JP2005083905A - Magnetic particle - Google Patents
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Abstract
Description
本発明は、生化学・医学分野での細胞の分離精製用の担体として磁性粒子に関する。 The present invention relates to magnetic particles as a carrier for separation and purification of cells in the biochemistry and medical fields.
近年、磁性粒子はその表面に抗体や抗原などのプローブを固定化し、多様な細胞群のなかから一種または複数種の細胞を特異的に捕捉し遺伝子検査などを行う細胞捕捉用担体として用いられている。このような細胞捕捉用担体においては、抗体、抗原、レクチン等のプローブが磁性粒子上に固定化され、サンプル中の細胞、特に動物細胞がプローブを介して磁性粒子上に捕捉される。続いて、細胞を免疫学的に染色することによりフローサイトメータを用いた解析に用いられたり、核酸抽出などの操作が行われた後、得られた細胞中の核酸（以下、検査対象核酸）をポリメラーゼ連鎖反応（ＰＣＲ）等の核酸増幅法などによる遺伝子検査に用いられる。近年、疾病の早期発見などの目的のため検査の高感度化が求められており、検査の感度向上は大きな課題となっている。磁性粒子を用いた細胞捕捉担体においては、血液中の癌細胞の検出等に用いることにより癌の早期発見が期待され、数個の細胞を捕捉・検出するための技術開発が行われている。 In recent years, magnetic particles have been used as cell capture carriers to immobilize probes such as antibodies and antigens on their surfaces, specifically capture one or more types of cells from various cell groups, and perform genetic testing and the like. Yes. In such a cell-capturing carrier, probes such as antibodies, antigens, and lectins are immobilized on the magnetic particles, and cells in the sample, particularly animal cells, are captured on the magnetic particles via the probes. Subsequently, the cells are used for analysis using a flow cytometer by immunologically staining the cells, or after nucleic acid extraction and other operations are performed, the nucleic acids in the cells obtained (hereinafter referred to as test target nucleic acids) Is used for genetic testing by a nucleic acid amplification method such as polymerase chain reaction (PCR). In recent years, for the purpose of early detection of diseases and the like, it has been demanded to increase the sensitivity of examinations, and improving the sensitivity of examinations has become a major issue. In cell capture carriers using magnetic particles, early detection of cancer is expected by using it for detection of cancer cells in blood and the like, and technical developments for capturing and detecting several cells have been carried out.
これらの技術の発展により理論上は一個の細胞の存在まで検出できるレベルに達しているといわれているが、実際には十分な感度は得られていない。その原因のひとつとして、磁性粒子への細胞の非特異的な吸着が上げられる。例えば、血液中の癌細胞を癌特異抗体を結合した磁性粒子で捕捉する場合、血液中には癌細胞以外にリンパ球、好中球、好酸球、好塩基球などの細胞が多数存在し、これらが磁性粒子に非特異的に吸着するとフローサイトメータでのノイズとなったり、遺伝子検査では核酸増幅に持ち込める核酸量の制限からサンプルの一部しか使えない結果となり感度低下を引き起こす。
また、遺伝子検査においては、磁性粒子表面への核酸の非特異的な吸着がおこると、細胞から抽出した核酸が磁性粒子に非特異的に吸着してしまえば検出が不可能となる場合がある。
このようなことから磁性粒子表面への細胞および核酸の非特異的な吸着の抑制が強く求められている。
The development of these technologies is theoretically said to have reached a level where even the presence of a single cell can be detected, but in reality, sufficient sensitivity has not been obtained. One of the causes is nonspecific adsorption of cells to magnetic particles. For example, when capturing cancer cells in blood with magnetic particles bound with cancer-specific antibodies, there are many cells such as lymphocytes, neutrophils, eosinophils, and basophils in addition to cancer cells in the blood. When these non-specifically adsorb to magnetic particles, noise is generated in the flow cytometer, and in genetic testing, only a part of the sample can be used due to the limitation of the amount of nucleic acid that can be brought into nucleic acid amplification, resulting in a decrease in sensitivity.
In addition, in genetic testing, if non-specific adsorption of nucleic acids to the surface of magnetic particles occurs, detection may be impossible if nucleic acids extracted from cells are non-specifically adsorbed to magnetic particles. .
For this reason, suppression of nonspecific adsorption of cells and nucleic acids to the surface of magnetic particles is strongly demanded.
従来、細胞の非特異吸着の抑制としては、粒子表面にポリウレタンのコーテイングする方法が取られてきた。しかし、細胞の非特異吸着の抑制としては十分ではなく、また核酸の非特異吸着の抑制法と両立するものではなかった。一方、核酸の非特異吸着の抑制法として、キャリアと呼ばれるサケ精子などから抽出したＤＮＡを遺伝子試薬に添加する方法がとられてきた。しかし、キャリアの非特異吸着効果が十分得られない場合や、生体物質であるキャリアの品質安定性の問題、キャリアと検査対象核酸の交差反応といった問題点があり、非特異吸着の抑制法としては十分なものではなかった。 Conventionally, as a method for suppressing nonspecific adsorption of cells, a method of coating polyurethane on the particle surface has been taken. However, it is not sufficient for suppressing nonspecific adsorption of cells, and is not compatible with a method for suppressing nonspecific adsorption of nucleic acids. On the other hand, as a method for suppressing nonspecific adsorption of nucleic acid, a method of adding DNA extracted from salmon sperm called a carrier to a gene reagent has been employed. However, there are problems such as when the non-specific adsorption effect of the carrier cannot be obtained sufficiently, the quality stability of the carrier that is a biological substance, and the cross reaction between the carrier and the nucleic acid to be tested. It was not enough.
本発明は、このような従来の問題点を解決するために、細胞および核酸に対して低吸着性表面をもつ磁性粒子により細胞および核酸の磁性粒子へ非特異吸着を抑制し、従来に比較して高純度の細胞の捕捉・検査を可能とする磁性粒子の提供を目的とするものである。 In order to solve such conventional problems, the present invention suppresses nonspecific adsorption of cells and nucleic acids to magnetic particles by using magnetic particles having a low adsorptive surface for cells and nucleic acids. It is intended to provide magnetic particles that can capture and inspect high-purity cells.
すなわち本発明は、磁性粒子であって少なくともその表面が細胞および核酸に対して低吸着性の材料で形成されており、粒子表面にプローブとの化学結合に使用可能な官能基を有していることを特徴とする磁性粒子である。 That is, the present invention is a magnetic particle, at least the surface of which is formed of a material having low adsorptivity to cells and nucleic acids, and has a functional group that can be used for chemical bonding with a probe on the particle surface. This is a magnetic particle.
本発明の磁性粒子において、少なくともその表面が細胞および核酸に対して低吸着性の材料を得るためには、磁性粒子の表面を非イオン性の親水性のポリマーで被覆することにより達成される。磁性粒子表面への細胞および核酸の非特異吸着は、疎水結合とイオン結合がその重要な役割を果たしており、粒子表面の非イオン性ポリマーよる親水化によって非特異吸着を大幅に低減できる。
本発明の免疫検査用磁性粒子において、基材となる磁性粒子には特に制限は無く、特公平０５−１０８０８や特開平７−８２３０１、特表平２−５０１７８５３などの方法で製造した粒子が利用可能であるが、好ましくは磁性粒子が核粒子の表面にＦｅ２Ｏ３およびＦｅ３Ｏ４の少なくとも一方を含む磁性体層が形成された母粒子に重合により該磁性体層上にポリマー層を形成する磁性粒子を用いることが出来る。磁性粒子の表面に非イオン性の親水性ポリマーを導入する方法としては、磁性粒子の存在下で、非イオン性の親水性モノマーおよびその他の共重合性モノマーを液体中で重合を行う方法をあげることができる。
すなわち、磁性粒子の存在下で、主原料としての非イオン性の親水性モノマーおよび必要に応じて官能基を有する共重合性モノマーや他の共重合性モノマー、副原料である重合開始剤、乳化剤、分散剤、界面活性剤、電解質、架橋剤、分子量調節剤などが必要に応じて添加し、液体中で重合を行う。非イオン性の親水性モノマーとしては、ヒドロキシアルキル（メタ）アクリレート、アルコキシアルキル（メタ）アクリレート、ポリオキシアルキレン（Ｃ２−Ｃ４）基含有（メタ）アクリレート、エポキシ基含有（メタ）アクリレート、ホスホリルコリン類似基含有単量体をあげることができる。官能基を有する共重合性モノマーとしては、アクリル酸、メタクリル酸、イタコン酸、無類マレイン酸、クロトン酸などのモノまたはジカルボン酸化合物をあげることができる。他の共重合性モノマーとしては、スチレン、ジビニルベンゼンなどの芳香族ビニル単量体、メチル（メタ）アクリレート、エチル（メタ）アクリレート、ブチル（メタ）アクリレート、シクロヘキシル（メタ）アクリレートなどのエチレン性不飽和カルボン酸アルキルエステルなどをあげることができる。
In the magnetic particle of the present invention, at least the surface of the magnetic particle can be obtained by coating the surface of the magnetic particle with a nonionic hydrophilic polymer in order to obtain a material having a low adsorptivity to cells and nucleic acids. Nonspecific adsorption of cells and nucleic acids to the surface of magnetic particles plays an important role in hydrophobic and ionic bonds, and nonspecific adsorption can be greatly reduced by hydrophilization of the particle surface with a nonionic polymer.
In the magnetic particle for immunological test of the present invention, there is no particular limitation on the magnetic particle used as a base material, and particles produced by a method such as Japanese Patent Publication No. 05-10808, Japanese Patent Application Laid-Open No. 7-82301, Japanese Patent Publication No. 2-5017853 are used. Although it is possible, the polymer layer is preferably formed on the magnetic layer by polymerizing the mother particle in which the magnetic particle is formed with a magnetic layer containing at least one of Fe 2 O 3 and Fe 3 O 4 on the surface of the core particle. Magnetic particles to be formed can be used. As a method for introducing a nonionic hydrophilic polymer onto the surface of magnetic particles, a method in which a nonionic hydrophilic monomer and other copolymerizable monomers are polymerized in a liquid in the presence of the magnetic particles is exemplified. be able to.
That is, in the presence of magnetic particles, a nonionic hydrophilic monomer as a main raw material, a copolymerizable monomer having a functional group as necessary, other copolymerizable monomers, a polymerization initiator as an auxiliary raw material, an emulsifier In addition, a dispersant, a surfactant, an electrolyte, a crosslinking agent, a molecular weight regulator and the like are added as necessary, and polymerization is performed in a liquid. Nonionic hydrophilic monomers include hydroxyalkyl (meth) acrylate, alkoxyalkyl (meth) acrylate, polyoxyalkylene (C2-C4) group-containing (meth) acrylate, epoxy group-containing (meth) acrylate, and phosphorylcholine-like group The containing monomer can be raised. Examples of the copolymerizable monomer having a functional group include mono- or dicarboxylic acid compounds such as acrylic acid, methacrylic acid, itaconic acid, non-maleic acid, and crotonic acid. Other copolymerizable monomers include aromatic vinyl monomers such as styrene and divinylbenzene, ethylenic monomers such as methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, and cyclohexyl (meth) acrylate. Examples thereof include saturated carboxylic acid alkyl esters.
本発明の磁性粒子において、捕捉対象の細胞およびそれに含まれる核酸の非特異吸着が完全に抑制されることが理想であるが、現実には核酸増幅検査に持ち込む核酸量が核酸増幅に影響を与えず、また核酸吸着が核酸増幅検査の感度に比較して十分低いレベルの吸着量であればよい。通常１ｍＬの血液から細胞の捕捉に１ｍｇ程度の粒子が使われ、核酸増幅検査に持ち込む核酸量の制限から、細胞の非特異吸着量が１０００個／ｍｇ粒子以下、核酸の非特異吸着量が１０コピー／ｍｇ粒子以下であれば問題はない。 In the magnetic particles of the present invention, it is ideal that nonspecific adsorption of cells to be captured and nucleic acids contained therein is completely suppressed. However, in reality, the amount of nucleic acid brought into the nucleic acid amplification test affects nucleic acid amplification. In addition, it is sufficient that the amount of adsorption of the nucleic acid is sufficiently lower than the sensitivity of the nucleic acid amplification test. Usually, about 1 mg of particles are used to capture cells from 1 mL of blood, and due to the limitation of the amount of nucleic acid brought into the nucleic acid amplification test, the nonspecific adsorption amount of cells is 1000 particles / mg particle or less, and the nonspecific adsorption amount of nucleic acid is 10 There is no problem as long as the copy / mg particle or less.
本発明の磁性粒子の実際の使用に当たって磁性粒子にプローブを結合させる方法としては、物理吸着法または化学結合法をあげることができるが、安定的な使用のためには通常一次プローブを磁性粒子表面の官能基と化学的に結合させる。この方法としては、例えば粒子表面のカルボキシル基とプローブのアミノ基を反応させる場合はカルボジイミド等の縮合剤を使用することができる。 In the actual use of the magnetic particle of the present invention, as a method of binding the probe to the magnetic particle, a physical adsorption method or a chemical bonding method can be mentioned, but for the stable use, the primary probe is usually used on the surface of the magnetic particle. It is chemically bonded to the functional group of As this method, for example, when a carboxyl group on the particle surface is reacted with an amino group of a probe, a condensing agent such as carbodiimide can be used.
本発明の磁性粒子は、細胞の非特異吸着が少ないため、生化学・医学分野での細胞の分離精製用のための細胞捕捉用担体として優れていることがわかる。 It can be seen that the magnetic particles of the present invention are excellent as a cell capture carrier for separating and purifying cells in the biochemistry and medical fields because of less non-specific adsorption of cells.
以下、実施例を挙げて本発明をさらに詳細に説明するが、本発明はこれらによって制限されるものではない。
参考例
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not restrict | limited by these.
Reference example
１．核粒子の作製
特公昭５７−２４３６９号公報記載の膨潤重合法、ジャーナル オブポリマーサイエンス ポリマーレター エディション（Ｊ．Ｐｏｌｙｍ．Ｓｃｉ．，Ｐｏｌｙｍｅｒ Ｌｅｔｔｅｒ Ｅｄ．）記載の重合方法、あるいは本発明者らが先に提案した重合方法（特開昭６１−２１５６０２、同６１−２１５６０３、同６１−２１５６０４）を参考に下記核粒子を作製した。下記核粒子は、重合後遠心分離により粒子のみ取り出したものをさらに水洗し、乾燥、粉砕した。
1. Preparation of Core Particles Swelling polymerization method described in Japanese Patent Publication No. 57-24369, polymerization method described in Journal of Polymer Science, Polymer Letter Edition (J. Polym. Sci., Polymer Letter Ed.), Or the present inventors The following core particles were prepared with reference to the proposed polymerization method (Japanese Patent Laid-Open Nos. 61-215602, 61-215603, and 61-215604). The following core particles were further washed with water after being polymerized by centrifugation, dried and pulverized.
核粒子１；メチルメタクリレート／ジビニルベンゼン＝８０／２０共重合体
(平均粒径１．５μｍ、ＣＶ値２．２％)
核粒子２；スチレン／ジビニルベンゼン＝８０／２０共重合体
（平均粒径３．５μｍ、ＣＶ値２．５％）
２．核粒子への磁性体の被覆（磁性体層の形成）
油性磁性流体「ＦＶ５５」［松本油脂（株）製］にアセトンを加えて粒子を析出沈殿させた後、これを乾燥することにより、疎水化処理された表面を有するフェライト系の超常磁性体（平均粒子径：０．０１μｍを得た。なおこの磁性体は界面活性剤により疎水化処理された表面を有するものである。得られた磁性体をトルエン／水（重量比１：１）に添加し、十分に攪拌した後静置したところ、磁性体はトルエンのみに分散されており、表面が疎水化されたことを確認した。ついで、核粒子５ｇに、疎水化された磁性体を５ｇ混合し、この混合物をハイブリダイゼーションシステムNHS−０型（奈良機械製作所（株）製）を使用して、羽根（撹拌翼）の周速度100m/秒（16200rpm）で３分間処理した。
Core particle 1; methyl methacrylate / divinylbenzene = 80/20 copolymer
(Average particle size 1.5μm, CV value 2.2%)
Core particle 2; styrene / divinylbenzene = 80/20 copolymer
(Average particle size 3.5μm, CV value 2.5%)
2. Coating of magnetic material on core particles (formation of magnetic layer)
A ferrite superparamagnetic substance (average) having a hydrophobized surface is obtained by adding acetone to the oil-based magnetic fluid “FV55” (manufactured by Matsumoto Yushi Co., Ltd.) to precipitate and precipitate particles. A particle size of 0.01 μm was obtained, which has a surface hydrophobized with a surfactant and added to toluene / water (weight ratio 1: 1). When the mixture was allowed to stand after sufficient stirring, it was confirmed that the magnetic substance was dispersed only in toluene and the surface was hydrophobized, and then 5 g of the hydrophobized magnetic substance was mixed with 5 g of the core particles. This mixture was treated for 3 minutes at a peripheral speed of 100 m / sec (16200 rpm) of a blade (stirring blade) using a hybridization system NHS-0 type (manufactured by Nara Machinery Co., Ltd.).
参考例の磁性体被覆粒子（核粒子１を使用）３０ｇと、分散剤としてノニオン性乳化剤「エマルゲン１５０」（花王製）の０．５％水溶液３７５ｇと、アニオン性乳化剤ラウリル硫酸ナトリウム（ＳＤＳ）の０．５％水溶液３７５ｇとを１Ｌセパラブルフラスコに投入し充分に分散させた。ついで、イカリ型撹拌羽２００ｒｐｍ撹拌、Ｎ２ガス気流下６０℃とした。これに、モノマーとしてシクロヘキシルメタクリレート１５ｇ、メタクリル酸１．５ｇ、ジビニルベンゼン０．６ｇ、２−ヒドロキシエチルメタクリレート１．５ｇ、重合開始剤としてターシャリーブチルペルオキシ２−エチルヘキサネート（日本油脂社製；パーブチルＯ）１．５ｇ、分散剤としてノニオン性乳化剤「エマルゲン１５０」（花王製）の０．５％水溶液７５ｇおよびアニオン性乳化剤ラウリル硫酸ナトリウム（ＳＤＳ）の０．５％水溶液７５ｇの混合物を１０℃以下において超音波微分散により乳化させて、２時間にわたり連続添加して反応させた。その後、さらに温度を８０℃とし３時間継続し反応を完結させた。その後、室温に冷却し５００メッシュステンレス製網で粗大物を除去し、さらに磁気精製において非磁性成分を除去した。粒径は２．１μm、粒子比重は１．６ｇ／ｃｍ３であった。 30 g of magnetic material-coated particles (using core particle 1) as a reference example, 375 g of a 0.5% aqueous solution of a nonionic emulsifier “Emulgen 150” (manufactured by Kao) as a dispersant, and anionic emulsifier sodium lauryl sulfate (SDS) 375 g of 0.5% aqueous solution was charged into a 1 L separable flask and sufficiently dispersed. Subsequently, it was set to 60 ° C. under a stream of N 2 gas and stirred at 200 rpm with a squid type stirring blade. To this, 15 g of cyclohexyl methacrylate, 1.5 g of methacrylic acid, 0.6 g of divinylbenzene, 1.5 g of 2-hydroxyethyl methacrylate, and tertiary butyl peroxy 2-ethylhexanate (manufactured by NOF Corporation; perbutyl) as a polymerization initiator O) 1.5 g, a mixture of a nonionic emulsifier “Emulgen 150” (manufactured by Kao) as a dispersant, 75 g of a 0.5% aqueous solution and a 0.5% aqueous solution of anionic emulsifier sodium lauryl sulfate (SDS) 75 g And emulsified by ultrasonic fine dispersion and continuously added for 2 hours to react. Thereafter, the temperature was further raised to 80 ° C. and continued for 3 hours to complete the reaction. Then, it cooled to room temperature and removed the coarse thing with the 500 mesh stainless steel net | network, and also removed the nonmagnetic component in the magnetic refinement | purification. The particle size was 2.1 μm and the particle specific gravity was 1.6 g / cm 3 .
参考例の磁性体被覆粒子（核粒子２を使用）３０ｇと、分散剤としてノニオン性乳化剤「エマルゲン１５０」（花王製）の０．５％水溶液３７５ｇと、アニオン性乳化剤ラウリル硫酸ナトリウム（ＳＤＳ）の０．５％水溶液３７５ｇとを１Ｌセパラブルフラスコに投入し充分に分散させた。ついで、イカリ型撹拌羽２００ｒｐｍ撹拌、Ｎ２ガス気流下６０℃とした。これに、モノマーとしてスチレン６ｇ、メタクリル酸１．５ｇ、ジビニルベンゼン６ｇ、グリシジルメタクリレート６ｇ、重合開始剤としてターシャリーブチルペルオキシ２−エチルヘキサネート（日本油脂社製；パーブチルＯ）１．５ｇ、分散剤としてノニオン性乳化剤「エマルゲン１５０」（花王製）の０．５％水溶液７５ｇおよびアニオン性乳化剤ラウリル硫酸ナトリウム（ＳＤＳ）の０．５％水溶液７５ｇの混合物を１０℃以下において超音波微分散により乳化させて、２時間にわたり連続添加して反応させた。その後、さらに温度を８０℃とし３時間継続し反応を完結させた。その後、室温に冷却し５００メッシュステンレス製網で粗大物を除去し、さらに磁気精製において非磁性成分を除去した。次に、この粒子10gを100mLの10mM HClに分散し、50℃で8時間転倒混和して粒子表面のエポキシ基を開環させた。磁気精製にて、液性が中性になるまで粒子分散液を蒸留水に置換した。得られた粒子の粒径は４．２μm、粒子比重は１．６ｇ／ｃｍ３であった。 30 g of magnetic material-coated particles (using core particles 2) as a reference example, 375 g of a 0.5% aqueous solution of a nonionic emulsifier “Emulgen 150” (manufactured by Kao) as a dispersant, and anionic emulsifier sodium lauryl sulfate (SDS) 375 g of 0.5% aqueous solution was charged into a 1 L separable flask and sufficiently dispersed. Subsequently, it was set to 60 ° C. under a stream of N 2 gas and stirred at 200 rpm with a squid type stirring blade. To this, 6 g of styrene as monomer, 1.5 g of methacrylic acid, 6 g of divinylbenzene, 6 g of glycidyl methacrylate, 1.5 g of tertiary butyl peroxy 2-ethylhexanate (manufactured by NOF Corporation; Perbutyl O) as a polymerization initiator, dispersant A mixture of 75 g of 0.5% aqueous solution of nonionic emulsifier “Emulgen 150” (manufactured by Kao) and 75 g of 0.5% aqueous solution of anionic emulsifier sodium lauryl sulfate (SDS) was emulsified at 10 ° C. or less by ultrasonic fine dispersion. The reaction was continued for 2 hours. Thereafter, the temperature was further raised to 80 ° C. and continued for 3 hours to complete the reaction. Then, it cooled to room temperature and removed the coarse thing with the 500 mesh stainless steel net | network, and also removed the nonmagnetic component in the magnetic refinement | purification. Next, 10 g of the particles were dispersed in 100 mL of 10 mM HCl, and mixed by inverting at 50 ° C. for 8 hours to open the epoxy groups on the particle surface. The particle dispersion was replaced with distilled water until the liquid became neutral by magnetic purification. The obtained particles had a particle size of 4.2 μm and a particle specific gravity of 1.6 g / cm 3 .
磁性粒子（Spherotech社、粒径２〜２.９μm）３０ｇと、分散剤としてノニオン性乳化剤「エマルゲン１５０」（花王製）の０．５％水溶液３７５ｇと、アニオン性乳化剤ラウリル硫酸ナトリウム（ＳＤＳ）の０．５％水溶液３７５ｇとを１Ｌセパラブルフラスコに投入し充分に分散させた。ついで、イカリ型撹拌羽２００ｒｐｍ撹拌、Ｎ２ガス気流下６０℃とした。これに、モノマーとしてスチレン８ｇ、メタクリル酸１．５ｇ、ジビニルベンゼン７ｇ、ヒドロキシ（オリゴエチレングリコールｎ＝４）メタクリレート１．５ｇ、重合開始剤としてターシャリーブチルペルオキシ２−エチルヘキサネート（日本油脂社製；パーブチルＯ）１．５ｇ、分散剤としてノニオン性乳化剤「エマルゲン１５０」（花王製）の０．５％水溶液７５ｇおよびアニオン性乳化剤ラウリル硫酸ナトリウム（ＳＤＳ）の０．５％水溶液７５ｇの混合物を１０℃以下において超音波微分散により乳化させて、２時間にわたり連続添加して反応させた。その後、さらに温度を８０℃とし３時間継続し反応を完結させた。その後、室温に冷却し５００メッシュステンレス製網で粗大物を除去し、さらに磁気精製において非磁性成分を除去した。粒径は２．６μm、粒子比重は１．３ｇ／ｃｍ３であった。
比較例１
30 g of magnetic particles (Spherotech, particle size 2 to 2.9 μm), 375 g of a 0.5% aqueous solution of a nonionic emulsifier “Emulgen 150” (manufactured by Kao) as a dispersant, and anionic emulsifier sodium lauryl sulfate (SDS) 375 g of 0.5% aqueous solution was charged into a 1 L separable flask and sufficiently dispersed. Subsequently, it was set to 60 ° C. under a stream of N 2 gas and stirred at 200 rpm with a squid type stirring blade. To this, 8 g of styrene as a monomer, 1.5 g of methacrylic acid, 7 g of divinylbenzene, 1.5 g of hydroxy (oligoethylene glycol n = 4) methacrylate, and tertiary butyl peroxy 2-ethylhexanate (manufactured by NOF Corporation) as a polymerization initiator 10 g of a mixture of 1.5 g of perbutyl O), 75 g of 0.5% aqueous solution of nonionic emulsifier “Emulgen 150” (manufactured by Kao) and 75 g of 0.5% aqueous solution of anionic emulsifier sodium lauryl sulfate (SDS) as a dispersant. The mixture was emulsified by ultrasonic fine dispersion at a temperature of 0 ° C. or lower and continuously added for 2 hours to react. Thereafter, the temperature was further raised to 80 ° C. and continued for 3 hours to complete the reaction. Then, it cooled to room temperature and removed the coarse thing with the 500 mesh stainless steel net | network, and also removed the nonmagnetic component in the magnetic refinement | purification. The particle size was 2.6 μm and the particle specific gravity was 1.3 g / cm 3 .
Comparative Example 1
実施例１で2−ヒドロキシエチルメタクリレートを用いなかった以外は全て実施例１の方法に基づいて実施した。粒径は２．１μm、粒子比重は１．６ｇ／ｃｍ３であった。
比較例２
All the steps were performed according to the method of Example 1 except that 2-hydroxyethyl methacrylate was not used in Example 1. The particle size was 2.1 μm and the particle specific gravity was 1.6 g / cm 3 .
Comparative Example 2
磁性粒子（Spherotech社、粒径２〜２.９μm）を用いた。粒径は２．５μm、粒子比重は１．３ｇ／ｃｍ３であった。
細胞吸着の評価
クエン酸採血した血液５ｍＬを遠心分離し、バフィーコートを採取した。これに０．１７Ｍ塩化アンモニウム水溶液１０ｍＬを加え室温で１０分放置したのち遠心分離し、得られた沈殿をリン酸緩衝塩溶液で２回洗浄し４．５ｘ１０６個の有核細胞を得た。これを４．５ｍＬの０．２％ウシ血清アルブミンを含有するリン酸緩衝塩溶液で浮遊させ、実施例１〜３および比較例１〜２の粒子各１ｍｇに加え、３０分間４℃で転倒混和を行った。磁気分離により磁性粒子を分離し、０．２％ウシ血清アルブミンを含有する緩衝リン酸塩溶液で２回洗浄ののち、磁性粒子に吸着した細胞からスマイテストＥＸ Ｒ＆ＤをもちいてゲノムＤＮＡを抽出した。このゲノムＤＮＡをβ−グロビン遺伝子を用いた定量ＰＣＲで定量することにより粒子に吸着した細胞数を評価した。結果を図１に示す。
核酸吸着の評価
クエン酸採血した血液１ｍＬに０．１７Ｍ塩化アンモニウム水溶液１０ｍＬを加え室温で１０分放置したのち遠心分離し、得られた沈殿をリン酸緩衝塩溶液で２回洗浄し１．２ｘ１０６個の有核細胞を得た。スマイテストＥＸ Ｒ＆Ｄ（ゲノムサイエンス社）を用いて、ゲノムＤＮＡの抽出を行った。得られたゲノムＤＮＡを１０００コピー/ｍＬとなるよう純水に溶解しその１００μL（１００コピー）を実施例１〜３および比較例１〜２の磁性粒子１ｍｇに、加えて攪拌し、室温で１０分放置した。磁気分離法により磁性粒子を除去し、上清中のゲノムＤＮＡ量をβ−グロビン遺伝子を用いた定量ＰＣＲ法により定量した。結果を図２に示す。
Magnetic particles (Spherotech, particle size 2 to 2.9 μm) were used. The particle size was 2.5 μm and the particle specific gravity was 1.3 g / cm 3 .
Evaluation of cell adsorption 5 mL of blood collected from citric acid was centrifuged, and a buffy coat was collected. To this was added 10 mL of 0.17 M ammonium chloride aqueous solution, and the mixture was allowed to stand at room temperature for 10 minutes and then centrifuged. The resulting precipitate was washed twice with a phosphate buffered salt solution to obtain 4.5 × 10 6 nucleated cells. This was suspended in a phosphate buffered salt solution containing 4.5 mL of 0.2% bovine serum albumin, added to each 1 mg of the particles of Examples 1 to 3 and Comparative Examples 1 and 2, and mixed by inverting at 4 ° C. for 30 minutes. Went. The magnetic particles were separated by magnetic separation, washed twice with a buffered phosphate solution containing 0.2% bovine serum albumin, and then genomic DNA was extracted from the cells adsorbed to the magnetic particles using Sumittest EX R & D. . The number of cells adsorbed on the particles was evaluated by quantifying this genomic DNA by quantitative PCR using a β-globin gene. The results are shown in FIG.
Evaluation of
Claims (5)
Of claim 1, wherein the magnetic particles have a polymer layer to the magnetic layer and the magnetic body layer containing at least one of Fe 2 O 3 and Fe 3 O 4 on the surface of core particles The magnetic particle in any one.
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