JP2020112486A - Detection method of composite body, and carrier and detection kit to be used in the method - Google Patents
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Abstract
Description
本発明は、複合体の検出方法、並びにそれに用いる担体及び検出キットに関する。本発明によれば、例えば酵素免疫法による抗原検出のシグナルの強度を向上させることができる。 The present invention relates to a method for detecting a complex, a carrier used for the method, and a detection kit. According to the present invention, it is possible to improve the signal intensity of antigen detection by enzyme immunoassay, for example.
疾患の診断方法として、疾患によって患者の検体中に増加又は出現するバイオマーカーを検出する方法がある。しかし、バイオマーカーは、患者の検体中に微量しか存在しないことも多く、低濃度のバイオマーカーを短時間で検出する方法の開発が求められている。
例えば、
As a method of diagnosing a disease, there is a method of detecting a biomarker that increases or appears in a sample of a patient due to the disease. However, biomarkers are often present only in trace amounts in patient samples, and development of a method for detecting low-concentration biomarkers in a short time is required.
For example,
バイオマーカーの検出法として、酵素免疫測定法（Enzyme-Linked ImmunoSorbent Assay：ＥＬＩＳＡ法）が汎用されている。ＥＬＩＳＡ法では、ウェルプレート内での抗原抗体反応によりバイオマーカーを捕捉し、続く酵素反応による発色で検出される。しかしながら、ＥＬＩＳＡ法は、検査に時間を要するといった問題がある。また、ウェルプレートに代えて、マイクロ流路を用いる抗原抗体反応があるが、再現性及び感度は十分ではなかった（非特許文献１）。
従って、本発明の目的は、短時間でバイオマーカーを検出できる方法を提供することである。
As a biomarker detection method, an enzyme immunoassay method (Enzyme-Linked ImmunoSorbent Assay: ELISA method) is widely used. In the ELISA method, a biomarker is captured by an antigen-antibody reaction in a well plate, and then the color is detected by a subsequent enzymatic reaction. However, the ELISA method has a problem that it takes time for inspection. Further, there is an antigen-antibody reaction that uses a microchannel instead of the well plate, but the reproducibility and sensitivity were not sufficient (Non-Patent Document 1).
Therefore, an object of the present invention is to provide a method capable of detecting a biomarker in a short time.
本発明者は、短時間でバイオマーカーを検出できる方法について、鋭意研究した結果、驚くべきことに、バイオマーカーを捕捉する捕捉成分が固相化された平均細孔直径１０μｍ以下の細孔を有する担体を用い、バイオマーカーを含む検体を前記細孔中に流入させることにより、短時間でバイオマーカー検出できることを見出した。
本発明は、こうした知見に基づくものである。
従って、本発明は、
［１］複合体の検出方法であって、
（Ａ）（１）捕捉成分が親水性のポリマーを介して固相化された平均細孔直径１０μｍ以下の細孔を有する担体において、前記捕捉成分に結合する被検出成分を含む可能性のある検体を、前記細孔に流入させる工程、（２）前記被検出成分に結合する検出成分を含む溶液を前記細孔に流入させ、捕捉成分／被検出成分／検出成分の複合体を形成させる工程、及び（３）前記複合体を検出する工程、を含むか、又は（Ｂ）（１）捕捉成分が親水性のポリマーを介して固相化された平均細孔直径１０μｍ以下の細孔を有する担体において、前記捕捉物質に結合する被検出成分を含む可能性のある検体、及び前記捕捉成分に結合する前記被検出成分と競合する競合検出成分を、前記細孔に流入させ、捕捉成分／競合検出成分の複合体を形成させる工程、及び（２）前記複合体を検出する工程、を含む、複合体の検出方法、
［２］前記細孔の細孔分布が、下記式（１）：（Ｄ１０−Ｄ９０）／Ｄ５０≦１ （１）（式中、Ｄ１０は累積体積百分率が１０％の細孔径であり、Ｄ９０は累積体積百分率が９０％の細孔径であり、Ｄ５０は累積体積百分率が５０％の細孔径である）を満たす、［１］に記載の検出方法、
［３］前記担体が、トラックエッチング膜である、［２］に記載の検出方法、
［４］前記捕捉成分が抗体又は抗原である、［１］〜［３］のいずれかに記載の検出方法、
［５］被検出成分を捕捉するための捕捉成分が親水性のポリマーを介して固相化された平均細孔直径１０μｍ以下の細孔を有する担体であって、前記細孔の細孔分布が、下記式（１）：（Ｄ１０−Ｄ９０）／Ｄ５０≦１ （１）（式中、Ｄ１０は累積体積百分率が１０％の細孔径であり、Ｄ９０は累積体積百分率が９０％の細孔径であり、Ｄ５０は累積体積百分率が５０％の細孔径である）を満たす担体、
［６］前記担体が、トラックエッチング膜である、［５］に記載の担体、
［７］前記捕捉成分が抗体又は抗原である、［５］又は［６］に記載の担体、又は
［８］（Ａ）［５］〜［７］のいずれかに記載の担体、及び被検出成分に結合する検出成分を含む溶液；又は（Ｂ）［５］〜［７］のいずれかに記載の担体、及び捕捉成分と結合する競合検出成分を含む溶液；を含む複合体の検出キット、
に関する。
なお、特許文献１には、「イ）細孔を有する多孔性基材であって外部環境変化により実質的に変化しない多孔性基材；ロ）前記細孔中に配置されるポリマーであって外部環境変化に応答するポリマー；及びハ）前記ポリマーと結合するように配置される捕捉物質であって被捕捉物質を特異的に捕捉する捕捉物質；を有する被捕捉物質認識装置」が記載されている。しかしながら、短時間でバイオマーカーを検出する方法については、開示も示唆もされていなかった。
As a result of diligent research on a method capable of detecting a biomarker in a short time, the present inventor has surprisingly found that a capture component for capturing a biomarker has pores with an average pore diameter of 10 μm or less immobilized on a solid phase. It was found that the biomarker can be detected in a short time by using a carrier and allowing a sample containing the biomarker to flow into the pores.
The present invention is based on these findings.
Therefore, the present invention provides
[1] A method for detecting a complex, comprising:
(A) (1) A carrier having a fine pore having an average fine pore diameter of 10 μm or less in which the capturing component is immobilized via a hydrophilic polymer may contain a component to be detected that binds to the capturing component. Flowing a sample into the pores, (2) Flowing a solution containing a detection component that binds to the detection target component into the pores, and forming a complex of capture component/detection component/detection component And (3) the step of detecting the complex, or (B) (1) the capture component has pores with an average pore diameter of 10 μm or less immobilized on a hydrophilic polymer. In the carrier, a sample that may contain a target component that binds to the capture substance and a competitive detection component that competes with the target component that binds to the capture component are allowed to flow into the pores to capture/compete A method for detecting a complex, comprising: forming a complex of a detection component; and (2) detecting the complex.
[2] The pore distribution of the pores is represented by the following formula (1): (D10-D90)/D50≦1 (1) (wherein, D10 is a pore diameter having a cumulative volume percentage of 10%, and D90 is The cumulative volume percentage has a pore size of 90%, and D50 has a cumulative volume percentage of 50%.) The detection method according to [1],
[3] The detection method according to [2], wherein the carrier is a track etching film.
[4] The detection method according to any one of [1] to [3], wherein the capture component is an antibody or an antigen,
[5] A carrier having a capture component for capturing a component to be detected, which has been immobilized through a hydrophilic polymer and has pores with an average pore diameter of 10 μm or less, and the pore distribution of the pores is The following formula (1): (D10-D90)/D50≦1 (1) (wherein, D10 is a pore diameter having a cumulative volume percentage of 10%, and D90 is a pore diameter having a cumulative volume percentage of 90%. , D50 is a pore size having a cumulative volume percentage of 50%),
[6] The carrier according to [5], wherein the carrier is a track etching film.
[7] The carrier according to [5] or [6], wherein the capture component is an antibody or an antigen, or the carrier according to any one of [8](A) [5] to [7], and the detection target. A kit containing a detection component that binds to a component; or (B) a carrier according to any one of [5] to [7], and a solution containing a competitive detection component that binds to a capture component;
Regarding
In addition, in Patent Document 1, "a) a porous base material having pores, which does not substantially change due to a change in external environment; b) a polymer arranged in the pores, C. a capture substance recognition device having a polymer that responds to changes in the external environment; and c) a capture substance that is arranged so as to bind to the polymer and that specifically captures the capture substance. There is. However, the method for detecting a biomarker in a short time has not been disclosed or suggested.
本発明の複合体の検出方法によれば、短時間で疾患を診断するバイオマーカーを検出することができる。また、本発明の複合体の検出方法は、生体中の疾患に関連する抗体を短時間で検出することができる。本発明の複合体の検出方法は、免疫測定法であるサンドイッチ法又は競合法に用いることができる。
本発明の担体及び前記担体を含む複合体の検出キットは、前記複合体の検出方法に用いることができる。
更に、本発明の複合体の検出方法においては、捕捉物質を固相化するポリマーが親水性であるために、捕捉物質の固相化の後に親水性物質によるブロッキングを行わなくても、十分な感度を得ることができる。また、本発明の複合体の検出方法においては、検出工程の発色又は発光を酵素反応で行う場合、ＥＬＩＳＡ法と異なり、発色又は発光停止液を添加しなくてもよく、工程が簡略化できる。
According to the method for detecting a complex of the present invention, a biomarker for diagnosing a disease can be detected in a short time. Moreover, the method for detecting a complex of the present invention can detect an antibody associated with a disease in a living body in a short time. The method for detecting the complex of the present invention can be used in a sandwich method or a competitive method which are immunoassay methods.
The carrier of the present invention and the complex detection kit including the carrier can be used in the method for detecting the complex.
Further, in the method for detecting a complex of the present invention, since the polymer for immobilizing the capture substance is hydrophilic, it is sufficient to perform no blocking with a hydrophilic substance after immobilizing the capture substance. Sensitivity can be obtained. Further, in the method for detecting a complex of the present invention, when color development or luminescence in the detection step is performed by an enzymatic reaction, unlike the ELISA method, it is not necessary to add a color development or luminescence termination solution, and the process can be simplified.
［１］複合体の検出方法
本発明の複合体の検出方法は、（Ａ）（１）捕捉成分が親水性のポリマーを介して固相化された平均細孔直径１０μｍ以下の細孔を有する担体において、前記捕捉成分に結合する被検出成分を含む可能性のある検体を、前記細孔に流入させる工程、（２）前記被検出成分に結合する検出成分を含む溶液を前記細孔に流入させ、捕捉成分／被検出成分／検出成分の複合体を形成させる工程、及び（３）前記複合体を検出する工程を含み得る（以下、サンドイッチ法と称することがある）。
また、本発明の複合体の検出方法は、（Ｂ）（１）捕捉成分が親水性のポリマーを介して固相化された平均細孔直径１０μｍ以下の細孔を有する担体において、前記捕捉物質に結合する被検出成分を含む可能性のある検体、及び前記捕捉成分に結合する前記被検出成分と競合する競合検出成分を、前記細孔に流入させ、捕捉成分／競合検出成分の複合体を形成させる工程、及び（２）前記複合体を検出する工程、を含み得る（以下、競合法と称することがある）。
[1] Method for detecting complex The method for detecting a complex according to the present invention comprises (A)(1) a capture component having pores with an average pore diameter of 10 μm or less immobilized on a hydrophilic polymer. In a carrier, a step of causing a sample that may contain a detected component that binds to the capture component to flow into the pores, (2) a solution that includes a detected component that binds to the detected components flows into the pores And a step of forming a complex of capture component/detected component/detected component, and (3) detecting the complex (hereinafter, may be referred to as a sandwich method).
In addition, the method for detecting a complex of the present invention is (B) (1) in which the capture component is immobilized on a carrier having a mean pore diameter of 10 μm or less through a hydrophilic polymer, An analyte that may contain a detected component that binds to the above, and a competitive detection component that competes with the detected component that binds to the capture component are allowed to flow into the pores to form a capture component/competitive detection component complex. It may include a step of forming and (2) a step of detecting the complex (hereinafter, sometimes referred to as a competitive method).
《担体》
本発明の検出方法に用いる担体は、平均細孔直径１０μｍ以下の細孔を有する限りにおいて特に限定されるものではないが、例えば多数の細孔を有する多孔質体、又はバイオリアクターが挙げられる。
担体がバイオリアクターの場合、１本のバイオリアクターを担体として用いてもよく、複数のバイオリアクターを組み合わせて担体として用いてもよい。
担体が多孔質体の場合、本発明の検出方法に用いるために、多孔質体は多孔質膜が好ましい。多孔質体の細孔は、本発明の検出方法において、被検出物質を含む検体、検出成分、及び競合検出成分などを通過させるために、基本的には連通孔である。多孔質膜の場合の形状は特に限定されず、方形及び円形等が挙げられるが、検体等を細孔に均等に通過させるため、好ましくは円形である。多孔質膜が円形の場合、本発明の効果が得られる限りにおいて、多孔質膜の直径は特に限定されるものではないが、例えば０．３〜５ｃｍの直径の多孔質膜を用いることができ、より好ましくは０．５〜２ｃｍの直径の多孔質膜を用いることができる。
《Carrier》
The carrier used in the detection method of the present invention is not particularly limited as long as it has pores having an average pore diameter of 10 μm or less, and examples thereof include a porous body having a large number of pores, or a bioreactor.
When the carrier is a bioreactor, one bioreactor may be used as the carrier, or a plurality of bioreactors may be combined and used as the carrier.
When the carrier is a porous body, the porous body is preferably a porous membrane for use in the detection method of the present invention. In the detection method of the present invention, the pores of the porous body are basically communication holes in order to allow the sample containing the substance to be detected, the detection component, and the competitive detection component to pass through. The shape of the porous membrane is not particularly limited, and examples thereof include a square shape and a circular shape, but a circular shape is preferable in order to allow the sample or the like to uniformly pass through the pores. When the porous membrane has a circular shape, the diameter of the porous membrane is not particularly limited as long as the effect of the present invention can be obtained, but for example, a porous membrane having a diameter of 0.3 to 5 cm can be used. , And more preferably, a porous membrane having a diameter of 0.5 to 2 cm can be used.
担体の平均細孔直径は、本発明の効果が得られる限りにおいて、特に限定されるものではないが、平均細孔直径の上限は好ましくは８μｍ以下であり、より好ましくは６μｍ以下であり、更に好ましくは４μｍ以下であり、最も好ましくは２μｍ以下である。平均細孔直径の下限は、好ましくは１０ｎｍ以上であり、より好ましくは３０ｎｍ以上であり、更に好ましくは５０ｎｍ以上であり、最も好ましくは１００ｎｍ以上である。 The average pore diameter of the carrier is not particularly limited as long as the effects of the present invention can be obtained, but the upper limit of the average pore diameter is preferably 8 μm or less, more preferably 6 μm or less, and It is preferably 4 μm or less, and most preferably 2 μm or less. The lower limit of the average pore diameter is preferably 10 nm or more, more preferably 30 nm or more, further preferably 50 nm or more, and most preferably 100 nm or more.
前記担体は、好ましくはシャープな細孔分布を有する。細孔分布がブロードである担体と比較すると、シャープな細孔分布を有する担体は、短時間で被検出成分を検出することができる。
従って、前記細孔の細孔分布は、好ましくは下記式（１）：
（Ｄ１０−Ｄ９０）／Ｄ５０≦１ （１）
（式中、Ｄ１０は累積体積百分率が１０％の細孔径であり、Ｄ９０は累積体積百分率が９０％の細孔径であり、Ｄ５０は累積体積百分率が５０％の細孔径である）
を満たす。
前記（Ｄ１０−Ｄ９０）／Ｄ５０の値は、１以下であるが、好ましくは０．８以下であり、より好ましくは０．６以下であり、更に好ましくは０．４以下であり、更に好ましくは０．２以下である。（Ｄ１０−Ｄ９０）／Ｄ５０が０．２以下である場合、細孔直径はほぼ均一である。
The carrier preferably has a sharp pore distribution. As compared with a carrier having a broad pore distribution, a carrier having a sharp pore distribution can detect a component to be detected in a short time.
Therefore, the pore distribution of the pores is preferably represented by the following formula (1):
(D10-D90)/D50≦1 (1)
(In the formula, D10 is a pore size with a cumulative volume percentage of 10%, D90 is a pore size with a cumulative volume percentage of 90%, and D50 is a pore size with a cumulative volume percentage of 50%).
Meet
The value of (D10-D90)/D50 is 1 or less, preferably 0.8 or less, more preferably 0.6 or less, further preferably 0.4 or less, and further preferably It is 0.2 or less. When (D10-D90)/D50 is 0.2 or less, the pore diameters are almost uniform.
前記累積体積百分率は、例えば水銀圧入法、又はガス吸着法によって測定することができる。
例えば、水銀圧入法による細孔容積の測定は、例えば水銀ポロシメーターを用い、以下のように実施することができる。
試料を試料容器に入れ、２．６７Ｐａ以下の圧力で３０分間脱気する。次いで、水銀を試料容器内に導入し、徐々に加圧して水銀を試料の細孔へ圧入する（最高圧力＝４１４ＭＰａ）。このときの圧力と水銀の圧入量との関係から以下の各計算式を用いて試料の細孔容積分布を測定する。例えば、細孔直径２２μｍに相当する圧力（０．０６ＭＰａ）から最高圧力（４１４ＭＰａ：細孔直径３ｎｍ相当）までに試料に圧入された水銀の体積を測定する。細孔直径の算出は、直径（Ｄ）の円筒形の細孔に水銀を圧力（Ｐ）で圧入する場合、水銀の表面張力を「γ」とし、水銀と細孔壁との接触角を「θ」とすると、表面張力と細孔断面に働く圧力の釣り合いから、次式：
−πＤγｃｏｓθ＝π（Ｄ／２）２・Ｐ
が成り立つ。従って
Ｄ＝（−４γｃｏｓθ）／Ｐ
となる。
The cumulative volume percentage can be measured, for example, by a mercury injection method or a gas adsorption method.
For example, the pore volume can be measured by the mercury porosimetry using a mercury porosimeter, for example, as follows.
The sample is placed in a sample container and degassed at a pressure of 2.67 Pa or less for 30 minutes. Then, mercury is introduced into the sample container and gradually pressurized to press the mercury into the pores of the sample (maximum pressure=414 MPa). From the relationship between the pressure at this time and the amount of mercury injected, the pore volume distribution of the sample is measured using the following formulas. For example, the volume of mercury injected into the sample from the pressure (0.06 MPa) corresponding to the pore diameter of 22 μm to the maximum pressure (414 MPa: pore diameter of 3 nm) is measured. The pore diameter is calculated by setting the surface tension of mercury to "γ" and inserting the contact angle between the mercury and the pore wall into "when the mercury is pressed into the cylindrical pore having the diameter (D) at a pressure (P)." θ”, the following equation can be calculated from the balance between the surface tension and the pressure acting on the pore cross section:
−πDγ cos θ=π(D/2)2·P
Holds. Therefore, D=(-4γ cos θ)/P
Becomes
ガス吸着法（ＢＥＴ法）による細孔容積の測定は、比表面積連続流通式の比表面積測定器を用いて、試料のガス吸着量を測定し、ＢＥＴの式により比表面積を計算することができる。具体的には、試料を試料管に充填し、その試料管に窒素３０ｖｏｌ％を含有するヘリウムガスを流しながら以下の操作を行い、試料への窒素吸着量を求める。すなわち、試料管を−１９６℃に冷却し、試料に窒素を吸着させる。次に、試料管を室温に戻す。このとき試料から脱離してくる窒素量を熱伝導度型検出器で測定し、吸着ガス量（ｖ）とする。ＢＥＴの式から誘導された近似式：ｖｍ＝１／（ｖ・（１−ｘ））
を用いて液体窒素温度における、窒素吸着による１点法（相対圧力ｘ＝０．３）によりｖｍを求め、次式：比表面積＝４．３５×ｖｍ（ｍ２／ｇ）
により試料の比表面積を計算する。前記の各計算式で、ｖｍは試料表面に単分子層を形成するのに必要な吸着量（ｃｍ３／ｇ）であり、ｖは実測される吸着量（ｃｍ３／ｇ）であり、ｘは相対圧力である。
In the measurement of the pore volume by the gas adsorption method (BET method), the gas adsorption amount of the sample can be measured using a specific surface area continuous flow type specific surface area measuring device, and the specific surface area can be calculated by the BET equation. .. Specifically, the sample is filled in a sample tube, and the following operation is performed while flowing a helium gas containing 30 vol% of nitrogen into the sample tube to determine the nitrogen adsorption amount to the sample. That is, the sample tube is cooled to −196° C. and nitrogen is adsorbed on the sample. Then, the sample tube is returned to room temperature. At this time, the amount of nitrogen desorbed from the sample is measured by a thermal conductivity type detector, and is set as the adsorbed gas amount (v). Approximate expression derived from equation BET: v m = 1 / ( v · (1-x))
1-point method in the liquid nitrogen temperature, by nitrogen adsorption using the search of v m by (relative pressure x = 0.3), the following equation: specific surface area = 4.35 × v m (m 2 / g)
Calculate the specific surface area of the sample by. In each formula of the, v m is an adsorption amount necessary to form a monomolecular layer on the surface of the sample (cm 3 / g), v is an adsorption amount is measured (cm 3 / g), x is the relative pressure.
なお、後述のトラックエッチング膜などの細孔がほぼ均一な担体の場合、水銀圧入法又はガス吸着法などの細孔容積の測定法以外に、電子顕微鏡写真の観察像から直接細孔直径を測定することができる。 In addition, in the case of a carrier with almost uniform pores such as the track etching film described below, the pore diameter is directly measured from the observation image of the electron microscope photograph in addition to the method of measuring the pore volume such as the mercury intrusion method or the gas adsorption method. can do.
（トラックエッチング膜）
細孔分布がシャープな多孔質膜として、高分子膜をエッチング処理することにより、均一なシリンダー様の細孔（連通孔）が形成されたトラックエッチング膜を挙げることができる（図１）。トラックエッチング膜は、例えば「ｉｐ ＰＯＲＥ」（登録商標）としてエア・ブラウン社から市販されている。
(Track etching film)
An example of the porous film having a sharp pore distribution is a track etching film in which uniform cylinder-like pores (communication holes) are formed by etching a polymer film (FIG. 1). The track etching film is commercially available from Air Brown, for example, as "ip PORE" (registered trademark).
前記担体の材質は、特に限定されるものではないが、例えばポリカーボネート、ポリエステル（例えば、ポリエチレンテレフタレート）、ポリスチレン、ポリイミド、ポリ塩化ビニル、ポリアセタール、ポリアクリロニトリル、スチレン／ジビニルベンゼン共重合体、ポリブチレンテレフタレート、ポリアリレート、ポリスルホン、ポリアミドイミド、エポキシ樹脂、ウレタン樹脂、ユリア樹脂、ジアリルフタレート樹脂、シリコン樹脂、ポリアセタール樹脂、ナイロン樹脂、フェノール樹脂、アミノ樹脂、多孔性ガラス、又は多孔性セラミックスなどを挙げることができる。 The material of the carrier is not particularly limited, but for example, polycarbonate, polyester (for example, polyethylene terephthalate), polystyrene, polyimide, polyvinyl chloride, polyacetal, polyacrylonitrile, styrene/divinylbenzene copolymer, polybutylene terephthalate. , Polyarylate, polysulfone, polyamideimide, epoxy resin, urethane resin, urea resin, diallyl phthalate resin, silicone resin, polyacetal resin, nylon resin, phenol resin, amino resin, porous glass, or porous ceramics. it can.
（空孔率）
前記担体の空孔率は、本発明の効果が得られる限りにおいて、特に限定されるものではないが、好ましくは５〜７０％であり、より好ましくは１０〜６０％である。前記担体がトラックエッチング膜の場合、空孔率の下限は、好ましくは５％であり、より好ましくは７％であり、更に好ましくは１０％であり、更に好ましくは１５％である。また、前記担体がトラックエッチング膜の場合、空孔率の上限は、好ましくは４０％であり、より好ましくは３５％であり、更に好ましくは３０％であり、更に好ましくは２５％である。前記空孔率のそれぞれの下限及び上限を組み合わせて、好ましい範囲とすることができる。
(Porosity)
The porosity of the carrier is not particularly limited as long as the effect of the present invention can be obtained, but is preferably 5 to 70%, more preferably 10 to 60%. When the carrier is a track etching film, the lower limit of the porosity is preferably 5%, more preferably 7%, further preferably 10%, further preferably 15%. Further, when the carrier is a track etching film, the upper limit of the porosity is preferably 40%, more preferably 35%, further preferably 30%, further preferably 25%. The respective lower and upper limits of the porosity can be combined to obtain a preferable range.
《親水性ポリマー》
前記捕捉成分は、親水性ポリマーを介して担体に固相化されている。親水性ポリマーとは、限定されるものではないが、親水基を分子内に有するポリマーを挙げることができる。親水性ポリマーに含まれる親水基としては、カルボキシル基、水酸基、スルホ基、又はアミノ基が挙げられる。具体的な親水性ポリマーとしては、ポリアクリル酸、ポリメタクリル酸、２−アクリルアミド−２−メチルプロパンスルホン酸、メタクリル酸２−（ジメチルアミノ）エチル、又はポリビニルアルコールが挙げられる。
<Hydrophilic polymer>
The capture component is immobilized on a carrier via a hydrophilic polymer. The hydrophilic polymer includes, but is not limited to, a polymer having a hydrophilic group in the molecule. Examples of the hydrophilic group contained in the hydrophilic polymer include a carboxyl group, a hydroxyl group, a sulfo group, or an amino group. Specific examples of hydrophilic polymers include polyacrylic acid, polymethacrylic acid, 2-acrylamido-2-methylpropanesulfonic acid, 2-(dimethylamino)ethyl methacrylate, and polyvinyl alcohol.
《捕捉成分》
前記捕捉成分は、被検出成分に結合できる物質である限りにおいて、特に限定されるものではなく、例えば抗体、抗原、又はレクチンを挙げることができるが、好ましくは抗体又は抗原である。例えば、抗体の重鎖及び軽鎖の超可変領域（ＣＤＲ）が、抗原のエピトープと特異的に結合する。また、抗原のエピトープとしては、特定のアミノ酸配列が挙げられるが、抗体のＣＤＲは、アミノ酸配列以外に、アミノ酸のメチル化、リン酸化、又はアセチル化を認識することもでき、アミノ酸に結合した糖鎖を認識することもできる。
また、レクチンを捕捉成分として用いた場合、抗原の特定の糖鎖を特異的に認識して、抗原を捕捉することができる。
<Captured component>
The capture component is not particularly limited as long as it is a substance that can bind to the component to be detected, and examples thereof include an antibody, an antigen, or a lectin, but an antibody or an antigen is preferable. For example, the heavy and light chain hypervariable regions (CDRs) of an antibody specifically bind to an epitope of an antigen. Specific examples of the epitope of the antigen include a specific amino acid sequence, but the CDR of the antibody can recognize amino acid methylation, phosphorylation, or acetylation in addition to the amino acid sequence, and the sugar bound to the amino acid. It can also recognize chains.
Moreover, when a lectin is used as a capturing component, it is possible to specifically recognize a specific sugar chain of the antigen and capture the antigen.
前記捕捉成分は、前記親水性ポリマーの官能基を介して親水性ポリマーと結合させることができる。官能基は、導入する捕捉成分によって、適宜選択することができるが、例えば捕捉成分のアミノ基（−ＮＨ２基）を用いて親水性ポリマーと結合させる場合、Ｎ−アクリルオキシスクシンイミド、又は炭酸ビス(ペンタフルオロフェニル)などの活性エステルを用いることができる。また、ジメチルスベリミデートまたはジメチルピメリミデート、又はジメチルアジピミデートなどのイミドエステル架橋剤を用いて、親水性ポリマーと結合することもできる。
具体的に、捕捉成分として抗体のアミノ基を用いて、担体のポリマーに結合させる場合、図２に示すように行うことができる。
担体にアクリル酸を、担体の細孔中でプラズマグラフト重合法により細孔表面に固定化する。固定化されたアクリル酸を足場にＮ−ヒドロキシスクシンイミド（以下、「ＮＨＳ」と称することがある）及び水溶性カルボジイミド（以下、ＥＤＣと称することがある）を用いて、溶媒下、例えばジメチルホルムアミド（以下、「ＤＭＦ」と称することがある）下で反応させ、アクリル酸をＮ−アクリルオキシスクシンイミド（以下、「ＮＡＳ」と称することがある）化する。さらに、リン酸緩衝液中でＮＡＳの活性エステル基と抗体のアミノ基とを反応させ、抗体固定グラフトポリマーを得ることができる。最後に、残ったＮＡＳの活性エステル基を不活化するために、イソプロピルアミンと反応させ、Ｎ−イソプロピルアクリルアミド化し、ＮＡＳと抗体との反応を完全に停止させる。
なお、抗原又はレクチンのアミノ基（−ＮＨ２基）を用いても、同様に親水性ポリマーと結合させることができる。
The capture component can be bound to the hydrophilic polymer via the functional group of the hydrophilic polymer. The functional group can be appropriately selected depending on the trapping component to be introduced. For example, when the amino group (—NH 2 group) of the trapping component is used to bind to the hydrophilic polymer, N-acryloxysuccinimide or biscarbonate Active esters such as (pentafluorophenyl) can be used. An imide ester crosslinker such as dimethylsuberimidate or dimethylpimelimidate, or dimethyladipimidate can also be used to bind hydrophilic polymers.
Specifically, when the amino group of the antibody is used as the capturing component to bind to the polymer of the carrier, it can be performed as shown in FIG.
Acrylic acid is immobilized on the surface of the carrier in the pores of the carrier by the plasma graft polymerization method. Using immobilized acrylic acid as a scaffold, N-hydroxysuccinimide (hereinafter sometimes referred to as “NHS”) and water-soluble carbodiimide (hereinafter sometimes referred to as EDC) are used in a solvent such as dimethylformamide ( Hereinafter, the acrylic acid is converted into N-acryloxysuccinimide (hereinafter, may be referred to as "NAS") under the reaction under "DMF". Furthermore, the active ester group of NAS and the amino group of the antibody are reacted in a phosphate buffer to obtain an antibody-immobilized graft polymer. Finally, in order to inactivate the active ester group of the remaining NAS, it is reacted with isopropylamine and converted into N-isopropylacrylamide to completely stop the reaction between the NAS and the antibody.
It should be noted that the amino group (—NH 2 group) of the antigen or lectin can be similarly used to bind to the hydrophilic polymer.
《被検出成分》
本発明の検出方法において、検出される被検出成分は、特に限定されるものではないが、抗原又は抗体が挙げられる。抗原としては、疾患によって体内で上昇する生体内タンパク質（例えば、酵素、受容体、リガンドなどの疾患のバイオマーカー）、又は病原体のタンパク質（例えば、ウイルスのタンパク質、細菌のタンパク質、真菌のタンパク質、寄生虫のタンパク質）が挙げられる。抗体としては、体内で外来抗原（例えば病原体）に対して産生される抗体、自己抗体、又はアレルギー抗体（ＩｇＥ抗体）などが挙げられる。
被検出成分は、基本的には疾患の発症時に生体内で増加又は減少するバイオマーカーであり、被検出成分に特異的に結合する捕捉成分によって、担体上に捕捉される。
疾患のバイオマーカーとしては、がんマーカー、感染症マーカー、又は生活習慣病マーカーなどが挙げられる。具体的ながんマーカーとしては、大腸癌、前立腺癌、肺癌、乳癌、胃癌、卵巣癌、腎癌、胸腺腫、小腸癌、膵臓癌、膀胱癌、骨癌、血液癌、脳癌、乳房、子宮頸部癌、結腸癌、子宮内膜癌、食道癌、肝癌、又は咽頭癌のマーカーが挙げられる。 具体的な感染症マーカーとしては、細菌感染症マーカー（肺炎球菌、ブドウ球菌、バチルス、レンサ球菌、髄膜炎菌、淋菌、大腸菌類、クレブシエラ、プロテウス、シュードモナス、サルモネラ、赤痢菌、ヘモフィルス、エルシニア、リステリア、コリネバクテリウム、ビブリオ、クロストリジウム、クラミジア、マイコバクテリウム、ヘリコバクター、およびトレポネーマ）、真菌感染症マーカー（カンジダ、アスペルギルス、クリプトコッカス・ネオフォルマンス、表在性および深在性真菌症を引き起こす真菌）、ウイルス感染症マーカー（ＨＩＶ、肝炎ウイルス、ヘルペスウイルス、ラブドウイルス、風疹ウイルス、痘瘡ウイルス、ポックスウイルス、パラミクソウイルス、およびモルビリウイルス）、又は寄生虫感染症マーカー（トリパノソーマ、シストソーマ、リーシュマニア、アメーバ、および条虫）が挙げられる。
具体的な生活習慣病マーカーとしては、肥満、脂質異常症、糖尿病、耐糖能異常、高血圧症、動脈硬化症、高尿酸血症、又は高インスリン血症のマーカーが挙げられる。
<Detected component>
In the detection method of the present invention, the component to be detected is not particularly limited, but may be an antigen or an antibody. As an antigen, an in-vivo protein (for example, a biomarker for a disease such as an enzyme, a receptor, or a ligand) that is increased in the body by a disease, or a pathogen protein (for example, a viral protein, a bacterial protein, a fungal protein, a parasite), Insect proteins). Examples of the antibody include an antibody produced in the body against a foreign antigen (for example, a pathogen), an autoantibody, an allergic antibody (IgE antibody), and the like.
The component to be detected is basically a biomarker that increases or decreases in vivo when a disease develops, and is captured on a carrier by a capture component that specifically binds to the component to be detected.
Examples of disease biomarkers include cancer markers, infectious disease markers, lifestyle-related disease markers, and the like. Specific cancer markers include colon cancer, prostate cancer, lung cancer, breast cancer, gastric cancer, ovarian cancer, renal cancer, thymoma, small intestine cancer, pancreatic cancer, bladder cancer, bone cancer, blood cancer, brain cancer, breast, Examples include markers of cervical cancer, colon cancer, endometrial cancer, esophageal cancer, liver cancer, or pharyngeal cancer. Specific infectious disease markers include bacterial infectious disease markers (pneumococcus, staphylococcus, bacillus, streptococcus, meningococcus, gonorrhea, Escherichia coli, Klebsiella, Proteus, Pseudomonas, Salmonella, Shigella, Haemophilus, Yersinia, Listeria, Corynebacterium, Vibrio, Clostridium, Chlamydia, Mycobacterium, Helicobacter, and Treponema), fungal infection markers (Candida, Aspergillus, Cryptococcus neoformans, fungi that cause superficial and deep-seated mycosis) , Viral infection markers (HIV, hepatitis virus, herpes virus, rhabdovirus, rubella virus, pox virus, poxvirus, paramyxovirus, and morbillivirus), or parasitic infection markers (trypanosomes, cystosomes, leishmania, Amoeba, and tapeworms).
Specific markers for lifestyle-related diseases include markers of obesity, dyslipidemia, diabetes, impaired glucose tolerance, hypertension, arteriosclerosis, hyperuricemia, or hyperinsulinemia.
《検出成分》
前記サンドイッチ法における検出成分は、被検出成分である抗原又は抗体に結合できる限りにおいて特に限定されるものではないが、例えば抗体又はレクチンを挙げることができる。被検出成分が抗原である場合、検出成分は抗原に結合することができる抗体、又はレクチンが好ましい。一方、被検出成分が抗体である場合、検出成分は被検出成分の抗体に結合することができる抗体が好ましい。
検出成分は、検出成分自体が酵素などの標識物によって標識されてもよいが、検出成分である抗体又はレクチンに対する二次抗体が酵素などの標識物によって標識されていてもよい。しかしながら、検出成分自体が酵素などの標識物によって標識されている、酵素標識抗体が好ましい。検出成分に対する標識二次抗体を用いる場合、工程が１つ増えるからである。
《Detection component》
The detection component in the sandwich method is not particularly limited as long as it can bind to the antigen or antibody that is the component to be detected, and examples thereof include an antibody or a lectin. When the component to be detected is an antigen, the detection component is preferably an antibody or lectin capable of binding to the antigen. On the other hand, when the component to be detected is an antibody, the detection component is preferably an antibody capable of binding to the antibody of the component to be detected.
Although the detection component itself may be labeled with a label such as an enzyme, the detection component itself or a secondary antibody against the lectin, which is the detection component, may be labeled with a label such as an enzyme. However, an enzyme-labeled antibody in which the detection component itself is labeled with a label such as an enzyme is preferable. This is because when the labeled secondary antibody against the detection component is used, the number of steps is increased by one.
《競合検出成分》
前記競合法における競合検出成分は、被検出成分と競合して捕捉成分と結合できる限りにおいて、特に限定されるものではないが、例えば抗原又は抗体が挙げられる。被検出成分が抗原である場合、競合検出成分は捕捉成分（例えば抗体又はレクチン）と結合できる抗原が好ましい。一方、被検出成分が抗体である場合、競合検出成分は捕捉成分（例えば、抗原）と結合できる抗原が好ましい。
競合検出成分は、好ましくは競合検出成分が酵素などの標識物によって標識される。
《Competitive detection component》
The competitive detection component in the competitive method is not particularly limited as long as it can compete with the component to be detected and bind to the capture component, and examples thereof include an antigen or an antibody. When the component to be detected is an antigen, the competitive detection component is preferably an antigen capable of binding to the capture component (eg antibody or lectin). On the other hand, when the component to be detected is an antibody, the competitive detection component is preferably an antigen that can bind to the capture component (eg, antigen).
The competitive detection component is preferably labeled with a label such as an enzyme.
検出成分又は競合検出成分の標識物としては、酵素又はアビジンを挙げることができる。前記標識物がアビジンの場合、アビジンと特異的に結合する酵素標識ビオチンによって、シグナルを検出することができる。
前記酵素としては、西洋ワサビペルオキシダーゼ（ＨＲＰ）、アルカリホスファターゼ（ＡＰ）、β−ガラクトシダーゼ、アセチルコリンエステラーゼ又はカタラーゼなどを用いることができる。これらの酵素と基質の組み合わせにより、発色又は発光によって、複合体を検出することができる。例えば、西洋ワサビペルオキシダーゼの基質としては、例えば３，３’，５，５’−テトラメチルベンジジン（3,3',5,5'-Tetramethylbenzidine，ＴＭＢ）、３，３’−ジアミノベンジジン（3,3'-Diaminobendizine，ＤＡＢ）、又はルミノール（発光）を用いることができる。例えば、アルカリホスファターゼの基質としては、ｐ−ニトロフェニルリン酸、又はジオキセタン（発光）を用いることができる。
Examples of the label of the detection component or the competitive detection component may include an enzyme or avidin. When the labeled substance is avidin, the signal can be detected by the enzyme-labeled biotin that specifically binds to avidin.
As the enzyme, horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, acetylcholinesterase or catalase can be used. The combination of these enzymes and the substrate allows the complex to be detected by color development or luminescence. For example, as a substrate for horseradish peroxidase, for example, 3,3',5,5'-tetramethylbenzidine (3,3',5,5'-Tetramethylbenzidine, TMB), 3,3'-diaminobenzidine (3,3'3'-Diaminobendizine, DAB), or luminol (luminescence) can be used. For example, p-nitrophenyl phosphate or dioxetane (luminescence) can be used as a substrate for alkaline phosphatase.
《サンドイッチ法》
前記サンドイッチ法は、（１）捕捉成分が親水性のポリマーを介して固相化された平均細孔直径１０μｍ以下の細孔を有する担体において、前記捕捉成分に結合する被検出成分を含む可能性のある検体を、前記細孔に流入させる工程（以下、検体流入工程（１）と称することがある）、（２）前記被検出成分に結合する検出成分を含む溶液を前記細孔に流入させ、捕捉成分／被検出成分／検出成分の複合体を形成させる工程（複合体形成工程（２））、及び（３）前記複合体を検出する工程（以下、サンドイッチ検出工程（３）と称することがある）を含む。サンドイッチ法としては、限定されるものではないが、被検出物質が抗原である抗原検出法、又は被検出物質が抗体である抗体検出法が挙げられる。
前記検体流入工程（１）においては、担体の細孔に検体を通過させ、捕捉成分に検体を接触させる。検体流入工程（１）において、捕捉成分と被検出成分との１次複合体（捕捉成分／被検出成分）が形成される。１次複合体は、例えば抗体／抗原、又は抗原／抗体の免疫複合体である。次に、複合体形成工程（２）において、担体の細孔に検出成分を通過させ、前記１複合体に検出成分を接触させ、２次複合体（捕捉成分／被検出成分／検出成分）を形成させる。前記２次複合体は、例えば抗体／抗原／抗体、又は抗原／抗体／抗体の免疫複合体である。複合体形成工程（２）において形成された、複合体がサンドイッチ検出工程（３）において検出される。検出は、前記の通り、酵素などの標識物と基質とを反応させ、発色又は発光などのシグナルを得ることによって検出できる。前記検体流入工程（１）及び複合体形成工程（２）の間、並びに複合体形成工程（２）及び検出工程（３）の間には、細孔内に残存している被検出成分又は検出成分を取り除くために、洗浄液によって洗浄することが好ましい。洗浄液としては、ＰＢＳ又はＴＢＳを用いることができる。
《Sandwich method》
The sandwich method may include (1) a carrier having a pore having an average pore diameter of 10 μm or less in which the capturing component is immobilized via a hydrophilic polymer, and the component to be detected may bind to the capturing component. A step of causing a certain sample to flow into the pores (hereinafter, may be referred to as a sample inflow step (1)), (2) causing a solution containing a detection component that binds to the detection target component to flow into the pores. A step of forming a complex of a capture component/a component to be detected/a detection component (complex forming step (2)), and (3) a step of detecting the complex (hereinafter referred to as a sandwich detection step (3) Is included). The sandwich method includes, but is not limited to, an antigen detection method in which the substance to be detected is an antigen, or an antibody detection method in which the substance to be detected is an antibody.
In the sample inflow step (1), the sample is passed through the pores of the carrier, and the sample is brought into contact with the capturing component. In the sample inflow step (1), a primary complex (capture component/detection component) of the capture component and the detection component is formed. The primary complex is, for example, an antibody/antigen or an antigen/antibody immune complex. Next, in the complex forming step (2), the detection component is passed through the pores of the carrier, the detection component is brought into contact with the one complex, and the secondary complex (capture component/detection component/detection component) is formed. Let it form. The secondary complex is, for example, an antibody/antigen/antibody or an antigen/antibody/antibody immune complex. The complex formed in the complex formation step (2) is detected in the sandwich detection step (3). As described above, the detection can be performed by reacting a label such as an enzyme with a substrate and obtaining a signal such as color development or luminescence. Between the sample inflow step (1) and the complex formation step (2), and between the complex formation step (2) and the detection step (3), the component to be detected or the detection remaining in the pores is detected. It is preferable to wash with a washing liquid in order to remove the components. PBS or TBS can be used as the washing liquid.
なお、ＥＬＩＳＡにおいては、ウェル中に酵素が存在しているため時間の経過とともに、発色又は発光が進行する為、一定の時間で発色又は発光を停止する必要がある。しかしながら、本発明においては、検出工程（３）において、担体の細孔に基質を通過させて反応させる。従って細孔を通過した基質は発色又は発光が停止しており、発色等の停止液を加えなくてもよい。
また、ＥＬＩＳＡにおいては、プレートのウェルに抗体又は抗原が非特異的に結合し、非特異反応が起きる。従って、抗体又は抗原を固相化した後に、ＢＳＡ、ＯＶＡ、又はスキムミルクなどの親水性タンパク質によってブロッキングを行う。しかしながら、本発明の検出方法においては、親水性ポリマーにより担体が覆われているため、ブロッキングなしでも担体が親水性となっている。そのため、ブロッキングなしで、抗体又は抗原の担体への非特異的吸着を抑制することができる。
In the case of ELISA, since the enzyme is present in the well and the color or luminescence progresses over time, it is necessary to stop the color or luminescence at a fixed time. However, in the present invention, in the detection step (3), the substrate is allowed to pass through the pores of the carrier to react. Therefore, the substrate that has passed through the pores has stopped coloring or emitting light, and it is not necessary to add a stop solution for coloring or the like.
Moreover, in ELISA, an antibody or an antigen nonspecifically binds to the well of the plate, and a nonspecific reaction occurs. Therefore, after immobilizing the antibody or antigen, blocking is performed with a hydrophilic protein such as BSA, OVA, or skim milk. However, in the detection method of the present invention, since the carrier is covered with the hydrophilic polymer, the carrier is hydrophilic even without blocking. Therefore, non-specific adsorption of the antibody or antigen to the carrier can be suppressed without blocking.
《競合法》
前記競合法は、（１）捕捉成分が親水性のポリマーを介して固相化された平均細孔直径１０μｍ以下の細孔を有する担体において、前記捕捉物質に結合する被検出成分を含む可能性のある検体、及び前記捕捉成分に結合する前記被検出成分と競合する競合検出成分を、前記細孔に流入させ、捕捉成分／競合検出成分の複合体を形成させる工程（以下、検体流入工程（１）と称することがある）、及び（２）前記複合体を検出する工程（以下、競合検出工程（２））を含む。競合法においては、捕捉成分／被検出成分の複合体、及び捕捉成分／競合検出成分の複合体が形成されるが、競合検出成分を標識することにより捕捉成分／競合検出成分の複合体を検出し、検体中の被検出成分の量を求める。
競合法としては、被検出成分が抗原である抗原検出法、又は被検出物質が抗体である抗体検出法が挙げられる。
競合法における検体流入工程（１）は、競合検出成分を被検出成分と一緒に細孔に流入させることを除いては、基本的にサンドイッチ法における検体流入工程（１）と同じように実施することができる。この検体流入工程（１）において、捕捉成分／競合検出成分の複合体が形成される。競合法における検出工程（２）も、基本的にサンドイッチ法における検出工程（３）に従って実施することができる。前記検体流入工程（１）及び検出工程（２）の間には、細孔内に残存している被検出成分又は検出成分を取り除くために、洗浄液によって洗浄することが好ましい。
更に、競合法においても、サンドイッチ法と同じように、発色等の停止液を加えなくてもよく、ブロッキングなしで、抗体又は抗原の担体への非特異的吸着を抑制することができる。
《Competitive law》
In the competitive method, (1) a carrier having a pore having an average pore diameter of 10 μm or less in which a capture component is immobilized through a hydrophilic polymer may contain a component to be detected that binds to the capture substance. And a step of causing a competitive detection component that competes with the detection target component that binds to the capture component to flow into the pores to form a capture component/competitive detection component complex (hereinafter, the sample inflow step ( 1)), and (2) a step of detecting the complex (hereinafter, competition detection step (2)). In the competitive method, a capture component/detected component complex and a capture component/competitive detection component complex are formed, and the capture component/competitive detection component complex is detected by labeling the competitive detection component. Then, the amount of the component to be detected in the sample is obtained.
Examples of the competition method include an antigen detection method in which the component to be detected is an antigen, or an antibody detection method in which the substance to be detected is an antibody.
The sample inflow step (1) in the competitive method is basically performed in the same manner as the sample inflow step (1) in the sandwich method, except that the competitive detection component and the detection target component are allowed to flow into the pores. be able to. In this sample inflow step (1), a capture component/competitive detection component complex is formed. The detection step (2) in the competitive method can also be basically performed according to the detection step (3) in the sandwich method. Between the sample inflow step (1) and the detection step (2), it is preferable to wash with a washing liquid in order to remove the component to be detected or the component to be detected remaining in the pores.
Further, in the competitive method as well as in the sandwich method, it is possible to suppress the non-specific adsorption of the antibody or antigen to the carrier without blocking, without adding a stop solution for color development or the like.
前記サンドイッチ法の検体流入工程（１）における検体、及び複合体形成工程（２）における検出成分（例えば、標識抗体）、並びに競合法の検体流入工程（１）における検体及び競合検出成分（例えば、標識抗体又は標識抗原）は、適当な緩衝液で希釈して細孔に流入させる。これらの細孔に流入される検体等の容量は、本発明の効果が得られる限りにおいて、特に限定されるものではないが、０．２〜５ｍＬであり、好ましくは０．５〜２ｍＬである。また、サンドイッチ法の検出工程（３）及び競合法の検出工程（２）における基質の容量も特に限定されるものではないが、０．２〜５ｍＬであり、好ましくは０．５〜２ｍＬである。また、使用する膜の面積が大きくなれば、容量も増加することが望ましいが、例えば前記検体等の容量は、０．２〜５ｍＬ／ｃｍ２であり、好ましくは０．５〜２ｍＬ／ｃｍ２である。
また、検体等及び基質の反応時間（流入時間）も、本発明の効果が得られる限りにおいて、特に限定されるものではないが、例えば１〜３０分であり、好ましくは２〜２０分であり、より好ましくは５〜１５分である。前記の時間の範囲であることにより、従来のＥＬＩＳＡ法と比較して短時間で、バイオマーカーの測定を行うことができる。
The sample in the sample inflow step (1) of the sandwich method, the detection component (for example, a labeled antibody) in the complex formation step (2), and the sample and the competitive detection component in the sample inflow step (1) of the competitive method (for example, The labeled antibody or labeled antigen) is diluted with an appropriate buffer and allowed to flow into the pores. The volume of the sample or the like flowing into these pores is not particularly limited as long as the effect of the present invention can be obtained, but is 0.2 to 5 mL, and preferably 0.5 to 2 mL. .. The volume of the substrate in the detection step (3) of the sandwich method and the detection step (2) of the competitive method is not particularly limited, but is 0.2 to 5 mL, preferably 0.5 to 2 mL. .. Further, it is desirable that the capacity increases as the area of the membrane used increases. For example, the capacity of the sample or the like is 0.2 to 5 mL/cm 2 , and preferably 0.5 to 2 mL/cm 2. Is.
The reaction time (inflow time) of the sample and the substrate is not particularly limited as long as the effect of the present invention can be obtained, but is, for example, 1 to 30 minutes, preferably 2 to 20 minutes. , And more preferably 5 to 15 minutes. When the time is within the above range, the biomarker can be measured in a shorter time as compared with the conventional ELISA method.
《作用》
本発明の複合体の検出方法において、短時間で疾患等のバイオマーカーを検出できる機構は、完全に解明されたわけではないが、以下のように推定できる。しかしながら、本発明は、以下の推定によって限定されるものではない。
本発明においては、図３に示すように、担体の細孔に捕捉抗体などの捕捉成分が固相化されており、その細孔に抗原などの被検出成分を通過させる。前記細孔の平均細孔直径は、１０μｍ以下と細いため、捕捉成分と被検出成分との接触の機会が増加し、捕捉成分と被検出成分との結合が促進されると考えられる。更に、細孔に被検出成分及び検出成分等を含む溶液を流入させることにより、捕捉成分と被検出成分との接触の機会が増加し、更に捕捉成分と被検出成分との結合が促進されると考えられる。
《Action》
In the method for detecting a complex of the present invention, the mechanism by which a biomarker such as a disease can be detected in a short time has not been completely elucidated, but it can be estimated as follows. However, the present invention is not limited by the following estimation.
In the present invention, as shown in FIG. 3, a capture component such as a capture antibody is immobilized on the pores of the carrier, and a component to be detected such as an antigen is allowed to pass through the pores. Since the average pore diameter of the pores is as small as 10 μm or less, it is considered that the chances of contact between the capture component and the detection target component are increased and the binding between the capture component and the detection component is promoted. Furthermore, by allowing a solution containing the component to be detected and the component to be detected to flow into the pores, the chances of contact between the capturing component and the component to be detected are increased, and further the binding between the capturing component and the component to be detected is promoted. it is conceivable that.
［２］担体
本発明の担体は、被検出成分を捕捉するための捕捉成分が親水性のポリマーを介して固相化された平均細孔直径１０μｍ以下の細孔を有する担体であって、前記細孔の細孔分布が、下記式（１）：
（Ｄ１０−Ｄ９０）／Ｄ５０≦１ （１）
（式中、Ｄ１０は累積体積百分率が１０％の細孔径であり、Ｄ９０は累積体積百分率が９０％の細孔径であり、Ｄ５０は累積体積百分率が５０％の細孔径である）
を満たす。
本発明の担体の、平均細孔直径、捕捉成分、空孔率、親水性ポリマー及び材質などは、前記「［１］複合体の検出方法」の項に記載したものと同様である。
[2] Carrier The carrier of the present invention is a carrier in which a capture component for capturing a component to be detected is immobilized through a hydrophilic polymer and has pores with an average pore diameter of 10 μm or less, The pore distribution of pores is represented by the following formula (1):
(D10-D90)/D50≦1 (1)
(In the formula, D10 is a pore size with a cumulative volume percentage of 10%, D90 is a pore size with a cumulative volume percentage of 90%, and D50 is a pore size with a cumulative volume percentage of 50%).
Meet
The average pore diameter, the trapping component, the porosity, the hydrophilic polymer, the material and the like of the carrier of the present invention are the same as those described in the above-mentioned "[1] Method for detecting complex".
［３］複合体の検出キット
本発明の複合体の検出キットは、（Ａ）本発明の担体、及び被検出成分に結合する検出成分を含む溶液を含む。また、本発明の複合体の検出キットは、（Ｂ）本発明の担体、及び捕捉成分と結合する競合検出成分を含む溶液を含む。
前記（Ａ）の複合体検出キットは、本発明のサンドイッチ法の複合体検出方法に用いることができる。また、前記（Ｂ）の複合体検出キットは、本発明の競合法の複合体検出方法に用いることができる。
[3] Complex detection kit The complex detection kit of the present invention comprises (A) the carrier of the present invention, and a solution containing a detection component that binds to the detection target component. The complex detection kit of the present invention comprises (B) a carrier of the present invention and a solution containing a competitive detection component that binds to a capture component.
The complex detection kit (A) can be used in the complex detection method of the sandwich method of the present invention. The complex detection kit (B) can be used in the complex detection method of the competitive method of the present invention.
複合体検出キットに含まれる担体は、前記「［２］担体」の項に記載の担体を用いることができる。
被検出成分に結合する検出成分を含む溶液は、被検出成分に結合する検出成分を含む限りにおいて、特に限定されるものではない。前記被検出成分及び検出成分は、前記「［１］複合体の検出方法」の項に記載したものと同様である。
また、捕捉成分と結合する競合検出成分を含む溶液は、捕捉成分と結合する競合検出成分を含む限りにおいて特に限定されるものではない。前記捕捉成分及び競合検出成分は、前記「［１］複合体の検出方法」の項に記載したものと同様である。
As the carrier included in the complex detection kit, the carrier described in the above section "[2] Carrier" can be used.
The solution containing the detection component that binds to the detection component is not particularly limited as long as it contains the detection component that binds to the detection component. The components to be detected and the components to be detected are the same as those described in the section "Method for detecting complex [1]".
The solution containing the competitive detection component that binds to the capture component is not particularly limited as long as it contains the competitive detection component that binds to the capture component. The capture component and the competitive detection component are the same as those described in the section “Method for detecting complex [1]”.
以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。 Hereinafter, the present invention will be described in detail with reference to Examples, but these do not limit the scope of the present invention.
《実施例１》
本実施例では、直径１μｍの細孔を有するトラックエッチング膜を用いて、細孔に親水性ポリマーを介して抗体を固相化した。用いたトラックエッチング膜は、平均細孔直径１．０μｍ、空孔率１９．６％、膜厚２２μｍであった。
溶媒を水としたアクリル酸モノマー濃度３．７５ｗｔ％、ドデシルベンゼンスルホン酸ナトリウム（ＳＤＢＳ）界面活性剤濃度５ｗｔ％を用い、遊離ラジカル法によりプラズマグラフト重合を行った。プラズマ照射条件はＡｒ １０Ｐａの雰囲気下で照射強度３０Ｗ、照射時間６０秒で行った。また重合は８０℃、３０分間振とうすることにより行った。多孔質細孔へ導入したアクリル酸は重量変化によりその固定量を評価した。
次に、Ｎ−ヒドロキシスクシンイミド（N-Hydroxysuccinimide；NHS)０．１１５ｇ、水溶性カルボジイミド（Water soluble carbodiimide；WSC）０．１９２ｇを超純水２０ｍＬに溶解し、５０ｍＭ ＮＨＳ／ＷＳＣ水溶液を調製した。この溶液に、前記ポリアクリル酸グラフト膜（φ＝４％）を入れて、振とう恒温槽内で３０℃で３時間反応させた。ポリアクリル酸グラフト膜は、反応前にエタノールと純水を用いて、溶媒を置換した。
ポリアクリル酸グラフト膜を直径１ｃｍに切り取り、洗浄バッファー（ＴＢＳ with Tween 20、ｐＨ８．０）で１ｍｇ／ｍＬに希釈したヒツジ由来抗ＢＳＡ抗体（ベチル・ラボラトリーズ）を１００μＬを滴下し、４℃で一昼夜、インキュベートした。インキュベート終了後、ポリアクリル酸グラフト膜を洗浄バッファー（ＴＢＳ with Tween 20、ｐＨ８．０）で１分、超純水で１分洗浄し、抗体固相化膜を得た。抗体固相化膜は、洗浄バッファー中に保存した。
<<Example 1>>
In this example, a track etching film having pores with a diameter of 1 μm was used to immobilize the antibody through the hydrophilic polymer in the pores. The track etching film used had an average pore diameter of 1.0 μm, a porosity of 19.6%, and a film thickness of 22 μm.
Plasma graft polymerization was performed by the free radical method using an acrylic acid monomer concentration of 3.75 wt% and a sodium dodecylbenzenesulfonate (SDBS) surfactant concentration of 5 wt% with water as the solvent. The plasma irradiation conditions were such that the irradiation intensity was 30 W and the irradiation time was 60 seconds in an atmosphere of Ar 10 Pa. The polymerization was performed by shaking at 80° C. for 30 minutes. The amount of acrylic acid introduced into the porous pores was evaluated by the weight change.
Next, 0.115 g of N-Hydroxysuccinimide (NHS) and 0.192 g of water-soluble carbodiimide (Water soluble carbodiimide; WSC) were dissolved in 20 mL of ultrapure water to prepare a 50 mM NHS/WSC aqueous solution. The polyacrylic acid graft film (φ=4%) was added to this solution, and the mixture was reacted in a shaking thermostat at 30° C. for 3 hours. For the polyacrylic acid graft film, the solvent was replaced with ethanol and pure water before the reaction.
Cut the polyacrylic acid graft membrane to a diameter of 1 cm, add 100 μL of sheep-derived anti-BSA antibody (Betyl Laboratories) diluted to 1 mg/mL with a washing buffer (TBS with Tween 20, pH 8.0), and drop at 4°C overnight. , Incubated. After completion of the incubation, the polyacrylic acid graft membrane was washed with a washing buffer (TBS with Tween 20, pH 8.0) for 1 minute and ultrapure water for 1 minute to obtain an antibody-immobilized membrane. The antibody-immobilized membrane was stored in the washing buffer.
《実施例２》
本実施例では、実施例１で得られた担体、及び被検出成分としてＢＳＡを用いて、本発明の検出方法によるＢＳＡの検出を行った。図４に検出方法の１つの実施態様を示す。
実施例１で得られた抗体固相化膜を、フィルターホルダー（メルク：Swinnex Filter Holder 13mm）にセットした。ＢＳＡ（ベチル・ラボラトリーズ）を洗浄バッファーを用いて、１０ｎｇ／ｍＬ、２５ｎｇ／ｍＬ、及び１００ｎｇ／ｍＬに希釈した。それぞれの濃度のＢＳＡ溶液を、シリンジポンプを用いて、１０分間抗体固相化膜に透過させた。ＢＳＡ溶液の流速は６ｍＬ／時間とした。抗体固相化膜をフィルターホルダーから取り出し１分間洗浄液（ＴＢＳ with Tween 20、ｐＨ８．０）中で洗浄し、そして膜ホルダにセットして５分間洗浄液で洗浄した。
次に、ＨＲＰ標識された抗ＢＳＡ抗体（二次抗体）を、シリンジポンプを用いて、１０分透過させた（６ｍＬ／時間）。抗体固相化膜を同様に洗浄した。ＴＭＢ溶液（ベチル・ラボラトリーズ）をシリンジポンプを用いて、１０分透過させた（６ｍＬ／時間）。基質溶液をサンプリングし、ＵＶ／ｖｉｓ分光計で６５０ｎｍの吸光度を測定した。
比較として、ベチル・ラボラトリーズの「Bovine Albumin ELISA Quantification Set（Cat. No. E10-113）」の説明書に記載のELISAの標準曲線のデータを示す。実施例２において使用した捕捉抗体、二次抗体、及び洗浄バッファー等は、ベチル・ラボラトリーズのELISAの標準曲線で使用されてものと同じものである。なお、ＥＬＩＳＡの１次反応及び２次反応は、それぞれ１時間であり、基質によるＨＲＰの発色は１５分間である。結果を図５に示す。
<<Example 2>>
In this example, BSA was detected by the detection method of the present invention using the carrier obtained in Example 1 and BSA as a component to be detected. FIG. 4 shows one embodiment of the detection method.
The antibody-immobilized membrane obtained in Example 1 was set in a filter holder (Merck: Swinnex Filter Holder 13 mm). BSA (Betyl Laboratories) was diluted to 10 ng/mL, 25 ng/mL, and 100 ng/mL with wash buffer. The BSA solution of each concentration was allowed to permeate the antibody-immobilized membrane for 10 minutes using a syringe pump. The flow rate of the BSA solution was 6 mL/hour. The antibody-immobilized membrane was taken out from the filter holder and washed in a washing solution (TBS with Tween 20, pH 8.0) for 1 minute, and then set on the membrane holder and washed with the washing solution for 5 minutes.
Next, the HRP-labeled anti-BSA antibody (secondary antibody) was allowed to permeate for 10 minutes using a syringe pump (6 mL/hour). The antibody-immobilized membrane was similarly washed. The TMB solution (Betyl Laboratories) was permeated with a syringe pump for 10 minutes (6 mL/hour). The substrate solution was sampled and the absorbance at 650 nm was measured with a UV/vis spectrometer.
For comparison, the data of the standard curve of ELISA shown in the instruction manual of "Bovine Albumin ELISA Quantification Set (Cat. No. E10-113)" by Betyl Laboratories is shown. The capture antibody, secondary antibody, washing buffer, etc. used in Example 2 are the same as those used in the standard curve of ELISA of Betyl Laboratories. The primary and secondary reactions of ELISA are each 1 hour, and the color development of HRP by the substrate is 15 minutes. Results are shown in FIG.
実施例２の本発明の検出方法では、比較例１のＥＬＩＳＡ法と比べて、高い発色シグナルが得られた。すなわち、反応時間を１／６にしているにも関わらず、迅速そして高感度に抗原を検出することができる。 In the detection method of the present invention of Example 2, a higher chromogenic signal was obtained as compared with the ELISA method of Comparative Example 1. That is, the antigen can be detected rapidly and with high sensitivity, even though the reaction time is set to 1/6.
《実施例３》
本実施例では、ポリエチレン多孔質膜を用いて、細孔にポリアクリル酸（親水性ポリマー）を形成し、その側鎖にＮ−アクリルオキシスクシンイミドを結合させ、グラフト重合体膜を得た（図６Ａ）。用いたポリエチレン多孔質膜は、平均細孔直径１５０ｎｍ、空孔率５０％、膜厚２７μｍであった。
水を溶媒としてＮＨＳとＥＤＣを溶解し、ＮＨＳ／ＥＤＣ水溶液（５０ｍＭ）を調製した。この溶液にグラフト率６％のアクリル酸グラフト膜を入れて、３０℃で２時間反応させた。膜は反応前に、エタノールと純水を用いて溶媒置換を行った。反応は振とう恒温槽内において行った。反応後はエタノールを用いて１分、続いて超純水を用いて１分反応後の膜を洗浄した。洗浄後の膜は乾燥機で真空乾燥し、ＦＴ−ＩＲによって分析を行った。
図６Ｂに示すように、重合の後にカルボキシル基（ＣＯＯＨ基）のシグナルが観察され、エステル化後に活性化エステルのシグナルが観察された。
<<Example 3>>
In this example, a polyethylene porous film was used to form polyacrylic acid (hydrophilic polymer) in the pores, and N-acryloxysuccinimide was bonded to its side chain to obtain a graft polymer film (Fig. 6A). The polyethylene porous film used had an average pore diameter of 150 nm, a porosity of 50%, and a film thickness of 27 μm.
NHS and EDC were dissolved using water as a solvent to prepare an NHS/EDC aqueous solution (50 mM). An acrylic acid graft membrane having a graft ratio of 6% was put into this solution and reacted at 30° C. for 2 hours. Before the reaction, the membrane was subjected to solvent substitution using ethanol and pure water. The reaction was carried out in a shaking constant temperature bath. After the reaction, ethanol was used for 1 minute, and then ultrapure water was used for 1 minute to wash the membrane after the reaction. The washed film was vacuum dried with a drier and analyzed by FT-IR.
As shown in FIG. 6B, a signal of a carboxyl group (COOH group) was observed after the polymerization, and a signal of the activated ester was observed after the esterification.
《実施例４及び比較例２》
本実施例では、細孔に捕捉抗体が固相化されていることを確認するために、蛍光標識された抗体を用いて、トラックエッチング膜に固相化した。
ヒツジ由来抗ＢＡＳ抗体に代えて、標識ヒツジ由来抗ＢＡＳ抗体を用いたことを除いては、実施例１の操作を繰り返して、標識抗体固相化膜を得た。得られた膜からミクロトームを用いて、３μｍの厚さの切片を作製し、その断面を蛍光観察した（図７）。
また、比較例として、活性化エステル化（ＮＨＳ及びＷＳＣによる処理）を行わなかったことを除いては、実施例１の操作を繰り返して、膜を得た（比較例２）。
図７（ａ）及び（ｂ）は、それぞれ実施例４の膜断面の透過像及び蛍光像である。抗体が膜厚方向にかなり均一に固定されていることがわかる。図７（ｃ）及び（ｄ）は、それぞれ比較例２の膜断面の透過像及び蛍光像である。比較例２においては、合蛍光標識抗体がほとんど固定されていなかった。実施例４の膜は、蛍光標識抗体は化学固定されていると考えられる。一方、比較例２では、膜細孔内における抗体の非特異的な残存や吸着がほとんど起こっていないことが分かる。
<<Example 4 and Comparative Example 2>>
In this example, in order to confirm that the capture antibody was immobilized on the pores, a fluorescently labeled antibody was used to immobilize it on the track etching film.
The procedure of Example 1 was repeated except that the labeled sheep-derived anti-BAS antibody was used in place of the sheep-derived anti-BAS antibody to obtain a labeled antibody-immobilized membrane. A section having a thickness of 3 μm was prepared from the obtained film using a microtome, and the cross section was observed by fluorescence (FIG. 7).
In addition, as a comparative example, a membrane was obtained by repeating the operation of Example 1 except that the activated esterification (treatment with NHS and WSC) was not performed (Comparative Example 2).
7A and 7B are a transmission image and a fluorescence image of the film cross section of Example 4, respectively. It can be seen that the antibody is immobilized fairly uniformly in the film thickness direction. 7C and 7D are a transmission image and a fluorescence image of the film cross section of Comparative Example 2, respectively. In Comparative Example 2, almost no fluorescent-labeled antibody was immobilized. The membrane of Example 4 is considered to have the fluorescently labeled antibody chemically immobilized. On the other hand, in Comparative Example 2, it can be seen that non-specific retention and adsorption of the antibody in the membrane pores hardly occur.
本発明の複合体の検出方法、担体、及び複合体の検出キットは、疾患等のバイオマーカーの検出に用いることができる。 INDUSTRIAL APPLICABILITY The complex detection method, carrier, and complex detection kit of the present invention can be used for detection of biomarkers such as diseases.
Claims (8)
（Ａ）（１）捕捉成分が親水性のポリマーを介して固相化された平均細孔直径１０μｍ以下の細孔を有する担体において、前記捕捉成分に結合する被検出成分を含む可能性のある検体を、前記細孔に流入させる工程、
（２）前記被検出成分に結合する検出成分を含む溶液を前記細孔に流入させ、捕捉成分／被検出成分／検出成分の複合体を形成させる工程、及び
（３）前記複合体を検出する工程、を含むか、又は
（Ｂ）（１）捕捉成分が親水性のポリマーを介して固相化された平均細孔直径１０μｍ以下の細孔を有する担体において、前記捕捉物質に結合する被検出成分を含む可能性のある検体、及び前記捕捉成分に結合する前記被検出成分と競合する競合検出成分を、前記細孔に流入させ、捕捉成分／競合検出成分の複合体を形成させる工程、及び
（２）前記複合体を検出する工程、を含む、
複合体の検出方法。 A method for detecting a complex, comprising:
(A) (1) A carrier having a fine pore having an average fine pore diameter of 10 μm or less in which the capturing component is immobilized via a hydrophilic polymer may contain a component to be detected that binds to the capturing component. Flowing a sample into the pores,
(2) Flowing a solution containing a detection component that binds to the detection component into the pores to form a complex of capture component/detection component/detection component, and (3) detecting the complex Or (B)(1) in which the capture component is immobilized through a hydrophilic polymer and has a pore having an average pore diameter of 10 μm or less, the carrier to be detected binds to the capture substance. Allowing a sample that may contain a component and a competitive detection component that competes with the detected component that binds to the capture component to flow into the pores to form a capture component/competitive detection component complex, and (2) detecting the complex,
Method of detecting complex.
（Ｄ１０−Ｄ９０）／Ｄ５０≦１ （１）
（式中、Ｄ１０は累積体積百分率が１０％の細孔径であり、Ｄ９０は累積体積百分率が９０％の細孔径であり、Ｄ５０は累積体積百分率が５０％の細孔径である）
を満たす、請求項１に記載の検出方法。 The pore distribution of the pores is represented by the following formula (1):
(D10-D90)/D50≦1 (1)
(In the formula, D10 is a pore size with a cumulative volume percentage of 10%, D90 is a pore size with a cumulative volume percentage of 90%, and D50 is a pore size with a cumulative volume percentage of 50%).
The detection method according to claim 1, which satisfies:
前記細孔の細孔分布が、下記式（１）：
（Ｄ１０−Ｄ９０）／Ｄ５０≦１ （１）
（式中、Ｄ１０は累積体積百分率が１０％の細孔径であり、Ｄ９０は累積体積百分率が９０％の細孔径であり、Ｄ５０は累積体積百分率が５０％の細孔径である）
を満たす担体。 A carrier having a fine pore having an average fine pore diameter of 10 μm or less in which a capture component for capturing a component to be detected is immobilized through a hydrophilic polymer,
The pore distribution of the pores is represented by the following formula (1):
(D10-D90)/D50≦1 (1)
(In the formula, D10 is a pore size with a cumulative volume percentage of 10%, D90 is a pore size with a cumulative volume percentage of 90%, and D50 is a pore size with a cumulative volume percentage of 50%).
A carrier that satisfies the requirements.
（Ｂ）請求項５〜７のいずれか一項に記載の担体、及び捕捉成分と結合する競合検出成分を含む溶液；
を含む複合体の検出キット。 (A) A carrier according to any one of claims 5 to 7, and a solution containing a detection component that binds to a detection target component; or (B) a carrier according to any one of claims 5 to 7, And a solution containing a competitive detection component that binds to the capture component;
A kit for detecting a complex containing:
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