JP4913454B2 - Method for measuring vitamin concentration and method for measuring PCB concentration - Google Patents
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Description
本発明は、免疫反応を利用した被験物質の定量に供するバイオセンサー装置、及びそれを用いた濃度測定方法に関する。 The present invention relates to a biosensor device used for quantification of a test substance using an immune reaction, and a concentration measurement method using the biosensor device.
近年、環境中の微量物質等の検出方法や定量方法として、従来の物理化学的な方法よりも極めて高い選択性を有し、簡便かつ迅速な免疫学的測定法（イムノアッセイ）が提案されている。 In recent years, simple and rapid immunoassay (immunoassay) has been proposed as a method for detecting and quantifying trace substances in the environment, which has a much higher selectivity than conventional physicochemical methods. .
例えば、環境汚染物質であるＰＣＢの分析方法としては、公定法として、高分解能ガスクロマトグラフ−高分解能質量分析（ＨＲＧＣ−ＨＲＭＳ）や、電子捕獲型検出器付きガスクロマトグラフ法（ＧＣ−ＥＣＤ法）が用いられている（非特許文献１参照）。これらの方法は分解能が高く、また定量下限も低いが、分析に要する時間が長く、分析の妨害となる夾雑成分を除去する試料の操作が煩雑であり、コスト負担が大きいという問題がある。このため、簡便かつ迅速な分析方法で、測定現場で簡易に測定可能な方法として、抗原抗体反応を利用した免疫学的測定法（イムノアッセイ）が提案されている。 For example, as an analysis method for PCB which is an environmental pollutant, official methods include high resolution gas chromatograph-high resolution mass spectrometry (HRGC-HRMS) and gas chromatograph method with electron capture detector (GC-ECD method). Used (see Non-Patent Document 1). These methods have high resolution and a low quantification limit, but have a problem that the time required for analysis is long, the operation of a sample for removing contaminant components that interfere with analysis is complicated, and the cost burden is large. For this reason, an immunological measurement method (immunoassay) using an antigen-antibody reaction has been proposed as a method that can be easily measured at a measurement site with a simple and rapid analysis method.
前記イムノアッセイのうち、最も一般的な測定方法は、酵素免疫測定法（ＥＬＩＳＡ）である。前記ＥＬＩＳＡ法としては、ラジオイムノアッセイ（ＲＩＡ）、蛍光イムノアッセイ（ＦＩＡ）、エンザイムイムノアッセイ（ＥＩＡ、ＥＬＩＳＡ）等が知られている。
しかしながら、前記ＥＬＩＳＡ法では、被験物質の検出に特殊な機器を必要としたり、試料の前処理や測定に長時間を要したりする等の問題点がある。このため、操作が簡単なイムノクロマト法が提案されている。
Among the immunoassays, the most common measurement method is enzyme immunoassay (ELISA). As the ELISA method, radioimmunoassay (RIA), fluorescent immunoassay (FIA), enzyme immunoassay (EIA, ELISA) and the like are known.
However, the ELISA method has problems such as requiring a special instrument for detection of a test substance and requiring a long time for sample pretreatment and measurement. For this reason, an immunochromatography method that is easy to operate has been proposed.
しかし、前記ＥＬＩＳＡ法、及びイムノクロマト法においては、検出に根本的な問題がある。両法において、高い検出感度を得るには、被験物質を担体上で２分子の抗体で挟み込む、いわゆる一般に知られるサンドイッチ法を用いるが、被験物質が、前記ＰＣＢ類のように低分子化合物である場合は、サンドイッチ法が成立しない。これは、被験物質である抗原が低分子であるため、生体高分子である抗体が被験物質に結合すると、抗体の結合部位に被験物質が埋もれてしまい、他の抗体が結合することができなくなるためである。
このため、通常ＥＬＩＳＡ法、及びイムノクロマト法を代表とするイムノアッセイの殆どの場合が、サンドイッチ法ではなく、競合法を採用している。
However, the ELISA method and the immunochromatography method have a fundamental problem in detection. In both methods, in order to obtain high detection sensitivity, a so-called generally known sandwich method in which a test substance is sandwiched between two molecules of antibody on a carrier is used, but the test substance is a low molecular weight compound such as the above-mentioned PCBs. If this is the case, the sandwich method is not approved. This is because the test substance antigen is a small molecule, and when the biopolymer antibody binds to the test substance, the test substance is buried in the binding site of the antibody, and other antibodies cannot bind. Because.
For this reason, most of the immunoassays represented by the ELISA method and the immunochromatography method adopt the competitive method instead of the sandwich method.
競合法は、前記被験物質を検出する際に、被験物質である前記低分子化合物と、前記低分子化合物又は前記低分子化合物に対する類似化合物とを、抗体に対する結合において競合させる方法である。しかし、この競合法は、抗体に対して試料中の被験物質と被験物質の類似化合物が競合的に結合する結果、本来の抗体の有する結合能力までの検出感度が得られにくいという原理的な短所がある。
また、競合法には、前記被験物質を検出する際に、担体上に前記低分子化合物又は前記低分子化合物に対する類似化合物を固定し、被験物質と抗体の結合溶液を担体と接触させる場合もある。しかし、この場合にも、抗体が液中の被験物質と担体上の低分子化合物又は前記低分子化合物に対する類似化合物とに競合的に結合するため、本来の抗体の有する結合能力までの検出感度が得られにくい。
The competition method is a method in which, when detecting the test substance, the low-molecular compound as the test substance competes with the low-molecular compound or a similar compound for the low-molecular compound in binding to the antibody. However, this competitive method has the principle disadvantage that it is difficult to obtain the detection sensitivity to the binding ability of the original antibody as a result of competitive binding of the test substance in the sample and the similar compound of the test substance to the antibody. There is.
In the competition method, when detecting the test substance, the low-molecular compound or a similar compound to the low-molecular compound is immobilized on a carrier, and the test substance and antibody binding solution may be contacted with the carrier. . However, in this case as well, since the antibody competitively binds to the test substance in the liquid and the low molecular compound on the carrier or a similar compound to the low molecular compound, the detection sensitivity up to the binding ability of the original antibody is high. It is difficult to obtain.
これに対し、例えば、被験物質を含む試料中に、前記被験物質に対する抗体を反応させ、前記試料中において前記被験物質を結合していないフリーの前記抗体を検出することにより、前記試料中の被験物質の検出を行う方法（例えば、特許文献１参照）や、被分析物質に特異的に結合する捕捉試薬を固定化した担体上に、被分析物質に特異的に結合するリガンドを含む標識試薬と検体とを接触させて、該被分析物質を検出する方法（例えば、特許文献２参照）等のフロースルー方式の測定法が提案されている。しかしながら、前記フロースルー免疫測定法においても、前記ＥＬＩＳＡ法と同様に、試料液中の被験物質あるいは捕捉用物質の間に競合的な結合が起きるため、本来の抗体の有する結合能力までの検出感度は得られていない。 In contrast, for example, by reacting an antibody against the test substance in a sample containing the test substance, and detecting the free antibody not bound to the test substance in the sample, the test in the sample is performed. A method for detecting a substance (for example, refer to Patent Document 1), and a labeling reagent containing a ligand that specifically binds to the analyte on a carrier on which a capture reagent that specifically binds to the analyte is immobilized A flow-through measurement method such as a method of detecting a substance to be analyzed by bringing it into contact with a specimen (for example, see Patent Document 2) has been proposed. However, in the flow-through immunoassay method, as in the ELISA method, competitive binding occurs between the test substance or the capture substance in the sample solution, so that the detection sensitivity up to the binding ability of the original antibody is detected. Is not obtained.
前記担体と前記試料の接触時間を極めて短くすることにより、前記担体上に固定した被験物質あるいは捕捉用物質（例えば、被測定物が抗体の場合、該抗体が特異的に認識する抗原又は擬似抗原であり、被測定物が抗原の場合、該抗原を特異的に認識する抗体）と、前記試料液中の被験物質あるいは捕捉用物質の間に競合的な結合が起きにくくなり、その結果良好な検出感度が得られると考えられる。 By significantly shortening the contact time between the carrier and the sample, the test substance or capture substance immobilized on the carrier (for example, when the analyte is an antibody, an antigen or pseudoantigen specifically recognized by the antibody) In the case where the object to be measured is an antigen, an antibody that specifically recognizes the antigen) and a test substance or a capturing substance in the sample solution are less likely to cause competitive binding, and as a result, good It is thought that detection sensitivity can be obtained.
前記フロースルー方式の測定方法に用いる装置においては、検出の感度や定量性を確保するために、限られた時間に多くの被測定物あるいは捕捉用物質を捕捉可能な担体を備えることが必要となる。このため、高密度に前記被測定物あるいは捕捉用物質が固定化され、かつ操作性のよい担体を備えたバイオセンサー装置が求められるが、そのような装置は未だ開発されていないのが現状である。 In the apparatus used for the flow-through measurement method, it is necessary to provide a carrier capable of capturing a large number of objects to be measured or capturing substances in a limited time in order to ensure detection sensitivity and quantitativeness. Become. For this reason, there is a need for a biosensor device in which the object to be measured or the substance to be captured is immobilized at a high density and a carrier having good operability is required, but such a device has not been developed yet. is there.
本発明は従来における前記問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、被験物質を高感度に検出可能であり、高密度に前記被測定物あるいは捕捉用物質が固定化可能であり、かつ操作性に優れた担体を備え、迅速に被験物質の濃度が測定可能なバイオセンサー装置、及び該バイオセンサー装置を用いた被験物質の濃度測定方法を提供することを目的とする。 An object of the present invention is to solve the above-described problems and achieve the following objects. That is, the present invention can detect a test substance with high sensitivity, can immobilize the analyte or capture substance at a high density, and has a carrier with excellent operability, so that the test substance can be rapidly detected. It is an object of the present invention to provide a biosensor device capable of measuring a concentration and a method for measuring the concentration of a test substance using the biosensor device.
前記課題を解決するための手段としては、以下の通りである。即ち、
＜１＞ 液体の被測定試料を通液可能な膜状担体を備える測定用セルと、
前記測定用セルを装着可能なセルホルダと、
前記セルホルダに装着された前記測定用セル中の前記膜状担体に対し、光を照射する光照射部と、
前記膜状担体からの反射光及び透過光の少なくともいずれかを受光し、受光した光量を測定する受光部と、
を備えることを特徴とするバイオセンサー装置である。
＜２＞ 測定用セルが、流路を有し、該流路中に膜状担体が固定されてなる前記＜１＞に記載のバイオセンサー装置である。
＜３＞ 膜状担体が、繊維状物質からなる前記＜１＞から＜２＞のいずれかに記載のバイオセンサー装置である。
＜４＞ 膜状担体が、被験物質、及びそのアナログ、並びに被験物質に対する抗体の少なくともいずれかが固定されてなる前記＜１＞から＜３＞のいずれかに記載のバイオセンサー装置である。
＜５＞ 受光部により受光した光量を、電気信号の信号強度として計測可能な測定手段を備える前記＜１＞から＜４＞のいずれかに記載のバイオセンサー装置である。
＜６＞ 光照射部が、膜状担体に単色光を照射する前記＜１＞から＜５＞のいずれかに記載のバイオセンサー装置である。
＜７＞ 光照射部が、複数の単色光からなる光を発光させる光源と、複数の単色光からなる光から、一の単色光のみを選択可能な単色光選択部とを備え、
前記単色光選択部によって選択された一の単色光を膜状担体に照射する前記＜１＞から＜５＞のいずれかに記載のバイオセンサー装置である。
＜８＞ 光照射部が、膜状担体に複数の単色光からなる光を照射し、
受光部が、複数の単色光からなる光から、一の単色光のみを選択可能な単色光選択部を備え、前記膜状担体からの反射光及び透過光の少なくともいずれかのうち、前記単色光選択部によって選択された一の単色光を受光する前記＜１＞から＜５＞のいずれかに記載のバイオセンサー装置である。
＜９＞ 光照射部が、互いに異なる単色光を発光する二以上の光源と、一の単色光のみを選択可能な単色光選択部とを備え、
前記二以上の光源から発光された二以上の単色光のうち、前記単色光選択部により選択された一の単色光を膜状担体に照射する前記＜１＞から＜５＞のいずれかに記載のバイオセンサー装置である。
＜１０＞ 光照射部と受光部との間に反射筒を備え、
前記反射筒が、膜状担体を内包するように光照射部から照射される光の照射方向に貫通し、その内面が前記膜状担体からの反射光を反射する前記＜１＞から＜９＞のいずれかに記載のバイオセンサー装置である。
＜１１＞ 被測定試料の測定により得られた光量と、基準試料の測定により得られた光量とから相対吸光度を計算する相対吸光度計算手段を備える前記＜１＞から＜１０＞のいずれかに記載のバイオセンサー装置である。
Means for solving the problems are as follows. That is,
<1> a measurement cell including a membrane-like carrier capable of passing a liquid sample to be measured;
A cell holder to which the measurement cell can be mounted;
A light irradiation unit for irradiating light to the film carrier in the measurement cell mounted on the cell holder;
A light receiving unit that receives at least one of reflected light and transmitted light from the film carrier and measures the amount of light received;
A biosensor device comprising:
<2> The biosensor device according to <1>, wherein the measurement cell has a flow path, and a membranous carrier is fixed in the flow path.
<3> The biosensor device according to any one of <1> to <2>, wherein the film carrier is made of a fibrous material.
<4> The biosensor device according to any one of <1> to <3>, wherein the membranous carrier is fixed with at least one of a test substance, an analog thereof, and an antibody against the test substance.
<5> The biosensor device according to any one of <1> to <4>, further including a measurement unit that can measure the amount of light received by the light receiving unit as the signal intensity of the electric signal.
<6> The biosensor device according to any one of <1> to <5>, wherein the light irradiation unit irradiates the film carrier with monochromatic light.
<7> The light irradiation unit includes a light source that emits light composed of a plurality of monochromatic lights, and a monochromatic light selection unit that can select only one monochromatic light from the light composed of a plurality of monochromatic lights,
The biosensor device according to any one of <1> to <5>, wherein the monochromatic light selected by the monochromatic light selection unit is irradiated onto the film carrier.
<8> The light irradiation unit irradiates the film carrier with light composed of a plurality of monochromatic lights,
The light receiving unit includes a monochromatic light selecting unit capable of selecting only one monochromatic light from light composed of a plurality of monochromatic lights, and the monochromatic light of at least one of reflected light and transmitted light from the film carrier The biosensor device according to any one of <1> to <5>, which receives one monochromatic light selected by a selection unit.
<9> The light irradiation unit includes two or more light sources that emit different monochromatic lights, and a monochromatic light selection unit that can select only one monochromatic light,
Any one of <1> to <5>, in which one monochromatic light selected by the monochromatic light selection unit among the two or more monochromatic lights emitted from the two or more light sources is irradiated to the film carrier. This is a biosensor device.
<10> A reflection cylinder is provided between the light irradiation unit and the light receiving unit,
<1> to <9> in which the reflection tube penetrates in the irradiation direction of the light irradiated from the light irradiation unit so as to enclose the film carrier, and the inner surface reflects the reflected light from the film carrier. The biosensor device according to any one of the above.
<11> The device according to any one of <1> to <10>, further including a relative absorbance calculation unit that calculates a relative absorbance from a light amount obtained by measurement of the sample to be measured and a light amount obtained by measurement of the reference sample. This is a biosensor device.
＜１２＞ 測定用セルに着脱可能に接続され、膜状担体に液体の被測定試料を供給して通液させる通液手段を備える前記＜１＞から＜１１＞のいずれかに記載のバイオセンサー装置である。
＜１３＞ 通液手段が、液体の被測定試料を加圧して膜状担体へ通液する手段である前記＜１２＞に記載のバイオセンサー装置である。
＜１４＞ 繊維状物質が、綿、絹、麻、ポリエステル、芳香族ポリアミド、ナイロン、及びポリオレフィンのいずれかである＜３＞から＜１３＞のいずれかに記載のバイオセンサー装置である。
<12> The biosensor according to any one of <1> to <11>, wherein the biosensor is detachably connected to the measurement cell and includes a liquid passing unit that supplies a liquid sample to be measured to the membrane carrier and allows the liquid to pass therethrough. Device.
<13> The biosensor device according to <12>, wherein the liquid passing unit is a unit that pressurizes a liquid sample to be measured and passes the sample through the membrane carrier.
<14> The biosensor device according to any one of <3> to <13>, wherein the fibrous substance is any one of cotton, silk, hemp, polyester, aromatic polyamide, nylon, and polyolefin.
＜１５＞ 前記＜１＞から＜１４＞のいずれかに記載のバイオセンサー装置を用い、被測定試料中の被験物質を定量することを特徴とする濃度測定方法である。
＜１６＞ 被測定試料を通液した後の測定用セルをセルホルダに装着し、前記セルホルダに装着された前記測定用セル中の膜状担体に対し、光照射部から光を照射し、受光部で前記膜状担体からの反射光及び透過光の少なくともいずれかを受光し、受光した光量を測定する前記＜１５＞に記載の濃度測定方法である。
＜１７＞ 被験物質Ｘに対する抗体Ｙを固定化した膜状担体に、
前記被験物質Ｘを標識物質で標識されてなる標識被験物質Ｘ’を添加した被測定試料を通液し、
前記膜状担体上の前記抗体Ｙに対し、前記被測定試料中の被験物質Ｘと、前記標識被験物質Ｘ’とを反応させ、
前記膜状担体上の前記抗体Ｙに結合した前記標識被験物質Ｘ’の量を、前記膜状担体の反射光量及び透過光量のいずれかとして検出し、該反射光量及び透過光量のいずれかから、前記被験物質Ｘと前記標識被験物質Ｘ’との反応の度合いを求め、
該反応の度合いから、前記被測定試料中の被験物質Ｘの濃度を算出する前記＜１５＞から＜１６＞の濃度測定方法である。
＜１８＞ 被験物質Ｘ及び該被験物質のアナログＺのいずれかを固定化した膜状担体に、
前記被験物質Ｘに対する抗体であって標識物質で標識されてなる標識抗体Ｙを添加した被測定試料を通液し、
前記膜状担体上の前記被験物質Ｘ及び前記被験物質のアナログＺのいずれかに対し、前記被測定試料中の前記被験物質Ｘと結合していない前記標識抗体Ｙを結合させ、
前記標識抗体Ｙの量を、前記膜状担体の反射光量及び透過光量のいずれかとして検出し、該反射光量及び透過光量のいずれかから、前記被測定試料中の前記被験物質Ｘの濃度を算出する前記＜１５＞から＜１６＞のいずれかに記載の濃度測定方法である。
＜１９＞ 被験物質Ｘ及び該被験物質のアナログＺのいずれかを固定化した膜状担体に、
前記被験物質に対する抗体Ｙを添加した被測定試料を通液し、
前記膜状担体上の前記被験物質Ｘ及び前記被験物質のアナログＺのいずれかに対し、前記被測定試料中の前記被験物質Ｘと結合していない前記抗体Ｙを結合させ、
前記抗体Ｙに、該抗体Ｙに対する二次抗体であって標識物質で標識されてなる標識抗体Ｙ’を結合させ、
前記標識抗体Ｙ’の量を、前記膜状担体の反射光量及び透過光量のいずれかとして検出し、該反射光量及び透過光量のいずれかから、前記被測定試料中の前記被験物質Ｘの濃度を算出する前記＜１５＞から＜１６＞のいずれかに記載の濃度測定方法である。
＜２０＞ 標識物質が酵素、放射性同位元素、蛍光物質、及び着色微粒子、並びにこれらにより標識された抗体のいずれかである前記＜１５＞から＜１９＞のいずれかに記載の免疫反応測定方法である。
＜２１＞ 被験物質が、ＰＣＢ、ダイオキシン、ホルモン、ビタミン類、農薬、及び重金属のいずれかである前記＜１５＞から＜２０＞のいずれかに記載の免疫反応測定方法である。
＜２２＞ 被験物質がＰＣＢであり、下記構造式（１）で表される擬似抗原を固定化した膜状担体に、抗ＰＣＢ抗体を添加した被測定試料を通液し、
前記膜状担体上の前記擬似抗原に対し、前記被測定試料中のＰＣＢと結合していない前記抗ＰＣＢ抗体を結合させた後、
前記抗ＰＣＢ抗体に対する二次抗体であって、標識物質で標識されてなる二次抗体を結合させ、
前記二次抗体の標識物質の量を、前記膜状担体の反射光量及び透過光量のいずれかとして検出し、該反射光量及び透過光量のいずれかから、前記被測定試料中のＰＣＢの濃度を算出する前記＜１５＞から＜２１＞のいずれかに記載の濃度測定方法である。
<16> The measurement cell after passing the sample to be measured is attached to the cell holder, the film carrier in the measurement cell attached to the cell holder is irradiated with light from the light irradiation unit, and the light receiving unit The concentration measurement method according to <15>, wherein at least one of reflected light and transmitted light from the film carrier is received and the received light amount is measured.
<17> A membranous carrier on which antibody Y against test substance X is immobilized,
Passing the sample to be measured to which the test substance X ′ labeled with the test substance X is added,
Reacting the antibody Y on the membranous carrier with the test substance X in the sample to be measured and the labeled test substance X ′;
The amount of the labeled test substance X ′ bound to the antibody Y on the membranous carrier is detected as either the reflected light amount or the transmitted light amount of the membranous carrier, and from either the reflected light amount or the transmitted light amount, Obtain the degree of reaction between the test substance X and the labeled test substance X ′,
The concentration measurement method according to <15> to <16>, wherein the concentration of the test substance X in the sample to be measured is calculated from the degree of the reaction.
<18> In a membranous carrier in which either test substance X or analog Z of the test substance is immobilized,
Passing a sample to be measured to which a labeled antibody Y, which is an antibody against the test substance X and is labeled with a labeling substance, is added;
Binding the labeled antibody Y not bound to the test substance X in the sample to be measured to either the test substance X or the test substance analog Z on the membranous carrier;
The amount of the labeled antibody Y is detected as either the amount of reflected light or the amount of transmitted light of the film carrier, and the concentration of the test substance X in the sample to be measured is calculated from either the amount of reflected light or the amount of transmitted light. The concentration measuring method according to any one of <15> to <16>.
<19> A membranous carrier in which either test substance X or analog Z of the test substance is immobilized,
Passing a sample to be measured to which an antibody Y against the test substance is added;
Binding the antibody Y not bound to the test substance X in the sample to be measured to either the test substance X or the analog Z of the test substance on the membranous carrier;
A labeled antibody Y ′ which is a secondary antibody against the antibody Y and labeled with a labeling substance is bound to the antibody Y;
The amount of the labeled antibody Y ′ is detected as either the amount of reflected light or the amount of transmitted light of the film carrier, and the concentration of the test substance X in the sample to be measured is determined from either the amount of reflected light or the amount of transmitted light. The concentration measurement method according to any one of <15> to <16>, wherein the concentration is calculated.
<20> The immunoreaction measurement method according to any one of <15> to <19>, wherein the labeling substance is any one of an enzyme, a radioisotope, a fluorescent substance, a colored fine particle, and an antibody labeled with these. is there.
<21> The immune reaction measurement method according to any one of <15> to <20>, wherein the test substance is any one of PCB, dioxin, hormones, vitamins, agricultural chemicals, and heavy metals.
<22> The test substance is PCB, and the sample to be measured is added to the membranous carrier on which the pseudoantigen represented by the following structural formula (1) is immobilized, and the anti-PCB antibody is added,
After binding the anti-PCB antibody not bound to PCB in the sample to be measured to the pseudoantigen on the membrane carrier,
A secondary antibody against the anti-PCB antibody, which is bound with a secondary antibody labeled with a labeling substance;
The amount of the secondary antibody labeling substance is detected as either the amount of reflected light or the amount of transmitted light of the film carrier, and the concentration of PCB in the sample to be measured is calculated from either the amount of reflected light or the amount of transmitted light. The concentration measuring method according to any one of <15> to <21>.
本発明によると、被験物質を高感度に検出可能であり、高密度に前記被測定物あるいは捕捉用物質が固定化可能であり、かつ操作性に優れた担体を備え、迅速に被験物質の濃度が測定可能なバイオセンサー装置、及び該バイオセンサー装置を用いた被験物質の濃度測定方法を提供することができる。 According to the present invention, the test substance can be detected with high sensitivity, the analyte or the capture substance can be immobilized at a high density, and the carrier has excellent operability, and the test substance concentration can be quickly obtained. Can be provided, and a test substance concentration measurement method using the biosensor apparatus can be provided.
（バイオセンサー装置）
本発明のバイオセンサー装置は、液体の被測定試料を通液可能な膜状担体を備える測定用セルと、前記測定用セルを装着可能なセルホルダと、前記セルホルダに装着された前記測定用セル中の前記膜状担体に対し、光を照射する光照射部と、前記膜状担体からの反射光及び透過光の少なくともいずれかを受光し、受光した光量を測定する受光部と、を備え、必要に応じて適宜選択した測定手段、通液手段、相対吸光度測定手段等のその他の手段を備える。
(Biosensor device)
The biosensor device of the present invention includes a measurement cell including a membrane-like carrier capable of passing a liquid sample to be measured, a cell holder in which the measurement cell can be mounted, and the measurement cell mounted in the cell holder. A light irradiating unit for irradiating light to the film carrier, and a light receiving unit for receiving the reflected light and / or transmitted light from the film carrier and measuring the received light amount And other means such as a measuring means, a liquid passing means, and a relative absorbance measuring means, which are appropriately selected according to the above.
前記バイオセンサー装置の概念図の一例を図１に、測定用セルの概念図を図２に示す。
前記バイオセンサー装置は、例えば、図２に平面図及び断面図を示すような、液体の被測定試料の流路１２及び前記被測定試料を通液可能な膜状担体１３を備える測定用セル１０と、前記測定用セル１０を装着可能なセルホルダ２１と、前記セルホルダ２１に装着された前記測定用セル１０中の前記膜状担体１３に対し、光を照射する光照射部３０と、前記膜状担体からの反射光及び透過光の少なくともいずれかを受光し、受光した光量を測定する受光部４０とを備え、光照射部３０及び受光部４０を遮光状態で収容するハウジング２０を備えているものが挙げられる。
An example of a conceptual diagram of the biosensor device is shown in FIG. 1, and a conceptual diagram of a measurement cell is shown in FIG.
The biosensor device includes, for example, a measurement cell 10 including a flow channel 12 of a liquid sample to be measured and a membrane carrier 13 through which the sample to be measured can pass, as shown in FIG. A cell holder 21 to which the measurement cell 10 can be attached, a light irradiation unit 30 for irradiating light to the film carrier 13 in the measurement cell 10 attached to the cell holder 21, and the film shape A light receiving unit 40 that receives at least one of reflected light and transmitted light from the carrier and measures the amount of received light, and includes a housing 20 that houses the light irradiation unit 30 and the light receiving unit 40 in a light-shielded state. Is mentioned.
＜測定用セル＞
前記測定用セルとしては、液体の被測定試料を通液可能な膜状担体を備える限り、特に制限はなく、目的に応じて適宜選択することができる。
<Measurement cell>
The measurement cell is not particularly limited as long as it includes a membrane-like carrier capable of passing a liquid sample to be measured, and can be appropriately selected according to the purpose.
前記測定用セルの形状としては、例えば、流路を有し、該流路中に膜状担体が配置されてなる形状が好ましく、例えば、図２に示すように、流路１２中に、前記膜状担体１３が、支持体１１に挟持されてなるものが挙げられる。
前記流路としては、少なくとも前記液体の被測定試料が通過可能である限り特に制限はなく、いわゆる開口部であってもよい。
The shape of the measurement cell is preferably, for example, a shape having a flow path and a membranous carrier disposed in the flow path. For example, as shown in FIG. An example is one in which the film carrier 13 is sandwiched between the supports 11.
The flow path is not particularly limited as long as at least the liquid sample to be measured can pass therethrough, and may be a so-called opening.
前記測定用セルの前記支持体の素材としては、液体の被測定試料を通液させるものであれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、ポリスチレン、ポリエステル、芳香族ポリアミド、ナイロン、ポリオレフィン等の合成樹脂、ステンレス等の金属、ガラスなどが挙げられる。 The material of the support of the measurement cell is not particularly limited as long as it allows liquid sample to be measured, and can be appropriately selected according to the purpose. For example, polystyrene, polyester, aromatic Examples thereof include synthetic resins such as group polyamide, nylon and polyolefin, metals such as stainless steel, and glass.
＜膜状担体＞
前記膜状担体としては、前記測定用セル中に備えられ、前記液体の被測定試料を通液可能である限り、特に制限はなく、目的に応じて適宜選択することができるが、繊維状物質からなることが好ましく、前記被験物質及び前記補足用物質のいずれかが固定化されてなることが好ましい。
<Membrane carrier>
The membrane carrier is not particularly limited as long as it is provided in the measurement cell and can pass the liquid sample to be measured, and can be appropriately selected according to the purpose. Preferably, any one of the test substance and the supplementary substance is immobilized.
前記繊維状物質としては、綿、絹、麻等の天然繊維、ポリエステル、芳香族ポリアミド、ナイロン、ポリオレフィン等の合成繊維、及びこれらの混合体などが挙げられる。
前記繊維状物質からなる前記膜状担体としては、例えば、織物、編物、不織布、並びにファイバやフィラメントを一定容積内に充填して膜状としたもの、及び単に絡合させて膜状としたものなどが挙げられる。
Examples of the fibrous material include natural fibers such as cotton, silk and hemp, synthetic fibers such as polyester, aromatic polyamide, nylon and polyolefin, and mixtures thereof.
Examples of the film carrier made of the fibrous material include a woven fabric, a knitted fabric, a non-woven fabric, and a film formed by filling fibers and filaments in a certain volume, and a film formed by simply entanglement. Etc.
前記繊維状物質からなる前記膜状担体としては、天然繊維からなる不織布が好ましい。前記天然繊維の中でも、繊維が数μｍ程度に細く、大きな比表面積を有し、高い固定化効率を示すとともに、親水性が高く、通液性等にも優れるため、綿がより好ましい。 The membrane carrier made of the fibrous material is preferably a nonwoven fabric made of natural fibers. Among the natural fibers, cotton is more preferable because the fibers are as thin as several μm, have a large specific surface area, exhibit high immobilization efficiency, high hydrophilicity, and excellent liquid permeability.
前記繊維状物質の繊維径としては、例えば、０．５〜１０μｍが好ましい。
前記膜状担体における前記繊維状物質の厚み、及び密度としては、対象となる被測定試料の粘度等に応じて適宜選択することができるが、例えば、均一な流速および高い固定化効率を得るためには例えば５０〜２００ｃｍ３／ｃｍ２／ｓｅｃ程度の通気性を得られる密度に調整することが好ましい。
As a fiber diameter of the said fibrous substance, 0.5-10 micrometers is preferable, for example.
The thickness and density of the fibrous substance in the membrane carrier can be appropriately selected according to the viscosity of the sample to be measured and the like. For example, in order to obtain a uniform flow rate and high immobilization efficiency. For example, it is preferable to adjust the density to obtain air permeability of about 50 to 200 cm 3 / cm 2 / sec.
前記膜状担体は、前記繊維状物質を所望の形状に成形して調製してもよく、シート状に成形された前記繊維状物質からなる材料を、所望のサイズ及び形状に公知の方法で裁断や打抜することにより調製してもよい。 The membrane-like carrier may be prepared by molding the fibrous substance into a desired shape, and the material comprising the fibrous substance formed into a sheet is cut into a desired size and shape by a known method. Alternatively, it may be prepared by punching.
また、前記膜状担体の形状としては、前記測定セル内に配置可能である限り、特に制限はなく目的に応じて適宜選択することができる。前記測定セル内においては、前記光照射部からの光が照射される位置に配置される。 Further, the shape of the film carrier is not particularly limited as long as it can be arranged in the measurement cell, and can be appropriately selected according to the purpose. In the measurement cell, it is arranged at a position where light from the light irradiation unit is irradiated.
前記繊維状物質からなる前記膜状担体を用いることにより、従来の固相担体として用いられる試験管壁やビーズ等に比べて、単位容積あたりの前記被測定溶液の接触面積を大きくすることができ、高密度かつ多量に擬似抗原を固定することができる。
また、前記被測定試料を前記繊維状物質からなる前記膜状担体に通液させることにより、前記膜状担体に固定化された擬似抗原に対し、抗体を累積的に結合させることができ、測定感度を高めることができる。
By using the membranous carrier made of the fibrous substance, the contact area of the solution to be measured per unit volume can be increased compared to a test tube wall or beads used as a conventional solid phase carrier. It is possible to immobilize pseudo antigens at high density and in large quantities.
Further, by passing the sample to be measured through the membranous carrier made of the fibrous substance, the antibody can be cumulatively bound to the pseudoantigen immobilized on the membranous carrier, and the measurement is performed. Sensitivity can be increased.
前記膜状担体には、前記被験物質及び前記補足用物質の少なくともいずれかが固定化されてなることが好ましく、前記補足用物質としては、検出対象（捕捉対象）である物質に結合親和性を有する物質であれば、特に制限はなく、目的に応じて適宜選択することができるが、例えば、前記被験物質のアナログ（以下、「擬似抗原」ということがある）、前記被験物質に対する抗体、２次抗体、抗体結合性蛋白質などが挙げられる。 It is preferable that at least one of the test substance and the supplementary substance is immobilized on the membranous carrier, and the supplementary substance has a binding affinity for a substance to be detected (capture target). The substance is not particularly limited as long as it is a substance, and can be appropriately selected according to the purpose. For example, an analog of the test substance (hereinafter sometimes referred to as “pseudoantigen”), an antibody against the test substance, 2 Examples include secondary antibodies and antibody binding proteins.
前記膜状担体に対し、前記被験物質及び前記補足用物質を固定化する方法としては、前記被験物質及び前記補足用物質を変性乃至失活化させない方法であれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記被験物質及び前記補足用物質を、前記膜状担体に直接自然吸着させる方法、イオン結合や共有結合などにより結合させる方法、適当なスペーサ物質を介して間接的に結合固定する方法等が挙げられる。前記自然吸着させる方法としては、例えば、前記被験物質及び前記補足用物質を、適宜タンパク質等と混合した溶液を調製し、該溶液中に前記膜状担体を投入して吸着させる方法が挙げられる。 The method for immobilizing the test substance and the supplementary substance on the membranous carrier is not particularly limited as long as it does not denature or inactivate the test substance and the supplementary substance. For example, the test substance and the supplementary substance can be directly spontaneously adsorbed to the membrane-like carrier, the ionic bond or the covalent bond can be used, and the appropriate spacer substance can be used. Examples include a method of indirectly binding and fixing. Examples of the method for natural adsorption include a method in which a solution in which the test substance and the supplementary substance are mixed with protein or the like is prepared as appropriate, and the membranous carrier is put into the solution and adsorbed.
前記膜状担体を備えた前記測定用セルは、一体型のディスポーザブルセルの態様としてもよく、前記膜状担体のみを交換可能な構造として支持体を繰り返し使用可能な態様としてもよい。 The measurement cell provided with the film carrier may be an integrated disposable cell, or may be a structure in which the support can be repeatedly used as a structure in which only the film carrier can be exchanged.
図３に、図１に示す前記バイオセンサー装置概念図の断面図の一例を示す。図３に示すように、光照射部３０は、光源３１及びスリット３２を備え、受光部４０は、フォトダイオード４１を備える。 FIG. 3 shows an example of a cross-sectional view of the biosensor device conceptual diagram shown in FIG. As shown in FIG. 3, the light irradiation unit 30 includes a light source 31 and a slit 32, and the light receiving unit 40 includes a photodiode 41.
＜光照射部＞
前記光照射部としては、少なくとも光源を備え、前記測定セル中の前記膜状担体に光を照射可能であれば、特に制限はなく、目的に応じて適宜選択することができ、必要に応じて選択する単色光を変更可能な前記単色光選択部を備える。
<Light irradiation part>
The light irradiator is not particularly limited as long as it includes at least a light source and can irradiate the film-like carrier in the measurement cell, and can be appropriately selected according to the purpose. The monochromatic light selection unit capable of changing monochromatic light to be selected is provided.
前記光源としては、例えば、レーザ、発光ダイオード、ハロゲンランプ、タングステンランプ、等が挙げられ、これらの中でも、発光ダイオードが好ましい。 Examples of the light source include a laser, a light emitting diode, a halogen lamp, a tungsten lamp, and the like. Among these, a light emitting diode is preferable.
前記光源から発光する光が二以上の単色光である場合、及び二以上の前記光源から二以上の単色光が発光する場合には、前記膜状担体に照射される光を、前記単色光選択部によって適宜選択した単色光とすることが好ましい。
このようにして、前記膜状担体に照射する光を単色光とし、さらにその単色光を他の単色光に変更することにより、前記被測定試料中の二以上の発色物質の反射光量又は透過光量を測定することができる。
When the light emitted from the light source is two or more monochromatic lights, and when two or more monochromatic lights are emitted from two or more light sources, the light irradiating the film carrier is selected as the monochromatic light selection. It is preferable to use monochromatic light appropriately selected according to the part.
In this way, the amount of reflected or transmitted light of two or more color developing substances in the sample to be measured is obtained by changing the light emitted to the film carrier to monochromatic light and changing the monochromatic light to another monochromatic light. Can be measured.
＜受光部＞
前記受光部としては、前記光照射部から前記膜状担体に照射された光のうち、前記膜状担体からの反射光及び透過光のいずれかを受光し、受光した光量を測定可能であれば、特に制限はなく、目的に応じて適宜選択することができ、必要に応じて選択する単色光を変更可能な前記単色光選択部を備える。
<Light receiver>
As the light receiving unit, it is possible to receive either reflected light or transmitted light from the film carrier out of the light irradiated to the film carrier from the light irradiation unit and measure the received light amount. There is no particular limitation, and the monochromatic light selection unit that can be appropriately selected according to the purpose and can change monochromatic light to be selected as necessary is provided.
前記光照射部から照射される光が二以上の単色光であり、前記膜状担体からの反射光又は透過光が二以上の単色光である場合には、前記単色光選択部によって適宜選択した単色光を受光することが好ましい。
このようにして、受光する光を単色光とし、さらにその単色光を他の単色光に変更することにより、前記被測定試料中の二以上の発色物質の反射光量又は透過光量を測定することができる。
When the light emitted from the light irradiation unit is two or more monochromatic lights and the reflected light or transmitted light from the film carrier is two or more monochromatic lights, the monochromatic light selection unit appropriately selected It is preferable to receive monochromatic light.
In this way, it is possible to measure the amount of reflected light or the amount of transmitted light of two or more color developing substances in the sample to be measured by making the received light monochromatic light and changing the monochromatic light to another monochromatic light. it can.
前記バイオセンサー装置は、前記受光部により受光した光量を、電気信号の信号強度として計測可能な測定手段を備えることが好ましい。
前記測定手段は、前記受光部と接続され、前記受光部により受光した光量を、電気信号の信号強度として変換し、出力する。
The biosensor device preferably includes a measuring unit capable of measuring the amount of light received by the light receiving unit as the signal intensity of an electric signal.
The measurement means is connected to the light receiving unit, converts the amount of light received by the light receiving unit as signal strength of an electric signal, and outputs the signal.
また、前記バイオセンサー装置は、前記光照射部と前記受光部との間に反射筒を備えることが好ましい。
前記反射筒は、前記膜状担体を内包するように光照射部から照射される光の照射方向に貫通し、その内面が前記膜状担体からの反射光を反射するように構成されており、前記膜状担体により反射された光を収束することができるため、該反射筒を備えることにより、測定感度が向上する。
Moreover, it is preferable that the said biosensor apparatus is provided with a reflection cylinder between the said light irradiation part and the said light-receiving part.
The reflection tube penetrates in the irradiation direction of the light irradiated from the light irradiation unit so as to contain the film carrier, and the inner surface thereof is configured to reflect the reflected light from the film carrier, Since the light reflected by the film carrier can be converged, the measurement sensitivity is improved by providing the reflecting cylinder.
さらに、前記バイオセンサー装置は、前記被測定試料の測定により得られた光量と、基準となる試料の測定により得られた光量とから相対吸光度を計算する相対吸光度計算手段を備えることが好ましい。 Furthermore, it is preferable that the biosensor device includes a relative absorbance calculation unit that calculates a relative absorbance from a light amount obtained by measuring the sample to be measured and a light amount obtained by measuring a reference sample.
＜通液手段＞
前記測定用セルは、該測定用セルに着脱可能に接続された通液手段により、被測定試料が前記膜状担体に通液されることが好ましい。
前記通液手段により被測定試料が通液された後、前記測定用セルは前記バイオセンサー装置中のセルホルダに設置される。
前記通液手段は、前記バイオセンサーに備えられていることが好ましい。
<Liquid passing means>
In the measurement cell, it is preferable that the sample to be measured is passed through the membrane-like carrier by liquid passing means detachably connected to the measurement cell.
After the sample to be measured is passed through the liquid passing means, the measurement cell is installed in a cell holder in the biosensor device.
The liquid passing means is preferably provided in the biosensor.
前記通液手段は、前記膜状担体に対し、前記液体の被測定試料が均一に流通するよう、前記液体の被測定試料を加圧して膜状担体へ通液する手段であることが好ましく、例えば、前記測定用セルの流路にシリンジ、ポンプ、遠心分離装置等を接続し、前記液体の被測定試料を加圧して送液する手段などが挙げられる。これらは、さらに流量を制御する制御部により、加圧の動作が制御されることが好ましい。 Preferably, the liquid passing means is a means for pressurizing the liquid sample to be measured and allowing the liquid sample to be passed through the film carrier so that the liquid sample to be uniformly distributed. For example, a means for connecting a syringe, a pump, a centrifuge, or the like to the flow path of the measurement cell and pressurizing and feeding the liquid sample to be measured may be used. It is preferable that the pressurizing operation is controlled by a control unit that further controls the flow rate.
（濃度測定方法）
本発明の濃度測定方法は、本発明のバイオセンサー装置を用い、前記被測定試料中の前記被験物質を定量する方法である。
前記被測定試料は液体であり、被測定試料を通液した後の測定用セルをセルホルダに装着し、前記セルホルダに装着された前記測定用セル中の膜状担体に対し、光照射部から光を照射し、受光部で前記膜状担体からの反射光及び透過光の少なくともいずれかを受光し、受光した光量を測定することにより、前記被測定試料中の前記被験物質を定量することが好ましい。
(Concentration measurement method)
The concentration measurement method of the present invention is a method for quantifying the test substance in the sample to be measured using the biosensor device of the present invention.
The sample to be measured is a liquid, the measurement cell after passing the sample to be measured is attached to a cell holder, and the film-like carrier in the measurement cell attached to the cell holder is irradiated with light from the light irradiation unit. It is preferable to quantify the test substance in the sample to be measured by receiving at least one of reflected light and transmitted light from the film-shaped carrier at the light receiving unit and measuring the amount of light received. .
前記被測定試料としては、液体の試料である限り、特に制限はなく、目的に応じて適宜選択することができ、例えば、各種排水、下水、河川水、海水、地下水等の環境試料、飲料水、清涼飲料水、酒類、牛乳等が挙げられる。
前記膜状担体は、擬似抗原等を高密度に固定することができ、さらに前記被測定試料中の前記被験物質を累積して結合させる効果が得られるため、前記被検試料が低濃度であっても、濃縮等の処理を行うことなく測定することができる。
The sample to be measured is not particularly limited as long as it is a liquid sample, and can be appropriately selected according to the purpose. For example, various drainage, sewage, river water, seawater, groundwater and other environmental samples, drinking water , Soft drinks, alcoholic beverages, milk and the like.
Since the membranous carrier can immobilize pseudoantigens and the like at a high density and further has an effect of accumulating and binding the test substance in the sample to be measured, the test sample has a low concentration. However, the measurement can be performed without performing a treatment such as concentration.
前記被測定試料の流速としては、例えば、０．１ｍＬ／ｍｉｎ以上であることが好ましく、０．１〜２０．０ｍＬ／ｍｉｎがより好ましい。 The flow rate of the sample to be measured is, for example, preferably 0.1 mL / min or more, and more preferably 0.1 to 20.0 mL / min.
前記の濃度測定方法としては、前記膜状担体に被験物質Ｘに対する抗体Ｙを固定化する第一の態様と、前記膜状担体に被験物質Ｘ及び該被験物質のアナログＺのいずれかを固定化する第二の態様及び第三の態様とが挙げられる。 As the concentration measuring method, the first embodiment in which the antibody Y against the test substance X is immobilized on the membranous carrier, and either the test substance X or the analog Z of the test substance is immobilized on the membranous carrier. The second embodiment and the third embodiment.
＜第一の態様＞
前記濃度測定方法の第一の態様は、被験物質Ｘに対する抗体Ｙを固定化した膜状担体に、前記被験物質Ｘを標識物質で標識されてなる標識被験物質Ｘ’を添加した被測定試料を通液し、前記膜状担体上の前記抗体Ｙに対し、前記被測定試料中の被験物質Ｘと、前記標識被験物質Ｘ’とを反応させ、前記膜状担体上の前記抗体Ｙに結合した前記標識被験物質Ｘ’の量を、前記膜状担体の反射光量及び透過光量のいずれかとして検出し、該反射光量及び透過光量のいずれかから、前記被験物質Ｘと前記標識被験物質Ｘ’との反応の度合いを求め、該反応の度合いから、前記被測定試料中の被験物質Ｘの濃度を算出する方法である。
<First aspect>
In the first aspect of the concentration measurement method, a sample to be measured is obtained by adding a labeled test substance X ′ obtained by labeling the test substance X with a labeling substance to a film carrier on which an antibody Y against the test substance X is immobilized. The test substance X in the sample to be measured and the labeled test substance X ′ were reacted with the antibody Y on the membranous carrier and bound to the antibody Y on the membranous carrier. The amount of the labeled test substance X ′ is detected as either the amount of reflected light or the amount of transmitted light of the film carrier, and the test substance X and the labeled test substance X ′ are detected from either the amount of reflected light or the amount of transmitted light. Is obtained, and the concentration of the test substance X in the sample to be measured is calculated from the degree of the reaction.
前記被験物質Ｘに対する前記抗体Ｙとしては、前記被験物質及びそのアナログのいずれかと結合可能である限り、特に制限は無く、目的に応じて適宜選択することができ、公知の方法により適宜調製してもよい。 The antibody Y against the test substance X is not particularly limited as long as it can bind to either the test substance or an analog thereof, and can be appropriately selected according to the purpose. Also good.
＜第二の態様＞
前記濃度測定方法の第二の態様は、被験物質Ｘ及び該被験物質のアナログＺのいずれかを固定化した膜状担体に、前記被験物質Ｘに対する抗体であって標識物質で標識されてなる標識抗体Ｙを添加した被測定試料を通液し、前記膜状担体上の前記被験物質Ｘ及び前記被験物質のアナログＺのいずれかに対し、前記被測定試料中の前記被験物質Ｘと結合していない前記標識抗体Ｙを結合させ、前記標識抗体Ｙの量を、前記膜状担体の反射光量及び透過光量のいずれかとして検出し、該反射光量及び透過光量のいずれかから、前記被測定試料中の前記被験物質Ｘの濃度を算出する方法である。
<Second aspect>
The second aspect of the concentration measurement method is a label formed by labeling a test substance X and an analog Z of the test substance with a labeling substance, which is an antibody against the test substance X, on a membranous carrier. A sample to be measured to which the antibody Y is added is passed through and bound to the test substance X in the sample to be measured with respect to either the test substance X or the analog Z of the test substance on the membranous carrier. The labeled antibody Y is not bound, and the amount of the labeled antibody Y is detected as either the amount of reflected light or the amount of transmitted light of the film carrier, and from either the amount of reflected light or the amount of transmitted light, In which the concentration of the test substance X is calculated.
＜第三の態様＞
前記濃度測定方法の第三の態様は、被験物質Ｘ及び該被験物質のアナログＺのいずれかを固定化した膜状担体に、前記被験物質に対する抗体Ｙを添加した被測定試料を通液し、前記膜状担体上の前記被験物質Ｘ及び前記被験物質のアナログＺのいずれかに対し、前記被測定試料中の前記被験物質Ｘと結合していない前記抗体Ｙを結合させ、前記抗体Ｙに、該抗体Ｙに対する二次抗体であって標識物質で標識されてなる標識抗体Ｙ’を結合させ、前記標識抗体Ｙ’の量を、前記膜状担体の反射光量及び透過光量のいずれかとして検出し、該反射光量及び透過光量のいずれかから、前記被測定試料中の前記被験物質Ｘの濃度を算出する方法である。
<Third embodiment>
In the third aspect of the concentration measurement method, a sample to be measured in which an antibody Y against the test substance is added to a membranous carrier on which either the test substance X or the analog Z of the test substance is immobilized is passed through, The antibody Y that is not bound to the test substance X in the sample to be measured is bound to either the test substance X or the analog Z of the test substance on the membranous carrier, A labeled antibody Y ′ that is a secondary antibody against the antibody Y and labeled with a labeling substance is bound, and the amount of the labeled antibody Y ′ is detected as either the amount of reflected light or the amount of transmitted light of the film carrier. The concentration of the test substance X in the sample to be measured is calculated from either the reflected light amount or the transmitted light amount.
前記膜状担体の反射光量及び透過光量のいずれかを結合した標識物質の量として測定する方法としては、特に制限はなく、前記標識物質の種類に応じて適宜選択することができる。
前記標識物質としては、特に制限はなく、目的に応じて適宜選択することができるが、例えば、ペルオキシターゼ、アルカリフォスファターゼ、チロシナーゼ、ガラクトシダーゼ、ルシフェラーゼ、グルコースオキシダーゼ等の酵素、Ｉ１２５、Ｉ１３１、Ｈ３等の放射性同位体、テトラメチルローダミン、ユーロピウムキレート、フルオレセインなどのイソシアネ―ト誘導物質等の蛍光物質、アクリジニウムエステル、アクリジニウムスルホン酸、ルシフェリン、ＮＡＤオキシドレダクターゼ、ルシゲニン、ロシフィン、金コロイド、セレンコロイド、着色ラテックス、磁性微粒子等の着色微粒子、ピペリジン−Ｎ−オキシド誘導体、ピロリヂン−Ｎ−オキシド誘導体、オキサゾリジン−Ｎ−オキシド誘導体のようなフリーラジカルを示す物質などが挙げられ、これらの中でも酵素、放射性同位元素、蛍光物質、及び着色微粒子、並びにこれらにより標識された抗体が好ましく、着色微粒子、及び着色微粒子により標識された抗体がより好ましい。
The method for measuring the amount of the labeled substance combined with either the reflected light amount or the transmitted light amount of the film carrier is not particularly limited, and can be appropriately selected according to the type of the labeling substance.
The labeling substance is not particularly limited and may be appropriately selected depending on the intended purpose. For example, enzymes such as peroxidase, alkaline phosphatase, tyrosinase, galactosidase, luciferase and glucose oxidase, I 125 , I 131 , H 3 Radioisotopes such as tetramethylrhodamine, europium chelate, fluorescent substances such as isocyanate derivatives such as fluorescein, acridinium ester, acridinium sulfonic acid, luciferin, NAD oxidoreductase, lucigenin, rosifine, colloidal gold, Free radicals such as selenium colloid, colored latex, colored fine particles such as magnetic fine particles, piperidine-N-oxide derivatives, pyrrolidine-N-oxide derivatives, oxazolidine-N-oxide derivatives Such as to material and the like, enzymes Of these, radioactive isotopes, fluorescent substances, and coloring fine particles, and an antibody is preferably labeled with these colored particles, and the antibody is more preferably labeled with colored particles.
前記測定方法としては、例えば、前記標識物質が酵素の場合、該酵素と基質とを反応させた後、反応生成物の反射吸光度、蛍光吸光度、発光強度等を前記膜状担体の反射光として測定する方法が挙げられ、前記標識物質が放射性同位体の場合は、放射線量を前記膜状担体の反射光として測定する方法、前記標識物質が蛍光物質の場合、蛍光強度を前記膜状担体の反射光として測定する方法、前記標識物質が発光物質の場合、発光強度を前記膜状担体の反射光として測定する方法、前記標識物質が着色微粒子の場合、着色により変化する前記膜状担体の透過光量を測定する方法が挙げられる。
これらの中でも、着色により変化する前記膜状担体の透過光量を測定する方法が好ましい。
As the measurement method, for example, when the labeling substance is an enzyme, the reaction product is reacted with the substrate, and then the reflected absorbance, fluorescence absorbance, emission intensity, etc. of the reaction product are measured as reflected light of the film carrier. When the labeling substance is a radioisotope, the radiation dose is measured as reflected light from the film-like carrier. When the labeling substance is a fluorescent substance, the fluorescence intensity is reflected from the film-like carrier. A method of measuring as light, a method of measuring the emission intensity as reflected light of the film carrier when the labeling substance is a luminescent substance, and a transmitted light amount of the film carrier changing by coloring when the labeling substance is colored fine particles The method of measuring is mentioned.
Among these, the method of measuring the transmitted light amount of the film-like carrier that changes due to coloring is preferable.
これらの測定された光量は、電気信号の信号強度として変換されることが好ましく、変換された信号強度は、例えば、１秒毎に測定し、コンピュータなどに記録し、最終的に得られた残存シグナル値と、ベースライン値との差を測定値としてボルト単位で表す方法が好ましい。 These measured light quantities are preferably converted as the signal strength of an electrical signal. The converted signal strength is measured, for example, every second, recorded in a computer, etc., and finally obtained residual A method of expressing the difference between the signal value and the baseline value as a measured value in volts is preferable.
前記第二の態様、すなわち、被験物質Ｘ又はそのアナログＺを固定した前記膜状担体上に捕捉される標識抗体Ｙ’の量から、前記被測定試料中の前記被験物質Ｘの濃度を求める方法の例を示す。
まず、ある濃度の前記標識抗体Ｙ’を前記膜状担体上に結合させた場合の信号強度（ブランク、例えば「Ｆ０」とする）を測定しておく。次に、前記ブランク測定時と同じ濃度の前記抗体Ｙを添加した被測定試料を、前記膜状担体に供給し、未結合の前記標識抗体Ｙ’を前記膜状担体上に結合させた場合の信号強度（例えば、「Ｆ１」とする）を測定する。
ここで、前記被測定試料中に被験物質Ｘが存在するとき、前記標識抗体Ｙ’は前記被測定試料中の被験物質Ｘと結合するため、前記膜状担体上に結合する未結合の前記標識抗体Ｙ’の量は少なくなる。したがって、信号強度がＦ１＜Ｆ０となるとき、前記被測定試料中に被験物質Ｘが存在すると判断することができ、Ｆ１の低下割合が、前記被測定試料中に含まれる前記被験物質Ｘの量と相関関係を有するため、既知の濃度の被験物質Ｘを用い、予め検量線を作成しておくことにより、Ｆ１の低下割合に応じて前記被験物質Ｘの定量を行うことができる。
In the second aspect, that is, a method for determining the concentration of the test substance X in the sample to be measured from the amount of the labeled antibody Y ′ captured on the membranous carrier on which the test substance X or its analog Z is fixed. An example of
First, the signal intensity (blank, for example, “F0”) when a certain concentration of the labeled antibody Y ′ is bound on the membrane-like carrier is measured. Next, the sample to be measured to which the antibody Y having the same concentration as that in the blank measurement was added is supplied to the membrane-like carrier, and the unbound labeled antibody Y ′ is bound to the membrane-like carrier. The signal strength (for example, “F1”) is measured.
Here, when the test substance X is present in the sample to be measured, the labeled antibody Y ′ binds to the test substance X in the sample to be measured, so that the unbound label that binds to the membranous carrier The amount of antibody Y ′ is reduced. Therefore, when the signal intensity satisfies F1 <F0, it can be determined that the test substance X is present in the sample to be measured, and the decrease rate of F1 is the amount of the test substance X contained in the sample to be measured. Therefore, the test substance X can be quantified according to the decrease rate of F1 by preparing a calibration curve in advance using a test substance X having a known concentration.
（ＰＣＢ濃度測定方法）
前記濃度測定方法による本発明のＰＣＢ濃度測定方法は、下記構造式（１）で表される擬似抗原を固定化した膜状担体に、抗ＰＣＢ抗体を添加した被測定試料を通液し、前記膜状担体上の前記擬似抗原に対し、前記被測定試料中のＰＣＢと結合していない前記抗ＰＣＢ抗体を結合させた後、前記抗ＰＣＢ抗体に対する二次抗体であって、標識物質で標識されてなる二次抗体を結合させ、前記二次抗体の標識物質の量を、前記膜状担体の反射光量及び透過光量のいずれかとして検出し、該反射光量及び透過光量のいずれかから、前記被測定試料中のＰＣＢの濃度を算出する方法である。
(PCB concentration measurement method)
The PCB concentration measurement method of the present invention by the concentration measurement method is such that a sample to be measured to which an anti-PCB antibody is added is passed through a membranous carrier on which a pseudoantigen represented by the following structural formula (1) is immobilized, A secondary antibody against the anti-PCB antibody, which is labeled with a labeling substance after binding the anti-PCB antibody not bound to PCB in the sample to be measured to the pseudoantigen on the membranous carrier. The amount of the labeling substance of the secondary antibody is detected as either the amount of reflected light or the amount of transmitted light of the film carrier, and the amount of the target substance is detected from either the amount of reflected light or the amount of transmitted light. This is a method for calculating the concentration of PCB in a measurement sample.
前記ＰＣＢ濃度測定方法としては、例えば、被験物質Ｘ（ＰＣＢ）のアナログ（ジクロロフェノール誘導体と牛血清アルブミン）を固定化した膜状担体に、前記被験物質に対する抗体Ｙ（抗ＰＣＢ抗体）を添加した被測定試料を通液し、前被験物質（ＰＣＢ）のアナログ（ジクロロフェノール誘導体と牛血清アルブミン）に対し、前記被測定試料中の前記被験物質Ｘ（ＰＣＢ）と結合していない前記抗体Ｙを結合させ、前記抗体Ｙに対する標識物質（金コロイド）で標識した二次抗体Ｙ’を結合させ、前記標識抗体Ｙ’の量を、前記膜状担体の透過光量として検出し、該透過光量から、前記被測定試料中の前記被験物質Ｘ（ＰＣＢ）の濃度を算出する方法が挙げられる。 As the method for measuring the PCB concentration, for example, an antibody Y (anti-PCB antibody) against the test substance was added to a membranous carrier on which an analog of the test substance X (PCB) (dichlorophenol derivative and bovine serum albumin) was immobilized. The antibody Y that is not bound to the test substance X (PCB) in the sample to be measured is passed through the sample to be measured, and the analog (dichlorophenol derivative and bovine serum albumin) of the previous test substance (PCB). The secondary antibody Y ′ labeled with a labeling substance (gold colloid) is bound to the antibody Y, and the amount of the labeled antibody Y ′ is detected as the amount of light transmitted through the membrane-like carrier, Examples include a method of calculating the concentration of the test substance X (PCB) in the sample to be measured.
本発明の濃度測定方法によるＰＣＢ濃度測定方法によれば、前記膜状担体上に前記擬似抗原を高密度に固定化可能であるため、被検試料中のＰＣＢを高感度に検出可能であり、また迅速に被験物質の濃度が測定可能である。 According to the PCB concentration measurement method according to the concentration measurement method of the present invention, the pseudoantigen can be immobilized at a high density on the membranous carrier, so that PCB in a test sample can be detected with high sensitivity, In addition, the concentration of the test substance can be measured quickly.
以下、本発明の実施例について説明するが、本発明はこの実施例に何ら限定されるものではない。 Hereinafter, although the Example of this invention is described, this invention is not limited to this Example at all.
（実施例１）
−膜状担体の調製−
ポリオレフィン繊維からなる不織布（厚み３．３ｍｍ）を、直径５ｍｍの円形に切り出し、前記膜状担体とした。
前記膜状担体１０枚を、擬似抗原としてビタミン−牛血清アルブミンの複合体を１００μg／ｍＬの濃度で含む生理食塩水３０ｍＬに入れ、一昼夜（１４時間）振とうした。次いで、牛血清アルブミンを１０ｍｇ／ｍＬとなるように添加し、２時間振とうした。
擬似抗原（ビタミン−牛血清アルブミン）を固定化した膜状担体フィルターは、生理食塩水で洗浄した後、前記測定用セルに設置した。
前記測定用セルには直径３ｍｍの孔部があり、該孔部に接続した注射器等の通液手段から、前記被測定試料を供給し、通液させることができる。
Example 1
-Preparation of membrane carrier-
A non-woven fabric (thickness: 3.3 mm) made of polyolefin fibers was cut into a circle with a diameter of 5 mm, and used as the film carrier.
Ten membrane-like carriers were placed in 30 mL of physiological saline containing a vitamin-bovine serum albumin complex at a concentration of 100 μg / mL as a pseudoantigen and shaken all day and night (14 hours). Next, bovine serum albumin was added to 10 mg / mL and shaken for 2 hours.
The membranous carrier filter on which the pseudo antigen (vitamin-bovine serum albumin) was immobilized was washed with physiological saline and then placed in the measurement cell.
The measurement cell has a hole having a diameter of 3 mm, and the sample to be measured can be supplied and allowed to flow from a liquid passing means such as a syringe connected to the hole.
−被測定試料の調製−
抗ビタミン抗体（生化学工業社製）を、牛血清アルブミンを１ｇ／Ｌ含む生理食塩水に０．８ｎＭ程度に溶解して抗体溶液を調製した。
粉末状ビタミン（和光純薬工業社製）を、牛血清アルブミンを１ｇ／Ｌ含む生理食塩水に溶解してビタミン溶液を調製した。
前記抗体溶液を１ｍＬ分取し、ここにビタミン溶液（１ｇ／ｍＬ）を牛血清アルブミンを１ｇ／Ｌ含む生理食塩水で希釈して添加し、0.001μＭ、0.1μＭ、10μＭ、100μＭの計５種の濃度の被測定試料を調製した。基準液として、ビタミンを含まない被測定試料も調製した。
-Preparation of sample to be measured-
An antibody solution was prepared by dissolving anti-vitamin antibody (manufactured by Seikagaku Corporation) in physiological saline containing 1 g / L of bovine serum albumin to about 0.8 nM.
A vitamin solution was prepared by dissolving powdered vitamin (manufactured by Wako Pure Chemical Industries, Ltd.) in physiological saline containing 1 g / L of bovine serum albumin.
Take 1 mL of the antibody solution, add vitamin solution (1 g / mL) diluted with physiological saline containing 1 g / L of bovine serum albumin, and add 5 kinds of 0.001 μM, 0.1 μM, 10 μM, and 100 μM in total. A sample to be measured having a concentration of 5 was prepared. As a reference solution, a sample to be measured not containing vitamins was also prepared.
−透過光量の測定−
調製した前記被測定試料1ｍＬを注射器にとり、０．２ｍＬ／ｍｉｎで前記測定用セル中に注射器からポンプによって一定流速で供給し、前記測定セル内の前記膜状担体に通液させた。
次いで、０．６ｍＬ／ｍｉｎで牛血清アルブミンを１ｇ／Ｌ含む生理食塩水に１ｍＬを通液した後、牛血清アルブミンを１ｇ／Ｌ含む生理食塩水に２ｎＭの濃度で溶解した金コロイド標識二次抗体溶液の１ｍＬを０．２ｍＬ／ｍｉｎで通液した。最後に、０．６ｍＬ／ｍｉｎで牛血清アルブミンを１ｇ／Ｌ含む生理食塩水に１ｍＬを通液して、前記膜状担体からの透過光量を測定した。
なお、前記膜状担体に対し、発光ダイオードの光を照射した。
受光した透過光量は、電気信号の信号強度として計測され、基準液による透過光量に対する信号強度を１００として、各濃度の被測定試料における透過光量に対する信号強度の割合を算出し、前記被測定試料中のビタミン濃度との相関を求めた。捕捉される抗体量が多いほど、標識金コロイドに由来する膜上の赤色が濃くなり、信号値としては小さくなる。結果を図４に示す。
-Measurement of transmitted light amount-
1 mL of the prepared sample to be measured was taken into a syringe, supplied at a constant flow rate from the syringe into the measurement cell at a constant flow rate at 0.2 mL / min, and passed through the membrane carrier in the measurement cell.
Next, after passing 1 mL through a physiological saline containing 1 g / L of bovine serum albumin at 0.6 mL / min, a secondary colloidal gold label dissolved in a physiological saline containing 1 g / L of bovine serum albumin at a concentration of 2 nM. 1 mL of the antibody solution was passed at 0.2 mL / min. Finally, 1 mL was passed through physiological saline containing 1 g / L of bovine serum albumin at 0.6 mL / min, and the amount of light transmitted from the membrane carrier was measured.
The film carrier was irradiated with light from a light emitting diode.
The amount of transmitted light received is measured as the signal intensity of the electrical signal, and the ratio of the signal intensity to the amount of transmitted light in the sample to be measured at each concentration is calculated with the signal intensity with respect to the amount of light transmitted by the reference solution being 100. Correlation with vitamin concentration was obtained. The greater the amount of antibody captured, the deeper the red color on the membrane derived from the labeled gold colloid, and the smaller the signal value. The results are shown in FIG.
（実施例２）
−膜状担体の調製−
ポリオレフィン繊維からなる不織布（厚み３．３ｍｍ）を、直径５ｍｍの円形に切り出し、前記膜状担体とした。
前記膜状担体１０枚を、擬似抗原として下記構造式であらわす化合物を１００μg／ｍＬの濃度で含む生理食塩水３０ｍＬに入れ、一昼夜（１４時間）振とうした。次いで、牛血清アルブミンを１０ｍｇ／ｍＬとなるように添加し、２時間振とうした。
擬似抗原（ジクロロフェノール誘導体−牛血清アルブミン）を固定化した膜状担体フィルターは、生理食塩水で洗浄した後、前記測定用セルに設置した。
前記測定用セルには直径３ｍｍの孔部があり、該孔部に接続した注射器等の通液手段から、前記被測定試料を供給し、通液させることができる。
(Example 2)
-Preparation of membrane carrier-
A non-woven fabric (thickness: 3.3 mm) made of polyolefin fibers was cut into a circle with a diameter of 5 mm, and used as the film carrier.
Ten membrane-like carriers were put in 30 mL of physiological saline containing a compound represented by the following structural formula as a pseudoantigen at a concentration of 100 μg / mL, and shaken all day and night (14 hours). Next, bovine serum albumin was added to 10 mg / mL and shaken for 2 hours.
The membranous carrier filter on which the pseudo antigen (dichlorophenol derivative-bovine serum albumin) was immobilized was washed with physiological saline and then placed in the measurement cell.
The measurement cell has a hole having a diameter of 3 mm, and the sample to be measured can be supplied and allowed to flow from a liquid passing means such as a syringe connected to the hole.
−被測定試料の調製−
−−抗ＰＣＢ抗体の調製−−
被験物質ＰＣＢに対する抗ＰＣＢモノクローナル抗体を調製するため、３塩素化物ＰＣＢ（三塩化ビフェニル）を、リンカーを介してキャリアータンパク質（スカシガイ由来のヘモシアニン（以下、「ＫＬＨ」と表す））に結合した複合体を合成した。
-Preparation of sample to be measured-
--Preparation of anti-PCB antibody--
In order to prepare an anti-PCB monoclonal antibody against the test substance PCB, a complex in which trichlorinated PCB (biphenyl trichloride) is bound to a carrier protein (hemiocyanin derived from mussel (hereinafter referred to as “KLH”)) via a linker. Was synthesized.
前記複合体を抗原として、マウス（Ｂｕｌｂ／ｃ、メス、５週齢）に免疫した。
初回免疫は、前記抗体調製用化合物（タンパク質量として約０．３ｍｇ）を完全アジュバントに混合後、皮下注射した。該初回免疫の２週間後と４週間後に、同量の前記抗体調製用化合物を、不完全アジュバントに混合後、皮下注射した。その後、１週間以上経過した後、同量の前記抗体調製用化合物を、腹腔又は尾部静脈に注射し、その４〜５日後に脾臓を摘出した。
Mice (Bulb / c, female, 5 weeks old) were immunized with the complex as an antigen.
For the first immunization, the compound for antibody preparation (about 0.3 mg as protein amount) was mixed with complete adjuvant and then injected subcutaneously. Two weeks and four weeks after the initial immunization, the same amount of the compound for antibody preparation was mixed with incomplete adjuvant and then injected subcutaneously. Thereafter, after one week or more had elapsed, the same amount of the compound for antibody preparation was injected into the abdominal cavity or tail vein, and the spleen was removed 4 to 5 days later.
摘出した前記脾臓から調製した脾臓細胞を、ミエローマ細胞とともにポリエチレングリコール溶液中で２分間混合し、細胞融合を行った。
融合反応後の融合細胞を培養し、細胞融合から２週間以上経過したハイブリドーマの培養上清を用い、所望の抗ＰＣＢモノクローナル抗体の有無をスクリーニングし、抗ＰＣＢモノクローナル抗体を産生する安定なハイブリドーマを得た。該ハイブリドーマを培養し、培養上清を精製し、抗ＰＣＢモノクローナル抗体を得た。
Spleen cells prepared from the extracted spleen were mixed with myeloma cells in a polyethylene glycol solution for 2 minutes to perform cell fusion.
After culturing the fused cells after the fusion reaction, the hybridoma culture supernatant that has passed for two weeks or more after cell fusion is used to screen for the presence of the desired anti-PCB monoclonal antibody to obtain a stable hybridoma that produces the anti-PCB monoclonal antibody. It was. The hybridoma was cultured, and the culture supernatant was purified to obtain an anti-PCB monoclonal antibody.
前記抗ＰＣＢ抗体を、牛血清アルブミンを１ｇ／Ｌ含む生理食塩水に０．５ｎＭ程度に溶解して抗体溶液を調製した。
ＰＣＢの異性体混合物であるカネクロール−５００（ＧＬサイエンス社製）のジメチルスルホキシド溶液を、牛血清アルブミンを１ｇ／Ｌ含む生理食塩水に溶解してＰＣＢ溶液を調製した。
前記抗体溶液を１ｍＬ分取し、ここにＰＣＢ溶液（２０ｐｐｍ）を、牛血清アルブミンを１ｇ／Ｌ含む生理食塩水で希釈して添加し、２ppm、8ppm、20ppm、80ppm、200ppmの計５種の濃度の被測定試料を調製した。基準液として、ＰＣＢを含まない被測定試料も調製した。
The anti-PCB antibody was dissolved to about 0.5 nM in physiological saline containing 1 g / L of bovine serum albumin to prepare an antibody solution.
A PCB solution was prepared by dissolving a dimethyl sulfoxide solution of Kanechlor-500 (manufactured by GL Science), which is an isomer mixture of PCB, in physiological saline containing 1 g / L of bovine serum albumin.
1 mL of the antibody solution is taken, and a PCB solution (20 ppm) is diluted with a physiological saline containing 1 g / L of bovine serum albumin and added thereto, and 5 kinds of total of 2 ppm, 8 ppm, 20 ppm, 80 ppm, and 200 ppm are added. A sample to be measured was prepared. As a reference solution, a sample to be measured that did not contain PCB was also prepared.
−透過光量の測定−
調製した前記被測定試料1ｍＬを注射器にとり、０．２ｍＬ／ｍｉｎで前記測定用セル中に注射器からポンプによって一定流速で供給し、前記測定セル内の前記膜状担体に通液させた。
次いで、０．２ｍＬ／ｍｉｎで牛血清アルブミンを１ｇ／Ｌ含む生理食塩水に１ｍＬを通液した後、牛血清アルブミンを１ｇ／Ｌ含む生理食塩水に２ｎＭの濃度で溶解した金コロイド標識二次抗体溶液の１ｍＬを０．２ｍＬ／ｍｉｎで通液した。最後に、０．６ｍＬ／ｍｉｎで牛血清アルブミンを１ｇ／Ｌ含む生理食塩水に１ｍＬを通液して、前記膜状担体からの透過光量を測定した。
なお、前記膜状担体に対し、発光ダイオードの光を照射した。
受光した透過光量は、電気信号の信号強度として計測され、基準液による透過光量に対する信号強度を１００として、各濃度の被測定試料における透過光量に対する信号強度の割合を算出し、前記被測定試料中のビタミン濃度との相関を求めた。捕捉される抗体量が多いほど、標識金コロイドに由来する膜上の赤色が濃くなり、信号値としては小さくなる。結果を図５に示す。
-Measurement of transmitted light amount-
1 mL of the prepared sample to be measured was taken into a syringe, supplied at a constant flow rate from the syringe into the measurement cell at a constant flow rate at 0.2 mL / min, and passed through the membrane carrier in the measurement cell.
Next, after passing 1 mL through a physiological saline containing 1 g / L of bovine serum albumin at 0.2 mL / min, a secondary colloidal gold label dissolved in a physiological saline containing 1 g / L of bovine serum albumin at a concentration of 2 nM. 1 mL of the antibody solution was passed at 0.2 mL / min. Finally, 1 mL was passed through physiological saline containing 1 g / L of bovine serum albumin at 0.6 mL / min, and the amount of light transmitted from the membrane carrier was measured.
The film carrier was irradiated with light from a light emitting diode.
The amount of transmitted light received is measured as the signal intensity of the electrical signal, and the ratio of the signal intensity to the amount of transmitted light in the sample to be measured at each concentration is calculated with the signal intensity with respect to the amount of light transmitted by the reference solution being 100. Correlation with vitamin concentration was obtained. The greater the amount of antibody captured, the deeper the red color on the membrane derived from the labeled gold colloid, and the smaller the signal value. The results are shown in FIG.
図４及び図５の結果から、本発明のバイオセンサー装置を用いた本発明の濃度測定方法は、被検試料中の被験物質を効率よく高感度に定量できることが明らかになった。 From the results of FIG. 4 and FIG. 5, it was revealed that the concentration measuring method of the present invention using the biosensor device of the present invention can quantitate the test substance in the test sample efficiently and with high sensitivity.
本発明のバイオセンサー装置は、被験物質を高感度に検出可能であり、低コストで効率よく調製可能な担体を備え、迅速に被験物質の濃度が測定可能であるため、例えば、環境汚染物質等の低分子量の有害物質や、ビタミン様物質等の低分子量の測定に好適である。
さらに、本発明の濃度測定方法は、本発明のバイオセンサー装置を用いるため、例えば、環境汚染物質等の低分子量の有害物質や、ビタミン様物質等の低分子量の測定方法として好適である。
The biosensor device of the present invention can detect a test substance with high sensitivity, has a carrier that can be efficiently prepared at low cost, and can quickly measure the concentration of the test substance. It is suitable for the measurement of low molecular weight substances such as low molecular weight harmful substances and vitamin-like substances.
Furthermore, the concentration measurement method of the present invention uses the biosensor device of the present invention, and is therefore suitable as a measurement method of low molecular weights such as low molecular weight harmful substances such as environmental pollutants and vitamin-like substances.
Claims (6)
前記膜状担体上の前記擬似抗原に対し、前記被測定試料中のビタミンと結合していない前記抗ビタミン抗体を結合させた後、
前記抗ビタミン抗体に対する二次抗体であって、標識物質で標識されてなる二次抗体を結合させ、
前記二次抗体の標識物質の量を、前記膜状担体の透過光量として検出し、該透過光量から、前記被測定試料中のビタミンの濃度を算出することを特徴とするビタミンの濃度測定方法。 Pass the sample to be measured to which an anti-vitamin antibody was added to a membrane carrier made of a fibrous material in which a complex of vitamin-bovine serum albumin was immobilized as a pseudoantigen,
After binding the anti-vitamin antibody not bound to the vitamin in the sample to be measured to the pseudoantigen on the membrane carrier,
A secondary antibody against the anti-vitamin antibody, wherein a secondary antibody labeled with a labeling substance is bound;
A method for measuring the concentration of vitamins, comprising detecting the amount of the labeling substance of the secondary antibody as the amount of light transmitted through the membranous carrier, and calculating the concentration of vitamin in the sample to be measured from the amount of light transmitted.
前記膜状担体上の前記擬似抗原に対し、前記被測定試料中のＰＣＢと結合していない前記抗ＰＣＢ抗体を結合させた後、
前記抗ＰＣＢ抗体に対する二次抗体であって、標識物質で標識されてなる二次抗体を結合させ、
前記二次抗体の標識物質の量を、前記膜状担体の透過光量として検出し、該透過光量から、前記被測定試料中のＰＣＢの濃度を算出することを特徴とするＰＣＢの濃度測定方法。
After binding the anti-PCB antibody not bound to PCB in the sample to be measured to the pseudoantigen on the membrane carrier,
A secondary antibody against the anti-PCB antibody, which is bound with a secondary antibody labeled with a labeling substance;
A method for measuring the concentration of PCB, comprising detecting the amount of labeling substance of the secondary antibody as the amount of light transmitted through the membranous carrier, and calculating the concentration of PCB in the sample to be measured from the amount of light transmitted.
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