JP2020141616A - Method for staining cancer cells - Google Patents
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Abstract
Description
本発明は、血液中のがん細胞の染色方法に関する。 The present invention relates to a method for staining cancer cells in blood.
がん細胞が発生するとやがて、血液・体液中に出て来ることが知られており、血液中に出て来たがん細胞は、血中循環腫瘍細胞（ＣＴＣ）と呼ばれている。そして、この血中循環腫瘍細胞を調べることによるがんの治療効果の確認、予後寿命、投与前の抗がん剤の効果予測、がん細胞の遺伝子解析を用いた治療方法の検討、等が期待されている。 It is known that when cancer cells develop, they eventually appear in blood and body fluids, and the cancer cells that appear in the blood are called circulating tumor cells (CTC). Then, confirmation of the therapeutic effect of cancer by examining these circulating tumor cells in the blood, prognosis lifespan, prediction of the effect of anticancer drugs before administration, examination of treatment methods using gene analysis of cancer cells, etc. It is expected.
ＣＴＣの捕捉方法として、例えば、特許文献１では、親水性ポリマー層にＣＴＣを捕捉する方法が開示されている。 As a method for capturing CTC, for example, Patent Document 1 discloses a method for capturing CTC in a hydrophilic polymer layer.
特許文献１では、親水性ポリマー層に捕捉したがん細胞を、蛍光抗体法によって染色し、観察している（特許文献１の［００５３］）。しかしながら、蛍光抗体法は、１０日〜２週間程度で染色が消失してしまう、観察時のバックグラウンドの影響が大きい、暗視野顕微鏡を用いるため背景組織の観察が困難である、といった点で改善の余地があった。 In Patent Document 1, cancer cells captured in a hydrophilic polymer layer are stained by a fluorescent antibody method and observed (Patent Document 1 [0053]). However, the fluorescent antibody method is improved in that the staining disappears in about 10 days to 2 weeks, the background influence during observation is large, and it is difficult to observe the background tissue because a dark field microscope is used. There was room for.
そこで、本発明者らが他の染色方法を検討したところ、酵素抗体法の一種である免疫ペルオキシダーゼ法に着目した。免疫ペルオキシダーゼ法では、ペルオキシダーゼ（酵素）で標識された抗体をがん細胞（抗原）に結合させた後、ジアミノベンジジン（ＤＡＢ）等の基質とペルオキシダーゼとを反応させることで、がん細胞を染色する。この方法は、蛍光抗体法と比較して、染色を長時間保持できる、観察時のバックグラウンドの影響が小さい、明視野顕微鏡を用いるため背景組織の観察が容易、といった利点がある。しかしながら、本発明者らが更に検討を進めたところ、以下の課題を見出した。 Therefore, when the present inventors examined other staining methods, they focused on the immunoperoxidase method, which is a kind of enzyme antibody method. In the immunoperoxidase method, an antibody labeled with peroxidase (enzyme) is bound to a cancer cell (antigen), and then the cancer cell is stained by reacting a substrate such as diaminobenzidine (DAB) with peroxidase. .. Compared with the fluorescent antibody method, this method has advantages that the staining can be retained for a long time, the influence of the background during observation is small, and the background tissue can be easily observed because a bright-field microscope is used. However, as a result of further studies by the present inventors, the following problems were found.
血液中のがん細胞を免疫ペルオキシダーゼ法で染色する場合、予め、血液中のペルオキシダーゼ（内因性ペルオキシダーゼ）を不活性化（ブロック）する必要がある。この不活性化は、通常、過酸化水素及びメタノールを用いて実施されるが、特許文献１に記載されたポリ２−メトキシエチルアクリレート（ＰＭＥＡ）等の親水性ポリマーは、メタノールで溶解する。そのため、特許文献１の方法において、蛍光抗体法の代わりに免疫ペルオキシダーゼ法を適用した場合、メタノールによって親水性ポリマーが溶解し、親水性ポリマー層に捕捉されたがん細胞が流れ出てしまうという点で改善の余地があった。 When staining cancer cells in blood by the immunoperoxidase method, it is necessary to inactivate (block) peroxidase (endogenous peroxidase) in blood in advance. This inactivation is usually carried out using hydrogen peroxide and methanol, but the hydrophilic polymer such as poly2-methoxyethyl acrylate (PMEA) described in Patent Document 1 is dissolved in methanol. Therefore, in the method of Patent Document 1, when the immunoperoxidase method is applied instead of the fluorescent antibody method, the hydrophilic polymer is dissolved by methanol and the cancer cells trapped in the hydrophilic polymer layer flow out. There was room for improvement.
本発明は、前記課題を解決し、血液中のがん細胞を親水性ポリマー層に捕捉し、免疫ペルオキシダーゼ法で染色する場合において、親水性ポリマーを溶解させることなく、血液中の内因性ペルオキシダーゼを不活性化できるがん細胞の染色方法を提供することを目的とする。 The present invention solves the above-mentioned problems, and when cancer cells in blood are captured in a hydrophilic polymer layer and stained by an immunoperoxidase method, the endogenous peroxidase in blood is removed without dissolving the hydrophilic polymer. An object of the present invention is to provide a method for staining cancer cells that can be inactivated.
前記課題を解決すべく本発明者らが更に検討を進めた結果、メタノールに代えてアジ化ナトリウムを用いることで、親水性ポリマーを溶解させることなく、血液中の内因性ペルオキシダーゼを不活性化できることを見出し、本発明に想到した。
すなわち、本発明は、血液中のがん細胞を親水性ポリマー層に捕捉する工程と、過酸化水素及びアジ化ナトリウムを用いて、前記血液中の内因性ペルオキシダーゼを不活性化する工程と、前記がん細胞を免疫ペルオキシダーゼ法によって染色する工程とを含むがん細胞の染色方法に関する。
As a result of further studies by the present inventors to solve the above problems, it is possible to inactivate the endogenous peroxidase in blood without dissolving the hydrophilic polymer by using sodium azide instead of methanol. And came up with the present invention.
That is, the present invention comprises a step of capturing cancer cells in blood in a hydrophilic polymer layer, a step of inactivating the endogenous peroxidase in blood using hydrogen peroxide and sodium azide, and the above-mentioned step. The present invention relates to a method for staining cancer cells, which comprises a step of staining cancer cells by an immunoperoxidase method.
前記免疫ペルオキシダーゼ法は、直接免疫ペルオキシダーゼ法であることが好ましい。 The immunoperoxidase method is preferably a direct immunoperoxidase method.
前記血液は、濃縮処理を施したものであることが好ましい。 The blood is preferably concentrated.
前記血液は、治療前の患者から採取したものであることが好ましい。 The blood is preferably collected from a patient before treatment.
前記親水性ポリマー層は、下記式（Ｉ）で表されるポリマー及びポリ（メタ）アクリロイルモルホリンからなる群より選択される少なくとも１種の親水性ポリマーで形成されていることが好ましい。
前記親水性ポリマー層は、下記式（Ｉ−１）で表される化合物及び（メタ）アクリロイルモルホリンからなる群より選択される少なくとも１種の親水性モノマーと、他のモノマーとの共重合体で形成されていることが好ましい。
本発明によれば、血液中のがん細胞を親水性ポリマー層に捕捉する工程と、過酸化水素及びアジ化ナトリウムを用いて、前記血液中の内因性ペルオキシダーゼを不活性化する工程と、前記がん細胞を免疫ペルオキシダーゼ法によって染色する工程とを含むがん細胞の染色方法であるため、親水性ポリマーを溶解させることなく、血液中の内因性ペルオキシダーゼを不活性化できる。 According to the present invention, a step of capturing cancer cells in blood in a hydrophilic polymer layer, a step of inactivating the endogenous peroxidase in blood using hydrogen peroxide and sodium azide, and the above-mentioned step. Since it is a method for staining cancer cells including a step of staining cancer cells by an immunoperoxidase method, it is possible to inactivate endogenous peroxidase in blood without dissolving a hydrophilic polymer.
本発明は、血液中のがん細胞を親水性ポリマー層に捕捉する工程（捕捉工程）と、過酸化水素及びアジ化ナトリウムを用いて、前記血液中の内因性ペルオキシダーゼを不活性化する工程（不活性化工程）と、前記がん細胞を免疫ペルオキシダーゼ法によって染色する工程（染色工程）とを含むがん細胞の染色方法である。 The present invention comprises a step of capturing cancer cells in blood on a hydrophilic polymer layer (capture step) and a step of inactivating the endogenous peroxidase in blood using hydrogen peroxide and sodium azide (capture step). This is a method for staining cancer cells, which comprises a step (inactivation step) and a step (staining step) of staining the cancer cells by an immunoperoxidase method.
上記方法では、血液中の内因性ペルオキシダーゼを不活性化する際、従来使用されていたメタノールに代えて、アジ化ナトリウムを使用する。これにより、親水性ポリマーを溶解させることなく、内因性ペルオキシダーゼを不活性化できる。そして、免疫ペルオキシダーゼ法によってがん細胞の染色を行うことで、蛍光抗体法と比較して、がん細胞やその背景組織の観察が容易であり、かつ長期保存が可能な試料を調製することができる。 In the above method, sodium azide is used in place of the conventionally used methanol when inactivating the endogenous peroxidase in blood. This allows the endogenous peroxidase to be inactivated without dissolving the hydrophilic polymer. Then, by staining the cancer cells by the immunoperoxidase method, it is possible to prepare a sample in which the cancer cells and their background tissues can be easily observed and can be stored for a long period of time as compared with the fluorescent antibody method. it can.
以下、上記方法を工程毎に説明する。 Hereinafter, the above method will be described for each step.
（捕捉工程）
この工程では、血液中のがん細胞を親水性ポリマー層に捕捉する。これは、例えば、血液と親水性ポリマー層とを接触させることで実施できる。
(Capture process)
In this step, cancer cells in the blood are trapped in a hydrophilic polymer layer. This can be done, for example, by bringing the blood into contact with the hydrophilic polymer layer.
親水性ポリマー層には、がん細胞は捕捉されるが、赤血球、白血球、血小板等の血球細胞は捕捉（吸着）されにくい。そのため、血液と親水性ポリマー層とを接触させた後に所定時間放置し、次いで、必要に応じてリン酸緩衝生理食塩水（ＰＢＳ）等で洗浄することで、がん細胞を選択的に親水性ポリマー層に捕捉することができる。 Cancer cells are captured in the hydrophilic polymer layer, but blood cells such as erythrocytes, white blood cells, and platelets are not easily captured (adsorbed). Therefore, the cancer cells are selectively hydrophilic by allowing the blood to come into contact with the hydrophilic polymer layer, leaving it for a predetermined time, and then washing it with phosphate buffered saline (PBS) or the like as necessary. It can be trapped in the polymer layer.
血液と親水性ポリマー層との接触方法は特に限定されないが、例えば、親水性ポリマー層への血液の注入、滴下、噴霧等が挙げられる。 The method of contacting the blood with the hydrophilic polymer layer is not particularly limited, and examples thereof include injection, dropping, and spraying of blood into the hydrophilic polymer layer.
血液は、親水性ポリマー層に接触させる前に、濃縮処理を施すことが好ましい。濃縮処理により、血球細胞を減少させ、血液中のがん細胞の割合を高めることで、より多くのがん細胞を親水性ポリマー層に捕捉させることができる。濃縮処理の方法は特に限定されないが、遠心分離が好ましく、濃度勾配遠心分離がより好ましい。濃度勾配遠心分離は、例えば、Ｇｒｅｉｎｅｒ Ｂｉｏ−Ｏｎｅ社のＯｎｃｏＱｕｉｃｋ（登録商標）によって行うことができる。 The blood is preferably concentrated prior to contact with the hydrophilic polymer layer. By the concentration treatment, the number of blood cells is reduced and the proportion of cancer cells in the blood is increased, so that more cancer cells can be captured by the hydrophilic polymer layer. The method of the concentration treatment is not particularly limited, but centrifugation is preferable, and concentration gradient centrifugation is more preferable. Concentration gradient centrifugation can be performed, for example, by OncoQuick® from Greener Bio-One.
なお、手術や化学療法等の治療を行った場合、通常、血液中のがん細胞は減少する。そのため、より多くのがん細胞を捕捉するためには、治療前の患者から採取した血液を使用することが好ましい。 When treatment such as surgery or chemotherapy is performed, cancer cells in the blood usually decrease. Therefore, in order to capture more cancer cells, it is preferable to use blood collected from the patient before treatment.
親水性ポリマー層（親水性ポリマーにより形成される層）は、所定の基材に形成できる。基材としては、ポリアクリル酸メチル、ポリメタクリル酸メチル、ポリアクリル酸、ポリメタクリル酸等のアクリル樹脂（ポリアクリル樹脂）、シクロオレフィン樹脂（ポリシクロオレフィン）、カーボネート樹脂（ポリカーボネート）、スチレン樹脂（ポリスチレン）、ポリエチレンテレフタレート（ＰＥＴ）等のポリエステル樹脂、ポリジメチルシロキサン、ソーダ石灰ガラス、ほうケイ酸ガラス等のガラス、等が挙げられる。 The hydrophilic polymer layer (layer formed by the hydrophilic polymer) can be formed on a predetermined substrate. As the base material, acrylic resin (polyacrylic resin) such as methyl polyacrylate, polymethyl methacrylate, polyacrylic acid, polymethacrylic acid, cycloolefin resin (polycycloolefin), carbonate resin (polycarbonate), styrene resin ( Polystyrene), polyester resins such as polyethylene terephthalate (PET), polydimethylsiloxane, soda lime glass, glass such as borosilicate glass, and the like.
親水性ポリマー層（親水性ポリマーにより形成される層）の膜厚は、好ましくは１０〜５００ｎｍ、より好ましくは３０〜４００ｎｍ、更に好ましくは５０〜３５０ｎｍである。上記範囲内に調整することで、がん細胞に対する選択的捕捉性が良好となる。 The film thickness of the hydrophilic polymer layer (layer formed by the hydrophilic polymer) is preferably 10 to 500 nm, more preferably 30 to 400 nm, and further preferably 50 to 350 nm. By adjusting within the above range, the selective capture property for cancer cells becomes good.
親水性ポリマーは、親水性を有するものを適宜選択できる。例えば、１種又は２種以上の親水性モノマーの単独重合体及び共重合体、１種又は２種以上の親水性モノマーと他のモノマーとの共重合体等が挙げられる。前記単独重合体、共重合体としては、ポリアクリル酸、ポリアクリル酸エステル、ポリメタクリル酸、ポリメタクリル酸エステル、ポリアクリロイルモルホリン、ポリメタクリロイルモルホリン、ポリアクリルアミド、ポリメタクリルアミド等が挙げられる。 As the hydrophilic polymer, one having hydrophilicity can be appropriately selected. For example, homopolymers and copolymers of one or more kinds of hydrophilic monomers and copolymers of one or more kinds of hydrophilic monomers and other monomers can be mentioned. Examples of the homopolymer and copolymer include polyacrylic acid, polyacrylic acid ester, polymethacrylic acid, polymethacrylic acid ester, polyacryloyl morpholine, polymethacryloyl morpholine, polyacrylamide, and polymethacrylamide.
親水性モノマーは、親水性基を有する各種モノマーを使用できる。親水性基は、例えば、アミド基、硫酸基、スルホン酸基、カルボン酸基、水酸基、アミノ基、アミド基、オキシエチレン基等、公知の親水性基が挙げられる。 As the hydrophilic monomer, various monomers having a hydrophilic group can be used. Examples of the hydrophilic group include known hydrophilic groups such as an amide group, a sulfate group, a sulfonic acid group, a carboxylic acid group, a hydroxyl group, an amino group, an amide group and an oxyethylene group.
親水性モノマーの具体例としては、（メタ）アクリル酸、（メタ）アクリル酸エステル、（メトキシエチル（メタ）アクリレート等のアルコキシアルキル（メタ）アクリレート、ヒドロキシエチル（メタ）アクリレート等のヒドロキシアルキル（メタ）アクリレート）、（メタ）アクリルアミド、環状基を有する（メタ）アクリルアミド誘導体（（メタ）アクリロイルモルホリン等）、などが挙げられる。なかでも、（メタ）アクリル酸、（メタ）アクリル酸エステル、アルコキシアルキル（メタ）アクリレート、（メタ）アクリロイルモルホリンが好ましく、アルコキシアルキル（メタ）アクリレートがより好ましく、２−メトキシエチルアクリレートが特に好ましい。 Specific examples of the hydrophilic monomer include (meth) acrylic acid, (meth) acrylic acid ester, alkoxyalkyl (meth) acrylate such as (methoxyethyl (meth) acrylate), and hydroxyalkyl (meth) such as hydroxyethyl (meth) acrylate. ) Acrylate), (meth) acrylamide, (meth) acrylamide derivative having a cyclic group ((meth) acryloylmorpholin, etc.), and the like. Of these, (meth) acrylic acid, (meth) acrylic acid ester, alkoxyalkyl (meth) acrylate, and (meth) acryloyl morpholine are preferable, alkoxyalkyl (meth) acrylate is more preferable, and 2-methoxyethyl acrylate is particularly preferable.
他のモノマーは、親水性ポリマーの作用効果を阻害しない範囲内で適宜選択すれば良い。例えば、スチレン等の芳香族モノマー、酢酸ビニル、温度応答性を付与できるＮ−イソプロピルアクリルアミドなどが挙げられる。 Other monomers may be appropriately selected within a range that does not interfere with the action and effect of the hydrophilic polymer. For example, aromatic monomers such as styrene, vinyl acetate, N-isopropylacrylamide that can impart temperature responsiveness, and the like can be mentioned.
なかでも、親水性ポリマーとしては、下記式（Ｉ）で表されるポリマー及びポリ（メタ）アクリロイルモルホリンからなる群より選択される少なくとも１種が好ましい。
Ｒ２のアルキル基の炭素数は、１〜１０が好ましく、１〜５がより好ましい。なかでも、Ｒ２は、メチル基又はエチル基が特に好ましい。ｍは、１〜３が好ましい。ｎ（繰り返し単位数）は、１５〜１５００が好ましく、４０〜１２００がより好ましい。 The carbon number of the alkyl group of R 2 is 1 to 10 preferably 1 to 5 is more preferable. Of these, R 2 is particularly preferably a methyl group or an ethyl group. m is preferably 1 to 3. n (number of repeating units) is preferably 15 to 1500, more preferably 40 to 1200.
また、親水性ポリマーとして、下記式（Ｉ−１）で表される化合物及び（メタ）アクリロイルモルホリンからなる群より選択される少なくとも１種の親水性モノマーと、他のモノマーとの共重合体も好適に使用できる。
親水性ポリマー層が形成された基材は、特開２０１８−５０５４８号公報に記載の方法等、公知の手法により、基材表面の全部又は一部に親水性ポリマー溶液・分散液をコーティングすることで、製造することができる。このような基材として、例えば、マルチウェルプレート、スライドチャンバー（チャンバー付スライドガラス）等が挙げられる。 The substrate on which the hydrophilic polymer layer is formed is coated with a hydrophilic polymer solution / dispersion on all or part of the surface of the substrate by a known method such as the method described in JP-A-2018-50548. Can be manufactured with. Examples of such a base material include a multi-well plate, a slide chamber (slide glass with a chamber), and the like.
親水性ポリマー層には、予め、フィブロネクチンを吸着させていてもよい。フィブロネクチンが吸着した親水性ポリマー層に血液を接触させることで、親水性ポリマー層により多くのがん細胞を捕捉することが可能となる。 Fibronectin may be adsorbed on the hydrophilic polymer layer in advance. By bringing blood into contact with the hydrophilic polymer layer on which fibronectin is adsorbed, it becomes possible to capture more cancer cells in the hydrophilic polymer layer.
捕捉工程後は、必要に応じて、ホルムアルデヒド等によるがん細胞の組織の固定化や、Ｔｒｉｔｏｎ Ｘ（オクチルフェノールエトキシレート）等の非イオン性界面活性剤によるがん細胞の細胞膜の透過処理等を実施した後、不活性化工程に移行する。 After the capture step, if necessary, immobilization of cancer cell tissues with formaldehyde or the like, and permeation treatment of cancer cell cell membranes with a nonionic surfactant such as Triton X (octylphenol ethoxylate) are carried out. After that, the process proceeds to the inactivation step.
（不活性化工程）
この工程では、過酸化水素及びアジ化ナトリウムを用いて、前記血液中の内因性ペルオキシダーゼを不活性化する。これは、例えば、捕捉工程を経た親水性ポリマー層を、過酸化水素及びアジ化ナトリウムの混合液と接触させ、所定の時間（５〜３０分程度）反応させることで実施できる。反応は、遮光下、室温（１〜３０℃程度）で行うことが好ましい。
(Inactivation process)
In this step, hydrogen peroxide and sodium azide are used to inactivate the endogenous peroxidase in the blood. This can be carried out, for example, by bringing the hydrophilic polymer layer that has undergone the capture step into contact with a mixed solution of hydrogen peroxide and sodium azide and reacting them for a predetermined time (about 5 to 30 minutes). The reaction is preferably carried out at room temperature (about 1 to 30 ° C.) under shading.
親水性ポリマー層と上記混合液との接触方法は特に限定されないが、例えば、親水性ポリマー層への上記混合液の注入、滴下、噴霧等が挙げられる。また、親水性ポリマー層が形成された基材を上記混合液中に浸漬させてもよい。 The method of contacting the hydrophilic polymer layer with the mixed solution is not particularly limited, and examples thereof include injection, dropping, and spraying of the mixed solution into the hydrophilic polymer layer. Further, the base material on which the hydrophilic polymer layer is formed may be immersed in the above-mentioned mixed solution.
上記混合液は、例えば、蒸留水にアジ化ナトリウムを溶解させた後、過酸化水素水溶液と混合することで調製することができる。上記混合液において、過酸化水素の濃度やアジ化ナトリウムの濃度は特に限定されず、不活性化を達成できる範囲で適宜変更可能である。 The mixed solution can be prepared, for example, by dissolving sodium azide in distilled water and then mixing it with an aqueous hydrogen peroxide solution. In the above mixed solution, the concentration of hydrogen peroxide and the concentration of sodium azide are not particularly limited and can be appropriately changed within the range in which inactivation can be achieved.
不活性化工程後は、必要に応じて、洗浄や、血清ブロッキング等を実施した後、染色工程に移行する。 After the inactivation step, if necessary, washing, serum blocking, etc. are performed, and then the staining step is started.
（染色工程）
この工程では、がん細胞を免疫ペルオキシダーゼ法で染色する。免疫ペルオキシダーゼ法には、直接免疫ペルオキシダーゼ法（直接法）、間接免疫ペルオキシダーゼ法（間接法）の二種類がある。直接免疫ペルオキシダーゼ法は、ペルオキシダーゼで標識した一次抗体により、抗原抗体反応を一度だけ行うものであり、間接免疫ペルオキシダーゼ法は、標識していない一次抗体で抗原抗体反応を行った後、ペルオキシダーゼで標識した別の抗体で更に抗原抗体反応を行うものである。
(Dyeing process)
In this step, cancer cells are stained with an immunoperoxidase method. There are two types of immunoperoxidase methods: direct immunoperoxidase method (direct method) and indirect immunoperoxidase method (indirect method). In the direct immunoperoxidase method, an antigen-antibody reaction is carried out only once with a primary antibody labeled with peroxidase, and in the indirect immunoperoxidase method, an antigen-antibody reaction is carried out with an unlabeled primary antibody and then labeled with peroxidase. An antigen-antibody reaction is further carried out with another antibody.
間接免疫ペルオキシダーゼ法は、発色のシグナルを増幅させることができるが、抗原抗体反応を実施する都度洗浄が必要で、洗浄によって親水性ポリマー層に捕捉されたがん細胞が離脱してしまうおそれがあり、また、処理に時間がかかる。一方、直接免疫ペルオキシダーゼ法は、抗原抗体反応を一度しか行わないため、洗浄の回数が少なく、また、間接免疫ペルオキシダーゼ法と比較して、短時間で処理できる。そのため、上記方法では、直接免疫ペルオキシダーゼ法で染色することが好ましい。 The indirect immunoperoxidase method can amplify the color-developing signal, but it requires washing each time the antigen-antibody reaction is performed, and the washing may cause the cancer cells trapped in the hydrophilic polymer layer to escape. Also, the processing takes time. On the other hand, in the direct immunoperoxidase method, since the antigen-antibody reaction is performed only once, the number of washings is small, and the treatment can be performed in a shorter time than in the indirect immunoperoxidase method. Therefore, in the above method, it is preferable to stain directly by the immunoperoxidase method.
直接免疫ペルオキシダーゼ法は、例えば、不活性化工程を経た親水性ポリマー層に、ペルオキシダーゼが結合した抗体（一次抗体）を添加し、所定の時間（１５〜６０分程度）反応させてから、洗浄した後、発色基質を更に添加し、所定の時間（１〜１０分程度）反応させることで実施できる。これらの反応は、遮光下、室温（１〜３０℃程度）で行うことが好ましい。 In the direct immunoperoxidase method, for example, an antibody (primary antibody) to which peroxidase is bound is added to the hydrophilic polymer layer that has undergone the inactivation step, reacted for a predetermined time (about 15 to 60 minutes), and then washed. After that, the color-developing substrate is further added and reacted for a predetermined time (about 1 to 10 minutes). These reactions are preferably carried out at room temperature (about 1 to 30 ° C.) under light shielding.
抗体は、がん細胞と反応し、血球細胞と反応しないものであればよく、ＥｐＣＡＭ、ＣＫ等を使用できる。細胞膜の透過処理が不要となるという点からは、ＥｐＣＡＭが好ましい。
また、抗体に結合させるペルオキシダーゼは、西洋わさび由来のペルオキシダーゼ（ＨＲＰ）等を使用できる。
The antibody may be any antibody that reacts with cancer cells and does not react with blood cells, and EpCAM, CK, or the like can be used. EpCAM is preferable from the viewpoint that the permeation treatment of the cell membrane becomes unnecessary.
As the peroxidase to be bound to the antibody, horseradish-derived peroxidase (HRP) or the like can be used.
発色基質としては、ジアミノベンジジン（ＤＡＢ）、Ｃｏ−ＤＡＢ等を使用することができる。 As the color-developing substrate, diaminobenzidine (DAB), Co-DAB and the like can be used.
染色工程後、必要に応じて、洗浄、ヘマトキシリン等による対比染色（後染色、核染色）、脱水、封入等を実施することで、がん細胞が染色された試料が得られる。 After the staining step, if necessary, washing, counterstaining with hematoxylin or the like (post-staining, nuclear staining), dehydration, encapsulation, etc. are performed to obtain a sample in which cancer cells are stained.
得られた試料は、がん細胞やその背景組織の観察が容易であり、かつ長期保存が可能である。そのため、この試料を観察することで、がん細胞の確定診断を簡便に行うことができる。 The obtained sample can be easily observed for cancer cells and their background tissues, and can be stored for a long period of time. Therefore, by observing this sample, a definitive diagnosis of cancer cells can be easily performed.
実施例に基づいて、本発明を具体的に説明するが、本発明はこれらのみに限定されるものではない。 Although the present invention will be specifically described based on Examples, the present invention is not limited to these.
（検査器具の作製）
ＡＩＢＮ（アゾビスイソブチロニトリル）を用いて、２−メトキシエチルアクリレートを８０℃で６時間熱重合し、ポリ２−メトキシエチルアクリレートを作製した（分子量Ｍｎ：約１５０００、Ｍｗ：約５００００）。そして、得られたポリ２−メトキシエチルアクリレートの０．２５Ｗ／Ｖ％メタノール溶液を作製した。
チャンバー付スライドガラスに、作製したポリ２−メトキシエチルアクリレート溶液（０．２５Ｗ／Ｖ％）を注入し、乾燥させることで、親水性ポリマー層を作製した。ＴＥＭ観察で測定した親水性ポリマー層の膜厚は１３５ｎｍであった。
次いで、ポリ２−メトキシエチルアクリレートがコーティングされた部分（親水性ポリマー層）にフィブロネクチンを吸着させ、４０℃で１時間放置した後、ＰＢＳで洗浄することで、検査器具を作製した。
(Making inspection equipment)
Using AIBN (azobisisobutyronitrile), 2-methoxyethyl acrylate was thermally polymerized at 80 ° C. for 6 hours to prepare poly2-methoxyethyl acrylate (molecular weight Mn: about 15,000, Mw: about 50,000). Then, a 0.25 W / V% methanol solution of the obtained poly2-methoxyethyl acrylate was prepared.
A hydrophilic polymer layer was prepared by injecting the prepared poly2-methoxyethyl acrylate solution (0.25 W / V%) into a slide glass with a chamber and drying it. The film thickness of the hydrophilic polymer layer measured by TEM observation was 135 nm.
Next, fibronectin was adsorbed on the portion coated with poly2-methoxyethyl acrylate (hydrophilic polymer layer), left at 40 ° C. for 1 hour, and then washed with PBS to prepare an inspection instrument.
（実施例）
大腸がんの患者から採取した血液を、ＯｎｃｏＱｕｉｃｋを用いて遠心分離し、がん細胞を分離（濃縮）した。次いで、がん細胞を含む分画を検査器具に注入し、室温で一晩放置した後、ＰＢＳで洗浄することで、がん細胞を選択的に親水性ポリマー層に捕捉した。
(Example)
Blood collected from a patient with colorectal cancer was centrifuged using OncoQuick to separate (concentrate) cancer cells. The fraction containing the cancer cells was then injected into a test instrument, allowed to stand overnight at room temperature, and then washed with PBS to selectively capture the cancer cells in a hydrophilic polymer layer.
検査器具を乾燥させた後、１０Ｖ／Ｖ％ホルムアルデヒド水溶液に浸漬させ、室温で１０分間放置した。その後、ＰＢＳで洗浄した。 After the inspection instrument was dried, it was immersed in a 10 V / V% formaldehyde aqueous solution and left at room temperature for 10 minutes. Then, it was washed with PBS.
蒸留水２００ｍｌにアジ化ナトリウム２００ｍｇを溶解させた後、３０Ｖ／Ｖ％過酸化水素水溶液と混合した。得られた混合液中に検査器具を浸漬させ、遮光下、室温で１０分間放置して反応させることで、内因性ペルオキシダーゼを不活性化した。その後、ＰＢＳで洗浄した。 After dissolving 200 mg of sodium azide in 200 ml of distilled water, it was mixed with a 30 V / V% hydrogen peroxide aqueous solution. The test instrument was immersed in the obtained mixed solution and allowed to react at room temperature for 10 minutes under shading to inactivate the endogenous peroxidase. Then, it was washed with PBS.
Ｖｅｃｔｏｒ Ｌａｂｏｒａｔｏｒｉｅｓ社のＶＥＣＴＡＳＴＡＩＮ Ｅｌｉｔｅ Ｋｉｔを用いて作製した血清を検査器具に滴下し、血清ブロッキングを実施した。 Serum prepared using VECTASTAIN Elite Kit manufactured by Vector Laboratories was dropped onto a test instrument to perform serum blocking.
ＨＲＰ（標識）が結合したＥｐＣＡＭ（抗体）をＰＢＳで希釈し、２００μｌずつ検査器具に滴下した。室温下で３０分間滴下し続けた後、ＰＢＳで洗浄した。次いで、ＤＡＢ（発色基質）を検査器具に滴下し、４分間放置して反応させることで、がん細胞を染色した。その後、流水で２分間洗浄した。 EpCAM (antibody) to which HRP (label) was bound was diluted with PBS and 200 μl each was added dropwise to the test instrument. After continuing to drop for 30 minutes at room temperature, the cells were washed with PBS. Next, DAB (color-developing substrate) was added dropwise to the test instrument, and the cells were stained by allowing them to react for 4 minutes. Then, it was washed with running water for 2 minutes.
後染色（核染色）のため、ヘマトキシリンを検査器具に滴下し、１５秒放置した。その後、流水で３分間洗浄した。 For post-staining (nuclear staining), hematoxylin was added dropwise to the test instrument and left for 15 seconds. Then, it was washed with running water for 3 minutes.
水溶性の封入剤で封入することで、試料を作製した。 A sample was prepared by encapsulating with a water-soluble encapsulant.
得られた試料を顕微鏡で観察し、図１に示す顕微鏡画像を得た。図１中の矢印で示した部分が、茶褐色に染色されたがん細胞である。 The obtained sample was observed with a microscope to obtain a microscope image shown in FIG. The portion indicated by the arrow in FIG. 1 is a cancer cell stained in brown.
Claims (6)
過酸化水素及びアジ化ナトリウムを用いて、前記血液中の内因性ペルオキシダーゼを不活性化する工程と、
前記がん細胞を免疫ペルオキシダーゼ法によって染色する工程とを含むがん細胞の染色方法。 The process of capturing cancer cells in the blood in a hydrophilic polymer layer,
The step of inactivating the endogenous peroxidase in blood using hydrogen peroxide and sodium azide, and
A method for staining cancer cells, which comprises a step of staining the cancer cells by an immunoperoxidase method.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61258173A (en) * | 1985-05-10 | 1986-11-15 | Akira Taniuchi | Monoclonal antibody and production and application thereof |
| WO1990002202A1 (en) * | 1988-08-16 | 1990-03-08 | Cetus Corporation | Reduction of peroxidatic and catalatic interference with assays of peroxidatic activity |
| JPH0875749A (en) * | 1994-09-01 | 1996-03-22 | Teijin Ltd | Detecting of human cytomegalovirus antigen positive cell in blood of renal transplantation patient |
| JP2018050548A (en) * | 2016-09-29 | 2018-04-05 | 住友ゴム工業株式会社 | Cancer cell catching method |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61258173A (en) * | 1985-05-10 | 1986-11-15 | Akira Taniuchi | Monoclonal antibody and production and application thereof |
| WO1990002202A1 (en) * | 1988-08-16 | 1990-03-08 | Cetus Corporation | Reduction of peroxidatic and catalatic interference with assays of peroxidatic activity |
| JPH0875749A (en) * | 1994-09-01 | 1996-03-22 | Teijin Ltd | Detecting of human cytomegalovirus antigen positive cell in blood of renal transplantation patient |
| JP2018050548A (en) * | 2016-09-29 | 2018-04-05 | 住友ゴム工業株式会社 | Cancer cell catching method |
Non-Patent Citations (2)
| Title |
|---|
| J HISTOCHEM CYTOCHEM, 1989, VOL. 37, NO. 2, PP. 257-263, JPN6022035495, ISSN: 0004861508 * |
| J IMMUNOL METHODS, 1988, VOL. 111, NO. 1, PP. 101-107, JPN6022035494, ISSN: 0005038138 * |
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