JP2014160061A - Electric field cleaning method - Google Patents
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Abstract
【課題】１次・２次抗原抗体反応工程間やその前後の工程に存在する洗浄工程を迅速化及び自動化することができる電界洗浄方法、電界洗浄装置、それを用いた電界免疫組織染色方法、電界免疫組織染色装置を提案する。
【解決手段】本発明の電界洗浄方法は、下部電極１０上に配置した、ガラスプレート１上に形成した液滴２の反応場に、貫通穴３を有する板状電極又は環状電極４を上方から臨ませ、印加電界強度の主電圧としてプラス側に０．４〜２．０ｋＶ／ｍｍ、これにオフセット電界強度０．１５〜１．０ｋＶ／ｍｍを加え、更に周波数０．１〜８００Ｈｚの範囲の交流電圧を印加することで、プラス側に偏った繰り返し方形波を生成し、発生する電界による撹拌現象を用いながら電界洗浄を行う方法であって、板状電極又は環状電極４の貫通穴３から液滴反応場２へ排出管５を挿入して試薬溶液６を排出する工程と、貫通穴３から液滴反応場２へ注入管７を挿入して洗浄液８を注入する工程と、を行うことで、液滴に浮遊する非特異反応物質を排出することを特徴とする。
【選択図】図１An electric field cleaning method, an electric field cleaning apparatus, and an electric field immunohistochemical staining method using the same, capable of speeding up and automating a cleaning process existing between steps before and after the primary and secondary antigen-antibody reaction steps, An electroimmunohistological staining apparatus is proposed.
In the electric field cleaning method of the present invention, a plate-like electrode or annular electrode 4 having a through hole 3 is placed from above on a reaction field of a droplet 2 formed on a glass plate 1 disposed on a lower electrode 10. As a main voltage of the applied electric field strength, 0.4 to 2.0 kV / mm on the plus side, an offset electric field strength of 0.15 to 1.0 kV / mm is added to this, and a frequency in the range of 0.1 to 800 Hz is further added. By applying an AC voltage, a repetitive square wave biased to the plus side is generated, and electric field cleaning is performed using the stirring phenomenon caused by the generated electric field. From the through hole 3 of the plate electrode or the annular electrode 4 Performing a step of inserting the discharge tube 5 into the droplet reaction field 2 to discharge the reagent solution 6 and a step of inserting the injection tube 7 from the through hole 3 into the droplet reaction field 2 and injecting the cleaning liquid 8. To discharge non-specific reactants floating in the droplets And wherein the Rukoto.
[Selection] Figure 1
Description
本発明は、１次・２次抗原抗体反応工程間やその前後の工程に存在する洗浄工程を迅速化、自動化することができる電界洗浄方法、電界洗浄装置、それを用いた電界免疫組織染色方法、電界免疫組織染色装置に関するものである。 The present invention relates to an electric field cleaning method, an electric field cleaning apparatus, and an electric field immunohistochemical staining method using the same, which can accelerate and automate the cleaning process existing between the steps before and after the primary / secondary antigen-antibody reaction step. The present invention relates to an electric field immunohistochemical staining apparatus.
本発明者らは、物理的な手段の１つである高電圧交流電界によって発生するクーロン力によって液滴が吸引される現象と与える高電圧の極性変化によって生じる液滴の振動現象によって、液滴内に存在する微細物が撹拌する現象と高圧電界を抗体液滴に与えることで、抗体はより分散する現象を示すことから、これらに着目して鋭意検討し、１ｍＬ以下、特に５０から数１００μＬオーダーの微少量の液滴を非接触に撹拌に適用し抗原定着工程、抗原抗体反応工程においても、著しく反応時間を短縮できる非接触撹拌方法として電界撹拌技術を特許文献１に提案した。
そして、この電界撹拌技術は、抗原抗体反応を約１０倍促進する技術であり、従来２時間を要していた１次・２次抗原抗体反応を合わせて１０分程度に完了する技術である。
The inventors of the present invention have proposed that a droplet is attracted by a phenomenon in which a droplet is attracted by a Coulomb force generated by a high voltage AC electric field, which is one of physical means, and a droplet vibration phenomenon caused by a change in polarity of a high voltage applied. Since the antibody exhibits a more dispersed phenomenon by applying a high voltage electric field to the antibody droplets and stirring phenomenon of the minute objects present in the inside, attention is focused on these, and 1 mL or less, especially 50 to several hundred μL Patent Document 1 proposes an electric field agitation technique as a non-contact agitation method that can significantly reduce the reaction time in the antigen fixing step and the antigen-antibody reaction step by applying a small amount of droplets of the order to the agitation without contact.
This electric field agitation technique is a technique for accelerating the antigen-antibody reaction by about 10 times, and is a technique for completing the primary and secondary antigen-antibody reactions, which conventionally required 2 hours, in about 10 minutes.
現在、術中迅速病理診断は時間の制約上、５分以内に染色可能なＨＥ染色（ヘマトキシリン・エオジン染色）が用いられている。しかし、ＨＥ染色では小さながん遺残やリンパ節微小転移が見逃されていることが多い。このがん遺残やリンパ節転移を見逃さずに縮小手術を施行するには免疫染色が必要である。従来の免疫染色には２時間以上を要するため、術中病理診断には適さない。術中病理診断に用いるためには４０分以内に診断を完了する必要がある。現在、Sysmex社から約３０分で遺伝子増幅するＯＳＮＡ法によるリンパ節転移診断機器が販売されているが、同法による転移診断は形態学的情報が欠如するため、正確な転移診断とは言えず、信頼性を欠いている。また、数社から自動免疫染色装置が市販されているが、これらは大量の免疫染色を自動化するために開発されたもので、最短でも９０分を要するため術中病理診断には適用できなかった。即ち従来の免疫染色を実施するため、繁忙な医療スタッフが手動の装置操作を行うには限界があった。
この問題を解決するため、本発明の発明者等は、免疫染色を迅速に行うために電界を試料に非接触に与える新たな撹拌技術を開発し、免疫染色技術を開発し、試作機レベルにて診断に要する時間を１３６分から２０分に短縮する研究成果を上げている。これが前記特許文献１に提案した非接触撹拌方法である。
Currently, HE staining (hematoxylin and eosin staining) that can be stained within 5 minutes is used for intraoperative rapid pathological diagnosis due to time constraints. However, HE cancer staining often misses small cancer residues and lymph node micrometastases. Immunostaining is necessary to perform reduction surgery without missing this cancer residue or lymph node metastasis. Since conventional immunostaining requires 2 hours or more, it is not suitable for intraoperative pathological diagnosis. In order to use it for intraoperative pathological diagnosis, it is necessary to complete the diagnosis within 40 minutes. Currently, Sysmex sells lymph node metastasis diagnostic equipment using the OSNA method, which amplifies the gene in about 30 minutes. However, metastasis diagnosis by this method is not an accurate metastasis diagnosis because of lack of morphological information. Lack of credibility. In addition, automatic immunostaining devices are commercially available from several companies, but these were developed to automate a large amount of immunostaining, and required 90 minutes at the shortest, and thus could not be applied to intraoperative pathological diagnosis. In other words, since conventional immunostaining is performed, there is a limit for a busy medical staff to perform manual device operation.
In order to solve this problem, the inventors of the present invention have developed a new stirring technique that applies an electric field to a sample in a non-contact manner in order to perform immunostaining quickly, developed an immunostaining technique, Research results have been shortened from 136 minutes to 20 minutes. This is the non-contact stirring method proposed in Patent Document 1.
しかしながら、免疫組織診断をより迅速化して広く普及させるためには、診断に関わる試料作製処理技術をより簡便に、しかも高精度に行う必要がある。前記特許文献１に提案した非接触撹拌方法（電界撹拌技術）は、１次・２次抗原抗体反応そのものを高速化できる技術であり、１次・２次抗原抗体反応工程間に存在する洗浄工程によって、電界撹拌装置より試料が搭載されるガラス等で構成されるプレートを一度装置外へ取り出して洗浄を行う必要があるため、この洗浄操作に人手を要してしまい、煩雑な病理現場では問題となっている。したがって、この洗浄工程を迅速化及び自動化する技術が希求されている。 However, in order to make immunohistological diagnosis faster and widely spread, it is necessary to perform a sample preparation processing technique related to diagnosis more easily and with high accuracy. The non-contact stirring method (electric field stirring technique) proposed in Patent Document 1 is a technique capable of speeding up the primary / secondary antigen-antibody reaction itself, and is a washing step existing between the primary / secondary antigen-antibody reaction steps. Therefore, it is necessary to take out a plate made of glass or the like on which the sample is mounted from the electric field stirring device and wash it once. It has become. Therefore, there is a need for a technique for speeding up and automating this cleaning process.
そこで、本発明は、１次・２次抗原抗体反応工程間やその前後の工程に存在する洗浄工程を迅速化、自動化することができる電界洗浄方法、電界洗浄装置、それを用いた電界免疫組織染色方法、電界免疫組織染色装置を提案することを目的とするものである。 Therefore, the present invention provides an electric field cleaning method, an electric field cleaning apparatus, and an electric field immune tissue using the same, which can speed up and automate the cleaning process existing between the steps before and after the primary / secondary antigen-antibody reaction step. An object of the present invention is to propose a staining method and an electroimmunohistological staining apparatus.
本発明は、上記に鑑み提案されたもので、抗原と抗体とを反応させるために基板上に形成した反応場と、前記反応場を挟むように上下に形成された上側電極と下側電極とを備え、前記反応場で抗原に１次抗体を含む液滴を添加して抗原と１次抗体とを反応させる１次抗原抗体反応工程（機構）と、前記１次抗原抗体反応工程（機構）の後に２次抗体を含む液滴を添加して２次抗体をさらに反応させる２次抗原抗体反応工程（機構）と、前記上側電極と下側電極との間に所定の電界を与えることにより前記反応場に形成された液滴を撹拌する工程（機構）と、前記１次抗原抗体反応工程と２次抗原抗体反応工程との後に、それぞれ未反応抗体を除去する未反応抗体洗浄工程（機構）と、を有することを特徴とする電界洗浄方法又は装置に関するものである。 The present invention has been proposed in view of the above, a reaction field formed on a substrate for reacting an antigen and an antibody, and an upper electrode and a lower electrode formed vertically so as to sandwich the reaction field. A primary antigen-antibody reaction step (mechanism) in which a droplet containing a primary antibody is added to the antigen in the reaction field to react the antigen with the primary antibody, and the primary antigen-antibody reaction step (mechanism) And a secondary antigen-antibody reaction step (mechanism) for further reacting the secondary antibody by adding a droplet containing the secondary antibody, and by applying a predetermined electric field between the upper electrode and the lower electrode, A step (mechanism) of stirring the droplets formed in the reaction field, and an unreacted antibody washing step (mechanism) for removing unreacted antibodies after the primary antigen-antibody reaction step and the secondary antigen-antibody reaction step, respectively. And an electric field cleaning method or apparatus characterized by comprising: Than is.
また、本発明は、前記電界洗浄方法又は装置において、前記所定の電界は、周波数０．１〜８００Ｈｚの方形波を発生させ、電界強度をプラス側に０．４〜２．０ｋＶ／ｍｍとし、オフセット電界強度が０．１５〜１．０ｋＶ／ｍｍであることを特徴とする電界洗浄方法又は装置をも提案する。
より望ましくは、周波数０．１〜３００Ｈｚの方形波を発生させ、電界強度をプラス側に０．４〜１．５ｋＶ／ｍｍとし、オフセット電界強度が０．１５〜０．７ｋＶ／ｍｍである。
Further, in the electric field cleaning method or apparatus according to the present invention, the predetermined electric field generates a square wave having a frequency of 0.1 to 800 Hz, and the electric field strength is set to 0.4 to 2.0 kV / mm on the plus side. An electric field cleaning method or apparatus characterized by an offset electric field strength of 0.15 to 1.0 kV / mm is also proposed.
More desirably, a square wave having a frequency of 0.1 to 300 Hz is generated, the electric field strength is 0.4 to 1.5 kV / mm on the plus side, and the offset electric field strength is 0.15 to 0.7 kV / mm.
さらに、本発明は、前記上側電極は貫通穴を備える板状電極又は環状電極であることを特徴とする電界洗浄方法又は装置をも提案する。 Furthermore, the present invention also proposes an electric field cleaning method or apparatus, wherein the upper electrode is a plate electrode or an annular electrode having a through hole.
また、本発明は、前記電界洗浄方法又は装置において、前記未反応抗体洗浄工程（機構）は、前記貫通穴から前記反応場へ排出管を挿入して未反応抗体を排出する工程（機構）と、前記貫通穴から前記反応場へ注入管を挿入して洗浄液を注入する工程（機構）と、をさらに備えることを特徴とする電界洗浄方法又は装置をも提案する。
さらに、基板上に複数の液滴の反応場を形成し、各反応場に対してそれぞれ液滴反応場の溶液を排出する排出管，洗浄液を注入する注入管を挿入可能な貫通穴を板状電極又は環状電極の上方から反応場となる液滴に臨ませるようにしてもよい。
Further, the present invention provides the electric field cleaning method or apparatus, wherein the unreacted antibody cleaning step (mechanism) includes a step (mechanism) for discharging the unreacted antibody by inserting a discharge pipe from the through hole into the reaction field. The present invention also proposes an electric field cleaning method or apparatus, further comprising a step (mechanism) of inserting an injection tube into the reaction field from the through hole and injecting a cleaning liquid.
In addition, a reaction field for a plurality of droplets is formed on the substrate, and a through hole into which a discharge pipe for discharging the solution in the droplet reaction field and an injection pipe for injecting cleaning liquid can be inserted into each reaction field. You may make it face the droplet used as a reaction field from the upper direction of an electrode or an annular electrode.
また、本発明は、前記電界洗浄方法又は装置において、基板上に耐アセトン性を有し、ポリビニル系、ポリ塩化ビニル系、シリコーン系又はフッ素系から１つ選ばれる樹脂からなり、電界撹拌の良好な撹拌動作を生む反応場液滴形状並びに領域を確保し、領域を形成するはっ水リングやはっ水枠を載置するか、はっ水処理剤塗布にて処理することにより液滴反応場を形成することを特徴とする電界洗浄方法又は装置をも提案する。
はっ水リングの内径範囲５〜２５ｍｍ，また、長方形のはっ水枠を採用することによって、滴下する液滴は、５〜１０００μＬである。
Further, the present invention provides the electric field cleaning method or apparatus, wherein the substrate has an acetone resistance and is made of a resin selected from polyvinyl, polyvinyl chloride, silicone, or fluorine, and has good electric field stirring. Reaction field that produces agitation action Droplet reaction by securing the shape and area of the droplet, placing a water-repellent ring or water-repellent frame to form the region, or processing with water-repellent treatment agent application An electric field cleaning method or apparatus characterized by forming a field is also proposed.
By employing a water-repellent ring with an inner diameter range of 5 to 25 mm and a rectangular water-repellent frame, a droplet to be dropped is 5 to 1000 μL.
さらに、本発明は、前記電界洗浄方法又は装置を用いて、１次抗原抗体反応工程（機構）前に抗原に電界を与えて賦活化させ、反応を加速化させる電界賦活化工程（機構）を行うことを特徴とする電界免疫組織染色方法又は装置をも提案するものである。 Furthermore, the present invention provides an electric field activation step (mechanism) for accelerating the reaction by applying an electric field to the antigen prior to the primary antigen-antibody reaction step (mechanism) using the electric field cleaning method or apparatus. The present invention also proposes an electroimmunohistological staining method or apparatus characterized in that it is performed.
なお、本発明の前記未反応抗体洗浄工程（機構）に関して詳細に説明すると、貫通穴を有する板状電極又は環状電極を上部電極とし、板状電極を下部電極とする電極間において、下部電極上に配置したガラスプレート等の基板上に、液滴を形成させて反応場とし、液滴と上部電極とは非接触状態を維持した状態で、これらの上下電極間に電界を与え、液滴は上部電極方向に振動し、撹拌を生じさせ、与える電界条件は、周波数０．１〜８００（望ましくは０．１〜３００）Ｈｚの繰り返し方形波を発生し昇圧させ、電界強度はプラス側に０．４〜２．０（望ましくは０．４〜１．５）ｋＶ／ｍｍ、これにオフセット電界強度０．１５〜１．０（望ましくは０．１５〜０．７）ｋＶ／ｍｍを加えてプラス側に偏らせて与える、電界条件によって液滴は非接触にそして活発に振動し、撹拌現象を生じ、この撹拌現象中に、前記貫通穴を有する板状電極又は環状電極の貫通穴から液滴反応場へ排出管を挿入して溶液を排出する工程（機構）と、貫通穴から液滴反応場へ注入管を挿入して洗浄液を注入する工程（機構）と、を行うことで、液滴に浮遊する未反応物等の非特異反応物質を排出することを特徴とする、となる。 The unreacted antibody washing step (mechanism) of the present invention will be described in detail. A plate-like or annular electrode having a through-hole is used as the upper electrode, and the plate-like electrode is used as the lower electrode. A droplet is formed on a substrate such as a glass plate as a reaction field, and an electric field is applied between the upper and lower electrodes while the droplet and the upper electrode are kept in a non-contact state. The electric field condition that oscillates in the direction of the upper electrode and causes agitation to be applied is that a repetitive square wave having a frequency of 0.1 to 800 (preferably 0.1 to 300) Hz is generated and boosted, and the electric field strength is 0 on the plus side. .4 to 2.0 (preferably 0.4 to 1.5) kV / mm, and offset electric field strength of 0.15 to 1.0 (preferably 0.15 to 0.7) kV / mm Depending on the electric field condition, biased toward the positive side The droplet vibrates in a non-contact and active manner to generate a stirring phenomenon. During this stirring phenomenon, a discharge pipe is inserted into the droplet reaction field from the plate-like electrode having the through-hole or the through-hole of the annular electrode, and the solution is poured. Non-specific reactions such as unreacted substances floating in the droplets by performing the discharge step (mechanism) and the step (mechanism) for injecting the cleaning liquid by inserting the injection tube from the through hole into the droplet reaction field. It is characterized by discharging substances.
本発明の電界洗浄方法及び電界洗浄装置は、図１に示すとおりであるが、高電圧交流電界によるクーロン力を１ｍＬ以下、特に５０〜６００μLオーダーの微量の液滴に与えることにより、液滴の内部にて撹拌子等を用いずに撹拌することができる電界撹拌技術を、１次・２次抗原抗体反応工程間やその前後の工程に存在する洗浄に利用するものであって、ガラスプレート等の基板上に複数の液滴の反応場を形成しても、それぞれの反応場に対して貫通穴を有する板状電極又は環状電極を臨ませればよく、貫通穴から液滴反応場へ臨ませた排出管と注入管とを的確に液滴の反応場に配置させ、所定の電界撹拌工程完了後に引き続き電界撹拌を与えながら適宜に溶液を排出したり、洗浄液を注入したりすることにより、洗浄工程の迅速化及び自動化を達成することができる方法である。 The electric field cleaning method and electric field cleaning apparatus of the present invention are as shown in FIG. 1, but by applying a Coulomb force by a high-voltage AC electric field to 1 mL or less, particularly a small amount of droplets on the order of 50 to 600 μL, Electric field agitation technology that can be agitated without using a stirrer inside is used for washing existing between the primary and secondary antigen-antibody reaction steps and the steps before and after that, such as glass plates Even if a reaction field for a plurality of droplets is formed on the substrate, a plate-like electrode or an annular electrode having a through hole may face each reaction field. By properly placing the discharge tube and the injection tube in the reaction field of the liquid droplets, the solution is appropriately discharged while injecting the electric field stirring after the completion of the predetermined electric field stirring step, or the cleaning liquid is injected, Fast and automatic cleaning process It is a method that can be achieved.
また、ガラスプレート等の基板上に耐アセトン性を有し、ポリビニル系、ポリ塩化ビニル系、シリコーン系又はフッ素系から１つ選ばれる樹脂からなり、電界撹拌の良好な撹拌動作を生む反応場液滴形状並びに領域を確保する領域を形成するはっ水リングやはっ水枠を載置するか、はっ水処理剤塗布にて処理することにより液滴反応場を形成することにより、ばらつきの少ない一定の量の液滴を滴下可能にすることによって、液滴反応場の高さばらつきが抑制される。すなわち電極間と液滴頭頂部との距離長ばらつきが抑制され、これにより、電界撹拌性のばらつきが抑制される。この電界撹拌を継続しながら、液滴を排水し、さらに新規に注入されたばらつきの少ない一定の量の洗浄液を電界撹拌後に電界撹拌中に排出することで、不要な抗体を除去して洗浄不良による非特異発色を抑制し易くし、すぐれた洗浄性を得る極めて簡易な液滴の反応場を形成することができる。 In addition, a reaction field solution that has acetone resistance on a substrate such as a glass plate and is made of a resin selected from polyvinyl, polyvinyl chloride, silicone, or fluorine, and produces a good stirring action of electric field stirring. By placing a water-repellent ring or water-repellent frame that forms a region that secures the droplet shape and region, or by forming a droplet reaction field by processing with a water-repellent treatment agent, variation can be reduced. By making it possible to drop a small and constant amount of droplets, variations in the height of the droplet reaction field are suppressed. That is, the variation in distance length between the electrodes and the top of the droplet is suppressed, thereby suppressing the variation in electric field agitation. While continuing this electric field agitation, the droplets are drained, and a certain amount of newly injected liquid with little variation is discharged after the electric field agitation to remove unwanted antibodies and poor cleaning. Therefore, it is possible to easily suppress non-specific coloration due to, and to form a very simple droplet reaction field that provides excellent detergency.
さらに、前記電界洗浄を電界免疫組織染色に適用することにより、電界による１次・２次抗原抗体反応工程間やその前後の工程に存在する洗浄工程を迅速化、自動化することができる電界洗浄方法を実施することができる。また、凍結切片に対する術中迅速免疫染色診断として、パラフィン切片を用いた免疫染色確定診にも適用可能である。この時、抗体試薬を希釈し用いることができる。さらに、核酸におけるハイブリダイゼーション工程にも適用可能であり、加えて抗原抗体反応においても適用可能である。なお、１次抗原抗体反応工程前に抗原に電界を与えて賦活化させ、反応を加速化させる電界賦活化工程を行うことにより、脳腫瘍のような反応性の低い抗原に用いると有効である。 Furthermore, by applying the electric field cleaning to the electroimmunohistological staining, the electric field cleaning method can accelerate and automate the cleaning process existing between the primary and secondary antigen-antibody reaction processes by the electric field and the processes before and after the reaction process. Can be implemented. Moreover, it can be applied to immunostaining confirmed diagnosis using a paraffin section as a rapid immunostaining diagnosis for a frozen section during an operation. At this time, the antibody reagent can be diluted and used. Furthermore, the present invention can be applied to a hybridization step using nucleic acid, and can also be applied to an antigen-antibody reaction. In addition, it is effective to use for the antigen with low reactivity like a brain tumor by performing the electric field activation process which gives an electric field to an antigen and activates it before a primary antigen antibody reaction process, and accelerates | stimulates reaction.
前記特許文献１に提案した電界撹拌の技術原理は図２に示すとおりであるが、これを、洗浄工程に適用しようとするものが本発明であり、電界を用いた撹拌現象を用いながら電界洗浄を行う方法（装置）であって、図３及び図４に示すとおりである。より具体的には、洗浄工程に電界洗浄工程を開発・導入し、電界撹拌装置に入った試料が洗浄工程のために装置より出入りする操作を無くし、医療スタッフの負担軽減を図る装置開発を行うものである。 The technical principle of electric field agitation proposed in Patent Document 1 is as shown in FIG. 2, but this is the present invention to be applied to the washing process, and electric field washing is performed using the agitation phenomenon using an electric field. The method (apparatus) for performing is as shown in FIG. 3 and FIG. More specifically, an electric field cleaning process is developed and introduced into the cleaning process, and an apparatus is developed to reduce the burden on medical staff by eliminating the operation of the sample entering the electric field stirring apparatus entering and exiting the cleaning process. Is.
本発明は、高電圧交流電界によるクーロン力を１ｍＬ以下、特にμＬオーダーの微少量液滴に与えることにより、液滴内部を非接触に撹拌することができる電界撹拌を洗浄に利用するものである。具体的には湿度６０±２０％の環境下で、印加電界強度の主電圧のとしてプラス側に０．４〜２．０（より望ましくは０．４〜１．５）ｋＶ／ｍｍ、これにオフセット電界強度０．１５〜１．０（より望ましくは０．１５〜０．７）ｋＶ／ｍｍを加えることで、プラス側に偏った繰り返し方形波を生成し、周波数０．１〜８００（より望ましくは０．１〜３００）Ｈｚの範囲で液滴が活発に振動する周波数を与えることで発生する電界の波に変化をもたせた（＝バースト波形を構成させた）高電圧交流電界を１ｍＬ以下、特にμＬオーダーの微少量液滴に非接触に与えることで発生する電界を用いた撹拌現象を用いながら電界洗浄を行う方法（装置）である。 The present invention utilizes electric field agitation that can stir the inside of a droplet in a non-contact manner by applying a Coulomb force generated by a high-voltage AC electric field to a small amount of droplet of 1 mL or less, particularly μL order. . Specifically, in an environment of 60 ± 20% humidity, the main voltage of the applied electric field strength is 0.4 to 2.0 (more preferably 0.4 to 1.5) kV / mm on the plus side, By adding an offset electric field strength of 0.15 to 1.0 (more preferably 0.15 to 0.7) kV / mm, a repetitive square wave biased to the plus side is generated, and a frequency of 0.1 to 800 (more Desirably, a high-voltage AC electric field generated by changing the wave of the electric field generated by giving a frequency at which the droplet vibrates actively in the range of 0.1 to 300 Hz (= a burst waveform) is 1 mL or less. In particular, this is a method (apparatus) for performing electric field cleaning while using a stirring phenomenon using an electric field generated by non-contacting a very small droplet on the order of μL.
前記電界撹拌において、印加電界強度（ｋＶ／ｍｍ）は、プラス側に２．０ｋＶ／ｍｍより強いと放電する可能性があり、０．４ｋＶ／ｍｍ未満より低いと撹拌が生じない。
また、オフセット電圧は、１．０ｋＶ／ｍｍより強いと放電する可能性があり、０．１５ｋＶ／ｍｍより低いと撹拌が生じない。
In the electric field stirring, when the applied electric field strength (kV / mm) is higher than 2.0 kV / mm on the plus side, there is a possibility of discharging, and when it is lower than 0.4 kV / mm, stirring does not occur.
Further, when the offset voltage is higher than 1.0 kV / mm, there is a possibility of discharging, and when it is lower than 0.15 kV / mm, stirring does not occur.
さらに、撹拌作用に影響を与える電界強度・周波数は液滴量に依存するが、この電界強度と液量、周波数と液量の関係を以下に説明する。
印加電界強度（ｋＶ／ｍｍ）は、試料の液量により最適化される。
炭酸カルシウム（観察のために粒径５０ｎｍ 濃度０．０４％）を入れた滅菌超純水の液滴をプラスチック製基板に滴下し、平行平板電極間において電界下の動きを観察したところ、０．６μＬでは０．７５ｋＶ／ｍｍ（電極間距離４ｍｍ）、１〜２μＬでは０．６３ｋＶ／ｍｍ（電極間距離４ｍｍ）、５〜１５μＬでは０．４ｋＶ／ｍｍ（電極間距離４ｍｍ）、１００〜１０００μＬでは０．４２ｋＶ／ｍｍ（電極間距離７ｍｍ）で液滴内部の炭酸カルシウム粒子が活発に動く様子を確認した。この結果を図５のグラフにして示した。
炭酸カルシウム（観察のために粒径５０ｎｍ 濃度０．０．４％）を入れた滅菌超純水１０μＬの液滴をプラスチック製基板に滴下し、電界下の動きを観察したところ、液量０．６μＬでは３８０〜４２０Ｈｚ、１μＬでは２６０〜３１０Ｈｚ、２μＬでは１９５〜２２０Ｈｚ、５μＬでは１２５〜１４０Ｈｚ、１０μＬでは８５〜９５Ｈｚ、１５μＬでは７０〜８５Ｈｚ、１００μＬでは３０Ｈｚ、５００μＬでは１３〜１５Ｈｚ、１０００μＬでは１０Ｈｚで、液滴内部の炭酸カルシウムが活発に動く様子を確認した。この結果を図６のグラフにして示した。
Furthermore, although the electric field strength and frequency that affect the stirring action depend on the amount of liquid droplets, the relationship between the electric field strength and the liquid amount, and the frequency and the liquid amount will be described below.
The applied electric field strength (kV / mm) is optimized depending on the liquid volume of the sample.
When a drop of sterilized ultrapure water containing calcium carbonate (for observation, particle size 50 nm, concentration 0.04%) was dropped on a plastic substrate, and the movement under an electric field was observed between parallel plate electrodes, 0. 6 μL is 0.75 kV / mm (distance between electrodes 4 mm), 1-2 μL is 0.63 kV / mm (distance between electrodes 4 mm), 5 to 15 μL is 0.4 kV / mm (distance between electrodes 4 mm), and 100 to 1000 μL. It was confirmed that the calcium carbonate particles inside the droplets actively moved at 0.42 kV / mm (distance between electrodes: 7 mm). The results are shown in the graph of FIG.
A drop of 10 μL of sterilized ultrapure water containing calcium carbonate (particle size: 50 nm, concentration: 0.04% for observation) was dropped on a plastic substrate, and the movement under an electric field was observed. 6 μL 380-420 Hz, 1 μL 260-310 Hz, 2 μL 195-220 Hz, 5 μL 125-140 Hz, 10 μL 85-95 Hz, 15 μL 70-85 Hz, 100 μL 30 Hz, 500 μL 13-15 Hz, 1000 μL 10 Hz It was confirmed that calcium carbonate inside the droplets moved actively. The results are shown in the graph of FIG.
本発明の電界洗浄では、ガラスプレート等の基板１上に形成した液滴反応場２に、貫通穴３を有する板状電極又は環状電極４を上方から臨ませ、貫通穴３から液滴反応場２へ排出管５を挿入して試薬溶液６を排出する工程と、貫通穴３から液滴反応場２へ注入管７を挿入して洗浄液８を注入する工程と、を行うと共に、必要に応じて上記工程を繰り返して行うようにしたものである。 In the electric field cleaning according to the present invention, a plate-like electrode or an annular electrode 4 having a through hole 3 faces a droplet reaction field 2 formed on a substrate 1 such as a glass plate from above, and a droplet reaction field from the through hole 3. 2 and the step of discharging the reagent solution 6 by inserting the discharge tube 5 into the step 2, and the step of inserting the injection tube 7 from the through-hole 3 to the droplet reaction field 2 and injecting the cleaning liquid 8 as necessary. The above process is repeated.
既に概略的には説明したが、免疫組織診断をより迅速化して広く普及させるためには、診断に関わる試料作製処理技術をより簡便に、しかも高い精度で行う必要があった。本発明は、前記電界撹拌の技術を１次・２次抗原抗体反応自体に限らず、煩雑な病理診断における洗浄工程にも利用しようとするものである。
例えば１次・２次抗原抗体反応を行わせるための前記電界撹拌では、平板状の電極を用いればよいが、この洗浄方法（装置）においては、貫通穴を有する板状電極又は環状電極を用いる。前者の電極は、盤状電極の中央に貫通穴を形成したものであり、貫通穴としては貫通スリット穴を含むものであり、後者の電極は、電極自体を環状（ドーナツ状又はリング状）に成形したものである。
As has already been schematically explained, in order to make immunohistological diagnosis faster and widely spread, it is necessary to perform a sample preparation processing technique related to diagnosis more simply and with high accuracy. The present invention intends to utilize the electric field agitation technique not only for the primary / secondary antigen-antibody reaction itself but also for the washing step in complicated pathological diagnosis.
For example, in the electric field stirring for causing the primary / secondary antigen-antibody reaction, a plate-like electrode may be used. In this cleaning method (apparatus), a plate-like electrode having a through hole or an annular electrode is used. . The former electrode has a through-hole formed at the center of the plate-like electrode, and the through-hole includes a through-slit hole. The latter electrode has an annular shape (doughnut shape or ring shape). Molded.
この電界洗浄における貫通穴を有する板状電極又は環状電極は、導体であればよく、アルミ又はステンレス等でもよく、ＩＴＯ（酸化インジウムスズ，透明電極）でもよい。また、その配置は、液滴からの溶液の排出時に液滴の頂部と電極間距離を一定にすることで撹拌性の維持向上を図るようにしてもよい。 The plate-like electrode or annular electrode having through holes in this electric field cleaning may be a conductor, and may be aluminum or stainless steel, or may be ITO (indium tin oxide, transparent electrode). Further, the arrangement may be such that the stirring property is maintained and improved by making the distance between the top of the droplet and the electrode constant when the solution is discharged from the droplet.
ガラスプレート表面に形成する液滴の反応場は、ガラスプレート表面を凸状に形成したものでも、平坦状の表面にはっ水リングやはっ水枠を載置（定着）したものでもよい。前者に比べて後者のはっ水リングやはっ水枠を用いた形成方法の方が極めて容易に作成でき、液滴の容量調整も容易に行うことができるという利点がある。さらに、はっ水機能を付与するはっ水ペンでガラスプレートに円や枠を描画して（＝はっ水処理剤塗布にて処理する）液滴を滴下する領域を形成するようにしてもよい。 The reaction field of droplets formed on the surface of the glass plate may be one in which the surface of the glass plate is formed in a convex shape, or one in which a water repellent ring or a water repellent frame is placed (fixed) on a flat surface. Compared to the former, the latter forming method using a water-repellent ring or a water-repellent frame can be produced very easily, and there is an advantage that the volume of a droplet can be easily adjusted. Furthermore, by drawing a circle or a frame on the glass plate with a water-repellent pen that imparts a water-repellent function (= processing by applying a water-repellent treatment agent), an area for dropping droplets may be formed. Good.
前記はっ水リング等は、耐アセトン性を有し、電界印加環境に影響を与えない樹脂から形成されるものである。したがって、ガラスプレート上のはっ水リング等の内側部に滴下された液滴は、良好なドーム形状を形成し、その最大高さ（頂点位置）が一定となるため、液滴の底面の径寸法のばらつきを抑制することができ、液滴の頂点位置と電極との間の距離のばらつきも抑制することができる。
ここで、はっ水リング等は、その材質としての電界印加環境に影響を与えない樹脂の例示として、ポリビニル系、ポリ塩化ビニル系、シリコーン系又はフッ素系から１つ選ばれる樹脂を挙げることができる。また、はっ水リングのリング内側部に滴下された液滴に対してリング幅が０．５〜５ｍｍ、リング内径が５〜２５ｍｍであることを挙げることができる。また、長方形のはっ水枠を採用することが可能なため、その滴下する液滴は、５〜１０００μＬとなる。
The water-repellent ring or the like is made of a resin that has acetone resistance and does not affect the electric field application environment. Therefore, the droplet dropped on the inner side of the water-repellent ring on the glass plate forms a good dome shape, and its maximum height (vertical position) is constant. Variations in size can be suppressed, and variations in the distance between the vertex position of the droplet and the electrode can also be suppressed.
Here, the water-repellent ring or the like includes a resin selected from polyvinyl, polyvinyl chloride, silicone, or fluorine as an example of a resin that does not affect the electric field application environment as its material. it can. Further, it can be mentioned that the ring width is 0.5 to 5 mm and the inner diameter of the ring is 5 to 25 mm with respect to the droplet dropped on the inner side of the ring of the water-repellent ring. Moreover, since it is possible to employ a rectangular water-repellent frame, the dropped liquid droplet is 5 to 1000 μL.
また、反応場に臨ませる注入管７及び排出管５の本数及び配置は、特に限定するものではないが、図３に示すようにそれぞれ１本ずつ設けてもよいが、図４に示すように注入管７は中央部に１本設け、排出管５は１本、又は注入管７の周囲に複数本設けることが好ましい。 Further, the number and arrangement of the injection pipes 7 and the discharge pipes 5 facing the reaction field are not particularly limited, but one each may be provided as shown in FIG. 3, but as shown in FIG. It is preferable to provide one injection tube 7 at the center and one discharge tube 5 or a plurality of discharge tubes 5 around the injection tube 7.
洗浄液としては、リン酸緩衝生理食塩水（ＰＢＳ）を用いることができる。このＰＢＳとしては、種々な組成のものがあり、カリウムを含む一例をあげると、ＮａＣｌ，ＫＣｌ、ＮａＨＰＯ4，ＫＨ2ＰＯ4を配合してなるものがあるが、カルシウムやマグネシウムを含む組成もある。 As the washing liquid, phosphate buffered saline (PBS) can be used. This PBS has various compositions, and examples of potassium include NaCl, KCl, NaHPO 4 , KH 2 PO 4 and other compositions containing calcium and magnesium. .
次に、本発明の電界洗浄を用いた迅速免疫染色法について図７に示す比較対照表を用いて説明する。
本発明の電界免疫組織染色技術の工程は、「５」と「７」が電界撹拌装置を用いて行う処理である。そのため、「６」のＰＢＳ洗浄の時に一度、試料が搭載されているスライドガラスを装置の外に出す必要がある。狙いは「５」〜「７」まで装置内で完了することである。
なお、工程「５」電界撹拌では上部に配置する電極形状は、平板電極、平板円盤電極、環状電極を採用し、電極形状が環状でも電界撹拌が成し得るかを確認した。これにより電界撹拌並びに洗浄にも同一の電極を用いることができ、装置を簡素化できることが確認された。
工程「６」〈電界洗浄技術〉電界撹拌印加中（０．４〜１．５ｋＶ／ｍｍ、０．1〜３００Ｈｚ，２５０秒）に、環状電極の貫通穴より排出管をガラスプレート上の液滴に差し込み、溶液をポンプを用いて排出し、洗浄工程を完了する場合、ここで工程「７」の処理に移行してもよい。染色発色品位を高める場合には、貫通穴よりＰＢＳ洗浄液を注入管にて注入した後、電界撹拌（０．４〜１．５ｋＶ／ｍｍ、０．1〜３００Ｈｚ，３０〜６０秒）し、その後、貫通穴から排出管をガラスプレート上の液滴に差し込み、溶液をポンプを用いて排出し、工程「７」へ移行する。（また、排出管１本、注入管２本を貫通穴から差し込み、排出と注入を同時に行うことによって、洗浄工程の時間を短縮化するようにしてもよい。）上記工程を１〜３回行うことで、非特異発色が発生しない免疫染色が得られる。即ち電界洗浄技術を導入することによって、免疫染色技術の迅速化及び自動化が可能になる。
なお、この電界洗浄技術は、免疫組織染色に限らず、核酸におけるハイブリダイゼーション工程にも適用可能であり、加えて抗原抗体反応においても適用可能である。
Next, the rapid immunostaining method using the electric field washing according to the present invention will be described with reference to a comparison table shown in FIG.
The process of the electroimmunohistological staining technique of the present invention is a process in which “5” and “7” are performed using an electric field stirring device. Therefore, it is necessary to take the slide glass on which the sample is mounted once out of the apparatus at the time of “6” PBS washing. The aim is to complete “5” to “7” within the device.
In addition, in the step “5” electric field stirring, a flat electrode, a flat disk electrode, and an annular electrode were adopted as the shape of the electrode disposed on the upper portion, and it was confirmed whether electric field stirring could be achieved even if the electrode shape was annular. Thus, it was confirmed that the same electrode can be used for electric field stirring and cleaning, and the apparatus can be simplified.
Process “6” <Electric field cleaning technique> During application of electric field stirring (0.4 to 1.5 kV / mm, 0.1 to 300 Hz, 250 seconds), the discharge tube is dropped on the glass plate through the through hole of the annular electrode. In the case where the solution is discharged using a pump and the washing process is completed, the process may be shifted to the process “7”. In order to improve the color quality of dyeing, a PBS cleaning solution is injected from the through hole with an injection tube, and then electric field stirring (0.4 to 1.5 kV / mm, 0.1 to 300 Hz, 30 to 60 seconds) is performed. Then, the discharge tube is inserted into the droplet on the glass plate from the through hole, and the solution is discharged using a pump, and the process proceeds to step “7”. (Alternatively, the time of the cleaning process may be shortened by inserting one discharge pipe and two injection pipes from the through hole and simultaneously performing the discharge and injection.) The above process is performed 1 to 3 times. Thus, immunostaining that does not cause non-specific color development is obtained. That is, by introducing the electric field cleaning technique, the immunostaining technique can be accelerated and automated.
This electric field washing technique is applicable not only to immunohistochemical staining but also to a nucleic acid hybridization step, and in addition, to an antigen-antibody reaction.
なお、本発明の電界洗浄方法を用いて、自動電界免疫組織染色方法を実施するには、以下に説明する電界賦活化工程、電界賦活化機構を実施することが望ましい。特にこの電界賦活化は脳腫瘍のような反応性の低い抗原に用いると有効である。
この電界賦活化工程（機構）は、１次・２次抗原抗体反応工程前に抗原に交流高電界を非接触に与えて、元来マイナスの電荷を有する抗原を賦活化（活性化）させ、抗原と抗体との接触頻度を向上させて染色の均一化と迅速化を図る工程（機構）である。
即ち本発明の電界洗浄方法を用いた自動電界免疫組織染色方法（装置）における電界非接触撹拌法による反応場の様子は図９に示すとおりであるが、その免疫染色メカニズムの特徴は、〔１〕撹拌により粒子速度が加速され、抗原と抗体の接触頻度が向上することで免疫染色反応が時短化される。〔２〕電界撹拌による溶液の温度上昇はない（図１０に示すように）ということである。そのため、室温で使用する限りタンパク質変性による非特異反応は生じ難い（タンパク質や組織の変性の恐れは無い）。
図２に示すように１次・２次抗原抗体反応工程前に抗原に交流高電界を非接触に与えて、マイナスの電荷を有する抗原を賦活化させる時間は３０秒〜３分とすることで反応性が１０〜３０％程度向上する。
印加電界強度の主電圧としてプラス側に０．４〜２ｋＶ／ｍｍ、これにオフセット電界強度０．１５〜１ｋＶ／ｍｍを加えることで、プラス側に偏った繰り返し方形波を生成し、周波数０．１〜８００Ｈｚにて活発に応答する周波数の高電圧交流電界を与える。
In order to carry out the automatic electroimmunohistological staining method using the electric field cleaning method of the present invention, it is desirable to carry out the electric field activation process and electric field activation mechanism described below. This electric field activation is particularly effective when used for antigens with low reactivity such as brain tumors.
In this electric field activation process (mechanism), an alternating high electric field is applied to the antigen in a non-contact manner before the primary / secondary antigen antibody reaction process to activate (activate) the antigen having a negative charge, This is a process (mechanism) for improving the frequency of contact between an antigen and an antibody to achieve uniform and rapid staining.
That is, the state of the reaction field by the electric field non-contact stirring method in the automatic electric field immunohistochemical staining method (apparatus) using the electric field washing method of the present invention is as shown in FIG. The particle velocity is accelerated by stirring, and the frequency of contact between the antigen and the antibody is improved, thereby shortening the immunostaining reaction. [2] The temperature of the solution does not increase due to electric field stirring (as shown in FIG. 10). Therefore, as long as it is used at room temperature, non-specific reaction due to protein denaturation hardly occurs (there is no fear of protein or tissue denaturation).
As shown in FIG. 2, the time for activating an antigen having a negative charge by applying an AC high electric field to the antigen in a non-contact manner before the primary / secondary antigen-antibody reaction step is 30 seconds to 3 minutes. The reactivity is improved by about 10 to 30%.
By adding 0.4 to 2 kV / mm as a main voltage of the applied electric field strength and offset electric field strength of 0.15 to 1 kV / mm to the plus side, a repetitive square wave biased to the plus side is generated, and a frequency of 0. A high voltage AC electric field with a frequency that actively responds at 1 to 800 Hz is applied.
前述の電界を反応場に与える電極としては、板状電極又は円板電極を用いることができる。前記電界洗浄で用いる環状電極の孔を塞いで板状とした状態で用いてもよい。
電極の厚さは４〜１０ｍｍ、材質は良導電材料の銅、アルミ合金などが用いられる。
なお、抗原を固定する基板の一例としては、通常の２６×７６×０．８ｍｍ大のスライドガラスを用い、はっ水フレームとしては、２穴型の内径形状が１０〜２０ｍｍ以下の貫通穴を有し、樹脂部の幅は０．５〜３ｍｍ以下、スライドガラスへ張りやすく、幅２〜３ｍｍを推奨する。また厚みは０．１５〜０．３ｍｍの２穴タイプはっ水リングを用いる。
As an electrode for applying the electric field to the reaction field, a plate electrode or a disk electrode can be used. You may use in the state which plugged up the hole of the annular electrode used by the said electric field washing | cleaning, and was made into plate shape.
The thickness of the electrode is 4 to 10 mm, and the material is copper, aluminum alloy or the like, which is a highly conductive material.
In addition, as an example of the substrate for immobilizing the antigen, a normal glass slide of 26 × 76 × 0.8 mm is used, and the water-repellent frame has a through-hole having a 2-hole inner diameter of 10 to 20 mm or less. The width of the resin part is 0.5 to 3 mm or less, and it is easy to stretch on the slide glass, and a width of 2 to 3 mm is recommended. A two-hole water-repellent ring with a thickness of 0.15 to 0.3 mm is used.
〈現状工程（比較例）〉
（Ａ）一次抗体滴下後 手動電界撹拌装置に試料を投入
→（Ｂ）一次抗体を電界撹拌
→（Ｃ）装置よりスライドガラスに配置されている試料を搬出
→（Ｄ）ＰＢＳ洗浄（技師）
→（Ｅ）手動電界撹拌装置にスライドガラスに配置されている試料を搬入後、二次抗体を滴下
→（Ｆ）二次抗体を電界撹拌
→（Ｇ）装置より試料取り出し
→（Ｈ）ＰＢＳ洗浄（用手法洗浄）
<Current process (comparative example)>
(A) After dropping the primary antibody, put the sample into the manual electric field agitator → (B) Electric field agitation of the primary antibody → (C) Unload the sample placed on the slide glass from the apparatus → (D) PBS cleaning (engineer)
→ (E) After loading the sample placed on the slide glass into the manual electric field stirrer, drop the secondary antibody → (F) Stir the secondary antibody into the electric field → (G) Remove the sample from the apparatus → (H) Wash with PBS (Use cleaning method)
〈実施例１の工程〉
前記の工程を下記に示す工程に変更し、殆どの工程を自動化し、装置より試料の取り出し投入工程を無くし、作業者の手間を省くと共に良好な染色を得ることを目標とした。
（Ａ）一次抗体を滴下後 電界撹拌装置に試料を設置
→（Ｂ）一次抗体を電界撹拌
→（Ｉ）ＰＢＳ洗浄（電界洗浄：電界撹拌中に抗体液を吸い上げ排出）
→（Ｅ）二次抗体を滴下 電界撹拌に試料を投入
→（Ｆ）二次抗体電界撹拌
→（Ｇ）試料は装置より自動搬出
→（Ｈ）ＰＢＳ洗浄（用手法洗浄）
このように、〈実施例１の工程〉と〈現状工程〉との相違は、現状工程における（Ｃ）装置よりスライドガラスに配置されている試料を搬出する工程、及び（Ｄ）ＰＢＳ洗浄（技師）の工程に代えて（Ｉ）ＰＢＳ洗浄（電界洗浄）を自動にて行った点である。
<Process of Example 1>
The above-mentioned process was changed to the process shown below, and most of the processes were automated, and the objective was to eliminate the sample taking-in and loading process from the apparatus, to save the labor of the operator, and to obtain good staining.
(A) After dropping the primary antibody Place the sample in the electric field stirring device → (B) Electric field stirring of the primary antibody → (I) PBS cleaning (electric field cleaning: sucking and discharging the antibody solution during electric field stirring)
→ (E) Add secondary antibody Drop sample into electric field agitation → (F) Secondary antibody electric field agitation → (G) Sample is automatically removed from the device → (H) PBS cleaning (method cleaning)
As described above, the difference between <Process of Example 1> and <Current Process> is that (C) in the current process, the process of unloading the sample placed on the slide glass from the apparatus, and (D) PBS cleaning (engineer) (I) PBS cleaning (electric field cleaning) was automatically performed instead of the step (1).
このように、〈現状工程（比較例）〉では、各工程毎に、一度装置外へ取り出して技師が洗浄作業を行う必要があり、人手を要してしまうため、煩雑な病理現場では極めて問題である。これに対して〈実施例１の工程〉では（Ｂ）から（Ｇ）までを装置内で電界洗浄を実施でき、洗浄工程の迅速化及び自動化を達成できることが確認した。 As described above, in <current process (comparative example)>, it is necessary to take out the apparatus once for each process and perform the cleaning work by the engineer. It is. On the other hand, in <Process of Example 1>, it was confirmed that electric field cleaning from (B) to (G) can be performed in the apparatus, and that the cleaning process can be speeded up and automated.
なお、前記工程における詳細な条件については、以下の通りである。
・一次抗体滴下後の電界撹拌条件：
はっ水リング φ２０ｍｍにて形成した液滴反応場に、一次抗体を滴下し、図１の電界洗浄装置を用いて４ｋＶ、オフセット２ｋＶ、１〜５０Ｈｚ、矩形波、４分３０秒、の条件で電界撹拌を行った。その後、同条件の電界撹拌中の液滴から排出管を用いて試験溶液を排出した。
・ＰＢＳ洗浄条件：
ＰＢＳ洗浄液４００〜６００μＬを注入管を用いて液滴反応場に注入し、電界撹拌４ｋＶ、オフセット２ｋＶ、１〜５０Ｈｚ、３０秒間後に排出した。電界洗浄時間〈回数〉については３０秒 ×１回でも ×２回でも、有為な差異は認められなかった。
・二次抗体滴下後の電界撹拌条件：
２次抗体を滴下し、４ｋＶ、オフセット２ｋＶ、１〜５０Ｈｚ、矩形波、５分の条件で電界撹拌を行った。その後、同条件の電界撹拌中の液滴から２次抗体液を排出管を用いて試験溶液を排出した。
その結果は、図８に示すように〈現状工程〉と同様の良好な染色性が得られた。
In addition, about the detailed conditions in the said process, it is as follows.
-Electric field agitation conditions after dropping primary antibody:
Water-repellent ring A primary antibody is dropped onto a droplet reaction field formed with a diameter of 20 mm, and the conditions are 4 kV, offset 2 kV, 1 to 50 Hz, rectangular wave, 4 minutes 30 seconds using the electric field cleaning apparatus of FIG. Electric field stirring was performed. Thereafter, the test solution was discharged from the droplet under electric field stirring under the same conditions using a discharge tube.
-PBS washing conditions:
PBS washing | cleaning liquid 400-600 microliters was inject | poured into the droplet reaction field using the injection tube, and it discharged | emitted after 30 seconds of electric field stirring 4kV, offset 2kV, 1-50Hz. Regarding the electric field cleaning time <number of times>, no significant difference was observed in either 30 seconds x 1 or x 2 times.
-Electric field agitation conditions after dropping secondary antibody:
The secondary antibody was dropped, and electric field stirring was performed under the conditions of 4 kV, offset 2 kV, 1 to 50 Hz, rectangular wave, and 5 minutes. Thereafter, the test solution was discharged from the droplet under electric field stirring under the same conditions using a discharge tube.
As a result, as shown in FIG. 8, the same good dyeability as in the <current process> was obtained.
本発明の電界洗浄方法を用いて、自動電界免疫組織染色方法を実施する際の基本プロトコールを表１に示す。また、表２には、研究背景となった免疫染色プロトコールを示す。
上表１，２より明らかなように、従来法では、この免疫染色に合計１５１分を要していたが、本発明の電界洗浄方法を用いた自動電界免疫組織染色方法（装置）では、合計２４分でこの免疫染色処理が完了し、迅速化が確認された。
詳しい内訳は、従来法では、それぞれ６０分程度、或いはそれ以上の時間を要していた一次抗体（１次抗原抗体反応）、二次抗体（２次抗原抗体反応）のそれぞれの工程が、本発明の電界洗浄方法を用いた自動電界免疫組織染色装置における一次抗体、二次抗体の工程では、それぞれ５分に短縮できた。
また、１次・２次抗原抗体反応後の洗浄工程は、従来法では、それぞれ５分×３であったが、本発明の一次抗体（１次抗原抗体反応）の後の洗浄工程（電界ＰＢＳ洗浄）では３０〜６０秒であり、二次抗体（２次抗原抗体反応）の後の洗浄工程（電界ＰＢＳ洗浄）では１分であった。
As is clear from Tables 1 and 2 above, in the conventional method, this immunostaining required a total of 151 minutes, but in the automatic electroimmunohistological staining method (apparatus) using the electric field washing method of the present invention, the total amount The immunostaining process was completed in 24 minutes, and speeding up was confirmed.
The detailed breakdown is as follows. The steps of the primary antibody (primary antigen-antibody reaction) and secondary antibody (secondary antigen-antibody reaction), which each required about 60 minutes or more in the conventional method, are as follows. In the step of primary antibody and secondary antibody in the automatic electric field immunohistochemical staining apparatus using the electric field washing method of the invention, each could be shortened to 5 minutes.
Further, the washing step after the primary and secondary antigen-antibody reaction was 5 minutes × 3 in the conventional method, but the washing step after the primary antibody (primary antigen-antibody reaction) of the present invention (electric field PBS) Washing) was 30 to 60 seconds, and it was 1 minute in the washing step (electric field PBS washing) after the secondary antibody (secondary antigen-antibody reaction).
図１１に示すように１次・２次抗原抗体反応工程前に抗原に交流高電界を非接触に与えて、マイナスの電荷を有する抗原を賦活化させる時間を３０秒〜３分にすることで反応性が１０〜３０％程度向上した。
印加電界強度の主電圧としてプラス側に０．４〜１．５ｋＶ／ｍｍ、これにオフセット電界強度０．１５〜０．７ｋＶ／ｍｍを加えることで、プラス側に偏った繰り返し方形波を生成し、周波数０．１〜２０Ｈｚにて３０秒から３分間高電圧交流電界を与える。
なお、ここで用いる電極としては、円板電極や板状電極を用いてもよい。
As shown in FIG. 11, by applying an AC high electric field to the antigen in a non-contact manner before the primary / secondary antigen antibody reaction step, the time for activating the negatively charged antigen is set to 30 seconds to 3 minutes. The reactivity was improved by about 10 to 30%.
By adding 0.4 to 1.5 kV / mm as the main voltage of the applied electric field strength on the plus side and offset electric field strength of 0.15 to 0.7 kV / mm to this, a repetitive square wave biased to the plus side is generated. A high voltage AC electric field is applied at a frequency of 0.1 to 20 Hz for 30 seconds to 3 minutes.
In addition, as an electrode used here, you may use a disk electrode and a plate-shaped electrode.
次に、前記表１に示すプロトコールを用いて本発明の電界洗浄方法を用いた自動電界免疫組織染色方法における各工程について説明する。
本発明の電界洗浄方法を用いた自動電界免疫組織染色方法において、「４」と「８」が電界非接触撹拌装置を用いて行う１次・２次抗原抗体反応工程である。そのため、従来は「５」のＰＢＳ洗浄の時に一度、試料が搭載されているスライドガラスを装置の外に出す必要がある。
それに対し、本発明では、電界洗浄を導入することで、「４」〜「９」まで装置内にて実施することができる。「６」はカセットより内因性ペルオキシダーゼ除去液を投入する工程である。
Next, each step in the automatic electroimmunohistological staining method using the electric field washing method of the present invention will be described using the protocol shown in Table 1.
In the automatic electroimmunohistological staining method using the electric field washing method of the present invention, “4” and “8” are primary and secondary antigen-antibody reaction steps performed using an electric field non-contact stirrer. Therefore, conventionally, it is necessary to take out the slide glass on which the sample is mounted once out of the apparatus at the time of “5” PBS washing.
On the other hand, in this invention, it can implement in an apparatus from "4" to "9" by introducing electric field cleaning. “6” is a step of introducing an endogenous peroxidase removal solution from the cassette.
図１２〜１５は、本発明の電界洗浄を用いた自動電界免疫組織染色装置の一例であるが、本発明の電界洗浄を用いた自動電界免疫染色方法は、このような装置により実施される。
工程「３」の電界賦活化では、１次・２次抗原抗体反応工程前に抗原に交流高電界を３０秒〜３分非接触に与えて、元来マイナスの電荷を有する抗原を賦活化（活性化）させるが、前述のように板状電極又は円板電極を用いることができる。
工程「４・８」の電界撹拌（１次・２次抗原抗体反応工程）では、上部に配置する電極形状として、中央部に穴３が開いている平板電極４、図１６に示す環状電極１３を採用し、電極形状が環状でも電界撹拌有効に作用する結果を得た。これは、環状電極によって、反応場に電界が集中する部位と電界が弱い（内径部洗浄ノズル挿入部位）とで液滴の挙動が変わり撹拌振動が生じやすいためと考えられる。これにより、電界撹拌にて実施される１次・２次抗原抗体反応工程及び洗浄工程に同一の電極を用いることができ、装置を簡素化できることが確認された。
上部に配置する電極形状は、電界を集中させる凸部１４を中心線に対称状に形成した環状集中型電極１３を用いる場合、中央部に貫通穴３が開いている平板電極、に電界を集中させる凸部１４をはっ水リング９内にたとえば２か所設けることで、液滴の動きを２か所に分けることができる。この効果により、吸引される液滴の高さが低くなり、より電極間距離を狭くすることが、可能となり、電界強度を高くすることができる。また、電界を集中させる凸部１４を２か所以上でもよい。
12 to 15 show an example of an automatic electroimmunohistological staining apparatus using the electric field cleaning according to the present invention. The automatic electroimmuno staining method using the electric field cleaning according to the present invention is carried out by such an apparatus.
In the electric field activation of the process “3”, an alternating high electric field is applied to the antigen for 30 seconds to 3 minutes in a non-contact manner before the primary / secondary antigen-antibody reaction step to activate the antigen originally having a negative charge ( However, as described above, a plate electrode or a disk electrode can be used.
In the electric field agitation (primary / secondary antigen-antibody reaction step) in the step “4 · 8”, the plate electrode 4 having a hole 3 in the central portion and the annular electrode 13 shown in FIG. As a result, even when the electrode shape is annular, the electric field stirring effect is obtained. This is presumably because the behavior of the liquid droplets changes due to the annular electrode, and the agitation vibration is likely to occur between the site where the electric field concentrates in the reaction field and the weak electric field (inner diameter portion cleaning nozzle insertion site). Thereby, it was confirmed that the same electrode can be used for the primary and secondary antigen-antibody reaction step and the washing step performed by electric field stirring, and the apparatus can be simplified.
When the annular concentrated electrode 13 in which the convex portion 14 for concentrating the electric field is formed symmetrically with respect to the center line is used, the electric field is concentrated on the flat plate electrode having the through hole 3 in the center. By providing, for example, two protrusions 14 in the water repellent ring 9, the movement of the droplets can be divided into two places. This effect makes it possible to reduce the height of the liquid droplets to be sucked, to further reduce the distance between the electrodes, and to increase the electric field strength. Moreover, the convex part 14 which concentrates an electric field may be two or more places.
１次・２次抗原抗体反応における抗原と抗体との接触頻度を考慮すると、抗原は、スライドガラスの上に固定化されているため、抗体が抗原により多く接触させるためにも、液滴の高さを下げる必要がある。また、電界の作用によって、抗体液滴は電極面に引きつけられ、電界作用の無い時には、吸引力がなくなり、スライドガラス方向に液滴は落下する。この動作によって、液滴は撹拌作用を生じる。はっ水リングの機能として、内面は防波堤のように抗体液が外周方向に流れを食い止め、内周方向に跳ね返すことでも乱流が生じ、撹拌機能は発生する。即ち図１７（ａ）は電界が抗体液等に印加され、電極４近傍の液滴が吸引されている様子を示し、図１７（ｂ）は電界が切られ、吸引されていた抗体液等が落下し、はっ水リング９の壁面に液が衝突することで、電極４の中央部に液等が流れ込んでいく様子を示す。また、液滴の動きを２か所以上に分けることで、抗原の移動距離は半分以下になるため、抗原と抗体の出会う距離が半分になるため撹拌の均一性の面から好ましい。また設けることで、抗原抗体反応時間が短くなる。 Considering the contact frequency between the antigen and the antibody in the primary / secondary antigen-antibody reaction, the antigen is immobilized on the slide glass. It is necessary to lower the height. Further, the antibody droplet is attracted to the electrode surface by the action of the electric field, and when there is no electric field effect, the suction force is lost and the droplet falls in the direction of the slide glass. This action causes the droplets to stir. As a function of the water-repellent ring, turbulent flow also occurs when the antibody solution stops the flow in the outer peripheral direction and rebounds in the inner peripheral direction like a breakwater, and the stirring function is generated. That is, FIG. 17A shows a state in which an electric field is applied to an antibody solution and the like, and a droplet in the vicinity of the electrode 4 is sucked, and FIG. A state in which the liquid or the like flows into the central portion of the electrode 4 by falling and colliding with the wall surface of the water-repellent ring 9 is shown. Further, dividing the movement of the droplet into two or more places makes the moving distance of the antigen less than half, so that the distance where the antigen and the antibody meet is reduced by half, which is preferable from the viewpoint of uniformity of stirring. Moreover, the antigen antibody reaction time is shortened by providing.
工程「４・８」の電界撹拌（１次・２次抗原抗体反応工程）では印加中（０．４〜１．５ｋＶ／ｍｍ、オフセット電界強度０．１５〜０．３０ｋＶ／ｍｍを加える、０．１〜３００Ｈｚ，３００秒）に２７０秒経過後に環状電極の貫通穴より排出管をガラスプレート上の液滴に差し込み、電界撹拌中に抗体液をポンプにて排出し、洗浄工程を完了する場合、この工程で洗浄工程が完了する場合には、工程「５・９」のＰＢＳ洗浄処理は不要となる。
さらに、抗原の種類によっては染色性が低いものもあるため、その場合には、貫通穴よりＰＢＳ洗浄液を注入管にて注入した後、工程「５・７・９」の電界ＰＢＳ洗浄工程を導入し、非特異発色が発生しない免疫染色が得られる。撹拌（０．４〜１．５ｋＶ／ｍｍ、オフセット電界強度０．１５〜０．７ｋＶ／ｍｍを加える、０．１〜５０Ｈｚ，３０秒）し、その後、貫通穴から排出管をガラスプレート上の液滴に差し込み、溶液をポンプにて排出し、工程「１０」へ移行し、装置からスライドガラスは排出される。このように免疫染色技術の迅速化及び自動化が可能になる。
In the electric field agitation (primary / secondary antigen-antibody reaction step) in the step “4.8”, during application (0.4 to 1.5 kV / mm, offset electric field strength of 0.15 to 0.30 kV / mm is added, 0 When 270 seconds elapse from 1 to 300 Hz, 300 seconds), the discharge tube is inserted into the droplet on the glass plate from the through hole of the annular electrode, and the antibody solution is discharged with a pump during electric field stirring to complete the washing process. When the cleaning process is completed in this process, the PBS cleaning process in the process “5, 9” is not necessary.
Furthermore, depending on the type of antigen, the staining property may be low, so in that case, the PBS washing solution is injected from the through-hole into the injection tube, and then the electric field PBS washing step of step “5, 7, 9” is introduced. Thus, immunostaining that does not cause non-specific color development is obtained. Stirring (0.4 to 1.5 kV / mm, applying offset electric field strength of 0.15 to 0.7 kV / mm, 0.1 to 50 Hz, 30 seconds), and then discharging the discharge tube from the through hole onto the glass plate The solution is inserted into the droplet, the solution is discharged by a pump, the process proceeds to step “10”, and the slide glass is discharged from the apparatus. Thus, the immunostaining technique can be speeded up and automated.
本発明の電界洗浄を用いた自動電界免疫組織染色方法における、表１中の「５」と「７」が電界撹拌を用いて行う電界ＰＢＳ洗浄工程である。
なお、工程「５」の電界撹拌（電界ＰＢＳ洗浄）工程では、上部に配置する電極形状は、図１４及び図１５に示すように貫通穴３を有する板状電極又は環状電極４であるが、図１６（ａ）に示す電界を集中させる凸部１４を形成させた環状集中型電極１３を用いてもよい。この電界集中型電極１３では、電界を集中させる凸部１４をはっ水リング９に沿った形状にたとえば２か所設けることで、液滴の動きを凸部１４の数と同数の２か所に分けることができる。この効果により、図１６（ｂ）に示すように吸引される液滴の高さが低くなり、より電極間距離を狭くすることが、可能となり、電界強度を高くすることができる。また、電界を集中させる凸部１４を２か所以上６か所以下でもよい。凸部数の数をＮ個とすると６＞Ｎ＞１ここで、Ｎ＝１は環状電極を示す。内径はφ１０＞、外径は２２＜φＤ＜３０ｍｍ。
In the automatic electroimmunohistological staining method using electric field washing according to the present invention, “5” and “7” in Table 1 are electric field PBS washing steps performed using electric field stirring.
In addition, in the electric field stirring (electric field PBS cleaning) step of the step “5”, the shape of the electrode disposed on the upper portion is a plate electrode or an annular electrode 4 having a through hole 3 as shown in FIGS. 14 and 15. An annular concentrated electrode 13 having convex portions 14 for concentrating the electric field shown in FIG. 16A may be used. In this electric field concentration type electrode 13, for example, two convex portions 14 for concentrating the electric field are provided in a shape along the water-repellent ring 9, so that the movement of the liquid droplets is equal to the number of the convex portions 14. Can be divided into By this effect, as shown in FIG. 16B, the height of the sucked droplet is reduced, the distance between the electrodes can be further reduced, and the electric field strength can be increased. Moreover, the convex part 14 which concentrates an electric field may be 2 or more and 6 or less. If the number of convex portions is N, 6>N> 1, where N = 1 indicates a ring electrode. The inner diameter is φ10> and the outer diameter is 22 <φD <30 mm.
環状集中型電極の狙いは、１次・２次抗原抗体反応の反応物とＰＢＳとの接触頻度を考慮すると、反応物は、スライドガラスの上に固定化されているため、ＰＢＳが反応物により多く接触させるためにも、液滴の高さを下げる必要がある。また、電界の作用によって、液滴は電極面に引きつけられ、電界作用の無い時には、吸引力がなくなり、スライドガラス方向に液滴は落下する。この動作によって、液滴は撹拌作用を生じる。はっ水リングの機能として、内面は防波堤のように抗体液が外周方向に流れを食い止め、内周方向に跳ね返すことでも乱流が生じ、撹拌機能は発生する。また、液滴の動きを２か所以上に分けることで、ＰＢＳの移動距離は半分以下になるため、反応物とＰＢＳの出会う距離が半分になるため撹拌の均一性の面から好ましい。また設けることで、電界洗浄時間が短くなる。即ち図１８（ａ）は電界が抗体液等に印加され、環状電極１３近傍の液滴が吸引されている様子を示し、図１８（ｂ）は電界が抗体液等に印加され、凸型電極１３に液滴が吸引されている様子を示し、図１８（ｃ）電界が切られ、吸引されていた抗体液等が落下し、はっ水リング９の壁面に液が衝突することで、電極１３の中央部に液等が流れ込んでいく様子を示す。 The aim of the circular concentrated electrode is to consider the contact frequency between the reaction product of the primary and secondary antigen-antibody reaction and PBS, and the reaction product is immobilized on the slide glass. In order to make many contact, it is necessary to reduce the height of the droplet. Further, the droplet is attracted to the electrode surface by the action of the electric field, and when there is no electric field effect, the suction force is lost and the droplet falls in the direction of the slide glass. This action causes the droplets to stir. As a function of the water-repellent ring, turbulent flow also occurs when the antibody solution stops the flow in the outer peripheral direction and rebounds in the inner peripheral direction like a breakwater, and the stirring function is generated. Further, by dividing the movement of the liquid droplets into two or more places, the moving distance of the PBS is reduced to half or less, and the distance between the reaction product and the PBS is halved, which is preferable from the viewpoint of uniformity of stirring. Moreover, the electric field cleaning time is shortened by providing. 18A shows a state in which an electric field is applied to an antibody solution and the like, and a droplet in the vicinity of the annular electrode 13 is sucked. FIG. 18B shows an electric field applied to the antibody solution and the like, and a convex electrode. FIG. 18 (c) shows the state in which the droplet is sucked, FIG. 18 (c), the electric field is turned off, the sucked antibody solution falls, and the liquid collides with the wall surface of the water-repellent ring 9, thereby A mode that liquid etc. flow in into the center part of 13 is shown.
さらに電極形状として、環状電極を採用すると、電極形状が環状でも電界撹拌が成し得た。これにより電界撹拌並びに洗浄にも同一の電極を用いることができ、装置を簡素化ができることが確認された。またより良い撹拌条件を環状電極に求めると環状電極にも凹凸部を設け電界が集中する箇所を設け電極間ギャップを狭め、電界強度を向上させることが可能となる。
なお、この電界洗浄技術は、免疫組織染色に限らず、核酸におけるハイブリダイゼーシ
ョン工程にも適用可能であり、加えて抗原抗体反応においても適用可能である。
Furthermore, when an annular electrode was employed as the electrode shape, electric field agitation could be achieved even if the electrode shape was annular. Thus, it was confirmed that the same electrode can be used for electric field stirring and cleaning, and the apparatus can be simplified. Further, when a better stirring condition is obtained for the annular electrode, the annular electrode is also provided with a concavo-convex portion to provide a portion where the electric field is concentrated, thereby narrowing the gap between the electrodes and improving the electric field strength.
This electric field washing technique is applicable not only to immunohistochemical staining but also to a nucleic acid hybridization step, and in addition, to an antigen-antibody reaction.
〈実施例２の工程〉
前記の工程を下記に示す工程に変更し、殆どの工程を自動化し、試料の取り出し投入工程を無くし、作業者の手間を省くと共に良好な染色を得ることを目標とした。即ち前記図１２〜１５、図２０に示す自動電界免疫組織染色装置を用いて処理を行った。
（Ａ’）例えば図２１（ｂ）に示すようにスライドガラス上のはっ水リング内にそれぞれ試料と陽性コントロールを固定し、自動電界撹拌装置に設置後、たとえば脳腫瘍の組織のように反応性の低い抗原に対しては電界賦活化を施すと効果的である。
→（Ｃ’）電界ＰＢＳ洗浄（電界洗浄：電界撹拌中に抗体液を吸い上げ排出）
→（Ｄ’）二次抗体を滴下後 電界撹拌 後半より抗体液を排出
→（Ｅ’）電界ＰＢＳ洗浄
→（Ｆ’）試料は装置より自動搬出
このように、〈実施例２の工程〉と〈現状工程〉との相違は、現状工程における（Ｃ）装置よりスライドガラスに配置されている試料を搬出する工程、及び（Ｄ）ＰＢＳ洗浄（装置外に取り出し技師が洗浄）の工程に代えて（Ｃ’）ＰＢＳ洗浄（電界洗浄）を自動にて行った点である。さらに品位向上のために、（Ａ’）電界賦活化を加えた点である。
<Process of Example 2>
The above-mentioned steps were changed to the steps shown below, and most of the steps were automated, the sample taking-out step was eliminated, the labor of the operator was saved, and good dyeing was obtained. That is, the treatment was performed using the automatic electroimmunohistological staining apparatus shown in FIGS.
(A ′) For example, as shown in FIG. 21 (b), the sample and the positive control are fixed in a water-repellent ring on a slide glass, respectively, and placed in an automatic electric field agitator, and then, for example, reactive like a brain tumor tissue. It is effective to apply electric field activation to antigens with low levels.
→ (C ') Electric field PBS cleaning (electric field cleaning: sucking up and discharging antibody solution during electric field stirring)
→ (D ′) After dropping secondary antibody Electric field agitation Discharge antibody solution from latter half → (E ′) Electric field PBS washing → (F ′) Sample is automatically carried out from the apparatus. The difference from the <current process> is that in the current process (C) the process of carrying out the sample placed on the slide glass from the apparatus, and (D) PBS cleaning (taken out of the apparatus and cleaned by an engineer) (C ') PBS cleaning (electric field cleaning) was automatically performed. Furthermore, in order to improve the quality, (A ′) electric field activation is added.
このように、〈現状工程（比較例）〉では、各工程毎に、一度装置外へ取り出して技師が洗浄作業を行う必要があり、人手を要してしまうため、煩雑な病理現場では極めて問題である。これに対して〈実施例２の工程〉では（Ｂ）から（Ｇ）までを装置が自動運転する。装置内に電界撹拌、電界洗浄を実施でき、洗浄工程の迅速化及び自動化を達成できることが確認した。また、図２１（ａ）に示すようにはっ水リングの内側面に撹拌液がぶつかり跳ね返り、内側に流体が流れることにで、スライドガラスに固定化されている抗原により抗体が接触する頻度が高まる。 As described above, in <current process (comparative example)>, it is necessary to take out the apparatus once for each process and perform the cleaning work by the engineer. It is. On the other hand, in <Process of Example 2>, the apparatus automatically operates from (B) to (G). It was confirmed that electric field agitation and electric field cleaning can be performed in the apparatus, and that the cleaning process can be speeded up and automated. Further, as shown in FIG. 21 (a), the stirring liquid splashes and splashes on the inner surface of the water-repellent ring, and the fluid flows inside, so that the frequency of the antibody contact with the antigen immobilized on the slide glass is increased. Rise.
なお、前記工程における詳細な条件については、以下の通りである。
・一次抗体滴下後の電界撹拌条件：
はっ水リング内径φ２０ｍｍにて形成した液滴反応場に、一次抗体を２００μＬ滴下し、図１５の電界洗浄装置を用いて４ｋＶ、オフセット２ｋＶ、1〜３０Ｈｚ、矩形波、４分３０秒、の条件で電界撹拌を行った。その後、同条件の電界撹拌中の液滴から排出管を用いて試験溶液を排出した。
・ＰＢＳ洗浄条件：
ＰＢＳ洗浄液４００μＬを注入管を用いて液滴反応場に注入し、電界撹拌４ｋＶ、オフセット２ｋＶ、1〜３０Ｈｚ、３０秒間後に排出した。電界洗浄時間〈回数〉については３０秒 ×１回で有為な差異は認められなかった。
・二次抗体滴下後の電界撹拌条件：
２次抗体を２００μＬ滴下し、４ｋＶ、オフセット２ｋＶ、１〜３０Ｈｚ、矩形波、５分の条件で電界撹拌を行った。その後、同条件の電界撹拌中の液滴から２次抗体液を排出管を用いて試験溶液を排出した。二次抗体の洗浄は２回以上行うことで良好な染色性が得られた。
その結果は、図２３に示すように〈現状工程（比較例）〉と同様の良好な染色性が得られた。即ち同図（ａ）は、従来法で得られた免疫組織染色（９０分）を示し、同図（ｂ）は、本発明の電界洗浄方法を用いた自動電界免疫組織染色方法で得られた免疫組織染色（１０分）を示すが、前述のように従来法では１５１分の処理時間に対し、本発明の電界洗浄方法を用いた自動電界免疫組織染色方法では２２．５分の短時間で処理できたにもかかわらず、むしろ本発明の方が免疫組織染色は明瞭であった。
In addition, about the detailed conditions in the said process, it is as follows.
-Electric field agitation conditions after dropping primary antibody:
200 μL of the primary antibody is dropped on a droplet reaction field formed with an inner diameter of water-repellent ring φ20 mm, and 4 kV, offset 2 kV, 1 to 30 Hz, rectangular wave, 4 minutes and 30 seconds using the electric field cleaning device of FIG. Electric field stirring was performed under the conditions. Thereafter, the test solution was discharged from the droplet under electric field stirring under the same conditions using a discharge tube.
-PBS washing conditions:
PBS washing | cleaning liquid 400microliter was inject | poured into the droplet reaction field using the injection tube, and it discharged | emitted after 30 seconds of electric field stirring 4kV, offset 2kV, 1-30Hz. The electric field cleaning time <number of times> was 30 seconds × 1 time, and no significant difference was observed.
-Electric field agitation conditions after dropping secondary antibody:
200 μL of the secondary antibody was dropped, and electric field stirring was performed under the conditions of 4 kV, offset 2 kV, 1 to 30 Hz, rectangular wave, and 5 minutes. Thereafter, the test solution was discharged from the droplet under electric field stirring under the same conditions using a discharge tube. The secondary antibody was washed twice or more to obtain good staining.
As a result, as shown in FIG. 23, good dyeability similar to that of the <current process (comparative example)> was obtained. That is, FIG. 5 (a) shows the immunohistochemical staining (90 minutes) obtained by the conventional method, and FIG. 6 (b) was obtained by the automatic electric field immunohistochemical staining method using the electric field washing method of the present invention. Although immunohistochemical staining (10 minutes) is shown, as described above, in the conventional method, the processing time of 151 minutes is compared with the automatic electric field immunohistochemical staining method using the electric field washing method of the present invention in 22.5 minutes. Despite being able to be processed, the immunohistochemical staining was clearer in the present invention.
図１９には、電界を非接触に抗体液に印加すると、発揮する現象についてゼータ電位を評価することで明らかにした。
即ち機械的な振動を生じるボルテックスミキサーにて抗体液を撹拌することにより分散が進行し、ゼータ電位はマイナス方向へシフトする。
一方、電界印加する周波数によっては、抗体液に撹拌振動が生じにくい状態がある。この場合において、抗体の運動は進行していないように観察されるが、通常の静置方法に比べると上記のような電界を印加した方が、染色性が加速することが明らかになっている。すなわち、電界印加によって抗体液が分散を生じるためと考えられる。この現象を明らかにするために、ゼータ電位を用いて評価した。
In FIG. 19, it was clarified by evaluating the zeta potential that the phenomenon is exhibited when an electric field is applied to the antibody solution in a non-contact manner.
That is, when the antibody solution is stirred with a vortex mixer that generates mechanical vibration, dispersion proceeds, and the zeta potential shifts in the negative direction.
On the other hand, depending on the frequency at which the electric field is applied, there is a state in which stirring vibration hardly occurs in the antibody solution. In this case, it is observed that antibody movement does not proceed, but it is clear that staining is accelerated when the electric field as described above is applied as compared to a normal standing method. . That is, it is considered that the antibody solution is dispersed by applying an electric field. In order to clarify this phenomenon, the zeta potential was used for evaluation.
〈実施例３の工程〉
反応性が高い肺腺癌の組織にて、同様な自動電界免疫組織染色を行った際の条件を表６に示し、その結果を図２２（抗原抗体反応が一次・二次計１０分）に示した。
Table 6 shows the conditions under which similar automatic electroimmunohistochemical staining was performed on highly reactive lung adenocarcinoma tissues, and the results are shown in FIG. 22 (antigen-antibody reaction is 10 minutes for primary and secondary reactions). Indicated.
１ 基板（ガラスプレート）
２ 液滴（反応場）
３ 貫通穴
４ 環状電極（上部電極）
５ 排出管
６ 試薬溶液
７ 注入管
８ 洗浄液
９ はっ水リング
１０ 下部電極
１１ 試薬用シリンジ
１２ 試薬格納カセット
１３ 電界集中型電極
１４ 洗浄液供給・排出ユニット
１５ 電界撹拌スライドガラス搬送ステージ
１６ 陽性コントロール
１７ 検体試料；抗原
１８ 取り外し用タグ
1 Substrate (glass plate)
2 droplet (reaction field)
3 Through hole 4 Ring electrode (upper electrode)
5 Discharge tube 6 Reagent solution 7 Injection tube 8 Wash solution 9 Water repellent ring 10 Lower electrode 11 Reagent syringe 12 Reagent storage cassette 13 Electric field concentrated electrode 14 Cleaning solution supply / discharge unit 15 Electric field stirring slide glass transport stage 16 Positive control 17 Sample Sample; Antigen 18 removal tag
Claims (12)
前記反応場を挟むように上下に形成された上側電極と下側電極とを備え、
前記反応場で抗原に１次抗体を含む液滴を添加して抗原と１次抗体とを反応させる１次抗原抗体反応工程と、
前記１次抗原抗体反応工程の後に２次抗体を含む液滴を添加して２次抗体をさらに反応させる２次抗原抗体反応工程と、
前記上側電極と下側電極との間に所定の電界を与えることにより前記反応場に形成された液滴を撹拌する工程と、
前記１次抗原抗体反応工程と２次抗原抗体反応工程との後に、それぞれ未反応抗体を除去する未反応抗体洗浄工程と、
を有することを特徴とする電界洗浄方法。 A reaction field formed on the substrate to react the antigen and the antibody;
An upper electrode and a lower electrode formed vertically so as to sandwich the reaction field,
A primary antigen-antibody reaction step of reacting the antigen and the primary antibody by adding a droplet containing the primary antibody to the antigen in the reaction field;
A secondary antigen-antibody reaction step in which a droplet containing a secondary antibody is added after the primary antigen-antibody reaction step to further react the secondary antibody;
Stirring the droplets formed in the reaction field by applying a predetermined electric field between the upper electrode and the lower electrode;
An unreacted antibody washing step for removing unreacted antibodies after the primary antigen-antibody reaction step and the secondary antigen-antibody reaction step, respectively;
An electric field cleaning method comprising:
をさらに備えることを特徴とする請求項３に記載の電界洗浄方法。 The unreacted antibody washing step includes a step of inserting a discharge tube from the through hole into the reaction field to discharge the unreacted antibody, and a step of inserting an injection tube from the through hole to the reaction field and injecting a cleaning liquid. When,
The electric field cleaning method according to claim 3, further comprising:
前記反応場を挟むように上下に形成された上側電極と下側電極とを備え、
前記反応場で抗原に１次抗体を含む液滴を添加して抗原と１次抗体とを反応させる１次抗原抗体反応機構と、
前記１次抗原抗体反応機構の後に２次抗体を含む液滴を添加して２次抗体をさらに反応させる２次抗原抗体反応機構と、
前記上側電極と下側電極との間に所定の電界を与えることにより前記反応場に形成された液滴を撹拌する機構と、
前記１次抗原抗体反応機構と２次抗原抗体反応機構との後に、それぞれ未反応抗体を除去する未反応抗体洗浄機構と、
を有することを特徴とする電界洗浄装置。 A reaction field formed on the substrate to react the antigen and the antibody;
An upper electrode and a lower electrode formed vertically so as to sandwich the reaction field,
A primary antigen-antibody reaction mechanism in which a droplet containing a primary antibody is added to the antigen in the reaction field to react the antigen and the primary antibody;
A secondary antigen-antibody reaction mechanism in which a droplet containing a secondary antibody is added after the primary antigen-antibody reaction mechanism to further react the secondary antibody;
A mechanism for stirring droplets formed in the reaction field by applying a predetermined electric field between the upper electrode and the lower electrode;
An unreacted antibody washing mechanism for removing unreacted antibodies after the primary antigen-antibody reaction mechanism and the secondary antigen-antibody reaction mechanism,
An electric field cleaning apparatus comprising:
をさらに備えることを特徴とする請求項８に記載の電界洗浄装置。 The unreacted antibody cleaning mechanism includes a mechanism that inserts a discharge tube from the through hole into the reaction field to discharge unreacted antibody, and a mechanism that inserts an injection tube from the through hole into the reaction field and injects a cleaning liquid. When,
The electric field cleaning apparatus according to claim 8, further comprising:
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