JPH05340945A - Method and kit for measuring micro substance - Google Patents
Method and kit for measuring micro substanceInfo
- Publication number
- JPH05340945A JPH05340945A JP17165892A JP17165892A JPH05340945A JP H05340945 A JPH05340945 A JP H05340945A JP 17165892 A JP17165892 A JP 17165892A JP 17165892 A JP17165892 A JP 17165892A JP H05340945 A JPH05340945 A JP H05340945A
- Authority
- JP
- Japan
- Prior art keywords
- substance
- measured
- binding
- measuring
- solid phase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は微量物質分析方法及び測
定キット、特に抗原−抗体反応等を利用した測定機構の
改良に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for analyzing trace substances and a measuring kit, and more particularly to improving a measuring mechanism utilizing an antigen-antibody reaction and the like.
【０００２】[0002]
【従来の技術】近年、バイオテクノロジーの長足な進歩
に伴い、生体内の各種微量生理活性物質の測定或いは機
能解明が極めて重要視されている。ところで、一般に生
体内の各種微量活性物質は精製が難しく、多くのステッ
プを経て精製する方法及びアフィニティークロマトグラ
フィー等による精製方法が用いられてきた。2. Description of the Related Art In recent years, with the great progress of biotechnology, it has been extremely important to measure or elucidate the functions of various trace physiologically active substances in the living body. By the way, in general, it is difficult to purify various trace amounts of active substances in the living body, and a method of purifying through a number of steps and a purification method such as affinity chromatography have been used.
【０００３】[0003]
【発明が解決しようとする課題】しかしながら、これら
の生理活性物質は、精製操作中に用いられる各種高濃度
の試薬或いは処理によって変性を受けることが知られて
いる。そして、一般にこれらの僅かに変性を受けた精製
品を用いて研究が行われているが、勿論これらの変性品
が未変性品と同様な生理活性を生体内で示すか否か不明
である。そこで、生体内の各種微量生理活性物質を、変
性を避けて純化し、その生理活性を生体内における存在
状態が反映された条件下で試験を行い、該生理活性を明
らかとする手法の重要性が指摘され、その必要性が高ま
っている。However, it is known that these physiologically active substances are modified by various high-concentration reagents or treatments used during the purification operation. In general, studies have been carried out using these slightly modified purified products, but it is of course unknown whether these modified products exhibit the same physiological activity in vivo as the unmodified products. Therefore, it is important to clarify various physiologically active substances in the living body by clarifying the biological activity by purifying the biologically active substances by avoiding denaturation. Has been pointed out, and the need for it is increasing.
【０００４】例えば、ヒトIV型コラーゲン標品に対して
凍結保存血清を共存させると、添加血清濃度に比例して
ＥＬＩＳＡ反応の抑制が認められる。これは血清中のフ
ィブロネクチンが凍結により構造変化を受け、IV型コラ
ーゲンと結合反応を生じることによると考えられるが、
フィブロネクチンの未変性体、変性体を分離・識別・測
定することは、たとえ抗原−抗体反応を用いても極めて
困難である。すなわち、フィブロネクチンの変性体、未
変性体の分離自体が困難であり、それぞれを個別に認識
する抗体を得ることはさらに困難であることによる。本
発明は前記従来技術の課題に鑑みなされたものであり、
その目的は各種微量物質を変性させることなく純化する
微量物質測定方法及び測定キットを提供することにあ
る。For example, when a cryopreserved serum coexists with a human type IV collagen preparation, suppression of the ELISA reaction is observed in proportion to the concentration of the added serum. It is thought that this is because fibronectin in serum undergoes a structural change due to freezing and causes a binding reaction with type IV collagen,
It is extremely difficult to separate, identify, and measure the non-denatured form and denatured form of fibronectin even if the antigen-antibody reaction is used. That is, it is difficult to separate the denatured form and the non-denatured form of fibronectin, and it is more difficult to obtain an antibody that individually recognizes each. The present invention has been made in view of the above problems of the prior art,
An object of the invention is to provide a trace substance measuring method and a measuring kit for purifying various trace substances without denaturing them.
【０００５】[0005]
【課題を解決するための手段】前記目的を達成するため
に本発明者らが鋭意検討した結果、特異抗体を固定化し
た固相を用いて不純物を含む試料より微量物質を純化
し、この純化した物質と特異的に相互作用する結合物質
を用いることにより、前記微量物質の解析、定量を行い
得ることを見出し、本発明を完成するに至った。すなわ
ち、請求項１記載の微量物質測定方法は、結合物質Ａを
固相に固定化する固定化工程と、前記結合物質Ａに特異
的に結合する被測定物質Ｂを結合させる結合工程と、前
記被測定物質Ｂを測定する測定工程と、を備えたことを
特徴とする。Means for Solving the Problems As a result of intensive studies by the present inventors in order to achieve the above-mentioned object, a trace amount substance was purified from a sample containing impurities using a solid phase on which a specific antibody was immobilized, and the purification was performed. It was found that the trace substance can be analyzed and quantified by using a binding substance that specifically interacts with the substance, and the present invention has been completed. That is, the method for measuring a trace substance according to claim 1 comprises an immobilization step of immobilizing the binding substance A on a solid phase, a binding step of binding a substance B to be measured that specifically binds to the binding substance A, And a measurement step of measuring the substance B to be measured.
【０００６】ここで、結合物質Ａは、該結合物質Ａに対
する抗体を用いて固相に固定されていることが好適であ
る。請求項３記載の微量物質測定方法は、結合物質Ａを
固相に固定化する固定化工程と、前記結合物質Ａと特異
的に反応する被測定物質Ｂ1及び前記結合物質Ａと反応
しない疑似被測定物質Ｂ2が混在する被測定液を前記固
相と反応させる結合工程と、前記結合物質Ａと結合した
被測定物質Ｂ1を、前記被測定物質Ｂ1及び疑似被測定物
質Ｂ2を共に測定可能な方法により測定する測定工程
と、を備え、被測定液中の被測定物質Ｂ1を特異的に測
定することを特徴とする。Here, the binding substance A is preferably immobilized on a solid phase using an antibody against the binding substance A. The method for measuring a trace substance according to claim 3, wherein an immobilization step of immobilizing the binding substance A on a solid phase, a substance to be measured B 1 that specifically reacts with the binding substance A and a pseudo substance that does not react with the binding substance A. The binding step of reacting the measured liquid in which the measured substance B 2 is mixed with the solid phase, the measured substance B 1 bound to the binding substance A, the measured substance B 1 and the pseudo measured substance B 2 And a measurement step in which both are measured by a measurable method, and the substance to be measured B 1 in the liquid to be measured is specifically measured.
【０００７】ここで、被測定物質Ｂ1及び疑似被測定物
質Ｂ2と共に測定する測定工程は、該被測定物質Ｂ1及び
疑似測定物Ｂ2を共に認識する酵素標識抗体と反応さ
せ、その酵素活性を測定するものであることが好適であ
る。請求項５記載の微量物質測定方法は、被測定物質Ｂ
を固相に固定化する固定化工程と、前記被測定物質Ｂに
特異的に結合する結合物質Ａを結合させる結合工程と、
前記結合物質Ａを測定する測定工程と、を備えたことを
特徴とする。ここで、被測定物質Ｂは、該被測定物質Ｂ
に対する抗体を用いて固相に固定されることを特徴とす
る。[0007] Here, the measurement step of measuring with the measured substance B 1 and the pseudo-measured substance B 2 are both reacted with recognizing the enzyme-labeled antibody該被analyte B 1 and the pseudo-measured object B 2, the enzyme It is preferable that the activity is measured. The trace substance measuring method according to claim 5 is a substance to be measured B.
An immobilization step of immobilizing a solid phase on a solid phase, and a binding step of binding a binding substance A that specifically binds to the substance to be measured B,
And a measurement step of measuring the binding substance A. Here, the substance B to be measured is the substance B to be measured.
It is characterized in that it is immobilized on a solid phase using an antibody against.
【０００８】請求項７記載の微量物質測定方法は、被測
定液中の被測定物質Ｂ1及び疑似被測定物質Ｂ2を固相に
固定化する固定化工程と、前記被測定物質Ｂ1と特異的
に反応する結合物質Ａを前記固相と反応させる結合工程
と、前記被測定物質Ｂ1と結合した結合物質Ａを測定す
る測定工程と、を備え、被測定液中の被測定物質Ｂ1を
特異的に測定することを特徴とする。ここで、被測定物
質Ｂ1及び疑似被測定物質Ｂ2を共に固定化する固定化工
程は、該被測定物質Ｂ1及び疑似測定物Ｂ2を共に認識す
る固定化抗体と反応させるものであることを特徴とす
る。The method for measuring a trace substance according to claim 7 comprises an immobilization step of immobilizing a substance to be measured B 1 and a pseudo substance to be measured B 2 in a liquid to be measured on a solid phase, and the substance to be measured B 1 The binding substance A which reacts specifically with the binding substance A reacts with the solid phase, and the measurement process which measures the binding substance A bound with the substance B 1 to be measured. It is characterized by specifically measuring 1 . Here, the immobilization step of both the immobilized substance to be measured B 1 and the pseudo-measured substance B 2 is to be reacted with recognizing immobilized antibody該被analyte B 1 and the pseudo-measured object B 2 together It is characterized by
【０００９】請求項９記載の微量物質測定キットは、結
合物質Ａが固定化される固相と、前記結合物質Ａに特異
的に結合する被測定物質Ｂを測定する測定試薬と、を備
えたことを特徴とする。請求項１０記載の微量物質測定
キットは、被測定物質Ｂを固定化する固相と、前記被測
定物質Ｂに特異的に結合された結合物質Ａを測定する測
定試薬と、を備えたことを特徴とする。以下、本発明の
構成を詳述する。本発明に係る測定方法において、使用
する抗体としては、モノクローナル抗体及び／又はポリ
クローナル抗体であり、抗原認識部位及び抗原の種差の
認識に関する情報の明らかな抗体を用いることが好適で
ある。The trace substance measuring kit according to claim 9 comprises a solid phase on which the binding substance A is immobilized, and a measuring reagent for measuring the substance B to be measured which specifically binds to the binding substance A. It is characterized by The trace substance measurement kit according to claim 10, comprising a solid phase on which the substance B to be measured is immobilized, and a measurement reagent for measuring the binding substance A specifically bound to the substance B to be measured. Characterize. Hereinafter, the constitution of the present invention will be described in detail. In the measuring method according to the present invention, the antibody used is a monoclonal antibody and / or a polyclonal antibody, and it is preferable to use an antibody having clear information on the recognition site of the antigen and the difference in species of the antigen.
【００１０】又、抗体は好ましくは精製抗体を用いる。
本発明に係る分析方法では抗体アフィニティークロマト
におけるような、変性剤による溶出操作を実施すること
なく、次の試験を実施することが可能であるため、リガ
ンド（例えば抗原物質）を変性させることなくその特性
を評価することが出来る。本発明に係る測定方法におい
て、反応するリガンドとしては蛋白質、ペプチド、糖蛋
白質、核酸等が好適である。The antibody is preferably a purified antibody.
In the analysis method according to the present invention, the following test can be carried out without carrying out elution operation with a denaturing agent such as in antibody affinity chromatography, so that the ligand (for example, an antigenic substance) can be prepared without denaturing it. Characteristic can be evaluated. In the measuring method according to the present invention, the reacting ligand is preferably a protein, peptide, glycoprotein, nucleic acid or the like.
【００１１】本発明に係る測定方法において、リガンド
と特異的に相互作用するものとしては、各種リセプータ
ーを介してリガンドと結合する各種細胞、リガンドを特
異的に認識・結合する抗体以外の蛋白質（レクチンを含
む）、リガンドを特異的に認識する病気との関連で生体
内に産生された抗体、リガンドと特異的に結合する糖、
ペプチド、核酸及び脂質、及びハプテンを介する結合を
利用した蛋白質、合成高分子化合物、合成低分子化合物
であることが好適である。尚、本測定方法により、生体
高分子と細胞及び生体高分子間の特異的相互作用の解
析、定量及び病的に産生された抗体の解析、定量、精製
が困難な物質に関しても可能となる。In the assay method according to the present invention, as a substance that specifically interacts with a ligand, various cells that bind to the ligand through various receptors, proteins other than antibodies that specifically recognize and bind to the ligand (lectin). Antibody, produced in vivo in the context of a disease that specifically recognizes the ligand, a sugar that specifically binds to the ligand,
A peptide, a nucleic acid and a lipid, and a protein utilizing a bond via a hapten, a synthetic high molecular compound, and a synthetic low molecular compound are preferable. In addition, the present measurement method enables analysis of specific interactions between biopolymers and cells and biopolymers, as well as quantification and analysis of pathologically produced antibodies, even for substances that are difficult to quantify and purify.
【００１２】又、本発明に係る測定方法において、リガ
ンドを認識するものとして各種細胞を用いる場合には、
抗体を固定化する固相として細胞培養用のフラスコ、シ
ャーレ、皿等の培養器及び培養液内で浮遊する粒子を用
いることが好適である。更に、本発明に係る測定方法に
おいて、捕捉抗原を特異的に認識して結合したものを定
性、定量する方法としては、抗体等の結合物を特異的に
認識するもので酵素標識を付与することが好適である。Further, in the assay method according to the present invention, when various cells are used to recognize the ligand,
As the solid phase on which the antibody is immobilized, it is preferable to use particles that float in a culture medium such as a cell culture flask, a dish, a dish, or the like. Furthermore, in the measuring method according to the present invention, as a method for qualitatively and quantitatively recognizing and binding a capture antigen specifically, a method of specifically recognizing a bound substance such as an antibody is to attach an enzyme label. Is preferred.
【００１３】[0013]
【実施例】以下、図面に基づき本発明の好適な実施例を
説明する。尚、本発明は以下の実施例によって限定され
るものではない。実施例１ まず、図１に従い本発明の第一実施例に係る測定方法に
ついて概念について説明する。即ち、図１（Ａ）に示す
ようにウエル壁（固相）１０に抗体１２を結合させる。
この抗体１２に対し、一定量の結合物質１４を結合さ
せ、更に結合物質１４に特異的に結合する被測定物質１
６を結合させる（図１（Ｂ））。そして、被測定物質１
６を特異的に認識する酵素標識抗体１８を結合させ（図
１（Ｃ））、酵素活性部位により化学反応を進行させ、
その化学発光量より被測定物質１６の量を測定する方法
である（図１（Ｄ））。DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT A preferred embodiment of the present invention will be described below with reference to the drawings. The present invention is not limited to the following examples. Example 1 First, the concept of a measuring method according to a first example of the present invention will be described with reference to FIG. That is, the antibody 12 is bound to the well wall (solid phase) 10 as shown in FIG.
To-be-measured substance 1 which binds a certain amount of binding substance 14 to this antibody 12 and further specifically binds to binding substance 14
6 is bound (FIG. 1 (B)). And the substance to be measured 1
An enzyme-labeled antibody 18 that specifically recognizes 6 is bound (FIG. 1 (C)), and a chemical reaction is allowed to proceed by the enzyme active site,
This is a method of measuring the amount of the substance 16 to be measured from the amount of chemiluminescence (FIG. 1 (D)).
【００１４】従って、被測定物質１６の変性体（疑似被
測定物質）２０が被測定物質１６と共存し、該変性体２
０も酵素標識抗体１８と結合反応を生起してしまう場合
であっても、結合物質１４を認識する被測定物質１６の
みを特異的に測定することが出来る。又、本実施例に係
る測定方法の場合、結合物質１４に対するポリクローナ
ル抗体ないしモノクローナル抗体１２により、該結合物
質１４を固相に固定しているので、被測定物質１６に対
する抗体は酵素標識抗体１８として用いる一種のみで足
り、一般的なサンドイッチ法のように一の被測定物質１
６に対し二種類の抗体が要求されることはない。Therefore, the denatured body 20 of the substance 16 to be measured (pseudo substance to be measured) coexists with the substance 16 to be measured,
Even when 0 also causes a binding reaction with the enzyme-labeled antibody 18, only the substance 16 to be measured that recognizes the binding substance 14 can be specifically measured. Further, in the measuring method according to this example, since the binding substance 14 is immobilized on the solid phase by the polyclonal antibody or the monoclonal antibody 12 against the binding substance 14, the antibody against the substance to be measured 16 is the enzyme-labeled antibody 18. Only one type used, one substance to be measured 1 like the general sandwich method
No two different antibodies to 6 are required.
【００１５】更に、結合物質１４を抗体１２に結合させ
る際に、該結合物質１４と共に混在物質２２が存在する
場合にも、該混在物質２２は抗体１２に結合されないた
め、結合物質１４を単離する必要もない。以上のように
本実施例に係る測定方法によれば、結合物質１４ないし
被測定物質１６を単離・精製することなく、被測定物質
１６の測定を行うことが可能となり、結合物質１４との
間で生理活性を有する被測定物質１６のみを、その変性
を避けつつ正確に測定することが可能となる。Further, when the binding substance 14 is bound to the antibody 12, even if the contaminant 22 is present together with the binding substance 14, the contaminant 22 is not bound to the antibody 12, so the binding substance 14 is isolated. You don't even have to. As described above, according to the measuring method of the present embodiment, it becomes possible to measure the substance to be measured 16 without isolating and purifying the substance to be bound 14 or the substance to be measured 16, and the binding substance 14 and It becomes possible to accurately measure only the substance 16 to be measured which has physiological activity during the period while avoiding its denaturation.
【００１６】次に本実施例に係る測定法の具体例を、IV
型コラーゲンと結合反応を生起するフィブロネクチン変
性体を被測定物質として測定する例に基づき説明する。 （ａ）固相担体としてポリスチレン性マイクロプレート
（Flow Laboratories製）の作成 特開昭６３−７８０６７及び特開昭６３−２４６３９６
に記載されたIV型コラーゲンに対するモノクローナル抗
体（ＪＫ１９９）をリン酸緩衝生理食塩液（ｐＨ７．
４）に溶解し、その濃度を１４．５μg/mlに調整した。
この抗体溶液を、固相担体としてのポリスチレン性マイ
クロプレートにコートするために、１ウェル当たり１０
０μl添加し、３７℃、２４時間処理後、ブロックエー
ス（大日本製薬）をリン酸緩衝生理食塩液（ｐＨ７．
４）で１／４希釈したブロッキング緩衝液を１ウェル当
たり３５０μｌ添加し、３７℃、１時間処理してブロッ
キングし、よく水を切ったのちに凍結保存する。使用時
に予め室温に戻してアッセイに使用する（図１
（Ａ））。Next, a specific example of the measuring method according to this embodiment will be described below.
A description will be given based on an example in which a modified fibronectin that causes a binding reaction with type collagen is measured as the substance to be measured. (A) Preparation of polystyrene microplate (manufactured by Flow Laboratories) as a solid phase carrier JP-A-63-78067 and JP-A-63-246396
The monoclonal antibody against type IV collagen (JK199) described in 1. is added to phosphate buffered saline (pH 7.
It was dissolved in 4) and its concentration was adjusted to 14.5 μg / ml.
To coat this antibody solution on a polystyrene microplate as a solid phase carrier, 10
After adding 0 μl and treating at 37 ° C. for 24 hours, Block Ace (Dainippon Pharmaceutical Co., Ltd.) was added to phosphate buffered saline (pH 7.
Blocking buffer solution diluted 1/4 in 4) was added at 350 μl per well, treated at 37 ° C. for 1 hour for blocking, well drained, and stored frozen. Before use, return to room temperature before use (see FIG. 1).
(A)).
【００１７】（ｂ）IV型コラーゲン特異結合プレートの
作成 抗体結合プレートにブロッキング緩衝液で希釈したIV型
コラーゲン（１２５ng/ml）溶液を１ウェル当たり１０
０μｌ添加し、３７℃、１．５時間反応させて結合させ
る（図１（Ｂ））。反応後、洗浄緩衝液（０．０５％Ｔ
ｗｅｅｎ２０含有リン酸緩衝生理食塩液（ｐＨ７．
４））でよく洗浄する。 （ｃ）フィブロネクチンの特異的結合反応 凍結保存血清及び凍結保存ＥＤＴＡ血漿を洗浄緩衝液と
ブロックエースを９：１に混和した溶液で各種濃度に希
釈した。この希釈液を１ウェル当たり１００μｌ添加
し、３７℃、３時間反応させて、前記IV型コラーゲンと
特異的に反応するフィブロネクチンを結合させる（図１
（Ｃ））。反応後、洗浄緩衝液をよく洗浄する。(B) Preparation of type IV collagen-specific binding plate An antibody binding plate was diluted with a blocking buffer solution of type IV collagen (125 ng / ml) solution at a rate of 10 per well.
0 μl was added, and the mixture was allowed to react at 37 ° C. for 1.5 hours for binding (FIG. 1 (B)). After the reaction, wash buffer (0.05% T
phosphate buffered saline containing ween 20 (pH 7.
Wash thoroughly with 4)). (C) Specific binding reaction of fibronectin Cryopreserved serum and cryopreserved EDTA plasma were diluted to various concentrations with a solution in which a wash buffer and Block Ace were mixed at 9: 1. 100 μl of this diluted solution was added to each well and reacted at 37 ° C. for 3 hours to bind fibronectin which specifically reacts with the type IV collagen (FIG. 1).
(C)). After the reaction, the washing buffer is thoroughly washed.
【００１８】（ｄ）結合フィブロネクチンの検出 結合したフィブロネクチンは、次のようにして検出し
た。すなわち、Cappel社製の西洋わさび由来ペルオキシ
ダーゼ結合抗ヒトフィブロネクチンポリクローナル抗体
を、洗浄緩衝液（０．０５％Ｔｗｅｅｎ２０含有リン酸
緩衝生理食塩液（ｐＨ７．４））とブロックエースを
９：１に混和した溶液で、７５０倍希釈し、該希釈溶液
を１ウェル当たり１００μｌ添加し、３７℃、１時間反
応させて特異的に反応したフィブロネクチンに結合させ
る。反応後、洗浄緩衝液で良く洗浄する。(D) Detection of bound fibronectin The bound fibronectin was detected as follows. That is, a horseradish-derived peroxidase-conjugated anti-human fibronectin polyclonal antibody manufactured by Cappel was mixed with a washing buffer (0.05% Tween20-containing phosphate buffered saline (pH 7.4)) and Block Ace at a ratio of 9: 1. The solution is diluted 750-fold, 100 μl of the diluted solution is added to each well, and the mixture is reacted at 37 ° C. for 1 hour to specifically bind to the reacted fibronectin. After the reaction, thoroughly wash with a wash buffer.
【００１９】次に2,2'-azino-di[3-ethyl-benzthiazoli
ne]sulfonate（ＡＢＴＳ）を基質とするペルオキシダー
ゼ基質システム（Kirregaard & Perry Laboratories）
を１ウェル当たり１００μｌ添加し、室温で１時間発色
反応後、マイクロプレートリーダーで波長４０５ｎｍの
吸光度を測定し、盲検と試料の吸光度の差を求める（図
１（Ｄ））。尚、図２にヒト凍結保存血清、ヘパリン血
漿、クエン酸血漿及び凍結保存ＥＤＴＡ血漿中のIV型コ
ラーゲンに特異的に結合するフィブロネクチン量に関し
て検討した結果を示す。同図より明らかなように、ヒト
凍結保存ＥＤＴＡ血漿と比較して、凍結保存血清中のフ
ィブロネクチンが著しい結合性を示した。この結果、IV
型コラーゲン非結合プレートを用いた実験より、この結
合反応はIV型コラーゲンに特異的結合反応であることが
明らかとなった。Next, 2,2'-azino-di [3-ethyl-benzthiazoli
Peroxidase substrate system using ne] sulfonate (ABTS) as substrate (Kirregaard & Perry Laboratories)
100 μl per well is added, and after color reaction for 1 hour at room temperature, the absorbance at a wavelength of 405 nm is measured with a microplate reader, and the difference between the absorbance in the blind and that in the sample is determined (FIG. 1 (D)). In addition, FIG. 2 shows the results of examination on the amount of fibronectin specifically binding to type IV collagen in human cryopreserved serum, heparin plasma, citrate plasma and cryopreserved EDTA plasma. As is clear from the figure, as compared with human cryopreserved EDTA plasma, fibronectin in cryopreserved serum showed remarkable binding. As a result, IV
From the experiment using the type IV collagen non-binding plate, it was revealed that the binding reaction was a type IV collagen-specific binding reaction.
【００２０】実施例２ 次に、本発明の第２実施例について説明する。図３は、
本実施例に係る測定法の概念の説明図であり、前記図１
と対応する部分には同一符合を付して説明を省略する。
本実施例においては、まず同図（Ａ）に示すように、ウ
ェル壁１０に被測定物質１６に対する抗体１２を結合さ
せる。そして、被測定物質１６を反応させると同図
（Ｂ）に示すように被測定物質１６が抗体１２と結合し
固定化される。この状態で結合物質１４を反応させる
と、同図（Ｃ）に示すように被測定物質１６と結合物質
１４が反応し、更に結合物質１４に対する酵素標識抗体
１８を作用させることにより、同図（Ｄ）に示すような
ウェル壁１０−抗体１２−被測定物質１６−結合物質１
４−酵素標識抗体１８の結合構造が完成し、酵素標識抗
体１８の酵素活性を測定することにより、被測定物質１
６の測定を行うことが出来る。[0020] Example 2 The following describes a second embodiment of the present invention. Figure 3
It is explanatory drawing of the concept of the measuring method which concerns on a present Example, and is the said FIG.
The parts corresponding to are given the same reference numerals and the description thereof will be omitted.
In this embodiment, first, as shown in FIG. 3A, the antibody 12 against the substance 16 to be measured is bound to the well wall 10. Then, when the substance 16 to be measured is reacted, the substance 16 to be measured binds to the antibody 12 and is immobilized as shown in FIG. When the binding substance 14 is reacted in this state, the substance 16 to be measured reacts with the binding substance 14 as shown in FIG. Well wall 10 as shown in D) -antibody 12-measured substance 16-binding substance 1
4-The binding structure of the enzyme-labeled antibody 18 is completed, and by measuring the enzyme activity of the enzyme-labeled antibody 18, the substance to be measured 1
6 measurements can be made.
【００２１】一方、被測定物質１６と同様の抗原−抗体
反応を示す変性体２０が存在した場合、同図（Ｂ）に示
すように変性体２０も抗体１２に捕捉されるが、該変性
体２０には結合物質１４が結合せず、この結果酵素標識
抗体１８が結合しないため、酵素活性は被測定物質１６
のみを特異的に示すこととなり、その変性体２０は測定
対象とはならない。次に、第二実施例に係る測定法の具
体例について説明する。On the other hand, when there is a modified substance 20 which shows the same antigen-antibody reaction as the substance to be measured 16, the modified substance 20 is also captured by the antibody 12 as shown in FIG. Since the binding substance 14 does not bind to 20 and the enzyme-labeled antibody 18 does not bind to 20 as a result, the enzyme activity is
Therefore, the modified body 20 is not a measurement target. Next, a specific example of the measuring method according to the second embodiment will be described.
【００２２】（ａ）ビオチン標識モノクローナル抗体の
調整 特開昭６３−７８０６７及び特開昭６３−２４６３９６
に記載されたIV型コラーゲンに対するモノクローナル抗
体を０．１Ｍ炭酸水素ナトリウム溶液に１ｍｇ／ｍｌに
なるように溶解させた。この溶液１ｍｌに対してＮ−ヒ
ドロキシサクシイミドビオチンを１ｍｇ／ｍｌの濃度と
なるようにジメチルスルオキサイドに溶解させた溶液を
０．０６ｍｌ加え、攪拌しながら室温で４時間反応させ
た。反応液をリン酸緩衝生理食塩液（ｐＨ７．４）に対
して充分に透析後、冷蔵又は凍結保存した。(A) Preparation of biotin-labeled monoclonal antibody JP-A-63-78067 and JP-A-63-246396
The monoclonal antibody against type IV collagen described in 1. was dissolved in a 0.1 M sodium hydrogen carbonate solution to a concentration of 1 mg / ml. 0.06 ml of a solution prepared by dissolving N-hydroxysuccinimidobiotin in dimethylsulfoxide to a concentration of 1 mg / ml was added to 1 ml of this solution, and reacted at room temperature for 4 hours while stirring. The reaction solution was thoroughly dialyzed against phosphate buffered saline (pH 7.4) and then refrigerated or stored frozen.
【００２３】（ｂ）ビオチン化抗体−アビジンＰＯＤ複
合体の調整法 前記（ａ）で調整したビオチン化抗体（０．２ｍｇ）に
アビジンＰＯＤ（２ｍｇ Vector Laboratories）を加
え、３７℃で１時間反応させる。反応液を遠心分離後、
上清をセファロース６Ｂカラム（直径１．５cm×長さ３
０cm 溶出液：０．１Ｍ ＨＥＰＥＳ緩衝液ｐＨ７．
０）にかけ、ビオチン化抗体−アビジンＰＯＤ複合体を
得た。 （ｃ）固相担体としてポリスチレン製マイクロプレート
（Ｆｌｏｗ Ｌａｂｏｒａｔｏｒｉｅｓ製）の作製 Ｃａｐｐｅｌ社製の抗ヒトフィブロネクチンポリクロー
ナル抗体をコーティング緩衝液（炭酸緩衝液）（ｐＨ
９．６）に溶解し、その濃度を１０μｇ／ｍｌに調製し
た。この抗体溶液を固相担体としてのポリスチレン製マ
イクロプレートにコートするために１ウェル当たり１０
０μｌ添加し、４℃、２４時間処理後、ブロックエース
（大日本製薬）をリン酸緩衝生理食塩液（ｐＨ７．４）
で１／４希釈したブッキング緩衝液を１ウェル当たり３
５０μｌ添加し、３７℃、１時間処理してブロッキング
し、よく水を切った後に凍結保存する。使用時に予め室
温に戻してアッセイに使用する（図３（Ａ））。(B) Method for preparing biotinylated antibody-avidin POD complex Avidin POD (2 mg Vector Laboratories) was added to the biotinylated antibody (0.2 mg) prepared in (a) above, and the mixture was reacted at 37 ° C. for 1 hour. . After centrifuging the reaction solution,
The supernatant was added to a Sepharose 6B column (diameter 1.5 cm x length 3
0 cm eluent: 0.1 M HEPES buffer pH 7.
0) to obtain a biotinylated antibody-avidin POD complex. (C) Preparation of polystyrene microplate (manufactured by Flow Laboratories) as a solid phase carrier An anti-human fibronectin polyclonal antibody manufactured by Cappel was used as a coating buffer (carbonate buffer) (pH).
It was dissolved in 9.6) and its concentration was adjusted to 10 μg / ml. To coat this antibody solution on a polystyrene microplate as a solid phase carrier, 10
After adding 0 μl and treating at 4 ° C. for 24 hours, Block Ace (Dainippon Pharmaceutical Co., Ltd.) is treated with phosphate buffered saline (pH 7.4).
Booking buffer diluted 1/4 with 3 per well
Add 50 μl, treat at 37 ° C. for 1 hour to block, drain well, and store frozen. It is returned to room temperature before use and used for the assay (FIG. 3 (A)).
【００２４】（ｄ）フィブロネクチンの特異的結合反応 凍結保存血清及び凍結保存ＥＤＴＡ血漿を、洗浄緩衝液
とブロックエースを９：１に混和した溶液で、各種濃度
に希釈した。この希釈液を１ウェル当たり１００μｌ添
加し、３７℃、３時間反応させて特異的に反応するフィ
ブロネクチンを結合させる（図３（ｃ））。反応後、洗
浄緩衝液で良く洗浄する。 （ｅ）IV型コラーゲン特異的結合 各プレートにブロッキング緩衝液で希釈したIV型コラー
ゲン（１．０ｍｇ／ｍｌ）溶液を１ウェル当たり１００
μｌ添加し、３７℃、１．５時間反応させて結合させる
（図３（Ｂ））。反応後、洗浄緩衝液（０．０５％Ｔｗ
ｅｅｎ２０含有リン酸緩衝生理食塩液（ｐＨ７．４））
で良く洗浄する。(D) Specific binding reaction of fibronectin Cryopreserved serum and cryopreserved EDTA plasma were diluted to various concentrations with a solution in which a washing buffer and Block Ace were mixed at a ratio of 9: 1. 100 μl of this diluted solution was added per well, and the mixture was allowed to react at 37 ° C. for 3 hours to bind specifically reacting fibronectin (FIG. 3 (c)). After the reaction, thoroughly wash with a wash buffer. (E) Type IV collagen-specific binding To each plate, a type IV collagen (1.0 mg / ml) solution diluted with a blocking buffer solution was added to 100 wells per well.
μl is added, and the mixture is allowed to react at 37 ° C. for 1.5 hours for binding (FIG. 3 (B)). After the reaction, washing buffer (0.05% Tw
phosphate buffered saline containing een20 (pH 7.4))
Wash well with.
【００２５】（ｆ）結合IV型コラーゲンの検出 フィブロネクチンと特異的に結合したIV型コラーゲンの
定量は、特開昭６３−７８０６７及び特開昭６３−２４
６３９６に記載されたIV型コラーゲンに対するモノクロ
ーナル抗体（ＪＫ１９９）を用いて行った。即ち、ビオ
チン化ＪＫ１９９抗体−アビジンＰＯＤ複合体を０．５
μg/ml濃度となるように、洗浄緩衝液（０．０５％Ｔｗ
ｅｅｎ２０含有リン酸緩衝生理食塩液（ｐＨ７．４））
とブロックエースの９：１混和液で希釈し、その希釈液
の０．５μｌ／ｍｌの溶液を１ウェル当たり１００μｌ
添加し、３７℃、１時間反応させてフィブロネクチンに
特異的に反応・結合したコラーゲンに結合させる（図３
（Ｄ））。反応後、洗浄緩衝液で良く洗浄する。(F) Detection of bound type IV collagen Quantification of type IV collagen specifically bound to fibronectin is carried out by the methods described in JP-A-63-78067 and JP-A-63-24.
It was performed using a monoclonal antibody against type IV collagen described in 6396 (JK199). That is, the biotinylated JK199 antibody-avidin POD complex was added to 0.5
Wash buffer (0.05% Tw) so that the concentration becomes μg / ml.
phosphate buffered saline containing een20 (pH 7.4))
Dilute with a 9: 1 mixture of Block Ace and Block Ace, and add 0.5 μl / ml of the diluted solution to 100 μl per well.
It is added and reacted at 37 ° C. for 1 hour to bind to collagen specifically reacted and bound to fibronectin (FIG. 3).
(D)). After the reaction, thoroughly wash with a wash buffer.
【００２６】次に、2,2'-azino-di[3-ethyl-benzthiazo
line] sulfonate（ＡＢＴＳ）を基質とするペルオキシ
ダーゼ基質システム（Ｋｉｒｋｅｇａａｒｄ ＆ Ｐｅ
ｒｒｙ Ｌａｂｏｒａｔｏｒｉｅｓ）を１ウェル当たり
１００μｌ添加し、室温で１時間発色反応後、マイクロ
プレートリーダーで波長４０５ｎｍの吸光度を測定し、
盲検と試料の吸光度の差を求める（図３（Ｄ））。尚、
図４にヒト凍結保存血清、ヘパリン血漿、クエン酸血漿
及び凍結保存ＥＤＴＡ血漿中のIV型コラーゲンに特異的
に結合するフィブロネクチン量に関して検討した結果を
示した。Next, 2,2'-azino-di [3-ethyl-benzthiazo
line] peroxidase substrate system (Kirkegaard & Pe) using sulfonate (ABTS) as a substrate
100 μl per well of rry Laboratories) was added, and after 1 hour of color reaction at room temperature, the absorbance at a wavelength of 405 nm was measured with a microplate reader,
The difference in the absorbance between the blind test and the sample is obtained (FIG. 3 (D)). still,
FIG. 4 shows the results of studies on the amount of fibronectin specifically binding to type IV collagen in human cryopreserved serum, heparin plasma, citrate plasma and cryopreserved EDTA plasma.
【００２７】同図より明らかなように、ヒト凍結保存Ｅ
ＤＴＡ血漿と比較して、凍結保存血清中のフィブロネク
チンが著しい結合性を示した。尚、以上の実施例におい
ては被測定物質をフィブロネクチンとした例について説
明したが、本発明は特に生体内の微量生理活性物質の測
定に好適であり、例えば抗原物質と特異的に相互作用す
るものが細胞である場合には、結合細胞数を、例えば試
薬を該細胞内に導入する等して細胞機能を利用した発色
反応を起こさせることにより定量することも可能であ
る。As is clear from the figure, human cryopreservation E
Fibronectin in cryopreserved serum showed significant binding compared to DTA plasma. In the above examples, an example in which the substance to be measured was fibronectin was described, but the present invention is particularly suitable for measuring a trace amount of physiologically active substance in the living body, for example, a substance which interacts specifically with an antigen substance. When is a cell, the number of bound cells can be quantified by, for example, introducing a reagent into the cell to cause a color reaction utilizing a cell function.
【００２８】[0028]
【発明の効果】以上説明したように本発明に係る微量物
質分析方法によれば、物質間の特異的相互作用を利用し
て微量成分の分析を行うこととしたので、微量活性物質
を複雑な精製工程等を得ることなく正確に測定すること
が可能となる。As described above, according to the method for analyzing a trace substance according to the present invention, since a trace component is analyzed by utilizing a specific interaction between substances, a trace active substance is complicated. It is possible to perform accurate measurement without obtaining a purification step or the like.
【図１】本発明の第一実施例に係る微量物質測定方法の
説明図である。FIG. 1 is an explanatory diagram of a trace substance measuring method according to a first embodiment of the present invention.
【図２】図１に示した方法による測定結果の説明図であ
る。FIG. 2 is an explanatory diagram of measurement results by the method shown in FIG.
【図３】本発明の第二実施例に係る微量物質測定方法の
説明図である。FIG. 3 is an explanatory diagram of a trace substance measuring method according to a second embodiment of the present invention.
【図４】図３に示した実施例による測定結果の説明図で
ある。FIG. 4 is an explanatory diagram of measurement results according to the example shown in FIG.
１２…抗体 １４…結合物質 １６…被測定物質 １８…酵素標識抗体 12 ... Antibody 14 ... Binding substance 16 ... Target substance 18 ... Enzyme-labeled antibody
Claims (10)
程と、 前記結合物質Ａに特異的に結合する被測定物質Ｂを結合
させる結合工程と、 前記被測定物質Ｂを測定する測定工程と、 を備えたことを特徴とする微量物質測定方法。1. An immobilization step of immobilizing a binding substance A on a solid phase, a binding step of binding a substance B to be measured that specifically binds to the binding substance A, and a measurement for measuring the substance B to be measured. A method for measuring trace substances, which comprises:
Ａは、該結合物質Ａに対する抗体を用いて固相に固定さ
れていることを特徴とする微量物質測定方法。2. The method for measuring a trace substance according to claim 1, wherein the binding substance A is immobilized on a solid phase by using an antibody against the binding substance A.
程と、 前記結合物質Ａと特異的に反応する被測定物質Ｂ1及び
前記結合物質Ａと反応しない疑似被測定物質Ｂ2が混在
する被測定液を前記固相と反応させる結合工程と、 前記結合物質Ａと結合した被測定物質Ｂ1を、前記被測
定物質Ｂ1及び疑似被測定物質Ｂ2を共に測定可能な方法
により測定する測定工程と、 を備え、被測定液中の被測定物質Ｂ1を特異的に測定す
ることを特徴とする微量物質測定方法。3. An immobilization step of immobilizing the binding substance A on a solid phase, and a substance to be measured B 1 which specifically reacts with the substance to be bound A and a pseudo substance to be measured B 2 which does not react with the substance to be bound A. A binding step of reacting a mixed solution to be measured with the solid phase, and a method capable of measuring the measured substance B 1 bound to the binding substance A together with the measured substance B 1 and the pseudo measured substance B 2. A method for measuring a trace substance, which comprises a measuring step for measuring, and specifically measures the substance B 1 to be measured in a liquid to be measured.
質Ｂ1及び疑似被測定物質Ｂ2と共に測定する測定工程
は、該被測定物質Ｂ1及び疑似測定物Ｂ2を共に認識する
酵素標識抗体と反応させ、その酵素活性を測定するもの
であることを特徴とする微量物質測定方法。4. A method according to claim 3, wherein the measuring step, both enzymes that recognize labeling該被analyte B 1 and the pseudo-measured object B 2 to be measured with the substance to be measured B 1 and the pseudo-measured substance B 2 A method for measuring a trace substance, which comprises reacting with an antibody and measuring the enzyme activity thereof.
工程と、 前記被測定物質Ｂに特異的に結合する結合物質Ａを結合
させる結合工程と、 前記結合物質Ａを測定する測定工程と、 を備えたことを特徴とする微量物質測定方法。5. An immobilization step of immobilizing a substance to be measured B on a solid phase, a binding step of binding a binding substance A that specifically binds to the substance to be measured B, and a measurement for measuring the binding substance A. A method for measuring trace substances, which comprises:
質Ｂは、該被測定物質Ｂに対する抗体を用いて固相に固
定されることを特徴とする微量物質測定方法。6. The method for measuring a trace substance according to claim 5, wherein the substance B to be measured is immobilized on a solid phase by using an antibody against the substance B to be measured.
測定物質Ｂ2を固相に固定化する固定化工程と、 前記被測定物質Ｂ1と特異的に反応する結合物質Ａを前
記固相と反応させる結合工程と、 前記被測定物質Ｂ1と結合した結合物質Ａを測定する測
定工程と、 を備え、被測定液中の被測定物質Ｂ1を特異的に測定す
ることを特徴とする微量物質測定方法。7. An immobilization step of immobilizing a substance B 1 to be measured and a pseudo substance B 2 to be measured in a liquid to be measured on a solid phase, and a binding substance A which specifically reacts with the substance B 1 to be measured. a bonding step of reacting with the solid phase, said measurement step of measuring a binding substance a bound to the substance to be measured B 1, comprises a specifically measuring the subject substance B 1 to be measured liquid Characteristic trace substance measurement method.
質Ｂ1及び疑似被測定物質Ｂ2を共に固定化する固定化工
程は、該被測定物質Ｂ1及び疑似測定物Ｂ2を共に認識す
る固定化抗体と反応させるものであることを特徴とする
微量物質測定方法。8. The method of claim 7, fixing step of fixing both the substance to be measured B 1 and the pseudo-measured substance B 2 is a該被analyte B 1 and the pseudo-measured object B 2 both recognition A method for measuring a trace substance, which comprises reacting with an immobilized antibody.
する測定試薬と、 を備えたことを特徴とする微量物質測定キット。9. A trace substance measurement kit comprising: a solid phase on which a binding substance A is immobilized; and a measurement reagent for measuring a substance B to be measured that specifically binds to the binding substance A. ..
定する測定試薬と、 を備えたことを特徴とする微量物質測定キット。10. A trace substance measurement, comprising: a solid phase on which the substance B to be measured is immobilized, and a measurement reagent for measuring the binding substance A specifically bound to the substance B to be measured. kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17165892A JPH05340945A (en) | 1992-06-05 | 1992-06-05 | Method and kit for measuring micro substance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17165892A JPH05340945A (en) | 1992-06-05 | 1992-06-05 | Method and kit for measuring micro substance |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH05340945A true JPH05340945A (en) | 1993-12-24 |
Family
ID=15927302
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP17165892A Withdrawn JPH05340945A (en) | 1992-06-05 | 1992-06-05 | Method and kit for measuring micro substance |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH05340945A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007534951A (en) * | 2004-04-29 | 2007-11-29 | ファーマ ディヴェロップメント ソシエタ ア レスポンサビリタ リミタータ | Monoclonal antibody, hybridoma, improved method for measuring protein PTX3, and kit for measurement |
-
1992
- 1992-06-05 JP JP17165892A patent/JPH05340945A/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007534951A (en) * | 2004-04-29 | 2007-11-29 | ファーマ ディヴェロップメント ソシエタ ア レスポンサビリタ リミタータ | Monoclonal antibody, hybridoma, improved method for measuring protein PTX3, and kit for measurement |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2006058295A (en) | Assay | |
| CN110196334A (en) | Thrombomodulin chemiluminescence immunoassay immue quantitative detection reagent box and preparation method thereof | |
| WO2006123789A1 (en) | Method of analyzing enzyme | |
| JP4197393B2 (en) | Test method for IgA nephropathy | |
| KR101255828B1 (en) | Qualitative and quantitative analytical method for analyzing the activity type of an enzyme that is activated by proteolysis | |
| RU2417375C2 (en) | Method and set for immune-enzyme assay of functional activity of human complement component c3 | |
| RU2232991C1 (en) | Method for assay of functional activity of human complement factor d | |
| JPWO1999050663A6 (en) | Testing methods for IgA nephropathy | |
| RU2380707C1 (en) | Method and set for immunoenzyme assay of functional activity of component c3 of human complement by alternative pathway of activation | |
| HU228077B1 (en) | Cobalamin assay | |
| JPH05340945A (en) | Method and kit for measuring micro substance | |
| Nechaeva et al. | Rapid Automatic Determination of Four Cardiomarkers in the Blood Plasma of Patients with Cardiopathologies | |
| AU703940B2 (en) | Regenerable solid phase for carrying out specific binding reactions | |
| RU2376605C1 (en) | Method and set for immune-enzyme detection of functuinal activity of factor d of human complement | |
| RU2506594C1 (en) | Method and kit for immune-enzyme assay of functional activity of human complement component c3 | |
| RU2251697C1 (en) | Method for detecting functional activity of human complement c3 component according to classical way of activation | |
| RU2425383C1 (en) | METHOD AND KIT FOR IMMUNE-ENZYME FUNCTIONAL ACTIVITY ASSAY ON C1r2s2 SUBCOMPONENTS OF FIRST COMPONENT OF HUMAN COMPLEMENT | |
| JPH01295165A (en) | Immunoassay | |
| SU1606940A1 (en) | Method of qualitative determination of catalitically active molecules of serine proteinases of bacillae | |
| RU2195670C1 (en) | Method of assay of functional activity of component c2 in human complement | |
| JPH11311624A (en) | Assay surface enabling analysis material liberation stage | |
| WO2000039586A2 (en) | Tetramethylbenzidine formulation for horseradish peroxidase enzyme assays | |
| RU2251698C1 (en) | METHOD FOR DETECTING FUNCTIONAL ACTIVITY OF SUBCOMPONENTS C1r2s2 OF THE FIRST COMPONENT OF HUMAN COMPLEMENT | |
| RU2235329C1 (en) | METHOD FOR DETERMINATION OF FUNCTIONAL ACTIVITY OF C1r2s2 SUBCOMPONENTS OF THE FIRST HUMAN COMPLEMENT COMPONENT | |
| JPH08509064A (en) | Immobilization of chemically crosslinked proteins on a solid support |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A300 | Withdrawal of application because of no request for examination |
Free format text: JAPANESE INTERMEDIATE CODE: A300 Effective date: 19990831 |