JP2016121940A - Test and diagnosis method using aptamer - Google Patents
Test and diagnosis method using aptamer Download PDFInfo
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- JP2016121940A JP2016121940A JP2014262084A JP2014262084A JP2016121940A JP 2016121940 A JP2016121940 A JP 2016121940A JP 2014262084 A JP2014262084 A JP 2014262084A JP 2014262084 A JP2014262084 A JP 2014262084A JP 2016121940 A JP2016121940 A JP 2016121940A
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Abstract
Description
本発明は、検出対象の検出方法及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行われてきた。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、流体と、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、抗原を検出又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 performed 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 the fluid with a latex carrying an antibody or fragment thereof that specifically binds to the antigen 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).
この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、その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 this latex agglutination method, an antigen added as a specimen crosslinks a plurality of latex-bound antibodies to promote latex agglutination. Since the procedure is simple as described above, the antigen can be detected easily and rapidly. However, when the antigen is in a trace amount, 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法やＣＬＥＩＡ法といった酵素基質反応を利用する方法も広く採用されている。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、この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、抗原を検出又は定量する。 Therefore, methods utilizing enzyme substrate reactions such as ELISA and CLEIA are also widely adopted. 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 be detected.
しかし、酵素基質反応を利用する方法では、二次抗体や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。 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.
また、この方法は、試料及び各試薬をインキュベーションする工程、洗浄する工程、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, this method has multiple steps such as a step of incubating the sample and each reagent, a step of washing, 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、近年、眼科領域においては人口の高齢化と生活の欧米化により加齢黄斑変性症の患者が著しく増加している。加齢黄斑変性症は血管内皮細胞増殖因子（Ｖａｓｃｕｌａｒ Ｅｎｄｏｔｈｅｌｉａｌ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ、ＶＥＧＦ）が関与しているとされ、治療にはＶＥＧＦ阻害剤等が用いられているが、国内で承認されているＶＥＧＦ阻害剤は薬効や高価格の影響で患者のリスクも高いとされている。早期発見で効果的な治療が行えるため視機能の維持や改善の可能性が高くなることから、ＶＥＧＦの迅速診断方法が望まれている。 On the other hand, in recent years, the number of patients with age-related macular degeneration has increased remarkably due to the aging of the population and the Westernization of life. Age-related macular degeneration is considered to involve vascular endothelial growth factor (VEGF), and VEGF inhibitors are used for treatment, but VEGF inhibitors are approved in Japan. Is said to have high patient risk due to the effects of drug efficacy and high price. Since effective treatment can be performed by early detection, the possibility of maintenance and improvement of visual function is increased. Therefore, a rapid diagnosis method for VEGF is desired.
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及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、刺激応答性ポリマーに有電荷物質又は親水性物質が結合すると、刺激応答性ポリマーの凝集が阻害または促進されることを見出し、本発明を完成するに至った。
具体的には、本発明は以下の構成を有する。
The present inventors have found that when a charged substance or a hydrophilic substance is bound to a stimulus-responsive polymer, aggregation of the stimulus-responsive polymer is inhibited or promoted, and the present invention has been completed.
Specifically, the present invention has the following configuration.
１．検体中の検出対象を検出する方法であって、前記検体と、前記検出対象と結合しうるアプタマーと、刺激応答性ポリマーを含有する凝集性物質とを混合し、得られた混合物を前記刺激応答性ポリマーが凝集する条件下で、前記刺激応答性ポリマーを含有する凝集性物質の凝集の程度を判定し、当該凝集の程度が、前記検体の非存在下に比して変化した場合に、前記検体中に検出対象が存在すると判別する手順を含む、検出対象の検出方法。
２．前記検出対象と結合しうるアプタマーが、ＤＮＡ、ＲＮＡまたはペプチドである前項１に記載の方法。
３．前記凝集性物質が、微粒子状の磁性物質を更に含有し、磁力を付加することで、凝集した磁性物質を分離することを更に含む前項１または２に記載の方法。
４．前記凝集の程度の変化が、前記刺激応答性ポリマーの凝集阻害が緩和する変化である前項１〜３のいずれか１項に記載の方法。
５．前記検出対象が、ＶＥＧＦである前項１〜４のいずれか１項に記載の方法。
６．前記検出対象が、トロンビンである前項１〜４のいずれか１項に記載の方法。
７．検体中の検出対象を定量する方法であって、前記検体と、前記検出対象と結合しうるアプタマーと、刺激応答性ポリマーを含有する凝集性物質とを混合し、当該混合物を前記刺激応答性ポリマーが凝集する条件下におき、前記混合物の濁度を測定し、前記検出対象の量と濁度との前記条件下における相関式に基づいて、前記検体中の検出対象の量を算出する手順を含む、検体対象の定量方法。
８．前記凝集性物質が、微粒子状の磁性物質を更に含有し、磁力を付加することで、凝集した前記磁性物質を分離することを更に含む前項７に記載の方法。
９．検出対象を検出及び／又は定量するためのキットであって、前記検出対象と結合しうるアプタマーと、刺激応答性ポリマーを含有する凝集性物質とを含むキット。
1. A method for detecting a detection target in a specimen, wherein the specimen, an aptamer that can bind to the detection target, and an aggregating substance containing a stimulus-responsive polymer are mixed, and the resulting mixture is used as the stimulus response. When the degree of aggregation of the aggregating substance containing the stimulus-responsive polymer is determined under the condition that the agglutinable polymer aggregates, and the degree of aggregation changes compared to the absence of the specimen, A method for detecting a detection target, including a procedure for determining that a detection target exists in a specimen.
2. 2. The method according to item 1 above, wherein the aptamer capable of binding to the detection target is DNA, RNA, or peptide.
3. 3. The method according to item 1 or 2, further comprising separating the agglomerated magnetic substance by applying a magnetic force, wherein the aggregating substance further contains a particulate magnetic substance.
4). 4. The method according to any one of items 1 to 3, wherein the change in the degree of aggregation is a change in which aggregation inhibition of the stimulus-responsive polymer is alleviated.
5). 5. The method according to any one of items 1 to 4, wherein the detection target is VEGF.
6). 5. The method according to any one of items 1 to 4, wherein the detection target is thrombin.
7). A method for quantifying a detection target in a sample, wherein the sample, an aptamer capable of binding to the detection target, and an aggregating substance containing a stimulus-responsive polymer are mixed, and the mixture is used as the stimulus-responsive polymer. Measuring the turbidity of the mixture, and calculating the amount of the detection target in the sample based on the correlation equation between the amount of the detection target and the turbidity under the condition Including a method for quantifying a specimen.
8). 8. The method according to item 7 above, wherein the aggregating substance further contains a fine particle magnetic substance, and further includes separating the agglomerated magnetic substance by applying a magnetic force.
9. A kit for detecting and / or quantifying a detection target, comprising an aptamer capable of binding to the detection target and an aggregating substance containing a stimulus-responsive polymer.
本発明によれば、検体中に検出対象が存在する場合、アプタマーと検出対象とが結合し、この複合体が刺激応答性ポリマーを含有する凝集性物質に接近すると、刺激応答性ポリマーが凝集する条件下において、検出対象の電荷部分又は疎水性部分の影響で、アプタマーによる刺激応答性ポリマーの凝集阻害が緩和または促進される。従って、この凝集阻害の有無を観察することで、検出対象の存否を検出できる。また、凝集阻害の程度を測定することで、検出対象を定量できる。 According to the present invention, when the detection target is present in the specimen, the aptamer and the detection target are combined, and when the complex approaches an aggregating substance containing the stimulation responsive polymer, the stimulation responsive polymer aggregates. Under the conditions, the inhibition of aggregation of the stimuli-responsive polymer by the aptamer is alleviated or promoted by the influence of the charge portion or the hydrophobic portion to be detected. Therefore, the presence or absence of the detection target can be detected by observing the presence or absence of this aggregation inhibition. Moreover, the detection target can be quantified by measuring the degree of aggregation inhibition.
以上の手順は、アプタマー及び刺激応答性ポリマーを含有する凝集性物質のみを用いて達成され、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。 The above procedure is achieved by using only an aggregating substance containing an aptamer and a stimulus-responsive polymer, and is performed without using any special reagent or device, so that 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.
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.
＜第１実施形態＞ 検出方法
（混合・凝集）
本発明の検出方法ではアプタマーと検体とを混合し、得られた混合物に刺激応答性ポリマーを含有する凝集性物質を混合した後、刺激応答性ポリマーが凝集する条件下におく。検出対象が存在しない場合は刺激応答性ポリマーの凝集が阻害されたままである一方、検出対象が存在する場合には、検出対象の非存在下よりも刺激応答性ポリマーの凝集の程度が変化する。具体的には、例えば、刺激応答性ポリマーの凝集阻害が緩和されることや、逆に刺激応答性ポリマーの凝集阻害がさらに促進される。好ましくは、検出対象の電荷または疎水性部分の影響で凝集阻害が緩和される。
<First embodiment> Detection method (mixing / aggregation)
In the detection method of the present invention, the aptamer and the specimen are mixed, and the obtained mixture is mixed with an aggregating substance containing a stimuli-responsive polymer, and then subjected to conditions under which the stimuli-responsive polymer aggregates. When the detection target is not present, the aggregation of the stimulus-responsive polymer remains inhibited, whereas when the detection target is present, the degree of aggregation of the stimulus-responsive polymer changes more than in the absence of the detection target. Specifically, for example, the inhibition of aggregation of the stimulus-responsive polymer is alleviated, and conversely, the inhibition of aggregation of the stimulus-responsive polymer is further promoted. Preferably, aggregation inhibition is mitigated by the influence of the charge to be detected or the hydrophobic portion.
（アプタマー）
アプタマーは、検出対象の特定部位を認識するアプタマーであってよく、検出対象の異なる部位を認識するアプタマーの混合物であってもよい。ここで用いるアプタマーは、いかなるタイプのアプタマーであってもよく、Ｈａｉｒｐｉｎ−ｌｏｏｐやＧ−ｑｕａｄｒｕｐｌｅｘ構造等を形成するＤＮＡアプタマー、ＲＮＡアプタマーであってもよい。その他、ペプチドアプタマーでもよい。また、検出対象の特定部位を認識するアプタマーの二分子をリンカーで連結させたｂｉｖａｌｅｎｔ型、検出対象に対して異なる部位を認識する二種類のアプタマーをリンカーで連結させたｂｉｓｐｅｃｉｆｉｃ型であってもよい。アプタマーを用いることによって、検出対象を特異的に認識する。抗体を用いた場合と比較して、例えば、ＤＮＡアプタマーは、５’末端及び３’末端にビオチン標識などの様々な修飾が容易であり、このような加工を施した後も抗原に対する結合能を高度に保持する。また、抗原に対して高い結合能と特異性（抗体より低い解離定数）を有するＤＮＡアプタマーを設計することが容易であるから、高感度かつ短時間で検出が可能となる。
(Aptamer)
The aptamer may be an aptamer that recognizes a specific site to be detected, or may be a mixture of aptamers that recognize different sites to be detected. The aptamer used here may be any type of aptamer, and may be a DNA aptamer or an RNA aptamer that forms a Hairpin-loop or G-quadruplex structure. In addition, a peptide aptamer may be used. Moreover, the bivalent type which connected two molecules of the aptamer which recognizes the specific site | part of a detection target with a linker, and the bispecific type which connected two types of aptamers which recognize a different site | part with respect to a detection target with a linker may be sufficient. . By using the aptamer, the detection target is specifically recognized. Compared to the case of using an antibody, for example, a DNA aptamer can be easily modified in various ways, such as biotin labeling at the 5 ′ end and 3 ′ end, and has the ability to bind to an antigen even after such processing. Hold high. Moreover, since it is easy to design a DNA aptamer having high binding ability and specificity for antigen (dissociation constant lower than that of an antibody), detection can be performed with high sensitivity and in a short time.
（凝集性物質）
本発明で用いられる凝集性物質は刺激応答性ポリマーを含有する。この刺激応答性ポリマーは、外的な刺激に応答して構造変化を起こし、凝集及び分散を調整できるポリマーである。刺激は、特に限定されないが、温度、光、酸、塩基、ｐＨ若しくは電気等の様々な物理的、または化学的信号であってよい。
(Aggregating substance)
The aggregating substance used in the present invention contains a stimulus-responsive polymer. This stimulus-responsive polymer is a polymer that undergoes structural changes in response to external stimuli and can adjust aggregation and dispersion. The stimulus may be any physical or chemical signal such as, but not limited to, temperature, light, acid, base, pH or electricity.
特に、本発明では、刺激応答性ポリマーとしては、温度変化によって凝集及び分散可能な温度応答性ポリマーが利用できる。なお、温度応答性ポリマーとしては、下限臨界溶液温度（以下、ＬＣＳＴとも称する）を有するポリマーや上限臨界溶液温度を有するポリマーが挙げられる。 In particular, in the present invention, as the stimulus-responsive polymer, a temperature-responsive polymer that can be aggregated and dispersed by temperature change can be used. Examples of the temperature-responsive polymer include a polymer having a lower critical solution temperature (hereinafter also referred to as LCST) and a polymer having an upper critical solution temperature.
本発明で用いられる下限臨界溶液温度を有するポリマーとしては、例えば、Ｎ−ｎ−プロピルアクリルアミド、Ｎ−イソプロピルアクリルアミド、Ｎ−エチルアクリルアミド、Ｎ，Ｎ−ジメチルアクリルアミド、Ｎ−アクリロイルピロリジン、Ｎ−アクリロイルピペリジン、Ｎ−アクリロイルモルホリン、Ｎ−ｎ−プロピルメタクリルアミド、Ｎ−イソプロピルメタクリルアミド、Ｎ−エチルメタクリルアミド、Ｎ，Ｎ−ジメチルメタクリルアミド、Ｎ−メタクリロイルピロリジン、Ｎ−メタクリロイルピペリジン、Ｎ−メタクリロイルモルホリン等のＮ置換（メタ）アクリルアミド誘導体からなるポリマー；ヒドロキシプロピルセルロース、ポリビニルアルコール部分酢化物、ポリビニルメチルエーテル、（ポリオキシエチレン−ポリオキシプロピレン）ブロックコポリマー、ポリオキシエチレンラウリルアミン等のポリオキシエチレンアルキルアミン誘導体；ポリオキシエチレンソルビタンラウレート等のポリオキシエチレンソルビタンエステル誘導体；（ポリオキシエチレンノニルフェニルエーテル）アクリレート、（ポリオキシエチレンオクチルフェニルエーテル）メタクリレート等の（ポリオキシエチレンアルキルフェニルエーテル）（メタ）アクリレート類；及び（ポリオキシエチレンラウリルエーテル）アクリレート、（ポリオキシエチレンオレイルエーテル）メタクリレート等の（ポリオキシエチレンアルキルエーテル）（メタ）アクリレート類等のポリオキシエチレン（メタ）アクリル酸エステル誘導体等が挙げられる。更に、例えば、これらのポリマー及びこれらの少なくとも２種のモノマーからなるコポリマーが挙げられる。また、例えば、Ｎ−イソプロピルアクリルアミドとＮ−ｔ−ブチルアクリルアミドのコポリマーも利用できるが挙げられる。（メタ）アクリルアミド誘導体を含むポリマーを使用する場合、このポリマーにその他の共重合可能なモノマーを、下限臨界溶液温度を有する範囲で共重合してもよい。本発明では、なかでも、Ｎ−ｎ−プロピルアクリルアミド、Ｎ−イソプロピルアクリルアミド、Ｎ−エチルアクリルアミド、Ｎ，Ｎ−ジメチルアクリルアミド、Ｎ−アクリロイルピロリジン、Ｎ−アクリロイルピペリジン、Ｎ−アクリロイルモルホリン、Ｎ−ｎ−プロピルメタクリルアミド、Ｎ−イソプロピルメタクリルアミド、Ｎ−エチルメタクリルアミド、Ｎ，Ｎ−ジメチルメタクリルアミド、Ｎ−メタクリロイルピロリジン、Ｎ−メタクリロイルピペリジン、Ｎ−メタクリロイルモルホリンからなる群から選ばれる少なくとも１種のモノマーからなるポリマー又はＮ−イソプロピルアクリルアミドとＮ−ｔ−ブチルアクリルアミドのコポリマーが好ましく利用できる。 Examples of the polymer having a lower critical solution temperature used in the present invention include Nn-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, and N-acryloylpiperidine. N-acryloylmorpholine, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine, N-methacryloylmorpholine, etc. Polymer consisting of N-substituted (meth) acrylamide derivative; hydroxypropyl cellulose, polyvinyl alcohol partially acetylated product, polyvinyl methyl ether, (polyoxyethylene-polyoxy Propylene) block copolymers, polyoxyethylene alkylamine derivatives such as polyoxyethylene laurylamine; polyoxyethylene sorbitan ester derivatives such as polyoxyethylene sorbitan laurate; (polyoxyethylene nonylphenyl ether) acrylate, (polyoxyethylene octylphenyl) (Polyoxyethylene alkylphenyl ether) (meth) acrylates such as ether) methacrylate; and (polyoxyethylene alkyl ether) (meth) acrylate such as (polyoxyethylene lauryl ether) acrylate and (polyoxyethylene oleyl ether) methacrylate And other polyoxyethylene (meth) acrylic acid ester derivatives. Furthermore, for example, a copolymer composed of these polymers and at least two kinds of these monomers can be mentioned. In addition, for example, a copolymer of N-isopropylacrylamide and Nt-butylacrylamide can also be used. When a polymer containing a (meth) acrylamide derivative is used, another copolymerizable monomer may be copolymerized with the polymer within a range having a lower critical solution temperature. In the present invention, among others, Nn-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, N-acryloylpiperidine, N-acryloylmorpholine, Nn- From at least one monomer selected from the group consisting of propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine, N-methacryloylmorpholine Or a copolymer of N-isopropylacrylamide and Nt-butylacrylamide can be preferably used.
本発明で用いられる上限臨界溶液温度を有するポリマーとしては、例えば、アクロイルグリシンアミド、アクロイルニペコタミド、アクリロイルアスパラギンアミド及びアクリロイルグルタミンアミド等からなる群から選ばれる少なくとも１種のモノマーからなるポリマーが挙げられる。また、これらの少なくとも２種のモノマーからなるコポリマーであってもよい。これらのポリマーには、アクリルアミド、アセチルアクリルアミド、ビオチノールアクリレート、Ｎ−ビオチニル−Ｎ’−メタクロイルトリメチレンアミド、アクロイルザルコシンアミド、メタクリルザルコシンアミド、アクロイルメチルウラシル等、その他の共重合可能なモノマーを、上限臨界溶液温度を有する範囲で共重合してもよい。 As the polymer having the upper critical solution temperature used in the present invention, for example, a polymer comprising at least one monomer selected from the group consisting of acryloylglycinamide, acryloylnipecotamide, acryloyl asparagine amide, acryloyl glutamine amide and the like. Is mentioned. Moreover, the copolymer which consists of these at least 2 types of monomers may be sufficient. These polymers include other copolymers such as acrylamide, acetylacrylamide, biotinol acrylate, N-biotinyl-N′-methacryloyl trimethylene amide, acroyl sarcosine amide, methacryl sarcosine amide, acroyl methyl uracil, etc. Possible monomers may be copolymerized in a range having an upper critical solution temperature.
また、本発明では、刺激応答性ポリマーとして、ｐＨ変化によって凝集及び分散可能なｐＨ応答性ポリマーが利用できる。
このようなｐＨ応答性ポリマーとしては、例えば、カルボキシル、リン酸、スルホニル、アミノ等の基を官能基として含有するポリマーが挙げられる。より具体的には、例えば、アクリル酸、メタクリル酸、マレイン酸、ビニルスルホン酸、ビニルベンゼンスルホン酸、ホスホリルエチル（メタ）アクリレート、アミノエチルメタクリレート、アミノプロピル（メタ）アクリルアミド、ジメチルアミノプロピル（メタ）アクリルアミドまたはこれらの塩を共重合成分として含むポリマーが挙げられる。
In the present invention, as the stimulus-responsive polymer, a pH-responsive polymer that can be aggregated and dispersed by pH change can be used.
Examples of such a pH-responsive polymer include a polymer containing a group such as carboxyl, phosphoric acid, sulfonyl, and amino as a functional group. More specifically, for example, acrylic acid, methacrylic acid, maleic acid, vinyl sulfonic acid, vinyl benzene sulfonic acid, phosphoryl ethyl (meth) acrylate, aminoethyl methacrylate, aminopropyl (meth) acrylamide, dimethylaminopropyl (meth) Examples include polymers containing acrylamide or a salt thereof as a copolymerization component.
凝集性物質は、後述の磁力付加により検出精度を向上できる点で、微粒子状の磁性物質を更に含有することが好ましい。かかる磁性物質は、多価アルコールとマグネタイトとで構成されてよい。 The aggregating substance preferably further contains a particulate magnetic substance in that detection accuracy can be improved by applying a magnetic force described later. Such a magnetic substance may be composed of a polyhydric alcohol and magnetite.
多価アルコールは、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アルコール構造体であれば特に限定されず、例えば、デキストラン、ポリビニルアルコール、マンニトール、ソルビトール及びシクロデキストリンが挙げられる。例えば特開２００５−８２５３８号公報には、デキストランを用いた微粒子状の磁性物質の製造方法が開示されている。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。 The polyhydric alcohol is not particularly limited as long as it is an alcohol structure having at least two hydroxyl groups as a structural unit and capable of binding to iron ions, and examples thereof include dextran, polyvinyl alcohol, mannitol, sorbitol, and cyclodextrin. For example, Japanese Patent Application Laid-Open No. 2005-82538 discloses a method for producing a particulate magnetic material using dextran. 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.
このような多価アルコールを用いて調製された微粒子状の磁性物質（磁性微粒子）は、良好な分散性を有するように、その平均粒径が０．９ｎｍ以上１０００ｎｍ未満であることが好ましい。平均粒径は、特に目的とする検出対象の検出感度を高めるためには、２．９ｎｍ以上２００ｎｍ未満であることが好ましい。 The fine particle magnetic material (magnetic fine particles) prepared using such a polyhydric alcohol preferably has an average particle size of 0.9 nm or more and less than 1000 nm so as to have good dispersibility. The average particle size is preferably 2.9 nm or more and less than 200 nm, in particular, in order to increase the detection sensitivity of the target detection target.
（アプタマーと凝集性物質の結合）
アプタマーと凝集性物質との結合方法は、特に限定されないが、例えば、アプタマー側及び凝集性物質側（例えば刺激応答性ポリマー部分）の双方に、互いに親和性の物質（例えば、ビオチン及びストレプトアビジンまたはアビジン）を結合させ、これら物質を介してアプタマー及び凝集性物質を結合させる。
(Bonding of aptamer and aggregating substance)
The binding method of the aptamer and the aggregating substance is not particularly limited. For example, both the aptamer side and the aggregating substance side (for example, the stimulus-responsive polymer moiety) have mutually affinity substances (for example, biotin and streptavidin or Avidin) is bound, and aptamer and aggregating substance are bound via these substances.
具体的には、刺激応答性ポリマーへのビオチンの結合は、国際公開第０１／０９１４１号に記載されているように、ビオチン等をメタクリルやアクリル等の重合性官能基と結合させて付加重合性モノマーとし、他のモノマーと共重合することにより行い、そこにストレプトアビジンを固定化する。また、アプタマーへのビオチン等の標識は常法に従って行い、オリゴＤＮＡ合成の際に５’側または３’側に標識しておく。ビオチン標識アプタマーとストレプトアビジン固定化刺激応答性ポリマーとを混合すると、ビオチンとストレプトアビジンとの結合を介して、アプタマー及び刺激応答性ポリマーが結合する。 Specifically, as described in International Publication No. 01/09141, the binding of biotin to the stimulus-responsive polymer is performed by adding biotin or the like to a polymerizable functional group such as methacryl or acrylic. A monomer is used and copolymerized with other monomers to immobilize streptavidin. The aptamer is labeled with biotin or the like according to a conventional method, and labeled on the 5 'side or 3' side during oligo DNA synthesis. When the biotin-labeled aptamer and the streptavidin-immobilized stimulus-responsive polymer are mixed, the aptamer and the stimulus-responsive polymer are bonded through the binding between biotin and streptavidin.
なお、本明細書では、刺激応答性ポリマーと微粒子状の磁性物質とが結合した材料を刺激応答性磁性微粒子と記し、その刺激が温度の場合には、温度応答性磁性微粒子と記している。 In this specification, a material in which a stimulus-responsive polymer and a particulate magnetic substance are combined is referred to as a stimulus-responsive magnetic fine particle, and when the stimulus is temperature, it is referred to as a temperature-responsive magnetic fine particle.
微粒子状の磁性物質と刺激応答性ポリマーとの結合は、反応性官能基を介して結合する方法や、磁性物質中の多価アルコール上の活性水素又は多価アルコールに重合性不飽和結合を導入してグラフト重合する方法等の当技術分野で周知の方法で行ってよい（例えば、ＡＤＶ．Ｐｏｌｙｍ．Ｓｃｉ．、Ｖｏｌ．４、ｐ１１１、１９６５やＪ．Ｐｏｌｙｍｅｒ Ｓｃｉ．、Ｐａｒｔ−Ａ、３、ｐ１０３１、１９６５参照）。 The fine magnetic substance can be bonded to the stimulus-responsive polymer via a reactive functional group, or a polymerizable unsaturated bond is introduced into the active hydrogen or polyhydric alcohol on the polyhydric alcohol in the magnetic substance. The graft polymerization may be carried out by a method known in the art such as graft polymerization (for example, ADV.Polym.Sci., Vol.4, p111, 1965, J. Polymer Sci., Part-A, 3, p1031). 1965).
以上のような凝集性物質、アプタマー、検体の混合物を凝集性物質に係る刺激応答性ポリマーが凝集する条件下におくと、検出対象が存在しない場合は刺激応答性ポリマーの凝集阻害が起こり、凝集が生じない。一方、検出対象が存在する場合には、検出対象の電荷部分又は疎水性部分によって刺激応答性ポリマーの凝集阻害が緩和され、凝集が生じる。 When a mixture of the aggregating substance, aptamer, and specimen as described above is placed under conditions where the stimuli-responsive polymer related to the aggregating substance aggregates, the aggregation of the stimuli-responsive polymer occurs when there is no target to be detected. Does not occur. On the other hand, when a detection target is present, aggregation inhibition of the stimulus-responsive polymer is mitigated by the charge portion or hydrophobic portion of the detection target, and aggregation occurs.
このうち、凝集阻害が緩和される現象を、図１および図２を参照しながら説明する。 Among these, the phenomenon in which aggregation inhibition is alleviated will be described with reference to FIGS. 1 and 2.
図１に示されるように、刺激応答性磁性微粒子１は微粒子状の磁性物質２を含み、この磁性物質２の表面に刺激応答性ポリマー３が結合されている。刺激応答性ポリマー３にはストレプトアビジン４が結合しており、ビオチン５を介して検出対象７に対するアプタマー６が結合する。これにより、検出対象７はアプタマー６を介して刺激応答性ポリマー３に接近し、検出対象７の正電荷部分または疎水性部分の影響で、刺激応答性ポリマー３の凝集阻害が緩和される。 As shown in FIG. 1, the stimulus-responsive magnetic fine particle 1 includes a fine particle-like magnetic substance 2, and a stimulus-responsive polymer 3 is bonded to the surface of the magnetic substance 2. Streptavidin 4 is bound to the stimulus-responsive polymer 3, and an aptamer 6 for the detection target 7 is bound via biotin 5. As a result, the detection target 7 approaches the stimulus-responsive polymer 3 via the aptamer 6, and the inhibition of aggregation of the stimulus-responsive polymer 3 is alleviated due to the influence of the positively charged portion or the hydrophobic portion of the detection target 7.
刺激応答性ポリマーを凝集させるためには、例えば温度応答性ポリマーを用いた場合、混合物の入った容器を温度応答性ポリマーの凝集する温度の恒温槽に移せばよい。温度応答性ポリマーには、上限臨界溶液温度（以下「ＵＣＳＴ」と略すことがある。）を有するポリマーと、下限臨界溶液温度（以下「ＬＣＳＴ」と略すことがある。）を有するポリマーの２種類がある。例えば、ＬＣＳＴが３７℃である下限臨界溶液温度を有するポリマーを用いた場合には、混合物の入った容器を３７℃以上の恒温槽に移すことで、温度応答性ポリマーを凝集させることができる。また、ＵＣＳＴが５℃である上限臨界溶液温度を有するポリマーを用いた場合には、混合物の入った容器を５℃未満の恒温槽に移すことで、温度応答性ポリマーを凝集させることができる。 In order to agglomerate the stimulus-responsive polymer, for example, when a temperature-responsive polymer is used, the container containing the mixture may be transferred to a thermostatic bath at a temperature at which the temperature-responsive polymer aggregates. There are two types of temperature-responsive polymers: a polymer having an upper critical solution temperature (hereinafter sometimes abbreviated as “UCST”) and a polymer having a lower critical solution temperature (hereinafter sometimes abbreviated as “LCST”). There is. For example, when a polymer having a lower critical solution temperature of LCST of 37 ° C. is used, the temperature-responsive polymer can be agglomerated by moving the container containing the mixture to a constant temperature bath of 37 ° C. or higher. When a polymer having an upper critical solution temperature with a UCST of 5 ° C. is used, the temperature-responsive polymer can be agglomerated by moving the container containing the mixture to a thermostatic bath of less than 5 ° C.
また、ｐＨ応答性ポリマーを用いた場合、混合物の入った容器に酸溶液又はアルカリ溶液を加えればよい。具体的には、ｐＨ応答性ポリマーが構造変化を起こすｐＨ範囲の外にある分散している混合物の入った容器に、酸溶液又はアルカリ溶液を加え、容器内をｐＨ応答性ポリマーが構造変化を起こすｐＨ範囲に変更すればよい。例えば、ｐＨ５以下で凝集、ｐＨ５超で分散するｐＨ応答性ポリマーを用いた場合、ｐＨ５超で分散している混合物の入った容器に、ｐＨが５以下になるように酸溶液を加えればよい。また、ｐＨ１０以上で凝集、ｐＨ１０未満で分散するｐＨ応答性ポリマーを用いた場合、ｐＨ１０未満で分散している混合物の入った容器に、ｐＨが１０以上になるようにアルカリ溶液を加えればよい。ｐＨ応答性ポリマーが構造変化を起こすｐＨは、特に限定されないが、ｐＨ４〜１０が好ましく、ｐＨ５〜９であることがさらに好ましい。 Moreover, what is necessary is just to add an acid solution or an alkaline solution to the container containing a mixture, when using a pH-responsive polymer. Specifically, an acid solution or an alkaline solution is added to a container containing a dispersed mixture outside the pH range where the pH-responsive polymer undergoes a structural change, and the pH-responsive polymer undergoes a structural change in the container. What is necessary is just to change to the pH range to raise. For example, when a pH-responsive polymer that aggregates at a pH of 5 or less and disperses at a pH of more than 5 is used, an acid solution may be added to a container containing a mixture dispersed at a pH of more than 5 so that the pH is 5 or less. When a pH-responsive polymer that aggregates at pH 10 or higher and disperses at a pH lower than 10 is used, an alkaline solution may be added to a container containing a mixture dispersed at a pH lower than 10 so that the pH becomes 10 or higher. The pH at which the pH-responsive polymer undergoes a structural change is not particularly limited, but is preferably pH 4-10, more preferably pH 5-9.
また、光応答性ポリマーを用いた場合、混合物の入った容器にポリマーを凝集できる波長の光を照射すればよい。凝集させるための好ましい光は、光応答性ポリマーに含まれる光応答性官能基の種類及び構造により異なるが、一般に波長１９０〜８００ｎｍの紫外光又は可視光が好適に使用できる。このとき、強度は０．１〜１０００ｍＷ／ｃｍ２が好ましい。なお、光応答性ポリマーは、測定精度を向上できる点で、濁度の測定に用いられる光が照射された際、分散を生じにくいもの、換言すれば凝集するものであることが好ましい。光応答性ポリマーとして、濁度の測定に用いられる光が照射された際に分散を生じるものを用いる場合、照射時間を短縮することで測定精度を向上できる。 Further, when a photoresponsive polymer is used, light having a wavelength capable of aggregating the polymer may be irradiated to a container containing the mixture. The preferred light for aggregation varies depending on the type and structure of the photoresponsive functional group contained in the photoresponsive polymer, but generally ultraviolet light or visible light having a wavelength of 190 to 800 nm can be suitably used. At this time, the strength is preferably 0.1 to 1000 mW / cm 2 . In addition, it is preferable that a photoresponsive polymer is a thing which is hard to produce dispersion | distribution in other words, when it is irradiated with the light used for a turbidity measurement in the point which can improve a measurement precision. When using a photoresponsive polymer that generates dispersion when irradiated with light used for turbidity measurement, the measurement accuracy can be improved by shortening the irradiation time.
アプタマーが刺激応答性ポリマーに結合し、この結合物を刺激応答性ポリマーが凝集する条件下におくと、アプタマーの負電荷の影響により凝集阻害が生じる。つまり、検出対象が存在しない場合は分散を維持することになる［図２の（Ａ）］。一方、検出対象が存在する場合には、検出対象の正電荷部分または疎水性部分の影響で、刺激応答性ポリマーの凝集阻害が緩和され、凝集が促進する［図２の（Ｂ）］。これは、図２に示されるように、（Ａ）では測定終了時に吸光度が（Ｂ）と比較して高くなっており、濁度が上昇していることからわかる。 When the aptamer binds to the stimulus-responsive polymer and this conjugate is subjected to a condition where the stimulus-responsive polymer aggregates, inhibition of aggregation occurs due to the negative charge of the aptamer. That is, when there is no detection target, dispersion is maintained [(A) of FIG. 2]. On the other hand, when the detection target exists, the inhibition of aggregation of the stimulus-responsive polymer is alleviated and the aggregation is promoted by the influence of the positively charged portion or the hydrophobic portion of the detection target [(B) of FIG. 2]. As can be seen from FIG. 2, the absorbance in (A) is higher than that in (B) at the end of the measurement, and the turbidity is increased.
ここで、下限臨界溶液温度及び上限臨界溶液温度は例えば以下のように決定する。まず、試料を吸光光度計のセルに入れ、１℃／分の速度で試料を昇温する。この間、５５０ｎｍにおける透過率変化を記録する。ここで、ポリマーが透明に溶解しているときの透過率を１００％、完全に凝集したときの透過率を０％としたとき、透過率が５０％になるときの温度をＬＣＳＴとして求める。ＵＣＳＴの場合は、１℃／分の速度で試料を冷却し、以下、ＬＣＳＴ同様の方法で求める。 Here, the lower critical solution temperature and the upper critical solution temperature are determined as follows, for example. First, a sample is put into a cell of an absorptiometer and the sample is heated at a rate of 1 ° C./min. During this time, the change in transmittance at 550 nm is recorded. Here, when the transmittance when the polymer is transparently dissolved is 100% and when the transmittance when the polymer is completely aggregated is 0%, the temperature at which the transmittance is 50% is obtained as LCST. In the case of UCST, the sample is cooled at a rate of 1 ° C./min.
（判定）
凝集の程度の判定、上記例で言えば、分散の有無の判定は、例えば目視又は濁度測定で行うことができる。濁度は光散乱装置での光透過率から算出でき、濁度が低ければ刺激応答性ポリマーの凝集が阻害されており、検出物質の存在が示唆される。ここで、使用する光の波長は、磁性物質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されてよい。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。
(Judgment)
The determination of the degree of aggregation, in the above example, can be performed by visual observation or turbidity measurement, for example. The turbidity can be calculated from the light transmittance of the light scattering device. If the turbidity is low, aggregation of the stimulus-responsive polymer is inhibited, which indicates the presence of the detection substance. Here, the wavelength of the light to be used may be appropriately set so as to obtain a desired detection sensitivity according to the particle size of the magnetic substance. The wavelength of light is preferably within the range of visible light in that a conventional general-purpose device can be used.
目視又は濁度測定は、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、ある時点における濁度測定値と、他の時点における濁度測定値との差に基づいて判定を行ってもよい。 Visual observation or turbidity 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 turbidity measurement value at a certain time point and the turbidity measurement value at another time point.
（分離）
凝集性物質が微粒子状の磁性物質を含有する場合、本発明の検出方法は、磁力を付加することで、凝集した磁性物質を分離することを更に含むことが好ましい。これによって、凝集した磁性物質が、非凝集状態の磁性物質を含む夾雑物から分離される。このため、分離した磁性物質の量、溶媒に分散した際の光透過率等の測定値は、夾雑物の影響が除外され、検出物質の存在をより忠実に反映したものとなる。
(Separation)
When the aggregating substance contains a particulate magnetic substance, it is preferable that the detection method of the present invention further includes separating the aggregated magnetic substance by applying a magnetic force. Thereby, the agglomerated magnetic substance is separated from impurities including the non-aggregated magnetic substance. For this reason, the measurement values such as the amount of the separated magnetic substance and the light transmittance when dispersed in the solvent exclude the influence of foreign substances and more accurately reflect the presence of the detection substance.
磁力の付加は磁性物質に磁石を接近させて行うことができる。この磁石の磁力は、用いる磁性物質が有する磁力の大きさによって異なる。磁石としては、例えばネオジム磁石（ネオマグ社製）が挙げられる。 The magnetic force can be applied by bringing a magnet close to the magnetic substance. The magnetic force of this magnet varies depending on the magnitude of the magnetic force of the magnetic substance used. An example of the magnet is a neodymium magnet (manufactured by Neomag).
また、磁力の付加は、判定の前又は判定と同時並行して行ってよいが、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。なお、磁力を付加すると、凝集した磁性物質は夾雑物を巻き込んで分離されるため、分離後における混合物の濁度は、夾雑物が存在していた場合の方がむしろ小さくなるものと推測される。 Further, the addition of magnetic force may be performed before the determination or in parallel with the determination. However, the simultaneous parallel is preferable in that the time spent for the process can be shortened. It should be noted that when magnetic force is applied, the aggregated magnetic substance is separated by inclusion of impurities, so the turbidity of the mixture after separation is presumed to be rather smaller when the impurities are present. .
（検出対象）
以上の検出方法で検出できる対象としては、臨床診断に利用される物質が挙げられ、具体的には、例えば、体液、尿、喀痰、糞便、唾液中等に含まれる血管内皮細胞増殖因子（ＶＥＧＦ）、トロンビン、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（フィブリン及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）、インスリン等）、薬剤等が挙げられる。その他、体のあらゆる部位から採取した分泌液なども対象とし、あらゆる疾患に関連する原因物質等も挙げられる。
(Detection target)
Examples of the target that can be detected by the above detection methods include substances used in clinical diagnosis. Specifically, for example, vascular endothelial growth factor (VEGF) contained in body fluid, urine, sputum, feces, saliva, etc. , Thrombin, human immunoglobulin G, human immunoglobulin M, human immunoglobulin A, human immunoglobulin E, human albumin, human fibrinogen (fibrin and degradation products thereof), α-fetoprotein (AFP), C-reactive protein (CRP) ), Myoglobin, carcinoembryonic antigen, hepatitis virus antigen, human chorionic gonadotropin (hCG), human placental lactogen (HPL), HIV virus antigen, allergen, bacterial toxin, bacterial antigen, enzyme, hormone (eg, human thyroid stimulation Hormones (TSH), insulin, etc.), drugs, etc. It is. In addition, secretory fluids collected from every part of the body are also targeted, and causative substances related to all diseases can be mentioned.
これらの検出対象が含まれると疑われる検体には、多種類且つ多量の夾雑物が混在する場合が多いが、測定される磁界は検出対象中の夾雑物に大きく影響されるものではない。このため、測定前に夾雑物を除去するといった予備手順を必ずしも行わなくてもよい。 In many cases, a sample suspected of containing these detection targets contains many types and a large amount of contaminants, but the measured magnetic field is not greatly affected by the contaminants in the detection targets. For this reason, it is not always necessary to perform a preliminary procedure of removing impurities before measurement.
（作用効果）
本発明の第１実施形態によれば、以下のような作用効果が得られる。
検体に検出対象が存在する場合、この検出対象にアプタマーが結合する。検出対象とアプタマーの複合体が刺激応答性ポリマーを有する凝集性物質に結合すると、検出対象の電荷部分又は疎水性部分が刺激応答性ポリマーの近傍に配置され、刺激応答性ポリマーの凝集阻害が緩和される。従って、この凝集阻害の変化の観察することで、検出対象の有無を判定できる。
(Function and effect)
According to 1st Embodiment of this invention, the following effects are obtained.
When the detection target exists in the specimen, the aptamer binds to the detection target. When the complex between the detection target and the aptamer binds to an aggregating substance having a stimulus-responsive polymer, the charge or hydrophobic portion of the detection target is placed in the vicinity of the stimulus-responsive polymer, and the aggregation inhibition of the stimulus-responsive polymer is alleviated. Is done. Therefore, the presence or absence of the detection target can be determined by observing this change in aggregation inhibition.
以上の手順は、アプタマー及び刺激応答性ポリマーを有する凝集性物質のみを用いて達成され、特殊な試薬、機器を特に使用することなく行われるので、安価且つ簡便である。また、凝集の程度の変化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しないため、迅速に行うことができる。 The above procedure is achieved using only an aggregating substance having an aptamer and a stimulus-responsive polymer, and is performed without using any special reagent or device, so that it is inexpensive and simple. In addition, only the change in the degree of aggregation is measured, and since the reaction catalyzed by the enzyme is not used, it can be performed rapidly.
＜第２実施形態＞
（定量方法）
本発明の定量方法では、まず、アプタマーと検体を混合する。次に、当該混合物と刺激応答性ポリマーを有する凝集性物質を混合し、刺激応答性ポリマーが凝集する所定条件下におく。続いて、この混合物の濁度を測定し、検出対象の量と濁度との所定条件下における相関式に基づいて、検体中の検出対象の量を算出する。前半部分の手順は前述した検出方法と類似するので、説明を省略する。
Second Embodiment
(Quantitative method)
In the quantification method of the present invention, first, an aptamer and a specimen are mixed. Next, the mixture and an aggregating substance having a stimulus-responsive polymer are mixed, and the mixture is subjected to predetermined conditions in which the stimulus-responsive polymer aggregates. Subsequently, the turbidity of the mixture is measured, and the amount of the detection target in the sample is calculated based on a correlation equation under a predetermined condition between the amount of the detection target and the turbidity. Since the procedure of the first half is similar to the detection method described above, the description is omitted.
（相関式）
上記所定条件と同一の条件における、検出対象の量と濁度との相関式を作成する。この相関式を構成する検出対象の量と濁度との測定は、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。
ここで、検出対象の量と濁度との相関式は、検出対象の量と濁度との直接的な相関を示す式のみならず、検出対象の量と濁度を反映するパラメータとの相関式であってもよい。
(Correlation formula)
A correlation equation between the amount of detection target and turbidity under the same condition as the predetermined condition is created. In the measurement of the amount of detection target and the turbidity constituting the correlation formula, the more reliable the correlation formula is obtained as the amount of data increases. 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.
Here, the correlation equation between the amount of detection target and turbidity is not only an equation showing a direct correlation between the amount of detection target and turbidity, but also the correlation between the amount of detection target and a parameter reflecting turbidity. It may be a formula.
（算出）
混合物の濁度測定値を、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できる。
なお、検出方法又は定量方法における「濁度測定」には、濁度を直接的に測定することのみならず、濁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、複数時点での濁度測定値の差異、分離された凝集物量、分離後の非凝集物の濁度等が挙げられる。ここで、複数時点のうちの１点は、例えば、検出対象が非存在下である陰性対照に磁力を付加した際、濁度が最大値となる時点であることが好ましい。これにより、別の時点での濁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。
(Calculation)
By substituting the turbidity measurement value of the mixture into the created correlation equation, the amount of the detection target in the sample can be calculated.
The “turbidity measurement” in the detection method or the quantitative method includes not only measuring the turbidity directly but also measuring a parameter reflecting the turbidity. Such parameters include differences in turbidity measurement values at multiple time points, the amount of separated aggregates, the turbidity of non-aggregates after separation, and the like. Here, one point of the plurality of time points is preferably a time point at which the turbidity reaches a maximum value when, for example, a magnetic force is applied to a negative control in which the detection target is absent. Thereby, the difference from the turbidity measurement value at another time point becomes large, and the amount of the detection target can be quantified more accurately.
（作用効果）
本発明の第２実施形態によれば、次の作用効果が得られる。第１実施形態と同様に、濁度の程度が検出対象の量に依存することになるので、検出対象量及び濁度の相関式に濁度測定値を代入することで、検出対象を定量できる。しかも、この手順は、アプタマー及び刺激応答性ポリマーを有する凝集性物質のみを用いて達成され、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ことから、迅速に行うことが可能である。
(Function and effect)
According to 2nd Embodiment of this invention, the following effect is obtained. As in the first embodiment, since the degree of turbidity depends on the amount of the detection target, the detection target can be quantified by substituting the turbidity measurement value into the correlation equation between the detection target amount and the turbidity. . In addition, this procedure is achieved using only an aggregating substance having an aptamer and a stimulus-responsive polymer, and is performed without using any special reagent or device, so that it is inexpensive and simple. Moreover, it is possible only to measure the degree of aggregation inhibition and not to use a reaction catalyzed by an enzyme.
＜実施例１＞
（トロンビン試料の調製）
ウシα−トロンビン（Ｔｈｒｏｍｂｉｎ；Ｔｈｅｒｍｏ ｓｃｉｅｎｔｉｆｉｃ社製）を０．１％（ｗ／ｖ）ＢＳＡ（和光純薬工業製）、１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５、和光純薬工業製）、１００ｍＭ ＮａＣｌ、５ｍＭ ＫＣｌを含有する溶液で、１００ｎＭ、８０ｎＭ、６０ｎＭ、４０ｎＭ、２０ｎＭ、１０ｎＭとなるように希釈したものを、それぞれトロンビン試料として調製した。
<Example 1>
(Preparation of thrombin sample)
Bovine α-thrombin (Thrombin; manufactured by Thermo scientific) 0.1% (w / v) BSA (manufactured by Wako Pure Chemical Industries), 10 mM Tris-HCl (pH 7.5, manufactured by Wako Pure Chemical Industries), 100 mM NaCl, Solutions containing 5 mM KCl diluted to 100 nM, 80 nM, 60 nM, 40 nM, 20 nM, and 10 nM were prepared as thrombin samples, respectively.
（トロンビン結合性ＤＮＡアプタマーの調製）
５’末端にビオチンを有するオリゴヌクレオチド（配列番号１）を１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５、和光純薬工業製）、１００ｍＭ ＮａＣｌ、５ｍＭ ＫＣｌを含有する溶液で、１μＭとなるように希釈し、サーマルサイクラー（ＤＮＡ Ｅｎｇｉｎｅ、Ｂｉｏ−Ｒａｄ社製）を用いて９５℃で１０分保温した後、３０分かけて２５℃にしたものをトロンビン結合性ＤＮＡアプタマーとして調製した。
(Preparation of thrombin-binding DNA aptamer)
An oligonucleotide having biotin at the 5 ′ end (SEQ ID NO: 1) was diluted to 1 μM with a solution containing 10 mM Tris-HCl (pH 7.5, manufactured by Wako Pure Chemical Industries), 100 mM NaCl, 5 mM KCl, A temperature cycler (DNA Engine, manufactured by Bio-Rad) was used to incubate at 95 ° C. for 10 minutes, and then a temperature of 25 ° C. over 30 minutes was prepared as a thrombin-binding DNA aptamer.
（配列番号１）
５’−ａｇｔｃｃｇｔｇｇｔａｇｇｇｃａｇｇｔｔｇｇｇｇｔｇａｃｔｔｔｔｔｔｇｇｔｔｇｇｔｇｔｇｇｔｔｇｇ−３’
(SEQ ID NO: 1)
5′-agtccgtggttagggcagggtggggtgactttttttggtttgtgtgtggtttgg-3 ′
（試料の混合）
トロンビン試料１００μＬとトロンビン結合性ＤＮＡアプタマー１０μＬを１．５ｍＬマイクロチューブに入れピペッティングで混合し、３７℃で５分間保温した。
(Sample mixing)
100 μL of the thrombin sample and 10 μL of the thrombin-binding DNA aptamer were placed in a 1.5 mL microtube and mixed by pipetting, and incubated at 37 ° C. for 5 minutes.
（試料と磁性微粒子の混合）
上記混合試料に、ストレプトアビジン及び温度応答性ポリマーが結合された磁性微粒子であるＪＮＣ石油化学社製のＴｈｅｒｍａ−Ｍａｘ（商品名） ＬＳＡ Ｓｔｒｅｐｔａｖｉｄｉｎ（０．０２質量％）１００μＬを加えピペッティングで混合し、２１℃で５分間保温した。
(Mixing of sample and magnetic fine particles)
To the above mixed sample, 100 μL of Thermo-Max (trade name) LSA Streptavidin (0.02 mass%) manufactured by JNC Petrochemical Co., Ltd., which is magnetic fine particles in which streptavidin and a temperature-responsive polymer are bonded, is mixed by pipetting. And kept at 21 ° C. for 5 minutes.
（試料の測定）
図３に示されるように、市販の分光光度計用マイクロセル（ＵＶｅｔｔｅ、ｅｐｐｅｎｄｏｒｆ社製）に付属のセルアダプターＡに内部に１０ｍｍ×５ｍｍ×３ｍｍのネオジム永久磁石Ｂ（ネオマグ社製）を取り付けた。このセルアダプターにマイクロセルＣを入れ、セル温度制御機が設けられた紫外可視分光光度計Ｖ−６６０ＤＳ（日本分光製）内に設置し、３７℃のもと１０分間以上保持した。
(Sample measurement)
As shown in FIG. 3, a 10 mm × 5 mm × 3 mm neodymium permanent magnet B (manufactured by Neomag) was attached to the cell adapter A attached to a commercially available microcell for spectrophotometer (UVette, manufactured by Eppendorf). . The microcell C was put into this cell adapter, installed in an ultraviolet-visible spectrophotometer V-660DS (manufactured by JASCO Corporation) equipped with a cell temperature controller, and kept at 37 ° C. for 10 minutes or more.
上記の試料と磁性微粒子の混合溶液２００μＬをマイクロセル内に分注し、直ちに、波長４２０ｎｍ、バンド幅２．０ｎｍで６００秒間に亘って連続して吸光度を測定した。磁性微粒子、磁性微粒子−アプタマー複合体、磁性微粒子−アプタマー−トロンビン（２０ｎＭ、６０ｎＭ、１００ｎＭ）複合体の測定結果を図４に示す。 200 μL of the mixed solution of the above sample and magnetic fine particles was dispensed into a microcell, and the absorbance was immediately measured continuously for 600 seconds at a wavelength of 420 nm and a bandwidth of 2.0 nm. FIG. 4 shows the measurement results of the magnetic fine particles, magnetic fine particle-aptamer complex, and magnetic fine particle-aptamer-thrombin (20 nM, 60 nM, 100 nM) complex.
図４に示されるように、測定開始後約２００秒後までは吸光度が上昇し、その後吸光度の低下が見られた。これは磁性微粒子の刺激応答性ポリマーが凝集することで濁度が上昇し、その後磁性微粒子が磁石に吸着されて分離されるからであると推測される。 As shown in FIG. 4, the absorbance increased until about 200 seconds after the start of measurement, and thereafter the absorbance decreased. This is presumably because the turbidity increases due to aggregation of the stimulus-responsive polymer of the magnetic fine particles, and then the magnetic fine particles are adsorbed and separated by the magnet.
また、測定開始後６００秒後の吸光度は、磁性微粒子のみに比べて磁性微粒子−アプタマー複合体のほうが高くなることがわかった。これは刺激応答性ポリマーがアプタマーによって凝集阻害を受けて分散し、磁石に吸着しにくくなったためであると推測される。 It was also found that the absorbance after 600 seconds after the start of measurement was higher for the magnetic fine particle-aptamer complex than for the magnetic fine particles alone. This is presumed to be because the stimulus-responsive polymer was dispersed due to aggregation inhibition by the aptamer and became difficult to adsorb to the magnet.
さらに、測定開始後６００秒後の吸光度は、トロンビン濃度が高いほど低くなることがわかった。これは磁性微粒子とアプタマーの複合体にトロンビンが結合することでアプタマーによる凝集阻害が緩和されたためであると推測される。 Furthermore, it was found that the absorbance after 600 seconds after the start of measurement decreases as the thrombin concentration increases. This is presumed to be because thrombin bound to the complex of magnetic fine particles and aptamer alleviated aggregation inhibition by the aptamer.
次に、各試料について、開始直後及び６００秒後の２点での測定値の差異を算出した。この結果を表１に示す。 Next, for each sample, the difference between the measured values at two points immediately after the start and after 600 seconds was calculated. The results are shown in Table 1.
表１に示されるように、開始直後及び６００秒後の２点での測定値の差は、トロンビンの量に依存するものであった。即ち、トロンビン濃度が高いほど、開始直後及び６００秒後の２点での測定値の差は大きかった。これにより、開始直後及び６００秒後の２点での測定値の差を測定することで、トロンビンを高感度に検出又は定量できることがわかった。 As shown in Table 1, the difference between the measured values at the two points immediately after the start and after 600 seconds was dependent on the amount of thrombin. That is, the higher the thrombin concentration, the greater the difference between the measured values at the two points immediately after the start and after 600 seconds. Thus, it was found that thrombin can be detected or quantified with high sensitivity by measuring the difference between the measured values at two points immediately after the start and after 600 seconds.
また、開始後６００秒以内という検出時間は、インキュベーション時間を含めても１５分以内で完了し、数時間を要していたＥＬＩＳＡなどの従来技術に比べて、格段に短いものである。また、アプタマーと検体の反応および温度応答性磁性微粒子の混合と濁度測定を行うだけで検出対象を検出又は定量できたことから手順が簡便である。 Further, the detection time of 600 seconds or less after the start is completed within 15 minutes including the incubation time, and is much shorter than conventional techniques such as ELISA that required several hours. In addition, the procedure is simple because the detection target can be detected or quantified simply by performing the reaction between the aptamer and the sample, mixing the temperature-responsive magnetic fine particles, and measuring the turbidity.
＜実施例２＞
（ＶＥＧＦ試料の調製）
ＶＥＧＦ（Ｒ＆Ｄ社製）を５０ｍＭ 酢酸、１００ｍＭ ＮａＣｌ、５ｍＭ ＫＣｌ（ｐＨ４．５）で０、１０、１００、４９５ｆＭとなるように希釈した。その際に、ヒト血清を最終濃度１０％となるように添加し、ＶＥＧＦ試料とした。
<Example 2>
(Preparation of VEGF sample)
VEGF (manufactured by R & D) was diluted with 50 mM acetic acid, 100 mM NaCl, 5 mM KCl (pH 4.5) to 0, 10, 100, 495 fM. At that time, human serum was added to a final concentration of 10% to prepare a VEGF sample.
（ＶＥＧＦ結合性ＤＮＡアプタマーの調製）
５’末端にビオチンを有するオリゴヌクレオチド（配列番号２）を５０ｍＭ 酢酸、１００ｍＭ ＮａＣｌ、５ｍＭ ＫＣｌ（ｐＨ４．５）で１μＭとなるように希釈し、９５℃で１０分加熱した後、３０分かけて２５℃まで緩やかに冷却した。これを５０ｍＭ 酢酸、１００ｍＭ ＮａＣｌ、５ｍＭ ＫＣｌ（ｐＨ４．５）で７５ｎＭになるように希釈し、ＶＥＧＦ結合性ＤＮＡアプタマーとした。
(Preparation of VEGF-binding DNA aptamer)
An oligonucleotide having biotin at the 5 ′ end (SEQ ID NO: 2) was diluted with 50 mM acetic acid, 100 mM NaCl, 5 mM KCl (pH 4.5) to 1 μM, heated at 95 ° C. for 10 minutes, and then over 30 minutes. Cooled slowly to 25 ° C. This was diluted with 50 mM acetic acid, 100 mM NaCl, 5 mM KCl (pH 4.5) to 75 nM to obtain a VEGF-binding DNA aptamer.
（配列番号２）
５’−ｔｇｔｇｇｇｇｇｔｇｇａｃｇｇｇｃｃｇｇｇｔａｇａ−３’
(SEQ ID NO: 2)
5′-tgtgggggggtggacggggccggggtag-3 ′
（試料の混合）
試料５０μＬとＶＥＧＦ結合性ＤＮＡアプタマー５０μＬを１．５ｍＬマイクロチューブに入れ、サーモミキサーを用いて２０℃、１，２００ｒｐｍ、１０分間振とうした。
(Sample mixing)
50 μL of the sample and 50 μL of the VEGF-binding DNA aptamer were put in a 1.5 mL microtube, and shaken at 20 ° C., 1,200 rpm for 10 minutes using a thermomixer.
（試料と磁性微粒子の混合）
上記混合試料に、５０ｍＭ 酢酸、１００ｍＭ ＮａＣｌ、５ｍＭ ＫＣｌ（ｐＨ４．５）で希釈したストレプトアビジン及び温度応答性ポリマーが結合された磁性微粒子であるＪＮＣ石油化学社製のＴｈｅｒｍａ−Ｍａｘ（商品名）ＬＳＡ Ｓｔｒｅｐｔａｖｉｄｎ（０．０２質量％）を５０μＬ添加し、サーモミキサーを用いて２０℃、１，２００ ｒｐｍ、１０秒間振とうした。
(Mixing of sample and magnetic fine particles)
Thermo-Max (trade name) LSA manufactured by JNC Petrochemical Co., Ltd., which is magnetic fine particles in which streptavidin diluted with 50 mM acetic acid, 100 mM NaCl, 5 mM KCl (pH 4.5) and a temperature-responsive polymer are bound to the above mixed sample. 50 μL of Streptavidn (0.02 mass%) was added, and shaken at 20 ° C., 1,200 rpm for 10 seconds using a thermomixer.
（試料の測定）
図３に示されるように、市販の分光光度計用マイクロセル（ＵＶｅｔｔｅ、ｅｐｐｅｎｄｏｒｆ社製）に付属のセルアダプターＡに内部に１０ｍｍ×５ｍｍ×３ｍｍのネオジム永久磁石Ｂ（ネオマグ社製）を取り付けた。このセルアダプターにマイクロセルＣを入れ、セル温度制御機が設けられた紫外可視分光光度計Ｖ−６６０ＤＳ（日本分光製）内に設置し、３７℃のもと１０分間以上保持した。
(Sample measurement)
As shown in FIG. 3, a 10 mm × 5 mm × 3 mm neodymium permanent magnet B (manufactured by Neomag) was attached to the cell adapter A attached to a commercially available microcell for spectrophotometer (UVette, manufactured by Eppendorf). . The microcell C was put into this cell adapter, installed in an ultraviolet-visible spectrophotometer V-660DS (manufactured by JASCO Corporation) equipped with a cell temperature controller, and kept at 37 ° C. for 10 minutes or more.
上記の試料と磁性微粒子の混合溶液１５０μＬをマイクロセル内に分注し、直ちに、波長４２０ｎｍ、バンド幅２．０ｎｍで６００秒間に亘って連続して吸光度を測定した。磁性微粒子−アプタマー複合体、磁性微粒子−アプタマー−ＶＥＧＦ複合体の測定結果を図５に示す。図５のグラフ横の数値は、ＶＥＧＦ濃度を示す。 150 μL of the mixed solution of the above sample and magnetic fine particles was dispensed into the microcell, and the absorbance was immediately measured continuously for 600 seconds at a wavelength of 420 nm and a bandwidth of 2.0 nm. The measurement results of the magnetic fine particle-aptamer complex and the magnetic fine particle-aptamer-VEGF complex are shown in FIG. The numerical value beside the graph in FIG. 5 indicates the VEGF concentration.
図５に示されるように、測定開始後３００秒後の吸光度は、ＶＥＧＦ濃度が高いほど低くなることがわかった。これは磁性微粒子とアプタマーの複合体にＶＥＧＦが結合することでアプタマーによる凝集阻害が緩和されたためであると推測される。 As shown in FIG. 5, it was found that the absorbance after 300 seconds from the start of the measurement decreases as the VEGF concentration increases. This is presumed to be because aggregation inhibition by the aptamer was alleviated by binding of VEGF to the complex of the magnetic fine particles and the aptamer.
次に、各試料について、開始直後及び６００秒後の２点での測定値の差異を算出した。この結果を表２に示す。 Next, for each sample, the difference between the measured values at two points immediately after the start and after 600 seconds was calculated. The results are shown in Table 2.
表２に示されるように、開始直後及び６００秒後の２点での測定値の差は、ＶＥＧＦの量に依存するものであった。即ち、ＶＥＧＦ濃度が高いほど、開始直後及び６００秒後の２点での測定値の差は大きかった。これにより、開始直後及び６００秒後の２点での測定値の差を測定することで、ＶＥＧＦを高感度に検出又は定量できることがわかった。 As shown in Table 2, the difference between the measured values at the two points immediately after the start and after 600 seconds was dependent on the amount of VEGF. That is, the higher the VEGF concentration, the greater the difference between the measured values at the two points immediately after the start and after 600 seconds. Thus, it was found that VEGF can be detected or quantified with high sensitivity by measuring the difference between the measured values at two points immediately after the start and after 600 seconds.
＜実施例３＞
実施例１と同様の手順で、ＳＡＲＳ Ｃｏｒｏｎａｖｉｒｕｓ Ｎｕｃｌｅｏｃａｐｓｉｄ Ｐｒｏｔｅｉｎ（ＲａｙＢｉｏｔｅｃｈ社製）の検出を行った。
<Example 3>
SARS Coronavirus Nucleocapsid Protein (manufactured by RayBiotech) was detected in the same procedure as in Example 1.
ＳＡＲＳ Ｃｏｒｏｎａｖｉｒｕｓ Ｎｕｃｌｅｏｃａｐｓｉｄ Ｐｒｏｔｅｉｎ結合アプタマーとして５’末端にビオチンを有するオリゴヌクレオチド（配列番号３）を用いた。 As a SARS Coronavirus Nucleocapsid Protein binding aptamer, an oligonucleotide having biotin at the 5 'end (SEQ ID NO: 3) was used.
（配列番号３）
５’− ｇｃａａｔｇｇｔａｃｇｇｔａｃｔｔｃｃｇｇａｔｇｃｇｇａａａｃｔｇｇｃｔａａｔｔｇｇｔｇａｇｇｃｔｇｇｇｇｃｇｇｔｃｇｔｇｃａｇｃａａａａｇｔｇｃａｃｇｃｔａｃｔｔｔｇｃｔａａ−３’
(SEQ ID NO: 3)
5'- gcaatggtacggtacttccggatgcgaaactggctaattggtgaggctggggcgtcgtgcagaaaagtgcacgctactttgcta-3 '
ＳＡＲＳ Ｃｏｒｏｎａｖｉｒｕｓ Ｎｕｃｌｅｏｃａｐｓｉｄ Ｐｒｏｔｅｉｎ試料濃度０ｎＭ，１０ｎＭ，２０ｎＭ，４０ｎＭ，６０ｎＭ，８０ｎＭ，１００ｎＭにおける、開始直後及び６００秒後の２点での測定値の差を表３に示す。 Table 3 shows the difference in measured values at two points immediately after the start and after 600 seconds at the SARS Coronavirus Nucleocapsid Protein sample concentrations of 0 nM, 10 nM, 20 nM, 40 nM, 60 nM, 80 nM, 100 nM.
表３に示されるように、開始直後及び６００秒後の２点での測定値の差は、ＳＡＲＳ Ｃｏｒｏｎａｖｉｒｕｓ Ｎｕｃｌｅｏｃａｐｓｉｄ Ｐｒｏｔｅｉｎの量に依存するものであった。即ち、ＳＡＲＳ Ｃｏｒｏｎａｖｉｒｕｓ Ｎｕｃｌｅｏｃａｐｓｉｄ Ｐｒｏｔｅｉｎ濃度が高いほど、開始直後及び６００秒後の２点での測定値の差は小さかった。これにより、開始直後及び６００秒後の２点での測定値の差を測定することで、ＳＡＲＳ Ｃｏｒｏｎａｖｉｒｕｓ Ｎｕｃｌｅｏｃａｐｓｉｄ Ｐｒｏｔｅｉｎを高感度に検出又は定量できることがわかった。 As shown in Table 3, the difference in the measured values at the two points immediately after the start and after 600 seconds was dependent on the amount of SARS Coronavirus Nucleocapsid Protein. That is, the higher the SARS Coronavirus Nucleocapsid Protein concentration, the smaller the difference between the measured values at two points immediately after the start and after 600 seconds. Thus, it was found that the SARS Coronavirus Nucleocapsid Protein can be detected or quantified with high sensitivity by measuring the difference between the measured values at two points immediately after the start and after 600 seconds.
＜実施例４＞
実施例１と同様の手順で、Ｉｎｆｌｕｅｎｚａ ｖｉｒｕｓ ｈｅｍａｇｇｌｕｔｉｎｉｎ（Ｈ５Ｎ１−ＨＡ，Ａ／Ａｎｈｕｉ／１／２００５，Ａｂｎｏｖａ社製）の検出を行った。
<Example 4>
Influenza virus hemagglutinin (H5N1-HA, A / Anhui / 1/2005, manufactured by Abnova) was detected in the same procedure as in Example 1.
Ｉｎｆｌｕｅｎｚａ ｖｉｒｕｓ ｈｅｍａｇｇｌｕｔｉｎｉｎ結合アプタマーとして５’末端にビオチンを有するオリゴヌクレオチド（配列番号４）を用いた。 As an Influenza virus hemagglutinin-binding aptamer, an oligonucleotide (SEQ ID NO: 4) having biotin at the 5 'end was used.
（配列番号４）
５’−ｇａａｔｔｃａｇｔｃｇｇａｃａｇｃｇｇｇｇｔｔｃｃｃａｔｇｃｇｇａｔｇｔｔａｔａａａｇｃａｇｔｃｇｃｔｔａｔａａｇｇｇａｔｇｇａｃｇａａｔａｔｃｇｔｃｔｃｃｃ−４’
(SEQ ID NO: 4)
5'-gaattcagtcggacagcgggggtcccatgcgatgtttaataagcagtcgcttataagggaggagatatatcgtccc-4 '
Ｉｎｆｌｕｅｎｚａ ｖｉｒｕｓ ｈｅｍａｇｇｌｕｔｉｎｉｎ試料濃度０ｎＭ，１００ｎＭにおける、開始直後及び６００秒後の２点での測定値の差を表４に示す。 Table 4 shows the difference between the measured values at two points immediately after the start and 600 seconds after the Influenza virus hemagglutinin sample concentrations of 0 nM and 100 nM.
表４に示されるように、測定開始６００秒後の測定値は試料濃度０ｎＭと１００ｎＭで明確な差が見られ、Ｉｎｆｌｕｅｎｚａ ｖｉｒｕｓ ｈｅｍａｇｇｌｕｔｉｎｉｎ存在下で磁性微粒子の凝集促進が見られた。これにより、Ｉｎｆｌｕｅｎｚａ ｖｉｒｕｓ ｈｅｍａｇｇｌｕｔｉｎｉｎを検出できることがわかった。 As shown in Table 4, the measured values 600 seconds after the start of measurement showed a clear difference between the sample concentrations of 0 nM and 100 nM, and the aggregation of magnetic fine particles was observed in the presence of Influenza virus hemagglutinin. Thus, it was found that Influenza virus hemagglutinin can be detected.
＜比較例１＞
実施例１と同様の手順で、アプタマーの代わりに抗体を使ってＶＥＧＦ（Ｈｕｍａｎ Ｒｅｃｏｍｂｉｎａｎｔ ＶＥＧＦ Ａ１６５：Ｈｕｍａｎ Ｚｙｍｅ社製）の検出を行った。
<Comparative Example 1>
In the same procedure as in Example 1, VEGF (Human Recombinant VEGF A165: manufactured by Human Zyme) was detected using an antibody instead of an aptamer.
ＶＥＧＦ結合抗体としてＡｎｔｉ−ＶＥＧＦ抗体（Ｂｉｏｔｉｎ）（ａｂｃａｍ社製）を用いた。Ａｎｔｉ−ＶＥＧＦ抗体（Ｂｉｏｔｉｎ）は、１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５、和光純薬工業製）、１００ｍＭ ＮａＣｌ、５ｍＭ ＫＣｌを含有する溶液で、１μＭとなるように希釈して使用した。 Anti-VEGF antibody (Biotin) (manufactured by abcam) was used as a VEGF binding antibody. Anti-VEGF antibody (Biotin) was used after diluting to 1 μM with a solution containing 10 mM Tris-HCl (pH 7.5, manufactured by Wako Pure Chemical Industries), 100 mM NaCl, 5 mM KCl.
ＶＥＧＦ試料濃度０ｎＭ，０．１ｎＭ，１ｎＭ，１０ｎＭ，１００ｎＭにおける、開始直後及び６００秒後の２点での測定値の差を表５に示す。 Table 5 shows the difference in measured values at two points immediately after the start and 600 seconds after the VEGF sample concentrations of 0 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM.
表５に示されるように、開始直後及び６００秒後の２点での測定値の差は、ＶＥＧＦの量に依存するものであった。即ち、ＶＥＧＦ濃度が高いほど、開始直後及び６００秒後の２点での測定値の差は大きかった。これにより、開始直後及び６００秒後の２点での測定値の差を測定することで、ＶＥＧＦを検出又は定量できることがわかった。 As shown in Table 5, the difference between the measured values at the two points immediately after the start and 600 seconds later was dependent on the amount of VEGF. That is, the higher the VEGF concentration, the greater the difference between the measured values at the two points immediately after the start and after 600 seconds. Thus, it was found that VEGF can be detected or quantified by measuring the difference between the measured values at two points immediately after the start and after 600 seconds.
一方、ＶＥＧＦ ０．１ｎＭの測定値の差はＶＥＧＦ ０ｎＭの測定値の差よりも小さくなった。このことから、この検出系における測定感度は０．１ｎＭ以上と考えられる。 On the other hand, the difference in the measured value of VEGF 0.1 nM was smaller than the difference in the measured value of VEGF 0 nM. From this, the measurement sensitivity in this detection system is considered to be 0.1 nM or more.
これにより、ＶＥＧＦ結合性ＤＮＡアプタマーを用いて１０ｆＭでも検出できた実施例２は、抗体を用いた比較例１に比べて格段に検出感度が高いことが確認された。 Thereby, it was confirmed that Example 2 which was able to detect even 10 fM using the VEGF binding DNA aptamer has remarkably higher detection sensitivity than Comparative Example 1 using the antibody.
以上の結果より、検出対象の濃度に応じて濁度が変化すること、濁度を測定することで検出対象の濃度を定量できることが示された。つまり、本発明に係る方法は、二次抗体、発光試薬、発光検出装置等の特殊な試薬、機器を必要とせず、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確認された。 From the above results, it was shown that the turbidity changes according to the concentration of the detection target, and that the concentration of the detection target can be quantified by measuring the turbidity. That is, the method according to the present invention is a novel method that can detect and quantify a detection target quickly, inexpensively, and easily without requiring special reagents and equipment such as secondary antibodies, luminescent reagents, and luminescence detection devices. Was confirmed.
本発明は前記実施形態に限定されるものではなく、本発明の目的を達成できる範囲での変形、改良等は本発明に含まれるものである。 The present invention is not limited to the above-described embodiment, and modifications, improvements, and the like within the scope that can achieve the object of the present invention are included in the present invention.
１ 刺激応答性磁性微粒子
２ 磁性物質
３ 刺激応答性ポリマー
４ ストレプトアビジン
５ ビオチン
６ アプタマー
７ 検出対象
Ａ セルアダプター
Ｂ ネオジム永久磁石
Ｃ マイクロセル
DESCRIPTION OF SYMBOLS 1 Stimulus responsive magnetic microparticle 2 Magnetic substance 3 Stimulus responsive polymer 4 Streptavidin 5 Biotin 6 Aptamer 7 Detection object A Cell adapter B Neodymium permanent magnet C Microcell
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| CN106290831A (en) * | 2016-08-02 | 2017-01-04 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of competition law test strip based on antigen aptamers and application thereof |
| CN111693712A (en) * | 2020-04-02 | 2020-09-22 | 苏州大学 | Method for detecting new coronavirus SARS-CoV-2N protein by adopting aptamer |
| WO2022072737A1 (en) * | 2020-09-30 | 2022-04-07 | Roswell Biotechnologies, Inc. | System, method and apparatus for personal virometer |
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