JP2006266970A - Polymer particle for immunodiagnostic drug - Google Patents
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本発明は、粒子表層部が凹凸することにより平滑表面粒子よりも多くの比表面積を有する診断用ポリマー粒子に関する。 The present invention relates to a diagnostic polymer particle having a specific surface area larger than that of a smooth surface particle due to unevenness of a particle surface layer portion.
ポリスチレン等からなるラテックスに抗原、抗体等の免疫 活性基を担持させ、ラテックスの凝集度で検体中の抗体、抗原等の濃度を測定する方法はラテックスイムノアッセイ法と呼ばれる。日本工業規格（ＪＩＳ）によると、濁度の光学的測定は、１）視覚濁度、２）透過光濁度、３）散乱光濁度、４）積分球濁度の４つの方法に分類されている。このうち、積分球濁度法では、前方散乱光と同時に平行透過光を測定し、その比率を求めて、抗原抗体反応本来の変化量以外の干渉因子に対する補償措置としている。これらの測定はその原理、操作が簡単で、測定時間が短く、低コストのラテックス、および自動測定ができるため、広く使用されている。 A method in which an immunoactive group such as an antigen or an antibody is supported on a latex made of polystyrene or the like and the concentration of the antibody or antigen in the sample is measured by the degree of latex aggregation is called a latex immunoassay method. According to the Japanese Industrial Standard (JIS), optical measurement of turbidity is classified into four methods: 1) visual turbidity, 2) transmitted light turbidity, 3) scattered light turbidity, and 4) integrating sphere turbidity. ing. Among these, in the integrating sphere turbidity method, the parallel transmitted light is measured simultaneously with the forward scattered light, and the ratio is obtained to compensate for the interference factor other than the original change amount of the antigen-antibody reaction. These measurements are widely used because of their simple principle and operation, short measurement time, low cost latex, and automatic measurement.
ラテックスイムノアッセイの簡便性から、高感度測定、および広い測定レンジの確保が常に解決すべき課題として努力されていた。通常のスチレンを主成分とし、乳化重合、懸濁重合、ソープフリー重合で調製されたポリマー粒子が平滑表面をもつものである。しかし、より高感度、広レンジの対象物を測定するために、粒子１個当たりにより多くの抗体が感作できることは必要である。これはつまり、粒子単位重量あたりにより多くの抗体が感作できることを意味する。 Due to the simplicity of latex immunoassay, high-sensitivity measurement and ensuring a wide measurement range have always been sought as problems to be solved. Normal styrene is the main component, and polymer particles prepared by emulsion polymerization, suspension polymerization, and soap-free polymerization have a smooth surface. However, it is necessary to be able to sensitize more antibodies per particle in order to measure higher sensitivity, wider range objects. This means that more antibodies can be sensitized per unit particle weight.
通常、抗体の分子サイズから、単位面積に感作できる抗体量が計算できるが、抗体が粒子表面に一層のみ感作することを想定する場合に、通常の平滑表面をもつポリスチレン粒子の抗体感作量がその幾何学計算された表面積以上の抗体量を感作することができない。抗体のアフィニティおよび粒子のコロイド性能、粒子の抗体吸着能が一定と仮定すれば、検出できる抗原量が感作抗体量に比例し、感作抗体量がまだ粒子表面積に依存する。つまり、高感度用ラテックス粒子の比表面積が大きいほど、感作抗体量の増大においては有利である。 Usually, the amount of antibody that can be sensitized to the unit area can be calculated from the molecular size of the antibody, but if it is assumed that the antibody is only sensitized to the particle surface, the antibody sensitization of polystyrene particles with a normal smooth surface An amount of antibody whose amount is greater than its geometrically calculated surface area cannot be sensitized. Assuming that the antibody affinity, the colloid performance of the particles, and the antibody adsorption capacity of the particles are constant, the amount of antigen that can be detected is proportional to the amount of sensitized antibody, and the amount of sensitized antibody still depends on the particle surface area. That is, the larger the specific surface area of the high sensitivity latex particles, the more advantageous the increase in the amount of sensitizing antibody.
一方、表面が凹凸である粒子であれば、同じ粒子の比表面積が平滑表面より増えるので、その分、抗体感作量アップが期待できる。
このような比表面積を大きくしようとする努力がクロマトグラフ用担体の分野でなされてきた。例えば、特開平１１−２７１２９４号や特開２００３−９３８０１号に記載の粒子などを挙げることができる。
しかし、これらの文献が低分子物質の分離を主な目的としたため、いずれのケースにおいても、粒子表面に一定サイズの細孔を必要とし、担体粒子も実質的に多孔質であることが必須である。
Efforts to increase the specific surface area have been made in the field of chromatographic supports. Examples thereof include particles described in JP-A Nos. 11-271294 and 2003-93801.
However, since these documents are mainly aimed at separation of low-molecular substances, in any case, it is essential that pores of a certain size are required on the particle surface and the carrier particles are also substantially porous. is there.
本発明の目的は、粒子表面の比表面積を増やしてより多くの抗体が感作できる粒子およびその製造方法を提供する。 An object of the present invention is to provide a particle capable of sensitizing more antibodies by increasing the specific surface area of the particle surface and a method for producing the same.
本発明は、ジビニルベンゼンの含有量が３０重量％を超える重合性不飽和化合物を重合した粒子であって、粒子径が０．０２〜２μｍ、粒径変動係数が２０％以下、かつ窒素吸着法で求められる粒子の比表面積が平均粒子径から計算される比表面積の１．３倍以上であることを特徴とする免疫診断薬用ポリマー粒子を提供するものである。
本発明診断薬用ポリマー粒子は、重合性不飽和化合物を、水系媒体での乳化重合あるいはソープフリー重合により重合して得られるものであるが、この際重合性不飽和化合物中のジビニルベンゼン含有量は３０重量％を超え、好ましくは５０重量％以上である。重合性不飽和化合物中のジビニルベンゼンの含有量が３０重量％以下になると、調製できた粒子表面の凹凸が浅くて所定の表面積アップに効果が少ないので好ましくない。また、形成されるポリマー粒子の屈折率が低くなるため、光学濁度が低下して、診断薬粒子としての感度が悪化するので好ましくない。
さらに、シード粒子の存在下に重合性不飽和化合物を添加して重合するシード重合法が粒子表面の凸凹を形成するために特に好ましい。
The present invention relates to particles obtained by polymerizing a polymerizable unsaturated compound having a divinylbenzene content exceeding 30% by weight, a particle size of 0.02 to 2 μm, a particle size variation coefficient of 20% or less, and a nitrogen adsorption method. The specific surface area of the particle | grain calculated | required by is 1.3 times or more of the specific surface area calculated from an average particle diameter, The polymer particle for immunodiagnostics characterized by the above-mentioned is provided.
The polymer particle for diagnostic medicine of the present invention is obtained by polymerizing a polymerizable unsaturated compound by emulsion polymerization or soap-free polymerization in an aqueous medium. In this case, the divinylbenzene content in the polymerizable unsaturated compound is It exceeds 30% by weight, preferably 50% by weight or more. If the content of divinylbenzene in the polymerizable unsaturated compound is 30% by weight or less, the unevenness of the prepared particle surface is shallow, and this is not preferable because the effect of increasing the predetermined surface area is small. Moreover, since the refractive index of the polymer particle formed becomes low, optical turbidity falls and the sensitivity as a diagnostic agent particle deteriorates, which is not preferable.
Further, a seed polymerization method in which a polymerizable unsaturated compound is added in the presence of seed particles for polymerization is particularly preferable for forming irregularities on the particle surface.
なお、本発明で平均粒子径とは表面積加重の算術平均径を言う。具体的には電子顕微鏡写真で少なくとも100個の粒子を計測し、各粒子の粒径値Xi、存在分率Niから次式で計算される。
平均粒子径＝（Σ Ni Xi3）／（Σ Ni Xi2）
また、粒径の変動係数は、粒子の粒径の標準偏差値を平均粒子径で割った値であり、パーセントで表す。
重合性不飽和化合物はジビニルベンゼンのみからなってもよいが、ジビニルベンゼン以外の重合性不飽和化合物として、スチレン、クロルスチレン、クロロメチルスチレン、α−メチルスチレン、スチレンスルホン酸ナトリウムなどの芳香族モノビニル化合物も含むことが好ましい。これらの芳香族モノビニル化合物は一種単独で、または二種以上を組合せて使用できる。また、これら芳香族ビニル化合物のほかに粒子表面への官能基を付与するために、アクリル酸、メタクリル酸、イタコン酸、フマル酸等のカルボキシル基含有重合性不飽和化合物、グリシジルメタクリレート等のエポキシ基含有重合性不飽和化合物、ビニルベンジルアミン、アリルアミン、Ｎ，Ｎ’−ジメチルアミノエチルメタクリレート等のアミノ基含有重合性不飽和化合物等を組み合わせることもできる。また、メチルメタクリレート、ｎ-ブチルアクリレート、ｔ−ブチルメタクリレート、シクロヘキシルメタクリレート、ポリエチレングリコールモノメタクリレート等の官能基を持たないアクリレートまたはメタクリレートも組み合わせることができる。しかし、これらの非芳香族の不飽和化合物は使用する不飽和化合物のなかの３０重量％以下である。
In the present invention, the average particle diameter refers to a surface area-weighted arithmetic average diameter. Specifically, at least 100 particles are measured with an electron micrograph, and the particle size value Xi and the abundance Ni of each particle are calculated by the following equation.
Average particle size = (ΣNi Xi 3 ) / (Σ Ni Xi 2 )
The variation coefficient of the particle diameter is a value obtained by dividing the standard deviation value of the particle diameter of the particle by the average particle diameter, and is expressed in percent.
The polymerizable unsaturated compound may consist only of divinylbenzene, but as the polymerizable unsaturated compound other than divinylbenzene, aromatic monovinyl such as styrene, chlorostyrene, chloromethylstyrene, α-methylstyrene, sodium styrenesulfonate, etc. It is preferable that a compound is also included. These aromatic monovinyl compounds can be used alone or in combination of two or more. In addition to these aromatic vinyl compounds, carboxyl groups-containing polymerizable unsaturated compounds such as acrylic acid, methacrylic acid, itaconic acid, fumaric acid, and epoxy groups such as glycidyl methacrylate are used to impart functional groups to the particle surface. An amino group-containing polymerizable unsaturated compound such as a containing polymerizable unsaturated compound, vinylbenzylamine, allylamine, or N, N′-dimethylaminoethyl methacrylate can also be combined. In addition, acrylates or methacrylates having no functional group such as methyl methacrylate, n-butyl acrylate, t-butyl methacrylate, cyclohexyl methacrylate, polyethylene glycol monomethacrylate and the like can also be combined. However, these non-aromatic unsaturated compounds are 30% by weight or less of the unsaturated compounds used.
本発明において、ポリマー粒子の重合方法としては、乳化重合法、懸濁重合法、ソープフリー重合法で粒子を調製することが出来る。さらにこれら粒子にシード重合法、モノマー後添加法で表面層を導入することができる。これらの重合方法は、公知の方法に従って行うことができ、例えば重合性不飽和化合物成分全量を反応系に一括して仕込んで重合する方法、重合性不飽和化合物成分の一部を仕込んで反応させた後、残りの重合性不飽和化合物成分を連続または分割して添加して重合する方法、重合性不飽和化合物成分全量を連続して仕込んで重合する方法などによって行うことができる。これらの重合方法において、ラジカル重合開始剤の添加方法も任意に選択でき、全量を一括して仕込む方法、連続的または分割して添加する方法などが採用できる。
またソープフリー重合法以外の方法で重合するとき、重合性不飽和化合物の重合時に乳化剤として、アニオン系、ノニオン系およびカチオン系の乳化剤などを使用することもできる。
本発明の重合に使用するラジカル重合開始剤としては、水溶性または油溶性のいずれのラジカル重合開始剤でも使用できる。粒子表面にアニオン碁を導入する目的で、水溶性開始剤が好ましい。上記水溶性のラジカル重合開始剤としては、例えば過硫酸カリウム、過硫酸アンモニウム、過硫酸ナトリウム等の過硫酸塩や過酸化水素などがあげられる。開始剤の使用量は重合性不飽和化合物に対して０．０１−１０重量％であることが好ましい。
また、油溶性ラジカル重合開始剤としては、例えば２，２’−アゾビスイソブチロニトリル、２，２’−アゾビス（４−メトキシ−２，４−ジメチルバレロニトリル）、２，２’−アゾビス−２，４−ジメチルバレロニトリル、１，１’−アゾビス−シクロヘキサン−１−カルボニトリル、過酸化ベンゾイル、過酸化ジブチル、クメンヒドロ過酸化物などがあげられる。
これらの油溶性のラジカル重合開始剤は、重合性不飽和化合物に溶解して、または水系媒体に分散して使用する。ラジカル重合開始剤の使用量は、重合性不飽和化合物成分に対して０．０１〜３０重量％、好ましくは０．１〜１０重量％とするのが望ましい。
In the present invention, the polymer particles can be prepared by an emulsion polymerization method, a suspension polymerization method, or a soap-free polymerization method. Furthermore, a surface layer can be introduced into these particles by a seed polymerization method or a monomer post-addition method. These polymerization methods can be carried out according to known methods, for example, a method in which the entire amount of the polymerizable unsaturated compound component is charged into the reaction system in a batch, and a part of the polymerizable unsaturated compound component is charged and reacted. Thereafter, the remaining polymerizable unsaturated compound component may be added continuously or in portions to polymerize, or the entire polymerizable unsaturated compound component may be charged continuously for polymerization. In these polymerization methods, the addition method of the radical polymerization initiator can also be arbitrarily selected, and a method of charging the whole amount at once, a method of adding continuously or dividedly, and the like can be employed.
In addition, when polymerizing by a method other than the soap-free polymerization method, anionic, nonionic, and cationic emulsifiers can be used as an emulsifier during polymerization of the polymerizable unsaturated compound.
As the radical polymerization initiator used in the polymerization of the present invention, any water-soluble or oil-soluble radical polymerization initiator can be used. A water-soluble initiator is preferred for the purpose of introducing anionic soot on the particle surface. Examples of the water-soluble radical polymerization initiator include persulfates such as potassium persulfate, ammonium persulfate, and sodium persulfate, and hydrogen peroxide. It is preferable that the usage-amount of an initiator is 0.01-10 weight% with respect to a polymerizable unsaturated compound.
Examples of the oil-soluble radical polymerization initiator include 2,2′-azobisisobutyronitrile, 2,2′-azobis (4-methoxy-2,4-dimethylvaleronitrile), and 2,2′-azobis. Examples include -2,4-dimethylvaleronitrile, 1,1'-azobis-cyclohexane-1-carbonitrile, benzoyl peroxide, dibutyl peroxide, cumene hydroperoxide and the like.
These oil-soluble radical polymerization initiators are used by dissolving in a polymerizable unsaturated compound or dispersing in an aqueous medium. The amount of radical polymerization initiator used is 0.01 to 30% by weight, preferably 0.1 to 10% by weight, based on the polymerizable unsaturated compound component.
水系媒体としては、水を単独で使用することもできるし、水に有機溶媒を混合した有機溶媒混合水を使用することもできる。このような有機溶媒としては、エタノール、メタノール、イソプロパノール、アセトン、テトラヒドロフラン、ジメチルホルムアミド等の水混和性有機溶媒などが使用できる。有機溶媒の添加量は、水に対して３０重量％以下とすることが好ましい。 As the aqueous medium, water can be used alone, or organic solvent mixed water obtained by mixing an organic solvent with water can also be used. As such an organic solvent, water-miscible organic solvents such as ethanol, methanol, isopropanol, acetone, tetrahydrofuran, dimethylformamide and the like can be used. The addition amount of the organic solvent is preferably 30% by weight or less with respect to water.
本発明品の免疫診断用ポリマー粒子を重合するとき、重合性不飽和化合物とともに不活性有機溶剤を加えて重合することができる。不活性有機溶剤として、例えば、シクロヘキサノール、ヘキサデカノール、トルエン、ヘキサン、イソオクタンなどの水溶性の低い有機溶剤が挙げられる。これら不活性有機溶剤の添加濃度によって得られる免疫診断薬用ポリマー粒子の表面凹凸の幅と深さを制御することができる。
水系媒体中の重合性不飽和化合物の濃度は、通常０．２〜４０重量％、好ましくは１〜３０重量％であり、重合正不飽和化合物の重合温度および重合時間は、ラジカル重合開始剤の種類等の合成条件により異なるが、通常５〜２０時間、３０〜１００℃である。
本発明において、抗体または抗原を化学結合で固定するためにポリマー粒子に官能基を導入してもよい。これらの官能基としては、カルボン酸基、アミノ基、チオール基、エポキシ基、トシル基、水酸基等が挙げられる。
カルボン酸基は、例えばα，β−不飽和カルボン酸を前記重合性不飽和化合物と共重合することによりポリマー粒子表面にカルボン酸基を導入することができる。α，β−不飽和カルボン酸としては、メタクリル酸、アクリル酸、イタコン酸などが上げられる。アミノ基、エポキシ基、チオール基、トシル基もこれら官能基有する化合物と重合性不飽和化合物との共重合で導入することができる。
これらの官能基は、前記ポリマー粒子の重合と同時に、または重合後に官能基を有する重合性化合物を反応系に添加することにより、ポリマー粒子表面に導入することができる。
When polymerizing the immunodiagnostic polymer particles of the present invention, polymerization can be performed by adding an inert organic solvent together with the polymerizable unsaturated compound. Examples of the inert organic solvent include organic solvents having low water solubility such as cyclohexanol, hexadecanol, toluene, hexane, and isooctane. The width and depth of the surface irregularities of the polymer particles for immunodiagnostic drugs obtained by the addition concentration of these inert organic solvents can be controlled.
The concentration of the polymerizable unsaturated compound in the aqueous medium is usually 0.2 to 40% by weight, preferably 1 to 30% by weight. The polymerization temperature and polymerization time of the polymerized unsaturated compound are determined by the radical polymerization initiator. Although it changes with synthesis conditions, such as a kind, it is 30-100 degreeC normally for 5 to 20 hours.
In the present invention, a functional group may be introduced into the polymer particle in order to immobilize the antibody or antigen by a chemical bond. Examples of these functional groups include carboxylic acid groups, amino groups, thiol groups, epoxy groups, tosyl groups, and hydroxyl groups.
The carboxylic acid group can be introduced into the surface of the polymer particle by copolymerizing, for example, an α, β-unsaturated carboxylic acid with the polymerizable unsaturated compound. Examples of the α, β-unsaturated carboxylic acid include methacrylic acid, acrylic acid, itaconic acid and the like. An amino group, an epoxy group, a thiol group, and a tosyl group can also be introduced by copolymerization of a compound having these functional groups and a polymerizable unsaturated compound.
These functional groups can be introduced to the surface of the polymer particles by adding a polymerizable compound having a functional group to the reaction system simultaneously with the polymerization of the polymer particles or after the polymerization.
本発明において、重合反応が終了した後にポリマー粒子を有機溶剤と接触させ、ポリマー粒子表面の前記有機溶媒への可溶成分や低分子量成分を有機溶剤で除去することが好ましい。
具体的な有機溶媒として、例えば、アセトン、イソプロピルアルコール、メチルエチルケトン、テトラヒドロフラン、ジメチルホルムアミドなどが挙げられる。
またこれら有機溶剤の添加濃度、分散時間、分散温度によって表面凹凸のサイズと深さを制御することができる。
In the present invention, it is preferable that after the polymerization reaction is completed, the polymer particles are brought into contact with an organic solvent, and soluble components and low molecular weight components in the organic solvent on the surface of the polymer particles are removed with the organic solvent.
Specific examples of the organic solvent include acetone, isopropyl alcohol, methyl ethyl ketone, tetrahydrofuran, dimethylformamide, and the like.
Further, the size and depth of the surface irregularities can be controlled by the addition concentration of these organic solvents, the dispersion time, and the dispersion temperature.
本発明の診断薬用ポリマー粒子の粒径は０．０２μｍ〜２μｍであり、０．０５μｍ〜０．８μｍであることがより好ましい。０．０２μｍ未満になると、免疫反応に基づく僅かな粒子凝集を検出できなくなる恐れがあり、結果的に低感度となるため、好ましくない。２μｍを超えると、粒子凝集に基づく吸光度変化があまりにも大きく、わずかの凝集でも、大きい吸光度変化をもたらし、結果的に測定レンジが狭くなるため好ましくない。 The particle size of the polymer particles for diagnostic agents of the present invention is 0.02 μm to 2 μm, and more preferably 0.05 μm to 0.8 μm. If it is less than 0.02 μm, there is a possibility that slight particle aggregation based on the immune reaction may not be detected, resulting in low sensitivity, which is not preferable. If it exceeds 2 μm, the absorbance change based on particle aggregation is too large, and even a slight aggregation results in a large absorbance change, resulting in a narrow measurement range.
本発明の診断薬用ポリマー粒子の粒径の変動係数（ＣＶ値）は２０％以下であり、１０％以下であることがより好ましい。ＣＶ値が２０％を超えると粒径分布の広さによる吸光度変化の不安定性が無視できなくなり、調製できた試薬の再現性に悪影響を与えるので好ましくない。
本発明の診断薬用ポリマー粒子の比表面積は、窒素吸着法で測定して得られる値が平均粒径で算出する値よりも１．３倍大きく、好ましくは１．５倍以上大きい。診断薬用ポリマー粒子の窒素吸着法で測定して得られる比表面積が平均粒径で算出する比表面積の１．３倍未満になると、粒度分布を考慮した比表面積との差が小さくなり、実質的な表面積アップに繋がらなくなり、診断薬粒子としての性能向上が期待できない。
The coefficient of variation (CV value) of the particle diameter of the diagnostic polymer particles of the present invention is 20% or less, and more preferably 10% or less. If the CV value exceeds 20%, the instability of the absorbance change due to the wide particle size distribution cannot be ignored, and this adversely affects the reproducibility of the prepared reagent.
The specific surface area of the polymer particles for diagnostic agents of the present invention is 1.3 times larger, preferably 1.5 times larger than the value calculated by the average particle size, as measured by the nitrogen adsorption method. When the specific surface area obtained by measuring the polymer particles for diagnostic drugs by the nitrogen adsorption method is less than 1.3 times the specific surface area calculated by the average particle diameter, the difference from the specific surface area considering the particle size distribution becomes small and substantially Therefore, the surface area cannot be increased, and the performance as diagnostic particles cannot be expected.
本発明の診断薬用ポリマー粒子の表面凹凸は、通常のスキャン式電子顕微鏡で観察することができる。この凹凸の平均サイズは、通常の抗体のサイズを考慮すれば、実質的に３〜７０ｎｍであることが好ましい。この凹凸が３ｎｍ未満になると抗体または、感作すべき抗原、ペプチドとサイズ的な差がつかなくなるので好ましく、また、７０ｎｍを超えると、粒子の形状異常となり診断薬粒子としての性能向上が期待できない。
また、本発明品の診断薬用ポリマー粒子は、クロマトグラフ分離用担体のような低分子物質も通過できる微小細孔を含む必要性がなく、多孔質である必要もない。これは低分子物質の分離精製に使うクロマト担体と基本的な相違点である。
The surface irregularities of the polymer particles for diagnostic agents of the present invention can be observed with a normal scanning electron microscope. It is preferable that the average size of the irregularities is substantially 3 to 70 nm considering the size of a normal antibody. If the unevenness is less than 3 nm, the size difference from the antibody or the antigen or peptide to be sensitized is not preferred, and if it exceeds 70 nm, the shape of the particle becomes abnormal and performance as a diagnostic agent particle cannot be expected. .
Further, the polymer particles for diagnostic agents of the present invention do not need to contain fine pores through which low molecular substances such as chromatographic separation carriers can pass, and need not be porous. This is a fundamental difference from chromatographic carriers used for separation and purification of low-molecular substances.
本発明品の診断薬用ポリマー粒子の表面への抗体結合量は、感作する抗体のサイズ、測定すべき抗原サイズ、または粒子表面の凹凸サイズによる。
例えば、診断薬用ポリマー粒子表面の凹部に結合する抗体が抗原と反応してからの分子サイズが相手側粒子の抗体と立体的に反応できる構造であれば、抗原の消費速度と粒子凝集速度の均衡が保たれるので、本発明品の粒子が凝集反応の高感度に寄与し、好適である。一方、もし、本発明の表面凹凸粒子の凹部に結合された抗体が抗原を捕獲しても、相手側粒子の抗体と反応できるスペーサーがなければ、抗原の消費速度が粒子同士の凝集速度より速いので、広いレンジの測定に好適である。
The amount of antibody bound to the surface of the diagnostic polymer particle of the present invention depends on the size of the sensitizing antibody, the antigen size to be measured, or the uneven size of the particle surface.
For example, if the molecular size after the antibody that binds to the recess on the surface of the diagnostic polymer particle reacts with the antigen can react sterically with the antibody of the partner particle, the balance between the antigen consumption rate and the particle aggregation rate is balanced. Therefore, the particles of the product of the present invention contribute to the high sensitivity of the agglutination reaction and are suitable. On the other hand, if the antibody bound to the concave portion of the surface irregularity particle of the present invention captures the antigen and there is no spacer capable of reacting with the antibody of the counterpart particle, the consumption rate of the antigen is faster than the aggregation rate of the particles. Therefore, it is suitable for a wide range measurement.
本発明品の診断用ポリマー粒子がラテックス凝集反応を原理とする全ての免疫反応に適合できる。そのときの測定時間、波長等が実際の粒子サイズ、反応速度に合わせて設定することができる。診断できる項目も特に限定されるものではなく、免疫反応によって粒子が凝集し、その変化が吸光度として取られるものであれば、すべて本発明の範疇にある。具体的に、たとえば、Ｃ-反応蛋白（ＣＲＰ）、リュウマチ因子（ＲＦ）、ＩｇＭ，抗ストレプトリジンＯ抗体、ヘモグロビン、βー２マイクログロブリン、α−フェトプロティン（ＡＦＰ）、ＩｇＥ、梅毒トレポネーマ抗体、ＨＢＳ抗体、ＨＢＳ抗原、ＨＢｃ抗体、又はＨＢｅ抗体などが挙げられる。
これら抗原を検出するために、例えば、抗体量が粒子重量の１〜３０重量％を添加し、物理吸着、または、粒子表面の官能基を利用して化学結合で固定させる方法が挙げられる。この場合、過剰に添加した抗体を固液分離で除去すれば、実際の診断反応に影響を与えない。
The diagnostic polymer particles of the present invention can be adapted to all immune reactions based on the principle of latex agglutination. The measurement time, wavelength, etc. at that time can be set according to the actual particle size and reaction rate. The items that can be diagnosed are not particularly limited, and any particles can be aggregated by an immune reaction and the change can be taken as absorbance. Specifically, for example, C-reactive protein (CRP), rheumatoid factor (RF), IgM, anti-streptolysin O antibody, hemoglobin, β-2 microglobulin, α-fetoprotein (AFP), IgE, syphilis treponema antibody, Examples include HBS antibody, HBS antigen, HBc antibody, and HBe antibody.
In order to detect these antigens, for example, an antibody amount of 1 to 30% by weight of the particle weight is added, and physical adsorption, or a method of fixing by chemical bonding using a functional group on the particle surface can be mentioned. In this case, if the excessively added antibody is removed by solid-liquid separation, the actual diagnostic reaction is not affected.
本発明の診断薬用ポリマー粒子は、表面を凹凸形状に設計することで、単位面積あたりに多くの抗体を結合することが可能となった。ラテックス凝集反応の高感度、広レンジ測定に好適である。 The polymer particles for diagnostic agents of the present invention can bind many antibodies per unit area by designing the surface to have an uneven shape. Suitable for high sensitivity and wide range measurement of latex agglutination reaction.
以下、本発明を実施例によりさら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。なお、本実施例において、部および％は重量換算である。
実施例
粒径測定
粒径測定は透過型電子顕微鏡で写真を撮影し、写真上で少なくとも１００個の粒子を計測して、平均粒子径と粒子径の変動係数を求めた。
表面積測定
本発明で調製された粒子の比表面積がＡＵＴＯＳＯＲＢ−１（アサユ（株））を用いて窒素ガス吸着法を使った。ＢＥＴプロット解析より表面積を求めた。
表面荷電測定
本発明で調製された粒子の表面ＣＯＯＨ量は、ＭｅｔｒｏＨｍ社製伝導度測定装置を用いて、酸・アルカリ滴定より求めた。
ラテックス凝集測定
本発明で調製された粒子のラテックス凝集試験は、日立分光光度計U2100を用いて測定した。反応温度は３７℃に設定した。
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited only to these Examples. In addition, in a present Example, a part and% are weight conversion.
Example Particle Size Measurement For particle size measurement, a photograph was taken with a transmission electron microscope, and at least 100 particles were measured on the photograph to determine the average particle diameter and the coefficient of variation of the particle diameter.
Surface area measurement The specific surface area of the particles prepared according to the present invention was measured using an ATOSORB-1 (Asayu Co., Ltd.) nitrogen gas adsorption method. The surface area was determined from BET plot analysis.
Surface Charge Measurement The surface COOH amount of the particles prepared in the present invention was determined by acid / alkali titration using a conductivity measuring device manufactured by MetroHm.
Latex aggregation measurement The latex aggregation test of the particles prepared in the present invention was measured using Hitachi spectrophotometer U2100. The reaction temperature was set to 37 ° C.
実施例１
撹拌装置つきガラス製フラスコに、窒素置換した後、イオン交換水６００部、ドデシルベンゼンスルホン酸ソーダ０．２部、過硫酸カリウム０．５部、スチレン５０部、ジビニルベンゼン５０部を入れ、８５℃で４時間の重合を行なった。４時間目で少量のラテックスをサンプリングしたところ、重合転化率９９％であった。ここで得られた粒子を粒子−１とする。
実施例２
実施例１で８５℃で４時間の加熱を終わった後に、加熱を中止せず、８５℃のまま、さらに水２０部、過硫酸カリウム０．２部、シクロヘキシルメタクリレート１９部、メタクリル酸１部を入れ、さらに８５℃で４時間の重合を行なった。重合後、室温に冷却したところ、重合転化率９９％、の粒子が得られた。この粒子を粒子―２とする。
Example 1
In a glass flask equipped with a stirrer, after substituting with nitrogen, 600 parts of ion-exchanged water, 0.2 part of sodium dodecylbenzenesulfonate, 0.5 part of potassium persulfate, 50 parts of styrene and 50 parts of divinylbenzene were placed at 85 ° C. For 4 hours. When a small amount of latex was sampled at 4 hours, the polymerization conversion was 99%. The particle | grains obtained here are set to particle-1.
Example 2
After finishing heating at 85 ° C. for 4 hours in Example 1, the heating was not stopped and the water was further kept at 85 ° C., 20 parts of water, 0.2 part of potassium persulfate, 19 parts of cyclohexyl methacrylate, and 1 part of methacrylic acid. Then, polymerization was further performed at 85 ° C. for 4 hours. After polymerization, when cooled to room temperature, particles having a polymerization conversion rate of 99% were obtained. This particle is referred to as particle-2.
実施例３
実施例１で得られた粒子を遠心管に入れ、１０，０００Ｇｘ２時間で遠心分離した。透明になった上澄を除去し、同容量のアセトンを添加し、粒子を分散した。粒子分散液をそのまま室温で一晩静置した。その後、同じ遠心条件で３回蒸留水に置換した。この粒子を粒子―３とする。
実施例４
実施例３のアセトンの代わりに、ＴＨＦを用いた以外は実施例３と全く同様な方法で実施した。得られた粒子を粒子―４とした。
実施例５
実施例２で得られた粒子を遠心管に入れ、１０，０００Ｇｘ２時間で遠心分離した。透明になった上澄を除去し、同容量のアセトンを添加し、粒子を分散した。粒子分散液をそのまま室温で一晩静置した。その後、同じ遠心条件で３回蒸留水に置換した。この粒子を粒子―５とする。
実施例６
実施例５のアセトンの代わりに、ＴＨＦを用いた以外は実施例３と全く同様な方法で実施した。得られた粒子を粒子―６とした。
Example 3
The particles obtained in Example 1 were put in a centrifuge tube and centrifuged at 10,000 G × 2 hours. The clear supernatant was removed and the same volume of acetone was added to disperse the particles. The particle dispersion was allowed to stand overnight at room temperature. Thereafter, it was replaced with distilled water three times under the same centrifugal conditions. This particle is referred to as particle-3.
Example 4
The procedure was exactly the same as in Example 3, except that THF was used instead of acetone in Example 3. The obtained particles were designated as Particle-4.
Example 5
The particles obtained in Example 2 were placed in a centrifuge tube and centrifuged at 10,000 G × 2 hours. The clear supernatant was removed and the same volume of acetone was added to disperse the particles. The particle dispersion was allowed to stand overnight at room temperature. Thereafter, it was replaced with distilled water three times under the same centrifugal conditions. This particle is referred to as particle-5.
Example 6
This was carried out in the same manner as in Example 3 except that THF was used instead of acetone in Example 5. The obtained particles were designated as Particle-6.
実施例７
撹拌装置つきガラス製フラスコに、窒素置換した後、シード粒子として別途に乳化重合して得た0.1μmのポリスチレン粒子の水分散体５００部（固形分で１００部）、イオン交換水３００部、ドデシルベンゼンスルホン酸ソーダ０．２部を入れ、８０℃に昇温した後、過硫酸カリウム０．５部を入れ、 スチレン５０部、ジビニルベンゼン５０部およびイソオクタン１０部の混合物を ２時間かけて連続的に添加し、さらに８０℃で２時間の重合を行なった。４時間目で少量のラテックスをサンプリングしたところ、重合転化率９９％であった。ここで得られた粒子を粒子−７とする。
Example 7
Into a glass flask equipped with a stirrer, after substituting with nitrogen, 500 parts of an aqueous dispersion of 0.1 μm polystyrene particles obtained separately by emulsion polymerization as seed particles (100 parts in solids), 300 parts of ion-exchanged water, dodecyl Add 0.2 part of sodium benzenesulfonate, raise the temperature to 80 ° C, add 0.5 part of potassium persulfate, and continuously add a mixture of 50 parts of styrene, 50 parts of divinylbenzene and 10 parts of isooctane over 2 hours. Was further polymerized at 80 ° C. for 2 hours. When a small amount of latex was sampled at 4 hours, the polymerization conversion was 99%. The particles obtained here are designated as Particle-7.
比較例１
撹拌装置つきガラス製フラスコに、窒素置換した後、イオン交換水６００部、ドデシルベンゼンスルホン酸ソーダ０．２部、過硫酸カリウム０．５部、スチレン１００部、８５℃で４時間の重合を行なった。４時間目で少量のラテックスをサンプリングしたところ、重合転化率９９％、であった。ここで得られた粒子を粒子−Ｒ１とする。
比較例２
比較例１で８５℃で４時間の加熱を終わった後に、加熱を中止せず、８５℃のまま、さらに水２０部、過硫酸カリウム０．２部、シクロヘキシルメタクリレート１９部、メタクリル酸１部を入れ、さらに８５℃で４時間の重合を行なった。重合後、室温に冷却したところ、重合転化率９９％、の粒子が得られた。この粒子を粒子−Ｒ２とする。
試験例
（表面積測定）
上記実施例および比較例で得られた粒子を遠心分離し、上澄を除去してから、ペレットを回収した。ペレットを振動で分散させ、４０℃の恒温槽で１週間静置し、時々振動をかけて、乾燥粒子の均一な粉末を得た。上記粒子粉末を窒素ガス吸着法で表面積を測定した。
Comparative Example 1
A glass flask with a stirrer was purged with nitrogen, and then subjected to polymerization at 600 parts of ion exchange water, 0.2 part of sodium dodecylbenzenesulfonate, 0.5 part of potassium persulfate, 100 parts of styrene, and 85 ° C. for 4 hours. It was. When a small amount of latex was sampled at 4 hours, the polymerization conversion was 99%. The particles obtained here are referred to as Particle-R1.
Comparative Example 2
In Comparative Example 1, after heating at 85 ° C. for 4 hours, the heating was not stopped and the water was kept at 85 ° C., and further 20 parts of water, 0.2 part of potassium persulfate, 19 parts of cyclohexyl methacrylate and 1 part of methacrylic acid were added. Then, polymerization was further performed at 85 ° C. for 4 hours. After polymerization, when cooled to room temperature, particles having a polymerization conversion rate of 99% were obtained. This particle is referred to as particle-R2.
Test example (surface area measurement)
The particles obtained in the above examples and comparative examples were centrifuged and the supernatant was removed, and then the pellets were collected. The pellets were dispersed by vibration, allowed to stand in a constant temperature bath at 40 ° C. for 1 week, and occasionally shaken to obtain a uniform powder of dry particles. The surface area of the particle powder was measured by a nitrogen gas adsorption method.
（ＩｇＧ物理吸着量）
上記実施例１，３，４および比較例１で得られた粒子分散液を固形分１０重量％に調整し、１００μｌを１．５ｍｌ遠心チューブにいれる。３０ｍＭリン酸緩衝液で２回洗浄した後に、固形分５％ラテックス分散液に、ウサギ抗CRP抗体（日本バイオテスト（株））１５０μｇ／ｍｇ−Ｌａｔｅｘ の割合で添加した。粒子分散液をそのまま室温で２時間ゆっくり攪拌し、抗体の物理吸着を行なった。その後、１５０００ｇで１０分遠心し、上澄液を２８０ｎｍで測定し、吸光度より吸着したＩｇＧを算出した。コントロールとして抗体を加えなくて粒子分散液のみ、および粒子を加えなくて、抗体を加えた各１点とした。
（ＩｇＧの化学結合量）
上記実施例２，５，６および比較例２で得られた粒子分散液を固形分１０重量％に調整し、１００μｌを１．５ｍｌ遠心チューブにいれる。３０ｍＭリン酸緩衝液で２回洗浄した後に、固形分５％ラテックス分散液に、水溶性カルボジイミド（同仁化学１０μｇ／ｍｇ の割合で添加し、室温で１時間ゆっくり転倒攪拌した。続いて１５０００ｇｘ１０分で遠心分離し、上澄除去してから、ウサギ抗CRP抗体（日本バイオテスト（株））１５０μｇ／ｍｇ−Ｌａｔｅｘの割合で加えて、一晩ゆっくり転倒攪拌した。その後、１５０００ｇで１０分遠心し、上澄液を２８０ｎｍで測定し、吸光度より吸着したＩｇＧを算出した。コントロールとして抗体を加えなくて粒子分散液のみ、および粒子を加えなくて、抗体を加えた各１点とした。
（ラテックスイムノアッセイ）
実施例８，９で感作したCRP抗体感作粒子をＰＢＳ／０．１％ＢＳＡで３回洗浄し、最終的に２０ｍＭリン酸緩衝液に分散させた。粒子固形分を０．１％とした。続いて間接超音波で１０分間照射し、粒径が感作前の１．０５倍以内にした。この粒子分散液２５０μｌにＰＢＳ／０．０５％ＢＳＡ／５ｍＭＣａＣｌ２溶液１２５μｌ、CRP抗原（0-2ｍｇ／ｍｌ）を含む血清１０μｌを加えて、室温で５分間反応させた。CRP抗原50μg/ml で0分と５分の６００ｎｍの吸光度変化値を測定し、吸光度変化とした。
また、測定レンジは、キャリブレーションカーブで吸光度変化の増大が見られなくなったときの抗原濃度とした。
上記測定値の結果を表１にまとめた。
(IgG physical adsorption amount)
The particle dispersions obtained in Examples 1, 3, 4 and Comparative Example 1 are adjusted to a solid content of 10% by weight, and 100 μl is placed in a 1.5 ml centrifuge tube. After washing twice with 30 mM phosphate buffer, rabbit anti-CRP antibody (Nippon Biotest Co., Ltd.) 150 μg / mg-Latex was added to the 5% solids latex dispersion. The particle dispersion was slowly stirred as it was at room temperature for 2 hours to perform physical adsorption of the antibody. Thereafter, the mixture was centrifuged at 15000 g for 10 minutes, the supernatant was measured at 280 nm, and the adsorbed IgG was calculated from the absorbance. As a control, no antibody was added, only the particle dispersion, and no particles were added, and one point was added for each antibody.
(Amount of chemically bound IgG)
The particle dispersions obtained in Examples 2, 5, 6 and Comparative Example 2 are adjusted to a solid content of 10% by weight, and 100 μl is placed in a 1.5 ml centrifuge tube. After washing twice with 30 mM phosphate buffer, water-soluble carbodiimide (Dojindo Chemical 10 μg / mg) was added to a 5% solid content latex dispersion, and the mixture was gently agitated for 1 hour at room temperature. After centrifuging and removing the supernatant, rabbit anti-CRP antibody (Nippon Biotest Co., Ltd.) was added at a ratio of 150 μg / mg-Latex, and slowly stirred by inverting overnight, then centrifuged at 15000 g for 10 minutes, The supernatant was measured at 280 nm, and the adsorbed IgG was calculated from the absorbance, and as a control, no antibody was added, only the particle dispersion, and no particles were added, and one point was added for each antibody.
(Latex immunoassay)
The CRP antibody-sensitized particles sensitized in Examples 8 and 9 were washed 3 times with PBS / 0.1% BSA and finally dispersed in 20 mM phosphate buffer. The particle solid content was 0.1%. Subsequently, irradiation was performed with indirect ultrasonic waves for 10 minutes, so that the particle size was within 1.05 times that before sensitization. To 250 μl of the particle dispersion, 125 μl of PBS / 0.05% BSA / 5 mM CaCl 2 solution and 10 μl of serum containing CRP antigen (0-2 mg / ml) were added and reacted at room temperature for 5 minutes. The absorbance change value at 600 nm at 0 min and 5 min at 50 μg / ml of CRP antigen was measured and used as the absorbance change.
The measurement range was the antigen concentration when no increase in absorbance change was observed in the calibration curve.
The results of the above measured values are summarized in Table 1.
Claims (4)
The polymerizable unsaturated compound having a divinylbenzene content of more than 30% by weight is polymerized in an aqueous medium in the presence of an inert organic solvent to form particles, and then obtained. The method for producing polymer particles for an immunodiagnostic drug according to claim 1, further comprising a step of bringing the particles into contact with an organic solvent.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2019319A2 (en) | 2007-07-25 | 2009-01-28 | JSR Corporation | Reagent for latex aggregation reaction and method for detecting target substance |
| CN102749457A (en) * | 2012-07-27 | 2012-10-24 | 北京恩济和生物科技有限公司 | Beta2-microglobulin detection kit and preparing method thereof |
| CN102749460A (en) * | 2012-07-27 | 2012-10-24 | 北京恩济和生物科技有限公司 | Troponin detection kit and preparing method thereof |
| JP5554247B2 (en) * | 2008-12-04 | 2014-07-23 | 積水メディカル株式会社 | Method for measuring cystatin C in human body fluid |
| CN110637232A (en) * | 2017-05-24 | 2019-12-31 | 积水化学工业株式会社 | Latex particle for measurement reagent, sensitized latex particle, and measurement reagent for immunoturbidimetry |
| US11493506B2 (en) | 2016-02-12 | 2022-11-08 | Jsr Corporation | Additive, surface treatment agent, surface-modified latex particles, method for producing surface-modified latex particles, reagent for latex agglutination reaction, kit, and method for detecting target substance |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05172817A (en) * | 1991-08-22 | 1993-07-13 | Sekisui Chem Co Ltd | Carrier for immunity diagnostic reagent and reagent for diagnosing immunity using the same |
| JPH089995A (en) * | 1983-05-12 | 1996-01-16 | Ciba Corning Diagnostics Corp | Implementation of enzyme reaction |
| JP2001289854A (en) * | 2000-04-11 | 2001-10-19 | Sekisui Chem Co Ltd | Polymer spherical body, immunoassay reagent and immunoassay method |
-
2005
- 2005-03-25 JP JP2005087719A patent/JP2006266970A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH089995A (en) * | 1983-05-12 | 1996-01-16 | Ciba Corning Diagnostics Corp | Implementation of enzyme reaction |
| JPH05172817A (en) * | 1991-08-22 | 1993-07-13 | Sekisui Chem Co Ltd | Carrier for immunity diagnostic reagent and reagent for diagnosing immunity using the same |
| JP2001289854A (en) * | 2000-04-11 | 2001-10-19 | Sekisui Chem Co Ltd | Polymer spherical body, immunoassay reagent and immunoassay method |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2019319A2 (en) | 2007-07-25 | 2009-01-28 | JSR Corporation | Reagent for latex aggregation reaction and method for detecting target substance |
| JP5554247B2 (en) * | 2008-12-04 | 2014-07-23 | 積水メディカル株式会社 | Method for measuring cystatin C in human body fluid |
| CN102749457A (en) * | 2012-07-27 | 2012-10-24 | 北京恩济和生物科技有限公司 | Beta2-microglobulin detection kit and preparing method thereof |
| CN102749460A (en) * | 2012-07-27 | 2012-10-24 | 北京恩济和生物科技有限公司 | Troponin detection kit and preparing method thereof |
| US11493506B2 (en) | 2016-02-12 | 2022-11-08 | Jsr Corporation | Additive, surface treatment agent, surface-modified latex particles, method for producing surface-modified latex particles, reagent for latex agglutination reaction, kit, and method for detecting target substance |
| CN110637232A (en) * | 2017-05-24 | 2019-12-31 | 积水化学工业株式会社 | Latex particle for measurement reagent, sensitized latex particle, and measurement reagent for immunoturbidimetry |
| CN110637232B (en) * | 2017-05-24 | 2023-08-18 | 积水医疗株式会社 | Latex particles for measurement reagent, sensitized latex particles, and measurement reagent for immunoturbidimetry |
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