JP2004163421A - Magnetic substance-including particle and its production method, and immunoassay particle using magnetic substance-including particle - Google Patents
Magnetic substance-including particle and its production method, and immunoassay particle using magnetic substance-including particle Download PDFInfo
- Publication number
- JP2004163421A JP2004163421A JP2003360986A JP2003360986A JP2004163421A JP 2004163421 A JP2004163421 A JP 2004163421A JP 2003360986 A JP2003360986 A JP 2003360986A JP 2003360986 A JP2003360986 A JP 2003360986A JP 2004163421 A JP2004163421 A JP 2004163421A
- Authority
- JP
- Japan
- Prior art keywords
- magnetic substance
- particles
- magnetic
- particle
- encapsulated
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
Description
本発明は、均一な磁性を有するとともに、粒径分布が狭く、かつ分散安定性に優れており、免疫測定用粒子として有用な磁性体を内包する高分子粒子とその製造方法、及びそれを用いた免疫測定用粒子に関する。 The present invention provides a polymer particle having a uniform magnetic property, a narrow particle size distribution and excellent dispersion stability, and encapsulating a magnetic material useful as an immunoassay particle, a method for producing the same, and a method for producing the same Related to immunoassay particles.
従来、磁性体内包粒子の作製法としては、（１）作製済みの高分子粒子に鉄イオンを含ませて磁性体内包粒子を作製する方法、（２）モノマーから粒子を重合する過程で作製済みの磁性体粒子を含ませる方法（特許文献１参照）、（３）別々に作製した高分子粒子と磁性体粒子とを複合化させる方法（特許文献２参照）が知られている。また、この他、（４）磁性体粒子を高分子等で被覆する方法（特許文献３参照）がある。 Conventionally, magnetic body-encapsulated particles are produced by (1) a method of producing magnetic body-encapsulated particles by including iron ions in the prepared polymer particles, and (2) a process of polymerizing particles from monomers. (3) A method of combining separately prepared polymer particles and magnetic particles (see Patent Document 2) is known. In addition, there is (4) a method of coating magnetic particles with a polymer or the like (see Patent Document 3).
（１）の方法は鉄イオンを高分子粒子に吸収させるため、表面に磁性体が露出し、磁性体が酸化するという課題があった。（２）の方法は磁性体粒子が均一に粒子に取り込まれないという課題や粒径の制御が困難で、粒径分布の広い物となるという課題があった。（３）の方法は高分子粒子が凝集するため、粒径の小さな粒子には使用できないという課題があった。（４）の方法は、被覆が均一にできないため、浮遊性や分散性が悪く、また、磁性体粒子表面の一部が露出している場合があるという課題があった。 In the method (1), since iron ions are absorbed by polymer particles, there is a problem that the magnetic material is exposed on the surface and the magnetic material is oxidized. The method (2) has a problem that the magnetic particles are not uniformly taken into the particles, and it is difficult to control the particle size, and there is a problem that the product has a wide particle size distribution. The method (3) has a problem that the polymer particles aggregate and cannot be used for particles having a small particle size. The method (4) has problems in that the coating cannot be made uniform, so that the floatability and dispersibility are poor, and a part of the surface of the magnetic particles may be exposed.
一方、従来から知られている微量免疫測定法としては、ラジオイムノアッセイ、酵素イムノアッセイ、蛍光イムノアッセイ等が既に実用化されている。これらの方法は、それぞれアイソトープ、酵素、蛍光物質を標識として付加した抗原又は抗体を用い、これと特異的に反応する抗体又は抗原の有無を検出する方法である。
このような免疫測定法に際して、磁性体内包粒子は効率よくかつ簡便にＢ／Ｆ分離を行うために用いられている。また、Ｂ／Ｆ分離以外の使用（特許文献４参照）や、磁性体内包粒子自体を標識材料とする免疫測定法（特許文献５、特許文献６、特許文献７参照）が開示されている。
On the other hand, radioimmunoassay, enzyme immunoassay, fluorescent immunoassay and the like have already been put to practical use as conventionally known microimmunoassay methods. These methods are methods for detecting the presence or absence of an antibody or antigen that specifically reacts with an antigen or an antibody to which an isotope, enzyme, or fluorescent substance is added as a label, respectively.
In such an immunoassay, the magnetic particles are used to perform B / F separation efficiently and simply. In addition, uses other than B / F separation (see Patent Document 4) and immunoassay methods (see Patent Document 5, Patent Document 6, and Patent Document 7) using magnetic inclusion particles themselves as labeling materials are disclosed.
本発明は、上記現状に鑑み、均一な磁性を有するとともに、粒径分布が狭く、かつ分散安定性に優れており、免疫測定用として有用な磁性体内包粒子とその製造方法、及びそれを用いた免疫測定用粒子を提供することを目的とする。 In view of the above situation, the present invention has a uniform magnetic property, a narrow particle size distribution and excellent dispersion stability, and is useful for immunoassay, a method for producing the same, and a method for producing the same. It is an object to provide particles for immunoassay.
本発明は、有機高分子物質と平均粒径１〜３０ｎｍの磁性体とからなる磁性体内包粒子であって、その内部に上記磁性体を分散状態で含有する磁性体内包粒子である。
以下に本発明を詳述する。
The present invention is a magnetic substance-encapsulated particle comprising an organic polymer substance and a magnetic substance having an average particle size of 1 to 30 nm, and containing the magnetic substance in a dispersed state therein.
The present invention is described in detail below.
本発明の磁性体内包粒子は、有機高分子物質と平均粒径１〜３０ｎｍの磁性体とからなるものである。
上記有機高分子物質は、高分子粒子のコアを形成するための疎水性モノマー、及び／又は、水中で安定に分散する高分子粒子を形成しつつ高分子粒子のシェルを形成するための親水性モノマーからなる重合体を主構成成分とするものである。
特に、有機高分子物質中の疎水性モノマーの量が少なくなると粒子重合中に後述する金属イオンを取り込み難くなり、また、親水性モノマーの量が少なくなると得られる磁性体内包粒子の分散安定性が低下するので、これらを併用するのが好ましく、その割合は、疎水性モノマーが５〜９７重量％、親水性モノマーが３〜９５重量％であるのが好ましく、この範囲で必要に応じて適宜調整するのがより好ましい。
The magnetic substance-encapsulated particles of the present invention are composed of an organic polymer substance and a magnetic substance having an average particle diameter of 1 to 30 nm.
The organic polymer material includes a hydrophobic monomer for forming a core of the polymer particle and / or a hydrophilic property for forming a polymer particle shell while forming the polymer particle stably dispersed in water. A polymer composed of monomers is used as a main constituent.
In particular, when the amount of the hydrophobic monomer in the organic polymer material is reduced, it becomes difficult to incorporate metal ions described later during particle polymerization, and when the amount of the hydrophilic monomer is reduced, the dispersion stability of the magnetic substance-encapsulated particles obtained is reduced. These are preferably used in combination, and the proportion is preferably 5 to 97% by weight for the hydrophobic monomer and 3 to 95% by weight for the hydrophilic monomer, and is adjusted as necessary within this range. More preferably.
＜疎水性モノマー＞
上記疎水性モノマーとしては、例えば、スチレン；α−メチルスチレン、ｐ−メチルスチレン、ｐ−クロロスチレン、クロロメチルスチレンなどのスチレン誘導体；塩化ビニル；酢酸ビニル、プロピオン酸ビニルなどのビニルエステル類；アクリロニトリルなどの不飽和ニトリル類；（メタ）アクリル酸メチル、（メタ）アクリル酸エチル、（メタ）アクリル酸ブチル、（メタ）アクリル酸２−エチルヘキシル、（メタ）アクリル酸ステアリル、エチレングリコール（メタ）アクリレート、トリフルオロエチル（メタ）アクリレート、ペンタフルオロプロピル（メタ）アクリレート、シクロヘキシル（メタ）アクリレート、グリシジルメタクリレート、テトラヒドロフルフリル（メタ）アクリレートなどの（メタ）アクリル酸エステル誘導体等が挙げられる。これら単量体は単独で用いてもよく、２種以上を併用しても良い。
<Hydrophobic monomer>
Examples of the hydrophobic monomer include styrene; styrene derivatives such as α-methylstyrene, p-methylstyrene, p-chlorostyrene, and chloromethylstyrene; vinyl chloride; vinyl esters such as vinyl acetate and vinyl propionate; acrylonitrile. Unsaturated nitriles such as: methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, stearyl (meth) acrylate, ethylene glycol (meth) acrylate , (Meth) acrylic acid ester derivatives such as trifluoroethyl (meth) acrylate, pentafluoropropyl (meth) acrylate, cyclohexyl (meth) acrylate, glycidyl methacrylate, tetrahydrofurfuryl (meth) acrylate And the like. These monomers may be used independently and may use 2 or more types together.
上記疎水性モノマーとしては、好ましくは、（メタ）アクリル酸メチル、（メタ）アクリル酸エチル、（メタ）アクリル酸ブチル、（メタ）アクリル酸２−エチルヘキシル、（メタ）アクリル酸ステアリル、エチレングリコール（メタ）アクリレート、トリフルオロエチル（メタ）アクリレート、ペンタフルオロプロピル（メタ）アクリレート、シクロヘキシル（メタ）アクリレート、グリシジルメタクリレート、テトラヒドロフルフリル（メタ）アクリレートなどの（メタ）アクリル酸エステル誘導体等のアクリル系モノマーが用いられる。 The hydrophobic monomer is preferably methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, stearyl (meth) acrylate, ethylene glycol ( Acrylic monomers such as (meth) acrylate derivatives such as (meth) acrylate, trifluoroethyl (meth) acrylate, pentafluoropropyl (meth) acrylate, cyclohexyl (meth) acrylate, glycidyl methacrylate, and tetrahydrofurfuryl (meth) acrylate Is used.
上記疎水性モノマーとしては、より好ましくは、後述するように重合による粒子形成と磁性体形成とを同時進行するため、粒子重合中に高濃度に金属イオンを取り込む能力に優れたグリシジル基を有するアクリル系モノマーが用いられる。即ち、上記アクリル系モノマーの中でも、特にグリシジルメタクリレート（ＧＭＡ）は鉄イオン及びマグネタイトとの親和性が高いため特に好適に使用される。 The hydrophobic monomer is more preferably an acryl having a glycidyl group having an excellent ability to take in metal ions at a high concentration during particle polymerization because particle formation by polymerization and magnetic substance formation proceed simultaneously as described later. System monomers are used. That is, among the acrylic monomers, glycidyl methacrylate (GMA) is particularly preferably used because of its high affinity with iron ions and magnetite.
上記疎水性モノマーとしては、架橋性単量体も使用できる。上記架橋性単量体としては、例えば、ジビニルベンゼン、ジビニルビフェニル、ジビニルナフタレン、エチレングリコールジ（メタ）アクリレート、１，６−ヘキサンジオールジ（メタ）アクリレート、ネオペンチルグリコールジ（メタ）アクリレート、トリメチロールプロパントリ（メタ）アクリレート、テトラメチロールメタントリ（メタ）アクリレート、テトラメチロールプロパンテトラ（メタ）アクリレート、ジアリルフタレート及びその異性体、トリアリルイソシアヌレート及びその誘導体等が挙げられる。これら架橋性単量体は単独で用いてもよく、２種以上を併用しても良い。
この中でも、エチレングリコールジ（メタ）アクリレートは鉄イオン及びマグネタイトとの親和性が高いため好適に使用される。
A crosslinkable monomer can also be used as the hydrophobic monomer. Examples of the crosslinkable monomer include divinylbenzene, divinylbiphenyl, divinylnaphthalene, ethylene glycol di (meth) acrylate, 1,6-hexanediol di (meth) acrylate, neopentyl glycol di (meth) acrylate, Examples include methylolpropane tri (meth) acrylate, tetramethylolmethane tri (meth) acrylate, tetramethylolpropane tetra (meth) acrylate, diallyl phthalate and isomers thereof, triallyl isocyanurate and derivatives thereof. These crosslinkable monomers may be used independently and may use 2 or more types together.
Among these, ethylene glycol di (meth) acrylate is preferably used because of its high affinity with iron ions and magnetite.
＜親水性モノマー＞
上記親水性モノマーとしては、例えば、アクリル酸、メタクリル酸、イタコン酸、フマル酸、マレイン酸などの重合性不飽和結合を有するカルボン酸；重合性不飽和結合を有するリン酸エステル；重合性不飽和結合を有するスルホン酸エステル；ジメチルアミノエチルメタクリレート４級塩、ジエチルアミノエチルメタクリレート４級塩などのアクリロイル基を有するアミンの塩；ビニルピリジン等のビニル基を有する含窒素化合物の塩などのカチオン基を有するビニル系単量体；２−ヒドロキシエチルメタクリレート、ポリエチレングリコール（メタ）アクリレート、（メタ）アクリルアミド、メチロールアクリルアミド、グリセロールメタクリレート（ＧＬＭ）などの非イオン性ビニル系単量体等が挙げられる。これら親水性モノマーは、単独で用いてもよく、２種以上を併用しても良い。この中でも、下記一般式で表されるポリエチレングリコール（メタ）アクリレートは、水中で粒子を安定に分散する能力が高く、磁性体の形成を妨げないので好適に使用される。
ＣＨ２＝ＣＲ−ＣＯＯ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−Ｈ
式中、ＲはＨ又はＣＨ３を表し、ｎは２〜２０の整数を表す。ｎの好ましい下限は２であり、好ましい上限は１０である。
<Hydrophilic monomer>
Examples of the hydrophilic monomer include carboxylic acids having a polymerizable unsaturated bond such as acrylic acid, methacrylic acid, itaconic acid, fumaric acid, and maleic acid; phosphate esters having a polymerizable unsaturated bond; Sulfonic acid ester having a bond; salt of an amine having an acryloyl group such as dimethylaminoethyl methacrylate quaternary salt or diethylaminoethyl methacrylate quaternary salt; a cationic group such as a salt of a nitrogen-containing compound having a vinyl group such as vinylpyridine Vinyl monomers: Nonionic vinyl monomers such as 2-hydroxyethyl methacrylate, polyethylene glycol (meth) acrylate, (meth) acrylamide, methylol acrylamide, and glycerol methacrylate (GLM). These hydrophilic monomers may be used independently and may use 2 or more types together. Among these, polyethylene glycol (meth) acrylate represented by the following general formula is preferably used because it has a high ability to stably disperse particles in water and does not hinder the formation of a magnetic substance.
CH 2 = CR-COO- (CH 2 -CH 2 -O) n-H
In the formula, R represents H or CH 3 , and n represents an integer of 2 to 20. The preferable lower limit of n is 2, and the preferable upper limit is 10.
上記磁性体は、粒子を形成させる重合過程において粒子内部で金属イオンが酸化して形成したものであるのが好ましい。
＜金属イオン＞
上記金属イオンは、磁性体を形成するものであれば特に限定されないが、好ましくは、鉄イオン、コバルトイオン、ニッケルイオン等であり、より好ましくは、鉄イオンである。磁性体であるマグネタイトは塩化第２鉄を酸化剤等で酸化して得られる。
The magnetic material is preferably formed by oxidation of metal ions inside the particles in the polymerization process for forming the particles.
<Metal ion>
Although the said metal ion will not be specifically limited if it forms a magnetic body, Preferably, they are an iron ion, cobalt ion, nickel ion, etc., More preferably, it is an iron ion. Magnetite, which is a magnetic substance, is obtained by oxidizing ferric chloride with an oxidizing agent or the like.
上記磁性体の平均粒径は１〜３０ｎｍである。１ｎｍ未満であると、磁性体の磁性応答特性が減少し、免疫測定に使用した際に測定感度が低下し、定性・定量分析が行い難くなる。また、３０ｎｍを超えると、磁性体内包粒子内での分散性が低下し、磁性が不均一になり、この場合も免疫測定に使用した際に定性・定量分析が行い難くなる。好ましい下限は２ｎｍであり、好ましい上限は２０ｎｍである。さらに、好ましい上限は１０ｎｍであり、このような微小な磁性体粒子を形成するのは通常困難であるが、後述する製造方法のように、粒子を形成する工程と金属イオンを粒子中に取り込みながら金属イオンを酸化して磁性体を形成する工程とを同時進行させることにより、微小な磁性体を形成することが可能となる。 The average particle diameter of the magnetic material is 1 to 30 nm. When the thickness is less than 1 nm, the magnetic response characteristics of the magnetic substance are reduced, and when used for immunoassay, the measurement sensitivity is lowered, making it difficult to perform qualitative and quantitative analysis. On the other hand, if the thickness exceeds 30 nm, the dispersibility in the magnetic substance-encapsulated particles decreases, and the magnetism becomes non-uniform. In this case also, it becomes difficult to perform qualitative and quantitative analysis when used for immunoassay. A preferred lower limit is 2 nm and a preferred upper limit is 20 nm. Further, the preferable upper limit is 10 nm, and it is usually difficult to form such fine magnetic particles, but as in the manufacturing method described later, while forming the particles and incorporating metal ions into the particles By simultaneously proceeding with the step of oxidizing the metal ions to form the magnetic body, it becomes possible to form a minute magnetic body.
本発明の磁性体内包粒子は、その内部に上記磁性体を分散状態で含有している。即ち、本発明の磁性体内包粒子においては、磁性体が粒子表面に露出することなく、粒子内部に分散した状態で存在している。 The magnetic substance-encapsulated particles of the present invention contain the above magnetic substance in a dispersed state. That is, in the magnetic substance-encapsulated particle of the present invention, the magnetic substance is present in a dispersed state inside the particle without being exposed on the particle surface.
本発明の磁性体内包粒子の磁性体含有量は、その重合組成により０．１〜４０重量％の範囲で調整するのが好ましい。０．１重量％未満であると、磁性体内包粒子の磁性応答特性が減少し、免疫測定に使用した際に測定感度が低下し、定性・定量分析が行い難くなる。また、４０重量％を超えると、粒子の重合操作性が低下し、粒子重合中に金属イオンを取り込み難くなる。好ましい下限は１重量％であり、好ましい上限は３０重量％である。 The magnetic substance content of the magnetic substance-encapsulated particles of the present invention is preferably adjusted in the range of 0.1 to 40% by weight depending on the polymerization composition. If it is less than 0.1% by weight, the magnetic response characteristics of the magnetic substance-encapsulated particles are reduced, and when used for immunoassay, the measurement sensitivity is lowered, and it becomes difficult to perform qualitative and quantitative analysis. On the other hand, if it exceeds 40% by weight, the polymerization operability of the particles is lowered, and it becomes difficult to incorporate metal ions during the particle polymerization. A preferred lower limit is 1% by weight and a preferred upper limit is 30% by weight.
本発明の磁性体内包粒子の平均粒径は、その重合条件により０．０５〜１μｍの範囲で調整するのが好ましい。０．０５μｍ未満や１μｍを超えると、磁性体内包粒子の粒子形状の制御及び重合操作がしづらくなり、また、０．０５μｍ未満では免疫測定法に適用した際に測定感度が低下し、定性・定量分析が行い難くなり、１μｍを超えると免疫測定法に適用した際に、分散液において凝集により経時で粒子が沈降し易くなり、この場合も定性・定量分析が行い難くなる。好ましい下限は０．０７μｍであり、好ましい上限は０．８μｍである。 The average particle diameter of the magnetic substance-encapsulated particles of the present invention is preferably adjusted in the range of 0.05 to 1 μm depending on the polymerization conditions. If it is less than 0.05 μm or more than 1 μm, it becomes difficult to control the particle shape and polymerization of the magnetic particles, and if it is less than 0.05 μm, the sensitivity of the measurement decreases when applied to an immunoassay. Quantitative analysis becomes difficult, and when it exceeds 1 μm, when applied to an immunoassay, particles are likely to settle over time due to aggregation in the dispersion, and in this case also, qualitative and quantitative analysis becomes difficult. A preferable lower limit is 0.07 μm, and a preferable upper limit is 0.8 μm.
本発明の磁性体内包粒子は、水系溶媒中において疎水性モノマー及び／又は親水性モノマーを重合して粒子を形成する工程と、上記粒子中に金属イオンを取り込みながら上記金属イオンを酸化して磁性体を形成する工程とからなり、上記粒子を形成する工程と上記磁性体を形成する工程とを同時に進行させる方法により製造される。このような製造方法もまた、本発明の１つである。 The magnetic substance-encapsulated particles of the present invention include a step of polymerizing a hydrophobic monomer and / or a hydrophilic monomer in an aqueous solvent to form particles, and oxidizing the metal ions while incorporating the metal ions into the particles. And the step of forming the particles, and the step of simultaneously forming the step of forming the particles and the step of forming the magnetic body. Such a manufacturing method is also one aspect of the present invention.
水系溶媒中において疎水性モノマー及び／又は親水性モノマーを重合して粒子を形成する工程においては、重合開始剤を添加するのが好ましい。
＜重合開始剤＞
上記重合開始剤としては特に限定されず、例えば、水溶性の有機アゾ化合物、無機過酸化物、有機過酸化物等が挙げられる。
上記重合開始剤の好適な例としては、過硫酸カリウム（ＫＰＳ；重合温度７０℃）、アゾビスアミジノプロパン塩酸塩（Ｖ−５０；重合温度７０℃）、２，２−アゾビス［２−（２−イミダゾリン−２−イル）プロパン］ジハイドロクロライド（ＶＡ−０４４；重合温度６０℃）等が挙げられる。このうち、過酸化物系重合開始剤であるＫＰＳは、重合開始とともに２価の鉄イオンの酸化に寄与することを期待して、モノマーと鉄イオンによる、重合とマグネタイト生成との同時進行を想定している。また、Ｖ−５０及びＶＡ−０４４は酸化力が弱く、２価の鉄イオンの緩やかな酸化反応に関与する重合開始剤となる。
上記重合開始剤は、Ｆｅ２＋の酸化による消費やＦｅ３＋によってラジカル活性を失う場合があるため、重合による粒子成長を促す目的で、粒子成長過程に後添加することが有効である。この場合、新たな２次粒子は形成されず、粒子表面がポリマーで被覆される。
In the step of forming particles by polymerizing a hydrophobic monomer and / or a hydrophilic monomer in an aqueous solvent, it is preferable to add a polymerization initiator.
<Polymerization initiator>
The polymerization initiator is not particularly limited, and examples thereof include water-soluble organic azo compounds, inorganic peroxides, and organic peroxides.
Preferable examples of the polymerization initiator include potassium persulfate (KPS; polymerization temperature 70 ° C.), azobisamidinopropane hydrochloride (V-50; polymerization temperature 70 ° C.), 2,2-azobis [2- (2 -Imidazolin-2-yl) propane] dihydrochloride (VA-044; polymerization temperature 60 ° C.) and the like. Among these, KPS, which is a peroxide polymerization initiator, is expected to contribute to the oxidation of divalent iron ions at the start of polymerization, and is assumed to proceed simultaneously with polymerization and magnetite formation by monomers and iron ions. doing. V-50 and VA-044 are weak in oxidizing power and become polymerization initiators involved in the mild oxidation reaction of divalent iron ions.
Since the polymerization initiator may lose its radical activity due to Fe 2+ oxidation or Fe 3+ , it is effective to add it later in the particle growth process for the purpose of promoting particle growth by polymerization. In this case, new secondary particles are not formed, and the particle surface is coated with the polymer.
＜ｐＨ調整剤＞
本発明において、重合と同時に磁性体を形成するにあたっては、重合系内のｐＨを塩基性に調整することが重要となる。例えば、重合開始剤としてＫＰＳを用いた系では、メリットとして水中での分散安定性がよく、粒径分布の狭い単分散粒子が得られるが、デメリットとして酸化力の制御ができず、重合系内が酸性になるために、得られる磁性体内包粒子が磁石への引き寄せられ方の弱い粒子になることがある。一方、重合開始剤として酸化力を持たないＶＡ−０４４を用いた系でのメリットは、重合系内のｐＨがほぼ中性であることである。
重合系内のｐＨを弱塩基性に保つには、ｐＨ調整剤として一般的な塩基を使用することができる。好適にはＮＨ４ＯＨがｐＨ調整剤として使用される。
上記ｐＨ調整剤は、必要に応じて数回添加することができる。
<PH adjuster>
In the present invention, when forming a magnetic material simultaneously with polymerization, it is important to adjust the pH in the polymerization system to basic. For example, in a system using KPS as a polymerization initiator, dispersion stability in water is good as a merit, and monodisperse particles with a narrow particle size distribution can be obtained. May become acidic, and the resulting magnetic inclusion particles may become particles that are weakly attracted to the magnet. On the other hand, a merit in a system using VA-044 having no oxidizing power as a polymerization initiator is that the pH in the polymerization system is almost neutral.
In order to keep the pH in the polymerization system weakly basic, a general base can be used as a pH adjuster. Preferably NH 4 OH is used as a pH adjuster.
The pH adjuster can be added several times as necessary.
＜重合方法＞
本発明の磁性体内包粒子は、懸濁重合、分散重合、乳化重合、ソープフリー乳化重合等の粒子重合法が使用できるが、得られる磁性体内包粒子のＣｖ値は５％以下であることが好ましいので、粒径分布の制御に優れたソープフリー乳化重合により好適に製造される。
以下、ソープフリー乳化重合による磁性体内包粒子の製造方法を例示するが、この方法に限定されるものではない。
<Polymerization method>
The magnetic body-encapsulated particles of the present invention can use particle polymerization methods such as suspension polymerization, dispersion polymerization, emulsion polymerization, soap-free emulsion polymerization, etc., but the Cv value of the obtained magnetic body-encapsulated particles should be 5% or less. Therefore, it is preferably produced by soap-free emulsion polymerization excellent in control of particle size distribution.
Hereinafter, although the manufacturing method of the magnetic body inclusion particle | grains by soap free emulsion polymerization is illustrated, it is not limited to this method.
代表的な重合組成は、以下のとおりである。
親水性モノマー／疎水性モノマー／反応性乳化剤からなるモノマー組成物：３ｇ
Ｈ２Ｏ：１００ｇ
四つ口フラスコに上記モノマー組成物及び水を秤量する。それぞれの口には攪拌棒、還流冷却管を取り付ける。次に、重合開始剤としてＫＰＳとＶ−５０を用いる系では７０℃の、ＶＡ−０４４を用いる系では５０℃〜６０℃の恒温槽に入れ、攪拌しながら系内を窒素置換する。その後、水に溶かした重合開始剤を注射筒で系内に注入する。この時点を重合開始とし、所定時間後に注射筒を用いて磁性源となるＦｅＣｌ２・４Ｈ２Ｏの水溶液を注入する。ＦｅＣｌ２・４Ｈ２Ｏは重合開始剤の１／３〜４倍モルを水５ｇに溶かしたものを使用する。即ち、重合開始剤により上述のモノマーの重合を開始するとともに２価の鉄イオンの酸化（マグネタイト化）を行うことにより磁性体内包粒子を製造する。
重合は開始から２時間〜２４時間行うことが好ましい。適度な酸化力を得るために、重合途中にＮＨ４ＯＨを加えてもよく、更に、重合による粒子の成長を促すために、重合途中に重合開始剤を加えてもよい。この様にして磁性体を内包した高分子粒子である磁性体内包粒子を得ることができる。
A typical polymerization composition is as follows.
Monomer composition comprising hydrophilic monomer / hydrophobic monomer / reactive emulsifier: 3 g
H 2 O: 100 g
The monomer composition and water are weighed into a four-necked flask. A stir bar and a reflux condenser are attached to each port. Next, in a system using KPS and V-50 as a polymerization initiator, a system using 70 ° C. and a system using VA-044 are placed in a thermostatic bath at 50 ° C. to 60 ° C., and the inside of the system is replaced with nitrogen while stirring. Thereafter, a polymerization initiator dissolved in water is injected into the system with a syringe. The polymerization is started at this time, and an aqueous solution of FeCl 2 .4H 2 O serving as a magnetic source is injected using a syringe after a predetermined time. FeCl 2 .4H 2 O is used by dissolving 1 to 3 to 4 moles of the polymerization initiator in 5 g of water. That is, a magnetic substance-encapsulated particle is produced by initiating polymerization of the above-described monomer with a polymerization initiator and oxidizing (magnetizing) divalent iron ions.
The polymerization is preferably performed for 2 to 24 hours from the start. In order to obtain an appropriate oxidizing power, NH 4 OH may be added during the polymerization, and further, a polymerization initiator may be added during the polymerization in order to promote particle growth by the polymerization. In this way, magnetic body-encapsulated particles, which are polymer particles encapsulating a magnetic body, can be obtained.
上記反応性乳化剤は共重合モノマーであり、必要に応じて添加してもよい。
＜反応性乳化剤＞
上記反応乳化剤としては、例えば、下記一般式で表される反応性乳化剤類が挙げられ、これら反応性乳化剤は、単独で用いてもよく、２種以上を併用しても良い。
The reactive emulsifier is a copolymerization monomer and may be added as necessary.
<Reactive emulsifier>
Examples of the reactive emulsifier include reactive emulsifiers represented by the following general formula, and these reactive emulsifiers may be used alone or in combination of two or more.
得られた磁性体内包粒子は、残存モノマー、重合開始剤、未反応の鉄イオン等を取り除くために遠心分離・再分散を蒸留水で繰り返し行うことで精製する。遠心分離を行った後、上澄みをデカンテーションにより捨て、蒸留水を加え、ガラス棒により再分散を行う。精製後、ガラス製バイアルに移し、ふた・パラフィルムで密閉・保存する。 The obtained magnetic substance-encapsulated particles are purified by repeated centrifugation and redispersion with distilled water in order to remove residual monomers, polymerization initiators, unreacted iron ions, and the like. After centrifugation, the supernatant is discarded by decantation, distilled water is added, and redispersion is performed with a glass rod. After purification, transfer to a glass vial, seal and store with a lid or parafilm.
本発明の磁性体内包粒子は免疫測定用に非常に適しており、磁性体内包粒子に抗原又は抗体を吸着又は結合させることにより免疫測定用粒子を得ることができる。このような免疫測定用粒子もまた、本発明の１つである。
本発明の磁性体内包粒子に抗体又は抗原を吸着又は結合させる方法としては特に限定されず、例えば、物理吸着法やカルボジイミドを用いた化学結合法等公知の方法を使用することができる。
The magnetic body-encapsulated particles of the present invention are very suitable for immunoassay, and the immunoassay particles can be obtained by adsorbing or binding an antigen or antibody to the magnetic body-encapsulated particles. Such immunoassay particles are also one aspect of the present invention.
The method for adsorbing or binding the antibody or antigen to the magnetic substance-encapsulated particle of the present invention is not particularly limited, and for example, a known method such as a physical adsorption method or a chemical bonding method using carbodiimide can be used.
本発明の磁性体内包粒子又は本発明の免疫測定用粒子は、免疫測定法に好適に用いることができる。
上記免疫測定法としては、例えば、磁性体内包粒子を坦体として用いたラジオイムノアッセイ、酵素イムノアッセイ等公知の方法が挙げられ、サンドイッチ法や競合法により、目的とする抗原又は抗体を測定することができる。また、上記方法の標識物質であるアイソトープ、酵素等の代わりに、磁性体内包粒子を標識として用いることができる。
The magnetic substance-encapsulated particle of the present invention or the immunoassay particle of the present invention can be suitably used for an immunoassay.
Examples of the immunoassay include known methods such as radioimmunoassay and enzyme immunoassay using magnetic inclusion particles as a carrier, and the target antigen or antibody can be measured by a sandwich method or a competitive method. it can. In addition, magnetic substance-encapsulated particles can be used as a label instead of isotopes, enzymes, and the like, which are labeling substances in the above method.
本発明の磁性体内包粒子は、磁性体を均一に分散含有する粒径分布の狭い粒子であるので、本発明の磁性体内包粒子を用いることにより磁性体を標識とする免疫測定法において感度よく精密に測定することができる。即ち、測定しようとする抗原又は抗体と特異的に反応する抗体又は抗原を磁性体内包粒子に結合させた免疫測定用粒子を用い、この免疫測定用粒子と測定しようとする抗原又は抗体とを反応させた後、反応した免疫測定用粒子における磁性体内包粒子の磁性量を測定することにより、測定しようとする抗原又は抗体を定性的かつ定量的に測定することができる。 Since the magnetic substance-encapsulated particle of the present invention is a particle having a narrow particle size distribution and containing a magnetic substance uniformly dispersed therein, the magnetic substance-encapsulated particle of the present invention is used to improve sensitivity in an immunoassay method using a magnetic substance as a label. It can be measured accurately. That is, immunoassay particles in which an antibody or antigen that specifically reacts with the antigen or antibody to be measured is bound to a magnetic substance-encapsulated particle are used, and this immunoassay particle reacts with the antigen or antibody to be measured. Then, the antigen or antibody to be measured can be qualitatively and quantitatively measured by measuring the magnetic content of the magnetic particles in the reacted immunoassay particles.
請求項１に記載の発明の磁性体内包粒子は、内部に平均粒径１〜３０ｎｍの磁性体を含有しているので、磁性体の磁性応答特性に優れているとともに、磁性体内包粒子内での磁性体の分散性にも優れており、即ち、磁性体内包粒子は、磁性が均一で、かつ、磁性応答特性に優れたものであり、さらに、粒径分布も狭く粒径が均一なものである。故に、測定感度等が要求される免疫測定用として好適に使用できるものであり、免疫測定法において免疫反応に寄与した磁性体内包粒子の磁性を測定することにより、優れた測定感度で、定性的のみならず定量的に免疫測定を行うことができる。また、表面が有機高分子物質から形成されているので、抗原又は抗体を確実に結合させることができる。
請求項２に記載の発明の磁性体内包粒子及び請求項３に記載の発明の磁性体内包粒子は、磁性体が粒子内部で金属イオン、好ましくは鉄イオンが酸化して形成されたものであるので、磁性体内包粒子内に確実かつ均一に磁性体が取り込まれており、免疫測定法に要求される測定感度を確実に発現することができる。
請求項４に記載の発明の磁性体内包粒子は、磁性体内包粒子を構成する有機高分子物質がアクリル系モノマーであるので、重合による粒子形成が確実に行われるとともに、重合による粒子形成と磁性体形成とを確実に同時進行させることができる。故に、磁性体内包粒子は、磁性が均一であり、かつ、粒径分布も狭く、免疫測定法においてより優れた測定感度を発現することができる。
請求項５に記載の発明の磁性体内包粒子は、磁性体内包粒子を構成する有機高分子物質がグリシジル基を有するアクリル系モノマーであるので、磁性体を形成するのに好適な鉄イオン及びマグネタイトとの親和性に優れており、故に、磁性体内包粒子は磁性体がより均一に分散されたものとなり、免疫測定法に要求される測定感度がより優れたものとなる。
請求項６に記載の発明の磁性体内包粒子は、磁性体内包粒子を構成する有機高分子物質が特定の疎水性アクリル系モノマーと親水性アクリル系モノマーとの組合せであるので、粒子重合中に金属イオンを均一に取り込み易いだけでなく、磁性体内包粒子の分散安定性にも優れており、故に、分散液において凝集により経時で粒子が沈降することがなく、かつ、超音波処理等の余分な工程を必要とせず、免疫測定法などにおいて優れた測定感度を発現することができる。
請求項７に記載の発明の磁性体内包粒子は、平均粒径が０．０５〜１μｍであるので、重合操作がし易く、即ち、上記のような磁性体内包粒子が確実に得られる。
請求項８に記載の発明の磁性体内包粒子は、磁性体の含有量が０．１〜４０重量％であるので、優れた磁性応答特性を均一かつ確実に発現することができ、免疫測定法により好適に適用することができる。また、重合操作もし易く、上記のような磁性体内包粒子が確実に得られる。
請求項９〜１３に記載の発明の製造方法は、粒子を形成する工程を磁性体を形成する工程とを同時進行させているので、上記の磁性体内包粒子を容易かつ確実に得ることができる。
請求項１４に記載の発明の免疫測定用粒子は、担体である磁性体内包粒子が内部に平均粒径１〜３０ｎｍの磁性体を含有しているので、磁性が均一で、かつ、磁性応答特性に優れたものであり、さらに、粒径分布も狭く粒径が均一なものである。また、表面が有機高分子物質で形成されているので、抗原又は抗体が確実に結合されている。故に、測定感度等が要求される免疫測定法に好適に使用できるものであり、免疫測定法において免疫反応に寄与した免疫測定用粒子における磁性体内包粒子の磁性を測定することにより、優れた測定感度で、定性的のみならず定量的に免疫測定を行うことができる。
Since the magnetic substance-encapsulated particle of the invention according to claim 1 contains a magnetic substance having an average particle diameter of 1 to 30 nm inside, it is excellent in magnetic response characteristics of the magnetic substance and is contained in the magnetic substance-encapsulated particle. In addition, the magnetic substance-encapsulated particles have a uniform magnetism, excellent magnetic response characteristics, and a narrow particle size distribution and a uniform particle size. It is. Therefore, it can be used suitably for immunoassay that requires measurement sensitivity, etc., and by measuring the magnetism of magnetic inclusion particles that contributed to the immune reaction in the immunoassay method, it is qualitative with excellent measurement sensitivity. Not only can quantitative immunoassay be performed. Moreover, since the surface is formed from an organic polymer substance, an antigen or an antibody can be reliably bound.
The magnetic substance-encapsulated particles of the invention described in claim 2 and the magnetic substance-encapsulated particles of the invention described in claim 3 are formed by oxidizing a magnetic substance with metal ions, preferably iron ions inside the particles. Therefore, the magnetic substance is reliably and uniformly incorporated in the magnetic substance-encapsulated particles, and the measurement sensitivity required for the immunoassay can be reliably expressed.
In the magnetic body-encapsulated particle of the invention according to claim 4, since the organic polymer substance constituting the magnetic body-encapsulated particle is an acrylic monomer, the particle formation by the polymerization is surely performed, and the particle formation by the polymerization and the magnetic The body formation can surely proceed simultaneously. Therefore, the magnetic substance-encapsulated particles have a uniform magnetism and a narrow particle size distribution, and can exhibit better measurement sensitivity in an immunoassay.
In the magnetic body-encapsulated particle of the invention according to claim 5, since the organic polymer substance constituting the magnetic body-encapsulated particle is an acrylic monomer having a glycidyl group, iron ions and magnetite suitable for forming a magnetic body Therefore, in the magnetic substance-encapsulated particles, the magnetic substance is more uniformly dispersed, and the measurement sensitivity required for the immunoassay is more excellent.
In the magnetic body-encapsulated particle of the invention described in claim 6, since the organic polymer material constituting the magnetic body-encapsulated particle is a combination of a specific hydrophobic acrylic monomer and a hydrophilic acrylic monomer, Not only is it easy to take up metal ions uniformly, but it also excels in dispersion stability of the magnetic particles, so that the particles do not settle over time due to aggregation in the dispersion, and there is no need for sonication, etc. Therefore, it is possible to express excellent measurement sensitivity in an immunoassay method and the like.
The magnetic substance-encapsulated particles according to the seventh aspect of the present invention have an average particle diameter of 0.05 to 1 μm, so that the polymerization operation is easy, that is, the magnetic substance-encapsulated particles as described above can be obtained with certainty.
Since the magnetic substance-encapsulated particle according to the eighth aspect of the present invention has a magnetic substance content of 0.1 to 40% by weight, an excellent magnetic response characteristic can be expressed uniformly and reliably. It can apply more suitably. In addition, the polymerization operation is easy, and the above-mentioned magnetic substance-encapsulated particles can be obtained with certainty.
In the manufacturing method of the invention according to claims 9 to 13, since the step of forming the particles and the step of forming the magnetic substance are simultaneously advanced, the above-mentioned magnetic substance-encapsulated particles can be obtained easily and reliably. .
In the immunoassay particle according to the fourteenth aspect of the invention, since the magnetic inclusion particles as a carrier contain a magnetic substance having an average particle diameter of 1 to 30 nm, the magnetism is uniform and the magnetic response characteristics In addition, the particle size distribution is narrow and the particle size is uniform. In addition, since the surface is formed of an organic polymer material, antigens or antibodies are reliably bound. Therefore, it can be used suitably for immunoassays that require measurement sensitivity, etc., and by measuring the magnetism of the magnetic particles in the immunoassay particles that contributed to the immune reaction in the immunoassay, an excellent measurement Sensitivity enables quantitative immunoassay as well as qualitative.
以下に実施例を掲げて本発明を更に詳しく説明するが、本発明はこれら実施例のみに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
（実施例１〜７）
２００ｍｌの四つ口フラスコに表１に示す各種モノマー及び水９０ｇを秤量した。それぞれの口には攪拌シールと攪拌棒、還流冷却管、セラムラバーを取り付けた。系を７０℃の恒温槽に入れ、２００ｒｐｍで攪拌しながら系内を３０分間窒素置換した。その後、水に溶かした重合開始剤であるＫＰＳ ０．０６ｇを１０ｇの水に溶解し注射筒で系内に注入した。この時点を重合開始とし、２分後に注射筒を用いて所定量のＦｅＣｌ２・４Ｈ２Ｏ水溶液を注入した。重合は重合開始から２０時間行った。適度な酸化力を得るために、重合途中にＮＨ4ＯＨ ０．１６５ｇを加えた。
作製した粒子は、遠心分離・再分散を蒸留水で４回繰り返し行うことで精製した。この際、遠心分離は２０℃、１３５００ｒｐｍで行った。遠心分離を行った後、上澄みをデカンテーションにより捨て、蒸留水を加え、ガラス棒により再分散を行って磁性体内包粒子を得た。
(Examples 1-7)
Various monomers shown in Table 1 and 90 g of water were weighed in a 200 ml four-necked flask. A stirring seal, a stirring rod, a reflux condenser, and a ceramic rubber were attached to each port. The system was placed in a constant temperature bath at 70 ° C., and the inside of the system was purged with nitrogen for 30 minutes while stirring at 200 rpm. Thereafter, 0.06 g of KPS, which is a polymerization initiator dissolved in water, was dissolved in 10 g of water and injected into the system with a syringe. At this time, the polymerization was started, and after 2 minutes, a predetermined amount of FeCl 2 .4H 2 O aqueous solution was injected using a syringe. The polymerization was carried out for 20 hours from the start of polymerization. In order to obtain an appropriate oxidizing power, 0.165 g of NH4OH was added during the polymerization.
The produced particles were purified by repeating centrifugation and redispersion four times with distilled water. At this time, centrifugation was performed at 20 ° C. and 13500 rpm. After centrifugation, the supernatant was discarded by decantation, distilled water was added, and redispersion was performed with a glass rod to obtain magnetic substance-encapsulated particles.
表中の記載は以下のとおりである。
ＧＭＡ：グリシジルメタクリレート
ＥＧＤＭ：エチレングリコールジメタクリレート
ＡＡｍ：アクリルアミド
ＰＥ−９０：ポリエチレングリコールメタクリレート（ｎ＝２）
ＰＥ−３５０：ポリエチレングリコールメタクリレート（ｎ＝８）
ＮＥ−２０：
The descriptions in the table are as follows.
GMA: Glycidyl methacrylate EGDM: Ethylene glycol dimethacrylate AAm: Acrylamide PE-90: Polyethylene glycol methacrylate (n = 2)
PE-350: Polyethylene glycol methacrylate (n = 8)
NE-20:
（式中、ＸはＨを表す。） (In the formula, X represents H.)
ＳＥ−２０： SE-20:
（式中、ＸはＳＯ４ＮＨ４を表す。） (In the formula, X represents SO 4 NH 4. )
得られた磁性体内包粒子分散液について、目視で粒子の分散状態を観察した。また、精製した磁性体内包粒子を水で希釈し、金属メッシュで支持したコロジオン膜上に沈着固定して、透過型電子顕微鏡（ＴＥＭ）により、粒子の形態を観察した。 With respect to the obtained magnetic substance-encapsulated particle dispersion, the particle dispersion state was visually observed. Further, the purified magnetic inclusion particles were diluted with water, deposited and fixed on a collodion film supported by a metal mesh, and the morphology of the particles was observed with a transmission electron microscope (TEM).
実施例１は、一部凝集塊が認められ、時間が経つにつれて粒子が沈降する分散安定性のやや低いものであったため、超音波処理により再分散した。一方、親水性モノマーとしてポリエチレングリコールメタクリレートを用いた実施例２〜５、及び、反応性乳化剤を用いた実施例６、７は、いずれも凝集塊が認められず、分散安定性の高い粒子が得られた。特に、反応性乳化剤を用いた実施例６、７は、粒子サイズが小さく、分散安定性が優れていることが認められた。また、実施例１〜７の粒子は、いずれも粒子内部に磁性体を含み、粒子表面がきれいな輪郭であることが観察された。図１に実施例２の磁性体内包粒子のＴＥＭ写真（平均粒径；磁性体内包粒子０．２１μｍ、磁性体５ｎｍ）を示した。 In Example 1, since some agglomerates were observed and the dispersion stability in which particles settled with time was somewhat low, the particles were redispersed by ultrasonic treatment. On the other hand, in Examples 2 to 5 using polyethylene glycol methacrylate as a hydrophilic monomer and Examples 6 and 7 using a reactive emulsifier, no agglomerates were observed, and particles having high dispersion stability were obtained. It was. In particular, Examples 6 and 7 using reactive emulsifiers were found to have a small particle size and excellent dispersion stability. Moreover, it was observed that all the particles of Examples 1 to 7 contained a magnetic substance inside the particle, and the particle surface had a clean outline. FIG. 1 shows a TEM photograph (average particle diameter: magnetic inclusion particles 0.21 μm, magnetic substance 5 nm) of magnetic inclusion particles of Example 2.
作製した磁性体内包粒子（実施例１〜７）は、磁石へ引き寄せられることの確認として１．５ｍｌのマイクロチューブに少量取り、蒸留水で適当に希釈して磁石つきマイクロチューブ立て（ＤＹＮＡＬ社製、ＭＰＣ（登録商標）＿Ｍ）にチューブを立てて、分散している粒子が磁石に引き寄せられることを目視により確認した。特に、親水性モノマーとしてポリエチレングリコールメタクリレートを用いた実施例２〜５は、他の実施例に比べ磁力が大きいことがうかがえた。各実施例における磁性体内包粒子の磁性体の平均粒径を表２に示した。 The produced magnetic substance-encapsulated particles (Examples 1 to 7) were taken in a small amount in a 1.5 ml microtube to confirm that they were attracted to the magnet, and diluted appropriately with distilled water to make a microtube stand with a magnet (manufactured by DYNAL). , MPC (registered trademark) _M), and it was visually confirmed that dispersed particles were attracted to the magnet. In particular, it was revealed that Examples 2 to 5 using polyethylene glycol methacrylate as the hydrophilic monomer had a larger magnetic force than other Examples. Table 2 shows the average particle size of the magnetic substance-encapsulated particles in each example.
（実施例８）
２００ｍｌの四つ口フラスコに下記に示す各種モノマー及び水を秤量した。
ＡＡｍ／ＧＭＡ／ＥＧＤＭ／Ｈ2Ｏ＝０．１５／２．８３５／０．０１５／９０（ｇ）
それぞれの口には攪拌シールと攪拌棒、還流冷却管、セラムラバーを取り付けた。系を７０℃の恒温槽に入れ、２００ｒｐｍで攪拌しながら系内を３０分間窒素置換した。その後、水に溶かした重合開始剤であるＫＰＳ ０．０６ｇを１０ｇの水に溶解し注射筒で系内に注入した。この時を重合開始とし、所定時間後に注射筒を用いてＦｅＣｌ２・４Ｈ２Ｏ水溶液（ＦｅＣｌ２・４Ｈ２Ｏ ０．１６５ｇを水５ｇに溶解）を注入した。重合開始１分後に適度な酸化力を得るためＮＨ４ＯＨ／Ｈ２Ｏ＝０．１６５／５（ｇ）を加え、２時間重合を行った。
(Example 8)
Various monomers and water shown below were weighed in a 200 ml four-necked flask.
AAm / GMA / EGDM / H2O = 0.15 / 2.835 / 0.015 / 90 (g)
A stirring seal, a stirring rod, a reflux condenser, and a ceramic rubber were attached to each port. The system was placed in a constant temperature bath at 70 ° C., and the inside of the system was purged with nitrogen for 30 minutes while stirring at 200 rpm. Thereafter, 0.06 g of KPS, which is a polymerization initiator dissolved in water, was dissolved in 10 g of water and injected into the system with a syringe. At this time, the polymerization was started, and after a predetermined time, an FeCl 2 .4H 2 O aqueous solution (0.165 g of FeCl 2 .4H 2 O was dissolved in 5 g of water) was injected using a syringe. NH 4 OH / H 2 O = 0.165 / 5 (g) was added to obtain an appropriate oxidizing power 1 minute after the start of polymerization, and polymerization was carried out for 2 hours.
作製した粒子は、遠心分離・再分散を蒸留水で４回繰り返し行うことで精製した。この際、遠心分離は２０℃、１３５００ｒｐｍで行った。遠心分離を行った後、上澄みをデカンテーションにより捨て、蒸留水を加え、ガラス棒により再分散を行って磁性体内包粒子を得た。 The produced particles were purified by repeating centrifugation and redispersion four times with distilled water. At this time, centrifugation was performed at 20 ° C. and 13500 rpm. After centrifugation, the supernatant was discarded by decantation, distilled water was added, and redispersion was performed with a glass rod to obtain magnetic substance-encapsulated particles.
（実施例９）
重合開始後、所定時間に下記物質を添加したこと、重合時間が３時間であること以外は実施例８と同様に磁性体内包粒子を得た。
重合開始３０分後 ＮＨ４ＯＨ／Ｈ２Ｏ＝０．１６５／５（ｇ）
重合開始６０分後 ＫＰＳ／Ｈ２Ｏ＝０．１６５／５（ｇ）
Example 9
Magnetic substance-encapsulated particles were obtained in the same manner as in Example 8 except that the following substances were added at a predetermined time after the start of polymerization and the polymerization time was 3 hours.
30 minutes after the start of polymerization, NH 4 OH / H 2 O = 0.165 / 5 (g)
60 minutes after the start of polymerization KPS / H 2 O = 0.165 / 5 (g)
（実施例１０）
重合開始後、所定時間に下記物質を添加したこと、重合時間が３時間であること以外は実施例８と同様に磁性体内包粒子を得た。
重合開始６０分後 ＫＰＳ／Ｈ２Ｏ＝０．１６５／５（ｇ）、ＦｅＣｌ２・４Ｈ２Ｏ／Ｈ２Ｏ＝０．０８８／５（ｇ）
重合開始１２０分後 ＧＭＡ＝０．５（ｇ）、ＮＨ４ＯＨ／Ｈ２Ｏ＝０．１６５／５（ｇ）
(Example 10)
Magnetic substance-encapsulated particles were obtained in the same manner as in Example 8 except that the following substances were added at a predetermined time after the start of polymerization and the polymerization time was 3 hours.
60 minutes after the start of polymerization KPS / H 2 O = 0.165 / 5 (g), FeCl 2 .4H 2 O / H 2 O = 0.088 / 5 (g)
120 minutes after the start of polymerization, GMA = 0.5 (g), NH 4 OH / H 2 O = 0.165 / 5 (g)
（実施例１１）
重合開始後、所定時間に下記物質を添加したこと、重合時間が４時間であること以外は実施例８と同様に磁性体内包粒子を得た。
重合開始１分後 ＮＨ４ＯＨ／Ｈ２Ｏ＝０．１６５／５（ｇ）
重合開始１２０分後 ＫＰＳ／Ｈ２Ｏ＝０．１６５／５（ｇ）
(Example 11)
Magnetic substance-encapsulated particles were obtained in the same manner as in Example 8 except that the following substances were added at a predetermined time after the start of polymerization and the polymerization time was 4 hours.
1 minute after the start of polymerization NH 4 OH / H 2 O = 0.165 / 5 (g)
120 minutes after the start of polymerization KPS / H 2 O = 0.165 / 5 (g)
得られた磁性体内包粒子（実施例８〜１１）に対し、ＴＥＭによる粒子の形態観察を行なった。
実施例９、１０は、いずれも、実施例８よりも多くの磁性体を含み、粒径が増大していることが認められた。また、実施例１１では、内部に実施例９、１０と同程度の磁性体を含み、粒子表面がきれいな輪郭であることが観察された。これまでの実験より、粒子成長の速い段階でＮＨ4ＯＨを加えることは、成長を阻害する要因になっていることが推測された。一方、重合開始剤の後添加は、重合率がほぼ１００％になり、また、２次粒子が形成されず粒子表面がポリマーで覆われていることが観察され、有効な手段であることが確認された。各実施例における磁性体内包粒子の磁性体の平均粒径を表３に示した。
The obtained magnetic substance-encapsulated particles (Examples 8 to 11) were observed for particle morphology by TEM.
Each of Examples 9 and 10 was found to contain more magnetic material than Example 8 and to have an increased particle size. Moreover, in Example 11, it was observed that the inside contains a magnetic material comparable to Examples 9 and 10, and the particle surface has a clean outline. From previous experiments, it was speculated that the addition of NH 4 OH in the early stage of particle growth is a factor inhibiting growth. On the other hand, post-addition of the polymerization initiator is confirmed to be an effective means that the polymerization rate is almost 100%, and secondary particles are not formed and the particle surface is covered with the polymer. It was done. Table 3 shows the average particle size of the magnetic substance-encapsulated particles in each example.
さらに、実施例２で得られた磁性体内包粒子から免疫測定用粒子を作製し、免疫測定を行った。
（免疫測定用粒子の作製）
実施例２で得られた磁性体内包粒子３０ｍｇにリン酸緩衝液（１００ｍｍｏｌ／ｌ、ｐＨ７．５）６ｍｌを加え、１５０００ｒｐｍにて２０分間遠心分離を行った。得られた沈渣に、抗ＨＢｓＡｇモノクローナル抗体を０．２５ｍｇ／ｍｌの濃度になるようにリン酸緩衝液（１００ｍｍｏｌ／ｌ、ｐＨ７．５）に溶解した溶液を１ｍｌ加え、室温にて２０時間撹拌混和した。その後、未反応の抗ＨＢｓＡｇモノクローナル抗体を除去するために１５０００ｒｐｍにて２０分間遠心分離を行い、さらに、得られた沈渣をリン酸緩衝液（１００ｍｍｏｌ／ｌ、ｐＨ７．５）６ｍｌに懸濁させ、再度１５０００ｒｐｍにて２０分間遠心分離を行った。その後、得られた沈渣を、牛血清アルブミンを１重量％の濃度になるようにリン酸緩衝液（１００ｍｍｏｌ／ｌ、ｐＨ７．５）に溶解した溶液６ｍｌに懸濁させ、室温で１時間撹拌してブロッキング処理を行い、抗ＨＢｓＡｇモノクローナル抗体が磁性体内包粒子に結合された免疫測定用粒子を得た。
次に、得られた免疫測定用粒子を冷蔵保存するため、１５０００ｒｐｍにて２０分間遠心分離を行い、得られた沈渣を、牛血清アルブミンの濃度が１重量％になるようにリン酸緩衝液（１００ｍｍｏｌ／ｌ、ｐＨ７．５）に溶解し、さらに、アジ化ナトリウムを０．０１重量％の濃度になるように溶解した溶液６ｍｌに懸濁させ、すぐに冷蔵保存した。
Furthermore, immunoassay particles were prepared from the magnetic substance-encapsulated particles obtained in Example 2, and immunoassay was performed.
(Preparation of immunoassay particles)
6 ml of a phosphate buffer (100 mmol / l, pH 7.5) was added to 30 mg of the magnetic substance-encapsulated particles obtained in Example 2, and centrifuged at 15000 rpm for 20 minutes. 1 ml of a solution obtained by dissolving anti-HBsAg monoclonal antibody in a phosphate buffer solution (100 mmol / l, pH 7.5) so as to have a concentration of 0.25 mg / ml is added to the resulting precipitate, and the mixture is stirred and mixed at room temperature for 20 hours. did. Thereafter, in order to remove the unreacted anti-HBsAg monoclonal antibody, centrifugation was performed at 15000 rpm for 20 minutes, and the resulting precipitate was suspended in 6 ml of a phosphate buffer (100 mmol / l, pH 7.5). Centrifugation was again performed at 15000 rpm for 20 minutes. Thereafter, the obtained precipitate is suspended in 6 ml of a solution obtained by dissolving bovine serum albumin in a phosphate buffer (100 mmol / l, pH 7.5) so as to have a concentration of 1% by weight, and stirred at room temperature for 1 hour. Then, blocking treatment was performed to obtain immunoassay particles in which the anti-HBsAg monoclonal antibody was bound to the magnetic particles.
Next, in order to store the obtained immunoassay particles in a refrigerated state, centrifugation was performed at 15000 rpm for 20 minutes, and the resulting precipitate was washed with a phosphate buffer solution (both with a bovine serum albumin concentration of 1% by weight). 100 mmol / l, pH 7.5) and suspended in 6 ml of a solution in which sodium azide was dissolved to a concentration of 0.01% by weight, and immediately stored in a refrigerator.
（試験片の作製）
ニトロセルロースメンブレン（ＳＲＨＦ、日本ミリポア社製）を幅３０ｃｍ×長さ６ｃｍに裁断し、その長さ方向上端より３ｃｍの部位（反応部位）に、上記免疫測定用粒子で用いたものとは異なる反応エピトープを有する抗ＨＢｓＡｇモノクローナル抗体を２．０ｍｇ／ｍｌの濃度になるようにトリス塩酸緩衝液（１０ｍｍｏｌ／ｌ、ｐＨ７．４）に溶解した溶液を幅０．７ｍｍの直線状に塗布した。その後、３７℃で２時間乾燥した後、牛血清アルブミン（和光純薬社製）を１重量％の濃度になるようにリン酸緩衝液（１００ｍｍｏｌ／ｌ、ｐＨ７．５）に溶解した溶液に１時間浸漬し、ブロッキング処理を行った。さらにその後、ラウリルベンゼンスルホン酸ナトリウムを０．１重量％の濃度になるようにリン酸緩衝液（１００ｍｍｏｌ／ｌ、ｐＨ７．５）に溶解した溶液にて洗浄後、シリカゲルデシケーター内で室温下にて乾燥し、抗ＨＢｓＡｇモノクローナル抗体固定化膜を得た。
得られた抗ＨＢｓＡｇモノクローナル抗体固定化膜を幅５ｍｍに裁断し、長さ方向上端に幅５ｍｍ×長さ２ｃｍの吸水用ろ紙（日本ミリポア社製）を重ね、透明なテープで固定して試験片とした。
(Preparation of test piece)
A nitrocellulose membrane (SRHF, manufactured by Nihon Millipore) is cut into a width of 30 cm and a length of 6 cm, and a reaction different from that used in the above immunoassay particles at a site 3 cm from the upper end in the length direction (reaction site) A solution prepared by dissolving an anti-HBsAg monoclonal antibody having an epitope in Tris-HCl buffer (10 mmol / l, pH 7.4) so as to have a concentration of 2.0 mg / ml was applied linearly with a width of 0.7 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 membrane was dried to obtain an anti-HBsAg monoclonal antibody-immobilized membrane.
The obtained anti-HBsAg monoclonal antibody-immobilized membrane was cut into a width of 5 mm, a water-absorbing filter paper (manufactured by Millipore Japan, Inc.) having a width of 5 mm and a length of 2 cm was stacked on the upper end in the length direction, and fixed with a transparent tape. It was.
（免疫測定の実施）
リン酸緩衝液（１００ｍｍｏｌ／ｌ、ｐＨ７．５）に上記免疫測定用粒子を０．１重量％の濃度になるように溶解し、さらに牛血清アルブミンを１重量％の濃度になるように溶解し、さらにアジ化ナトリウムを０．０１重量％の濃度になるように溶解した溶液を作製し、該溶液２０μｌを９６ウェルマイクロプレート（ナルジェヌンクインターナショナル社製）の各ウェルに添加した。
次に、ＨＢｓ抗原標準品（５０ＩＵ／ｍｌ）を所定の濃度になるようにリン酸緩衝液（１００ｍｍｏｌ／ｌ、ｐＨ７．５）で希釈し、各々１００μｌをウェルに添加混合後、試験片をウェル内に入れ、立位するように立てた。
３０分後、試験片を取り出した所、反応部位においてＨＢｓ抗原濃度に応じた磁性が確認され、磁性を標識とする免疫測定法に有用であることが示された。
(Immunity measurement)
The above immunoassay particles are dissolved in a phosphate buffer (100 mmol / l, pH 7.5) to a concentration of 0.1% by weight, and bovine serum albumin is dissolved to a concentration of 1% by weight. Further, a solution in which sodium azide was dissolved to a concentration of 0.01% by weight was prepared, and 20 μl of the solution was added to each well of a 96-well microplate (manufactured by Nargenunk International).
Next, the HBs antigen standard (50 IU / ml) was diluted with a phosphate buffer (100 mmol / l, pH 7.5) to a predetermined concentration, and 100 μl of each was added to the well and mixed. I stood inside and stood to stand.
After 30 minutes, when the test piece was taken out, magnetism corresponding to the HBs antigen concentration was confirmed at the reaction site, and it was shown to be useful for an immunoassay method using magnetism as a label.
Claims (14)
ＣＨ２＝ＣＲ−ＣＯＯ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−Ｈ
（式中、ＲはＨ又はＣＨ３を表し、ｎは２〜２０の整数を表す。） 5. The magnetic substance-encapsulated particle according to claim 4, wherein the acrylic monomer is a monomer having a glycidyl group and polyethylene glycol (meth) acrylate represented by the following general formula.
CH 2 = CR-COO- (CH 2 -CH 2 -O) n-H
(In the formula, R represents H or CH 3 , and n represents an integer of 2 to 20)
前記粒子中に金属イオンを取り込みながら前記金属イオンを酸化して磁性体を形成する工程とからなり、
前記粒子を形成する工程と前記磁性体を形成する工程とを同時に進行させることを特徴とする磁性体内包粒子の製造方法。 A step of polymerizing a hydrophobic monomer and / or a hydrophilic monomer in an aqueous solvent to form particles;
The step of oxidizing the metal ions while incorporating the metal ions into the particles to form a magnetic material,
A method for producing a magnetic substance-encapsulated particle, wherein the step of forming the particle and the step of forming the magnetic body are simultaneously performed.
ＣＨ２＝ＣＲ−ＣＯＯ−（ＣＨ２−ＣＨ２−Ｏ）ｎ−Ｈ
（式中、ＲはＨ又はＣＨ３を表し、ｎは２〜２０の整数を表す。） The method for producing magnetic body-encapsulated particles according to claim 9, wherein the hydrophilic monomer is polyethylene glycol (meth) acrylate represented by the following general formula.
CH 2 = CR-COO- (CH 2 -CH 2 -O) n-H
(In the formula, R represents H or CH 3 , and n represents an integer of 2 to 20)
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2003360986A JP4426819B2 (en) | 2002-10-21 | 2003-10-21 | Method for producing magnetic inclusion particles |
| US10/553,100 US7632688B2 (en) | 2003-04-16 | 2004-04-16 | Particle having magnetic material incorporated therein, process for producing the same, particle for immunoassay and method of immunoassay |
| DE602004022395T DE602004022395D1 (en) | 2003-04-16 | 2004-04-16 | METHOD FOR PRODUCING A PARTICLE WITH ITS INTEGRATED MAGNETIC MATERIAL |
| AT04727432T ATE438854T1 (en) | 2003-04-16 | 2004-04-16 | METHOD FOR PRODUCING A PARTICLE HAVING A MAGNETIC MATERIAL INCORPORATED THEREIN |
| EP04727432A EP1617220B1 (en) | 2003-04-16 | 2004-04-16 | Process for producing a particle having magnetic material incorporated therein |
| PCT/JP2004/005515 WO2004092732A1 (en) | 2003-04-16 | 2004-04-16 | Particle having magnetic material incorporated therein, process for producing the same, particle for immunoassay and method of immunoassay |
| KR1020057019505A KR101115903B1 (en) | 2003-04-16 | 2004-04-16 | Particle Having Magnetic Material Incorporated Therein, Process for Producing the Same, Particle for Immunoassay and Method of Immunoassay |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2002305969 | 2002-10-21 | ||
| JP2003360986A JP4426819B2 (en) | 2002-10-21 | 2003-10-21 | Method for producing magnetic inclusion particles |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2004163421A true JP2004163421A (en) | 2004-06-10 |
| JP4426819B2 JP4426819B2 (en) | 2010-03-03 |
Family
ID=32828021
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2003360986A Expired - Fee Related JP4426819B2 (en) | 2002-10-21 | 2003-10-21 | Method for producing magnetic inclusion particles |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4426819B2 (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004092732A1 (en) * | 2003-04-16 | 2004-10-28 | Sekisui Chemical Co., Ltd. | Particle having magnetic material incorporated therein, process for producing the same, particle for immunoassay and method of immunoassay |
| JP2004331953A (en) * | 2003-04-16 | 2004-11-25 | Sekisui Chem Co Ltd | Magnetic material encapsulating particle, immunity measuring particle and immunity measuring method |
| JP2006131771A (en) * | 2004-11-05 | 2006-05-25 | Tokyo Institute Of Technology | Polymer-coated magnetic bead and method for producing the same |
| JP2006234417A (en) * | 2005-02-22 | 2006-09-07 | Jsr Corp | Magnetic particle dispersant, and particles for diagnostic agent |
| JP2006292721A (en) * | 2005-03-15 | 2006-10-26 | Sekisui Chem Co Ltd | Magnetic body-containing particle, manufacturing method of magnetic body-containing particle, particle for measuring immunity, and immunity measuring method |
| JP2007145999A (en) * | 2005-11-28 | 2007-06-14 | Fuji Xerox Co Ltd | Hydroxyl-containing magnetic polymer and method for producing the same |
| WO2012015125A1 (en) * | 2010-07-29 | 2012-02-02 | 연세대학교 산학협력단 | Stimulus sensitive magnetic nanocomposite using pyrene polymer, and contrast medium composition containing the nanocomposite |
| JP2017120242A (en) * | 2015-12-29 | 2017-07-06 | 新日鉄住金化学株式会社 | Resin-metal composite body, labeling substance, immunological measurement method, reagent for immunological measurement, analyte measurement method, and lateral flow type chromatographic test strip |
| JP2017223604A (en) * | 2016-06-17 | 2017-12-21 | 新日鉄住金化学株式会社 | Labeled antibody and method for producing the same, and immunological measurement method |
| JP2018136350A (en) * | 2014-07-01 | 2018-08-30 | 新日鉄住金化学株式会社 | Labeling substance, immunological measuring method, reagent for immunological measurement, analyte measuring method, analyte measuring kit, and lateral flow type chromatographic test strip |
| JP2018194332A (en) * | 2017-05-12 | 2018-12-06 | 株式会社Ｌｓｉメディエンス | Magnetic particle for supporting physiological active substance and production process for the same |
-
2003
- 2003-10-21 JP JP2003360986A patent/JP4426819B2/en not_active Expired - Fee Related
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7632688B2 (en) | 2003-04-16 | 2009-12-15 | Sekisui Chemical Co., Ltd. | Particle having magnetic material incorporated therein, process for producing the same, particle for immunoassay and method of immunoassay |
| JP2004331953A (en) * | 2003-04-16 | 2004-11-25 | Sekisui Chem Co Ltd | Magnetic material encapsulating particle, immunity measuring particle and immunity measuring method |
| WO2004092732A1 (en) * | 2003-04-16 | 2004-10-28 | Sekisui Chemical Co., Ltd. | Particle having magnetic material incorporated therein, process for producing the same, particle for immunoassay and method of immunoassay |
| JP4593146B2 (en) * | 2003-04-16 | 2010-12-08 | 積水化学工業株式会社 | Method for producing magnetic inclusion particles |
| JP2006131771A (en) * | 2004-11-05 | 2006-05-25 | Tokyo Institute Of Technology | Polymer-coated magnetic bead and method for producing the same |
| JP2006234417A (en) * | 2005-02-22 | 2006-09-07 | Jsr Corp | Magnetic particle dispersant, and particles for diagnostic agent |
| JP4653652B2 (en) * | 2005-03-15 | 2011-03-16 | 積水化学工業株式会社 | Magnetic inclusion particles, method for producing magnetic inclusion particles, immunoassay particles and immunoassay method |
| JP2006292721A (en) * | 2005-03-15 | 2006-10-26 | Sekisui Chem Co Ltd | Magnetic body-containing particle, manufacturing method of magnetic body-containing particle, particle for measuring immunity, and immunity measuring method |
| JP2007145999A (en) * | 2005-11-28 | 2007-06-14 | Fuji Xerox Co Ltd | Hydroxyl-containing magnetic polymer and method for producing the same |
| WO2012015125A1 (en) * | 2010-07-29 | 2012-02-02 | 연세대학교 산학협력단 | Stimulus sensitive magnetic nanocomposite using pyrene polymer, and contrast medium composition containing the nanocomposite |
| JP2018136350A (en) * | 2014-07-01 | 2018-08-30 | 新日鉄住金化学株式会社 | Labeling substance, immunological measuring method, reagent for immunological measurement, analyte measuring method, analyte measuring kit, and lateral flow type chromatographic test strip |
| US10690665B2 (en) | 2014-07-01 | 2020-06-23 | Nippon Steel Chemical & Materials Co., Ltd. | Marker, immunoassay method, immunoassay reagent, method for assaying analyte, analyte measurement kit, and lateral-flow chromatographic test strip |
| JP2020177035A (en) * | 2014-07-01 | 2020-10-29 | 日鉄ケミカル＆マテリアル株式会社 | Labeling substance, immunological measuring method, reagent for immunological measurement, analyte measuring method, analyte measuring kit, and lateral flow type chromatographic test strip |
| US11733241B2 (en) | 2014-07-01 | 2023-08-22 | Nippon Steel Chemical & Material Co., Ltd. | Marker, immunoassay method, immunoassay reagent, method for assaying analyte, analyte measurement kit, and lateral-flow chromatographic test strip |
| JP2017120242A (en) * | 2015-12-29 | 2017-07-06 | 新日鉄住金化学株式会社 | Resin-metal composite body, labeling substance, immunological measurement method, reagent for immunological measurement, analyte measurement method, and lateral flow type chromatographic test strip |
| JP2017223604A (en) * | 2016-06-17 | 2017-12-21 | 新日鉄住金化学株式会社 | Labeled antibody and method for producing the same, and immunological measurement method |
| JP2018194332A (en) * | 2017-05-12 | 2018-12-06 | 株式会社Ｌｓｉメディエンス | Magnetic particle for supporting physiological active substance and production process for the same |
| JP6998004B2 (en) | 2017-05-12 | 2022-01-18 | 株式会社Ｌｓｉメディエンス | Magnetic particles for supporting physiologically active substances and their manufacturing methods |
Also Published As
| Publication number | Publication date |
|---|---|
| JP4426819B2 (en) | 2010-03-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101115903B1 (en) | Particle Having Magnetic Material Incorporated Therein, Process for Producing the Same, Particle for Immunoassay and Method of Immunoassay | |
| US5814687A (en) | Magnetic polymer particle and process for manufacturing the same | |
| JP4593146B2 (en) | Method for producing magnetic inclusion particles | |
| KR100206159B1 (en) | Immunoassay using magnetic particle | |
| JP5279357B2 (en) | Composite particle, method for producing the same, dispersion, magnetic biosensing device, and magnetic biosensing method | |
| JP3738847B2 (en) | Method for producing diagnostic particles | |
| US20090099342A1 (en) | Process for Preparing Composite Particles, Composite Particles Obtained, and Their Use in a Diagnostic Test | |
| TW200903531A (en) | Magnetic particles and fabrication method thereof | |
| JP2003513093A (en) | Composite nanospheres and their complexes with biomolecules | |
| JP4426819B2 (en) | Method for producing magnetic inclusion particles | |
| JP2008164488A (en) | Magnetic particle, its producing method, and carrier for biochemistry | |
| JP6913908B2 (en) | Magnetic composite particles, their manufacturing methods, and immunoassay particles | |
| JP5428166B2 (en) | Aggregation and dispersion method of magnetic particles and separation, detection method and detection kit using the same | |
| CN111393574A (en) | Magnetic microsphere with functional groups on surface and preparation method and application thereof | |
| JP3646461B2 (en) | Magnetic polymer particles and method for producing the same | |
| JPH10505630A (en) | Novel latexes of calibrated monodisperse magnetizable microspheres, their preparation and their use in chemistry or biology | |
| JP4548598B2 (en) | Magnetic particle, method for producing the same, and carrier for biochemistry | |
| US20030181606A1 (en) | Process for producing particles for diagnostic reagent | |
| JP4485823B2 (en) | Magnetic inclusion particles and immunoassay particles | |
| JPH06231957A (en) | Particle coated with ferrite and its manufacture | |
| JP4359181B2 (en) | Method for producing magnetic inclusion particles | |
| JP2005098974A (en) | Metal-containing resin particles for immunity measurement, manufacturing method thereof, and particles for the immunity measurement | |
| CN100523815C (en) | Particle having magnetic material incorporated therein, process for producing the same, particle for immunoassay and method of immunoassay | |
| JP7363327B2 (en) | Magnetic particles for antibody binding and their manufacturing method | |
| JPH07316466A (en) | Particle protected with polymer and its production |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20060809 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20090715 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20090914 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20091118 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20091211 |
|
| R151 | Written notification of patent or utility model registration |
Ref document number: 4426819 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R151 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121218 Year of fee payment: 3 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121218 Year of fee payment: 3 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20131218 Year of fee payment: 4 |
|
| LAPS | Cancellation because of no payment of annual fees |