JP4653652B2 - Magnetic inclusion particles, method for producing magnetic inclusion particles, immunoassay particles and immunoassay method - Google Patents

Description

The present invention relates to a magnetic substance-encapsulated particle excellent in chromatographic development and capable of performing highly sensitive immunoassay, a method for producing magnetic substance-encapsulated particle, an immunoassay particle, and an immunoassay method using the same.

Examples of a method for detecting a test substance contained in a measurement sample include biological reactions such as enzyme immunoassay, fluorescence immunoassay, latex agglutination, and immunochromatography using an antigen-antibody reaction. Various methods used have been proposed. Among these, immunochromatography has been frequently used because it is simple and quick.

In immunochromatography, a sandwich method using at least two kinds of antibodies is usually employed. That is, using a metal colloid or colored particles as a support, a reagent containing an antibody labeled with an isotope, enzyme, fluorescent substance, or the like is reacted with a measurement sample, and the antigen contained in the measurement sample is bound to the labeled antibody. By passing this on a chromatographic carrier on which another antibody is immobilized, the antigen is captured in the chromatographic carrier, and the captured antigen is analyzed based on the label.

Magnetic encapsulated particles have attracted attention as a support for use in such immunochromatography. Magnetic encapsulated particles have been proposed for use as a support for separating B / F efficiently and easily by magnetism in radioimmunoassay and the like, but the magnetic content of magnetic encapsulated particles should be labeled. Therefore, there is an advantage that analysis can be performed without labeling with another labeling substance (Patent Documents 1 to 3).

However, when magnetic particles are actually used in immunochromatography, the magnetic particles may stay in the chromatographic carrier, or may remain unevenly near the chromatographic tip of the developing side. There is a problem that chromatographic developability is greatly inferior to metal colloids and colored particles.
JP-A-6-148189 JP 7-225233 A JP 2001-524675 A

In view of the above-described situation, the present invention provides magnetic inclusion particles that are excellent in chromatographic development and capable of performing highly sensitive immunoassay, a method for producing magnetic inclusion particles, immunoassay particles, and immunoassay using the same. The purpose is to provide the law.

The present invention relates to a magnetic substance-encapsulated particle having an average particle diameter of 50 to 500 nm, comprising an organic polymer substance and a magnetic substance dispersed in the organic polymer substance, wherein the magnetic substance in the organic polymer substance This is a magnetic substance-encapsulated particle having a body dispersion diameter of 1 to 30 nm and containing 50 to 80% by weight of the magnetic substance.
The present invention is described in detail below.

The present inventors diligently investigated the cause of poor chromatographic developability when magnetic inclusion particles were used for immunochromatography. As a result, conventional magnetic inclusion particles are prone to self-aggregation. It has been found that this is caused by the fact that it becomes difficult to pass through the pores of the chromatographic carrier and that it is easily adsorbed non-specifically on the chromatographic carrier itself. As a result of further intensive studies, the present inventors used magnetic inclusion particles in which the average particle diameter of the magnetic inclusion particles was a predetermined size, and the dispersion diameter and content of the magnetic substance were within a specific range. In this case, it was found that self-aggregation of magnetic substance-encapsulated particles was suppressed, chromatographic development was improved, and highly sensitive immunoassay became possible, and the present invention was completed.

The magnetic substance-encapsulated particle of the present invention comprises an organic polymer substance and a magnetic substance dispersed in the organic polymer substance.
The organic polymer substance has a role as a matrix of the magnetic substance-encapsulated particles of the present invention.
The organic polymer substance is preferably a copolymer having a segment derived from a styrene monomer. By having a segment derived from a styrene monomer, the dispersibility of the magnetic substance-encapsulated particles of the present invention in an aqueous medium is improved.

The styrene monomer is not particularly limited, and examples thereof include styrene, α-methylstyrene, p-methylstyrene, p-chlorostyrene, chloromethylstyrene, and bifunctional divinylbenzene. These styrenic monomers may be used alone or in combination of two or more.

The minimum with preferable content of the segment derived from the said styrene-type monomer is 60 weight%. If it is less than 60% by weight, the dispersibility of the obtained magnetic substance-encapsulated particles of the present invention in an aqueous medium may be inferior.

The organic polymer substance may have a segment derived from a vinyl monomer containing a reactive functional group in addition to a segment derived from a styrene monomer. By having a segment derived from a vinyl monomer containing a reactive functional group, it is possible to easily bind an antigen or an antibody via the reactive functional group.

The reactive functional group of the vinyl monomer containing the reactive functional group is not particularly limited as long as it can bind an antigen or an antibody by a covalent bond. For example, a carboxyl group, a hydroxyl group, an epoxy group, an amino group , Triethylammonium group, dimethylamino group, sulfonic acid group and the like. The vinyl monomer containing such a reactive functional group is not particularly limited. For example, (meth) acrylic acid, (meth) acrylic acid-2-hydroxyethyl, glycidyl (meth) acrylate, triethylammonium (meth) acrylate And dimethylamino (meth) acrylate. The vinyl monomer containing these reactive functional groups may be used independently, and 2 or more types may be used together.

The copolymer may have a segment derived from another vinyl monomer.
Other vinyl monomers are not particularly limited. For example, vinyl chloride; vinyl esters such as vinyl acetate and vinyl propionate; unsaturated nitriles such as acrylonitrile; methyl (meth) acrylate, ethyl (meth) acrylate, Butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, stearyl (meth) acrylate, ethylene glycol (meth) acrylate, trifluoroethyl (meth) acrylate, pentafluoropropyl (meth) acrylate, cyclohexyl (meth) And (meth) acrylic acid ester derivatives such as acrylate, glycidyl methacrylate, and tetrahydrofurfuryl (meth) acrylate. These monomers may be used independently and 2 or more types may be used together.

The copolymer may have segments derived from a crosslinkable monomer, and may be crosslinked by these segments.
The crosslinkable monomer is not particularly limited. For example, ethylene glycol di (meth) acrylate, 1,6-hexanediol di (meth) acrylate, neopentylglycol di (meth) acrylate, trimethylolpropane tri (meth) acrylate , Tetramethylol methane tritri (meth) acrylate, tetramethylolpropane tetra (meth) acrylate, diallyl phthalate and isomers thereof, triallyl isocyanurate and derivatives thereof, and the like. These crosslinkable monomers may be used independently and 2 or more types may be used together.

The magnetic substance is dispersed in the organic polymer substance.
Although it does not specifically limit as said magnetic body, The thing which has a superparamagnetism without a residual magnetism is suitable. If there is residual magnetism, self-aggregation tends to occur and chromatographic developability may be poor.
The magnetic body having superparamagnetism is not particularly limited, and various ferrites such as iron trioxide (Fe ₃ O ₄ ) and γ-diiron trioxide (γ-Fe ₂ O ₃ ); iron, manganese , Metals such as cobalt, and alloys thereof. Of these, ferrites are preferable, and triiron tetroxide (Fe ₃ O ₄ ) is preferable.
Examples of such a magnetic material include those prepared by a coprecipitation reaction method in which Fe ₃ O ₄ is obtained by dropping a mixed solution containing Fe ²⁺ and Fe ^{3+ at} a ratio of 1: 2 into a basic solution. Can be used. Commercial products such as ferricolloid HC-50 (manufactured by Taiho Kogyo Co., Ltd.) and HX-20 (manufactured by Sigma High Chemical Co., Ltd.) can also be used.

The lower limit of the dispersion diameter of the magnetic substance in the organic polymer substance is 1 nm, and the upper limit is 30 nm. When the thickness is less than 1 nm, in addition to the difficulty in producing the magnetic material itself, the magnetic response characteristics of the magnetic material are reduced, and the sensitivity when used as a label is lowered. If it exceeds 30 nm, residual magnetism tends to occur, and self-aggregation tends to occur, and in addition, the magnetic substance is likely to be exposed on the surface of the magnetic substance-encapsulated particles. A preferred lower limit is 5 nm and a preferred upper limit is 20 nm.

The lower limit of the content of the magnetic substance in the magnetic substance-encapsulated particles of the present invention is 50% by weight, and the upper limit is 80% by weight. If the amount is less than 50% by weight, the amount of magnetism is low, and it becomes difficult to detect when analyzing a very small amount of the measurement target component. If the amount exceeds 80% by weight, self-aggregation tends to occur or the total weight of the magnetic substance-encapsulated particles The chromatographic developability is inferior due to the excessively large. A preferred lower limit is 60% by weight and a preferred upper limit is 70% by weight.

The magnetic substance-encapsulated particles containing 50 to 80% by weight of the magnetic substance have a high content of the magnetic substance that is attracted to the magnet in one minute by using a neodymium magnet in the magnetic substance-encapsulated particle dispersion obtained in the manufacturing process, for example. The fraction can be collected as magnetic inclusion particles.
When particles having a magnetic substance content of more than 80% by weight are produced, they become a magnetic agglomerate and adhere to the wall surface of the polymerizer and are not mixed.

In the magnetic substance-encapsulated particles of the present invention, the lower limit of the average particle diameter is 50 nm, and the upper limit is 500 nm. When the thickness is less than 50 nm, the dispersion stability when suspended in a medium is deteriorated and the self-aggregation is liable to occur. When the thickness exceeds 500 nm, it is difficult to pass through the pores of the chromatographic carrier and the chromatographic developability is poor. A preferred lower limit is 100 nm and a preferred upper limit is 400 nm.

The magnetic substance-encapsulated particles of the present invention preferably have a particle diameter CV value of less than 50%. When it is 50% or more, particles having a large particle diameter are difficult to pass through the pores of the chromatographic carrier and may remain in the chromatographic carrier.

In the magnetic substance-encapsulated particles of the present invention, the preferable upper limit of the absolute deviation of the constituent ratio between the carbon element constituting the organic polymer substance and the metal element constituting the magnetic substance is 0.3.
In the present specification, the absolute deviation means the synchronous emission of the carbon element that constitutes the organic polymer substance and the metal element that constitutes the magnetic substance, and the mixture ratio of the carbon element and the metal element for each particle. It is a deviation value indicating the dispersion state of the measurement data calculated from the variation, and is a parameter indicating the variation of the magnetic substance content of the magnetic substance-encapsulated particles. The smaller the absolute deviation value is, the smaller the variation in the magnetic substance content, that is, the higher the uniformity of the magnetic substance-encapsulated particles, and the larger the value, the greater the variation in the magnetic substance content, that is, the uniformity of the magnetic substance-encapsulated particles. Indicates low.
When the absolute deviation exceeds 0.3, when used in an immunoassay, measurement reproducibility and quantitativeness are lowered and measurement accuracy may be deteriorated, and the reliability of measurement data obtained is lowered. A more preferred upper limit is 0.27, a still more preferred upper limit is 0.25, and a particularly preferred upper limit is 0.20.

The method for producing the magnetic substance-encapsulated particles of the present invention is not particularly limited, and examples thereof include a method applying a suspension polymerization method, a micro suspension polymerization method, a miniemulsion polymerization method, a dispersion polymerization method and the like. Among these, a method using the miniemulsion polymerization method is preferable because particles having a small particle diameter can be easily produced.
Specifically, a step of preparing a monomer mixture by mixing a magnetic dispersion in which a magnetic substance is dispersed in an organic solvent, and a monomer solution containing a monomer, a polymerization initiator, and a co-surfactant. 1. A step of preparing a mini-emulsion solution in which non-uniform monomer droplets are formed by dripping the monomer mixture into an aqueous medium in which a surfactant is dissolved and finely dispersing the mixture. Step 3 of polymerizing and preparing a magnetic substance-encapsulated particle dispersion, and a step of fractionating and recovering particles having a magnetic substance content of 50 to 80% by weight from the magnetic substance-encapsulated particle dispersion by a magnetic separation method 4 is a method for producing a magnetic substance-encapsulated particle having 4. Such a method for producing a magnetic substance-encapsulated particle is also one aspect of the present invention.
Hereinafter, the method for producing the magnetic substance-encapsulated particles of the present invention using the miniemulsion polymerization method will be described in detail.

In the method for producing a magnetic substance-encapsulated particle of the present invention, first, a magnetic substance dispersion liquid in which a magnetic substance is dispersed in an organic solvent and a monomer solution containing a monomer, a polymerization initiator, and a co-surfactant are mixed. Then, Step 1 for preparing the monomer mixture is performed.
Thus, once the magnetic dispersion is prepared and mixed with the monomer to prepare the monomer mixture, the magnetic substance is finely dispersed in the matrix made of the organic polymer material with the above-mentioned dispersion diameter. In addition, it is possible to obtain magnetic substance-encapsulated particles having a uniform magnetic substance content that achieves the absolute deviation value described above.

The organic solvent used in the magnetic dispersion liquid is not particularly limited as long as it is excellent in dispersibility of the magnetic substance, does not dissolve the magnetic substance, and can be mixed with the monomer.
As such an organic solvent, a solvent containing butanol or an aliphatic hydrocarbon solvent is suitable. As the aliphatic hydrocarbon solvent, a linear or branched one having 5 to 20 carbon atoms is preferable because the dispersibility of the magnetic substance is particularly excellent, and a straight or branched one having 5 to 7 carbon atoms. Is more preferred. Specific examples include pentane, hexane, heptane, isobutane, isopentane and the like. These aliphatic hydrocarbon solvents may be used alone or in combination of two or more.

The content of the aliphatic hydrocarbon solvent in the organic solvent is preferably 80% by weight or more. If it is less than 80% by weight, the magnetic substance cannot be sufficiently dispersed, and as a result, the dispersion diameter of the magnetic substance in the obtained magnetic substance-encapsulated particles becomes large, and the variation in the content of the magnetic substance becomes large. There are things to do.

The content of the organic solvent is preferably 20% by weight and preferably 500% by weight with respect to the magnetic substance. If the amount is less than 20% by weight, the magnetic substance may not be sufficiently dispersed. If the amount exceeds 500% by weight, the residual solvent may need to be removed after the polymerization step, and the operation may be complicated. A more preferred lower limit is 30% by weight, and a more preferred upper limit is 300% by weight. In addition, when using an organic solvent with a high boiling point, it is preferable to use a smaller amount.

Examples of the polymerization initiator include 2,2′-azobisisobutyronitrile, 2,2′-azobis- (2-methylpropanenitrile), 2,2′-azobis- (2,4-dimethylpentane). Nitrile), 2,2′-azobis- (2-methylbutanenitrile), 1,1′-azobis- (cyclohexanecarbonitrile), 2,2′-azobis- (2,4-dimethyl-4-methoxyvaleronitrile) ), 2,2′-azobis- (2,4-dimethylvaleronitrile), 2,2′-azobis- (2-amidinopropane) hydrochloride and other azo (azobisnitrile) type initiators, benzoyl peroxide , Cumene hydroperoxide, hydrogen peroxide, acetyl peroxide, lauroyl peroxide, persulfates (eg ammonium persulfate), peracid esters (eg t-butyl peroxide) Tate, alpha-cumyl peroxypivalate and t- butyl per octoate) peroxide type radical polymerization initiators such as and the like.
These polymerization initiators may be added when preparing the dispersion liquid of the monomer mixture droplets, or may be added separately after the adjustment.
Although it does not specifically limit as content of the said polymerization initiator, A preferable minimum is 0.1 weight part with respect to 100 weight part of monomers, and a preferable upper limit is 30 weight part.

The co-surfactant is not particularly limited as long as it is generally used in miniemulsion polymerization. For example, C ₈ -C ₃₀ straight chain, branched chain such as hexadecane, squalane, cyclooctane, etc. cyclic alkanes; _C 8 _{-C 30} alkyl thiols such as dodecyl mercaptan;; _C 8 _{-C 30} alkyl alcohols such as cetyl alcohol; stearyl methacrylate, _C 8 _{-C 30} alkyl acrylate and dodecyl methacrylate polyurethane, polyester, polystyrene, etc. Polymers; long chain aliphatic or aromatic carboxylic acids, long chain aliphatic or aromatic carboxylic esters, long chain aliphatic or aromatic amines, ketones, halogenated alkanes, silanes, siloxanes, isocyanate And the like. Of these, alkanes having 12 or more carbon atoms are preferable, and alkanes having 12 to 20 carbon atoms are more preferable. These cosurfactants may be used alone or in combination of two or more.
Although it does not specifically limit as content of the said co-surfactant, A preferable minimum is 0.1 weight part with respect to 100 weight part of monomers, and a preferable upper limit is 50 weight part.

When the monomer dispersion is prepared by mixing the magnetic dispersion liquid and the monomer, a surfactant as described later may be used as necessary.

In the method for producing magnetic substance-encapsulated particles of the present invention, the above-mentioned monomer mixture is then dropped onto an aqueous medium in which a surfactant is dissolved and finely dispersed to form a miniemulsion in which non-uniform monomer droplets are formed. Step 2 of preparing the solution is performed.

It does not specifically limit as said aqueous medium, Usually, distilled water, ion-exchange water, etc. are used.

The surfactant is not particularly limited, and any of an anionic surfactant, a cationic surfactant, and a nonionic surfactant can be used. Of these, anionic surfactants are preferred.

The anionic surfactant is not particularly limited, and examples thereof include dodecyl benzene sulfonate, decyl benzene sulfonate, undecyl benzene sulfonate, tridecyl benzene sulfonate, nonyl benzene sulfonate, and sodium, potassium, and ammonium salts thereof.

The cationic surfactant is not particularly limited, and examples thereof include cetyltrimethylammonium promide, hexadecylpyridinium chloride, hexadecyltrimethylammonium chloride and the like.

The nonionic surfactant is not particularly limited, and examples thereof include polyvinyl alcohol. Examples of the nonionic surfactant include Triton X-100, X-114, X-305, N-101 (above, manufactured by Union Carbide), Tween 20, 40, 60, 80, 85 (above. Manufactured by ICI), Brij 35, 58, 76, 98 (above, manufactured by ICI), Nonidet P-40 (manufactured by Shell), Igepol CO530, CO630, CO720, CO730 (Rhone) A commercially available product such as (made by Pulan Co., Ltd.) can be used. Of these, anionic surfactants are preferred.

As the surfactant, a reactive surfactant having a reactive group polymerizable with the monomer can also be used. As said reactive group, ethylenically unsaturated groups, such as a vinyl group, an allyl group, and a (meth) acryloyl group, are suitable, for example.
Such a reactive surfactant is not particularly limited, and examples thereof include those described in JP-A-9-279073. Specifically, examples of the anionic surfactant include alkylbenzene sulfonic acids such as lauryl (allylbenzene) sulfonate, lauryl styrene sulfonate, stearyl (allyl benzene) sulfonate, and stearyl styrene sulfonate. Salts or their polyethylene oxide adducts; alkyl sulfosuccinates such as lauryl allyl sulfosuccinate, lauryl vinyl sulfosuccinate, stearyl allyl sulfosuccinate, stearyl vinyl sulfosuccinate, or their polyethylene oxide Adducts; alkyl or alkenyl sulfonates such as (meth) acrylic acid lauryl sulfonate and oleyl sulfonate; alkyl such as (meth) acrylic acid stearyl sulfate and oleyl sulfate; Alkenyl sulfates or polyethylene oxide adducts thereof, and the like.

Examples of the cationic surfactant include quaternary ammonium salts such as lauryl triallyl ammonium chloride, stearyl triallyl ammonium chloride, and distearyl diallyl ammonium chloride.

Examples of the nonionic surfactant include polyethylene glycol alkyls or alkenyl phenyl ethers such as polyethylene glycol octyl (allylphenyl) ether, polyethylene glycol nonyl (allylphenyl) ether, polyethylene glycol oleyl phenyl ether; monoallyl monoallylate Glycerin fatty acid esters such as glyceryl and monoallyl glyceryl distearate or polyethylene oxide adducts thereof; sorbitan fatty acid esters such as monoallyl sorbitan monostearate and monoallyl sorbitan tristearate or polyethylene oxide adducts thereof; Polyethylene glycol mono (meth) acrylate, polyethylene glycol di (meth) acrylate, etc. Polyethylene oxide esters of acrylic acid and the like.

Examples of the reactive surfactant include Aqualon HS-10 (Daiichi Kogyo Seiyaku Co., Ltd.), Antox-MS-60, RA-1000 series, and Antox-MS-2N (Nippon Emulsifier) which are anionic surfactants. Adekaria soap SE-10N (Asahi Denka Kogyo Co., Ltd.), Teramuru S-180A (Kao Co., Ltd.), Eleminol JS-2 (Sanyo Kasei Kogyo Co., Ltd.); RF-751, a cationic surfactant (Manufactured by Nippon Emulsifier Co., Ltd.); Adekaria soap NE-10 (manufactured by Asahi Denka Kogyo Co., Ltd.) which is a nonionic surfactant, Blemmer PE-200, Blemmer PE-350, Blemmer PE-400 (manufactured by NOF Corporation), etc. A commercial item is mentioned.

These surfactants may be used alone or in combination of two or more.
As the content of the surfactant, a preferable lower limit is 0.01 part by weight and a preferable upper limit is 5 parts by weight with respect to 100 parts by weight of the monomer, a more preferable lower limit is 0.1 part by weight, and a more preferable upper limit is 30 parts by weight. Part.

The method for finely dispersing the droplets composed of the monomer mixture is not particularly limited. For example, the monomer mixture is added to an aqueous medium containing a surfactant and emulsified by a shear mixing device that generates a high shear force. Conventionally known methods such as a method of making them can be used.
The shear mixer is not particularly limited, and examples thereof include a piston homogenizer, a microfluidizer (for example, Microfluidizer (manufactured by Mizuho Kogyo Co., Ltd.)), an ultrasonic disperser, and the like. Of these, a probe-type ultrasonic disperser is preferably used.

When an ultrasonic disperser is used for fine dispersion, the preferable lower limit of the ultrasonic output is 5 W, and the preferable upper limit is 200 W. If it is less than 5 W, large droplets are generated due to insufficient dispersion force, and the polymerization reaction in the next step may be difficult. If it exceeds 200 W, desired magnetic encapsulated particles may not be obtained.
Moreover, although it is based also on an ultrasonic output as an ultrasonic irradiation time, it is preferable to carry out in the range for 10 to 30 seconds.

Here, in the case of a general mini-emulsion polymerization method in which completely uniform monomer droplets are formed, it is not possible to obtain magnetic content-encapsulated particles having a high content.
In this specification, the term “non-uniform” means that the dispersion step is stopped before the droplets become uniform. For magnetic inclusion particles recovered by the magnetic separation method, the particle diameter and magnetic substance content are the same for each particle. This is not to say that it is uneven. It is difficult to quantify the non-uniform condition, but it can be realized under the condition that the output of the ultrasonic homogenizer is low and the processing time is short.
Non-uniform monomer droplets use the fact that the droplet splitting efficiency changes depending on the amount of magnetic inclusions during the ultrasonic dispersion process even if the monomer droplets have the same ultrasonic output. Can be obtained by finishing the treatment in a non-uniform state until the droplet diameter becomes uniform. Further, the monomer droplets obtained by this method are droplets having a higher magnetic substance content than when the droplet diameter is made uniform. The magnetic substance-encapsulated particles can be obtained by polymerizing the monomer droplets in a non-uniform state and then collecting a fraction having a high magnetic substance content and high uniformity by a magnetic separation method.

In the method for producing magnetic particles for immunoassay of the present invention, the ultrasonic output and the emulsification time are adjusted, and the fine dispersion process is terminated in a non-uniform emulsified state. By avoiding such a completely uniform monomer droplet, it is possible to obtain magnetic substance-encapsulated particles having a high magnetic substance content.

In the method for producing a magnetic substance-encapsulated particle according to the present invention, next, Step 3 of polymerizing the monomer droplets to prepare a magnetic substance-encapsulated particle dispersion is performed.
The polymerization is usually carried out by heating at 50 to 95 ° C. for about 5 to 24 hours.

In the method for producing a magnetic substance-encapsulated particle according to the present invention, the step 4 of fractionating and recovering particles having a magnetic substance content of 50 to 80% by weight from the magnetic substance-encapsulated particle dispersion by a magnetic separation method is then performed. .

The fractionation method is not particularly limited, and examples thereof include purification methods such as a centrifugal separation method and a magnetic separation method. Thereby, what has a desired average particle diameter, a magnetic substance dispersion diameter, and magnetic substance content can be extract | collected, and the magnetic substance inclusion particle | grains of this invention can be obtained.
In particular, the magnetic separation method is preferably used among the purification methods. By shortening the treatment time of magnetic separation, magnetic substance-encapsulated particles having a high magnetic substance content can be recovered.

Since the magnetic substance-encapsulated particle of the present invention has a large content of magnetic substance, an extremely small amount of magnetic substance is obtained by binding or adsorbing an antigen or an antibody to the magnetic substance-encapsulated particle of the present invention to form an immunoassay particle. Even in the case of encapsulated particles, the amount of magnetism can be detected, and highly sensitive immunoassay can be performed.
Such immunoassay particles are also one aspect of the present invention.

In addition, since the magnetic substance-encapsulated particles of the present invention have an average particle diameter of 50 to 500 nm and a magnetic substance dispersion diameter of 1 to 30 nm, self-aggregation can be suppressed, and the magnetic particles of the present invention can be suppressed. The immunoassay particles to which antigens or antibodies are bound or adsorbed using the encapsulated particles as a support can be easily developed (moved) in a chromatographic carrier having a pore size of usually 5 to 20 μm. Furthermore, since it does not adsorb non-specifically to the chromatographic carrier, it is extremely excellent in chromatographic developability. Therefore, when the immunoassay particles of the present invention are used, an immunoassay method, particularly an immunochromatography method, in which analysis is performed using the magnetic content of the magnetic substance-encapsulated particles as a label can be suitably performed.
Such an immunoassay method using the immunoassay particles of the present invention is also one aspect of the present invention.

According to the present invention, when the magnetic substance-encapsulated particles are used in which the average particle diameter of the magnetic substance-encapsulated particles is a predetermined size and the dispersion diameter and content of the magnetic substance are within a specific range. Self-aggregation of particles is suppressed, chromatographic developability is improved, and highly sensitive immunoassay is possible.
According to the present invention, there are provided a magnetic substance-encapsulated particle having excellent chromatographic developability and capable of performing highly sensitive immunoassay, a method for producing a magnetic substance-encapsulated particle, an immunoassay particle, and an immunoassay method using the same. can do.

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

Example 1
(1) Preparation of magnetic body-encapsulated particles 10.0 g (containing 5 g of magnetic substance) of a magnetic fluid “Ferricolloid HC50 (made by Taiho Kogyo Co., Ltd.)” was dried in an incubator at 80 ° C. for 12 hours, and concentrated magnetic fluid 7 0.0 g was obtained. 3 g of hexane was added to the obtained magnetic fluid and left overnight to disperse the magnetic material to obtain a magnetic material dispersion.
10 g of styrene, 0.8 g of hexadecane and 0.05 g of 2,2′-azobisisobutyronitrile are added to the total amount of the obtained magnetic dispersion, and the mixture is mixed under ice cooling using a stirrer. A mixture was obtained.

Next, an aqueous solution obtained by dissolving 0.8 g of sodium dodecylbenzenesulfonate in 100 g of water was added to the obtained monomer mixture, and a probe type ultrasonic homogenizer (Sonifier model 250, manufactured by Branson) with an output of 200 W was used. Under ice-cooling, the mixture was treated for 2 minutes at an output scale of 5 (70 W) to prepare a mini-emulsion solution in which droplets of a monomer mixture containing a magnetic substance were dispersed in water.
The obtained mini-emulsion solution was polymerized at 80 ° C. for 24 hours under a nitrogen atmosphere to obtain a magnetic substance-encapsulated particle dispersion.
Next, of the obtained magnetic substance-encapsulated particle dispersion, a fraction having a high magnetic substance content that was attracted to the magnet in one minute using a neodymium magnet was fractionated as magnetic substance-encapsulated particles.

When the particle diameter of the obtained magnetic substance-encapsulated particles was measured by a dynamic light scattering method using a dynamic light scattering photometer (Photo PAR-IIIS, manufactured by Otsuka Electronics Co., Ltd.), the average particle diameter was 263 nm. . Moreover, after diluting the obtained magnetic substance-encapsulated particles with water and depositing and fixing on a collodion film supported by a metal mesh, the dispersion diameter of the magnetic substance was measured using a transmission electron microscope (TEM). It was 10-25 nm. Furthermore, the magnetic substance content in the magnetic substance-encapsulated particles was measured by TG-DTA analysis and found to be 67% by weight.

(2) Preparation of immunoassay particles 1 mL of aqueous potassium hydroxide solution at pH 9.5 was added to 5.0 mg of the magnetically encapsulated particles obtained, centrifuged at 15000 RPM for 20 minutes, the supernatant was removed, and the dispersion was added. Any surfactant present was removed. Subsequently, 1.0 mL of 0.02 M phosphate buffer was added to the obtained magnetic substance-encapsulated particles, redispersed with ultrasound, and centrifuged at 15000 RPM for 20 minutes to remove the supernatant. This centrifugal washing operation was repeated three times.

Subsequently, 1.0 mL of 0.02 M phosphate buffer was added to the obtained magnetic substance-encapsulated particles, 100 μg of anti-α-hCG monoclonal antibody was added, and the mixture was stirred in a 37 ° C. constant temperature bath for 2 hours. Thereafter, centrifugation was performed at 15000 RPM for 20 minutes to remove unreacted anti-α-hCG monoclonal antibody. The amount of anti-α-hCG monoclonal antibody bound to the particles was confirmed to be 72% of the preparation based on the protein concentration measurement of the supernatant.

The obtained magnetic substance-encapsulated particles were suspended in 1 mL of 100 mM phosphate buffer (pH 6.5) and centrifuged again. Thereafter, the magnetic substance-encapsulated particles are suspended in 1 mL of 100 mM phosphate buffer (pH 6.5) adjusted so that bovine serum albumin has a concentration of 1% (w / v) and stirred in a 37 ° C. constant temperature bath for 1 hour. Then, blocking treatment was performed. Next, centrifugation was carried out at 15000 RPM for 20 minutes, and 100 mM phosphoric acid adjusted so that bovine serum albumin and glycerol each had a concentration of 1% (w / v) and sodium azide to a concentration of 0.01% (w / v). It was dispersed in 1 mL of a buffer solution (pH 7.5) to obtain immunoassay particles.

(Example 2)
The average particle size was the same as in Example 1 except that it was treated with an output scale 3 (30 W) for 5 minutes under ice cooling using a probe-type ultrasonic homogenizer (Sonifer model 250, manufactured by Branson) with an output of 200 W. Magnetic body-encapsulated particles and immunoassay particles having a diameter of 310 nm, a magnetic dispersion diameter of 10 to 25 nm, and a magnetic substance content of 62% by weight were obtained.

(Comparative Example 1)
The magnetic fluid “Ferricolloid HC50” (4.0 g, containing 2 g of magnetic substance) was dried in an incubator at 80 ° C. for 12 hours to obtain 3.0 g of concentrated magnetic fluid. Next, 1 g of hexane was added to the obtained magnetic fluid and allowed to stand overnight to obtain a magnetic dispersion.
The obtained magnetic dispersion liquid was treated with an output scale 5 (110 W) for 10 minutes using a probe type ultrasonic homogenizer (Sonifier model 450, manufactured by Branson) with an output of 400 W, and the magnetic substance-encapsulated particle dispersion liquid Of these, the average particle diameter was 180 nm and the dispersion diameter of the magnetic substance was 10 to 25 nm, in the same manner as in Example 1, except that the fraction attracted to the magnet in 5 minutes using a neodymium magnet was collected as magnetic substance-encapsulated particles. Thus, a magnetic substance-encapsulated particle and an immunoassay particle having a magnetic substance content of 36% by weight were obtained.

(Comparative Example 2)
A mixed solution of 50 mL of 0.5 mol / L ferrous sulfate aqueous solution and 50 mL of 0.5 mol / L ferric sulfate aqueous solution was further mixed with 110 mL of 2 mol / L sodium hydroxide to obtain magnetite. 40 g of a 5 wt% sodium oleate solution was added to 5 g of the obtained magnetite to obtain a magnetic material coated with oleic acid. 1 g of hexane was added to 5 g of the obtained magnetic material to obtain a magnetic material dispersion.
The obtained magnetic dispersion liquid was treated with an output scale of 5 (110 W) for 10 minutes using a probe type ultrasonic homogenizer (Sonifier model 450, manufactured by Branson) with an output of 400 W, and dispersion of the magnetic substance encapsulated particles In the same manner as in Example 1 except that the fraction attracted to the magnet in 5 minutes by using a neodymium magnet was separated as magnetic substance-encapsulated particles, the average particle diameter was 230 nm, and the dispersion diameter of the magnetic substance was 20 to 20 A magnetic substance-encapsulated particle and an immunoassay particle having a content of 40 nm and a magnetic substance content of 41% by weight were obtained.

(Comparative Example 3)
The magnetic dispersion obtained in Example 1 was treated with an output scale of 7 (140 W) for 10 minutes using a probe-type ultrasonic homogenizer (Sonifier model 450, manufactured by Branson) with an output of 400 W, and magnetic An average particle diameter of 110 nm, magnetic material, as in Example 1, except that the fraction entrained in the encapsulated particle dispersion using a neodymium magnet was collected as a magnetic encapsulated particle in 60 minutes. Magnetic particle-containing particles and immunoassay particles having a dispersion diameter of 10 to 30 nm and a magnetic substance content of 28% by weight were obtained.

(Comparative Example 4)
Using commercially available magnetic substance-encapsulated particles (Estapol M1-030 / 40, manufactured by Merck & Co., Inc., average particle diameter 420 nm, magnetic dispersion diameter 15 to 35 nm, magnetic substance content 40.2% by weight) To 12.5 mg of the particles, 1 mL of a potassium hydroxide aqueous solution having a pH of 9.5 was added and centrifuged at 15000 RPM for 10 minutes, and then the supernatant was removed to remove the surfactant added to the dispersion. Subsequently, 625 μL of 0.02M phosphate buffer and 0.625 mL of a 2% carbodiimide solution (PBS buffer) prepared in advance were added to the resulting precipitate, and the mixture was stirred for 1.5 hours in a 37 ° C. constant temperature bath. . The reaction solution was centrifuged at 15000 RPM for 10 minutes, and after removing the supernatant, 1.2 mL of 0.02M phosphate buffer was added and redispersed with ultrasound. This centrifugal washing operation was repeated three times to remove unreacted carbodiimide.

Subsequently, 1.2 mL of 0.1 M borate buffer was added to the resulting precipitate, 200 μg of anti-α-hCG monoclonal antibody was added, and the mixture was stirred overnight in a 37 ° C. constant temperature bath. On the next day, 50 μL of a 30 mM glycine solution (borate buffer) was added to the reaction solution, and the mixture was stirred in a 37 ° C. constant temperature bath for 30 minutes. Thereafter, the mixture was centrifuged at 15000 RPM for 10 minutes to remove unreacted anti-α-hCG monoclonal antibody. The amount of anti-α-hCG monoclonal antibody bound to the particles was confirmed to be 65% of the amount prepared from the protein concentration measurement of the supernatant.

The obtained sediment was suspended in 1 mL of 100 mM phosphate buffer (pH 6.5), and centrifuged again. The obtained sediment was suspended in 1 mL of 100 mM phosphate buffer (pH 6.5) adjusted to a bovine serum albumin concentration of 1% (w / v), and stirred for 30 minutes in a 37 ° C. constant temperature bath. Blocking treatment was performed. Thereafter, the mixture was centrifuged at 15000 RPM for 10 minutes, and the sediment was adjusted so that bovine serum albumin and glycerol each had a concentration of 5% (w / v) and sodium azide had a concentration of 0.01% (w / v). Particles for immunoassay were obtained by dispersing in 1 mL of 100 mM phosphate buffer (pH 7.5).

(Evaluation)
About the particle | grains for immunoassay obtained in Examples 1-2 and Comparative Examples 1-4, performance evaluation was performed with the following method.

A nitrocellulose membrane (SRHF P70, manufactured by Nippon Millipore) is cut into a width of 20 cm and a length of 6 cm, and an anti-β-hCG monoclonal antibody is 2.0 mg / mL at a site (reaction site) 2 cm from the upper end in the length direction. A solution dissolved in Tris-HCl buffer (10 mmol / L, pH 7.4) was applied in a straight line having a width of 0.7 mm so as to have a concentration of 0.5 mm. Thereafter, after drying at 37 ° C. for 2 hours, 1 in a solution obtained by dissolving bovine serum albumin (manufactured by Wako Pure Chemical Industries, Ltd.) in a phosphate buffer solution (100 mmol / L, pH 7.5) to a concentration of 1% by weight. It was immersed for a period of time and subjected to a blocking treatment. Further, after washing with a solution obtained by dissolving sodium laurylbenzenesulfonate in a phosphate buffer solution (100 mmol / L, pH 7.5) so as to have a concentration of 0.1% by weight, the mixture was washed at room temperature in a silica gel desiccator. The test piece which dried and fixed the anti- beta-hCG monoclonal antibody was obtained.

The obtained test piece was cut into a width of 5 mm, a water absorption pad (AP22, manufactured by Nihon Millipore) with a width of 5 mm × length 20 mm at the upper end in the length direction, and a conjugate pad (glass) with a width of 5 mm × length 15 mm at the lower end. Fiber, manufactured by Nihon Millipore) and fixed with a transparent tape to obtain a test piece.

As test solutions, bovine serum albumin 1% (w / v), Triton-100 0.03% (w / v), and hCG concentrations become 0 mIU / mL, 10 mIU / mL, 50 mIU / mL, 100 mIU / mL Such a physiological saline was prepared.
Next, 10 μg of immunoassay particles were added to and mixed with 200 μL of each test solution, and then 100 μL was dropped onto the conjugate pad of the prepared test piece.

After 20 minutes from the dropping, the amount of magnetism at the reaction site of the test piece was measured using a commercially available GMR sensor (differential magnetic field sensor, manufactured by NVE).
The measurement results are shown in Table 1.

As shown in Table 1, in the immunoassay particles prepared in Examples 1 and 2, a constant magnetic amount was obtained in all the test pieces except for the case where the hCG concentration was 0 mIU / mL 20 minutes after the dropping. Was detected. Moreover, it was confirmed that the detected amount of magnetism depends on the hCG concentration. Thus, it was found that the immunoassay particles produced in Example 1 are useful for immunoassay methods using magnetism as a label.

On the other hand, in the immunoassay particles prepared in Comparative Examples 1 and 3, when hCG was at a high concentration, the amount of magnetism could be confirmed, but hCG was at a low concentration (Comparative Example 1: 10 mIU / mL, Comparative Example 3: 50 mIU / In the case of mL), since the amount of magnetism was below the detection limit, it could not be detected.
In addition, in the immunoassay particles prepared in Comparative Examples 2 and 4, coloring was observed based on the immunoassay particles in any test piece at the reaction site and the site up to the reaction site. In particular, there was a coloration in the vicinity of the site where the conjugate pad was superimposed, and it was confirmed that the particles for immunoassay were retained. Further, even when the test substance was not present (0 mIU / mL), it was found that the immunoassay particles were captured nonspecifically at the reaction site because the magnetic amount was detected.

According to the present invention, there are provided a magnetic substance-encapsulated particle having excellent chromatographic developability and capable of performing highly sensitive immunoassay, a method for producing a magnetic substance-encapsulated particle, an immunoassay particle, and an immunoassay method using the same. can do.

Claims (1)

It consists of an organic polymer material and a magnetic material dispersed in the organic polymer material, the dispersion diameter of the magnetic material in the organic polymer material is 1 to 30 nm, and the magnetic material is 50 to 80 wt. %, A method for producing magnetic inclusion particles having an average particle diameter of 50 to 500 nm,
Step 1 of preparing a monomer mixture by mixing a magnetic dispersion in which a magnetic substance is dispersed in an organic solvent, and a monomer solution containing a monomer, a polymerization initiator, and a cosurfactant.
A step of preparing a mini-emulsion solution in which non-uniform monomer droplets are formed by dropping the monomer mixture on an aqueous medium in which a surfactant is dissolved and finely dispersing the mixture with ultrasonic waves having an output of 5 to 200 W. 2,
Step 3 of polymerizing the monomer droplets to prepare a magnetic substance-encapsulated particle dispersion, and
A method for producing magnetic inclusion particles, comprising a step 4 of fractionating and collecting particles having a magnetic substance content of 50 to 80% by weight from the magnetic inclusion particle dispersion by magnetic separation.