JP5720318B2 - Detection method and quantification method of detection target - Google Patents
Detection method and quantification method of detection target Download PDFInfo
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- JP5720318B2 JP5720318B2 JP2011052081A JP2011052081A JP5720318B2 JP 5720318 B2 JP5720318 B2 JP 5720318B2 JP 2011052081 A JP2011052081 A JP 2011052081A JP 2011052081 A JP2011052081 A JP 2011052081A JP 5720318 B2 JP5720318 B2 JP 5720318B2
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Description
本発明は、検出対象の検出方法および定量方法に関する。 The present invention relates to a detection method and a quantification method of a detection target.
従来から、被検体中の検出対象を検出する方法として、ラテックス凝集法が利用されてきた。ラテックス凝集法とは、生体試料等の流体中における抗原を検出する場合、抗原に特異的に結合する抗体もしくはそのフラグメントを担持させたラテックスと、流体とを混合して、ラテックスの凝集の程度を測定することにより、抗原を検出または定量する方法である（例えば、特許文献１参照）。 Conventionally, a latex agglutination method has been used as a method for detecting a detection target in a specimen. The latex agglutination method is a method for detecting the antigen in a fluid such as a biological sample by mixing a latex carrying an antibody or a fragment thereof that specifically binds to the antigen with the fluid to determine the degree of latex aggregation. This is a method for detecting or quantifying an antigen by measuring (for example, see Patent Document 1).
ラテックス凝集法によれば、検体として添加された抗原が複数のラテックス結合抗体を架橋させ、ラテックスの凝集を促す。このように手順が単純であるから、簡便且つ迅速に抗原を検出できる。しかし、抗原が微量の場合、該架橋が起こりにくいため、ラテックスが十分に凝集しない。このため、微量の抗原を検出することが困難であった。 According to the latex agglutination method, an antigen added as a specimen crosslinks a plurality of latex-bound antibodies to promote latex aggregation. Since the procedure is simple as described above, the antigen can be detected easily and rapidly. However, when the amount of antigen is very small, the crosslinking is difficult to occur, so that the latex does not sufficiently aggregate. For this reason, it was difficult to detect a trace amount of antigen.
そこで、ＥＬＩＳＡ法およびＣＬＥＩＡ法といった酵素基質反応を利用する方法も広く利用されている。これらの方法では、例えば、抗原に特異的に結合する一次抗体を抗原に結合させ、該一次抗体に酵素を有する二次抗体を結合させる。ここで、酵素の基質を添加し、酵素が触媒する反応の程度を測定することで、抗原を検出または定量する。 Therefore, methods using enzyme substrate reactions such as ELISA and CLEIA are also widely used. In these methods, for example, a primary antibody that specifically binds to an antigen is bound to the antigen, and a secondary antibody having an enzyme is bound to the primary antibody. Here, the antigen is detected or quantified by adding the enzyme substrate and measuring the degree of reaction catalyzed by the enzyme.
これらの方法によれば、例えば、基質として発光試薬を用いると、基質添加後の発光の検出感度が高いため、微量の抗原も検出できる。 According to these methods, for example, when a luminescent reagent is used as the substrate, the detection sensitivity of luminescence after the addition of the substrate is high, so that a trace amount of antigen can also be detected.
しかし、酵素基質反応を利用する方法では、二次抗体および発光試薬等の特殊な試薬が多数必須であり、作業コストが高い。また、発光試薬の退色（ブリーチング現象）を抑制する必要から、測定工程を極めて短時間に終了せざるを得ないため、測定精度が不充分になることが懸念される。 However, in the method using the enzyme substrate reaction, a large number of special reagents such as secondary antibodies and luminescent reagents are essential, and the operation cost is high. Moreover, since it is necessary to suppress discoloration (bleaching phenomenon) of the luminescent reagent, the measurement process must be completed in a very short time, and there is a concern that the measurement accuracy may be insufficient.
また、酵素基質反応を利用する方法は、試料および各試薬をインキュベーションする工程、系を洗浄する工程、並びに発光を測定する工程等の多段階からなっており、操作が煩雑である。しかも、各段階に要する時間が極めて長く、大規模処理には適さない。 In addition, the method utilizing the enzyme substrate reaction is composed of multiple steps such as a step of incubating the sample and each reagent, a step of washing the system, and a step of measuring luminescence, and the operation is complicated. Moreover, the time required for each stage is extremely long, and is not suitable for large-scale processing.
一方、刺激応答性ポリマーを含有する物質と検出対象に対する親和性物質とが結合した結合物、並びに電荷を有する物質と検出対象に対する親和性物質とが結合した結合物を用いて、検出対象の検出および定量する方法が開発されている（例えば、特許文献２参照）。 On the other hand, a detection target is detected using a binding substance in which a substance containing a stimulus-responsive polymer and an affinity substance for the detection target are bound, and a binding substance in which a substance having a charge and an affinity substance for the detection target are bound. And a method for quantification has been developed (see, for example, Patent Document 2).
特許文献２に記載の方法は、上記２種類の結合物と検体とを混合した混合物を刺激応答性ポリマーが凝集する条件下に置いた後、濁度測定等によって刺激応答性ポリマーの凝集の程度が低下したと判定された場合には、検体中に検出対象が存在すると判別する方法である。 In the method described in Patent Document 2, the degree of aggregation of the stimulus-responsive polymer is measured by measuring the turbidity after placing the mixture obtained by mixing the above-mentioned two kinds of conjugates and the specimen under conditions where the stimulus-responsive polymer aggregates. This is a method for determining that the detection target is present in the sample when it is determined that the decrease has occurred.
特許文献２に記載の方法によれば、刺激応答性ポリマーを含有する物質、親和性物質および電荷を有する物質のみを用いて検出対象を検出および定量することができ、特殊な試薬を特に使用しなくてもよいので、安価且つ簡便である。また、凝集阻害の程度を測定するだけであり、酵素によって触媒される反応を利用する系ではないから、迅速に行うことができる。 According to the method described in Patent Document 2, a detection target can be detected and quantified using only a substance containing a stimulus-responsive polymer, an affinity substance, and a substance having a charge, and a special reagent is used in particular. Since it is not necessary, it is inexpensive and simple. In addition, it is only a measure of the degree of aggregation inhibition, and since it is not a system that utilizes a reaction catalyzed by an enzyme, it can be carried out rapidly.
しかし、上記技術では、検出対象の検出および定量のために、例えば温度を変化させなければならず、そのための専用の装置も必要となる。 However, in the above technique, for example, the temperature must be changed for detection and quantification of the detection target, and a dedicated device for that purpose is also required.
本発明は、以上の実情に鑑みてなされたものであり、検出対象を迅速、安価、簡便且つ高精度に検出、定量できる検出対象の検出方法および定量方法を提供することを課題とする。 This invention is made | formed in view of the above situation, and makes it a subject to provide the detection method and quantification method of the detection target which can detect and quantitate a detection target rapidly, cheaply, simply and with high precision.
本発明者らは、磁性体を含有する磁性物質と重量平均分子量が１，０００〜１，０００，０００である高分子とを接近させると、磁性体の磁気分離速度が変化することを見出し、本発明を完成させるに至った。具体的には、本発明の要旨は以下のとおりである。 The present inventors have found that when a magnetic substance containing a magnetic substance is brought close to a polymer having a weight average molecular weight of 1,000 to 1,000,000, the magnetic separation rate of the magnetic substance changes. The present invention has been completed. Specifically, the gist of the present invention is as follows.
［１］検体中の検出対象を検出および／または定量するためのキットであって、
該検出対象に対する第１の親和性物質と磁性体を含有する磁性物質とが結合した第１の結合物と、該検出対象に対する第２の親和性物質と重量平均分子量が１，０００〜１，０００，０００である高分子とが結合した第２の結合物とを含み、
第１の親和性物質と第２の親和性物質が、該検出対象の異なる部位において結合できることを特徴とするキット。
［２］前記磁性物質の平均粒子径が１００ｎｍ以上１μｍ未満であることを特徴とする［１］に記載のキット。
［３］前記検出対象が抗原であり、第１の親和性物質および第２の親和性物質が該抗原に対する抗体である［１］または［２］に記載のキット。
［４］検体中の検出対象を検出および／または定量する方法であって、
該検出対象に対する第１の親和性物質と磁性体を含有する磁性物質とが結合した第１の結合物と、該検出対象に対する第２の親和性物質と重量平均分子量が１，０００〜１，０００，０００である高分子とが結合した第２の結合物と、該検体とを混合し、第１の結合物を磁気分離する条件下で、第１の結合物の磁気分離速度を判定する工程を含み、
第１の親和性物質と第２の親和性物質が、該検出対象の異なる部位において結合できることを特徴とする方法。
［５］磁性物質の平均粒子径が１００ｎｍ以上１μｍ未満であることを特徴とする［４］に記載の方法。
［６］前記検出対象が抗原であり、第１の親和性物質および第２の親和性物質が該抗原に対する抗体である［４］または［５］に記載の方法。
[1] A kit for detecting and / or quantifying a detection target in a specimen,
A first binding substance in which a first affinity substance for the detection target and a magnetic substance containing a magnetic substance are bound; a second affinity substance for the detection target; and a weight average molecular weight of 1,000 to 1, A second bonded product bonded to a polymer that is 1,000,000,
A kit characterized in that a first affinity substance and a second affinity substance can bind at different sites of the detection target.
[2] The kit according to [1], wherein an average particle size of the magnetic substance is 100 nm or more and less than 1 μm.
[3] The kit according to [1] or [2], wherein the detection target is an antigen, and the first affinity substance and the second affinity substance are antibodies against the antigen.
[4] A method for detecting and / or quantifying a detection target in a specimen,
A first binding substance in which a first affinity substance for the detection target and a magnetic substance containing a magnetic substance are bound; a second affinity substance for the detection target; and a weight average molecular weight of 1,000 to 1, A second binding substance to which a polymer of 000,000 is bound is mixed with the specimen, and the magnetic separation speed of the first binding substance is determined under the condition of magnetically separating the first binding substance. Including steps,
A method characterized in that the first affinity substance and the second affinity substance can bind at different sites to be detected.
[5] The method according to [4], wherein the magnetic substance has an average particle size of 100 nm or more and less than 1 μm.
[6] The method according to [4] or [5], wherein the detection target is an antigen, and the first affinity substance and the second affinity substance are antibodies against the antigen.
本発明によれば、検体中に検出対象が存在する場合、該検出対象の異なる部位に第１の結合物中の第１の親和性物質と第２の結合物中の第２の親和性物質が結合する。そのため、第１の親和性物質に結合した磁性物質と、第２の親和性物質に結合した重量平均分子量が１，０００〜１，０００，０００である高分子が接近する。これにより、磁性物質を含む第１の結合物の磁気分離速度が阻害または促進されるため、検体中の検出対象の存在量に応じて磁性物質を含む第１の結合物の磁気分離速度が変化する。この変化の有無または程度に基づいて、検体中の検出対象を簡便に検出または定量することができる。 According to the present invention, when the detection target exists in the specimen, the first affinity substance in the first binding substance and the second affinity substance in the second binding substance at different sites of the detection target. Join. Therefore, a magnetic substance bound to the first affinity substance and a polymer having a weight average molecular weight of 1,000 to 1,000,000 bound to the second affinity substance approach each other. This inhibits or accelerates the magnetic separation rate of the first binding substance containing the magnetic substance, so that the magnetic separation rate of the first binding substance containing the magnetic substance changes according to the amount of the detection target in the sample. To do. Based on the presence or absence or degree of this change, the detection target in the sample can be easily detected or quantified.
以上の手順は、いずれも特殊な試薬、機器を特に使用することなく行われるので、安価且つ簡便に実施できる。 All of the above procedures can be carried out inexpensively and easily because they are carried out without using special reagents and equipment.
以下、本発明の一実施形態について、図面を参照しながら説明する。 Hereinafter, an embodiment of the present invention will be described with reference to the drawings.
＜キット＞
本発明のキットは、検体中の検出対象を検出および／または定量するためのキットであって、第１の結合物と、第２の結合物とを含有する。
各構成について、以下詳細に説明する。
<Kit>
The kit of the present invention is a kit for detecting and / or quantifying a detection target in a specimen, and contains a first binding substance and a second binding substance.
Each configuration will be described in detail below.
［検体］
検体としては、人または動物の体液、尿、喀痰および糞便等の生物学的物質、飲食品、水道水、並びに河川等の環境から採取した試料等が挙げられる。
[Sample]
Samples include human or animal body fluids, biological materials such as urine, sputum and feces, food and drinks, tap water, and samples collected from environments such as rivers.
［検出対象］
検出対象としては、例えば、環境汚染物質、飲食品汚染物質および臨床診断に利用される物質が挙げられる。このような物質としては、具体的には、例えば、ダイオキシン、環境ホルモン、農薬、ＰＣＢ（ｐｏｌｙｃｈｌｏｒｏｂｉｐｈｅｎｙｌ）、有機水銀等、プリオン、カビ毒、フグ毒、抗生物質、防カビ剤、ヒトイムノグロブリンＧ、ヒトイムノグロブリンＭ、ヒトイムノグロブリンＡ、ヒトイムノグロブリンＥ、ヒトアルブミン、ヒトフィブリノーゲン（フィブリンおよびそれらの分解産物）、α−フェトプロテイン（ＡＦＰ）、Ｃ反応性タンパク質（ＣＲＰ）、ミオグロビン、ガン胎児性抗原、肝炎ウイルス抗原、ヒト絨毛性ゴナドトロピン（ｈＣＧ）、ヒト胎盤性ラクトーゲン（ＨＰＬ）、ＨＩＶウイルス抗原、アレルゲン、細菌毒素、細菌抗原、酵素、ホルモン（［例えば、ヒト甲状腺刺激ホルモン（ＴＳＨ）およびインスリン等］および薬剤が挙げられる。
[Detection target]
Examples of detection targets include environmental pollutants, food and drink contaminants, and substances used for clinical diagnosis. Specific examples of such substances include, for example, dioxins, environmental hormones, agricultural chemicals, PCBs (polychlorobiphenyl), organic mercury, prions, mold toxins, puffer poisons, antibiotics, fungicides, human immunoglobulin G, Human immunoglobulin M, human immunoglobulin A, human immunoglobulin E, human albumin, human fibrinogen (fibrin and their degradation products), α-fetoprotein (AFP), C-reactive protein (CRP), myoglobin, carcinoembryonic antigen , Hepatitis virus antigens, human chorionic gonadotropin (hCG), human placental lactogen (HPL), HIV virus antigens, allergens, bacterial toxins, bacterial antigens, enzymes, hormones (eg, human thyroid stimulating hormone (TSH) and insulin, etc. ] Fine drug, and the like.
［第１の結合物］
第１の結合物は、検出対象に対する第１の親和性物質と、磁性体を含有する磁性物質とが結合したものである。
[First combined product]
The first binding substance is a combination of a first affinity substance for a detection target and a magnetic substance containing a magnetic substance.
（第１の親和性物質）
検出対象に対する第１の親和性物質は、検出対象に対して親和性を有する物質であれば、特に限定されない。ここで、「親和性」とは、ある物質が他の物質と特異的に結合する性質をいう。第１の親和性物質としては、例えば、検出対象が抗原である場合は該抗原に対する抗体、検出対象が抗体である場合は該抗体に結合する抗原、検出対象がＧＳＴタグ付のタンパク質である場合はグルタチオン、検出対象がヒスチジンタグ付のタンパク質の場合は金属イオンを配位したキレート剤、検出対象が核酸の場合は相補的な配列を持つ核酸が挙げられる。
(First affinity substance)
The first affinity substance for the detection target is not particularly limited as long as it is a substance having affinity for the detection target. Here, “affinity” refers to the property that a certain substance specifically binds to another substance. As the first affinity substance, for example, when the detection target is an antigen, an antibody against the antigen, when the detection target is an antibody, an antigen that binds to the antibody, and when the detection target is a protein with a GST tag Includes glutathione, a chelating agent coordinated with a metal ion when the detection target is a histidine-tagged protein, and a nucleic acid having a complementary sequence when the detection target is a nucleic acid.
前記抗体は、いかなるタイプの免疫グロブリン分子であってもよく、Ｆａｂ等の抗原結合部位を有する免疫グロブリン分子断片であってもよい。また、抗体は、モノクローナル抗体でもポリクローナル抗体でもよい。 The antibody may be any type of immunoglobulin molecule, and may be an immunoglobulin molecule fragment having an antigen binding site such as Fab. The antibody may be a monoclonal antibody or a polyclonal antibody.
第１の親和性物質と第２の親和性物質としては、検出対象の異なる部位において非競合的に結合できる物質を用いる。例えば、検出対象が抗原である場合、第１の親和性物質および第２の親和性物質は、該抗原において互いに異なる抗原決定基を認識するモノクローナル抗体であることが好ましい。 As the first affinity substance and the second affinity substance, substances that can bind non-competitively at different sites to be detected are used. For example, when the detection target is an antigen, the first affinity substance and the second affinity substance are preferably monoclonal antibodies that recognize different antigenic determinants in the antigen.
（磁性物質）
本発明に用いる磁性物質は磁性体を含有する物質である。磁性物質としては、磁性体のみでもよく、磁性体と親水性物質との複合体、ポリスチレンおよびポリメチルアクリレートなどのようなラテックスにより表面を被覆した磁性体であってもよい。
(Magnetic substance)
The magnetic substance used in the present invention is a substance containing a magnetic substance. The magnetic substance may be only a magnetic substance, a complex of a magnetic substance and a hydrophilic substance, or a magnetic substance whose surface is coated with a latex such as polystyrene and polymethyl acrylate.
磁性物質としては、国際公開第２００４−０８３１２４号に示すような金磁性粒子なども利用できる。なお、本発明に用いる磁性物質は、刺激応答性ポリマーによる凝集性の機能が必要ではなく、また、磁性物質の構成の簡易化の観点から、刺激応答性ポリマーを含有する必要はなく、刺激応答性ポリマーを含有しないことが特に好ましい。 As the magnetic substance, gold magnetic particles as shown in International Publication No. 2004-083124 can also be used. The magnetic substance used in the present invention does not need an aggregating function by the stimulus-responsive polymer, and it is not necessary to contain a stimulus-responsive polymer from the viewpoint of simplification of the configuration of the magnetic substance. It is particularly preferred not to contain a functional polymer.
磁性体としては、例えば、磁性金属微粒子および磁性酸化物微粒子などの磁性微粒子を挙げることができる。これらの磁性微粒子は、必要に応じて、希土類元素または遷移金属元素を含有していてもよい。 Examples of the magnetic material include magnetic fine particles such as magnetic metal fine particles and magnetic oxide fine particles. These magnetic fine particles may contain a rare earth element or a transition metal element as necessary.
磁性微粒子の素材としては、例えば、マグネタイト、酸化ニッケル、フェライト、コバルト鉄酸化物、バリウムフェライト、炭素鋼、タングステン鋼、ＫＳ鋼、希土類コバルト磁石、マグヘマイト、ヘマタイトおよびゲーサイト等の微粒子を挙げることができる。これらの中でも、マグネタイト、マグヘマイト、ヘマタイトおよびゲーサイトが好ましい。これらの磁性微粒子の形状は、球状、針状、紡錘状および無定形のいずれでもよい。 Examples of the magnetic fine particle material include fine particles such as magnetite, nickel oxide, ferrite, cobalt iron oxide, barium ferrite, carbon steel, tungsten steel, KS steel, rare earth cobalt magnet, maghemite, hematite, and goethite. it can. Among these, magnetite, maghemite, hematite and goethite are preferable. The shape of these magnetic fine particles may be any of a spherical shape, a needle shape, a spindle shape, and an amorphous shape.
磁性金属微粒子としては、例えば、Ｆｅ−Ｃｏ、Ｆｅ−Ｎｉ、Ｆｅ−Ａｌ、Ｆｅ−Ｎｉ−Ａｌ、Ｆｅ−Ｃｏ−Ｎｉ、Ｆｅ−Ｎｉ−Ａｌ−ＺｎおよびＦｅ−Ａｌ−Ｓｉなどの金属微粒子を挙げることができる。 Examples of magnetic metal fine particles include metal fine particles such as Fe—Co, Fe—Ni, Fe—Al, Fe—Ni—Al, Fe—Co—Ni, Fe—Ni—Al—Zn, and Fe—Al—Si. Can be mentioned.
磁性酸化物微粒子としては、例えば、ＦｅＯｘ（４／３≦ｘ≦３／２）で表される酸化鉄（フェライト）型の強磁性微粒子およびＦｅの一部がＮｉまたはＣｏで一部置換されたフェライトを挙げることができる。 Examples of magnetic oxide fine particles include iron oxide (ferrite) type ferromagnetic fine particles represented by FeOx (4/3 ≦ x ≦ 3/2) and a part of Fe partially substituted with Ni or Co. Mention may be made of ferrite.
磁性体と親水性物質との複合体に用いる親水性物質は、構成単位に水酸基を少なくとも２個有し且つ鉄イオンと結合可能な多価アルコールであれば特に限定されない。このような親水性物質としては、例えば、デキストラン、デキストリン、セルロース、アガロース、澱粉およびジェラン等のポリサッカライド類、カルボキシメチルセルロース、ジエチルアミノセルロース、ヒドロキシアセチルセルロース、ヒドロキシアセチルセルロース、カルボキシメチルデキストラン、ジエチルアミノエチルセルロースおよびジエチルアミノエチルデキストラン等のポリサッカライド誘導体、並びにポリビニルアルコールおよびポリアリルアルコールなどの合成ポリオールが挙げられる。 The hydrophilic substance used for the complex of the magnetic substance and the hydrophilic substance is not particularly limited as long as it is a polyhydric alcohol having at least two hydroxyl groups in the structural unit and capable of binding to iron ions. Examples of such hydrophilic substances include polysaccharides such as dextran, dextrin, cellulose, agarose, starch and gellan, carboxymethylcellulose, diethylaminocellulose, hydroxyacetylcellulose, hydroxyacetylcellulose, carboxymethyldextran, diethylaminoethylcellulose and diethylamino. Examples thereof include polysaccharide derivatives such as ethyl dextran, and synthetic polyols such as polyvinyl alcohol and polyallyl alcohol.
また、前記親水性物質としては、例えば、モノマーとして、アリルアルコール、２−ヒドロキシエチル（メタ）アクリレート、グリセロール−モノ（メタ）アクリレートおよび２−ヒドロキシエチル（メタ）アクリルアミド等の水酸基を有するモノマーの少なくとも一種を重合成分として含有するポリマー、並びに酢酸エステル型、トリメチルシリルエーテル型およびｔ−ブトキシカルボニルオキシ型などの保護された水酸基を有するビニルアルコールを含むモノマーの少なくとも一種を重合成分として含有するポリマーから水酸基の保護を除去して得られるポリビニルアルコールランダムコポリマーを挙げることができる。 Examples of the hydrophilic substance include at least monomers having a hydroxyl group such as allyl alcohol, 2-hydroxyethyl (meth) acrylate, glycerol-mono (meth) acrylate, and 2-hydroxyethyl (meth) acrylamide as monomers. A polymer containing one kind as a polymerization component and a polymer containing at least one monomer containing a vinyl alcohol having a protected hydroxyl group, such as an acetate ester type, a trimethylsilyl ether type and a t-butoxycarbonyloxy type, as a polymerization component. Mention may be made of polyvinyl alcohol random copolymers obtained by removing the protection.
これらの親水性物質は、１種または２種以上組み合わせて使用することができる。また、グリシジルメタクリレート重合体のようにエポキシを有し、開環後多価アルコール構造体を形成する化合物も使用できる。 These hydrophilic substances can be used alone or in combination of two or more. Moreover, the compound which has an epoxy like a glycidyl methacrylate polymer and forms a polyhydric alcohol structure after ring-opening can also be used.
磁性体と親水性物質との複合体の複合化様式としては、例えば、物理的な吸着および共有結合形成を挙げることができる。 Examples of the complexing mode of the complex of the magnetic substance and the hydrophilic substance include physical adsorption and covalent bond formation.
また、磁性体と親水性物質との複合体としては、例えば、多価アルコールを含む鉄イオン水溶液に、アンモニアおよび水酸化ナトリウムなどのアルカリを添加する共沈法により得られる、ポリオールにより被覆されたフェライト微粒子を用いてもよい（例えば、特開平６−９２６４０号公報参照）。 In addition, as a complex of a magnetic substance and a hydrophilic substance, for example, it is coated with a polyol obtained by a coprecipitation method in which an alkali such as ammonia and sodium hydroxide is added to an iron ion aqueous solution containing a polyhydric alcohol. Ferrite fine particles may be used (for example, see JP-A-6-92640).
磁性体と親水性物質との複合体の調製方法としては、具体的には、例えば、米国特許第４４５２７７３号に記載されているように、デキストラン５０質量％水溶液（１０ｍｌ）中に、塩化第二鉄・六水和物（１．５１ｇ）および塩化第一鉄・四水和物（０．６４ｇ）混合水溶液（１０ｍｌ）を加えて撹拌し、６０〜６５℃に水浴中で７．４（Ｖ／Ｖ）％アンモニア水溶液をｐＨ１０〜１１程度になるように滴下しながら加熱し、１５分反応させる方法により得ることができる。
As a method for preparing a complex of a magnetic substance and a hydrophilic substance, specifically, as described in, for example, U.S. Pat. No. 4,452,773,
磁性体は親水性物質などへの親和性を高めるために表面処理を施してもよい。表面処理としては、例えば、シラン系カップリング処理、チタン系カップリング処理、リン酸系カップリング処理、塩酸および硫酸などによる酸処理、並びに水酸化ナトリウムなどによるアルカリ処理が挙げられる。 The magnetic material may be subjected to a surface treatment in order to increase the affinity for a hydrophilic substance or the like. Examples of the surface treatment include silane coupling treatment, titanium coupling treatment, phosphoric acid coupling treatment, acid treatment with hydrochloric acid and sulfuric acid, and alkali treatment with sodium hydroxide.
磁性体と親水性物質との複合体における磁性体含有率は、磁気分離および磁性体の分散性の観点から、１０〜９０重量％とすることが好ましく、４０〜８０重量％とすることがより好ましい。 The magnetic substance content in the composite of magnetic substance and hydrophilic substance is preferably 10 to 90% by weight and more preferably 40 to 80% by weight from the viewpoint of magnetic separation and dispersibility of the magnetic substance. preferable.
ラテックスにより表面を被覆した磁性体は、Ｊｏｕｒｎａｌ ｏｆ Ｍａｇｎｅｔｉｓｍ ａｎｄ Ｍａｇｎｅｔｉｃ Ｍａｔｅｒｉａｌｓ １２２ （１９９３）３７−４１に記載の方法により調製することができる。 A magnetic material whose surface is coated with latex can be prepared by the method described in Journal of Magnetics and Magnetic Materials 122 (1993) 37-41.
磁性体を含有する磁性物質の平均粒子径は、１００ｎｍ以上１μｍ未満であることが好ましく、２００ｎｍ以上５００ｎｍ以下であることがより好ましい。磁性体を含有する第１の磁性物質の平均粒子径をこの範囲内とすることで、該複合体を水溶液中で均一に分散させることができ、長時間において沈降物が生じず、かつ短時間で磁気回収することができる。磁性体を含有する磁性物質の平均粒子径は、動的光散乱法により測定する。 The average particle size of the magnetic substance containing the magnetic material is preferably 100 nm or more and less than 1 μm, and more preferably 200 nm or more and 500 nm or less. By setting the average particle size of the first magnetic substance containing the magnetic substance within this range, the complex can be uniformly dispersed in the aqueous solution, no precipitate is formed for a long time, and the short time is short. Can be magnetically recovered. The average particle diameter of the magnetic substance containing the magnetic material is measured by a dynamic light scattering method.
［第１の結合物の作製］
第１の結合物は、磁性物質と検出対象に対する第１の親和性物質とを結合することによって作製する。磁性物質と検出対象に対する第１の親和性物質との結合方法は、特に限定されないが、例えば、磁性物質側（例えば、磁性体部分）および第１の親和性物質（例えば、第１の抗体）側の双方に、互いに親和性の物質（例えば、アビジンおよびビオチン、並びにグルタチオンおよびグルタチオンＳトランスフェラーゼ）を結合させ、これら物質を介して磁性物質と検出対象に対する第１の親和性物質とを結合させる方法が挙げられる。
[Preparation of first bonded product]
The first binding substance is prepared by binding the magnetic substance and the first affinity substance for the detection target. The method for binding the magnetic substance and the first affinity substance to the detection target is not particularly limited. For example, the magnetic substance side (for example, the magnetic part) and the first affinity substance (for example, the first antibody) are used. A substance having affinity for each other (for example, avidin and biotin, and glutathione and glutathione S-transferase) is bound to both sides, and the magnetic substance and the first affinity substance for the detection target are bound via these substances Is mentioned.
磁性物質と検出対象に対する第１の親和性物質とをビオチンを介して結合させる方法としては、例えば、ビオチンの末端をＮ−ヒドロキシスクシンイミドでエステル化したｓｕｌｆｏ−ＥＭＣＳ（商品名）［（株）同仁化学研究所製）］と第１の親和性物質とを反応させてビオチンを導入し、ビオチンを介してアビジンを結合した磁性物質と第１の親和性物質とを結合することができる。 Examples of a method for binding a magnetic substance and a first affinity substance for a detection target via biotin include, for example, sulfo-EMCS (trade name) [Dojindo Co., Ltd.] in which the end of biotin is esterified with N-hydroxysuccinimide. Chemical Laboratories)] and a first affinity substance are reacted to introduce biotin, and the magnetic substance bound with avidin and the first affinity substance can be bound via biotin.
また、磁性物質と検出対象に対する第１の親和性物質とを直接的に結合させる場合、官能基を介して結合させてもよい。 Further, when the magnetic substance and the first affinity substance for the detection target are directly bonded, they may be bonded via a functional group.
磁性物質と検出対象に対する第１の親和性物質とを官能基を介して結合させる方法としては、例えば、磁性物質としてアルデヒド基を有する親水性物質と磁性体との複合体を用い、検出対象に対する第１の親和性物質として１級アミンを有する物質（例えば、抗体）を含む物質を用いて、該アルデヒド基と１級アミンとの結合を介して磁性物質と第１の親和性物質とを結合させる方法が挙げられる。 As a method for binding the magnetic substance and the first affinity substance for the detection target through a functional group, for example, a complex of a hydrophilic substance having an aldehyde group and a magnetic substance is used as the magnetic substance, and the detection target is used. Using a substance including a primary amine-containing substance (for example, an antibody) as the first affinity substance, the magnetic substance and the first affinity substance are bound via the bond between the aldehyde group and the primary amine. The method of letting it be mentioned.
前記アルデヒド基を有する親水性物質と磁性体との複合体は、アルカリ処理などの操作により親水性物質のグリコシド結合を開裂させて低分子量の末端にアルデヒド基を有する親水性物質を発生させるか、または過ヨウ素酸ナトリウムなどの酸化試薬を用いて、親水性物質の構造中のビシナルジオールを発生することにより得られる。 The complex of a hydrophilic substance having an aldehyde group and a magnetic substance generates a hydrophilic substance having an aldehyde group at a low molecular weight end by cleaving the glycoside bond of the hydrophilic substance by an operation such as alkali treatment. Alternatively, it can be obtained by generating vicinal diol in the structure of the hydrophilic substance using an oxidizing reagent such as sodium periodate.
また、例えば、検出対象に対する第１の親和性物質として第１の抗体を用いる場合は、第１の抗体のＦｃ部分と親和性が高い物質（以下、「抗体親和性物質」と略す。）を用い、親水性物質を介して抗体親和性物質を磁性物質に結合させることにより、該抗体親和性物質を介して磁性物質と第１の抗体とを結合させることができる。 Further, for example, when the first antibody is used as the first affinity substance for the detection target, a substance having high affinity with the Fc portion of the first antibody (hereinafter abbreviated as “antibody affinity substance”). By using and binding an antibody affinity substance to a magnetic substance via a hydrophilic substance, the magnetic substance and the first antibody can be bound via the antibody affinity substance.
具体的には、抗体親和性物質として、例えば、メロンゲル、プロテインＡおよびプロテインＧを用いる。親水性物質として、例えば、グリセロール−モノ（メタ）アクリレートなどのモノマーを重合して得られるポリマーを用いる。該ポリマーとしてカルボキシル、アミノおよびエポキシ等の官能基を持つモノマーを他のモノマーとを共重合させたポリマーを用いることが好ましく、該官能基を介して当該技術分野で周知の方法に従って前記抗体親和性物質を親水性物質であるポリマーに結合させ、該親水性物質を磁性体に結合させ、磁性物質とすることができる。 Specifically, for example, melon gel, protein A, and protein G are used as antibody affinity substances. As the hydrophilic substance, for example, a polymer obtained by polymerizing monomers such as glycerol-mono (meth) acrylate is used. As the polymer, it is preferable to use a polymer obtained by copolymerizing a monomer having a functional group such as carboxyl, amino and epoxy with another monomer, and the affinity of the antibody according to a method well known in the art via the functional group. A substance can be bonded to a polymer that is a hydrophilic substance, and the hydrophilic substance can be bonded to a magnetic substance to form a magnetic substance.
このようにして得られた抗体親和性物質を含む磁性物質を、該抗体親和性物質を介して第１の抗体に結合させることにより、磁性物質と第１の抗体との第１の結合物を作製することができる。 By binding the magnetic substance containing the antibody affinity substance thus obtained to the first antibody via the antibody affinity substance, the first bound substance of the magnetic substance and the first antibody is obtained. Can be produced.
あるいは、磁性物質に含まれる磁性体と、検出対象に対する第１の親和性物質とを結合させて、第１の結合物としてもよい。 Alternatively, a magnetic substance contained in the magnetic substance and a first affinity substance for the detection target may be bound to form a first bound substance.
［第１の結合物の精製］
第１の結合物を精製する方法としては、例えば、磁性物質と検出対象に対する第１の親和性物質を結合させた後、磁力を付加して磁性物質を含む第１の結合物を回収する方法、および遠心分離によって未結合の第１の親和性物質を分離して精製する方法が挙げられる。
[Purification of first conjugate]
As a method for purifying the first bound substance, for example, after the magnetic substance and the first affinity substance for the detection target are bound, a magnetic force is applied to recover the first bound substance containing the magnetic substance. And a method of separating and purifying unbound first affinity substance by centrifugation.
［第２の結合物］
本発明の方法では、第１の結合物に加えて、検出対象に対する第２の親和性物質と、重量平均分子量が１，０００〜１，０００，０００である高分子とが結合した第２の結合物を用いる。これにより、検出感度を向上することができる。
[Second combined product]
In the method of the present invention, in addition to the first binding substance, the second affinity substance for the detection target and the second polymer having a weight average molecular weight of 1,000 to 1,000,000 bound to each other. Use conjugate. Thereby, detection sensitivity can be improved.
（第２の親和性物質）
検出対象に対する第２の親和性物質は、第１の親和性物質とは異なる部位において、第１の親和性物質と同じ検出対象に結合できる物質である。第１の親和性物質と同様に、第２の親和性物質としては、例えば、検出対象が抗原である場合は該抗原に対する抗体、検出対象が抗体である場合は該抗体に結合する抗原、検出対象がＧＳＴタグ付のタンパク質である場合はグルタチオン、検出対象がヒスチジンタグ付のタンパク質の場合は金属イオンを配位したキレート剤および検出対象が核酸の場合は相補的な配列を持つ核酸などが挙げられる。
(Second affinity substance)
The second affinity substance for the detection target is a substance that can bind to the same detection target as the first affinity substance at a site different from the first affinity substance. Similar to the first affinity substance, examples of the second affinity substance include an antibody against the antigen when the detection target is an antigen, an antigen that binds to the antibody when the detection target is an antibody, and detection. Examples include glutathione when the target is a GST-tagged protein, chelating agents coordinated with metal ions when the target to be detected is a histidine-tagged protein, and nucleic acids with complementary sequences when the target is a nucleic acid. It is done.
前記抗体は、いかなるタイプの免疫グロブリン分子であってもよく、Ｆａｂ等の抗原結合部位を有する免疫グロブリン分子断片であってもよい。また、抗体は、モノクローナル抗体でもポリクローナル抗体でもよい。上記したように、第１の親和性物質および第２の親和性物質は、例えば、検出対象（抗原）の異なる抗原決定基を認識するモノクローナル抗体であることが好ましい。 The antibody may be any type of immunoglobulin molecule, and may be an immunoglobulin molecule fragment having an antigen binding site such as Fab. The antibody may be a monoclonal antibody or a polyclonal antibody. As described above, the first affinity substance and the second affinity substance are preferably monoclonal antibodies that recognize different antigenic determinants of the detection target (antigen), for example.
（高分子）
第２の親和性物質と結合して第２の結合物を構成する高分子の重量平均分子量は、１，０００〜１，０００，０００である。高分子の重量平均分子量が１，０００以上であると、磁気分離速度の差が大きくなるため好ましい。また、高分子の重量平均分子量が１，０００，０００以下であると、第２の親和性物質と高分子との結合が可能であるため好ましい。高分子の重量平均分子量は、ゲル浸透クロマトグラフィー（ＧＰＣ、Ｇｅｌ Ｐｅｒｍｅａｔｉｏｎ Ｃｈｒｏｍａｔｏｇｒａｐｈｙ）により測定する。
(High molecular)
The weight average molecular weight of the polymer constituting the second bound substance by binding to the second affinity substance is 1,000 to 1,000,000. It is preferable that the weight average molecular weight of the polymer is 1,000 or more because the difference in magnetic separation speed is increased. Further, it is preferable that the polymer has a weight average molecular weight of 1,000,000 or less because the second affinity substance can be bonded to the polymer. The weight average molecular weight of the polymer is measured by gel permeation chromatography (GPC, Gel Permeation Chromatography).
例えば、高分子としてポリアクリル酸の重量平均分子量を測定する場合、次のように行えばよい。測定対象のポリアクリル酸の濃度が約０．５質量％になるように、０．２Ｍの硝酸ナトリウムで希釈し、これをサンプルとして用いる。ＧＰＣ装置として島津製作所製、示差屈折率計 ＲＩＤ−１０Ａを用い、カラムとして東ソー（株）製カラムＳｕｐｅｒ ＡＷ５，０００およびＳｕｐｅｒ ＡＷ４０００を用いる。ここで、ＧＰＣ装置にカラムをＳｕｐｅｒ ＡＷ５，０００、Ｓｕｐｅｒ ＡＷ４０００の順で、直列に取り付け、カラム温度４０℃、流速１．０ｍｌ／ｍｉｎとし、０．２Ｍの硝酸ナトリウムを展開剤として用いて、サンプル中のポリアクリル酸の溶出時間を測定し、ポリエチレンオキシド換算することにより、重量平均分子量を求めることができる。 For example, when measuring the weight average molecular weight of polyacrylic acid as a polymer, it may be performed as follows. The sample is diluted with 0.2 M sodium nitrate so that the concentration of polyacrylic acid to be measured is about 0.5% by mass, and this is used as a sample. A differential refractometer RID-10A manufactured by Shimadzu Corporation is used as the GPC apparatus, and columns Super AW5,000 and Super AW4000 manufactured by Tosoh Corporation are used as the columns. Here, a column was attached to the GPC apparatus in the order of Super AW 5,000 and Super AW 4000 in series, the column temperature was 40 ° C., the flow rate was 1.0 ml / min, and 0.2 M sodium nitrate was used as a developing agent. The weight average molecular weight can be determined by measuring the elution time of polyacrylic acid therein and converting it to polyethylene oxide.
重量平均分子量が１，０００〜１，０００，０００である高分子（以下、単に「高分子」ともいう）は、磁性物質の磁気分離速度を変化させる高分子であることが好ましい。このような高分子としては、例えば、磁気分離を阻害する高分子および磁気分離を促進する高分子が利用できる。磁気分離を阻害する高分子としては、例えば、水溶性の高分子化合物および電荷を有する高分子化合物が挙げられる。また、磁気分離を促進する高分子としては、例えば、水に対する溶解性の低い高分子化合物が挙げられる。 The polymer having a weight average molecular weight of 1,000 to 1,000,000 (hereinafter also simply referred to as “polymer”) is preferably a polymer that changes the magnetic separation rate of the magnetic substance. As such a polymer, for example, a polymer that inhibits magnetic separation and a polymer that promotes magnetic separation can be used. Examples of the polymer that inhibits magnetic separation include a water-soluble polymer compound and a charged polymer compound. Examples of the polymer that promotes magnetic separation include a polymer compound having low solubility in water.
水溶性の高分子化合物としては、例えば、ポリエチレングリコール、ポリプロピレングリコール、ポリエチレンオキシドおよびポリプロピレンオキシド等のエーテル結合を含有する高分子、ポリビニルアルコール等のアルコール性水酸基を含有する高分子、デキストラン、シクロデキストリン、アガロースおよびヒドロキシプロピルセルロース等の水溶性多糖類並びに中性アミノ酸を含むポリペプチドが挙げられる。 Examples of the water-soluble polymer compound include polymers containing an ether bond such as polyethylene glycol, polypropylene glycol, polyethylene oxide and polypropylene oxide, polymers containing an alcoholic hydroxyl group such as polyvinyl alcohol, dextran, cyclodextrin, Examples include water-soluble polysaccharides such as agarose and hydroxypropylcellulose, and polypeptides containing neutral amino acids.
電荷を有する高分子化合物としては、例えば、ポリアニオンおよびポリカチオンが挙げられる。ポリアニオンとは、複数のアニオン基を有する物質を意味する。また、ポリカチオンとは、複数のカチオン基を有する物質を意味する。 Examples of the polymer compound having a charge include a polyanion and a polycation. The polyanion means a substance having a plurality of anion groups. The polycation means a substance having a plurality of cationic groups.
ポリアニオンとしては、例えば、ＤＮＡおよびＲＮＡ等の核酸が挙げられる。これらの核酸は、核酸骨恪に沿って複数個のホスホジエステル基が存在することにより、ポリアニオンの性質を有する。 Examples of the polyanion include nucleic acids such as DNA and RNA. These nucleic acids have the properties of polyanions due to the presence of a plurality of phosphodiester groups along the nucleic acid urn.
また、ポリアニオンには、多数のカルボキシルを含むポリペプチド（例えば、グルタミン酸およびアスパラギン酸等のアミノ酸からなるポリペプチド）、ポリアクリル酸、ポリメタクリル酸、ポリスルホン酸、アクリル酸およびメタクリル酸等を重合成分として含有するポリマー、カルボキシメチルセルロース、ヒアルロン酸並びにヘパリン等の多糖等も含まれる。 In addition, polyanions include polypeptides containing many carboxyls (for example, polypeptides consisting of amino acids such as glutamic acid and aspartic acid), polyacrylic acid, polymethacrylic acid, polysulfonic acid, acrylic acid and methacrylic acid as polymerization components. Also included are polymers, polysaccharides such as carboxymethylcellulose, hyaluronic acid and heparin.
一方、ポリカチオンとしては、例えば、ポリリジン、ポリアルギニン、ポリオルニチン、ポリアルキルアミン、ポリエチレンイミンおよびポリプロピルエチレンイミンが挙げられる。なお、ポリアニオン（カルボキシル）またはポリカチオン（アミノ）の官能基数は、２５個以上が好ましい。また、カルボキシル基を持つラテックス粒子なども挙げられる。 On the other hand, examples of the polycation include polylysine, polyarginine, polyornithine, polyalkylamine, polyethyleneimine, and polypropylethyleneimine. The number of functional groups of the polyanion (carboxyl) or polycation (amino) is preferably 25 or more. In addition, latex particles having a carboxyl group are also included.
水に対する溶解性の低い高分子化合物としてはポリペプチドまたはアクリル系高分子化合物が挙げられる。 Examples of the polymer compound having low solubility in water include polypeptides and acrylic polymer compounds.
ポリペプチドとしては、例えば、バリン（Ｖａｌ）、イソロイシン（Ｉｌｅ）、ロイシン（Ｌｅｕ）、フェニルアラニン（Ｐｈｅ）、システイン（Ｃｙｓ）、メチオニン（Ｍｅｔ）およびアラニン（Ａｌａ）等の疎水性アミノ酸を多く含むポリペプチドが挙げられる。 Polypeptides, for example, are those containing many hydrophobic amino acids such as valine (Val), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), cysteine (Cys), methionine (Met) and alanine (Ala). Peptides are mentioned.
ポリペプチド全体において疎水性アミノ酸が占める割合は、例えば、Ｋｙｔｅ Ｊ．，Ｄｏｏｌｉｔｔｌｅ Ｒ．Ｆ．，Ｊ．Ｍｏｌ．Ｂｉｏｌ．１５７：１０５−１３２（１９８２）に記載された各アミノ酸の疎水性度の値を用いたとき、ポリペプチドを構成する各アミノ酸の疎水性度の相加平均が０以上であることが好ましい。なかでも、アミノ酸２０残基以上のポリペプチドユニットを構成する各アミノ酸の疎水性度の相加平均が０．５以上である部分構造を含むポリペプチドが、より好ましい。このようなポリペプチドとしては、ベータ・アミロイドペプチドが例示できる。また、当該疎水性度を示すポリペプチドは、人工的に合成してもよい。 The proportion of hydrophobic amino acids in the whole polypeptide is, for example, Kyte J. et al. , Doolittle R.D. F. , J .; Mol. Biol. 157: 105-132 (1982), when the value of the hydrophobicity of each amino acid is used, the arithmetic average of the hydrophobicity of each amino acid constituting the polypeptide is preferably 0 or more. Among these, a polypeptide containing a partial structure in which the arithmetic average of the hydrophobicity of each amino acid constituting a polypeptide unit having 20 or more amino acid residues is 0.5 or more is more preferable. An example of such a polypeptide is beta amyloid peptide. Moreover, you may synthesize | combine the polypeptide which shows the said hydrophobicity artificially.
アクリル系高分子化合物としては、高分子を構成するモノマーの構造中に、例えば、ｔ−ブチル、イソプロピル、プロピル、エチルおよびフェニル等の疎水性官能基を有するモノマーを多く含む水溶性ポリマーが挙げられる。 Examples of the acrylic polymer compound include a water-soluble polymer containing a large amount of monomers having a hydrophobic functional group such as t-butyl, isopropyl, propyl, ethyl and phenyl in the structure of the monomer constituting the polymer. .
ポリマー全体において疎水性官能基を有するモノマーが占める割合は、例えば、ポリマーを構成するモノマーのｌｏｇＰ値（Ｐは分配係数）の相加平均が０．２以上であることが好ましい。 As for the ratio of the monomer having a hydrophobic functional group in the whole polymer, for example, the arithmetic average of log P values (P is a distribution coefficient) of the monomer constituting the polymer is preferably 0.2 or more.
これら高分子は、高分子鎖の中または末端に、第２の親和性物質を結合させるための官能基等を有していてもよい。また、該高分子は、一種で用いてもよいし、複数を混合して用いてもよい。 These polymers may have a functional group or the like for binding the second affinity substance in or at the terminal of the polymer chain. In addition, the polymers may be used alone or in combination.
［作製方法］
第２の結合物は、高分子と第２の親和性物質とを直接または間接的に結合させることによって作製する。
[Production method]
The second binding substance is prepared by binding the polymer and the second affinity substance directly or indirectly.
高分子と第２の親和性物質とを間接的に結合させる方法としては、例えば、高分子および第２の親和性物質（例えば、第２の抗体）側の双方に、互いに親和性の物質（例えば、アビジンおよびビオチン、並びにグルタチオンおよびグルタチオンＳトランスフェラーゼ）を結合させる。当該物質を介して、および前記高分子および第２の親和性物質を間接的に結合させることができる。 Examples of a method for indirectly binding the polymer and the second affinity substance include, for example, substances having affinity for each other (both the polymer and the second affinity substance (for example, the second antibody)) ( For example, avidin and biotin, and glutathione and glutathione S transferase) are conjugated. The polymer and the second affinity substance can be indirectly bound via the substance.
高分子と第２の親和性物質とを直接的に結合させる方法としては、例えば、高分子の末端をＮ−ヒドロキシスクシンイミドでエステル化して第２の親和性物質と結合させる方法（ＣＡＮＣＥＲ ＲＥＳＥＡＲＣＨ ５１、ｐ．４３１０−４３１５、１９９１）、官能基を介して高分子と第２の親和性物質とを結合させる方法が挙げられる。 As a method of directly binding the polymer and the second affinity substance, for example, a method of esterifying the terminal of the polymer with N-hydroxysuccinimide and binding to the second affinity substance (CANCER RESEARCH 51, p. 4310-4315, 1991), and a method of binding a polymer and a second affinity substance via a functional group.
高分子の末端をＮ−ヒドロキシスクシンイミドでエステル化して第２の親和性物質と結合させる方法としては、具体的には、例えば、次の方法が挙げられる。高分子であるポリエチレングリコールの末端をＮ−ヒドロキシスクシンイミドでエステル化し、ＮＨＳ−ポリエチレングリコールのエステルとする。該ＮＨＳ−ポリエチレングリコールのエステルと第２の親和性物質（抗体）のアミノ基とを反応させて、ポリエチレングリコールと抗体とを結合させて、アミドを得ることができる。 Specific examples of the method of esterifying the terminal of the polymer with N-hydroxysuccinimide and binding to the second affinity substance include the following methods. The terminal of polyethylene glycol, which is a polymer, is esterified with N-hydroxysuccinimide to form an ester of NHS-polyethylene glycol. By reacting the ester of the NHS-polyethylene glycol with the amino group of the second affinity substance (antibody), the polyethylene glycol and the antibody are bound to obtain an amide.
高分子と第２の親和性物質とを直接的に結合させる場合、官能基を介して結合させてもよい。官能基を介して結合させる方法としては、例えば、ゴッシュらの方法（Ｇｈｏｓｈ ｅｔ ａｌ．：Ｂｉｏｃｏｎｊｕｇａｔｅ Ｃｈｅｍ．１、７１−７６、１９９０）のマレイミド−チオールカップリングが挙げられる。 When the polymer and the second affinity substance are directly bonded, they may be bonded via a functional group. Examples of the method of bonding via a functional group include maleimide-thiol coupling according to the method of Gosh et al. (Bioconjugate Chem. 1, 71-76, 1990).
マレイミド−チオールカップリングによる方法としては、具体的には、例えば、次の２つの方法が挙げられる。第１の方法では、まず、高分子（核酸）の５’末端にメルカプト基（別名、スルフヒドリル基）を導入する。一方、第２の親和性物質（抗体）に６−マレイミドヘキサノイックアシッドスクシンイミドエステル（例えば、「ＥＭＣＳ（商品名）」［（株）同仁化学研究所製］を反応させてマレイミド基を導入する。次に、導入したメルカプト基およびマレイミド基を介して、これら２種の物質を結合させる。 Specific examples of the method by maleimide-thiol coupling include the following two methods. In the first method, first, a mercapto group (also called a sulfhydryl group) is introduced at the 5 'end of a polymer (nucleic acid). On the other hand, 6-maleimidohexanoic acid succinimide ester (for example, “EMCS (trade name)” [manufactured by Dojindo Laboratories Co., Ltd.) is reacted with the second affinity substance (antibody) to introduce a maleimide group. Next, these two substances are bonded through the introduced mercapto group and maleimide group.
第２の方法では、まず、第１の方法と同様にして高分子（核酸）の５’末端にメルカプト基を導入する。該メルカプト基に更にホモ二官能性試薬であるＮ，Ｎ−１，２−フェニレンジマレイミドを反応させることによって、核酸の５’末端にマレイミド基を導入する。一方、第２の親和性物質（抗体）にメルカプト基を導入する。次に、導入したメルカプト基およびマレイミド基を介して、これら２種の物質を結合させる。 In the second method, first, a mercapto group is introduced into the 5 'end of the polymer (nucleic acid) in the same manner as in the first method. The mercapto group is further reacted with N, N-1,2-phenylenedimaleimide, which is a homobifunctional reagent, to introduce a maleimide group at the 5 'end of the nucleic acid. On the other hand, a mercapto group is introduced into the second affinity substance (antibody). Next, these two substances are bonded through the introduced mercapto group and maleimide group.
［第２の結合物の精製］
第２の結合物を精製する方法としては、例えば、高分子と検出対象に対する第２の親和性物質を結合させた後、ゲルろ過、イオン交換、アフィニティークロマトグラフィーおよび遠心分離からなる少なくとも１種の分離方法を用いることによって精製する方法が挙げられる。
[Purification of second conjugate]
As a method for purifying the second binding substance, for example, after binding the second affinity substance to the detection target with the polymer, at least one kind consisting of gel filtration, ion exchange, affinity chromatography and centrifugation is used. The method of refine | purifying by using a separation method is mentioned.
上記のようにして製造される本発明のキットは、例えば以下のような方法で、検出対象を検出および／または定量するために使用できる。 The kit of the present invention produced as described above can be used for detecting and / or quantifying the detection target by, for example, the following method.
本発明の検出方法および／または定量方法は、以下の工程（１）および（２）を含む。
（１）第１の結合物、第２の結合物および検体の混合物を得る工程
（２）工程（１）で得られた混合物に磁力を付加して第１の結合物を回収する条件下で、第１の結合物が磁気分離速度を調べる工程
The detection method and / or quantification method of the present invention includes the following steps (1) and (2).
(1) A step of obtaining a mixture of the first conjugate, the second conjugate and the specimen (2) Under a condition where a magnetic force is applied to the mixture obtained in step (1) to recover the first conjugate. , The step of examining the magnetic separation speed of the first combination
（１）第１の結合物、第２の結合物および検体の混合物を得る工程
工程（１）では、第１の結合物と第２の結合物とを容器内で混合し、更に検体を添加して混合物を得る。なお、第１の結合物、第２の結合物および検体は、すべてを同時に混合してもよく、１種ずつを別々に混合してもよい。
(1) Step of obtaining a mixture of the first binding substance, the second binding substance and the specimen In the step (1), the first binding substance and the second binding substance are mixed in a container, and the specimen is further added. To obtain a mixture. The first bound substance, the second bound substance, and the specimen may be mixed at the same time or may be mixed separately one by one.
（２）工程（１）で得られた混合物に磁力を付加して第１の結合物を回収する条件下で、第１の結合物が磁気分離速度を調べる工程
工程（２）では、工程（１）で得られた混合物を、磁力を付加して磁性物質を含む第１の結合物を回収する条件下に置く。
(2) A step of examining the magnetic separation rate of the first combined substance under the condition of collecting the first combined substance by applying a magnetic force to the mixture obtained in the step (1). In the step (2), the process ( The mixture obtained in 1) is subjected to conditions for applying a magnetic force to recover the first combined product containing the magnetic substance.
第１の結合物、第２の結合物および検体の混合物に対する磁力の付加は、第１の結合物に含まれる磁性物質に磁石を接近させて行うことができる。用いる磁石の磁力は、用いる磁性物質が有する磁力の大きさおよび磁性物質との距離によって任意に設定することができる。磁石の種類としては、例えば、ＮｅｏＭａｇ社製のネオジム、フェライト、サマコバ、アルニコおよびネオジムボンド磁石が挙げられる。 The magnetic force can be applied to the mixture of the first binding substance, the second binding substance and the specimen by bringing the magnet close to the magnetic substance contained in the first binding substance. The magnetic force of the magnet used can be arbitrarily set depending on the magnitude of the magnetic force of the magnetic material used and the distance from the magnetic material. Examples of the type of magnet include neodymium, ferrite, samakoba, alnico, and neodymium bonded magnets manufactured by NeoMag.
また、磁力の付加は、磁気分離速度の判定の前、または判定と同時並行で行ってよいが、工程に費やされる時間を短縮化できる点で同時並行が好ましい。 The addition of the magnetic force may be performed before the determination of the magnetic separation speed or in parallel with the determination, but the simultaneous parallel is preferable in that the time spent for the process can be shortened.
第１の結合物の磁気分離速度は、例えば、第１の結合物、第２の結合物および検体の混合物に対して磁力を付加し、一定時間内に目視または吸光度を測定することにより調べることができる。 The magnetic separation rate of the first binding substance is examined by, for example, applying a magnetic force to the mixture of the first binding substance, the second binding substance, and the specimen, and measuring the absorbance or measuring the absorbance within a predetermined time. Can do.
吸光度は分光光度計を用いて測定し、回収した第１の結合物の量を測定してもよく、溶媒に分散した第１の結合物の吸光度を測定してもよい。ここで、使用する光の波長は、磁性体の粒径等に応じ所望の検出感度が得られるよう適宜設定できる。光の波長は、従来汎用の装置を利用できる点で、可視光の範囲内であることが好ましい。 The absorbance is measured using a spectrophotometer, and the amount of the first bound substance recovered may be measured, or the absorbance of the first bound substance dispersed in the solvent may be measured. Here, the wavelength of the light to be used can be appropriately set so as to obtain a desired detection sensitivity in accordance with the particle size of the magnetic material. The wavelength of light is preferably within the range of visible light in that a conventional general-purpose device can be used.
目視または吸光度の測定は、一定の時点で断続的に行ってもよいし、経時的に連続して行ってもよい。また、ある時点における吸光度測定値と、他の時点における吸光度測定値との差に基づいて判定を行ってもよい。 The visual or absorbance measurement may be performed intermittently at a certain point in time or continuously over time. Further, the determination may be made based on the difference between the absorbance measurement value at a certain time point and the absorbance measurement value at another time point.
また、吸光度の測定には、吸光度を直接的に測定することのみならず、吸光度を反映するパラメータを測定することも包含される。かかるパラメータとしては、複数時点での吸光度測定値の差異、分離された第１の結合物の量および分離後の第１の結合物の吸光度等が挙げられる。 Further, the measurement of the absorbance includes not only measuring the absorbance directly but also measuring a parameter reflecting the absorbance. Examples of such parameters include differences in absorbance measurement values at a plurality of time points, the amount of the first bound substance separated, and the absorbance of the first bound substance after separation.
ここで、複数時点のうちの１点は、例えば、検出対象が非存在である陰性対照に磁力を付加した際、吸光度が最大値となる時点近傍であることが好ましい。これにより、別の時点での吸光度測定値との差異が大きくなり、検出対象の量をより正確に定量できることになる。 Here, it is preferable that one point of the plurality of time points is, for example, in the vicinity of the time point at which the absorbance reaches the maximum value when a magnetic force is applied to the negative control in which the detection target is not present. Thereby, the difference from the absorbance measurement value at another time point becomes large, and the amount of the detection target can be quantified more accurately.
＜検出方法＞
本発明の検出方法は、前記工程（１）および工程（２）に加えて、さらに以下の工程（３−１）を含むことが好ましい。
（３−１）第１の結合物と第２の結合物との混合物に検体を添加しない場合と比較して、磁気分離速度が変化する場合、検体中に検出対象が存在すると判定する工程
各工程の詳細を以下に説明する。
工程（３−１）は、第１の結合物と第２の結合物との混合物に検体を添加しない場合と比較して、第１の結合物の磁気分離速度が変化するか否かを調べることにより、検体中の検出対象の有無を判定する工程である。
<Detection method>
The detection method of the present invention preferably further includes the following step (3-1) in addition to the step (1) and the step (2).
(3-1) A step of determining that the detection target is present in the sample when the magnetic separation speed is changed as compared with the case where the sample is not added to the mixture of the first binding substance and the second binding substance. Details of the process will be described below.
In step (3-1), it is examined whether or not the magnetic separation rate of the first binding substance is changed as compared with the case where the sample is not added to the mixture of the first binding substance and the second binding substance. This is a step of determining the presence or absence of the detection target in the sample.
検体中に検出対象が存在する場合には、検出対象に結合した第２の結合物中の高分子によって、磁性物質を含む第１の結合物の磁気分離が阻害または促進されて、第１の結合物と第２の結合物との混合物に検体を添加しない場合と比較して、第１の結合物の磁気分離速度が変化する。 When the detection target exists in the sample, the magnetic separation of the first binding substance containing the magnetic substance is inhibited or promoted by the polymer in the second binding substance bound to the detection target, so that the first The magnetic separation rate of the first binding substance is changed as compared with the case where the specimen is not added to the mixture of the binding substance and the second binding substance.
一方、検出対象が存在しない場合には、第１の結合物の磁気分離が阻害または促進されずに、前記第１の結合物と第２の結合物との混合物に検体を添加しない場合と比較して、第１の結合物の磁気分離速度が変化しないことになる。 On the other hand, when there is no detection target, the magnetic separation of the first binding substance is not inhibited or promoted, and compared with the case where the specimen is not added to the mixture of the first binding substance and the second binding substance. As a result, the magnetic separation speed of the first combined substance does not change.
例えば、第２の結合物中の高分子が、磁性物質の磁気分離を阻害する高分子である場合、第１の結合物と第２の結合物との混合物に検体を添加しない場合と比較して、第１の結合物と第２の結合物との混合物に検体を添加した場合の吸光度が高ければ、第１の結合物の磁気回収が阻害されており、検体中に検出対象の存在が示唆される。 For example, when the polymer in the second binding substance is a polymer that inhibits magnetic separation of the magnetic substance, the sample is not added to the mixture of the first binding substance and the second binding substance. If the absorbance when the sample is added to the mixture of the first bound substance and the second bound substance is high, the magnetic recovery of the first bound substance is inhibited, and the presence of the detection target is present in the specimen. It is suggested.
また、例えば、第２の結合物中の高分子が、磁性物質の磁気分離を促進する高分子である場合、第１の結合物と第２の結合物との混合物に検体を添加しない場合と比較して、第１の結合物と第２の結合物との混合物に検体を添加した場合の吸光度が低ければ、第１の結合物の磁気回収が促進されており、検体中に検出対象の存在が示唆される。 For example, when the polymer in the second binding substance is a polymer that promotes magnetic separation of the magnetic substance, the sample is not added to the mixture of the first binding substance and the second binding substance. In comparison, if the absorbance when the specimen is added to the mixture of the first bound substance and the second bound substance is low, the magnetic recovery of the first bound substance is promoted, and the detection target is detected in the specimen. Existence is suggested.
この現象を、図１〜図２を参照しながら説明する。なお、図２は、例として、第２の結合物中の高分子によって、第１の結合物中の磁性物質の磁気分離が阻害される場合を説明する。 This phenomenon will be described with reference to FIGS. In addition, FIG. 2 demonstrates the case where the magnetic separation of the magnetic substance in a 1st conjugate | bonded_material is inhibited by the polymer in a 2nd conjugate | bonded material as an example.
図１に示されるように、第１の結合物１０は、アビジン１３およびビオチン１５を介して、磁性物質１１と検出対象（抗原）５０に対する第１の親和性物質（抗体）１７とが結合することにより構成されている。一方、第２の結合物２０は、検出対象（抗原）５０に対する第２の親和性物質（抗体）２３と高分子２１とが結合することにより構成されている。
As shown in FIG. 1, the first bound
そして、第１の親和性物質１７および第２の親和性物質２３は、検出対象５０の異なる部位に結合できることから、同じ検出対象５０に結合できる。第２の結合物２０は検出対象５０とアビジンを介して磁性物質１１に接近でき、このとき第２の結合物２０中の高分子２１が第１の結合物１０中の磁性物質１１の近傍に位置することになる。
Since the
図２に示されるように、第１の結合物１０、第２の結合物２０および検体の混合物を所定条件下におくと、検出対象５０が存在する場合には、第２の結合物２０中の高分子によって第１の結合物１０に含まれる磁性物質１１の磁気分離が阻害され、第１の結合物１０の磁気回収が阻害される。［図２（Ａ）］。一方、検出対象５０が存在しない場合には第１の結合物１０に含まれる磁性物質１１の磁気分離が阻害されず、第１の結合物１０が磁気回収されることになる［図２（Ｂ）］。
As shown in FIG. 2, when the mixture of the first binding
＜定量方法＞
本発明の定量方法は、前記工程（１）および（２）に加えて、さらに以下の工程（３−２）および工程（４）を含むことが好ましい。
（３−２）工程（２）と同一の条件下における磁気分離速度と検出対象の量との相関式を作成する工程
（４）工程（２）で調べた磁気分離速度から、工程（３−２）で作成した相関式に基づいて、検体中の検出対象の量を算出する工程
以下、各工程について説明する。
<Quantitative method>
The quantitative method of the present invention preferably further includes the following steps (3-2) and (4) in addition to the steps (1) and (2).
(3-2) Step (4) for creating a correlation between the magnetic separation speed and the amount of detection target under the same conditions as in step (2). From the magnetic separation speed examined in step (2), the step (3- Step of calculating amount of detection target in sample based on correlation equation created in 2) Each step will be described below.
（３−２）工程（２）と同一の条件下における磁気分離速度と検出対象の量との相関式を作成する工程
工程（３−２）は、工程（２）と同一の条件下における検出対象の量と磁気分離速度（例えば、吸光度）との所定条件下における相関式を作成する工程である。ここで、工程（２）と同一の条件とは、工程（１）で得られた混合物を、磁力を付加して磁性物質を含む第１の結合物を回収する条件をいう。
(3-2) Step of creating a correlation equation between the magnetic separation speed and the amount of detection target under the same conditions as in step (2) Step (3-2) is detection under the same conditions as in step (2) This is a step of creating a correlation equation between a target amount and a magnetic separation rate (for example, absorbance) under a predetermined condition. Here, the same condition as in the step (2) refers to a condition in which the mixture obtained in the step (1) is subjected to a magnetic force to recover the first combined substance containing the magnetic substance.
前記相関式を構成する検出対象の量と磁気分離速度との測定は、データが多い程に信頼性の高い相関式が得られる。そこでデータは、２以上の検出対象の量に関するものであればよく、３点以上の検出対象の量に関するものであることが好ましい。 For the measurement of the amount of detection target and the magnetic separation speed constituting the correlation equation, the more reliable the data, the more reliable the correlation equation is obtained. Therefore, the data may be related to the amount of two or more detection targets, and is preferably related to the amount of three or more detection targets.
ここで、検出対象の量と磁気分離速度との相関式は、検出対象の量と吸光度との直接的な相関を示す式のみならず、検出対象の量と磁気分離速度を反映するパラメータとの相関式であってもよい。 Here, the correlation equation between the amount of the detection target and the magnetic separation speed is not only an expression indicating a direct correlation between the amount of the detection target and the absorbance, but also a parameter reflecting the amount of the detection target and the magnetic separation speed. It may be a correlation equation.
（４）工程（２）で調べた磁気分離速度から、工程（３−２）で作成した相関式に基づいて、検体中の検出対象の量を算出する工程
工程（４）は、工程（２）で調べた磁気分離速度の測定値を、工程（３−２）で作成した相関式に代入することによって、検体中の検出対象の量を算出する工程である。
(4) A step of calculating the amount of the detection target in the sample based on the correlation equation created in the step (3-2) from the magnetic separation speed examined in the step (2). This is a step of calculating the amount of the detection target in the sample by substituting the measured value of the magnetic separation speed investigated in () into the correlation equation created in step (3-2).
［キットの構成とその使用方法の例］
以下に、本発明の方法を利用するためのキットの構成とその使用方法の例を検出対象が抗原の場合で説明する。
[Example of kit configuration and usage]
Hereinafter, an example of the structure of a kit for using the method of the present invention and the method of using the kit will be described in the case where the detection target is an antigen.
試薬キットとしては、例えば、下記の試薬から構成される。
抗原検出用試薬キット：
試薬Ａ：検出対象の抗原に特異的に結合する第１の抗体が結合した磁性物質
試薬Ｂ：第１の抗体とは異なる部位を認識し、検出対象の抗原に非競合的に結合しうる第２の抗体が結合した、重量平均分子量が１，０００〜１，０００，０００である高分子
試薬Ｃ：被測定物質の標準品（具体例として、精製抗原が挙げられる。）
試薬Ｄ：希釈用バッファー（上記試薬の希釈用、並びに被測定試料の希釈用に使用可能なバッファーであって、例えば、トリス塩酸バッファーおよびリン酸バッファーが挙げられる。）
The reagent kit includes, for example, the following reagents.
Antigen detection reagent kit:
Reagent A: a magnetic substance to which a first antibody that specifically binds to the antigen to be detected is bound Reagent B: a magnetic substance that recognizes a site different from the first antibody and can bind non-competitively to the antigen to be detected A polymer having a weight average molecular weight of 1,000 to 1,000,000 to which two antibodies are bound. Reagent C: Standard product of a substance to be measured (a specific example is a purified antigen).
Reagent D: Buffer for dilution (a buffer that can be used for dilution of the above-mentioned reagent and dilution of the sample to be measured, and examples thereof include Tris-HCl buffer and phosphate buffer.)
また、吸光度を測定する装置としては、２００ｎｍ〜９００ｎｍの透過光を照射できる従来周知の装置が使用できる。 Moreover, as a device for measuring absorbance, a conventionally known device that can irradiate transmitted light of 200 nm to 900 nm can be used.
上記した試薬からなるキットは、例えば、以下の方法で使用できる。 A kit comprising the above-described reagents can be used, for example, by the following method.
まず、試薬Ａ ５〜１０００μｌと試薬Ｂ ５〜１０００μｌとを混合する。試薬Ａと試薬Ｂの入った溶液中に（１）被測定物質の標準品を添加した陽性対照、（２）何も添加しない陰性対照、（３）被検液の５〜１０００μｌを添加したサンプル、を準備し、一定時間反応させる。反応後、磁力を付加した容器に反応液を入れ、５５０ｎｍの透過光を照射して吸光度を測定し、被検液中の抗原の有無の判定または抗原の定量を行う。 First, 5-1000 [mu] l of reagent A and 5-1000 [mu] l of reagent B are mixed. In a solution containing Reagent A and Reagent B, (1) a positive control to which a standard substance to be measured is added, (2) a negative control to which nothing is added, and (3) a sample to which 5-1000 μl of the test solution is added , And react for a certain time. After the reaction, the reaction solution is put into a container to which magnetic force is applied, and the absorbance is measured by irradiating with 550 nm transmitted light to determine the presence or absence of the antigen in the test solution or to quantify the antigen.
上記とは別の使用方法として、試薬Ａと試薬Ｃを一定時間反応後、試薬Ｂを添加し、一定時間反応させ、磁力を付加した容器に反応液を入れ、５５０ｎｍの透過光を照射して濁度を測定し、被検液中の抗原の有無の判定または抗原の定量をしてもよい。
［実施例］
As another usage, the reagent A and the reagent C are reacted for a certain time, then the reagent B is added, the reaction is performed for a certain time, the reaction solution is put into a container to which a magnetic force is applied, and a transmitted light of 550 nm is irradiated. The turbidity may be measured to determine the presence or absence of the antigen in the test solution or to quantify the antigen.
[Example]
本発明の実施例で用いた代表的な試薬は次のとおりである。
ＰＢＳバッファー：１０倍濃度の市販のＰＢＳ［８１ｍＭ Ｎａ２ＨＰＯ４、１５ｍＭ ＫＨ２ＰＯ４、２７ｍＭ ＫＣｌ、１３７０ｍＭ ＮａＣｌ、ｐＨ７．４、ニッポンジーン（株）製］を精製水で１／１０（Ｖ／Ｖ）に希釈して用いた。
精製水：ＭＩＬＬＩＰＯＲＥ社製超純水製造装置「Ｄｉｒｅｃｔ−Ｑ」（商品名）で精製した水。
The typical reagents used in the examples of the present invention are as follows.
PBS buffer: 10 times concentration of commercially available PBS [81 mM Na 2 HPO 4 , 15 mM KH 2 PO 4 , 27 mM KCl, 1370 mM NaCl, pH 7.4, manufactured by Nippon Gene Co., Ltd.] with purified water 1/10 (V / V ) And diluted.
Purified water: Water purified by an ultrapure water production apparatus “Direct-Q” (trade name) manufactured by MILLIPORE.
［キットの作製］
（第１の結合物の調製）
検出対象としてのヒト甲状腺刺激ホルモン（ＴＳＨ）に対する第１の親和性物質として、Ｌｅｉｎｃｏ Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｉｎｃ．製の抗ヒトＴＳＨβ抗体（Ａｎｔｉ−Ｈｕｍａｎ Ｔｈｙｒｏｉｄ Ｓｔｉｍｕｌａｔｉｎｇ Ｈｏｒｍｏｎｅ Ｂｅｔａ、クローン：１９５マウス、クラス：マウスＩｇＧ）を用いた。抗ヒトＴＳＨβ抗体を、ｓｕｌｆｏ−ＮＨＳ−Ｂｉｏｔｉｎ（Ｖｅｃｔｏｒ社製）を用いて、スクラム社においてビオチン化し、ビオチン化抗ヒトＴＳＨβ抗体を調製した。
[Production of kit]
(Preparation of first conjugate)
As a first affinity substance for human thyroid stimulating hormone (TSH) as a detection target, Leinco Technologies, Inc. Anti-human TSHβ antibody (Anti-Human Thyroid Stimulating Hormone Beta, clone: 195 mouse, class: mouse IgG) was used. The anti-human TSHβ antibody was biotinylated at Scrum using sulfo-NHS-Biotin (manufactured by Vector) to prepare a biotinylated anti-human TSHβ antibody.
磁性物質として、ストレプトアビジンを結合させた磁性粒子（ストレプトアビジン結合−磁性粒子）として、ＢＤ社製のＳｔｒｅｐｔａｖｉｄｉｎ ｐａｒｔｉｃｌｅｓ ｐｌｕｓ ＤＭ（１ｍｇ／ｍｌ）を用いた。 Streptavidin particles plus DM (1 mg / ml) manufactured by BD was used as a magnetic substance to which streptavidin was bound as a magnetic substance (streptavidin bond-magnetic particle).
ストレプトアビジン結合−磁性粒子（濃度１ｍｇ／ｍｌ）５０μｌを１．５ｍｌマイクロチューブに取り、これにビオチン化抗ヒトＴＳＨβ抗体（０．３７ｍｇ／ｍｌ）３０μｌを加え、４℃で３０分間反応させて、磁性粒子と抗ヒトＴＳＨβ抗体をビオチンとストレプトアビジンを介して結合させた。得られた抗ヒトＴＳＨβ抗体化磁性粒子を第１の結合物とした。光散乱測定装置 ＥＬＳ−８０００（大塚電子製）を用いて第１の結合物の平均粒子径を測定したところ、２４６ｎｍであった。 Streptavidin-binding-magnetic particles (concentration 1 mg / ml) 50 μl is taken into a 1.5 ml microtube, biotinylated anti-human TSHβ antibody (0.37 mg / ml) 30 μl is added, and reacted at 4 ° C. for 30 minutes. Magnetic particles and anti-human TSHβ antibody were bound via biotin and streptavidin. The obtained anti-human TSHβ antibody-conjugated magnetic particles were used as the first conjugate. It was 246 nm when the average particle diameter of the 1st conjugate | bonded_material was measured using the light-scattering measuring apparatus ELS-8000 (made by Otsuka Electronics).
（第２の結合物の調製）
検出対象としてのヒト甲状腺刺激ホルモン（ＴＳＨ）に対する第２の親和性物質として、抗ヒトＴＳＨα抗体（Ａｎｔｉ−Ｈｕｍａｎ Ｔｈｙｒｏｉｄ Ｓｔｉｍｕｌａｔｉｎｇ Ｈｏｒｍｏｎｅ Ａｌｐｈａ、クローン：１７６マウス、マウスＩｇＧ、Ｌｅｉｎｃｏ Ｔｅｃｈｎｏｌｏｇｙ，Ｉｎｃ．製、１ｍｇ／ｍｌ）を用いた。
(Preparation of second conjugate)
As a second affinity substance for human thyroid-stimulating hormone (TSH) as a detection target, anti-human TSHα antibody (Anti-Human Thyroid Stimulating Home Alpha, clone: 176 mouse, mouse IgG, manufactured by Leinco Technology, Inc., 1 mg / ml).
高分子として、ＰＥＧ［ＳＵＮＢＲＩＧＨＴ ＭＥ−４００ＣＳ（商品名）日油（株）製 重量平均分子量４０，０００（カタログ値）］を用いた。 As the polymer, PEG [SUNBRIGHT ME-400CS (trade name), NOF Corporation, weight average molecular weight 40,000 (catalog value)] was used.
抗ヒトＴＳＨα抗体（１ｍｇ／ｍｌ）１ｍｌにＰＥＧを２．５ｍｇ加え、４℃で１５時間反応させて、抗ヒトＴＳＨα抗体にＰＥＧを結合させ、得られた粗ＰＥＧ化抗ヒトＴＳＨα抗体をＧＥヘルスケア社製ゲルろ過クロマトグラフィーカラム（ＨｉＰｒｅｐ １６／６０（プラスチックカラム）に、Ｓｅｐｈａｃｒｙｌ Ｓ−３００ ＨＲ（ゲルろ過担体）を充填したカラム）を用いてゲル濾過精製した。得られたＰＥＧ化抗ヒトＴＳＨα抗体を第２の結合物とした。 2.5 mg of PEG was added to 1 ml of anti-human TSHα antibody (1 mg / ml), reacted at 4 ° C. for 15 hours to bind PEG to anti-human TSHα antibody, and the resulting crude PEGylated anti-human TSHα antibody was treated with GE Health Gel filtration purification was performed using a gel filtration chromatography column manufactured by Care (HiPrep 16/60 (plastic column) packed with Sephacryl S-300 HR (gel filtration carrier)). The obtained PEGylated anti-human TSHα antibody was used as the second conjugate.
（検体の調製）
検体として、ヒト甲状腺刺激ホルモン（ＴＳＨ）［Ａｓｐｅｎ Ｂｉｏ Ｐｈａｒｍａ，Ｉｎｃ．製ヒト甲状腺刺激ホルモン（活性８．５ＩＵ／ｍｇ、ＷＨＯ８０／５５８）］を用いた。ＴＳＨの溶液（濃度１ｍｇ／ｍｌ）を、ＰＢＳバッファーで０．０１ｍｇ／ｍｌ、０．１ｍｇ／ｍｌとなるよう希釈したものを、それぞれ検体とした。なお、ヒト甲状腺刺激ホルモンを含有しないことを除き、同様の手順で調製したものを対照とした。
(Sample preparation)
As a specimen, human thyroid stimulating hormone (TSH) [Aspen Bio Pharma, Inc. Human thyroid stimulating hormone (activity 8.5 IU / mg, WHO 80/558)] was used. Samples were prepared by diluting a TSH solution (concentration 1 mg / ml) with PBS buffer to 0.01 mg / ml and 0.1 mg / ml, respectively. A control prepared in the same manner except that it did not contain human thyroid stimulating hormone was used as a control.
＜実施例１＞
第１の結合物として前記抗ヒトＴＳＨβ抗体化磁性粒子を、第２の結合物として前記ＰＥＧ化抗ヒトＴＳＨα抗体を用いて、ヒト甲状腺刺激ホルモン（ＴＳＨ）を検出した。
<Example 1>
Human thyroid-stimulating hormone (TSH) was detected using the anti-human TSHβ antibody-conjugated magnetic particles as the first conjugate and the PEGylated anti-human TSHα antibody as the second conjugate.
［定量］
図３に示すように、従来汎用されている分光光度計用セミミクロセル７１の光路外に、寸法４．６ｍｍ×９ｍｍ×２ｍｍのネオジム磁石７３（ネオマグ社製）を取り付けた。このセル７１を分光光度計Ｖ−６６０（日本分光製）のセルホルダ内に設置した。
[Quantitative]
As shown in FIG. 3, a neodymium magnet 73 (manufactured by Neomag) having dimensions of 4.6 mm × 9 mm × 2 mm was attached outside the optical path of a conventionally used
図４は、実施例に係る定量方法の手順を示すフローチャートである。定量方法は、上記の第１の結合物、第２の結合物および試料を混合する工程（ＳＴ１０）と、混合物の吸光度を測定する工程（ＳＴ２０）とを含む。 FIG. 4 is a flowchart illustrating the procedure of the quantification method according to the embodiment. The quantification method includes a step of mixing the first bound substance, the second bound substance and the sample (ST10), and a step of measuring the absorbance of the mixture (ST20).
（混合）
第１の結合物８０μｌおよび第２の結合物４０μｌと各検体５０μｌとＰＢＳバッファー１０３０μｌとを、マイクロチューブ内で混合し、ボルテックスミキサで１分間撹拌し、混合液を得た。
(mixture)
80 μl of the first binding substance and 40 μl of the second binding substance, 50 μl of each specimen, and 1030 μl of PBS buffer were mixed in a microtube, and stirred for 1 minute with a vortex mixer to obtain a mixed solution.
（相関式の作製）
前記混合液１０００μｌをセル７１内に分注し、波長４２０ｎｍの光を用いて、分光光度計による吸光度の測定を開始し、スリット幅１０ｍｍで、１０００秒間にわたって連続して測定した。この結果を図５に示す。この時の分光光度計の温度は２５℃であった。
(Production of correlation equation)
1000 μl of the mixed solution was dispensed into the
次に、各試料について、０秒、６００秒および１０００秒の測定値の差異を表した。この結果を図６および図７に示す。 Next, for each sample, the difference in measured values at 0 seconds, 600 seconds, and 1000 seconds was expressed. The results are shown in FIG. 6 and FIG.
図６および図７に示されるように、０秒、６００秒および１０００秒のうちの２点間の測定値の差異は、いずれもＴＳＨの量に依存するものであった。 As shown in FIG. 6 and FIG. 7, the difference in measured values between two points of 0 seconds, 600 seconds, and 1000 seconds was all dependent on the amount of TSH.
［再現性評価］
上記の第１の結合物、第２の結合物および試料について、前記と同様の手順で吸光度を測定し、３回の平均および変動係数を求めた結果を表１に示す。
[Reproducibility evaluation]
Table 1 shows the results obtained by measuring the absorbance of the first bound product, the second bound product, and the sample in the same manner as described above, and calculating the average and coefficient of variation three times.
表１に示すように、ＣＶ（変動係数）は１．８以下という低い値であった。よって、本発明の系によれば高い再現性が得られることが確認された。 As shown in Table 1, CV (coefficient of variation) was a low value of 1.8 or less. Therefore, it was confirmed that high reproducibility can be obtained according to the system of the present invention.
＜実施例２＞
磁性粒子にＤｙｎａｂｅａｄｓ Ｍｙｏｎｅ Ｓｔｒｅｐｔａｖｉｄｉｎ Ｃ１（Ｉｎｖｉｔｒｏｇｅｎ製）を１０μｌ用いた以外は実施例１と同様の方法でヒト甲状腺刺激ホルモン（ＴＳＨ）を検出した。その結果を図８および表２に示す。Ｄｙｎａｂｅａｄｓ Ｍｙｏｎｅ Ｓｔｒｅｐｔａｖｉｄｉｎ Ｃ１の平均粒子径は１１００ｎｍであった。
Human thyroid stimulating hormone (TSH) was detected in the same manner as in Example 1 except that 10 μl of Dynabeads Myone Streptavidin C1 (manufactured by Invitrogen) was used as the magnetic particles. The results are shown in FIG. The average particle size of Dynabeads Myone Streptavidin C1 was 1100 nm.
図８および表２に示すように、第１の結合物を構成する磁性物質の平均粒子径を１μｍ未満とする方が、第１の結合物の磁気分離が阻害されやすく、再現性もよいことが確認された。 As shown in FIG. 8 and Table 2, when the average particle size of the magnetic substance constituting the first combined substance is less than 1 μm, the magnetic separation of the first combined substance is more easily inhibited and the reproducibility is good. Was confirmed.
＜比較例１＞
第１の結合物として前記抗ヒトＴＳＨβ抗体化磁性粒子を、第２の結合物の代わりに抗ヒトＴＳＨα抗体を用いた以外は、実施例１と同様の方法で、ヒト甲状腺刺激ホルモン（ＴＳＨ）を検出した。その結果を図９および表３に示す。表３は、実施例１と同様の手順で吸光度を測定し、３回の平均を求めた結果である。
<Comparative Example 1>
Human Thyroid Stimulating Hormone (TSH) in the same manner as in Example 1 except that the anti-human TSHβ antibody-conjugated magnetic particles were used as the first conjugate and the anti-human TSHα antibody was used instead of the second conjugate. Was detected. The results are shown in FIG. Table 3 shows the results of measuring the absorbance in the same procedure as in Example 1 and obtaining the average of three times.
図９および表３に示すように、第２の結合物の代わりに抗ヒトＴＳＨα抗体を用いた比較例１は、表１の実施例１の結果と比較して磁気分離の阻害の程度が低かった。この結果から、第１の結合物とともに第２の結合物を用いる本発明の方法によれば、検出対象の存在による第１の結合物の磁気分離速度の変化が大きく、検出対象を迅速かつ高精度に検出できることがわかった。 As shown in FIG. 9 and Table 3, Comparative Example 1 using an anti-human TSHα antibody instead of the second conjugate had a lower degree of inhibition of magnetic separation than the results of Example 1 in Table 1. It was. From this result, according to the method of the present invention using the second binding substance together with the first binding substance, the change in the magnetic separation speed of the first binding substance due to the presence of the detection target is large, and the detection target is quickly and highly It was found that it can be detected accurately.
以上の結果から、本発明の方法は、二次抗体、発光試薬および発光検出装置等の特殊な試薬、機器を必要とせず、種種の環境において検出対象を迅速、安価且つ簡便に検出、定量できる新規な方法であることが示された。 From the above results, the method of the present invention does not require special reagents and equipment such as secondary antibodies, luminescent reagents and luminescence detection devices, and can detect and quantify detection targets quickly, inexpensively and easily in various environments. It was shown to be a new method.
１０ 第１の結合物
１１ 磁性物質
１３ アビジン
１５ ビオチン
１７ 第１の親和性物質（抗体）
２０ 第２の結合物
２１ 高分子
２３ 第２の親和性物質（抗体）
５０ 検出対象（抗原）
６０ 磁石
DESCRIPTION OF
20 second binding
50 Detection target (antigen)
60 magnets
Claims (6)
該検出対象に対する第１の親和性物質と磁性体を含有する磁性物質とが結合し、ｐＨ応答性ポリマーを有しない第１の結合物と、該検出対象に対する第２の親和性物質と重量平均分子量が１，０００〜１，０００，０００である高分子とが結合した第２の結合物とを含み、
第１の親和性物質と第２の親和性物質が、該検出対象の異なる部位において結合でき、
酵素基質反応を利用する発色用の基質を含まないことを特徴とするキット。 A kit for detecting and / or quantifying a detection target in a sample,
The first affinity substance for the detection target and the magnetic substance containing the magnetic substance bind to each other, the first binding substance having no pH-responsive polymer, the second affinity substance for the detection target and the weight average A second bonded product bonded to a polymer having a molecular weight of 1,000 to 1,000,000,
A first affinity substance and a second affinity substance can bind at different sites of the detection target ;
A kit that does not contain a substrate for color development utilizing an enzyme substrate reaction .
該検出対象に対する第１の親和性物質と磁性体を含有する磁性物質とが結合し、ｐＨ応答性ポリマーを有しない第１の結合物と、該検出対象に対する第２の親和性物質と重量平均分子量が１，０００〜１，０００，０００である高分子とが結合した第２の結合物と、該検体とを混合し、第１の結合物を磁気分離する条件下で、第１の結合物の磁気分離速度を調べる工程を含み、
第１の親和性物質と第２の親和性物質が、該検出対象の異なる部位において結合できることを特徴とする方法。 A method for detecting and / or quantifying a detection target in a specimen,
The first affinity substance for the detection target and the magnetic substance containing the magnetic substance bind to each other, the first binding substance having no pH-responsive polymer, the second affinity substance for the detection target and the weight average The first binding is performed under a condition in which the second binding substance bound to the polymer having a molecular weight of 1,000 to 1,000,000 is mixed with the specimen and the first binding substance is magnetically separated. Including the step of examining the magnetic separation rate of the object,
A method characterized in that the first affinity substance and the second affinity substance can bind at different sites to be detected.
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