JP2017062145A - Immunoassay kit and immunoassay - Google Patents
Immunoassay kit and immunoassay Download PDFInfo
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- JP2017062145A JP2017062145A JP2015186581A JP2015186581A JP2017062145A JP 2017062145 A JP2017062145 A JP 2017062145A JP 2015186581 A JP2015186581 A JP 2015186581A JP 2015186581 A JP2015186581 A JP 2015186581A JP 2017062145 A JP2017062145 A JP 2017062145A
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Abstract
Description
本発明は、免疫測定用キット及び免疫測定方法に関し、特に、洗浄工程を不要とすることで、短時間で検体中の測定対象物質の有無を検出又は測定対象物質を定量することができる免疫測定用キット及び免疫測定方法に関する。 The present invention relates to an immunoassay kit and an immunoassay method, and in particular, an immunoassay capable of detecting the presence or absence of a measurement target substance in a sample or quantifying the measurement target substance in a short time by eliminating a washing step. The present invention relates to a kit and an immunoassay method.
農畜産物、食品、食品添加物、飲料、医薬品、医薬部外品、化粧品などの製品または原料における菌学的安全性は、常に保証されている必要がある。そのため、製品または原料中の菌学的分析手法を利用した菌検査・定量が日常的に行われている。また、人間や動物等の健康状態を調べるために、血液及び尿等の体液や細胞等の生体組織（以下、「生体サンプル」と記載することがある。）に含まれる種々のアレルギー物質、ウィルス、タンパク質等の検査・定量も日常的に行われている。 The bacteriological safety of products or raw materials such as agricultural and livestock products, foods, food additives, beverages, pharmaceuticals, quasi-drugs and cosmetics must always be guaranteed. For this reason, bacterial inspection and quantification using a mycological analysis technique in products or raw materials are routinely performed. In addition, in order to examine the health condition of humans and animals, various allergens and viruses contained in body fluids such as blood and urine, and biological tissues such as cells (hereinafter sometimes referred to as “biological samples”). In addition, inspection and quantification of proteins and the like are also routinely performed.
製品または原料に含まれる菌検査・定量、生体サンプルの検査・定量方法としては、例えば、ＥＬＩＳＡ（Ｅｎｚｙｍｅ ｌｉｎｋｅｄ ｉｍｍｕｎｏｓｏｒｂｅｎｔ ａｓｓａｙ）、イムノクロマト法等の抗原−抗体反応を利用した物質の検査・定量方法が知られている。 Methods for testing and quantifying bacteria contained in products or raw materials and methods for testing and quantifying biological samples include, for example, methods for testing and quantifying substances using antigen-antibody reactions such as ELISA (Enzyme linked immunosorbent assay) and immunochromatography. It has been.
ＥＬＩＳＡとは、以下の手順により、検体中の測定対象物質の有無を検出、又は測定対象物質を定量する方法である（非特許文献１参照）。
（１）マイクロプレート上に固定した１次抗体に抗原を結合させる。
（２）緩衝液を加えて洗浄した後、酵素等により修飾されている２次抗体を抗原に結合させる。
（３）非結合の２次抗体を緩衝液で洗浄・除去後、基質を加えることで２次抗体の酵素により発色する。
ELISA is a method for detecting the presence or absence of a measurement target substance in a sample or quantifying the measurement target substance by the following procedure (see Non-Patent Document 1).
(1) The antigen is bound to the primary antibody immobilized on the microplate.
(2) After adding a buffer solution and washing, a secondary antibody modified with an enzyme or the like is bound to an antigen.
(3) After washing / removing unbound secondary antibody with a buffer, color is developed by the secondary antibody enzyme by adding a substrate.
また、イムノクロマト法とは、以下の手順により、検体中の測定対象物質の有無を検出、又は測定対象物質を定量する方法である（非特許文献２参照）。
（１）メンブラン上に滴下された検体中の測定対象物質が、金属コロイド等で標識された抗体と複合体を形成し、毛細管現象によってメンブラン上を移動する。
（２）測定対象物質が存在する時は、検出抗体によって複合体の移動が途中でトラップされ、その呈色を目視によって判断する。
Further, the immunochromatography method is a method for detecting the presence or absence of a measurement target substance in a sample or quantifying the measurement target substance by the following procedure (see Non-Patent Document 2).
(1) A substance to be measured in a sample dropped on a membrane forms a complex with an antibody labeled with a metal colloid or the like, and moves on the membrane by capillary action.
(2) When the measurement target substance exists, the movement of the complex is trapped in the middle by the detection antibody, and the coloration is visually determined.
しかしながら、ＥＬＩＳＡは、非結合の抗原や２次抗体を洗浄・除去する工程が必要であり、人手や時間がかかるという問題がある。一方、イムノクロマト法は、ＥＬＩＳＡと比較して短時間で検出が可能であるが、感度が低く、夾雑物に弱いという問題がある。 However, ELISA requires a step of washing and removing unbound antigens and secondary antibodies, and has the problem that it takes manpower and time. On the other hand, the immunochromatography method can be detected in a short time as compared with the ELISA, but has a problem that the sensitivity is low and it is weak against impurities.
本発明は、上記問題点を解決するためになされたもので、鋭意研究を行ったところ、（１）発光することができる標識成分で標識した抗体（２次抗体）から発する波長域の光を吸収する化合物を抗原―抗体反応を行う媒体中に添加すると、（２）検体中に測定対象物質が含まれない場合は、媒体中に分散している２次抗体からの発光が化合物に吸収されることで、検出機による発光の検出が抑えられること、（３）一方、検体中に測定対象物質が含まれる場合は抗原−抗体反応により測定対象物質及び２次抗体が複合体を形成し、該複合体を容器の一部に偏在させることで、検体中に測定対象物質が含まれない場合と比較して、検出機による光の検出量を多くできること、（４）そして、媒体中に分散している２次抗体からの発光は化合物に吸収されることから、２次抗体を洗浄・除去する工程が無くても検体中の測定対象物質の有無を検出、又は測定対象物質を定量できることから、測定対象物質の検出・定量時間を短縮できること、を新たに見出した。 The present invention has been made to solve the above-mentioned problems, and as a result of extensive research, (1) light in a wavelength range emitted from an antibody (secondary antibody) labeled with a labeling component capable of emitting light (secondary antibody). When a compound to be absorbed is added to the medium in which the antigen-antibody reaction is performed, (2) if the analyte is not contained in the sample, light emitted from the secondary antibody dispersed in the medium is absorbed by the compound. (3) On the other hand, when the measurement target substance is contained in the sample, the measurement target substance and the secondary antibody form a complex by the antigen-antibody reaction, By making the complex unevenly distributed in a part of the container, the amount of light detected by the detector can be increased compared to the case where the sample does not contain the measurement target substance, and (4) and dispersed in the medium Light emitted from the secondary antibody Therefore, the detection / quantification time of the measurement target substance can be shortened because the presence or absence of the measurement target substance in the sample can be detected or the measurement target substance can be quantified without the step of washing / removing the secondary antibody. , Newly found.
すなわち、本発明の目的は、短時間で検体中の測定対象物質を検出・定量するための免疫測定用キット及び免疫測定方法を提供することにある。 That is, an object of the present invention is to provide an immunoassay kit and an immunoassay method for detecting and quantifying a measurement target substance in a specimen in a short time.
本発明は以下に示す免疫測定用キット及び免疫測定方法に関する。 The present invention relates to the following immunoassay kit and immunoassay method.
（１）発光することができる標識成分で標識した抗体、及び、
前記標識成分が発する波長域の光を吸収する化合物、
を少なくとも含む免疫測定用キット。
（２）前記発光することができる標識成分が酵素である上記（１）に記載の免疫測定用キット。
（３）前記酵素がルシフェラーゼである上記（２）に記載の免疫測定用キット。
（４）前記光を吸収する化合物がブルーデキストランである上記（１）〜（３）の何れか一に記載の免疫測定用キット。
（５）検体中の測定対象物質と特異的に結合する抗体を固定した容器又はビーズを含む上記（１）〜（４）の何れか一に記載の免疫測定用キット。
（６）標識成分が発する波長域の光を、近赤外波長領域の光に変換する材料で形成された変換部を含む容器、又は前記材料で形成された近赤外蛍光ガラス体を含む上記（１）〜（４）の何れか一に記載の免疫測定用キット。
（７）検体中の測定対象物質の有無を検出、又は測定対象物質を定量する免疫測定方法であって、
検体、前記検体中の測定対象物質と特異的に結合し且つ発光することができる標識成分で標識した抗体、及び前記標識成分から発する波長域の光を吸収する化合物、を媒体中で混合する工程、
前記標識成分から発した光を測定する工程、
測定した光量から、検体中の測定対象物質の有無を検出、又は測定対象物質を定量する工程、
を少なくとも含む免疫測定方法。
（８）前記混合する工程が、検体中の測定対象物質と特異的に結合する抗体を固定した容器で行われる上記（７）に記載の免疫測定方法。
（９）前記混合する工程が、媒体中に検体中の測定対象物質と特異的に結合する抗体を固定したビーズを更に含む上記（７）に記載の免疫測定方法。
（１０）前記混合する工程の後に、遠心分離を行う工程を含む上記（７）〜（９）の何れか一に記載の免疫測定方法。
(1) an antibody labeled with a labeling component capable of emitting light, and
A compound that absorbs light in a wavelength range emitted by the labeling component,
An immunoassay kit comprising at least
(2) The immunoassay kit according to (1), wherein the labeling component capable of emitting light is an enzyme.
(3) The immunoassay kit according to (2), wherein the enzyme is luciferase.
(4) The immunoassay kit according to any one of (1) to (3), wherein the compound that absorbs light is blue dextran.
(5) The immunoassay kit according to any one of the above (1) to (4), comprising a container or a bead on which an antibody that specifically binds to a measurement target substance in a sample is immobilized.
(6) A container including a conversion part formed of a material that converts light in a wavelength region emitted from a labeling component into light in a near infrared wavelength region, or the above-mentioned including a near infrared fluorescent glass body formed of the material The immunoassay kit according to any one of (1) to (4).
(7) An immunoassay method for detecting the presence or absence of a measurement target substance in a sample or quantifying the measurement target substance,
Mixing in a medium a sample, an antibody labeled with a labeling component that can specifically bind to and emit light from the analyte in the sample, and a compound that absorbs light in the wavelength region emitted from the labeling component ,
Measuring light emitted from the labeling component,
Detecting the presence or absence of the measurement target substance in the sample from the measured light quantity, or quantifying the measurement target substance,
An immunoassay method comprising at least
(8) The immunoassay method according to (7), wherein the mixing step is performed in a container in which an antibody that specifically binds to a measurement target substance in a specimen is fixed.
(9) The immunoassay method according to (7), wherein the mixing step further includes beads in which an antibody that specifically binds to a measurement target substance in a specimen is immobilized in a medium.
(10) The immunoassay method according to any one of (7) to (9), including a step of performing centrifugation after the step of mixing.
本発明の免疫測定用キット及び免疫測定方法を用いると、２次抗体の洗浄・除去が不要であることから、操作が簡単で且つ短時間で検体中の測定対象物質を検出・定量することができる。したがって、食中毒菌等に汚染された食品等を早期に発見することが可能となる。また、生体サンプルに含まれる種々のタンパク質等の検査・定量も迅速に行うことができる。 When the immunoassay kit and the immunoassay method of the present invention are used, it is not necessary to wash and remove the secondary antibody, so that the operation can be easily performed and the measurement target substance in the sample can be detected and quantified in a short time. it can. Therefore, it becomes possible to discover foods contaminated with food poisoning bacteria at an early stage. In addition, various proteins and the like contained in the biological sample can be rapidly examined and quantified.
以下に、本発明の免疫測定用キット及び免疫測定方法について詳しく説明する。図１は、本発明の免疫測定の原理を説明する図である。図１（１）は検体中に検出対象物質が含まれる場合の例を示している。本発明の免疫測定用キットは、少なくとも、発光することができる標識成分１で標識した抗体（２次抗体）２、標識成分１が発する波長域の光を吸収する化合物３を含んでいる。検体中に測定対象物質４が含まれていると、容器５の一部に固定されている測定対象物質４を特異的に認識する抗体（１次抗体）６に測定対象物質４が結合し、更に測定対象物質４に２次抗体２が結合して複合体を形成する。そして、複合体が容器５の壁面に偏在することから２次抗体２が発する光も容器壁面に偏在し、検出機７により２次抗体２が発する光を検出し易くなる。以下、本発明の免疫測定方法を「生物発光色素吸収法」と記載することがある。 The immunoassay kit and immunoassay method of the present invention will be described in detail below. FIG. 1 is a diagram for explaining the principle of immunoassay of the present invention. FIG. 1 (1) shows an example in which a detection target substance is contained in a specimen. The immunoassay kit of the present invention includes at least an antibody (secondary antibody) 2 labeled with a labeling component 1 capable of emitting light, and a compound 3 that absorbs light in the wavelength region emitted by the labeling component 1. When the measurement target substance 4 is contained in the sample, the measurement target substance 4 binds to an antibody (primary antibody) 6 that specifically recognizes the measurement target substance 4 fixed to a part of the container 5; Further, the secondary antibody 2 is bound to the measurement target substance 4 to form a complex. Since the complex is unevenly distributed on the wall surface of the container 5, the light emitted from the secondary antibody 2 is also unevenly distributed on the container wall surface, and the detector 7 can easily detect the light emitted from the secondary antibody 2. Hereinafter, the immunoassay method of the present invention may be referred to as “bioluminescent dye absorption method”.
一方、図１（２）は検体中に測定対象物質４が含まれない場合の例を示している。測定対象物質４が含まれない場合は、２次抗体２は、測定対象物質４と複合体を形成しないことから、化合物３を含む媒体中に分散した状態となる。２次抗体２が発する光は化合物３に吸収されるので、図１（１）と比較して検出機７により検出され難くなる。 On the other hand, FIG. 1 (2) shows an example in which the measurement target substance 4 is not contained in the specimen. When the measurement target substance 4 is not included, the secondary antibody 2 does not form a complex with the measurement target substance 4 and thus is dispersed in a medium containing the compound 3. Since the light emitted from the secondary antibody 2 is absorbed by the compound 3, it is difficult to be detected by the detector 7 as compared with FIG.
本発明の生物発光色素吸収法では、２次抗体２が発する光を化合物３で吸収することが特徴である。そのため、測定対象物質４と２次抗体２が複合体を形成した場合でも、当該複合体が溶媒中に分散した状態であると２次抗体２が発する光を化合物３が吸収してしまい、検出感度が悪くなる。そのため、測定対象物質４と２次抗体２の複合体が可能な限り容器５の壁面に偏在し、２次抗体２が発する光が化合物３に吸収され難くすることが好ましい。図１（１）に示す例では、容器５の壁面の一部（底面）に１次抗体６を固定することで複合体を容器５の底面付近に偏在させているが、複合体を容器５の壁面の特定部分に偏在できれば、特に制限は無い。 The bioluminescent dye absorption method of the present invention is characterized in that the light emitted from the secondary antibody 2 is absorbed by the compound 3. Therefore, even when the measurement target substance 4 and the secondary antibody 2 form a complex, if the complex is dispersed in the solvent, the compound 3 absorbs light emitted from the secondary antibody 2 and is detected. Sensitivity deteriorates. Therefore, it is preferable that the complex of the substance to be measured 4 and the secondary antibody 2 is unevenly distributed on the wall surface of the container 5 as much as possible so that the light emitted from the secondary antibody 2 is not easily absorbed by the compound 3. In the example shown in FIG. 1 (1), the complex is unevenly distributed near the bottom surface of the container 5 by fixing the primary antibody 6 to a part (bottom surface) of the wall surface of the container 5. There is no particular limitation as long as it can be unevenly distributed on a specific portion of the wall surface.
例えば、測定対象物質４が菌体等の媒体に不溶性の物質の場合、容器５として遠心分離機に装着可能な容器を用い、菌体と２次抗体２を結合させて複合体を形成した後、遠心分離により複合体を容器５の底部に偏在させてもよい。その場合、１次抗体６を容器５に固定しなくてもよい。また、１次抗体６を容器５ではなく磁性ビーズ等に固定することで、１次抗体６−測定対象物質４−２次抗体２の複合体を、磁力等を用いて容器５の壁面に引き寄せることで偏在してもよい。更に、１次抗体６をガラスビーズ等の固体物質に固定して、１次抗体６−測定対象物質４−２次抗体２の複合体を、遠心分離機を用いて容器５の底部に偏在してもよい。 For example, when the measurement target substance 4 is a substance that is insoluble in a medium such as a microbial cell, a container that can be attached to a centrifuge is used as the container 5 and the microbial cell and the secondary antibody 2 are combined to form a complex. The composite may be unevenly distributed at the bottom of the container 5 by centrifugation. In that case, the primary antibody 6 may not be fixed to the container 5. In addition, by fixing the primary antibody 6 not to the container 5 but to a magnetic bead or the like, the complex of the primary antibody 6-the substance to be measured 4-2 and the secondary antibody 2 is attracted to the wall surface of the container 5 using magnetic force or the like. It may be unevenly distributed. Further, the primary antibody 6 is fixed to a solid substance such as glass beads, and the complex of the primary antibody 6-the substance to be measured 4-secondary antibody 2 is unevenly distributed at the bottom of the container 5 using a centrifuge. May be.
標識成分１は、２次抗体２に結合することができ、発光できる成分であれば特に制限は無い。例えば、ルシフェラーゼ（以下、「Ｌｕｃ」と記載することがある。）、βガラクトシダーゼ、クロラムフェニコール・アセチルトランスフェラーゼ（ＣＡＴ）、アルカリフォスファターゼ（ＡＰ）、βグルクロニダーゼ、ホースラディッシュペルオキシダーゼ（ＨＲＰ）等の基質と反応することで発光する酵素；緑色蛍光タンパク質（Ｇｒｅｅｎ Ｆｌｕｏｒｅｓｃｅｎｔ Ｐｒｏｔｅｉｎ，ＧＦＰ）、ｐｈｙｃｏｅｒｙｔｈｒｉｎ（ＰＥ）、ａｌｌｏｐｈｙｃｏｃｙａｎｉｎ（ＡＰＣ）等の基質が無くても発光する蛍光タンパク質；フルオレセインイソチオシアネート（ＦＩＴＣ）、ローダミン、テキサスレッド、Ｃｙ３、Ｃｙ５等の蛍光色素：量子ドット（Ｑｕａｎｔｕｍ Ｄｏｔ：ＱＤ）と呼ばれる半導体結晶；等が挙げられる。 The labeling component 1 is not particularly limited as long as it can bind to the secondary antibody 2 and can emit light. For example, substrates such as luciferase (hereinafter sometimes referred to as “Luc”), β-galactosidase, chloramphenicol acetyltransferase (CAT), alkaline phosphatase (AP), β-glucuronidase, horseradish peroxidase (HRP), etc. Enzyme that emits light by reacting with it; green fluorescent protein (Green Fluorescent Protein, GFP), fluorescein isothiocyanate (FITC), rhodamine, fluorescent protein that emits light even without a substrate such as phycoerythrin (PE), allophycocyanin (APC), etc. Fluorescent dyes such as Texas Red, Cy3, and Cy5: semiconductor crystals called Quantum Dots (QD); and the like.
標識成分１として酵素を用いる場合、当該酵素に特異的な公知の基質を用いて発光すればばよい。例えば、ホタルルシフェラーゼであれば甲虫ルシフェリン、ウミシイタケルシフェラーゼであればセレンテラジン、βガラクトシダーゼであれば１，２−ｄｉｏｘｅｔａｎｅ類、ＨＲＰであればルミノール系基質等、公知の酵素と基質の組み合わせを用いればよい。 When an enzyme is used as the labeling component 1, light emission may be performed using a known substrate specific to the enzyme. For example, a combination of a known enzyme and a substrate such as beetle luciferin for firefly luciferase, coelenterazine for Renilla luciferase, 1,2-dioxetanes for β-galactosidase, and luminol substrate for HRP may be used. .
２次抗体２は、測定対象物質４を特異的に認識できるものであれば特に制限は無い。例えば、市販されている抗体、又は、測定対象物質４を用いた公知の抗体製造方法により製造した抗体を用いればよい。標識成分１で標識した抗体（２次抗体）２は、標識成分１と抗体２が結合できれば特に制限はなく、公知の方法で結合すればよい。例えば、標識成分１をビオチン化し、２次抗体２をストレプトアビジン化し、ビオチン−ストレプトアビジン結合により両者を結合すればよい。ビオチン化、ストレプトアビジン化は、市販されている公知のキットを用いて行えばよい。 The secondary antibody 2 is not particularly limited as long as it can specifically recognize the measurement target substance 4. For example, a commercially available antibody or an antibody produced by a known antibody production method using the measurement target substance 4 may be used. The antibody (secondary antibody) 2 labeled with the labeling component 1 is not particularly limited as long as the labeling component 1 and the antibody 2 can be bound, and may be bound by a known method. For example, the labeling component 1 may be biotinized, the secondary antibody 2 may be streptavidin, and both may be bound by a biotin-streptavidin bond. Biotinylation and streptavidinization may be performed using a commercially available kit.
本発明の化合物３は、標識成分１が発する光の波長域を吸収できるものであれば特に制限は無い。化合物３が吸収できる光の波長域が、標識成分１が発する光の波長域と少なくとも一部が重複していることが好ましく、標識成分１が発する光のピーク波長を含んでいることがより好ましく、標識成分１が発する光の波長域を全て含んでいることが特に好ましい。 The compound 3 of the present invention is not particularly limited as long as it can absorb the wavelength range of light emitted from the labeling component 1. The wavelength range of light that can be absorbed by the compound 3 is preferably at least partially overlapped with the wavelength range of the light emitted by the labeling component 1, and more preferably includes the peak wavelength of the light emitted by the labeling component 1. It is particularly preferable that the entire wavelength range of light emitted from the labeling component 1 is included.
化合物３の具体例としては、ブルーデキストラン、ブロムフェノールブルー、アントシアニン等が挙げられる。例えば、ルシフェラーゼは生物種により異なるものの、ホタルルシフェラーゼは約５５６ｎｍ、ウミシイタケルシフェラーゼは約４８０ｎｍがピーク波長を有するので、化合物３としては、ルシフェラーゼの上記波長域の光を収できるブルーデキストランを選択すればよい。なお、化合物３は単独で用いることもできるが、標識成分１が発する光の波長域を単独の化合物３で吸収できない場合は、異なる吸収波長域を持つ化合物３を組み合わせて用いてもよい。 Specific examples of compound 3 include blue dextran, bromophenol blue, anthocyanin and the like. For example, although luciferase varies depending on the species, firefly luciferase has a peak wavelength of about 556 nm, and Renilla luciferase has a peak wavelength of about 480 nm. Therefore, as compound 3, blue dextran capable of collecting light in the above wavelength range of luciferase can be selected. Good. In addition, although the compound 3 can also be used independently, when the wavelength range of the light which the labeling component 1 emits cannot be absorbed with the single compound 3, you may use combining the compound 3 which has a different absorption wavelength range.
化合物３の濃度は、媒体中に遊離した２次抗体２が発する光を吸収し、検出機７に届き難くするために必要な量であればよい。媒体中の標識成分１の濃度にもよるが、例えば、ＡＢＳに換算して２以上が好ましく、２．５以上がより好ましく、３以上が特に好ましい。化合物３の濃度が薄すぎると、媒体中に遊離している２次抗体２が発する光が検出機に届くので好ましくない。一方、化合物３の濃度の上限は特になく、媒体中に溶解可能な範囲とすればよい。 The concentration of the compound 3 may be an amount necessary for absorbing light emitted from the secondary antibody 2 released into the medium and making it difficult to reach the detector 7. Although depending on the concentration of the labeling component 1 in the medium, for example, 2 or more is preferable in terms of ABS, 2.5 or more is more preferable, and 3 or more is particularly preferable. If the concentration of the compound 3 is too low, light emitted from the secondary antibody 2 released in the medium reaches the detector, which is not preferable. On the other hand, the upper limit of the concentration of compound 3 is not particularly limited, and may be in a range that can be dissolved in the medium.
本発明の生物発光色素吸収法により測定できる測定対象物質４としては、測定対象物質４を抗原として、抗原―抗体反応できるものであれば特に制限は無い。例えば、食品等に含まれる菌を検出する場合、細菌、真菌及び放線菌等が挙げられる。 The measurement target substance 4 that can be measured by the bioluminescent dye absorption method of the present invention is not particularly limited as long as it can undergo an antigen-antibody reaction using the measurement target substance 4 as an antigen. For example, when detecting bacteria contained in food or the like, bacteria, fungi, actinomycetes and the like can be mentioned.
細菌としては、グラム陽性細菌やグラム陰性細菌がある。グラム陽性細菌としては、腸球菌、連鎖球菌、ブドウ球菌、バチルス属、パエニバチルス属、乳酸桿菌属、リステリア属、ペプトストレプトコッカス属、プロピオン酸菌属、クロストリジウム属、バクテロイデス属、ガードネレラ属、コクリア属、ラクトコッカス属、ロイコノストック属、ミクロコッカス、マイコバクテリウム属、コリネバクテリウム属などに属する細菌等が挙げられる。グラム陰性細菌としては、シュードモナス属、エシェリキア属、サルモネラ属、赤痢菌属、エンテロバクター属、クレブシエラ属、セラチア属、プロテウス属、カンピロバクター属、ヘモフィルス属、モルガネラ属、ビブリオ属、エルシニア属、アシネトバクター属、ステノトロフォモナス属、ブレブンディモナス属、ラルストニア属、アクロモバクター属、フゾバクテリウム属、プレボテラ属、ブランハメラ亜属、ナイセリア属、バークホルデリア属、シトロバクター属、ハフニア属、エドワードシエラ属、アエロモナス属、モラクセラ属、ブルセラ属、パスツレラ属、プロビデンシア属、レジオネラ属などに属する細菌等が挙げられる。 Bacteria include gram positive bacteria and gram negative bacteria. Gram-positive bacteria include enterococci, streptococci, staphylococci, Bacillus, Paenibacillus, Lactobacillus, Listeria, Peptostreptococcus, Propionic acid, Clostridium, Bacteroides, Gardnerella, Coclear, Examples include bacteria belonging to the genus Lactococcus, Leuconostoc, Micrococcus, Mycobacterium, Corynebacterium and the like. Gram-negative bacteria include Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Hemophilus, Morganella, Vibrio, Yersinia, Acinetobacter, Stenotrophomonas, Brevendimonas, Ralstonia, Achromobacter, Fusobacterium, Prevotella, Blanchamella, Neisseria, Burkholderia, Citrobacter, Hafnia, Edward Sierra, Aeromonas And bacteria belonging to the genus Moraxella, Brucella, Pasteurella, Providencia, Legionella and the like.
真菌としては、酵母、カビ（糸状菌）、キノコを挙げることができ、具体的には、カンジダ属、クリプトコックス属、ノカルジア属、アオカビ属、アルタナリア属、ロドトルラ属、アスペルギルス属、フザリウム属、サッカロミセス属、トリコスポロン属などに属する菌が挙げられる。放線菌の例としては、ストレプトマイセス属、フランキア属、マイコバクテリウム属、コリネバクテリウム属、アクチノマイセズ属などに属する菌が挙げられる。 Examples of fungi include yeast, mold (filamentous fungi), and mushrooms. Specifically, Candida, Cryptococcus, Nocardia, Blue mold, Alternaria, Rhodotorula, Aspergillus, Fusarium, Saccharomyces Examples include bacteria belonging to the genus, Trichosporon. Examples of actinomycetes include bacteria belonging to the genera Streptomyces, Francia, Mycobacterium, Corynebacterium, Actinomyces, and the like.
本発明の方法では、ＥＬＩＳＡ等の従来の測定方法と比較して簡単且つ短時間で測定することが可能であることから、特定の病原性細菌を検出する際に特に有用である。病原性細菌の具体例としては、サルモネラ菌、リステリア菌、病原性大腸菌、Ｏ１５７等に代表される腸管出血性大腸菌、腸炎ビブリオ、チフス菌、パラチフス菌、コレラ菌、赤痢菌、セレウス菌、カンピロバクター菌、ボツリヌス菌、黄色ブドウ球菌などが挙げられる。 The method of the present invention is particularly useful when detecting a specific pathogenic bacterium because it can be measured easily and in a short time as compared with a conventional measuring method such as ELISA. Specific examples of pathogenic bacteria include Salmonella, Listeria, pathogenic Escherichia coli, enterohemorrhagic Escherichia coli represented by O157, Vibrio parahaemolyticus, Salmonella typhi, Paratyphi, Vibrio cholerae, Shigella, Cereus, Campylobacter, Examples include Clostridium botulinum and Staphylococcus aureus.
上記の測定対象物質４を含む検体としては、農畜産物、食品、食品添加物、飲料、医薬品、医薬部外品、化粧品、水（例えば飲料水、非飲料水及び廃水）、海水バラスト、空気、土壌、汚水、血液製剤（例えば血小板、血清、血漿、白血球分画等）等が挙げられる。 Samples containing the above-described measurement target substance 4 include agricultural and livestock products, foods, food additives, beverages, pharmaceuticals, quasi drugs, cosmetics, water (for example, drinking water, non-drinking water and wastewater), seawater ballast, air , Soil, sewage, blood products (for example, platelets, serum, plasma, leukocyte fraction, etc.) and the like.
また、生体サンプルを検体とした場合の測定対象物質４としては、花粉、ダニ等のアレルギー物質；ＨＩＶ、肝炎等のウィルス；ＰＳＡ等の癌マーカー；各種タンパク質；等が挙げられる。 Examples of the measurement target substance 4 when using a biological sample as a specimen include allergens such as pollen and mites; viruses such as HIV and hepatitis; cancer markers such as PSA; various proteins;
検体、２次抗体２、化合物３を混合するための媒体としては、従来から免疫測定で用いられている媒体であれば特に制限は無く、例えば、リン酸バッファー等が挙げられる。 The medium for mixing the specimen, the secondary antibody 2 and the compound 3 is not particularly limited as long as it is a medium conventionally used in immunoassays, and examples thereof include a phosphate buffer.
容器５は、免疫測定に使用できる容器であれば特に制限は無いが、本発明では容器５を通過する光を検出する。そのため、検出感度を上げるためには標識成分１が発する光の透過性が高い透明な材料で容器を作製することが好ましい。測定対象物質４を捕捉する為の１次抗体６を容器５に固定しない場合は、エッペンドルフチューブ等、遠心分離機に装着し遠心分離できる容器が好ましい。一方、１次抗体６を用いる場合は、容器５は特に制限は無く、９６ウェルプレート等、免疫測定の分野で使用されている一般的な容器を用いればよい。 Although there will be no restriction | limiting in particular if the container 5 can be used for an immunoassay, In this invention, the light which passes the container 5 is detected. Therefore, in order to increase the detection sensitivity, it is preferable that the container is made of a transparent material having a high transmittance for the light emitted from the labeling component 1. In the case where the primary antibody 6 for capturing the substance 4 to be measured is not fixed to the container 5, a container such as an Eppendorf tube that can be attached to a centrifuge and centrifuged is preferable. On the other hand, when the primary antibody 6 is used, the container 5 is not particularly limited, and a general container used in the field of immunoassay such as a 96-well plate may be used.
なお、本発明では、標識成分１が発する光をそのまま検出機７で検出してもよいが、標識成分１から発する光の波長域を近赤外波長領域、例えば、８５０〜１７００ｎｍ程度に変換部で変換し、当該波長域を検出できる検出機７を用いてもよい。変換部を構成する部材としては、例えば、Ｙｂイオン、Ｎｄイオン、Ｔｍイオン、Ｓｍイオン、Ｈｏイオン、Ｅｒイオン、Ｄｙイオン、Ｐｒイオン等の希土類イオンを、硼酸系ガラス、ゲルマン酸系ガラス、リン酸系ガラス、フッ化物ガラス等に添加したガラス等が挙げられる。具体的には、Ｂｉ2Ｏ3−Ｂ2Ｏ3系ガラス、Ｂｉ2Ｏ3−ＧｅＯ2系ガラス、ＺｎＯ−Ｂ2Ｏ3系ガラス、ＣａＯ−Ｂ2Ｏ3系ガラス及びＣａＯ−Ｐ2Ｏ5系等の公知のガラスが挙げられる。 In the present invention, the light emitted from the labeling component 1 may be detected by the detector 7 as it is, but the wavelength region of the light emitted from the labeling component 1 is converted into a near infrared wavelength region, for example, about 850 to 1700 nm. It is also possible to use a detector 7 that can convert the signal and detect the wavelength range. Examples of the member constituting the conversion unit include rare earth ions such as Yb ion, Nd ion, Tm ion, Sm ion, Ho ion, Er ion, Dy ion, and Pr ion, boric acid glass, germanic acid glass, phosphorus The glass etc. which were added to acid type glass, fluoride glass, etc. are mentioned. Specifically, Bi 2 O 3 —B 2 O 3 glass, Bi 2 O 3 —GeO 2 glass, ZnO—B 2 O 3 glass, CaO—B 2 O 3 glass and CaO—P 2 O Well-known glass of 5 series etc. is mentioned.
可視光の波長領域を検出する場合と異なり、標識成分１から発する光の波長領域を近赤外波長領域に変換する場合、容器５は透明である必要はない。したがって、容器５は不透明な材料で形成してもよく、容器５を作製する材料の選択範囲を広くすることができる。光の波長領域を変える場合は、上記成分で作製した近赤外蛍光ガラス体に１次抗体６を固定して、媒体中に添加すればよい。近赤外蛍光ガラス体は１次抗体６−測定対象物質４−２次抗体２と複合体を形成することから、標識成分１が発する光を近赤外蛍光ガラス体で近赤外波長領域に変換することができ、容器５を通過した近赤外波長領域の光を検出機で検出すればよい。 Unlike the case of detecting the wavelength region of visible light, the container 5 does not need to be transparent when the wavelength region of light emitted from the labeling component 1 is converted to the near-infrared wavelength region. Therefore, the container 5 may be formed of an opaque material, and the selection range of the material for producing the container 5 can be widened. When changing the wavelength region of light, the primary antibody 6 may be fixed to the near-infrared fluorescent glass body prepared with the above components and added to the medium. Since the near-infrared fluorescent glass body forms a complex with the primary antibody 6-substance to be measured 4-secondary antibody 2, the light emitted from the labeling component 1 is placed in the near-infrared wavelength region with the near-infrared fluorescent glass body. The light in the near-infrared wavelength region that can be converted and has passed through the container 5 may be detected by a detector.
近赤外蛍光ガラス体を用いた場合は、容器５全体を不透明な材料で作製することができるが、容器５の一部に上記成分で作製した変換部（近赤外蛍光ガラス体）を組み込んでもよい。変換部に一次抗体６を固定しておくことで、変換部及び／又は変換部周辺の容器５を通して、近赤外波長領域の光を検出機７で検出することができる。 When the near-infrared fluorescent glass body is used, the entire container 5 can be made of an opaque material, but a conversion part (near-infrared fluorescent glass body) made of the above components is incorporated in a part of the container 5. But you can. By fixing the primary antibody 6 to the conversion unit, light in the near-infrared wavelength region can be detected by the detector 7 through the conversion unit and / or the container 5 around the conversion unit.
１次抗体６は、２次抗体２と同様、測定対象物質４を特異的に認識して結合できるものであれば特に制限は無く、市販されている抗体、又は、測定対象物質４を用いた公知の抗体製造方法により製造した抗体を用いればよい。なお、上記のとおり１次抗体６を用いることは必須ではないが、用いる場合は、容器５の壁面に公知の方法で固定すればよい。また、容器５に変え、免疫測定分野で従来より使用されている磁性ビーズ、ガラスビーズ等に固定してもよい。 As with the secondary antibody 2, the primary antibody 6 is not particularly limited as long as it can specifically recognize and bind to the measurement target substance 4, and a commercially available antibody or the measurement target substance 4 was used. An antibody produced by a known antibody production method may be used. Although it is not essential to use the primary antibody 6 as described above, when used, it may be fixed to the wall surface of the container 5 by a known method. Further, the container 5 may be replaced with a magnetic bead or glass bead conventionally used in the immunoassay field.
検出機７は、標識成分１から発する光が可視光の場合は可視光領域の波長に感度がある検出機であれば特に制限は無く、例えば、Ｓｉ系のＣＣＤ、ＣＭＯＳカメラ等が挙げられる。また、近赤外光領域に変換して検出する場合には、例えば、８５０〜１７００ｎｍ程度の波長に感度があり、且つ８５０ｎｍ以下の波長には感度がないものが好ましく、例えば、ＩｎＧａＡｓカメラ、ゲルマニウムカメラ、ビジコンカメラ等が挙げられる。また、検出機７には、標識成分１が発する波長域以外の波長域の光を除去するためのフィルターを設けることで周囲のノイズを除去し、検出感度を高めてもよい。 If the light emitted from the labeling component 1 is visible light, the detector 7 is not particularly limited as long as it is sensitive to the wavelength in the visible light region, and examples thereof include a Si-based CCD and a CMOS camera. In the case of detecting by converting into the near infrared light region, for example, those having sensitivity at a wavelength of about 850 to 1700 nm and insensitive at a wavelength of 850 nm or less are preferable. For example, an InGaAs camera, germanium, etc. Examples include cameras and vidicon cameras. In addition, the detector 7 may be provided with a filter for removing light in a wavelength region other than the wavelength region emitted by the labeling component 1, thereby removing ambient noise and increasing detection sensitivity.
本発明の免疫測定方法は、必要に応じて１次抗体を固定した容器５に、検体、標識成分１で標識した２次抗体２、標識成分１が発する光の波長域の光を吸収する化合物３を媒体中で混合する工程、標識成分１から発光した光を検出機７で測定する工程を少なくとも含んでいる。なお、標識成分１が酵素の場合は、当該酵素に特異的な基質を媒体中に添加しておけばよい。また、１次抗体６を容器５に固定した場合、又は、１次抗体６を固定したビーズを容器５に入れる場合は、遠心分離工程は必要に応じて実施すればよいが、１次抗体６を用いない場合は、測定する工程の前に、遠心分離をする工程を設けることが好ましい。 The immunoassay method of the present invention is a compound that absorbs light in a wavelength region of light emitted from a specimen, a secondary antibody 2 labeled with a labeling component 1 and a labeling component 1 in a container 5 in which a primary antibody is immobilized as necessary. 3 in the medium, and at least the step of measuring the light emitted from the labeling component 1 with the detector 7. When the labeling component 1 is an enzyme, a substrate specific for the enzyme may be added to the medium. In addition, when the primary antibody 6 is fixed to the container 5 or when beads having the primary antibody 6 fixed are put into the container 5, the centrifugation step may be performed as necessary. When not using, it is preferable to provide the process of centrifuging before the process of measuring.
以下に実施例を掲げ、本発明を具体的に説明するが、この実施例は単に本発明の説明のため、その具体的な態様の参考のために提供されているものである。これらの例示は本発明の特定の具体的な態様を説明するためのものであるが、本願で開示する発明の範囲を限定したり、あるいは制限することを表すものではない。 The present invention will be described in detail with reference to the following examples, which are provided merely for the purpose of illustrating the present invention and for reference to specific embodiments thereof. These exemplifications are for explaining specific specific embodiments of the present invention, but are not intended to limit or limit the scope of the invention disclosed in the present application.
＜ルシフェラーゼ（Ｌｕｃ）の作製＞
本発明の実施例で用いるオレンジ色Ｌｕｃと緑色Ｌｕｃを以下の手順で作製した。
１．オレンジ色Ｌｕｃ
（１）発現に用いるＬｕｃのＤＮＡ配列の決定
ＮＣＢＩのデータベース（ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ｎｕｃｃｏｒｅ／Ｅ０５４４７．１）から、Ｌｕｃのアミノ酸配列を取得し、以下の２つの変異を加えた配列を設計した。
・２１７番目：Ｔｈｒ→Ｉｌｅ
なお、当該変異は耐熱性向上が目的である（“Ｋａｊｉｙａｍａ ｅｔ ａｌ．，“Ｔｈｅｒｍｏｓｔａｂｉｌｉｚａｔｉｏｎ ｏｆ Ｆｉｒｅｆｌｙ Ｌｕｃｉｆｅｒａｓｅ ｂｙ ａ Ｓｉｎｇｌｅ Ａｍｉｎｏ Ａｃｉｄ Ｓｕｂｓｔｉｔｕｔｉｏｎ ａｔ Ｐｏｓｉｔｉｏｎ ２１７”，Ｂｉｏｃｈｅｍｉｓｔｒｙ １９９３，３２，１３７９５−１３７９９、参照）。
・２５７番目：Ｔｙｒ→Ａｒｇ
なお、当該変異はＬｕｃの色を、緑からオレンジ色に変えるのが目的である（Ｙｕ Ｗａｎｇ ｅｔ ａｌ．，”Ｉｍｐａｃｔ ｏｆ Ｓｉｔｅ−Ｄｉｒｅｃｔｅｄ Ｍｕｔａｎｔ Ｌｕｃｉｆｅｒａｓｅｏｎ Ｑｕａｎｔｉｔａｔｉｖｅ Ｇｒｅｅｎ ａｎｄ Ｏｒａｎｇｅ／Ｒｅｄ Ｅｍｉｓｓｉｏｎ Ｉｎｔｅｎｓｉｔｉｅｓ ｉｎ Ｆｉｒｅｆｌｙ Ｂｉｏｌｕｍｉｎｅｓｃｅｎｃｅ”，ＳＣＩＥＮＴＩＦＩＣ ＲＥＰＯＲＴ，２０１３、参照）。
次に、決定したアミノ酸配列をＯｐｔｉｍｉｚｅｒ（ｈｔｔｐ：／／ｇｅｎｏｍｅｓ．ｕｒｖ．ｅｓ／ＯＰＴＩＭＩＺＥＲ／）よって、大腸菌に発現させるのに最適なＤＮＡ配列に変換した。
次に、５’末端側にＨｉｓ ｔａｇ及び制限酵素Ｎｄｅ Ｉの認識配列、３’末端にＸｂａ Ｉの認識配列を付加した。
<Production of luciferase (Luc)>
An orange Luc and a green Luc used in the examples of the present invention were produced by the following procedure.
1. Orange Luc
(1) Determination of Luc DNA sequence used for expression Obtaining the amino acid sequence of Luc from the NCBI database (http://www.ncbi.nlm.nih.gov/nuccore/E0547.1), the following two A sequence with mutation was designed.
・ 217th: Thr → Ile
Note that the mutation is intended to improve heat resistance (see “Kajiyama et al.,“ Thermostabilization of Firefly Luciferase by Single Amino Acid Substitution at Position 217 ”, Biochim 37, 1999, Biochem.
・ 257th: Tyr → Arg
The purpose of the mutation is to change the color of Luc from green to orange (Yu Wang et al., “Impact of Site-Directed Mutant Luciferous Quantitative Green Intensive Green and Red Intensive Green / Red Intensive Green / Red Intensive Green / Red Intensive Green / Red Intensive Green and Red Intensive Green / Red Intensive Green / Red Intensive Green / Red Emission REPORT, 2013).
Next, the determined amino acid sequence was converted into an optimal DNA sequence for expression in Escherichia coli by an optimizer (http://genomes.urv.es/OPTIMIZER/).
Next, a recognition sequence for His tag and restriction enzyme Nde I was added to the 5 ′ end, and a recognition sequence for Xba I was added to the 3 ′ end.
（２）ＤＮＡ配列の合成
ＩＤＴ社のＤＮＡ合成サービス（ｈｔｔｐ：／／ｒｕｏ．ｍｂｌ．ｃｏ．ｊｐ／ｃｕｓｔｏｍ／ｃｕｓｔｏｍ＿ｇｅｎｅ．ｈｔｍｌ）により、上記（１）で配列決定したＤＮＡの合成を行った。
(2) DNA sequence synthesis The DNA sequenced in (1) above was synthesized by IDT's DNA synthesis service (http://ruo.mbl.co.jp/custom/custom_gene.html).
（３）オレンジ色Ｌｕｃ遺伝子の発現ベクターへのクローニング
制限酵素ＮｄｅＩ及びＸｂａＩ（いずれもＴａｋａｒａ ｂｉｏ社製）により、ＬｕｃのＤＮＡ配列及び低温発現ベクターｐＣｏｌｄ Ｉ（Ｔａｋａｒａ ｂｉｏ社製）を処理した。次に、Ｔａｋａｒａ ＤＮＡ ｌｉｇａｔｉｏｎ ｋｉｔ ｖｅｒ２を用いて、２つのＤＮＡ断片を結合し、大腸菌ＤＨ５α（Ｔｏｙｏｂｏ 社製 ＤＮＡ−９０１）に形質転換後、発生したコロニーを培養してプラスミドを抽出した。次に、シーケンス解析により狙い通り発現ベクターにＬｕｃの遺伝子が導入されているのを確認した。
(3) Cloning of orange Luc gene into expression vector Luc DNA sequence and low temperature expression vector pCold I (Takara bio) were treated with restriction enzymes NdeI and XbaI (both manufactured by Takara bio). Next, using Takara DNA ligation kit ver2, the two DNA fragments were combined, transformed into E. coli DH5α (DNA-901 manufactured by Toyobo), and the resulting colonies were cultured to extract a plasmid. Next, it was confirmed by sequence analysis that the Luc gene was introduced into the expression vector as intended.
（４）オレンジ色Ｌｕｃの発現、精製
大腸菌ＢＬ２１（ＤＥ３）（Ｎｏｖａｇｅｎ社製（現メルクミリポア社製）、６９４５０−３ＣＮ）に、上記（３）で配列を確認したプラスミドを導入した。次に、セルを２００ｍＬのＬＢ培地中、３７℃で対数増殖期まで培養し、１６℃で３０ｍｉｎ静置後、終濃度１ＭになるようにＩＰＴＧ（Ｗａｋｏ製）を加えた。更に、２４時間培養後、Ｎｉ−ＮＴＡ−Ａｇａｒｏｓｅ（Ｑｉａｇｅｎ社製）を使って、Ｈｉｓ ｔａｇ精製を行った。各画分をＳＤＳ−ＰＡＧＥにより解析し、Ｌｕｃが含まれる画分を回収した。次に、ＰＢＳ（ｐＨ７．４）で透析することで、オレンジ色Ｌｕｃを得た。
(4) Expression and purification of orange Luc The plasmid whose sequence was confirmed in (3) above was introduced into E. coli BL21 (DE3) (Novagen (currently Merck Millipore), 69450-3CN). Next, the cells were cultured in 200 mL of LB medium at 37 ° C. until the logarithmic growth phase, allowed to stand at 16 ° C. for 30 min, and then IPTG (manufactured by Wako) was added to a final concentration of 1M. Furthermore, after culturing for 24 hours, His tag purification was performed using Ni-NTA-Agarose (manufactured by Qiagen). Each fraction was analyzed by SDS-PAGE, and a fraction containing Luc was collected. Next, an orange Luc was obtained by dialysis against PBS (pH 7.4).
２．緑色Ｌｕｃ
（１）緑色Ｌｕｃの配列の作製
上記「１．オレンジ色Ｌｕｃ」に記載の（３）で合成したＤＮＡをＱｕｉｃｋ ｃｈａｎｇｅ法を用いて、２５７番目のＡｒｇをＴｙｒに変換した。
2. Green Luc
(1) Preparation of sequence of green Luc The DNA synthesized in (3) described in “1. Orange Luc” was converted to Arg at the 257th Arg using the Quick change method.
（２）緑色Ｌｕｃの作製
以下の２つのＰＣＲプライマーを用いて、ｐＣｏｌｄ ｏｒａｎｇｅ Ｌｕｃを鋳型として、２５７番目のＡｒｇがＴｙｒに変換したプラスミドを合成した。増幅は、Ｐｆｕ ｔｕｒｂｏ（アジレントテクノロジー社製）を用い、以下の条件で実施した。
・２５７ｔｈ Ｒ Ｌｕｃ Ｙ ｆｏｒ ＱＣ Ｆ：
ＧＧＣＡＴＧＴＴＣＡＣＣＡＣＴＣＴＧＧＧＴＴＡＴＣＴＧＡＴＣＴＧＣＧＧＣＴＴＣＣ（配列番号１）
・２５７ｔｈ Ｒ Ｌｕｃ Ｙ ｆｏｒ ＱＣ Ｒ：
ＧＧＡＡＧＣＣＧＣＡＧＡＴＣＡＧＡＴＡＡＣＣＣＡＧＡＧＴＧＧＴＧＡＡＣＡＴＧＣＣ（配列番号２）
増幅条件：９５℃で２分→（９５℃で１０秒→５５℃で１５秒→７２℃で６分）×２０回→７２℃で２０分→１６℃
(2) Production of Green Luc Using the following two PCR primers, a plasmid in which the 257th Arg was converted to Tyr was synthesized using pCold orange Luc as a template. Amplification was performed using Pfu turbo (manufactured by Agilent Technologies) under the following conditions.
257th R Luc Y for QC F:
GGCATGTTCACCACTCTGGGTTATCTGATCTGCGCTTCC (SEQ ID NO: 1)
257th R Luc Y for QC R:
GGAAGCCGCAGATCAGATAACCCAGAGTGGTGAACATGCCC (SEQ ID NO: 2)
Amplification condition: 95 ° C. for 2 minutes → (95 ° C. for 10 seconds → 55 ° C. for 15 seconds → 72 ° C. for 6 minutes) × 20 times → 72 ° C. for 20 minutes → 16 ° C.
次に、制限酵素Ｄｐｎ Ｉ（Ｔａｋａｒａ ｂｉｏ社製）を加えて鋳型を分解した。そして、大腸菌ＤＨ５αに形質転換した後は、上記１．（４）と同様の手順で、緑色Ｌｕｃの発現、精製を行った。 Next, the restriction enzyme Dpn I (manufactured by Takara bio) was added to decompose the template. After transformation into E. coli DH5α, the above 1. Green Luc was expressed and purified in the same procedure as (4).
［モデル系による生物発光色素吸収法の実証］
＜実施例１＞
ビオチンとストレプトアビジンは自然界で最も強固に結合することが知られており、様々な生物化学の研究において用いられている。この結合を利用して、生物発光色素吸収法のモデル系を構築し、本発明の免疫測定方法の実証を行った。
上記の手順で作製した緑色Ｌｕｃを、同人化学研究所製Ｂｉｏｔｉｎ ｌａｂｅｌｉｎｇ ｋｉｔ−ＮＨ２を用いてビオチン化した。
次に、ＰＣＲチューブ（ＢＭ機器株式会社製 ＰＣＲ−０２Ｆ２）に、
・緑色ビオチン化Ｌｕｃを１０ｎｇ、
・ビオチンと結合するストレプトアビジンが固定されたＳＡビーズ（Ｌｉｆｅ ｔｅｃｈｎｏｌｏｇｉｅｓ社製、Ｄｙｎａｂｅａｄｓ（登録商標） Ｍ２７０ ｓｔｒｅｐｔａｖｉｄｉｎ）を６−７×１０6ｂｅａｄｓ、
・ブルーデキストラン（ＧＥ Ｈｅａｌｔｈ ｃａｒｅ ｌｉｆｅ ｓｃｉｅｎｃｅ社製 １７−０３６０−０１）、
・Ｌｕｃの基質が含まれている定量試薬（Ｐｒｏｍｅｇａ社製、Ｏｎｅ Ｇｌｏ Ｌｕｃｉｆｅｒａｓｅ ａｓｓａｙ ｓｙｓｔｅｍ）を１００μＬ、
加え、更に、ＰＢＳ（ｐＨ７．４）を加えて合計２００μＬとすることで反応系を作製した。なお、ブルーデキストランは、反応系中の濃度が１．０ｗｔ％となるように添加した。反応系をピペッティングによって混合後、磁石上に１０秒間静置し、浜松ホトニクス社製Ｈ７３６０−０１を用いて３０秒間測定した。なお、株式会社島津製作所製紫外可視分光光度計ＵＶ２４５０を用いて混合時の反応系のＡＢＳを測定した値は、６．９８（トップピーク）であった。
[Demonstration of bioluminescent dye absorption method using model system]
<Example 1>
Biotin and streptavidin are known to bind most tightly in nature and are used in various biochemical studies. Using this binding, a model system of a bioluminescent dye absorption method was constructed, and the immunoassay method of the present invention was demonstrated.
Green Luc produced by the above procedure was biotinylated using Biotin labeling kit-NH2 manufactured by Doujin Chemical Laboratory.
Next, in the PCR tube (BM-02 Co., Ltd. PCR-02F2)
10 ng of green biotinylated Luc,
-SA beads to which streptavidin that binds to biotin was immobilized (Life technologies, Dynabeads (registered trademark) M270 streptavidin) was 6-7 × 10 6 beads,
-Blue dextran (GE Healthcare life science company 17-0360-01),
100 μL of a quantitative reagent (Promega, One Glo Luciferase assay system) containing a Luc substrate,
In addition, PBS (pH 7.4) was further added to make a total of 200 μL to prepare a reaction system. Blue dextran was added so that the concentration in the reaction system was 1.0 wt%. The reaction system was mixed by pipetting, then allowed to stand on the magnet for 10 seconds, and measured for 30 seconds using H7360-01 manufactured by Hamamatsu Photonics. In addition, the value which measured ABS of the reaction system at the time of mixing using Shimadzu Corporation ultraviolet visible spectrophotometer UV2450 was 6.98 (top peak).
＜比較例１＞
実施例１のＳＡビーズの代わりに、ネガティブコントロールとしてビオチンと結合しないＣＡビーズ（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ社製、Ｄｙｎａｂｅａｄｓ（登録商標） Ｍ２７０ ｃａｒｂｏｘｙｌｉｃ ａｃｉｄ）を用いた以外は、実施例１と同様の手順で実験を行った。
<Comparative Example 1>
Instead of the SA beads of Example 1, CA beads that do not bind to biotin (Life Technologies, Dynabeads (registered trademark) M270 carboxylic acid) were used as a negative control. went.
図２は、実施例１及び比較例１の測定結果を表すグラフである。ＳＡビーズを用いた実施例１は、ネガティブコントロールであるＣＡビーズを用いた比較例１より、有意に高いシグナルを得ることができた。これは、比較例１ではＬｕｃの発光がブルーデキストランに吸収されたのに対して、実施例１ではビオチン−ストレプトアビジン相互作用によってビオチン化Ｌｕｃと結合したビーズが、磁石により容器底部に偏在したため、より多くのルシフェラーゼの発光が検出機により検出されたためと考えられる。 FIG. 2 is a graph showing the measurement results of Example 1 and Comparative Example 1. Example 1 using SA beads was able to obtain a significantly higher signal than Comparative Example 1 using CA beads as a negative control. This is because, in Comparative Example 1, Luc luminescence was absorbed by blue dextran, whereas in Example 1, beads bound to biotinylated Luc by biotin-streptavidin interaction were unevenly distributed at the bottom of the container by a magnet. It is thought that more luminescence of luciferase was detected by the detector.
［標識成分から発光する波長域と光を吸収する化合物の関係］
＜比較例２＞
実施例１（ＳＡビーズ）のブルーデキストランに代え、ＡＢＳのピークが同じとなるようにタートラジン（Ｗａｋｏ社製 ２０４−００１０２）を添加（５．５４×１０-3ｗｔ％）した以外は、実施例１と同様の手順で実験を行った。また、比較例１（ＣＡビーズ）についても、上記と同様の手順でタートラジンに変えた実験を行った。
[Relationship between wavelength range of light emitted from labeling component and compound that absorbs light]
<Comparative example 2>
Example 1 except that tartrazine (204-000102 manufactured by Wako) was added (5.54 × 10 −3 wt%) so that the peak of ABS was the same instead of blue dextran of Example 1 (SA beads) The experiment was performed in the same procedure as in 1. Moreover, also about the comparative example 1 (CA bead), the experiment changed into the tartrazine in the same procedure as the above was conducted.
図３は、ＳＡビーズ及びＣＡビーズを用いた場合の測定結果を表すグラフである。化合物として実施例１及び比較例１のブルーデキストランに代え、タートラジンを用いた場合、ＳＡビーズとＣＡビーズで大きな差は無く、且つ、ｐｈｏｔｏｎ ｃｏｕｎｔｓ／ｓの値も実施例１及び比較例１より高かった。これは、ＣＡビーズを用いた場合、ビオチン化Ｌｕｃは遊離しているものの、タートラジンでほとんど吸収されなかった為と考えられる。なお、ＳＡビーズを用いたものの方が若干高い値が得られたが、これはＳＡビーズと結合したビオチン化Ｌｕｃが容器底部に集まっていることに起因するものと考えられる。 FIG. 3 is a graph showing measurement results when SA beads and CA beads are used. When tartrazine was used in place of blue dextran of Example 1 and Comparative Example 1 as a compound, there was no significant difference between SA beads and CA beads, and the value of phototon counts / s was higher than that of Example 1 and Comparative Example 1 It was. This is probably because when CA beads were used, biotinylated Luc was released, but was hardly absorbed by tartrazine. A slightly higher value was obtained using the SA beads, which is considered to be due to the biotinylated Luc bound to the SA beads gathering at the bottom of the container.
次に、Ｌｕｃの発光波長域と、光を吸収する化合物であるブルーデキストラン及びタートラジンの吸収波長域の関係を調べる実験を行った。 Next, an experiment was conducted to examine the relationship between the emission wavelength range of Luc and the absorption wavelength range of blue dextran and tartrazine, which are compounds that absorb light.
＜光を吸収する化合物の吸収波長域の測定＞
ブルーデキストラン（ＰＢＳで濃度を０．１ｗｔ％となるように調整）及びタートラジン（ＰＢＳで濃度を０．１×１０-2ｗｔ％となるように調整）の吸収波長域を、株式会社島津製作所製紫外可視分光光度計ＵＶ２４５０を用いて測定した。図４（１）はブルーデキストランの吸収波長域を表し、図４（２）はタートラジンの吸収波長域を表す。ブルーデキストランの吸収波長域は約４５０〜７５０ｎｍで吸収のピークは６１７ｎｍ、タートラジンの吸収波長域は約３００〜５００ｎｍで吸収のピークは４２６ｎｍであった。
<Measurement of absorption wavelength range of compound that absorbs light>
The absorption wavelength region of blue dextran (adjusted to a concentration of 0.1 wt% with PBS) and tartrazine (adjusted to a concentration of 0.1 × 10 −2 wt% with PBS) was manufactured by Shimadzu Corporation It measured using ultraviolet visible spectrophotometer UV2450. FIG. 4 (1) represents the absorption wavelength region of blue dextran, and FIG. 4 (2) represents the absorption wavelength region of tartrazine. The absorption wavelength range of blue dextran was about 450 to 750 nm, the absorption peak was 617 nm, the absorption wavelength range of tartrazine was about 300 to 500 nm, and the absorption peak was 426 nm.
＜Ｌｕｃの発光波長域の測定＞
オレンジ色Ｌｕｃを含むＰＢＳバッファー（ｐＨ７．４）１００μＬ（Ｌｕｃ濃度：１０μｇ／ｍＬ）及びＯｎｅ Ｇｌｏ Ｌｕｃｉｆｅｒａｓｅ ａｓｓａｙ ｓｙｓｔｅｍ １００μＬを混合し、日本分光株式会社製Ｓｐｅｃｔｒｏｆｌｕｏｒｏｍｅｔｅｒ ＦＰ−８５００を用いて発光波長域の測定を行った。緑色Ｌｕｃについても同様の手順で測定を行った。
<Measurement of emission wavelength range of Luc>
PBS buffer (pH 7.4) containing orange Luc (100 μL (Luc concentration: 10 μg / mL)) and One Glo Luciferase assay system (100 μL) were mixed, and the emission wavelength region was measured using Spectrofluorometer FP-8500 manufactured by JASCO Corporation. went. Measurement was also performed for green Luc in the same procedure.
図５はオレンジ色Ｌｕｃ及び緑色Ｌｕｃの発光波長域を表す図である。オレンジ色Ｌｕｃの発光波長域は約５００〜６５０ｎｍで発光のピークは約５７５ｎｍ、緑色Ｌｕｃの発光波長域は約４８０〜６３０ｎｍで発光のピークは約５４５ｎｍであった。上記の測定結果より、実施例１及び比較例１において、緑色Ｌｕｃの発光波長域（約４８０〜６３０ｎｍ）はブルーデキストランの吸収波長域（約４５０〜７５０ｎｍ）に含まれていた。一方、比較例２において、緑色Ｌｕｃの発光波長域（約４８０〜６３０ｎｍ）は、タートラジンの吸収波長域（約３００〜５００ｎｍ）とはほぼ異なっていた。以上の結果より、本発明の生物発光色素吸収法では、抗原−抗体反応する媒体中に加える化合物が吸収する光の波長領域が、標識成分１が発する光の波長域と少なくとも一部が重複、より好ましくは標識成分１が発する光の波長域を含むと、反応しなかった２次抗体２の標識成分１から発する光を効率的に吸収できることが明らかとなった。 FIG. 5 is a diagram illustrating the emission wavelength ranges of orange Luc and green Luc. The emission wavelength range of orange Luc was about 500 to 650 nm, the emission peak was about 575 nm, the emission wavelength range of green Luc was about 480 to 630 nm, and the emission peak was about 545 nm. From the above measurement results, in Example 1 and Comparative Example 1, the emission wavelength range of green Luc (about 480 to 630 nm) was included in the absorption wavelength range of blue dextran (about 450 to 750 nm). On the other hand, in Comparative Example 2, the emission wavelength range of green Luc (about 480 to 630 nm) was almost different from the absorption wavelength range of tartrazine (about 300 to 500 nm). From the above results, in the bioluminescent dye absorption method of the present invention, the wavelength range of the light absorbed by the compound added to the antigen-antibody reaction medium is at least partially overlapped with the wavelength range of the light emitted by the labeling component 1, More preferably, the light emitted from the label component 1 of the secondary antibody 2 that did not react can be efficiently absorbed when the wavelength range of the light emitted by the label component 1 is included.
［生物発光色素吸収法による菌の検出］
上記実施例１により、本発明の生物発光色素吸収法の有用性が確認できたことから、大腸菌を用いた検出を行った。
[Detection of bacteria by bioluminescent dye absorption method]
Since the usefulness of the bioluminescent dye absorption method of the present invention was confirmed in Example 1, detection using Escherichia coli was performed.
＜実施例２＞
１μｇ／ｍＬの緑色ビオチン化Ｌｕｃを２０μＬ、ストレプトアビジン化ａｎｔｉ Ｏ１５７抗体を５μＬ、４×１０5ｃｅｌｌｓ（１ ＯＤ６００／ｍＬ＝８×１０8で換算）を含む菌液５μＬ、を１．５ｍＬエッペンドルフチューブに加えてピペッティングにより混合し、３０ｍｉｎローテーターで混合した。緑色ビオチン化Ｌｕｃとストレプトアビジン化ａｎｔｉ Ｏ１５７抗体は、ビオチン−ストレプトアビジンの相互作用によって結合し、抗体−Ｌｕｃ複合体となる。ストレプトアビジン化ａｎｔｉ Ｏ１５７抗体は、ＫＰＬ社製Ａｆｆｉｎｉｔｙ Ｐｕｒｉｆｉｅｄ Ａｎｔｉｂｏｄｙ Ｔｏ Ｅ．ｃｏｌｉ Ｏ１５７：Ｈ７ Ｂａｃ Ｔｒａｃｅ Ａｎｔｉｂｏｄｙを、Ａｂｃａｍ社製Ｓｔｒｅｐｔａｖｉｄｉｎ ｃｏｎｊｕｇａｔｉｏｎ ｋｉｔによってストレプトアビジン化することで作製した。続いて、Ｏｎｅ Ｇｌｏ Ｌｕｃｉｆｅｒａｓｅ ａｓｓａｙ ｓｙｓｔｅｍ ４０μＬ、ブルーデキストラン、更にＰＢＳを加え、合計２００μＬの反応系を作製した。なお、反応系中のブルーデキストランの濃度は１．６２５ｗｔ％となるように添加した。ピペッティングにより混合し、遠心分離機により２０，８１７×ｇでＦｌａｓｈｉｎｇ後、浜松ホトニクス社製Ｈ７３６０−０１を用いて３０秒間測定した。
<Example 2>
20 μL of 1 μg / mL green biotinylated Luc, 5 μL of streptavidinated anti O157 antibody, 5 μL of 4 × 10 5 cells (converted to 1 OD600 / mL = 8 × 10 8 ), 1.5 mL Eppendorf tube In addition to the above, it was mixed by pipetting and mixed with a rotator for 30 minutes. Green biotinylated Luc and streptavidinated anti O157 antibody are combined by biotin-streptavidin interaction to form an antibody-Luc complex. Streptavidinated anti O157 antibody is an Affinity Purified Antibody To E. E. coli O157: H7 Bac Trace Antibody was produced by streptavidinization using the Abcam company Streptavidin conjugation kit. Subsequently, One Glo Luciferase assay system 40 μL, blue dextran, and PBS were further added to prepare a total reaction system of 200 μL. In addition, it added so that the density | concentration of the blue dextran in a reaction system might be 1.625 wt%. After mixing by pipetting and flashing with a centrifugal separator at 20,817 × g, measurement was performed for 30 seconds using H7360-01 manufactured by Hamamatsu Photonics.
＜比較例３＞
菌体を加えなかった以外は、実施例２と同様の手順で実験を行った。
<Comparative Example 3>
The experiment was performed in the same procedure as in Example 2 except that the cells were not added.
図６は、実施例２及び比較例３の測定結果を表すグラフである。菌を添加しなかった比較例３では、シグナルが４６４ｃｏｕｎｔｓ／ｓだったのに対し、菌を添加した実施例２では、２１８９ｃｏｕｎｔｓ／ｓのシグナルを検出することができた。以上の結果から、本発明の生物発光色素吸収法を用いることで、従来のＥＬＩＳＡと異なり、結合しなかった抗原、抗体等の洗浄工程を要せず、簡単且つ短時間で検体中の測定対象物質を測定できることを確認した。 FIG. 6 is a graph showing the measurement results of Example 2 and Comparative Example 3. In Comparative Example 3 in which no fungus was added, the signal was 464 counts / s, whereas in Example 2 in which the fungus was added, a signal of 2189 counts / s could be detected. From the above results, by using the bioluminescent dye absorption method of the present invention, unlike conventional ELISA, no washing step for unbound antigen, antibody, etc. is required, and the measurement target in the sample is simple and short. It was confirmed that the substance could be measured.
次に、生物発光色素吸収法の実施形態として、ルシフェラーゼの光を蛍光ガラスによって他の波長に変換した上で計測できるか確認を行った。
＜実施例３＞
蛍光ガラスを組み込んだ容器に、４００μｇ／ｍＬの緑色ビオチン化Ｌｕｃを１μＬ、実施例２で作製したストレプトアビジン化ａｎｔｉ Ｏ１５７抗体を５μＬ、８×１０5ｃｅｌｌｓ（１ ＯＤ６００／ｍＬ＝８×１０8で換算）を含む菌液を１０μＬ、加えてピペッティングにより撹拌し、３０ｍｉｎローテーターで混合した。続いて、Ｏｎｅ Ｇｌｏ Ｌｕｃｉｆｅｒａｓｅ ａｓｓａｙ ｓｙｓｔｅｍ ３６μＬ、ブルーデキストラン、更にＰＢＳを加え、合計２００μＬの反応系を作製した、なお、反応系中のブルーデキストランの濃度１．８ｗｔ％となるように添加した。ピペッティングにより混合し、３，０００×ｇで５ｍｉｎ遠心分離し、浜松ホトニクス社製近赤外光電子増倍管Ｈ１０３３０Ｂ−４５を用いて３０秒間測定した。
Next, as an embodiment of the bioluminescent dye absorption method, it was confirmed whether luciferase light could be measured after being converted to another wavelength by fluorescent glass.
<Example 3>
In a container incorporating fluorescent glass, 1 μL of 400 μg / mL green biotinylated Luc, 5 μL of the streptavidinated anti O157 antibody prepared in Example 2, 8 × 10 5 cells (1 OD600 / mL = 8 × 10 8 10 μL of the bacterial solution containing (converted) was added, stirred by pipetting, and mixed with a rotator for 30 min. Subsequently, One Glo Luciferase assay system 36 μL, blue dextran, and PBS were further added to prepare a total reaction system of 200 μL. The concentration of blue dextran in the reaction system was added to a concentration of 1.8 wt%. The mixture was mixed by pipetting, centrifuged at 3,000 × g for 5 min, and measured for 30 seconds using a near infrared photomultiplier tube H10330B-45 manufactured by Hamamatsu Photonics.
なお、蛍光ガラスを組み込んだ容器は、次の手順で作製した。（１）先ず、４９．５ｍｏｌ％Ｂｉ2Ｏ3粉末（関東化学社製 素材研究用試薬）、Ｂ2Ｏ3換算で４９．５ｍｏｌ％となる量のＨ3ＢＯ3粉末（ナカライテスク社製 特級試薬）、１ｍｏｌ％Ｓｂ2Ｏ3粉末（和光純薬工業株式会社製 試薬）を母体としたガラス原料、並びに、３ｍｏｌ％Ｙｂ2Ｏ3粉末（関東化学社製 素材研究用試薬）及び１ｍｏｌ％Ｎｄ2Ｏ3粉末（関東化学社製 素材研究用試薬）をチャック袋の中で混合し、蛍光ガラスの原料である混合粉末を調整した。次いで、混合粉末をアルミナ坩堝に投入し、大気雰囲気化、１２５０℃で１０分間加熱をして、溶融させた。続いて、ステンレス鋳型に溶融物（融液）を流し出して、室温（約２０℃）で空冷させて、台形状の蛍光ガラス（変換部）を作製した。図７（１）は作製した蛍光ガラスの写真である。
次に、３Ｄプリンター（Ｓｔｒａｔａｓｙｓ ＯＢＪＥＴ３０ Ｐｒｏ）を使用し、樹脂としてＯＢＪＥＴ Ｖｅｌｏ ｃｌｅａｒ ＲＧＤ８１０（Ｓｔｒａｔａｓｙｓ社製）を用いて、図７（２）に示す形状の容器を作製した。容器の筒状部分の内径（Ｌ）は６ｍｍ、筒状部分の高さ（Ｈ）は１１ｍｍであった。次いで、作製した容器の底面の内径部分に、図７（１）に示す台形状のガラス組成物を嵌めこんだ。
The container incorporating the fluorescent glass was prepared by the following procedure. (1) First, 49.5 mol% Bi 2 O 3 powder (material research reagent manufactured by Kanto Chemical Co., Inc.), H 3 BO 3 powder in an amount of 49.5 mol% in terms of B 2 O 3 (special grade manufactured by Nacalai Tesque) Reagent) 1 mol% Sb 2 O 3 powder (reagent manufactured by Wako Pure Chemical Industries, Ltd.) as a base material, 3 mol% Yb 2 O 3 powder (material research reagent manufactured by Kanto Chemical Co., Inc.) and 1 mol% Nd 2 O 3 powder (material research reagent manufactured by Kanto Chemical Co., Inc.) was mixed in a chuck bag to prepare a mixed powder as a raw material of fluorescent glass. Next, the mixed powder was put into an alumina crucible, and was heated to 1250 ° C. for 10 minutes to be melted by being atmospheric. Subsequently, the melt (melt) was poured out into a stainless steel mold and air-cooled at room temperature (about 20 ° C.) to produce a trapezoidal fluorescent glass (conversion part). FIG. 7 (1) is a photograph of the produced fluorescent glass.
Next, using a 3D printer (Stratasys OBJET30 Pro) and using OBJET Veloclear RGD810 (manufactured by Stratasys) as a resin, a container having the shape shown in FIG. 7 (2) was produced. The inner diameter (L) of the cylindrical portion of the container was 6 mm, and the height (H) of the cylindrical portion was 11 mm. Next, the trapezoidal glass composition shown in FIG. 7 (1) was fitted into the inner diameter portion of the bottom surface of the produced container.
＜比較例４＞
菌体を加えなかった以外は、実施例３と同様の手順で実験を行った。
<Comparative example 4>
The experiment was performed in the same procedure as in Example 3 except that the cells were not added.
図８は、実施例３及び比較例４の測定結果を表すグラフである。菌を添加しなかった比較例４では、シグナルが６０ｃｏｕｎｔｓ／ｓであったのに対し、菌を添加した実施例３では、２５３ｃｏｕｎｔｓ／ｓのシグナルを検出することができ、有意な差が見られた。以上の結果から、本発明の生物発光色素吸収法は、標識成分１からの発光を直接検出するのみでなく、標識成分１が発する光を他の波長域に変換して測定できることを確認した。 FIG. 8 is a graph showing the measurement results of Example 3 and Comparative Example 4. In Comparative Example 4 in which no bacterium was added, the signal was 60 counts / s, whereas in Example 3 in which the bacterium was added, a signal of 253 counts / s could be detected, showing a significant difference. It was. From the above results, it was confirmed that the bioluminescent dye absorption method of the present invention can not only directly detect the light emission from the labeling component 1 but also convert the light emitted from the labeling component 1 into another wavelength range for measurement.
本発明の免疫測定用キット及び免疫測定方法を用いると、簡単且つ短時間で検体中の測定対象物質を検出・測定することができる。したがって、食中毒菌等に汚染された食品等の検査、医療機関等での検査・定量に有用である。 By using the immunoassay kit and immunoassay method of the present invention, it is possible to detect and measure a measurement target substance in a sample easily and in a short time. Therefore, it is useful for inspection of foods contaminated with food poisoning bacteria, etc., and inspection / quantification in medical institutions.
Claims (10)
前記標識成分が発する波長域の光を吸収する化合物、
を少なくとも含む免疫測定用キット。 An antibody labeled with a labeling component capable of emitting light, and
A compound that absorbs light in a wavelength range emitted by the labeling component,
An immunoassay kit comprising at least
検体、前記検体中の測定対象物質と特異的に結合し且つ発光することができる標識成分で標識した抗体、及び前記標識成分から発する波長域の光を吸収する化合物、を媒体中で混合する工程、
前記標識成分から発した光を測定する工程、
測定した光量から、検体中の測定対象物質の有無を検出、又は測定対象物質を定量する工程、
を少なくとも含む免疫測定方法。 An immunoassay method for detecting the presence or absence of a measurement target substance in a sample or quantifying the measurement target substance,
Mixing in a medium a sample, an antibody labeled with a labeling component that can specifically bind to and emit light from the analyte in the sample, and a compound that absorbs light in the wavelength region emitted from the labeling component ,
Measuring light emitted from the labeling component,
Detecting the presence or absence of the measurement target substance in the sample from the measured light quantity, or quantifying the measurement target substance,
An immunoassay method comprising at least
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| US5017473A (en) * | 1987-05-26 | 1991-05-21 | Becton, Dickinson And Company | Homogeneous chemiluminescence immunoassay using a light absorbing material |
| JP2002034594A (en) * | 2000-07-24 | 2002-02-05 | National Institute Of Advanced Industrial & Technology | Method for living cell detection |
| WO2010036827A1 (en) * | 2008-09-24 | 2010-04-01 | Straus Holdings Inc. | Method for detecting analytes |
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| US5017473A (en) * | 1987-05-26 | 1991-05-21 | Becton, Dickinson And Company | Homogeneous chemiluminescence immunoassay using a light absorbing material |
| JP2002034594A (en) * | 2000-07-24 | 2002-02-05 | National Institute Of Advanced Industrial & Technology | Method for living cell detection |
| WO2010036827A1 (en) * | 2008-09-24 | 2010-04-01 | Straus Holdings Inc. | Method for detecting analytes |
| JP2012503780A (en) * | 2008-09-24 | 2012-02-09 | ストラウス ホールディングス インコーポレイテッド | Methods for detecting analytes |
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