JP2005232237A - Carrier particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a polymer particle for bioassay or bioseparation, which specifically extracts a bioactive substance such as a targeted protein, peptide, nucleic acid or small molecule compound or which is used for immunodiagnosis, molecular biology study, screening of a pharmaceuticals-use candidate material or separation of a useful biomaterial. <P>SOLUTION: The carrier particle is formed by copolymerizing (a) a monomer selected from glycidyl methacrylate and glycidyl acrylate and (b) a crosslinkable monomer. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention specifically extracts a physiologically active substance such as a target protein, peptide, nucleic acid or low molecular weight compound, or is used for immunodiagnosis, molecular biological research, screening of drug candidate substances, separation of useful biomaterials, etc. Related cross-linked polymer particles for bioassay and bioseparation.

In recent years, diagnosis is performed by separating / purifying specific markers such as genes, proteins, sugar chains, ligands, and receptors from B / F separation from various specimens such as cells, blood, urine, etc. Various assays such as analysis and screening are performed. Alternatively, treatments such as amplification, biomanipulation, biosynthesis and the like, and production of useful substances such as pharmaceuticals and various bio-produced chemicals are starting to be performed from the specimen based on the separated and purified product by bioseparation.
In these bioassays or bioseparations, an immobilized carrier that is solidified using a physiologically active substance that specifically acts on in vivo substances as a probe is used, and so-called B / F separation operations using the immobilized carrier are widely performed. Yes. Various plates, affinity chromatography column fillers, immunoassay particles, and the like are used as the immobilization carrier used here. Among these, particles are frequently used because of their large surface area and high reactivity with specimens when used in solution.
As for the particle | grains with a probe used here, it is desirable for a probe particle to have the function to capture | acquire only the specific substance which interacts, and it is desirable for a support particle not to interact with a non-specific substance.
This is because when non-specific substances in the sample substance interact with the carrier particles, it becomes a background and the S / N ratio is lowered or the separation efficiency is lowered. However, it is difficult for a carrier to have a function of recognizing the ability to discriminate between specific substances and non-specific substances, and it is a carrier that does not interact with all specimens including both specific substances and non-specific substances. This is required as the next best measure.
Conventionally, cellulosic gels such as Sepharose, silica, beads such as polystyrene / divinylbenzene, hybrid particles of styrene divinylbenzene and magnetic particles, and the like have been used as carrier particles for B / F separation.
Currently used carrier particles have a problem in that they interact with analytes. Background and separation / purification have been pointed out as a problem of reduced purification efficiency, and carrier particles with less interaction are required. ing. In order to improve these problems, for example, Japanese Patent Application Laid-Open No. 58-171670 discloses an immunologically active substance in which an immunoactive substance having an amino group is immobilized on a glycidyl (meth) acrylate fine particle by a covalent bond. . This fine particle is described as solving the problem of non-specific adsorption of proteins of conventional styrene beads.
However, these particles have a problem that glycidyl methacrylate has a low glass point transfer of XX ° C. and thus tends to agglomerate, lacks mechanical strength, and is difficult to stabilize and redisperse.
JP-A-55-110118 and JP-A-4-218772 disclose copolymer particles of styrene and glycidyl methacrylate. These particles have a non-specific bond reducing effect as compared with conventional styrene divinylbenzene particles, but according to the above-mentioned JP-A-58-171670, the non-specific binding property is inferior to a glycidyl methacrylate group homopolymer. Has been.

Japanese Patent Nos. 2753762 and 3086427 disclose particles in which the surfaces of styrene glycidyl methacrylate particles are further covered with glycidyl methacrylate. This particle is presumed that the problem of mechanical strength and aggregation is solved because styrene is copolymerized with the core particle, and that non-specific protein adsorption is also reduced because the surface is polyglycyridyl methacrylate. The It is said that the particle | grains which couple | bonded the chemical | medical agent through this spacer have an extremely low background compared with the styrene glycidyl methacrylate whose surface is not covered with glycidyl methacrylate.
These particles are obtained by so-called two-stage polymerization in which styrene glycidyl methacrylate particles are prepared and then the surface of the polymer obtained by adding glycidyl methacrylate is completely covered.
In general, in the second-stage polymerization, it is difficult to confirm whether or not the shell polymer is uniformly covered on the surface of the core particles, and it is impossible to deny the possibility that some particles are not completely covered. If such particles are present, the background and separability are reduced.
JP 58-171670 A JP-A-55-111018 JP-A-4-218772 Patent 2753762 Patent No. 3086427

In this way, the current carrier particles (1) have low interaction with analytes such as proteins and have stable performance. (2) The mechanical strength and stability of the particles (1) ) There is a problem in coexistence of (2), and particles having both of these characteristics are demanded.

The present inventors have realized that mechanical strength and stability can be cleared by copolymerizing a non-aromatic vinyl compound and an epoxy group-containing (meth) acrylate in one step and crosslinking the resulting particles. The present invention has been completed by finding that stable particles having a stable reduction in adsorption of proteins and the like, and mechanical strength and stability can be obtained.
The present invention provides carrier particles obtained by copolymerizing a) a monomer selected from glycidyl methacrylate and glycidyl acrylate and b) a crosslinkable monomer.
Examples of the crosslinkable monomer include civinylbenzene, ethylene glycol dimethacrylate, oligoethylene glycol dimethacrylate, and tetramethylene glycol dimethacrylate. As the crosslinkable monomer, non-aromatic ethylene glycol is more aromatic than aromatic. Dimethacrylate, tetramethylene glycol dimethacrylate and the like are preferable, and the addition amount is 30 mol% or less, preferably 10 mol% or less, based on the total monomer amount.
However, polyfunctional acrylates such as ethylene glycol dimethacrylate, tetramethylene glycol dimethacrylate, and oligoethylene glycol dimethacrylate can be used not only as a crosslinkable monomer but also as a monomer constituting the carrier particles. These monomers can be used beyond the above range.

Examples of the polymerization method of these monomers include emulsion polymerization, soap-free polymerization, suspension polymerization, precipitation polymerization, dispersion polymerization, miniemulsion polymerization, two-stage swelling polymerization of Ugelstad et al., And film emulsion polymerization. As a method for obtaining polymer particles, emulsion polymerization, soap-free polymerization, and two-stage swelling polymerization are preferable. Hereinafter, the emulsion polymerization will be described as an example. The addition method of the monomer used here is not particularly limited, and can be divided addition or continuous addition in addition to the polymerization method of batch addition.
The emulsifier used here can be so-called soap-free polymerization, and any of an anionic emulsifier, a positive on emulsifier, and a non-on emulsifier can be used. Anionic emulsifiers such as sodium dodecylbenzenesulfonate, sodium lauryl sulfate, sodium polyoxyethylene lauryl ether sulfate, cetyl trimethyl ammonium chloride as cationic emulsifier, and polyoxyethylene lauryl ether as nonionic emulsifier can be used. is there.

As the polymerization initiator, a polymerization initiator usually used in emulsion polymerization can be used. Examples of these polymerization initiators include persulfates such as potassium persulfate and ammonium persulfate, redox polymerization catalysts such as hydrogen peroxide / ferrous chloride, azo initiators such as azobisisobutyronitrile, benzoyl peroxide, and the like. A peroxide initiator such as t-butyl hydroperoxide can be used.

The conditions for these emulsion polymerizations are usually preferably in the temperature range of 20 ° C. to 80 ° C., and the concentration of the monomer in the polymerization solution is usually preferably 1 to 50% by weight. However, if it is less than 1% by weight, the particle concentration is dilute and the productivity is poor.
After the emulsion polymerization, it is preferable to remove the monomer by steam stripping if necessary, and adjust the particle surface by pH adjustment, dialysis, ultrafiltration / centrifugation or the like.

In the present invention, when polymerizing the carrier particles, c) a non-aromatic vinyl compound may be further used as a monomer other than the components a) and b).
These non-aromatic vinyl compounds include methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, propyl (meth) acrylate, methoxyethyl (meth) acrylate, methoxypropyl (meth) acrylate, methoxybutyl (Meth) acrylate, ethoxymethyl (meth) acrylate, ethoxyethyl (meth) acrylate, ethoxypropyl (meth) acrylate, ethoxybutyl (meth) acrylate, propoxymethyl (meth) acrylate, propoxyethyl (meth) acrylate, propoxypropyl ( As meth) acrylate, propoxybutyl (meth) acrylate, alkoxyalkyl (meth) acrylate, methoxymethyl (meth) acrylate, methoxyethyl (meth) Acrylate, methoxypropyl (meth) acrylate, methoxybutyl (meth) acrylate, ethoxymethyl (meth) acrylate, ethoxyethyl (meth) acrylate, ethoxypropyl (meth) acrylate, ethoxybutyl (meth) acrylate, propoxymethyl (meth) acrylate , Propoxyethyl (meth) acrylate, propoxypropyl (meth) acrylate, propoxybutyl (meth) acrylate, and glycerin mono (meth) acrylate. These may be used alone or in combination of two or more. These non-aromatic vinyl compounds can be copolymerized with the component a), and further, unlike styrene monomers, non-specific adsorption with proteins is required to be as little as homopolyglycidyl methacrylate.

Among the non-aromatic vinyl compounds, cyclohexyl (meth) acrylate and 2-methoxyethyl (meth) acrylate have extremely low interaction with proteins as compared with styrene monomers. Furthermore, cyclohexyl (meth) acrylate has a high Tg, and a mechanical strength equivalent to that of a styrene monomer can be realized.
It should also be noted that cyclohexyl (meth) acrylate and 2-methoxyethyl (meth) acrylate are hydrophobic acrylates, but have less non-specific protein adsorption, similar to glycidyl methacrylate homopolymers. is there.

In the present invention, the epoxy group possessed by the carrier particles can be used directly as a chemical bonding group when the physiologically active substance has an amino group. It is also possible to combine with a spacer having an epoxy group at the terminal.
In addition, after binding a physiologically active substance directly or via a spacer or the like to a carrier particle, hydrochloric acid is blocked with various blocking agents in order to prevent non-bonded epoxy groups from binding nonspecifically to proteins. It is desirable to use a method such as treatment with ethanolamine or hydrolysis to convert to a glycol group as described below. As a blocking agent generally used here, a nucleic acid having a sequence that does not interact with a specimen, bovine serum albumin (BSA), gelatin, glycine, alanine, or the like can be used.

In the present invention, it is preferable that the carrier particles further have either a carboxyl group or an amino group.
These groups can be introduced by producing carrier particles by copolymerizing carboxylic acid monomers such as methacrylic acid, acrylic acid, fumaric acid and itaconic acid, and amino group-containing monomers such as vinylbenzylamine.

In the present invention, the physiologically active substance and the carrier particles can be bound using an epoxy group and other polar groups such as carboxylic acid and amino acid.
Examples of physiologically active substances that can be bound to the carrier particles of the present invention include oligonucleotides, proteins, glycoproteins, lipids, sugar chains, lectins, cells, protein-expressing cells, aptamers, viruses, enzymes, transcription factors, and pharmacological activities. It is possible to use a lead compound having, or a chemical substance having or having a specific physiological activity.
In the present invention, it is preferable that the method for binding the polar group of the particle carrier and the physiologically active substance can be basically performed by chemical bonding. For example, the epoxy group on the surface of the carrier particles using the carboxylic acid, amino 碁, SH group, OH group, aldehyde group of the lead compound capable of affinity reaction with the target substance to be separated, such as protein, nucleic acid, etc. It can be fixed by chemically reacting with a carboxyl group, an amino group or a hydroxyl group. In order to maintain the physiological activity of the probe substance, a predetermined functional group can be introduced through a spacer as necessary.
In addition, after binding the physiologically active substance and the particles using these polar groups, the residual epoxy group is prevented from binding to the protein in a non-specific manner as described above in order to prevent the residual epoxy groups on the particle surface from binding nonspecifically. It is desirable to perform an activation process.

The carrier particles of the present invention can be converted into glycol groups by adding water and, if necessary, hydrolyzing.
This hydrolysis can be carried out by reducing the pH to 3 or less with an acid or to pH 9.5 or more with an alkali. For example, specifically, the pH is adjusted to pH 2 with hydrochloric acid in an aqueous dispersion of carrier particles. It is possible to adjust and treat at room temperature for 24 hours. It is also possible to accelerate the hydrolysis reaction by heating to 60 to 100 ° C.
By hydrolyzing the epoxy group into a glycol group, it is possible to further reduce non-specific protein adsorption than the epoxy group-containing particles.
Epoxy groups are generally unstable in solution and may gradually open, and particles having an epoxy group on the surface are generally stored as powder particles to delay the ring opening (Dynabeads M270 Epoxy (Dynal Corporation)), In the reaction with the specimen, it is further dispersed in an aqueous solution. However, additional operations such as a powder particle process and a redispersion process are necessary, and there are concerns about poor redispersibility due to aggregation during storage of powder particles. Additional operations or concerns are dispelled.

As these spacers, double-stranded DNA having 2 to 20 bases, ethylene glycol derivatives having 2 to 40 linkages, silane coupling agents having 5 to 30 carbon chains, and the like can be used.
By using these spacers to bind a bioactive substance to the end of the spacer, it is possible to minimize the decrease in the interaction between the bioactive substance and the specific substance in the sample due to steric hindrance of the polymer particles. It is.
Specifically, for example, double-stranded DNA has amino and thiol ends, and ethylene glycol derivatives include ethylene glycol diglycidyl ether (EGDA (Wako Pure Chemical Industries, Ltd.)), Denacol (Nagase Chemtech Co., Ltd.). Ethyne glycol (manufactured by Nippon Oil & Fats Co., Ltd.) having an amino group, a carboxyl group, a thiol group, an aldehyde group, and a succinimide group can be used.

In the present invention, a physiologically active substance can be chemically bonded to the carrier particle surface or the spacer end.
The physiologically active substance that can be supported on the carrier particles in the present invention is not particularly limited as long as it has physiological activity, and specifically, the physiologically active substance described above can be used. As for the binding method with these physiologically active substances, in general, when binding to the epoxy group on the particle surface, binding with the amino group of the physiologically active substance is used. A carbonyl group of a physiologically active substance can be used.
When a physiologically active substance is attached via a spacer, an epoxy, carboxyl group, thiol group, aldehyde group, succinimide group, or the like is used at the end of the spacer.

Based on the above results, the product of the present invention has almost no non-specific adsorption by the protein. On the other hand, the binding reaction with the probe to be immobilized is efficient, and a large amount of the probe can be bound, so that a high signal intensity is obtained in the immunoassay. It can be seen that the carrier is suitable for efficient extraction, separation, and detection of target substances from contaminants, biological specimens containing a large amount of impurities, and bioassay specimens.
Example 1
In a four-necked glass flask equipped with a stirrer, 3000 parts of ion exchange water and 0.2 part of sodium dodecylbenzenesulfonate were added, and the pH of the system was adjusted to 6.5. The temperature was raised to 75 ° C with nitrogen substitution, 99 parts of glycidyl methacrylate, 1 part of ethylene glycol dimethacrylate and 2 parts of 2,2'-azobisisobutyronitrile (AIBN) were added, and polymerization was carried out at 75 ° C for 6 hours. Was done. When examined after cooling, the pH of the system was 6.2, the polymerization conversion rate was 99.5%, and the particle size of the polymer particles obtained was 0.35 μm. Let the particle | grains obtained here be the particle | grains of Example 1. FIG.

Example 2
Particles were obtained in the same manner as in Example 1 except that the monomer composition and initiator of Example 1 were 98 parts of glycidyl methacrylate, 2 parts of ethylene glycol dimethacrylate, and 2 parts of BPO as the initiator.

Example 3
Particles were obtained in the same manner as in Example 1 except that 70 parts of glycidyl methacrylate and 30 parts of cyclohexyl methacrylate were used as the monomers in Example 1.

Example 4
The composition of the monomer in Example 1 was the same as in Example 1 except that 70 parts of glycidyl methacrylate, 30 parts of oligoethylene glycol (n = 20) dimethacrylate, and 2 parts of benzoyl peroxide (BPO) instead of AIBN were used. Particles were obtained.

Example 5
Particles were obtained in the same manner as in Example 1 except that the monomer composition in Example 1 was 90 parts of glycidyl acrylate, 10 parts of ethylene glycol dimethacrylate, and 3 parts of AIBN.

Comparative Example 1
Particles were obtained in the same manner as in Example 1 except that the monomer composition in Example 1 was changed to 70 parts of glycidyl methacrylate and 30 parts of styrene.
Comparative Example 2
Particles were obtained in the same manner as in Example 1 except that the monomer composition in Example 1 was 90 parts of glycidyl acrylate and 10 parts of divinylbenzene.

Example 6
Particles were obtained in the same manner as in Example 1 except that the monomer composition in Example 1 was 95 parts of glycidyl methacrylate and 5 parts of methacrylic acid.
Example 7
Particles were obtained in the same manner as in Example 1 except that the monomer composition in Example 1 was changed to 70 parts of glycidyl methacrylate and 30 parts of methoxyethyl acrylate.
Example 8
Particles were obtained in the same manner as in Example 1 except that the monomer composition in Example 1 was changed to 70 parts glycidyl methacrylate and 30 parts glycerin monomethacrylate (GLM).
Example 9
The dispersion liquid of the particles of Examples 1, 6, and 7 was adjusted to pH 11 with 1% by weight sodium hydroxide, and subjected to a heat treatment at 98 ° C. for 6 hours to hydrolyze the epoxy groups of the particles. After cooling, the pH was adjusted to 8 to obtain particles.

Comparative Example 3
The dispersion liquid of the particles of Comparative Examples 1 and 2 was adjusted to pH 11 with 1% by weight sodium hydroxide and subjected to a heat treatment at 98 ° C. for 6 hours to hydrolyze the epoxy groups of the particles. After cooling, the pH was adjusted to 8 to obtain particles.

Example 10
The particle dispersions of Examples 1, 2, 3 and 4 were adjusted to pH 2 with 1N aqueous hydrochloric acid, and acid treatment was performed at 25 ° C. for 48 hours to hydrolyze the epoxy groups of the particles. After cooling, the pH was adjusted to 8 to obtain the particles of Example 3.
Comparative Example 4
The dispersions of the particles of Comparative Examples 1 and 2 were adjusted to pH 2 with a 1N hydrochloric acid aqueous solution and subjected to acid treatment at 25 ° C. for 48 hours to hydrolyze the epoxy groups of the particles. After cooling, the pH was adjusted to 8 to obtain particles 14.
Comparative Example 5
Particles were obtained in the same manner as in Example 1 except that the monomer composition in Example 1 was changed to 100 parts of styrene.

The particles obtained by the above operation are as shown in Table 1 below.

Example 10 Evaluation of Nonspecific Adsorption of γIgG by Particle Carrier and Protein Take 2 ml of 10% dispersion of each particle (No1-20) obtained in the above Example and Comparative Example (particle dry amount 0.2g), 15000rpmx1 Centrifuge at time to remove the clear supernatant, make up to 2 ml with sterile distilled water, disperse by vortex, and then centrifuge again under the same conditions. This operation was repeated three times to remove soluble components contained in the particle dispersion.
Each of the purified particles was finally dispersed in 1 ml of physiological saline (PBS, pH7), and 1 ml of 30 mg-γG / ml-PBS (Rabbit, Sigma FrII, III) solution was added thereto, and vortexed. The mixture was rotationally stirred for 10 minutes at a rate of 1 rpm / min in a constant temperature bath at ° C. In addition, in order to exclude the adsorption amount of the tube container wall of γG, a tube without particles was simultaneously prepared as a control, and 1 ml of sterilized distilled water was added in place of the 1 ml particle dispersion, and the same operation was performed. . The particle dispersion incubated at 37 ° C. for 1 hour was centrifuged at 15000 rpm × 1 hour, and 1.5 ml of the supernatant was collected so as not to break the particle pellet, transferred to a new tube, and centrifuged again at 15000 rpm × 1 hour. Subsequently, 1 ml of the supernatant was carefully transferred to a new tube and centrifuged a third time at 15000 rpm × 1 hour. Finally, 0.5 ml of a transparent supernatant was collected and the absorbance was measured at 280 nm. The amount of γG adsorbed on each particle was calculated by comparing and calculating the absorbance of the 30 mg-γG / ml solution before addition to the particles and the absorbance of the control. The results are shown in Table 3. In the calculation, an extinction coefficient of 1.4 of 1 mg γG / ml was adopted. The particle surface area was obtained geometrically using the particle size obtained by the photodynamic scattering method (Otsuka Electronics Co., Ltd., Photoal LPA3100).
Example 11
Nonspecific adsorption evaluation of particle carrier and serum protein Nonspecific adsorption of particle carrier was evaluated in the same manner as in Example 10 except that calf serum (BSA) was used instead of γG. The results are shown in Table 3.

実施例１２
粒子担体の免疫測定への応用-1
実施例１１と同様な方法で全粒子を洗浄し、粒子固形分１０％分散液0.1mlに、０．１M硫酸アンモニア0.1mlを加え、ボルテックスしてから、抗ＡＦＰマウスIgG抗体溶液（０．１ｍｇ/ｍL、１０ｍＭ ＭＥＳ−ＮａＯＨ（ｐＨ６））を0.1ｍL加え、３７℃で1rpm/分で一晩回転攪拌した。続いて、１ｍｌPBSで実施例１０と同様な条件で3回洗浄した。次に、粒子に０．１％ＢＳＡ含有リン酸緩衝塩溶液（ｐH 7.4）を１ｍL加え室温で回転攪拌機にて2時間攪拌した。その後、実施例、比較例で得られた粒子をすべてリン酸緩衝液（ｐH 7.4）で２回洗浄後、粒子濃度が０．５重量％となるように０．１％ＢＳＡ含有リン酸緩衝液（ｐH 7.4）に再分散し、抗ＡＦＰＩｇＧ結合粒子とした。
この免疫測定用粒子１０μLに１００ｎｇ／ｍＬのＡＦＰを含むサンプル５０μLを加えて攪拌し、室温で５分放置した。遠心分離法により免疫測定用粒子をリン酸緩衝塩溶液（ＰＢＳ）で２回洗浄を行った。
次に、アルカリフォスファターゼコンジュゲート抗ＡＦＰ抗体（抗体濃度２．５μg／ｍＬ、０．１％ＢＳＡ／ＰＢＳ）を１００μL加えて攪拌し、室温で１０分放置した。同様な方法により洗浄を行った。この粒子にＡＭＰＰＤ２００μg／ｍＬを含む基質液１００μLを加え攪拌の後、１０分間放置後、ルミノメータ（ベルトールド社製）で測定した。表４に抗原濃度０、100ng/mlでの抗体結合量と化学発光強度を示す。

Example 12
Application of particle carrier to immunoassay-1
All particles were washed in the same manner as in Example 11, 0.1 ml of 0.1M ammonia sulfate was added to 0.1 ml of a 10% solids content dispersion, vortexed, and then anti-AFP mouse IgG antibody solution (0.1 mg). / mL, 0.1 mM of 10 mM MES-NaOH (pH 6)) was added, and the mixture was rotated and stirred overnight at 37 ° C. at 1 rpm / min. Subsequently, the plate was washed 3 times with 1 ml PBS under the same conditions as in Example 10. Next, 1 mL of 0.1% BSA-containing phosphate buffer solution (pH 7.4) was added to the particles, and the mixture was stirred at room temperature with a rotary stirrer for 2 hours. Thereafter, all the particles obtained in Examples and Comparative Examples were washed twice with a phosphate buffer (pH 7.4), and then a phosphate buffer solution containing 0.1% BSA so that the particle concentration became 0.5% by weight. Re-dispersed in (pH 7.4) to obtain anti-AFP IgG binding particles.
50 μL of a sample containing 100 ng / mL AFP was added to 10 μL of the immunoassay particles, and the mixture was stirred and allowed to stand at room temperature for 5 minutes. The immunoassay particles were washed twice with a phosphate buffered saline (PBS) by centrifugation.
Next, 100 μL of alkaline phosphatase-conjugated anti-AFP antibody (antibody concentration 2.5 μg / mL, 0.1% BSA / PBS) was added and stirred, and left at room temperature for 10 minutes. Washing was performed in the same manner. To this particle, 100 μL of a substrate solution containing 200 μg / mL of AMPPD was added, stirred, allowed to stand for 10 minutes, and then measured with a luminometer (Berthold). Table 4 shows the antibody binding amount and chemiluminescence intensity at antigen concentrations of 0 and 100 ng / ml.

実施例１２ 粒子担体の免疫測定への応用-2
実施例６にて得られた粒子１０％分散液１０ｍｌを70ｍｌ0.01NNaOHに希釈し、0.05N H2SO4を用いて伝道度滴定法で粒子表面のカルボン酸量を測定した。その結果128ｎｍｏｌ／gであることを確認した。
この粒子の１０％分散液１ｍｌを１０ｍＭ ＭＥＳ−ＮａＯＨ（ｐＨ６）５ｍLに分散させ、実施例１同様な抗ＡＦＰマウスIgG抗体溶液を同量に加え、室温で回転攪拌機にて３０分間攪拌した。これに水溶性カルボジイミド ５ｍｇを加え、室温で回転攪拌機にて2時間攪拌した。2時間後、上清を除去した。次に、粒子に０．１％ＢＳＡ含有リン酸緩衝塩溶液（ｐH 7.4）を１０ｍL加え室温で回転攪拌機にて2時間攪拌し、リン酸緩衝液（ｐH 7.4）で２回洗浄後、粒子濃度が０．５重量％となるように０．１％ＢＳＡ含有リン酸緩衝液（ｐH 7.4）に再分散し、抗ＡＦＰＩｇＧ結合磁性粒子とした。実施例１１同様な方法でアッセイを行い、その結果抗原０ng/mlのとき、発光値は１２９、抗原100ng/mlのとき、発光値は477000であった。

Example 12 Application of Particle Carrier to Immunoassay-2
10 ml of the 10% particle dispersion obtained in Example 6 was diluted to 70 ml 0.01 N NaOH and the amount of carboxylic acid on the particle surface was measured by the conductance titration method using 0.05N H2SO4. As a result, it was confirmed that it was 128 nmol / g.
1 ml of a 10% dispersion of these particles was dispersed in 5 mL of 10 mM MES-NaOH (pH 6), the same amount of the same anti-AFP mouse IgG antibody solution as in Example 1 was added, and the mixture was stirred at room temperature with a rotary stirrer for 30 minutes. To this was added 5 mg of water-soluble carbodiimide, and the mixture was stirred at room temperature with a rotary stirrer for 2 hours. After 2 hours, the supernatant was removed. Next, 10 mL of 0.1% BSA-containing phosphate buffer solution (pH 7.4) was added to the particles, stirred at room temperature for 2 hours with a rotary stirrer, washed twice with phosphate buffer (pH 7.4), and then the particle concentration Was re-dispersed in a phosphate buffer solution (pH 7.4) containing 0.1% BSA to give 0.5 wt% to obtain anti-AFP IgG-binding magnetic particles. The assay was conducted in the same manner as in Example 11. As a result, when the antigen was 0 ng / ml, the luminescence value was 129, and when the antigen was 100 ng / ml, the luminescence value was 477000.

Claims (7)

Carrier particles obtained by copolymerizing a) a monomer selected from glycidyl methacrylate and glycidyl acrylate and b) a crosslinkable monomer. The carrier particle according to claim 1, wherein the crosslinkable monomer is ethylene glycol dimethacrylate or tetramethylene glycol dimethacrylate. Further, c) at least one selected from cyclohexyl (meth) acrylate, ethylene glycol di (meth) acrylate, oligoethylene glycol di (meth) acrylate, 2-methoxyethyl (meth) acrylate, and glycerin mono (meth) acrylate 2. The carrier particles according to claim 1, which are copolymerized. The carrier particle according to any one of claims 1 to 3, wherein d) is further copolymerized with a monomer having either a carboxyl group or an amino group. The carrier particle formed by hydrolyzing the carrier particle in any one of Claims 1-4. A carrier particle obtained by chemically bonding a spacer to the carrier particle according to claim 1. A carrier particle obtained by chemically bonding a physiologically active substance to the carrier particle according to claim 1.