JP2001000197A - Measurement of protease activity - Google Patents
Measurement of protease activityInfo
- Publication number
- JP2001000197A JP2001000197A JP11174826A JP17482699A JP2001000197A JP 2001000197 A JP2001000197 A JP 2001000197A JP 11174826 A JP11174826 A JP 11174826A JP 17482699 A JP17482699 A JP 17482699A JP 2001000197 A JP2001000197 A JP 2001000197A
- Authority
- JP
- Japan
- Prior art keywords
- thin film
- protease
- antibody
- sample
- labeled
- 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.)
- Pending
Links
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- 102220201851 rs143406017 Human genes 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000003001 serine protease inhibitor Substances 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- YZORUOZKRBVLEG-UHFFFAOYSA-M sodium;4-[[4-(diethylamino)phenyl]diazenyl]benzenesulfonate Chemical compound [Na+].C1=CC(N(CC)CC)=CC=C1N=NC1=CC=C(S([O-])(=O)=O)C=C1 YZORUOZKRBVLEG-UHFFFAOYSA-M 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 210000001258 synovial membrane Anatomy 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
- 239000001043 yellow dye Substances 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【0001】[0001]
【発明の属する技術分野】本発明は、プロテアーゼの測
定方法に関するものである。より具体的には、動脈粥状
硬化プラークの破綻の危険性、癌細胞の浸潤活性や転移
活性などの癌の悪性度、歯周炎などの歯周病の進行度、
リウマチ性関節炎などにおける破壊性病変などの正確な
診断を可能にするプロテアーゼ測定方法に関するもので
ある。[0001] The present invention relates to a method for measuring a protease. More specifically, the danger of atherosclerotic plaque rupture, malignancy of cancer such as invasive activity and metastatic activity of cancer cells, progression of periodontal disease such as periodontitis,
The present invention relates to a method for measuring a protease that enables accurate diagnosis of a destructive lesion or the like in rheumatoid arthritis or the like.
【0002】[0002]
【従来の技術】動脈粥状硬化の発生進展やプラークの破
綻の危険性、癌細胞の浸潤や転移、歯周炎などの歯周病
の進行、リウマチ性関節炎などにおける組織破壊の進
行、創傷治癒過程、個体発生過程などにおいて、マトリ
ックスメタロプロテアーゼ、プラスミノーゲンアクティ
ベーターなど種々のプロテアーゼが関与することが知ら
れており、それらのプロテアーゼの検出および定量方法
として、抗体を用いたイミュノアッセイ法、免疫組織化
学的方法、イミュノブロッティング法、電気泳動ザイモ
グラフィー法などが知られている。また、組織中におけ
るプロテアーゼの活性を測定する方法として、The FASE
B Journal, Vol.9, July, pp.974-980, 1995あるいはWO
97/32035に示されるいわゆるin situ zymography法があ
る。2. Description of the Related Art The development and development of arterial atherosclerosis and the risk of plaque rupture, invasion and metastasis of cancer cells, progression of periodontal disease such as periodontitis, progression of tissue destruction in rheumatoid arthritis, etc., wound healing It is known that various proteases such as a matrix metalloprotease and a plasminogen activator are involved in the process and the ontogeny process, and the detection and quantification of those proteases include immunoassay using antibodies, immunoassay using an antibody, and the like. Histochemical methods, immunoblotting, electrophoretic zymography and the like are known. As a method for measuring protease activity in tissues, The FASE
B Journal, Vol.9, July, pp.974-980, 1995 or WO
There is a so-called in situ zymography method shown in 97/32035.
【0003】[0003]
【発明が解決しようとする課題】生理現象あるいは病態
を詳しく解析するためには、in situ zymography法によ
るプロテアーゼ活性の測定と同時に、プロテアーゼ蛋白
あるいは組織中に存在するその他の物質の存在位置に関
しても情報を得たい場合がある。しかしながら、The FA
SEB Journal, Vol.9, July, pp.974-980, 1995又はWO97
/32035に示された方法ではプロテアーゼ活性の測定のみ
が可能であり、プロテアーゼ自体やその他の物質の存在
量、分布に関しての情報は得られなかった。本発明の課
題は、プロテアーゼ活性の測定方法において、同じ組織
上でプロテアーゼ自体やその他の物質の存在部位をプロ
テアーゼと同時に解析できる方法を提供することにあ
る。In order to analyze physiological phenomena or pathological conditions in detail, it is necessary to measure the protease activity by in situ zymography, and at the same time, to obtain information on the location of the protease protein or other substances present in the tissue. Sometimes you want to get. However, The FA
SEB Journal, Vol.9, July, pp.974-980, 1995 or WO97
In the method described in US Pat. No. 3,320,35, only the protease activity can be measured, and no information on the abundance and distribution of the protease itself and other substances was obtained. An object of the present invention is to provide a method for measuring protease activity in which the presence site of the protease itself and other substances can be simultaneously analyzed on the same tissue.
【0004】[0004]
【課題を解決するための手段】本発明者らは上記の課題
を解決すべく鋭意努力した結果、ゼラチン等の薄膜の染
色により消化痕を観察すると同時に、膜上の組織に含ま
れる目的物質に対する免疫組織化学的観察を併用する
と、プロテアーゼ活性の測定と同時に他の目的物質の存
在部位などに関する情報を入手でき、診断などをより正
確に行うことができることを見いだした。本発明はこれ
らの知見を基にして完成されたものである。Means for Solving the Problems The present inventors have made intensive efforts to solve the above-mentioned problems, and as a result, while observing digestion marks by staining a thin film of gelatin or the like, the present inventors have studied the target substance contained in the tissue on the film. When immunohistochemical observation was used together, it was found that information on the location of other target substances and the like can be obtained at the same time as the measurement of protease activity, so that diagnosis and the like can be performed more accurately. The present invention has been completed based on these findings.
【0005】すなわち本発明は、プロテアーゼ活性測定
と免疫組織化学的観察とを同時に行う方法であって、
(1)プロテアーゼ基質を含み支持体表面に形成された
薄膜に対して、プロテアーゼを含む試料を接触させる工
程;(2)プロテアーゼの作用により該薄膜に形成され
た消化痕を、該薄膜を染料で染色することにより検出す
る工程;及び(3)試料中の特定の物質に反応する抗体
を用いて、該物質を可視化する工程を含む方法を提供す
るものである。That is, the present invention provides a method for simultaneously performing protease activity measurement and immunohistochemical observation,
(1) a step of contacting a sample containing a protease with a thin film formed on the surface of a support containing a protease substrate; (2) a digestion mark formed on the thin film by the action of protease is removed by dyeing the thin film with a dye And (3) using an antibody that reacts with a specific substance in a sample to visualize the substance.
【0006】また、本発明の別の態様によれば、プロテ
アーゼ活性測定と免疫組織化学的観察とを同時に行う方
法であって、(1)プロテアーゼ基質と硬膜剤とを含み
支持体表面に形成された薄膜に対して、プロテアーゼを
含む試料を接触させる工程;(2)プロテアーゼの作用
により該薄膜に形成された消化痕を、該薄膜を染料で染
色することにより検出する工程;及び(3)試料中の特
定の物質に反応する抗体を用いて、該物質を可視化する
工程を含む方法が提供される。According to another aspect of the present invention, there is provided a method for simultaneously performing protease activity measurement and immunohistochemical observation, comprising the steps of: (1) forming a protease containing a protease substrate and a hardening agent on the surface of a support; (2) detecting a digestion mark formed on the thin film by the action of the protease by staining the thin film with a dye; and (3) detecting the digestion mark formed on the thin film by the action of the protease. A method is provided that includes the step of visualizing a specific substance in a sample using an antibody that reacts with the substance.
【0007】さらに、本発明の別の態様によれば、プロ
テアーゼ活性測定と免疫組織化学的観察とを同時に行う
方法であって、(1)プロテアーゼ基質と硬膜剤とを含
み支持体表面に形成された薄膜に対して生体試料の実質
的に連続した2以上の切片のうちの一つを接触させる工
程;(2)プロテアーゼ基質、硬膜剤、及びプロテアー
ゼ・インヒビターを含み支持体表面に形成された薄膜に
対して残りの切片を接触させる工程;(3)プロテアー
ゼの作用により該薄膜に形成された消化痕を、該薄膜を
染料で染色することにより検出する工程;(4)工程
(1)で用いた薄膜の消化痕と工程(2)で用いた薄膜
の消化痕とを対比する工程;及び(5)試料中の特定の
物質に反応する抗体を用いて、該物質を可視化する工程
を含む方法が提供される。この発明の好ましい態様で
は、工程(2)においてプロテアーゼ・インヒビターが
キレート剤、マトリックスメタロプロテアーゼ阻害剤、
セリンプロテアーゼ阻害剤である上記方法が提供され
る。Further, according to another aspect of the present invention, there is provided a method for simultaneously performing protease activity measurement and immunohistochemical observation, comprising: (1) a method comprising forming a protease substrate and a hardening agent on the surface of a support; Contacting one of two or more substantially continuous sections of the biological sample with the thin film obtained; (2) forming a substrate substrate, a hardening agent, and a protease inhibitor formed on a support surface; Contacting the remaining section with the thin film obtained; (3) detecting a digestion mark formed on the thin film by the action of a protease by staining the thin film with a dye; (4) step (1) Comparing the digestion mark of the thin film used in the above with the digestion mark of the thin film used in step (2); and (5) visualizing the substance by using an antibody that reacts with a specific substance in the sample. Including methods provided That. In a preferred embodiment of the present invention, in step (2), the protease inhibitor comprises a chelating agent, a matrix metalloprotease inhibitor,
Provided is a method as described above which is a serine protease inhibitor.
【0008】これらの発明の好ましい態様によれば、該
試料が患者又は疾患が疑われるヒトおよび他の哺乳動物
から分離・採取した生体試料、あるいは実験動物から分
離・採取した生体試料である上記方法;生体試料が組織
又は体液中の細胞である上記方法;生体試料が動脈粥状
硬化組織、癌組織、リンパ節、歯周病組織、破壊性病変
組織、皮膚、子宮内膜、子宮粘膜組織、喀痰、尿沈査で
ある上記方法;プロテアーゼがマトリックスメタロプロ
テアーゼ又はセリンプロテアーゼである上記方法;薄膜
がマトリックスメタロプロテアーゼ基質又はセリンプロ
テアーゼ基質を含む薄膜である上記方法;薄膜の消化を
可視染料又は蛍光染料により可視化する上記方法;染料
が赤色、橙色、及び黄色からなる群から選ばれる染料
(最大吸収波長が400nm〜580nm)である上記
方法;染料がポンソー3Rである上記方法;抗体がモノ
クローナル抗体である上記方法が提供される。According to a preferred embodiment of the present invention, the above-mentioned method, wherein the sample is a biological sample separated or collected from a patient or a mammal suspected of having a disease, or a biological sample separated or collected from an experimental animal. The above-mentioned method, wherein the biological sample is a cell in a tissue or a body fluid; the biological sample is atherosclerotic tissue, cancer tissue, lymph node, periodontal disease tissue, destructive lesion tissue, skin, endometrium, uterine mucosal tissue, The above method of sputum and urine sedimentation; The above method wherein the protease is a matrix metalloprotease or a serine protease; The above method wherein the thin film contains a matrix metalloprotease substrate or a serine protease substrate; The above method for visualizing; a dye selected from the group consisting of red, orange, and yellow (having a maximum absorption wavelength of 4 The method is 0nm~580nm); the method the dye is Ponceau 3R; the method the antibody is a monoclonal antibody is provided.
【0009】別の好ましい態様によれば、試料中の特定
の物質に反応する抗体が酵素標識抗体、蛍光標識抗体、
又は金コロイド標識抗体である上記方法;試料中の特定
の物質に反応する抗体に対して特異的に結合する標識抗
体又は抗体酵素複合体を用いて試料中の特定の物質の存
在部位を可視化する上記方法;試料中の特定の物質に反
応する抗体と特異的に反応する酵素標識抗体、蛍光標識
抗体、又は金コロイド標識抗体を2次抗体として用いる
上記方法;2次抗体としてビオチン標識抗体を用い、該
2次抗体を酵素標識アビジン、蛍光標識アビジン、又は
金コロイド標識アビジンを用いて可視化する上記方法;
プロテアーゼ基質がゼラチン又はカゼインである上記方
法:画像処理装置を用いて消化痕の定量又は数値化、あ
るいは特定の物質の定量又は数値化を行う上記方法が提
供される。また、別の観点から、これらの方法に使用す
る薄膜が本発明により提供される。According to another preferred embodiment, the antibody reacting with a specific substance in the sample is an enzyme-labeled antibody, a fluorescent-labeled antibody,
Or the method described above, which is a colloidal gold-labeled antibody; the presence of a specific substance in a sample is visualized using a labeled antibody or an antibody-enzyme complex that specifically binds to an antibody that reacts with the specific substance in the sample. The above-mentioned method; an enzyme-labeled antibody, a fluorescent-labeled antibody, or a colloidal gold-labeled antibody that specifically reacts with an antibody that reacts with a specific substance in a sample as the secondary antibody; a biotin-labeled antibody as a secondary antibody The above method of visualizing the secondary antibody using enzyme-labeled avidin, fluorescently-labeled avidin, or colloidal gold-labeled avidin;
The above-mentioned method, wherein the protease substrate is gelatin or casein: The above-mentioned method for quantifying or quantifying a digestion mark or quantifying or quantifying a specific substance using an image processing apparatus is provided. In another aspect, the present invention provides a thin film used in these methods.
【0010】[0010]
【発明の実施の形態】本明細書において用いられる測定
方法という用語は、定性及び定量を含めて最も広義に解
釈されるべきである。本発明のプロテアーゼの測定方法
では、試料中に含まれるプロテアーゼによってプロテア
ーゼ基質が消化され、薄膜中に消化痕が形成される。こ
の消化痕は染料による薄膜の染色により、濃度の低い部
分として顕微鏡下で検出することができ、試料中のプロ
テアーゼ活性の存在を証明することができる。また、同
じ試料中に含まれる特定の物質を、該物質に対して結合
する抗体、好ましくは該物質に特定的に結合するモノク
ローナル抗体を用いた免疫組織化学的に検出することに
より、プロテアーゼ活性測定と免疫組織化学的観察とを
同時に行い、プロテアーゼ活性の存在と特定の物質の存
在との関係を調べることができる。本発明の方法に用い
る薄膜としては、WO97/32035に記載されたものを用いる
ことができる。硬膜剤又はプロテアーゼインヒビターな
ども上記文献に記載のものを用いることができる。上記
WO97/32035の開示を参考として本明細書の開示に含め
る。DETAILED DESCRIPTION OF THE INVENTION The term measurement method as used herein should be interpreted in the broadest sense, including qualitative and quantitative. In the protease measuring method of the present invention, the protease substrate is digested by the protease contained in the sample, and a digestion mark is formed in the thin film. This digestion mark can be detected under a microscope as a low concentration portion by staining the thin film with a dye, and the presence of protease activity in the sample can be proved. In addition, protease activity is measured by detecting a specific substance contained in the same sample by immunohistochemistry using an antibody that binds to the substance, preferably a monoclonal antibody that specifically binds to the substance. And immunohistochemical observation at the same time, it is possible to examine the relationship between the presence of protease activity and the presence of a specific substance. As the thin film used in the method of the present invention, those described in WO97 / 32035 can be used. As the hardening agent or the protease inhibitor, those described in the above literature can be used. the above
The disclosure of WO 97/32035 is incorporated herein by reference.
【0011】本発明の対象となるプロテアーゼとして
は、例えばマトリックス・メタロプロテアーゼ及びセリ
ンプロテアーゼを挙げることができ、これらの酵素につ
いては、鶴尾隆編「癌転移の分子機構」、pp.92-107、
メジカルビュー社、1993年発行に詳細に説明されてい
る。本発明の方法に特に好適なプロテアーゼとして、例
えば、間質型コラーゲナーゼ(MMP-1)、ゼラチナーゼA
(MMP-2)、及びゼラチナーゼB (MMP-9)などのマトリッ
クス・メタロプロテアーゼ;及びプラスミノーゲン・ア
クティベーター(PA)、トリプシンなどのセリンプロテア
ーゼを挙げることができるが、本発明の方法の対象は上
記の特定のプロテアーゼに限定されることはない。The proteases to be used in the present invention include, for example, matrix metalloproteases and serine proteases. These enzymes are described in Takaru Tsuruo, "Molecular Mechanism of Cancer Metastasis", pp. 92-107. ,
This is described in detail in Medical View, 1993. Particularly suitable proteases for the method of the present invention include, for example, stromal collagenase (MMP-1), gelatinase A
Matrix metalloproteases such as (MMP-2) and gelatinase B (MMP-9); and serine proteases such as plasminogen activator (PA) and trypsin. It is not limited to the specific protease described above.
【0012】プロテアーゼ基質は、プロテアーゼの基質
として分解される高分子化合物であれば特に限定されな
い。例えばコラーゲン、ゼラチン、プロテオグリカン、
フィブロネクチン、ラミニン、エラスチン、またはカゼ
インなどを用いることができる。好ましくは、コラーゲ
ン、ゼラチン、フィブロネクチン、又はカゼインを用い
ることができ、より好ましくはゼラチンまたはカゼイン
を用いることができる。2種以上の異なるプロテアーゼ
基質を組み合わせて用いることにより、生体試料中に含
まれるプロテアーゼの種類を正確に特定できる場合があ
る。例えば、カゼインとプラスミノーゲンを混合して基
質薄膜を作製することにより、プラスミノーゲンアクテ
ィベーター類の活性を測定することができる。プロテア
ーゼ基質を含む薄膜の膜厚は、乾燥後の膜厚が1〜10
μ、好ましくは4〜7μのものを用いることが好適であ
る。The protease substrate is not particularly limited as long as it is a high molecular compound that can be decomposed as a protease substrate. For example, collagen, gelatin, proteoglycan,
Fibronectin, laminin, elastin, or casein can be used. Preferably, collagen, gelatin, fibronectin, or casein can be used, and more preferably, gelatin or casein can be used. By using a combination of two or more different protease substrates, the type of protease contained in a biological sample may be accurately specified in some cases. For example, by mixing casein and plasminogen to prepare a substrate thin film, the activity of plasminogen activators can be measured. The thickness of the thin film containing the protease substrate is 1 to 10 after drying.
μ, preferably 4 to 7 μ.
【0013】本発明の方法に用いる試料としては、患者
又は疾患が疑われるヒト及びそれ以外の哺乳動物から分
離・採取した生体試料、あるいは実験動物から分離・採
取した生体試料を用いることができる。例えば、動脈粥
状硬化性病変の他、肺癌、胃癌、食道癌、大腸癌、乳
癌、子宮癌、卵巣癌、甲状腺癌、肝臓癌、口腔癌、前立
腺癌、腎臓癌、膀胱癌などの癌組織から手術や組織検査
などにより分離・採取した病変部組織、またはリンパ
節、歯周病組織、リウマチ性関節炎の滑膜や骨組織など
の組織から手術や組織検査などにより分離・採取した組
織、または乳腺異常分泌液、尿、喀痰などに含まれる細
胞を用いることができる。As the sample used in the method of the present invention, a biological sample separated and collected from a patient or a mammal suspected of having a disease or other mammal, or a biological sample separated and collected from an experimental animal can be used. For example, in addition to arterial atherosclerotic lesions, cancer tissues such as lung cancer, stomach cancer, esophageal cancer, colon cancer, breast cancer, uterine cancer, ovarian cancer, thyroid cancer, liver cancer, oral cancer, prostate cancer, kidney cancer, and bladder cancer Tissue isolated or collected by surgery or histological examination from tissues such as lymph node, periodontal disease tissue, synovium or bone tissue of rheumatoid arthritis, Cells contained in abnormal mammary gland secretion, urine, sputum and the like can be used.
【0014】試料が組織の場合には、例えば、液体窒素
で急速凍結した試料から凍結切片作成装置を用いて厚さ
1〜10μm、好ましくは4〜6μmの切片を調製し、
この切片を薄膜に貼付することによって試料と薄膜とを
接触させることができる。穿刺吸引により採取した組織
検体についても、コンパウンドと共に液体窒素で急速凍
結し、同様に切片を作製して用いることができる。ま
た、穿刺吸引により採取した組織検体について細胞診を
行う場合には、吸引した検体をプロテアーゼ基質を含む
薄膜の上に吐出して分散状態で接着させることで試料と
薄膜とを接触させることができる。さらに、組織検体の
場合は、採取した組織の水分を軽く拭った後、プロテア
ーゼ基質を含む薄膜の上に軽く押しつけることで試料の
細胞の一部を薄膜表面に転写させ薄膜と接触させること
ができる。When the sample is a tissue, for example, a slice having a thickness of 1 to 10 μm, preferably 4 to 6 μm is prepared from a sample which has been rapidly frozen with liquid nitrogen by using a cryosectioning apparatus.
By attaching this section to the thin film, the sample and the thin film can be brought into contact. Tissue samples collected by fine aspiration can also be rapidly frozen in liquid nitrogen together with the compound, and similarly prepared and used for sectioning. When performing cytodiagnosis on a tissue sample collected by fine needle aspiration, the sample and the thin film can be brought into contact by discharging the suctioned sample onto a thin film containing a protease substrate and adhering them in a dispersed state. . Furthermore, in the case of a tissue sample, after gently wiping the moisture of the collected tissue, a part of the cells of the sample can be transferred to the thin film surface by lightly pressing it on the thin film containing the protease substrate, and the thin film can be brought into contact with the thin film. .
【0015】本発明の方法では、例えば、プロテアーゼ
基質を含む薄膜にプロテアーゼを含む試料を接触させた
後、プロテアーゼ活性の発現に適した温度、例えば37
℃の飽和湿度条件下で基質の消化に必要な時間インキュ
ベートする。必要な時間は試料によって異なるが、好ま
しくは、例えば組織切片あるいは細胞については37℃
で1〜48時間、さらに好ましくは6〜30時間インキ
ュベートし、試料中のプロテアーゼによって薄膜中に消
化痕を形成させる。その後、薄膜を染料で染色し光学顕
微鏡などを用いて消化痕を観察することができる。さら
に、試料中の特定の物質を検出するために、該物質に特
異的に反応する抗体を用いて免疫組織化学的な染色を行
う。In the method of the present invention, for example, after a sample containing a protease is brought into contact with a thin film containing a protease substrate, a temperature suitable for expressing protease activity, for example, 37 ° C.
Incubate under conditions of saturated humidity at ℃ C for the time required for digestion of the substrate. The time required varies depending on the sample, but is preferably 37 ° C. for tissue sections or cells, for example.
For 1 to 48 hours, more preferably 6 to 30 hours, and the protease in the sample causes a digestion mark to be formed in the thin film. Thereafter, the thin film is stained with a dye, and digestion marks can be observed using an optical microscope or the like. Furthermore, in order to detect a specific substance in a sample, immunohistochemical staining is performed using an antibody that specifically reacts with the substance.
【0016】プロテアーゼ基質を含む薄膜の染色に好ま
しい染料として、赤色、橙色、及び黄色からなる群から
選ばれる1種又は2種以上の染料を用いることができ
る。本明細書において用いられる赤色、橙色、又は黄色
という用語はもっとも広義に解釈すべきであり、いかな
る意味においても限定的に解釈してはならない。このよ
うな染料は、一般的には、吸収極大が400 nm〜580 nmの
範囲である。赤色染料としてはAcid Red 1 (C.I.1805
0), Acid Red 4(C.I.14710), Acid Red 8 (C.I.1490
0), Acid Red 37 (C.I.17045), Acid Red 40(C.I.180
70), Acid Red 44 ,Acid Red 106 (C.I.18110), Acid
Red 183 (C.I.18800), Xylidin Ponceau 2R(C.I.1615
0), Mordant Red 19, Nitro Red、およびポンソー3
Rをあげることができ、特にポンソー3Rが好ましい。
橙色の染料としてはMethyl Orange, Ethyl Orange, C
rocein Orange G, Orange II, Orange G, Acid Oran
ge 8 (C.I.15575), Acid Orange 74 (C.I.18745)が挙
げられ、黄色の染料としてはMordant Yellow 10, Morda
nt Yellow 7, Acid Yellow 99(C.I.13900), Acid Yello
w 65(C.I.14170), Acid Yellow 17 (C.I.18965), Nit
razine Yellow(C.I.14890)が好ましく使用できる。One or more dyes selected from the group consisting of red, orange, and yellow can be used as dyes preferable for staining a thin film containing a protease substrate. The terms red, orange or yellow as used herein should be interpreted in the broadest sense and not in any sense in a limiting sense. Such dyes generally have an absorption maximum in the range of 400 nm to 580 nm. Acid Red 1 (CI1805
0), Acid Red 4 (CI14710), Acid Red 8 (CI1490
0), Acid Red 37 (CI17045), Acid Red 40 (CI180
70), Acid Red 44, Acid Red 106 (CI18110), Acid
Red 183 (CI18800), Xylidin Ponceau 2R (CI1615
0), Mordant Red 19, Nitro Red, and Ponceau 3
R can be given, and Ponceau 3R is particularly preferable.
Methyl Orange, Ethyl Orange, C
rocein Orange G, Orange II, Orange G, Acid Oran
ge 8 (CI15575), Acid Orange 74 (CI18745), and as the yellow dye, Mordant Yellow 10, Morda
nt Yellow 7, Acid Yellow 99 (CI13900), Acid Yello
w 65 (CI14170), Acid Yellow 17 (CI18965), Nit
Razine Yellow (CI14890) can be preferably used.
【0017】抗体を用いて可視化する試料中の物質の種
類は特に限定されないが、例えば、細胞表面あるいは細
胞内の各種レセプターや抗原などを挙げることができ、
癌細胞、免疫系細胞、繊維芽細胞、上皮細胞、平滑筋細
胞などに特異的に存在する物質を対象とすることができ
る。また、MMPやPAその他の酵素を対象とすることがで
き、各種コラーゲン、フィブロネクチン、ビトロネクチ
ンなどの細胞外マトリックスや接着分子を対象としても
よい。The type of the substance in the sample to be visualized using the antibody is not particularly limited, and examples thereof include various receptors and antigens on the cell surface or in the cell.
Substances specifically present in cancer cells, immune system cells, fibroblasts, epithelial cells, smooth muscle cells, and the like can be targeted. In addition, MMP, PA, and other enzymes can be targeted, and extracellular matrices such as various collagens, fibronectin, and vitronectin, and adhesion molecules may be targeted.
【0018】これらの物質に特異的に反応する抗体とし
てはポリクローナル抗体又はモノクローナル抗体のいず
れを用いてもよいが、好ましくはモノクローナル抗体を
用いることができる。抗体としては、マウス、ラット、
ウサギ、犬、羊、ヤギ、馬等の動物で産生されたIgGが
好ましい。さらにこれらの抗体を検出するための2次抗
体として、抗マウスIgG、抗ラットIgG、抗ウサギIgG 、
抗イヌIgG 、抗ヒツジIgG 、抗ヤギIgG 、抗ウマIgGな
どの抗体を、金コロイド、蛍光色素、酵素、又はビオチ
ンなどで修飾したものを好適に用いることができる。2
次抗体としてビオチン標識抗体を用いる場合には、それ
を検出するために金コロイド、蛍光色素、酵素などと結
合させたアビジンを用いることができる。Either a polyclonal antibody or a monoclonal antibody may be used as an antibody specifically reacting with these substances, but a monoclonal antibody can be preferably used. Antibodies include mice, rats,
IgGs produced in animals such as rabbits, dogs, sheep, goats, and horses are preferred. Further, as secondary antibodies for detecting these antibodies, anti-mouse IgG, anti-rat IgG, anti-rabbit IgG,
Antibodies such as anti-dog IgG, anti-sheep IgG, anti-goat IgG, and anti-horse IgG, which are modified with colloidal gold, a fluorescent dye, an enzyme, biotin, or the like, can be suitably used. 2
When a biotin-labeled antibody is used as the secondary antibody, avidin conjugated with colloidal gold, a fluorescent dye, an enzyme, or the like can be used to detect it.
【0019】金コロイドを結合させた抗体を用いる場合
には、光学顕微鏡でその存在を検出することができる
が、さらに銀による信号の増強を行うことができ、例え
ばAmersham社よりSilver Enhancement Kitの名称で市販
されているものを用いることができる。蛍光色素を結合
させた抗体を用いる場合には蛍光顕微鏡でその存在を検
出することができる。また、酵素を結合させた抗体を用
いる場合には、その酵素に適した発色基質を用いて発色
反応を行わせることによりその存在を検出することがで
きる。代表的な基質として、例えば、酵素としてペルオ
キシダーゼを用いる場合には3-amino-9-ethylcarbazol
e, 3,3'-diaminobenzizine, 3,3',5,5'-tetramethylben
zizineなど、アルカリフォスファターゼを用いる場合に
は5-bromo-4-chloro-3-indolylphosphate p-toluidine
saltとnitroblue tetrazolium chlorideとの混合物、na
phthol-AS-phosphateなど、βーガラクトシダーゼを用
いる場合には5-bromo-4-chloro-3-indolyl-β-D-galact
oside, halogenated indolyl-β-D-galactosideなどを
用いることができる。When an antibody to which gold colloid is bound is used, its presence can be detected by an optical microscope, but the signal can be further enhanced by silver. For example, the name of Silver Enhancement Kit from Amersham can be used. Can be used. When an antibody to which a fluorescent dye is bound is used, its presence can be detected with a fluorescence microscope. When an antibody to which an enzyme is bound is used, the presence of the antibody can be detected by performing a coloring reaction using a coloring substrate suitable for the enzyme. As a typical substrate, for example, when using peroxidase as an enzyme, 3-amino-9-ethylcarbazol
e, 3,3'-diaminobenzizine, 3,3 ', 5,5'-tetramethylben
When using alkaline phosphatase such as zizine, 5-bromo-4-chloro-3-indolylphosphate p-toluidine
A mixture of salt and nitroblue tetrazolium chloride, na
When using β-galactosidase, such as phthol-AS-phosphate, 5-bromo-4-chloro-3-indolyl-β-D-galact
oside, halogenated indolyl-β-D-galactoside and the like can be used.
【0020】本発明の方法で試料に含まれるプロテアー
ゼ活性を測定し、同時に試料中の特定の物質の存在位置
を可視化して観察することができるが、プロテアーゼ活
性及び特定の物質の免疫組織化学的観察には、光学顕微
鏡下で目視で判定する方法、分光器により消化痕の光学
濃度を測定する方法、光学顕微鏡で得られる画像をコン
ピューターに取り込み、画像解析の方法により消化痕に
おける各種の数値化を行う方法などを利用することがで
きる。According to the method of the present invention, the protease activity contained in a sample can be measured, and at the same time, the location of a specific substance in the sample can be visualized and observed. For observation, a method of visual judgment under an optical microscope, a method of measuring the optical density of digestive marks using a spectroscope, and taking a computer image of the image obtained with an optical microscope and digitizing various types of digestive marks by an image analysis method And the like.
【0021】[0021]
【実施例】以下、本発明を実施例によりさらに具体的に
説明するが、本発明の範囲はこれらの実施例に限定され
ることはない。 例1:動脈粥状硬化巣のプロテアーゼ活性とマクロファ
ージの分布観察 (1)動脈粥状硬化巣組織とゼラチン薄膜との反応 外科手術あるいは剖検により摘出し凍結した動脈検体
を、凍結切片作製装置を用いて-25℃で厚さ4μに薄
切しWO97/32035に開示されたゼラチン薄膜に接着させ
た。ゼラチンの膜厚は6μのものを用いた。このゼラチ
ン膜を37℃、相対湿度100%で30時間インキュベ
ートした。EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples, but the scope of the present invention is not limited to these Examples. Example 1: Observation of protease activity in atherosclerotic lesion and distribution of macrophages (1) Reaction between atherosclerotic lesion tissue and gelatin thin film An artery specimen extracted and frozen by surgical operation or necropsy using a cryosection preparation device At -25 ° C. and sliced to a thickness of 4 μm and adhered to a gelatin thin film disclosed in WO97 / 32035. The gelatin having a thickness of 6 μm was used. This gelatin film was incubated at 37 ° C. and 100% relative humidity for 30 hours.
【0022】(2)マクロファージの免疫染色 インキュベートを完了した薄膜をPBSで洗浄し、1%
過酸化水素メタノール溶液に10分間浸漬した。PBS
で洗浄後、10%正常ウサギ血清に30分間浸漬した。
余分な水分を拭き取った後、抗マクロファージモノクロ
ーナル抗体(HAM56)のPBS溶液を動脈切片上に滴下
し、4℃で一晩反応させた。PBSで洗浄後余分な水分
を拭き取り、ビオチン標識抗マウスIgG抗体のPBS
溶液を滴下した。室温で10分間反応させた後、PBS
で洗浄し、ペルオキシダーゼ標識ストレプトアビジンの
PBS溶液を滴下した。5分間の反応の後PBSで洗浄
し、3,3'-diaminobenzidine tetrahydrochlorideと過酸
化水素を含む溶液を滴下した。10分間の反応の後水洗
した。(2) Immunostaining of Macrophages The incubated thin film was washed with PBS, and then washed with 1%
It was immersed in a methanol solution of hydrogen peroxide for 10 minutes. PBS
And then immersed in 10% normal rabbit serum for 30 minutes.
After wiping off excess water, a PBS solution of anti-macrophage monoclonal antibody (HAM56) was dropped on the arterial section and allowed to react at 4 ° C. overnight. After washing with PBS, excess water was wiped off, and PBS containing biotin-labeled anti-mouse IgG antibody was removed.
The solution was added dropwise. After reacting at room temperature for 10 minutes, PBS
And a PBS solution of peroxidase-labeled streptavidin was added dropwise. After the reaction for 5 minutes, the mixture was washed with PBS, and a solution containing 3,3′-diaminobenzidine tetrahydrochloride and hydrogen peroxide was added dropwise. After the reaction for 10 minutes, it was washed with water.
【0023】(3)ゼラチン膜の染色 染色液としては6%のトリクロロ酢酸水溶液に濃度0.
8%になるようにポンソー3Rを溶解させた液とエタノ
ールを3対7で混合した液を用い、薄膜を室温で3分間
浸漬して染色した。10分間水洗した後70%エタノー
ル、純エタノールの順番で各3分間ずつ浸漬、組織切片
を覆うようにカバーグラスをキシレンで溶いた封入剤を
用いて貼り付け動脈切片を封入した。このフィルムの裏
面(組織接着面の裏面)を組織切片用スライドガラスに封
入剤を用いて張り付け,光学顕微鏡を用いて観察すると
動脈粥状硬化病変組織切片中、ゼラチンの分解によりポ
ンソーの赤色が薄くなった部分と、マクロファージの存
在を示す茶色の発色が同時に観察できた。(3) Staining of Gelatin Film As a staining solution, a 6% aqueous solution of trichloroacetic acid was used at a concentration of 0.5%.
Using a solution in which Ponceau 3R was dissolved to a concentration of 8% and ethanol in a ratio of 3: 7, the thin film was immersed at room temperature for 3 minutes and stained. After washing with water for 10 minutes, immersion was performed for 3 minutes each in the order of 70% ethanol and pure ethanol, and a cover glass was stuck so as to cover the tissue section using a mounting medium dissolved in xylene, and the arterial section was enclosed. The back side of this film (the back side of the tissue-adhesive side) was adhered to a glass slide for tissue section using an encapsulating agent, and when observed using an optical microscope, the red color of Ponceau was faint due to the decomposition of gelatin in the atherosclerotic lesion tissue section. A brown portion indicating the presence of macrophages was observed at the same time.
【0024】例2:動脈粥状硬化巣のプロテアーゼ活性
と平滑筋アクチンの分布観察 (1)動脈粥状硬化巣組織とゼラチン薄膜との反応 外科手術あるいは剖検により摘出し凍結した動脈粥状硬
化巣を有する血管組織を、凍結切片作製装置を用いて-
25℃で厚さ5μに薄切しWO97/32035に開示されたゼラ
チン薄膜に接着させた。ゼラチンの膜厚は6μのものを
用いた。このゼラチン膜を37℃、相対湿度100%で
30時間インキュベートした。Example 2: Observation of protease activity of arterial atherosclerotic lesion and distribution of smooth muscle actin (1) Reaction between arterial atherosclerotic tissue and gelatin thin film Arterial atherosclerotic tissue isolated and frozen by surgical operation or autopsy Using a frozen section preparation device-
The slices were sliced at 25 ° C. to a thickness of 5 μm and adhered to a gelatin thin film disclosed in WO97 / 32035. The gelatin having a thickness of 6 μm was used. This gelatin film is heated at 37 ° C and 100% relative humidity.
Incubated for 30 hours.
【0025】(2)平滑筋アクチンの免疫染色 インキュベートを完了した薄膜を0.8%牛血清アルブ
ミンとゼラチン(アマシャム社、IGSS仕様)を含むPBS
で10分間洗浄し、0.2%正常ウサギ血清に30分間浸
漬した。余分な水分を拭き取った後、抗アクチンマウス
モノクローナル抗体の1%牛血清アルブミン含有PBS
溶液を動脈切片上に滴下し、室温で90分反応させた。
10分間3回の洗浄の後金コロイド標識された抗マウス
IgGヤギ抗体と60分間ずつ液を新しく換えて2回反
応、PBSで洗浄後蒸留水で洗浄し、フィルム上で切片
を2%グルタルアルデヒドで10分間固定した。蒸留水
で洗浄した後、アマシャム社のIntenSETM M Silver Enh
ancement Kitを用いてアルミホイル遮光下で10分間反
応させ、蒸留水で洗浄した。(2) Immunostaining of Actin of Smooth Muscle The incubated thin film was washed with PBS containing 0.8% bovine serum albumin and gelatin (Amersham, IGSS specification).
For 10 minutes, and immersed in 0.2% normal rabbit serum for 30 minutes. After wiping off excess water, PBS containing 1% bovine serum albumin of anti-actin mouse monoclonal antibody
The solution was dropped on the arterial section and reacted at room temperature for 90 minutes.
After washing three times for 10 minutes, the solution was newly changed with anti-mouse IgG goat antibody labeled with colloidal gold for 60 minutes, and reacted twice. After washing with PBS, washing with distilled water, the section on the film was subjected to 2% glutaraldehyde. For 10 minutes. After washing with distilled water, Amersham IntenSETM M Silver Enh
Using an ancement Kit, the reaction was performed for 10 minutes under the light shielding of aluminum foil, and washed with distilled water.
【0026】(3)ゼラチン膜の染色 染色液としては6%のトリクロロ酢酸水溶液に濃度0.
8%になるようにポンソー3Rを溶解させた液とエタノ
ールを3対7で混合した液を用い、薄膜を室温で3分間
浸漬して染色した。10分間水洗した後70%エタノー
ル、純エタノールの順番で各3分間ずつ浸漬、組織切片
を覆うようにカバーグラスをキシレンで溶いた封入剤を
用いて貼り付け動脈切片を封入した。このフィルムの裏
面(組織接着面の裏面)を組織切片用スライドガラスに封
入剤を用いて張り付け,光学顕微鏡を用いて観察すると
動脈粥状硬化病変組織切片中、ゼラチンの分解によりポ
ンソーの赤色が薄くなった部分と、平滑筋アクチンの存
在を示す黒色の発色が同時に観察できた。(3) Staining of gelatin film As a staining solution, a 6% aqueous solution of trichloroacetic acid was used at a concentration of 0.5%.
Using a solution in which Ponceau 3R was dissolved to a concentration of 8% and ethanol in a ratio of 3: 7, the thin film was immersed at room temperature for 3 minutes and stained. After washing with water for 10 minutes, immersion was performed for 3 minutes each in the order of 70% ethanol and pure ethanol, and a cover glass was stuck so as to cover the tissue section using a mounting medium dissolved in xylene, and the arterial section was enclosed. The back side of this film (the back side of the tissue-adhesive side) was adhered to a glass slide for tissue section using an encapsulating agent, and when observed using an optical microscope, the red color of Ponceau was faint due to the decomposition of gelatin in the atherosclerotic lesion tissue section. The blackened part and the black color indicating the presence of smooth muscle actin were simultaneously observed.
【0027】例3:乳癌組織のプロテアーゼ活性とサイ
トケラチン19の分布観察 (1)乳癌組織とゼラチン薄膜との反応 外科手術あるいは剖検により摘出し凍結した乳癌検体
を、凍結切片作製装置を用いて−25℃で厚さ4μに薄
切しWO97/32035に開示されたゼラチン薄膜に接着させ
た。ゼラチンの膜厚は6μのものを用いた。このゼラチ
ン膜を37℃、相対湿度100%で16時間インキュベ
ートした。Example 3 Observation of Protease Activity of Breast Cancer Tissue and Distribution of Cytokeratin 19 (1) Reaction between Breast Cancer Tissue and Thin Film of Gelatin A breast cancer specimen which has been removed by surgery or necropsy and frozen using a cryosection preparation apparatus. The slices were sliced at 25 ° C. to a thickness of 4 μm and adhered to a gelatin thin film disclosed in WO97 / 32035. The gelatin having a thickness of 6 μm was used. The gelatin film was incubated at 37 ° C. and 100% relative humidity for 16 hours.
【0028】(2)サイトケラチン19の免疫染色 インキュベートを完了した薄膜をPBSで洗浄し、1%
過酸化水素メタノール溶液に10分間浸漬した。PBS
で洗浄後、10%正常ウサギ血清に30分間浸漬した。
余分な水分を拭き取った後、抗サイトケラチン19マウ
スモノクローナル抗体のPBS溶液を乳癌切片上に滴下
し、4℃で一晩反応させた。PBSで洗浄後余分な水分
を拭き取り、ビオチン標識抗マウスIgG抗体のPBS
溶液を滴下した。10分間3回の洗浄の後金コロイド標
識された抗マウスIgGヤギ抗体と60分間ずつ液を新
しく換えて2回反応、PBSで洗浄後蒸留水で洗浄し、
フィルム上で切片を2%グルタルアルデヒドで10分間
固定した。蒸留水で洗浄した後、アマシャム社のIntenS
ETM M Silver Enhancement Kitを用いてアルミホイル遮
光下で10分間反応させ、蒸留水で洗浄した。(2) Immunostaining of cytokeratin 19 The incubated thin film was washed with PBS and 1%
It was immersed in a methanol solution of hydrogen peroxide for 10 minutes. PBS
And then immersed in 10% normal rabbit serum for 30 minutes.
After wiping off excess water, a PBS solution of anti-cytokeratin 19 mouse monoclonal antibody was dropped on the breast cancer section, and reacted at 4 ° C. overnight. After washing with PBS, excess water was wiped off, and PBS containing biotin-labeled anti-mouse IgG antibody was removed.
The solution was added dropwise. After washing three times for 10 minutes, the solution was renewed with the anti-mouse IgG goat antibody labeled with colloidal gold for 60 minutes, and reacted twice. After washing with PBS, washing with distilled water,
Sections were fixed on film with 2% glutaraldehyde for 10 minutes. After washing with distilled water, Amersham IntenS
Using an ETM M Silver Enhancement Kit, the reaction was performed for 10 minutes under the light shielding of aluminum foil, followed by washing with distilled water.
【0029】(3)ゼラチン膜の染色 染色液としては6%のトリクロロ酢酸水溶液に濃度0.
8%になるようにポンソー3Rを溶解させた液とエタノ
ールを3対7で混合した液を用い、薄膜を室温で3分間
浸漬して染色した。10分間水洗した後70%エタノー
ル、純エタノールの順番で各3分間ずつ浸漬、組織切片
を覆うようにカバーグラスをキシレンで溶いた封入剤を
用いて貼り付け動脈切片を封入した。このフィルムの裏
面(組織接着面の裏面)を組織切片用スライドガラスに封
入剤を用いて張り付け,光学顕微鏡を用いて観察すると
動脈粥状硬化病変組織切片中、ゼラチンの分解によりポ
ンソーの赤色が薄くなった部分と、サイトケラチン19
の存在を示す黒色の発色が同時に観察できた。(3) Staining of gelatin film As a staining solution, a 6% aqueous solution of trichloroacetic acid was used at a concentration of 0.5%.
Using a solution in which Ponceau 3R was dissolved to a concentration of 8% and ethanol in a ratio of 3: 7, the thin film was immersed at room temperature for 3 minutes and stained. After washing with water for 10 minutes, immersion was performed for 3 minutes each in the order of 70% ethanol and pure ethanol, and a cover glass was stuck so as to cover the tissue section using a mounting medium dissolved in xylene, and the arterial section was enclosed. The back side of this film (the back side of the tissue-adhesive side) was adhered to a glass slide for tissue section using an encapsulating agent, and when observed using an optical microscope, the red color of Ponceau was faint due to the decomposition of gelatin in the atherosclerotic lesion tissue section. Part and the cytokeratin 19
Was simultaneously observed.
【0030】[0030]
【発明の効果】本発明の方法によれば、試料に含まれる
プロテアーゼ活性を測定し、同時にプロテアーゼ蛋白や
他の物質の存在に関して存在量や分布の情報を得ること
ができる。According to the method of the present invention, the protease activity contained in a sample can be measured, and at the same time, information on the amount and distribution of the protease protein and other substances can be obtained.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 FI テーマコート゛(参考) G01N 33/543 521 G01N 33/543 521 525 525W 525U 33/573 33/573 A 33/577 33/577 A 33/68 33/68 //(C12Q 1/37 C12R 1:91) Fターム(参考) 2G045 AA25 AA26 BA14 BB01 BB14 BB22 BB24 BB46 BB50 BB51 CA11 CB17 CB26 DA77 DA78 FA16 FA18 FB01 FB03 FB07 FB11 FB12 GC12 GC15 JA01 4B063 QA01 QA19 QQ02 QQ79 QR16 QS03 QS33 QX02 ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 7 Identification symbol FI theme coat ゛ (Reference) G01N 33/543 521 G01N 33/543 521 525 525W 525U 33/573 33/573 A 33/577 33/577 A 33/68 33/68 // (C12Q 1/37 C12R 1:91) F term (reference) 2G045 AA25 AA26 BA14 BB01 BB14 BB22 BB24 BB46 BB50 BB51 CA11 CB17 CB26 DA77 DA78 FA16 FA18 FB01 FB03 FB07 FB11 FB12 GC12 GC15 JA01 QA01 QA19 QQ02 QQ79 QR16 QS03 QS33 QX02
Claims (12)
観察とを同時に行う方法であって、 (1)プロテアーゼ基質を含み支持体表面に形成された
薄膜に対して、プロテアーゼを含む試料を接触させる工
程; (2)プロテアーゼの作用により該薄膜に形成された消
化痕を、該薄膜を染料で染色することにより検出する工
程;及び (3)試料中の特定の物質に反応する抗体を用いて、該
物質を可視化する工程を含む方法。1. A method for simultaneously performing protease activity measurement and immunohistochemical observation, comprising: (1) a step of bringing a protease-containing sample into contact with a thin film containing a protease substrate and formed on the surface of a support; (2) a step of detecting a digestion mark formed on the thin film by the action of a protease by staining the thin film with a dye; and (3) using an antibody that reacts with a specific substance in a sample, A method comprising the step of visualizing a substance.
観察とを同時に行う方法であって、 (1)プロテアーゼ基質と硬膜剤とを含み支持体表面に
形成された薄膜に対して、プロテアーゼを含む試料を接
触させる工程; (2)プロテアーゼの作用により該薄膜に形成された消
化痕を、該薄膜を染料で染色することにより検出する工
程;及び (3)試料中の特定の物質に反応する抗体を用いて、該
物質を可視化する工程を含む方法。2. A method for simultaneously performing a protease activity measurement and an immunohistochemical observation, wherein (1) a protease is contained in a thin film formed on the surface of a support containing a protease substrate and a hardening agent. (2) a step of detecting a digestion mark formed on the thin film by the action of a protease by staining the thin film with a dye; and (3) an antibody that reacts with a specific substance in the sample. A method for visualizing the substance using the method.
観察とを同時に行う方法であって、 (1)プロテアーゼ基質と硬膜剤とを含み支持体表面に
形成された薄膜に対して、生体試料の実質的に連続した
2以上の切片の内の一つを接触させる工程; (2)プロテアーゼ基質、硬膜剤、及びプロテアーゼ・
インヒビターを含み支持体表面に形成された薄膜に対し
て、残りの切片を接触させる工程; (3)プロテアーゼの作用により該薄膜に形成された消
化痕を、該薄膜を染料で染色することにより検出する工
程; (4)工程(1)で用いた薄膜の消化痕と工程(2)で
用いた薄膜の消化痕とを対比する工程;及び (5)試料中の特定の物質に反応する抗体を用いて、該
物質を可視化する工程含む方法。3. A method for simultaneously performing protease activity measurement and immunohistochemical observation, comprising: (1) applying a biological sample to a thin film containing a protease substrate and a hardening agent and formed on the surface of a support; Contacting one of the two or more substantially continuous sections; (2) a protease substrate, a hardener, and a protease.
Contacting the remaining slice with a thin film formed on the surface of the support containing the inhibitor; (3) detecting a digestion mark formed on the thin film by the action of a protease by staining the thin film with a dye (4) a step of comparing the digestion mark of the thin film used in step (1) with the digestion mark of the thin film used in step (2); and (5) an antibody that reacts with a specific substance in the sample. Using the method for visualizing the substance.
応するモノクローナル抗体である請求項1から3のいず
れか1項に記載の方法。4. The method according to claim 1, wherein the antibody is a monoclonal antibody that specifically reacts with a specific substance in a sample.
素標識抗体、蛍光標識抗体、又は金コロイド標識抗体で
ある請求項1から4のいずれか1項に記載の方法。5. The method according to claim 1, wherein the antibody that reacts with a specific substance in the sample is an enzyme-labeled antibody, a fluorescently-labeled antibody, or a colloidal gold-labeled antibody.
異的に反応する酵素標識抗体、蛍光標識抗体、又は金コ
ロイド標識抗体を2次抗体として用いる請求項1から4
のいずれか1項に記載の方法。6. The secondary antibody is an enzyme-labeled antibody, a fluorescently-labeled antibody, or a colloidal gold-labeled antibody that specifically reacts with an antibody that reacts with a specific substance in a sample.
The method according to any one of claims 1 to 4.
い、該2次抗体を酵素標識アビジン、蛍光標識アビジ
ン、又は金コロイド標識アビジンを用いて可視化する請
求項6に記載の方法。7. The method according to claim 6, wherein a biotin-labeled antibody is used as the secondary antibody, and the secondary antibody is visualized using enzyme-labeled avidin, fluorescent-labeled avidin, or colloidal gold-labeled avidin.
ンである請求項1から7のいずれか1項に記載の方法。8. The method according to claim 1, wherein the protease substrate is gelatin or casein.
から選ばれる1種又は2種以上の染料である請求項1か
ら8のいずれか1項に記載の方法。9. The method according to claim 1, wherein the dye is one or more dyes selected from the group consisting of red, orange, and yellow.
記載の方法。10. The method according to claim 9, wherein the dye is Ponceau 3R.
れた組織又は体液中の細胞である請求項1から10のいず
れか1項に記載の方法。11. The method according to claim 1, wherein the biological sample is a cell in a tissue or a body fluid obtained from a mammal including a human.
の方法に使用するための薄膜。12. A thin film for use in the method according to any one of claims 1 to 11.
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JP11174826A JP2001000197A (en) | 1999-06-22 | 1999-06-22 | Measurement of protease activity |
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Cited By (8)
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GB2435511A (en) * | 2006-02-23 | 2007-08-29 | Mologic Ltd | Protease detection |
JP2010513856A (en) * | 2006-12-15 | 2010-04-30 | キンバリー クラーク ワールドワイド インコーポレイテッド | Enzyme detection technology |
JP2011090009A (en) * | 2004-01-26 | 2011-05-06 | President & Fellows Of Harvard College | System and method for fluid delivery |
US8241588B2 (en) | 2006-02-23 | 2012-08-14 | Mologic Ltd | Binding assay |
US8361386B2 (en) | 2006-02-23 | 2013-01-29 | Mologic Ltd | Enzyme detection |
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US10082507B2 (en) | 2003-12-31 | 2018-09-25 | President And Fellows Of Harvard College | Assay device and method |
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1999
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US10082507B2 (en) | 2003-12-31 | 2018-09-25 | President And Fellows Of Harvard College | Assay device and method |
US9116148B2 (en) | 2004-01-26 | 2015-08-25 | President And Fellows Of Harvard College | Fluid delivery system and method |
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JP2011090009A (en) * | 2004-01-26 | 2011-05-06 | President & Fellows Of Harvard College | System and method for fluid delivery |
US8361386B2 (en) | 2006-02-23 | 2013-01-29 | Mologic Ltd | Enzyme detection |
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JP2010513856A (en) * | 2006-12-15 | 2010-04-30 | キンバリー クラーク ワールドワイド インコーポレイテッド | Enzyme detection technology |
US9255866B2 (en) | 2013-03-13 | 2016-02-09 | Opko Diagnostics, Llc | Mixing of fluids in fluidic systems |
US9588027B2 (en) | 2013-03-13 | 2017-03-07 | UPKO Diagnostics, LLC | Mixing of fluids in fluidic systems |
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