of the carboxy group is an organic amine, and * indicates a portion of the copolymer that binds to a unit derived from the glycidyl group-containing monomer.) Particles for the agglutination method containing a copolymer having a unit derived from a styrene-based monomer and a unit derived from a glycidyl group-containing monomer.
Particles in which the copolymer has a partial structure represented by the following formula (1) and a partial structure represented by the following formula (2).

(In the formula (1), the counter ion R ⁺ of the carboxy group is a hydrogen ion or an organic amine, and in the formulas (1) and (2), * is the glycidyl group-containing monomer of the copolymer. Indicates the part that binds to the unit from which it is derived.) The agglutination method particle according to claim 1 or 2, wherein the organic amine of the counter ion R ^{+ is triethylamine.} A ligand-sensitized particle in which a ligand is fixed to the agglutination method particle according to any one of claims 1 to 3. The ligand sensitized particle according to claim 4, wherein the ligand is fixed to the carboxy group of the agglutination method particle. A ligand sensitized particle in which polyethylene glycol or trishydroxymethylaminomethane is bound to the ligand sensitized particle according to claim 4 or 5. The ligand sensitized particle according to claim 6, wherein polyethylene glycol or trishydroxymethylaminomethane is bonded to the carboxy group of the agglutination method particle. The ligand sensitized particle according to any one of claims 4 to 7, wherein the ligand-immobilized amount of the ligand is 1 μg to 500 μg with respect to 1 mg of the agglutination method particle. A particle dispersion in which the agglutination method particles or ligand sensitized particles according to any one of claims 1 to 8 are dispersed in an aqueous solution. The particle dispersion liquid according to claim 9, wherein the particle dispersion liquid further contains a surfactant. The method for detecting a target substance in a sample by an agglutination method using the ligand-sensitized particles or the particle dispersion according to any one of claims 4 to 10. A reagent for use in detecting a target substance in a sample by an agglutination method using the ligand-sensitized particles or the particle dispersion according to any one of claims 4 to 10. A kit for use in detecting a target substance in a sample by an agglutination method, which comprises at least the reagent according to claim 12.

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