JP2000009733A - Measuring method and measurement kit for sugar chain coupling physiological active material - Google Patents
Measuring method and measurement kit for sugar chain coupling physiological active materialInfo
- Publication number
- JP2000009733A JP2000009733A JP10192312A JP19231298A JP2000009733A JP 2000009733 A JP2000009733 A JP 2000009733A JP 10192312 A JP10192312 A JP 10192312A JP 19231298 A JP19231298 A JP 19231298A JP 2000009733 A JP2000009733 A JP 2000009733A
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- Prior art keywords
- substance
- sugar chain
- physiologically active
- binding
- antibody
- 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.)
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は糖鎖結合性生理活性
物質の測定法に関する。また、更に本発明は上記測定法
に使用するための測定キットに関する。The present invention relates to a method for measuring a sugar chain-binding physiologically active substance. Further, the present invention relates to a measurement kit for use in the above-mentioned measurement method.
【０００２】[0002]
【従来の技術】近年、生体内において生理活性を誘導す
る働きがある生理活性物質（例えば肝炎などの疾患にお
いて著しく増加し、肝実質細胞の成長を促進することが
知られている肝細胞成長因子（ＨＧＦ）などのサイトカ
イン等）が糖鎖と特異的親和性を示し、結合性を有する
ことが明らかになってきている。2. Description of the Related Art In recent years, a physiologically active substance which has a function of inducing a biological activity in a living body (for example, a hepatocyte growth factor which is known to increase remarkably in diseases such as hepatitis and promote the growth of hepatocytes). (E.g., cytokines such as (HGF)) have been shown to exhibit specific affinity for sugar chains and have binding properties.
【０００３】このような生体内に存在する様々な生理活
性物質と糖鎖の結合性を利用して生理活性物質の測定を
行う方法としては、例えば、線維芽細胞増殖因子に対す
る抗体を固相担体に固着し、線維芽細胞増殖因子を含む
検体をこの固相担体に接触させて抗原抗体反応に供し、
抗体を介して固相担体に固着した線維芽細胞増殖因子を
標識物質により標識したヘパリンと結合させることで検
出する方法が知られている（特開平３−１５７５８
号）。また、特開平４−２６２２５７号では、ヘパリン
を固相担体に固着させ、標識物質により標識した抗線維
芽細胞増殖因子抗体を用いて、ヘパリンを介して固相担
体に結合した線維芽細胞増殖因子を測定する方法が記載
されている。[0003] As a method of measuring a physiologically active substance by utilizing the binding properties of various physiologically active substances present in a living body and a sugar chain, for example, an antibody against fibroblast growth factor is applied to a solid support. The specimen containing fibroblast growth factor is contacted with this solid phase carrier and subjected to an antigen-antibody reaction.
A method is known in which fibroblast growth factor fixed to a solid support via an antibody is detected by binding to heparin labeled with a labeling substance (JP-A-3-15758).
issue). JP-A-4-262257 discloses a fibroblast growth factor bound to a solid phase carrier via heparin using an anti-fibroblast growth factor antibody labeled with a heparin fixed to a solid phase carrier and labeled with a labeling substance. Is described.
【０００４】[0004]
【発明が解決しようとする課題】固相担体に抗体を固着
させる生理活性物質の従来の測定法は、抗体を固相担体
に結合させる際、及び長期保存の際に抗体の力価が低下
することから、使用する固相担体の調製は測定の直前に
行うか、抗体を固着した固相担体の保存に工夫を要した
ため、予め生理活性物質を特異的に結合できる物質を固
着させ、長期保存が可能な固相担体を用いる測定法の開
発が必要とされていた。In the conventional method for measuring a physiologically active substance for immobilizing an antibody on a solid support, the titer of the antibody decreases when the antibody is bound to the solid support and during long-term storage. Therefore, the solid phase carrier to be used should be prepared immediately before the measurement, or the storage of the solid phase carrier to which the antibody was immobilized required some contrivance. It has been required to develop a measurement method using a solid support capable of performing the measurement.
【０００５】また、糖鎖を固相担体に結合させる従来の
測定方法においては、抗体を標識物質により標識するこ
とから、抗体が糖鎖結合性生理活性物質に対する親和性
を失うこともあり、正確な測定を行うことができない場
合も多かった。[0005] Further, in the conventional measurement method in which a sugar chain is bound to a solid support, since the antibody is labeled with a labeling substance, the antibody may lose its affinity for a sugar chain-binding physiologically active substance. In many cases could not be performed.
【０００６】[0006]
【課題を解決するための手段】本発明者らは、上記課題
を解決するべく鋭意検討を重ねた結果、固相担体に糖鎖
を結合することにより、従来知られていた生理活性物質
の測定法と比較して、より簡便で正確な測定が可能であ
ることを見いだした。Means for Solving the Problems The present inventors have found, after intensive studies to solve the above problems, by binding a sugar chain to a solid support, the physiologically active substance has been known traditional It has been found that simpler and more accurate measurement is possible as compared with the measurement method.
【０００７】すなわち、本発明は、「生理活性物質に特
異的に結合する糖鎖が結合した固相担体に検体を接触さ
せ、検体中の糖鎖結合性生理活性物質を固相担体上の該
糖鎖に結合させ、固相担体に結合した当該糖鎖結合性生
理活性物質を検出することを特徴とする測定法」であ
り、糖鎖を結合した固相担体の使用による糖鎖結合性生
理活性物質の測定法に特徴付けられる。[0007] That is, the present invention provides a method wherein "a sample is brought into contact with a solid support to which a sugar chain specifically binding to a physiologically active substance is bound, and the sugar chain-binding bioactive substance in the sample is applied to the solid support. A sugar chain-bound physiologically active substance bound to a solid phase carrier and detecting the sugar chain-bound physiologically active substance bound to the sugar chain. Characterized by the method of measuring the active substance.
【０００８】また、更に本発明は、「検体中に含まれる
二種以上の糖鎖結合性生理活性物質を測定する方法であ
って、糖鎖が結合した固相担体に検体を接触させて該糖
鎖を介して固相担体に前記生理活性物質を結合させ、固
相担体に結合した糖鎖結合性生理活性物質を、各糖鎖結
合性生理活性物質に対する二種以上の抗体によって検出
する糖鎖結合性生理活性物質の測定法」を提供し、単一
の固相担体を使用して複数の糖鎖結合性生理活性物質を
同時に測定することを可能とした。さらに、本発明は上
述の測定法それぞれに適した糖鎖結合性生理活性物質の
測定キットを提供する。Further, the present invention provides a method for measuring two or more sugar chain-binding physiologically active substances contained in a sample, the method comprising: The bioactive substance is bound to the solid support via a sugar chain, and the sugar chain-binding bioactive substance bound to the solid support is detected by two or more antibodies against each sugar chain-binding bioactive substance. The present invention provides a "method of measuring a chain-binding bioactive substance", thereby making it possible to simultaneously measure a plurality of sugar chain-binding bioactive substances using a single solid support. Further, the present invention provides a kit for measuring a sugar chain-binding physiologically active substance, which is suitable for each of the above-mentioned measuring methods.
【０００９】[0009]
【発明の実施の形態】以下に本発明を実施の形態により
詳説する。 本発明方法１ 本発明方法１は、生理活性物質に特異的に結合する糖鎖
が結合した固相担体に検体を接触させ、検体中の糖鎖結
合性生理活性物質を固相担体上の糖鎖に特異的に結合さ
せ、固相担体に結合した当該糖鎖結合性生理活性物質を
検出することを特徴とする測定法である。DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to embodiments. Method 1 of the Present Invention In the method 1 of the present invention, a sample is brought into contact with a solid support to which a sugar chain specifically binding to a physiologically active substance is bound, and the sugar chain-binding bioactive substance in the sample is converted to a sugar on the solid support. This is a measurement method characterized by specifically binding to a chain and detecting the sugar chain-binding physiologically active substance bound to a solid phase carrier.
【００１０】本明細書において、糖鎖とは生理活性物質
が結合しうる糖鎖であれば特に限定はされないが、糖鎖
としてはグリコサミノグリカン、多くのサイトカインは
ヘパリン又はヘパラン硫酸に特異的に結合することか
ら、その中でも特にヘパリン又はヘパラン硫酸が好まし
い。また本発明において上記糖鎖を結合するための固相
担体としては、マイクロプレート、ビーズ、チューブ、
メンブレン、ゲル、微粒子状固相担体〔例えばアガロー
ス粒子、ゼラチン粒子、カオリン粒子、合成ポリマー粒
子（ラテックス粒子等）〕等が挙げられ、これらの固相
担体の中でも、特に正確な定量性とその実施に際しての
簡便性を考慮すると、マイクロプレート、ビーズ、チュ
ーブ又は微粒子状固相担体を固相担体として用いること
が好ましい。糖鎖を上記の固相担体に結合する方法は、
アフィニティークロマトグラフィー担体の調製などで常
用されている公知の結合法を用いることができる。糖鎖
の結合が困難な素材（例えばポリスチレン製）の固相担
体の場合、その素材とよく吸着し、併せて糖鎖にも吸着
性を示すタンパク質など（例えば糖鎖としてヘパリン又
はヘパラン硫酸を使用する場合には、フィブロネクチ
ン、或いはその還元誘導体又はアルキル化誘導体などが
例示される）を最初に固相担体に結合させ、前記タンパ
ク質に糖鎖を結合させる方法が例示される。In the present specification, the sugar chain is not particularly limited as long as it is a sugar chain to which a physiologically active substance can bind, but the sugar chain is glycosaminoglycan, and many cytokines are specific to heparin or heparan sulfate. Among them, heparin or heparan sulfate is particularly preferable. Further, in the present invention, as a solid phase carrier for binding the sugar chain, a microplate, beads, tubes,
Membrane, gel, fine-particle solid support [eg agarose particles, gelatin particles, kaolin particles, synthetic polymer particles (latex particles, etc.)] and the like. Considering the simplicity of the process, it is preferable to use a microplate, a bead, a tube, or a particulate solid carrier as the solid carrier. The method of binding a sugar chain to the solid support is as follows.
A known binding method commonly used in the preparation of affinity chromatography carriers and the like can be used. In the case of a solid phase carrier made of a material (for example, made of polystyrene) to which sugar chains are difficult to bind, a protein that adsorbs well to the material and also has an adsorptivity to sugar chains (for example, using heparin or heparan sulfate as sugar chains) In this case, fibronectin or a reduced derivative or an alkylated derivative thereof is exemplified), which is first bound to a solid support, and a sugar chain is bound to the protein.
【００１１】本発明方法により測定される糖鎖結合性生
理活性物質としては、上述の糖鎖に結合性を有する生理
活性物質であれば特に限定はされないが、例えばサイト
カインが挙げられる。例えば上述の好ましい糖鎖として
挙げたヘパリン及びヘパラン硫酸を用いる場合には、線
維芽細胞増殖因子（ＦＧＦ）、肝細胞成長因子（ＨＧ
Ｆ）、及び血管内皮細胞成長因子（ＶＥＧＦ）、血管内
皮細胞増殖因子（ＥＣＧＦ）、トランスフォーミング成
長因子（ＴＧＦ）、上皮細胞成長因子（ＥＧＦ）、ミッ
ドカイン（ＭＫ）、インターロイキン８（ＩＬ８）、ビ
トロネクチン（ＶＮ）、ヘパリン結合性脳細胞分裂誘発
因子（ＨＢＢＭ）、ヘパリン結合性神経突起伸長促進因
子（ＨＢＮＦ）等のサイトカインが挙げられるが、その
中でも特に広い反応条件下で安定した結合性を示す線維
芽細胞増殖因子、肝細胞成長因子及び血管内皮細胞成長
因子が好ましく、より正確な測定が可能であるため肝細
胞成長因子及び血管内皮細胞成長因子が最も好ましい。[0011] The sugar chain-binding physiologically active substance measured by the method of the present invention is not particularly limited as long as it is a physiologically active substance having the above-mentioned sugar chain binding property, and examples thereof include cytokines. For example, when heparin and heparan sulfate mentioned above as preferred sugar chains are used, fibroblast growth factor (FGF), hepatocyte growth factor (HG
F) and vascular endothelial growth factor (VEGF), vascular endothelial growth factor (ECGF), transforming growth factor (TGF), epidermal growth factor (EGF), midkine (MK), interleukin 8 (IL8) , Vitronectin (VN), heparin-binding brain cell mitogen (HBBM), heparin-binding neurite outgrowth promoting factor (HBNF), and other cytokines. The indicated fibroblast growth factor, hepatocyte growth factor and vascular endothelial cell growth factor are preferred, and hepatocyte growth factor and vascular endothelial cell growth factor are most preferred because more accurate measurement is possible.
【００１２】固相担体に糖鎖を介して結合した上記の糖
鎖結合性生理活性物質の検出は、該糖鎖結合性生理活性
物質に特異的に結合する物質を用いて行うことが好まし
い。そのような物質としては、例えば前記糖鎖結合性生
理活性物質に対する抗体等が親和性の強さから好まし
い。前記抗体としてはポリクローナル抗体又はモノクロ
ーナル抗体を用いることが可能であり特に限定はされな
い。上記検出において、上記糖鎖結合性生理活性物質に
対する抗体を用いる場合は、当該抗体又は当該抗体に対
する二次抗体を標識物質により標識することが、より正
確な糖鎖結合性生理活性物質の測定が可能となるため好
ましい。当該標識物質としては、例えば特異的結合対
（例えばビオチンとストレプトアビジン等のアビジン
類、又はレクチンとそのレクチンが認識する糖鎖等）の
一方の物質；ＦＩＴＣ、フィコエリトリン、ユーロピウ
ム、フィコシアニン、ローダミン、テキサスレッド、ウ
ンベリフェロン、トリカラー、シアニン又は７−アミノ
−４−メチルクマリン−３−酢酸（ＡＭＣＡ）等の蛍光
物質類；ルミノール、アクリジニウム又はルシゲニン等
の発光物質類、アルカリホスファターゼ、β−ガラクト
シダーゼ、ペルオキシダーゼ又はグルコースオキシダー
ゼ等の酵素類；ジニトロフルオロベンゼン、ＡＭＰ（ア
デノシン一リン酸）又は２，４−ジニトロアニリン等の
ハプテン類；及び12 5Ｉ、131Ｉ、3Ｈ等のラジオアイソ
トープ類等を用いることが可能であり、特に限定される
ものではない。標識物質の検出は、使用する標識物質に
適した通常実施される方法により行うことが可能であ
る。例えば、標識物質として上記酵素に分類されるペル
オキシダーゼを用いた場合は、テトラメチルベンジジン
（ＴＭＢ）などの酸素受容体と過酸化水素（H2O2）など
の酸素供与体を反応させることにより、溶液の着色によ
り検出することが可能である。The detection of the sugar chain-binding physiologically active substance bound to the solid support via a sugar chain is preferably performed using a substance that specifically binds to the sugar chain-binding physiologically active substance. As such a substance, for example, an antibody or the like to the sugar chain-binding physiologically active substance is preferable because of its high affinity. The antibody may be a polyclonal antibody or a monoclonal antibody, and is not particularly limited. In the detection, when an antibody against the sugar chain-binding physiologically active substance is used, labeling the antibody or a secondary antibody against the antibody with a labeling substance enables more accurate measurement of the sugar chain-binding physiologically active substance. It is preferable because it becomes possible. As the labeling substance, for example, one substance of a specific binding pair (eg, avidins such as biotin and streptavidin, or lectin and a sugar chain recognized by the lectin); FITC, phycoerythrin, europium, phycocyanin, rhodamine, Texas Fluorescent substances such as red, umbelliferone, tricolor, cyanine or 7-amino-4-methylcoumarin-3-acetic acid (AMCA); luminescent substances such as luminol, acridinium or lucigenin, alkaline phosphatase, β-galactosidase, enzymes such as peroxidase or glucose oxidase; used and 12 5 I, 131 I, 3 H radioisotopes such as such; dinitrofluorobenzene, AMP (adenosine monophosphate) or 2,4-dinitrophenyl hapten such as aniline Possible There, it is not particularly limited. The detection of the labeling substance can be performed by a commonly practiced method suitable for the labeling substance to be used. For example, when peroxidase classified as the above enzyme is used as a labeling substance, by reacting an oxygen acceptor such as tetramethylbenzidine (TMB) with an oxygen donor such as hydrogen peroxide (H 2 O 2 ), It can be detected by coloring the solution.
【００１３】本明細書において検体とは、糖鎖結合性生
理活性物質を含みうる液体であれば特に限定はされない
が、特に生体内から取り出した体液検体（尿、血液、血
漿、血清、組織液、リンパ液、腹水等）、臓器抽出液、
細胞抽出液又は培養上清等が挙げられる。[0013] In the present specification, the sample is not particularly limited as long as it can contain a sugar chain-binding physiologically active substance. In particular, a body fluid sample (urine, blood, plasma, serum, tissue fluid, Lymph, ascites, etc.), organ extract,
Cell extract, culture supernatant and the like can be mentioned.
【００１４】本発明方法２ 本発明方法２は、検体中に含まれる二種以上の糖鎖結合
性生理活性物質を測定する方法であって、糖鎖が結合し
た固相担体に検体を接触させ、糖鎖を介して固相担体に
結合した二種以上の糖鎖結合性生理活性物質を、糖鎖結
合性生理活性物質に対する二種以上の抗体により測定す
る方法であり、且つ二種以上の糖鎖結合性生理活性物質
に対する抗体、あるいはこれらの抗体に対する二次抗体
がそれぞれ異なる標識物質により標識されていることが
好ましい。Method 2 of the Present Invention Method 2 of the present invention is a method for measuring two or more kinds of sugar chain-binding physiologically active substances contained in a sample, wherein the sample is brought into contact with a solid support to which a sugar chain is bound. A method for measuring two or more kinds of sugar chain-binding physiologically active substances bound to a solid phase carrier via sugar chains by using two or more kinds of antibodies against the sugar chain-binding physiologically active substances, and two or more kinds. It is preferable that the antibodies against the sugar chain-binding physiologically active substance or the secondary antibodies against these antibodies are labeled with different labeling substances.
【００１５】本発明における、検体、糖鎖及び固相担体
は上述の本発明方法１に記載したものと同様である。本
発明方法２は検体中に含まれる複数の糖鎖結合性生理活
性物質を測定するための方法であるが、その測定の対象
となる糖鎖結合性生理活性物質は、本発明方法によって
測定できる限り限定されないが、前記の通りサイトカイ
ンが好ましい。例えば、糖鎖として上述の最も好ましい
形態であるヘパリン又はヘパラン硫酸を用いた場合に
は、それに対して親和性が高く、且つ抗体の作成が可能
である物質であるＦＧＦ、ＨＧＦ又はＶＥＧＦなどが挙
げられるが、その中でも本発明方法１と同様、安定した
測定が可能となるためＨＧＦ及びＶＥＧＦが最も好まし
いが、特に限定はされない。例えば前記ＨＧＦ及びＶＥ
ＧＦを同時に測定する場合は、それらに対する抗体（抗
ＨＧＦ抗体及び抗ＶＥＧＦ抗体）を使用できる。これら
の抗体としてはポリクローナル抗体又はモノクローナル
抗体を利用することが可能である。本発明方法２で使用
する生理活性物質に対する二種以上の抗体は、それぞれ
判別して検出することが可能であるように、例えば一方
はマウス由来のＩｇＧ抗体、他方はヤギ由来のＩｇＧ抗
体と、それぞれ異なる生物種由来の抗体を使用する。例
えば一方の抗体をマウス由来のＩｇＧ抗体、他方をヤギ
由来のＩｇＧ抗体を用いた場合には、それらを判別、検
出する手段としては、それぞれ異なる標識物質により標
識した二次抗体である抗マウスＩｇＧ抗体及び抗ヤギＩ
ｇＧ抗体を使用する。また、生理活性物質に対する二種
以上の抗体は、それぞれ異なるサブクラスに属する抗体
であってもよい。複数の二次抗体それぞれを標識する標
識物質としては、上述の本発明方法１で列挙した物質が
挙げられるが、その中でも特に一方の二次抗体を標識す
る標識物質としてラジオアイソトープ類又は蛍光物質類
を使用することが好ましい。それぞれの標識物質を検出
する方法は、選択する標識物質にあわせ、公知の方法か
ら適宜選択することが可能である。In the present invention, the specimen, sugar chain and solid phase carrier are the same as those described in the above-mentioned method 1 of the present invention. The method 2 of the present invention is a method for measuring a plurality of sugar chain-binding bioactive substances contained in a sample, and the sugar chain-binding bioactive substance to be measured can be measured by the method of the present invention. Although not limited, cytokines are preferred as described above. For example, when heparin or heparan sulfate, which is the most preferable form described above, is used as the sugar chain, FGF, HGF, or VEGF, which is a substance having a high affinity for the sugar chain and capable of producing an antibody, may be used. Among them, HGF and VEGF are most preferable because stable measurement is possible as in the method 1 of the present invention, but there is no particular limitation. For example, the HGF and VE
When GF is measured simultaneously, antibodies against them (anti-HGF antibody and anti-VEGF antibody) can be used. As these antibodies, polyclonal antibodies or monoclonal antibodies can be used. The two or more antibodies against the physiologically active substance used in the method 2 of the present invention can be distinguished and detected, for example, one is a mouse-derived IgG antibody, the other is a goat-derived IgG antibody, Antibodies from different species are used. For example, when one antibody is a mouse-derived IgG antibody and the other is a goat-derived IgG antibody, the means for discriminating and detecting them is anti-mouse IgG, which is a secondary antibody labeled with a different labeling substance. Antibodies and anti-goat I
Use the gG antibody. Further, the two or more antibodies against the physiologically active substance may be antibodies belonging to different subclasses. Examples of the labeling substance for labeling each of a plurality of secondary antibodies include the substances listed in the above-mentioned method 1 of the present invention. Among them, radioisotopes or fluorescent substances are particularly preferred as labeling substances for labeling one secondary antibody. It is preferred to use The method for detecting each labeling substance can be appropriately selected from known methods according to the labeling substance to be selected.
【００１６】本発明キット１ 本発明キット１は、少なくとも下記の〜の構成要素
を含む糖鎖結合性生理活性物質の測定キットである。 糖鎖を結合した固相担体； 糖鎖結合性生理活性物質に対する抗体； 標識物質により標識した抗糖鎖結合性生理活性物質抗
体に対する二次抗体。Kit 1 of the Present Invention Kit 1 of the present invention is a kit for measuring a sugar chain-binding physiologically active substance containing at least the following components: A solid-phase carrier having a sugar chain bound thereto; an antibody against a sugar chain-binding bioactive substance; a secondary antibody against an anti-sugar chain binding bioactive substance antibody labeled with a labeling substance.
【００１７】本発明キット１は本発明方法１に記載され
た測定法を行うためのキットである。従って、本発明キ
ット１における糖鎖、糖鎖結合性生理活性物質、糖鎖結
合性生理活性物質に対する抗体、二次抗体は本発明方法
１に記載したものと同様である。本発明キット１におけ
る標識物質としては、本発明方法１に列挙した標識物質
を使用することが可能であるが、特にキットとしての操
作の簡便性を考慮すると、酵素類を使用することが好ま
しい。標識物質の検出は、使用する標識物質により適宜
選択するべきであるが、例えば標識物質としてペルオキ
シダーゼを選択した際は、それを検出する手段として
は、通常は過酸化水素及びＴＭＢの反応による発色が挙
げられる。The kit 1 of the present invention is a kit for performing the measuring method described in the method 1 of the present invention. Therefore, the sugar chain, the sugar chain-binding physiologically active substance, the antibody against the sugar chain-binding physiologically active substance, and the secondary antibody in the kit 1 of the present invention are the same as those described in the method 1 of the present invention. As the labeling substance in the kit 1 of the present invention, the labeling substances listed in the method 1 of the present invention can be used. However, considering the simplicity of the operation as a kit, it is preferable to use enzymes. The detection of the labeling substance should be appropriately selected depending on the labeling substance to be used.For example, when peroxidase is selected as the labeling substance, as a means for detecting it, color development by the reaction of hydrogen peroxide and TMB is usually used. No.
【００１８】本発明キット２ 本発明キット２は、少なくとも下記の〜の構成要素
を含む糖鎖結合性生理活性物質の測定キットである。 糖鎖を結合した固相担体； 第一の糖鎖結合性生理活性物質に対する抗体（抗体
１）； 第二の糖鎖結合性生理活性物質に対する抗体（抗体
２）； 第一の標識物質により標識した抗体１に対する二次抗
体（二次抗体１）； 第二の標識物質により標識した抗体２に対する二次抗
体（二次抗体２）。Kit 2 of the Present Invention Kit 2 of the present invention is a kit for measuring a sugar chain-binding physiologically active substance, which contains at least the following components: A solid phase carrier to which a sugar chain is bound; an antibody against the first sugar chain-binding bioactive substance (antibody 1); an antibody against the second sugar chain-binding bioactive substance (antibody 2); labeled with the first labeling substance A secondary antibody against the antibody 1 (secondary antibody 1); a secondary antibody against the antibody 2 labeled with the second labeling substance (secondary antibody 2).
【００１９】本発明キット２は本発明方法２に記載され
た測定法を行うためのキットである。従って、本発明キ
ット２における糖鎖、固相担体、測定対象とする糖鎖結
合性生理活性物質及びそれぞれの糖鎖結合性生理活性物
質に対する二次抗体は本発明方法２の記載と同様であ
る。本発明キット２における標識物質としては、本発明
方法２で列挙したものを使用することが可能であるが、
その中でも特に二種の抗体（抗体１、抗体２）を明確に
判別して検出することが可能であるように、異なる検出
法により検出される標識物質の組み合わせを適宜選択す
ることが好ましい。少なくとも、二種の二次抗体の一方
がラジオアイソトープ又は蛍光物質により標識されたキ
ットが、検出及び測定の正確性から好ましい。これらの
標識物質により、検出の手段は適宜選択される。検出の
手段としては、本発明方法１、本発明方法２及び本発明
キット１で記載したものと同様である。The kit 2 of the present invention is a kit for performing the measuring method described in the method 2 of the present invention. Therefore, the sugar chain, the solid phase carrier, the sugar chain-binding physiologically active substance to be measured, and the secondary antibody against each of the sugar chain-binding physiologically active substances in Kit 2 of the present invention are the same as those described in Method 2 of the present invention. . As the labeling substance in the kit 2 of the present invention, those listed in the method 2 of the present invention can be used.
Among them, it is particularly preferable to appropriately select a combination of labeling substances detected by different detection methods so that two types of antibodies (antibody 1 and antibody 2) can be clearly distinguished and detected. A kit in which at least one of the two secondary antibodies is labeled with a radioisotope or a fluorescent substance is preferable in terms of detection and measurement accuracy. The detection means is appropriately selected depending on these labeling substances. The means for detection is the same as that described in the method 1, the method 2 and the kit 1 of the present invention.
【００２０】[0020]
【実施例】以下に実施例により、本発明をより具体的に
詳説する。 ＜１＞ヘパリンを使用したＨＧＦの測定 １２本つながった0.25mlのマイクロチューブ（ヌンク社
製）に、牛血清アルブミンを1%含む100μlの生理的リン
酸緩衝液（ＢＳＡ−ＰＢＳと記載する）にヒト組換えＨ
ＧＦを各種濃度（0μg、0.13μg、0.25μg、0.5μg、1
μg、2μg、4μg、8μg）で含むように加えた溶液を分
注した。このマイクロチューブに、洗浄したヘパリン結
合粒子（ヘパリンアガロースビーズ（タイプI：シグマ
社製）50μlを添加し、室温に30分間放置した。各チュ
ーブ中のヘパリン結合粒子をＢＳＡ−ＰＢＡで４回遠心
により洗浄後、0.02%のTween-20を含むＰＢＳ（ＰＢＳ
Ｔと記載する）で、遠心により４回洗浄した。各チュー
ブに抗ＨＧＦ抗体（ポリクローナルヤギＩｇＧ抗体：Ｒ
＆Ｄ システムス社製）を含むＢＳＡ−ＰＢＳ（抗ＨＧ
Ｆ抗体：ＢＳＡ−ＰＢＳ＝１：５００）を添加し、60分
間室温に放置した。ヘパリン結合粒子をＢＳＡ−ＰＢＳ
Ｔで４回遠心により洗浄し、１：１０００で西洋ワサビ
ペルオキシダーゼ結合抗ＩｇＧ抗体（バイオラッド社
製）を含むＢＳＡ−ＰＢＳを100μl添加し、室温で60分
間放置した。その後、ヘパリン結合粒子をＢＳＡ−ＰＢ
ＳＴで４回、ＰＢＳＴで４回、遠心により洗浄し、洗浄
後のヘパリン結合粒子に100μlの西洋ワサビペルオキシ
ダーゼ基質溶液（1.25mM TMB、2.21mM過酸化水素、1% D
MSO、0.08M 酢酸緩衝液（pH4.9）：バイオラッド社製）
を添加して室温で３０分間放置して発色させた。ヘパリ
ン結合粒子を遠沈後、上清を９６穴マイクロタイタープ
レート（ファルコン社製）に移し、イムノミニプレート
リーダー（ヌンク社製）を使用して、414nmの吸光度を
測定した（図１）。The present invention will be described in more detail with reference to the following examples. <1> Measurement of HGF using heparin Into 12 connected 0.25 ml microtubes (manufactured by Nunc), 100 μl of a physiological phosphate buffer containing 1% of bovine serum albumin (described as BSA-PBS) Human recombinant H
Various concentrations of GF (0 μg, 0.13 μg, 0.25 μg, 0.5 μg, 1 μg
μg, 2 μg, 4 μg, and 8 μg). 50 μl of the washed heparin-bound particles (heparin agarose beads (Type I: manufactured by Sigma)) was added to the microtube, and left at room temperature for 30 minutes.The heparin-bound particles in each tube were centrifuged four times with BSA-PBA. After washing, PBS containing 0.02% Tween-20 (PBS
(Described as T)) and washed four times by centrifugation. Add an anti-HGF antibody (polyclonal goat IgG antibody: R
& D Systems Inc.) containing BSA-PBS (anti-HG
F antibody: BSA-PBS = 1: 500) and left at room temperature for 60 minutes. Heparin-binding particles were converted to BSA-PBS
After washing by centrifugation four times at T, 100 μl of BSA-PBS containing horseradish peroxidase-conjugated anti-IgG antibody (manufactured by Bio-Rad) at 1: 1000 was added and left at room temperature for 60 minutes. Thereafter, the heparin-bound particles were transferred to BSA-PB.
After washing by centrifugation four times with ST and four times with PBST, 100 μl of a horseradish peroxidase substrate solution (1.25 mM TMB, 2.21 mM hydrogen peroxide, 1% D
MSO, 0.08M acetate buffer (pH4.9): Bio-Rad)
Was added and left at room temperature for 30 minutes to develop color. After centrifuging the heparin-bound particles, the supernatant was transferred to a 96-well microtiter plate (Falcon), and the absorbance at 414 nm was measured using an immunominiplate reader (Nunc) (FIG. 1).
【００２１】＜２＞ヘパリンを使用したＶＥＧＦの測定 ＜１＞の方法と同様に、ＨＧＦをＶＥＧＦ１６５に変え
て、ヘパリン結合粒子を用によるＶＥＧＦの測定を行っ
た（図２）。<2> Measurement of VEGF using heparin In the same manner as in <1>, HGF was changed to VEGF165, and VEGF was measured using heparin-binding particles (FIG. 2).
【００２２】＜３＞ＨＧＦ測定キット（本発明キット
１） 本発明キット１の構成及び使用法の一例を以下に記載す
る。本実施例における本発明キット１は、次の１〜８の
要素からなる。 １．ヘパリンを固着させたイムノプレート（ブロッキン
グ剤でブロッキング済）：１枚 ２．標準ＨＧＦ溶液（1.3、2.5、5、10、20、40、80μg
/mL、各0.1mL）：１バイアルずつ ３．抗ＨＧＦヤギポリクローナルＩｇＧ抗体（Ｒ＆Ｄシ
ステムス社製）グリセロール溶液（10μL：PBSで100倍
希釈して使用）：１バイアル ４．ペルオキシダーゼ標識抗ヤギＩｇＧ抗体（バイオラ
ッド社製）（100μg/mL、150μl：PBSで100倍希釈して
使用）：１バイアル ５．テトラメチルベンジジン溶液（1.25mM TMB、2.21m
M H2O2、1% DMSO、0.08M 酢酸緩衝液、pH4.9、15m
L）：１バイアル ６．反応液（1% BSAを含むPBS(-)、40mL）：１バイア
ル ７．洗浄液（0.05% Tween-20を含むPBS(-)、500mL）：
１バイアル ８．反応停止液（1N HCl、15mL）：１バイアル<3> HGF Measurement Kit (Kit 1 of the Present Invention) An example of the structure and use of the kit 1 of the present invention is described below. The kit 1 of the present invention in this embodiment is composed of the following components 1 to 8. 1. 1. Immunoplate to which heparin is fixed (blocked with a blocking agent): 1 sheet Standard HGF solution (1.3, 2.5, 5, 10, 20, 40, 80 μg
/ mL, 0.1mL each): 1 vial 3. Anti-HGF goat polyclonal IgG antibody (manufactured by R & D Systems) glycerol solution (10 μL: used after diluting 100-fold with PBS): 1 vial 4. Peroxidase-labeled anti-goat IgG antibody (manufactured by Bio-Rad) (100 μg / mL, 150 μl: used after diluting 100-fold with PBS): 1 vial Tetramethylbenzidine solution (1.25 mM TMB, 2.21 m
MH 2 O 2 , 1% DMSO, 0.08M acetate buffer, pH 4.9, 15m
L): 1 vial 6. Reaction solution (PBS (-) containing 1% BSA, 40 mL): 1 vial Washing solution (PBS (-) containing 0.05% Tween-20, 500mL):
One vial 8. Stop solution (1N HCl, 15mL): 1 vial
【００２３】この本発明キット１の使用に際しては、イ
ムノプレートの各ウェルを予め洗浄液で洗浄し、各ウェ
ルに反応液を100μLずつ分注した。その後、各種濃度の
標準ＨＧＦ溶液、劇症肝炎（血清中のＨＧＦ量が激増す
ることが知られている）患者の血清又は健常人の血清を
10μLずつ添加して室温で30分間静置した。その後、洗
浄液で洗浄後、抗ＨＧＦヤギポリクローナル抗体溶液を
100μLずつ添加して37℃に60分間静置した。洗浄液で2
回洗浄した後、ペルオキシダーゼ標識抗ヤギＩｇＧ抗体
を100μLずつ添加して37℃に30分間静置して反応させ
た。When using the kit 1 of the present invention, each well of the immunoplate was washed in advance with a washing solution, and 100 μL of the reaction solution was dispensed into each well. Then, various concentrations of standard HGF solution, serum of a patient with fulminant hepatitis (the amount of HGF in the serum is known to drastically increase) or serum of a healthy person are added.
Each 10 μL was added, and the mixture was allowed to stand at room temperature for 30 minutes. Then, after washing with a washing solution, an anti-HGF goat polyclonal antibody solution is added.
100 μL of each was added, and the mixture was allowed to stand at 37 ° C. for 60 minutes. 2 with cleaning solution
After washing twice, 100 μL of peroxidase-labeled anti-goat IgG antibody was added thereto, and the mixture was allowed to stand at 37 ° C. for 30 minutes to react.
【００２４】静置後、各ウェルを洗浄液で3回洗浄し、
その後各ウェルにテトラメチルベンジジン溶液100μLを
添加し、37℃で15分間反応させ、発色させた。発色後、
各ウェルに反応停止液を100μL添加して反応を停止し、
ペルオキシダーゼによるTMBとH2O2との反応生成物の着
色波長である450nmの吸光度（対照波長630nm）をウェル
リーダー（SK601：生化学工業株式会社販売）で定量し
た。その結果、劇症肝炎患者の血清中のＨＧＦ量が健常
人の血清中のＨＧＦ量を大きく上回ることが確認され
た。After standing, each well is washed three times with a washing solution,
Thereafter, 100 μL of a tetramethylbenzidine solution was added to each well, and reacted at 37 ° C. for 15 minutes to develop color. After coloring,
Stop the reaction by adding 100 μL of reaction stop solution to each well,
The absorbance at 450 nm (control wavelength: 630 nm), which is the coloring wavelength of the reaction product of TMB and H 2 O 2 by peroxidase, was quantified using a well reader (SK601: sold by Seikagaku Corporation). As a result, it was confirmed that the amount of HGF in the serum of a patient with fulminant hepatitis greatly exceeded the amount of HGF in the serum of a healthy person.
【００２５】＜４＞ＨＧＦ、ＶＥＧＦ同時測定用キット
（本発明キット２） 本実施例における本発明キット１は、次の１〜１１の要
素からなる。 １．ヘパリンを固着させたイムノプレート（ブロッキン
グ剤でブロッキング済）：１枚 ２．標準ＨＧＦ溶液（1.3、2.5、5、10、20、40、80μg
/mL、各0.1mL）：１バイアルずつ ３．標準ＶＥＧＦ溶液（1.3、2.5、5、10、20、40、80
μg/mL、各0.1mL）：１バイアルずつ ４．抗ＨＧＦヤギポリクローナル抗体グリセロール溶液
（10μL：PBSで50倍希釈して使用）：１バイアル ５．抗ＶＥＧＦマウスポリクローナル抗体グリセロール
溶液（10μL：PBSで50倍希釈して使用）：１バイアル ６．ペルオキシダーゼ標識抗ヤギＩｇＧ抗体（100μg/m
L、150μl：PBSで50倍希釈して使用）：１バイアル ７．131I標識抗マウスＩｇＧ抗体（100μg/mL、150μ
l：PBSで50倍希釈して使用）：１バイアル ８．テトラメチルベンジジン溶液（1.25mM TMB、2.21m
M H2O2、1% DMSO、0.08M 酢酸緩衝液、pH4.9、15m
L）：１バイアル ９．反応液（1% BSAを含むPBS(-)、40mL）：１バイア
ル １０．洗浄液（0.05% Tween-20を含むPBS(-)、500m
L）：１バイアル １１．反応停止液（1N HCl、15mL）：１バイアル<4> Kit for Simultaneous Measurement of HGF and VEGF (Kit 2 of the Present Invention) Kit 1 of the present invention in this embodiment comprises the following elements 1 to 11. 1. 1. Immunoplate to which heparin is fixed (blocked with a blocking agent): 1 sheet Standard HGF solution (1.3, 2.5, 5, 10, 20, 40, 80 μg
/ mL, 0.1mL each): 1 vial Standard VEGF solution (1.3, 2.5, 5, 10, 20, 40, 80
3. μg / mL, 0.1 mL each): 1 vial 4. Anti-HGF goat polyclonal antibody glycerol solution (10 μL: used by diluting 50-fold with PBS): 1 vial 5. Anti-VEGF mouse polyclonal antibody glycerol solution (10 μL: used after diluting 50-fold with PBS): 1 vial Peroxidase-labeled anti-goat IgG antibody (100 μg / m
L, 150 μl: diluted 50-fold with PBS): 1 vial 131 I-labeled anti-mouse IgG antibody (100 μg / mL, 150 μg
l: Used 50-fold diluted with PBS): 1 vial Tetramethylbenzidine solution (1.25 mM TMB, 2.21 m
MH 2 O 2 , 1% DMSO, 0.08M acetate buffer, pH 4.9, 15m
L): 1 vial 9. Reaction solution (PBS (-) containing 1% BSA, 40 mL): 1 vial Washing solution (PBS (-) containing 0.05% Tween-20, 500m
L): 1 vial Stop solution (1N HCl, 15mL): 1 vial
【００２６】この本発明キット２の使用に際しては、イ
ムノプレートの各ウェルを予め洗浄液で洗浄し、各ウェ
ルに反応液を100μLずつ分注した。その後、各種濃度の
標準ＨＧＦ溶液、各種濃度の標準ＶＥＧＦ溶液、劇症肝
炎患者の血清又は健常人の血清を10μLずつ添加して室
温で30分間静置した。その後、洗浄液で洗浄後、抗ＨＧ
Ｆヤギポリクローナル抗体溶液及び抗ＶＥＧＦマウスポ
リクローナル抗体を50μLずつ添加して37℃に60分間静
置した。洗浄液で2回洗浄した後、ペルオキシダーゼ標
識抗ヤギＩｇＧ抗体及び131I標識抗マウスＩｇＧ抗体を
50μLずつ添加して37℃に30分間静置して反応させた。When using the kit 2 of the present invention, each well of the immunoplate was washed in advance with a washing solution, and 100 μL of the reaction solution was dispensed into each well. Thereafter, 10 μL of standard HGF solutions of various concentrations, standard VEGF solutions of various concentrations, serum of fulminant hepatitis patients or serum of healthy persons were added thereto, and the mixture was allowed to stand at room temperature for 30 minutes. Then, after washing with a washing solution, anti-HG
F goat polyclonal antibody solution and anti-VEGF mouse polyclonal antibody were added in an amount of 50 µL each, and the mixture was allowed to stand at 37 ° C for 60 minutes. After washing twice with a washing solution, a peroxidase-labeled anti-goat IgG antibody and a 131 I-labeled anti-mouse IgG antibody were added.
The reaction was carried out by adding 50 μL each and leaving still at 37 ° C. for 30 minutes.
【００２７】静置後、各ウェルを洗浄液で3回洗浄し、
その後各ウェルにテトラメチルベンジジン溶液100μLを
添加し、37℃で15分間反応させ、発色させた。発色後、
各ウェルに反応停止液を100μLずつ添加して反応を停止
し、ペルオキシダーゼによるTMBとH2O2との反応生成物
の着色波長である450nmの吸光度（対照波長630nm）をウ
ェルリーダー（SK601：生化学工業株式会社販売）で定
量した。さらに、各ウェルの放射能をシンチレーション
カウンターを使用して測定した。その結果、劇症肝炎患
者の血清中のＨＧＦ量が著しく上昇し、ＶＥＧＦ量はほ
とんど変化していないことが明らかとなった。After standing, each well is washed three times with a washing solution,
Thereafter, 100 μL of a tetramethylbenzidine solution was added to each well, and reacted at 37 ° C. for 15 minutes to develop color. After coloring,
The reaction was stopped by adding 100 μL of a reaction stop solution to each well, and the absorbance at 450 nm (control wavelength: 630 nm), which is the coloration wavelength of the reaction product of TMB and H 2 O 2 by peroxidase, was measured using a well reader (SK601: raw). Chemical Industry Co., Ltd.). Further, the radioactivity of each well was measured using a scintillation counter. As a result, it became clear that the amount of HGF in the serum of a patient with fulminant hepatitis increased remarkably, and the amount of VEGF hardly changed.
【００２８】[0028]
【発明の効果】本発明によれば、臨床現場などで必要と
されている検体中に含まれる糖鎖結合性生理活性物質を
正確且つ簡便に測定の方法が提供され、また、前記測定
法を実施するためのキットが提供される。According to the present invention, there is provided a method for accurately and easily measuring a sugar chain-binding physiologically active substance contained in a specimen required at a clinical site or the like. A kit for performing is provided.
【図】ヘパリンを固着したアガロース粒子を用いて作成
した、ＨＧＦの標準曲線を示した図面である。FIG. 1 is a drawing showing a standard curve of HGF prepared using agarose particles to which heparin is fixed.
【図】ヘパリンを固着したアガロース粒子を用いて作成
した、ＶＥＧＦの標準曲線を示した図面である。FIG. 5 is a drawing showing a standard curve of VEGF prepared using agarose particles to which heparin is fixed.
Claims (29)
結合した固相担体に検体を接触させ、検体中の糖鎖結合
性生理活性物質を固相担体上の該糖鎖に特異的に結合さ
せ、固相担体に結合した当該糖鎖結合性生理活性物質を
検出することを特徴とする測定法。1. A sample is brought into contact with a solid support to which a sugar chain specifically binding to a physiologically active substance is bound, and the sugar chain-binding bioactive substance in the sample is specifically bound to the sugar chain on the solid support. And detecting the sugar chain-binding physiologically active substance bound to the solid phase carrier.
的に結合する糖鎖がグリコサミノグリカンであることを
特徴とする請求項１記載の測定法。2. The method according to claim 1, wherein the sugar chain specifically binding to the physiologically active substance bound to the solid phase carrier is glycosaminoglycan.
が、ヘパリン又はヘパラン硫酸である請求項２記載の測
定法。3. The method according to claim 2, wherein the sugar chain that specifically binds to a physiologically active substance is heparin or heparan sulfate.
である請求項１乃至３いずれか１記載の測定法。4. The method according to claim 1, wherein the sugar chain-binding physiologically active substance is a cytokine.
子又は血管内皮細胞成長因子である請求項１乃至３いず
れか１項記載の測定法。5. The method according to claim 1, wherein the sugar chain-binding physiologically active substance is hepatocyte growth factor or vascular endothelial cell growth factor.
物質の検出を、該生理活性物質に特異的に結合する物質
を用いて行う請求項１乃至５いずれか１項記載の測定
法。6. The method according to claim 1, wherein the detection of the sugar chain-binding physiologically active substance bound to the solid phase carrier is performed using a substance that specifically binds to the physiologically active substance. .
理活性物質に対する抗体と、該抗体に対する二次抗体で
あって標識物質により標識された二次抗体を用いて行う
請求項１乃至６いずれか１項記載の測定法。7. The method for detecting a sugar chain-binding physiologically active substance using an antibody against the physiologically active substance and a secondary antibody against the antibody, the secondary antibody being labeled with a labeling substance. 6. The method according to any one of claims 6 to 7.
物質の検出を、標識物質により標識された該生理活性物
質に対する抗体を用いることを特徴とする請求項１乃至
６いずれか１項記載の測定法。8. The method according to claim 1, wherein the detection of the sugar chain-binding physiologically active substance bound to the solid phase carrier is carried out using an antibody against the physiologically active substance labeled with a labeling substance. The described measurement method.
相担体に検体を接触させ、検体中の線維芽細胞増殖因
子、肝細胞成長因子及び血管内皮細胞成長因子からなる
群から選択される糖鎖結合性生理活性物質を固相担体上
の該ヘパリン又はヘパラン硫酸に特異的に結合させ、固
相担体に結合した該生理活性物質を、該生理活性物質に
対する抗体と、前記抗体に対する二次抗体であって標識
物質により標識された二次抗体を用いることを特徴とす
る糖鎖結合性生理活性物質の測定法。9. A sugar chain binding selected from the group consisting of fibroblast growth factor, hepatocyte growth factor and vascular endothelial cell growth factor in a sample by contacting the sample with a solid support to which heparin or heparan sulfate is bound. A bioactive substance specifically bound to the heparin or heparan sulfate on the solid support, and the bioactive substance bound to the solid support is an antibody against the bioactive substance and a secondary antibody against the antibody. A method for measuring a sugar chain-binding physiologically active substance, comprising using a secondary antibody labeled with a labeling substance.
質、蛍光物質、発光物質、酵素、ハプテン及びラジオア
イソトープからなる群から選択される物質である、請求
項７乃至９いずれか１項記載の測定法。10. The labeling substance according to claim 7, wherein the labeling substance is a substance selected from the group consisting of one substance of a specific binding pair, a fluorescent substance, a luminescent substance, an enzyme, a hapten, and a radioisotope. The described measurement method.
性生理活性物質をそれぞれ測定する方法であって、糖鎖
が結合した固相担体に検体を接触させて該糖鎖を介して
固相担体に前記生理活性物質を結合させ、固相担体に結
合した糖鎖結合性生理活性物質を、各糖鎖結合性生理活
性物質に対する二種以上の抗体によって検出する糖鎖結
合性生理活性物質の測定法。11. A method for measuring two or more sugar chain-binding physiologically active substances contained in a sample, wherein the sample is brought into contact with a solid support to which a sugar chain is bound, and the sugar chain is bound via the sugar chain. The above-mentioned physiologically active substance is bound to a solid phase carrier, and the sugar chain-binding physiologically active substance bound to the solid phase carrier is detected by two or more antibodies against each of the sugar chain-binding physiologically active substances. How to measure a substance.
合した異なる種類の標識物質をそれぞれ測定することに
よって行うことを特徴とする請求項１１記載の測定法。12. The method according to claim 11, wherein the detection of two or more antibodies is performed by measuring different types of labeling substances bound to the antibodies.
各抗体に対する二次抗体であって別異の標識物質により
標識された二次抗体によって行うことを特徴とする請求
項１１記載の測定法。13. The method according to claim 11, wherein the detection of the two or more kinds of antibodies is performed using a secondary antibody to each of the two or more kinds of antibodies and labeled with a different labeling substance. Measurement method.
質、蛍光物質、発光物質、酵素、ハプテン及びラジオア
イソトープからなる群から選択される物質である、請求
項１２又は１３記載の測定法。14. The method according to claim 12, wherein the labeling substance is a substance selected from the group consisting of one substance of a specific binding pair, a fluorescent substance, a luminescent substance, an enzyme, a hapten, and a radioisotope. .
アイソトープが使用されることを特徴とする請求項１２
又は１３記載の測定法。15. The method according to claim 12, wherein a fluorescent substance or a radioisotope is used as the labeling substance.
Or the measurement method according to 13.
ノグリカンであることを特徴とする請求項１１乃至１５
いずれか１項記載の測定法。16. The method according to claim 11, wherein the sugar chain bound to the solid support is glycosaminoglycan.
The measurement method according to any one of the preceding claims.
ヘパラン硫酸である請求項１６記載の測定法。17. The method according to claim 16, wherein the glycosaminoglycan is heparin or heparan sulfate.
ンである請求項１１乃至１７いずれか１項記載の測定
法。18. The method according to claim 11, wherein the sugar chain-binding physiologically active substance is a cytokine.
因子又は血管内皮細胞成長因子である請求項１１乃至１
７いずれか１項記載の測定法。19. The sugar chain-binding physiologically active substance is hepatocyte growth factor or vascular endothelial cell growth factor.
7. The measuring method according to any one of the above items 7.
含む糖鎖結合性生理活性物質の測定キット。 糖鎖を結合した固相担体； 糖鎖結合性生理活性物質に対する抗体； 標識物質により標識した、上記の抗体に対する二次
抗体。20. A kit for measuring a sugar chain-binding physiologically active substance comprising at least the following components: A solid phase carrier having a sugar chain bound thereto; an antibody against a sugar chain-binding physiologically active substance; a secondary antibody to the above antibody, which is labeled with a labeling substance.
ズ、チューブ又は微粒子状固相担体であることを特徴と
する請求項２０記載の測定キット。21. The measurement kit according to claim 20, wherein the solid phase carrier is a microplate, a bead, a tube, or a particulate solid phase carrier.
増殖因子、肝細胞成長因子又は血管内皮細胞成長因子で
ある請求項２０又は２１記載の測定キット。22. The assay kit according to claim 20, wherein the sugar chain-binding physiologically active substance is fibroblast growth factor, hepatocyte growth factor, or vascular endothelial cell growth factor.
質、蛍光物質、発光物質、酵素、ハプテン及びラジオア
イソトープからなる群から選択される物質である、請求
項２０乃至２２いずれか１項記載の測定キット。23. The labeling substance according to claim 20, wherein the labeling substance is a substance selected from the group consisting of one substance of a specific binding pair, a fluorescent substance, a luminescent substance, an enzyme, a hapten and a radioisotope. The measurement kit as described.
含む糖鎖結合性生理活性物質の測定キット。 糖鎖を結合した固相担体； 第一の糖鎖結合性生理活性物質に対する抗体（抗体
１）； 第二の糖鎖結合性生理活性物質に対する抗体（抗体
２）； 第一の標識物質により標識した抗体１に対する二次抗
体（二次抗体１）； 第二の標識物質により標識した抗体２に対する二次抗
体（二次抗体２）。24. A kit for measuring a sugar chain-binding physiologically active substance comprising at least the following components: A solid phase carrier to which a sugar chain is bound; an antibody against the first sugar chain-binding bioactive substance (antibody 1); an antibody against the second sugar chain-binding bioactive substance (antibody 2); labeled with the first labeling substance A secondary antibody against the antibody 1 (secondary antibody 1); a secondary antibody against the antibody 2 labeled with the second labeling substance (secondary antibody 2).
ズ、チューブ又は微粒子状固相担体である請求項２４記
載の測定キット。25. The measurement kit according to claim 24, wherein the solid phase carrier is a microplate, a bead, a tube, or a particulate solid phase carrier.
質が、線維芽細胞増殖因子、肝細胞成長因子及び血管内
皮細胞成長因子からなる群からそれぞれ選択される異な
る物質である特徴とする請求項２４又は２５記載の測定
キット。26. The method according to claim 26, wherein the first and second physiologically active sugar chain-binding substances are different substances selected from the group consisting of fibroblast growth factor, hepatocyte growth factor and vascular endothelial cell growth factor. The measurement kit according to claim 24 or 25.
であることを特徴とする請求項２４乃至２６いずれか１
項記載の測定キット。27. The antibody according to claim 24, wherein the antibodies 1 and 2 are derived from different species.
The measurement kit according to the item.
合対の一方の物質、蛍光物質、発光物質、酵素、ハプテ
ン及びラジオアイソトープからなる群からそれぞれ選択
される異なる物質である、請求項２４乃至２７いずれか
１項記載の測定キット。28. The first and second labeling substances are different substances each selected from the group consisting of one substance of a specific binding pair, a fluorescent substance, a luminescent substance, an enzyme, a hapten and a radioisotope. Item 28. The measurement kit according to any one of items 24 to 27.
方が、ラジオアイソトープ又は蛍光物質であることを特
徴とする請求項２４乃至２７いずれか１項記載の測定キ
ット。29. The measurement kit according to claim 24, wherein one of the first and second labeling substances is a radioisotope or a fluorescent substance.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP19231298A JP4002345B2 (en) | 1998-06-24 | 1998-06-24 | Method and kit for measuring sugar chain-binding physiologically active substance |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP19231298A JP4002345B2 (en) | 1998-06-24 | 1998-06-24 | Method and kit for measuring sugar chain-binding physiologically active substance |
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| Publication Number | Publication Date |
|---|---|
| JP2000009733A true JP2000009733A (en) | 2000-01-14 |
| JP2000009733A5 JP2000009733A5 (en) | 2005-10-27 |
| JP4002345B2 JP4002345B2 (en) | 2007-10-31 |
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| JP19231298A Expired - Fee Related JP4002345B2 (en) | 1998-06-24 | 1998-06-24 | Method and kit for measuring sugar chain-binding physiologically active substance |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002544485A (en) * | 1999-05-06 | 2002-12-24 | グレイコデータ、リミテッド | Polysaccharide structure and sequencing |
| JP2009229351A (en) * | 2008-03-25 | 2009-10-08 | Sumitomo Bakelite Co Ltd | Method of detecting physiologically active substance, and measurement kit |
| WO2010134225A1 (en) * | 2009-05-20 | 2010-11-25 | 国立大学法人鳥取大学 | Method for detection of infection by pathogenic neisseria bacteria using partial sugar chain epitope, and vaccine against the bacteria |
| WO2019026870A1 (en) * | 2017-08-01 | 2019-02-07 | 学校法人学文館 | Novel method for measuring sflt-1 (soluble vascular endothelial growth factor receptor-1) |
-
1998
- 1998-06-24 JP JP19231298A patent/JP4002345B2/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002544485A (en) * | 1999-05-06 | 2002-12-24 | グレイコデータ、リミテッド | Polysaccharide structure and sequencing |
| JP4656731B2 (en) * | 1999-05-06 | 2011-03-23 | プロコグニア （イスラエル） リミテッド | Polysaccharide structure and sequencing |
| JP2009229351A (en) * | 2008-03-25 | 2009-10-08 | Sumitomo Bakelite Co Ltd | Method of detecting physiologically active substance, and measurement kit |
| WO2010134225A1 (en) * | 2009-05-20 | 2010-11-25 | 国立大学法人鳥取大学 | Method for detection of infection by pathogenic neisseria bacteria using partial sugar chain epitope, and vaccine against the bacteria |
| JPWO2010134225A1 (en) * | 2009-05-20 | 2012-11-08 | 国立大学法人鳥取大学 | Method for detecting pathogenic Neisseria infection using partial sugar chain epitope and vaccine against these bacteria |
| JP5536765B2 (en) * | 2009-05-20 | 2014-07-02 | 国立大学法人鳥取大学 | Method for detecting pathogenic Neisseria infection using partial sugar chain epitope and vaccine against these bacteria |
| WO2019026870A1 (en) * | 2017-08-01 | 2019-02-07 | 学校法人学文館 | Novel method for measuring sflt-1 (soluble vascular endothelial growth factor receptor-1) |
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| Publication number | Publication date |
|---|---|
| JP4002345B2 (en) | 2007-10-31 |
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