JP2020030180A - Hemolytic agent for blood sample - Google Patents
Hemolytic agent for blood sample Download PDFInfo
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- JP2020030180A JP2020030180A JP2018157678A JP2018157678A JP2020030180A JP 2020030180 A JP2020030180 A JP 2020030180A JP 2018157678 A JP2018157678 A JP 2018157678A JP 2018157678 A JP2018157678 A JP 2018157678A JP 2020030180 A JP2020030180 A JP 2020030180A
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- JP
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- Prior art keywords
- cells
- blood sample
- blood
- saponin
- hemolytic agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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Images
Abstract
Description
本発明は、血液試料を溶血処理するために用いる溶血剤に関する。特に本発明は、血液中に含まれる細胞内または細胞膜表面タンパク質の細胞外への放出を抑制し、前記タンパク質を利用した細胞の検出を精度高く実施可能な溶血剤に関する。 The present invention relates to a hemolytic agent used for hemolyzing a blood sample. In particular, the present invention relates to a hemolytic agent capable of suppressing the release of intracellular or cell membrane surface proteins contained in blood to the outside of cells, and capable of accurately detecting cells using the proteins.
近年、血液などの体液や、臓器などの組織を溶液に懸濁もしくは分散して得られる組織懸濁液や、細胞培養液などから標的細胞を選択的に分離回収し、当該分離回収した細胞を基礎研究や臨床診断、治療へ応用する研究が進められている。例えば、がん患者より採取した血液から腫瘍細胞（Ｃｉｒｃｕｌａｔｉｎｇ Ｔｕｍｏｒ Ｃｅｌｌ、以下ＣＴＣ）を採取し、当該細胞について形態学的分析、組織型分析や遺伝子分析を行ない、前記分析により得られた知見に基づき治療方針を判断する研究が進められている。 In recent years, target cells have been selectively separated and collected from body fluids such as blood, tissue suspensions obtained by suspending or dispersing tissues such as organs in a solution, and cell culture solutions. Basic research, clinical diagnosis, and research applied to treatment are underway. For example, a tumor cell (Circuling Tumor Cell, hereinafter referred to as CTC) is collected from blood collected from a cancer patient, and the cells are subjected to morphological analysis, histological analysis and gene analysis, and based on the knowledge obtained by the analysis. Research to determine treatment strategies is ongoing.
全血、組織懸濁液といった血液成分を含む試料（以下、血液試料）中に含まれる腫瘍細胞を検出する方法として、細胞質内タンパク質であるサイトケラチンを検出する方法が広く用いられている。しかしながら細胞質内に存在するサイトケラチン（特にサイトケラチン１８）は、細胞の死滅により細胞質外に放出され、細胞内に存在するサイトケラチン量が低下するため、腫瘍細胞の検出精度を低下させる要因となっていた（非特許文献１）。
前記腫瘍細胞を検出する別の方法として、腫瘍細胞の細胞膜タンパク質であるＥｐＣＡＭ（Ｅｐｉｔｈｅｌｉａｌ ｃｅｌｌ ａｄｈｅｓｉｏｎ ｍｏｌｅｃｕｌｅ）（特許文献１）や白血球の細胞膜タンパク質であるＣＤ４５（特許文献２）を検出することで、腫瘍細胞を検出する方法が用いられている。しかしながら細胞損傷等によって、細胞膜に存在するタンパク質が分解または細胞から漏出することがあるため、腫瘍細胞の検出精度を低下させる要因となっていた。
As a method for detecting tumor cells contained in a sample containing a blood component such as whole blood or a tissue suspension (hereinafter, referred to as a blood sample), a method for detecting cytokeratin, a cytoplasmic protein, is widely used. However, cytokeratin (especially cytokeratin 18) present in the cytoplasm is released outside the cytoplasm due to cell death, and the amount of cytokeratin present in the cell decreases, which is a factor that reduces the detection accuracy of tumor cells. (Non-Patent Document 1).
As another method for detecting the above-mentioned tumor cells, by detecting EpCAM (Epithelial cell adhesion molecule) (Patent Document 1) which is a cell membrane protein of a tumor cell and CD45 (Patent Document 2) which is a cell membrane protein of a leukocyte, A method for detecting cells has been used. However, protein present in the cell membrane may be decomposed or leaked out of the cell due to cell damage or the like, which has been a factor in reducing the detection accuracy of tumor cells.
血液試料中に含まれる腫瘍細胞を検出しようとする場合、血液試料中には赤血球など不要な細胞が多く含まれているため、溶血操作により赤血球を破壊し、除去する操作が必要である。血液試料を溶血させる方法として、従来より塩化アンモニウム溶液を用いた方法が知られている（特許文献３）。しかしながら前記方法は、血液試料中に含まれる腫瘍細胞に対する損傷が大きい。そのため、サイトケラチンのような腫瘍細胞内で発現するタンパク質を利用して腫瘍細胞を検出しようとした場合、腫瘍細胞の損傷により前記タンパク質が細胞質外に放出されるため、血液中に含まれる腫瘍細胞を精度高く検出するのは困難であった。 When trying to detect tumor cells contained in a blood sample, an unnecessary cell such as red blood cells is often contained in the blood sample, so that an operation of destroying and removing red blood cells by a hemolysis operation is required. As a method for hemolyzing a blood sample, a method using an ammonium chloride solution has been conventionally known (Patent Document 3). However, this method has a large damage to tumor cells contained in the blood sample. Therefore, when an attempt is made to detect a tumor cell using a protein expressed in the tumor cell, such as cytokeratin, since the protein is released outside the cytoplasm due to the damage of the tumor cell, the tumor cell contained in the blood Was difficult to detect with high accuracy.
また、溶血法として界面活性剤を用いた方法も知られている。特許文献４では界面活性剤を用いた溶血方法が記載されており、特許文献５では非イオン性界面活性剤とイオン性界面活性剤とホルムアルデヒドまたはパラホルムアルデヒドと糖または糖アルコールとを含む溶血剤に関して記載されている。しかしながら前記方法は、界面活性剤を用いることで細胞膜表面に存在する細胞膜タンパク質を細胞外へ漏出させることに繋がり、前記腫瘍細胞を精度高く検出するのは困難であった。 A method using a surfactant is also known as a hemolysis method. Patent Document 4 describes a hemolysis method using a surfactant, and Patent Document 5 relates to a hemolysis agent containing a nonionic surfactant, an ionic surfactant, and formaldehyde or paraformaldehyde and a sugar or a sugar alcohol. Has been described. However, the above method has led to the leakage of cell membrane proteins existing on the cell membrane surface to the outside of cells by using a surfactant, and it has been difficult to detect the tumor cells with high accuracy.
本発明の課題は、血液試料を溶血処理する際、血液中に含まれる細胞内または細胞膜表面タンパク質の細胞外への放出を抑制し、前記タンパク質を利用した細胞の検出を精度高く実施可能な溶血剤を提供することにある。 It is an object of the present invention to provide a hemolysis treatment of a blood sample, which suppresses the release of intracellular or cell membrane surface proteins contained in blood to the outside of cells, and enables highly accurate detection of cells using the proteins. To provide an agent.
上記課題を解決するために、本発明者らは鋭意検討を重ねた結果、本発明に到達した。 Means for Solving the Problems In order to solve the above problems, the present inventors have conducted intensive studies, and as a result, have reached the present invention.
すなわち本発明の第一の態様は、アルデヒド架橋剤とポリエチレングリコールとサポニンとを少なくとも含む、血液試料の溶血剤である。 That is, a first aspect of the present invention is a hemolytic agent for a blood sample, which contains at least an aldehyde crosslinking agent, polyethylene glycol, and saponin.
また本発明の第二の態様は、溶血剤に含まれる各成分の重量濃度の比率として、サポニンに対するポリエチレングリコールの比率が１以上５以下である、および／もしくはアルデヒド架橋剤に対するポリエチレングリコールの比率が０．２以上１以下である、、前記第一の態様に記載の溶血剤である。 In a second aspect of the present invention, the ratio of polyethylene glycol to saponin is 1 or more and 5 or less, and / or the ratio of polyethylene glycol to aldehyde crosslinking agent is the weight concentration ratio of each component contained in the hemolytic agent. The hemolytic agent according to the first aspect, which is 0.2 or more and 1 or less.
さらに本発明の第三の態様は、アルデヒド架橋剤、ポリエチレングリコール、およびサポニンを血液試料に添加する工程を含む、血液試料を溶血する方法である。 Further, a third aspect of the present invention is a method for hemolyzing a blood sample, the method including a step of adding an aldehyde crosslinking agent, polyethylene glycol, and saponin to the blood sample.
さらに本発明の第四の態様は、添加後の血液試料中に含まれるポリエチレングリコールの濃度が０．１％（ｗ／ｖ）以上１％（ｗ／ｖ）以下である、前記第三の態様に記載の方法である。 Further, the fourth aspect of the present invention is the third aspect, wherein the concentration of polyethylene glycol contained in the blood sample after the addition is 0.1% (w / v) or more and 1% (w / v) or less. It is a method of description.
さらに本発明の五の態様は、血液試料中に含まれる標的細胞を検出する方法であって、前記血液試料を前記第三または四の態様に記載の方法で溶血させる工程、および
溶血した血液試料中の標的細胞を光学的に検出する工程、を含む前記方法である。
Further, a fifth aspect of the present invention is a method for detecting target cells contained in a blood sample, wherein the blood sample is hemolyzed by the method according to the third or fourth aspect, and a hemolyzed blood sample Optically detecting a target cell therein.
また本発明の第六の態様は、血液試料中に含まれる標的細胞を検出する方法であって、前記血液試料を前記第三または四の態様に記載の方法で溶血させる工程、および
溶血した血液試料中の標的細胞および／もしくは標的細胞以外の不要細胞を光学的に検出する工程、を含む前記方法である。
A sixth aspect of the present invention is a method for detecting target cells contained in a blood sample, wherein the blood sample is hemolyzed by the method according to the third or fourth aspect, and the hemolyzed blood is provided. Optically detecting target cells and / or unnecessary cells other than the target cells in the sample.
また本発明の第七の態様は、不要細胞が白血球である、前記第六の態様に記載の方法である。 A seventh aspect of the present invention is the method according to the sixth aspect, wherein the unnecessary cells are leukocytes.
また本発明の第八の態様は、標的細胞の光学的検出を、当該細胞の膜表面タンパク質を光学的に検出することで実施する、前記第五から第七の態様のいずれかに記載の方法である。 The eighth aspect of the present invention is the method according to any one of the fifth to seventh aspects, wherein the optical detection of the target cell is performed by optically detecting a membrane surface protein of the cell. It is.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明において血液試料とは、赤血球を含むまたは赤血球を含み得る試料のことをいう。具体的には、全血、希釈血、血清、血漿、臍帯血、成分採血液といった血液由来成分や、肝臓、肺、脾臓、腎臓、腫瘍、リンパ節といった血液由来成分を含む組織の一片を適切な緩衝液で懸濁させた懸濁液や、尿、羊水、腹水といった血液由来成分を含み得る生体試料などがあげられる。またこれらの試料や懸濁液を遠心分離などにより分離回収して得られた、血液由来成分を含む細胞の画分も、本発明における血液試料に含まれる。 In the present invention, a blood sample refers to a sample containing or possibly containing red blood cells. Specifically, a piece of tissue containing blood-derived components, such as whole blood, diluted blood, serum, plasma, umbilical cord blood, and component blood, or a blood-containing component, such as liver, lung, spleen, kidney, tumor, or lymph node, is appropriately used. And a biological sample that may contain blood-derived components such as urine, amniotic fluid, and ascites. The blood sample of the present invention also includes a fraction of cells containing blood-derived components obtained by separating and collecting these samples and suspensions by centrifugation or the like.
本発明における血液試料中に含まれる標的細胞の一例としては、血中循環がん細胞（ＣＴＣ）などの腫瘍細胞、循環血液内皮細胞（ＣＥＣ）、循環血管内皮細胞（ＣＥＰ）、循環胎児細胞（ＣＦＣ）、各種幹細胞があげられる。また本発明における血液試料中に含まれる不要細胞とは、前述した標的細胞以外の細胞を指し、具体例としては、血液試料中に含まれる細胞である白血球、赤血球、血小板および小胞、ならびにこれら細胞または前述した標的細胞由来のデブリ（ｄｅｂｒｉｓ）があげられる。 Examples of target cells contained in the blood sample in the present invention include tumor cells such as circulating cancer cells (CTC), circulating blood endothelial cells (CEC), circulating vascular endothelial cells (CEP), and circulating fetal cells ( CFC) and various stem cells. Further, the unnecessary cells contained in the blood sample in the present invention refers to cells other than the above-described target cells, and specific examples thereof include leukocytes, erythrocytes, platelets and vesicles which are cells contained in the blood sample, and Debris derived from the cells or the target cells described above.
本発明は、血液試料中に含まれる標的細胞を検出するにあたり、血液試料を溶血する工程を、アルデヒド架橋剤とポリエチレングリコール（ＰＥＧ）とサポニンとを少なくとも含む溶血剤で行なうことを特徴としている。 The present invention is characterized in that in detecting target cells contained in a blood sample, the step of hemolyzing the blood sample is performed with a hemolytic agent containing at least an aldehyde cross-linking agent, polyethylene glycol (PEG) and saponin.
本発明におけるアルデヒド架橋剤とは、アルデヒド類を含む化合物または分解することでアルデヒド類を放出するアルデヒドドナー化合物のことをいう。アルデヒド類を含む化合物としてはホルムアルデヒド、グルタルアルデヒド、グリオキサールが例示できる。アルデヒドドナー化合物としては、イミダゾリジニル尿素、ベンジルヘミホルマール（フェニルメトキシメタノール）、５−ブロモ−５−ニトロ−１，３−ジオキサン、ブロノポール（２−ブロモ−２−ニトロプロペイン−１，３−ジオール）、ジアゾリジニル尿素、ＤＭＤＭヒダントイン（１，３−ジメチロール−５，５−ジメチルヒダントイン）、メセナミン（ヘキサメチレンテトラミン）、クオタニウム−１５（メセナミン ３−クロロアリロクロリド）、ヒドロキシメチルグリシンナトリウム、アミンやアミドのメチロール、ヒドロキシメチル誘導体、メチロール、メテンアミン、パラホルムアルデヒドが例示できる。中でもホルムアルデヒドおよびパラホルムアルデヒドは、溶血効果を維持しながら、標的細胞および／または不要細胞の内部ならびに膜表面に有するタンパク質の保持にも効果がある点で最も好ましいアルデヒド架橋剤の一つといえる。アルデヒド架橋剤の添加量は、血液試料中に含まれる標的細胞および／もしくは不要細胞の検出に用いるタンパク質の性質を考慮して適宜決定すればよいが、アルデヒド架橋剤としてホルマリン（ホルムアルデヒド水溶液）を用いる場合は、血液試料中のホルムアルデヒド濃度として０．０１％（ｗ／ｖ）から１０％（ｗ／ｖ）の間とすればよく、０．１％（ｗ／ｖ）から４％（ｗ／ｖ）の間とすると好ましく、０．５％（ｗ／ｖ）から２％（ｗ／ｖ）の間とするとさらに好ましい。 The aldehyde cross-linking agent in the present invention refers to a compound containing aldehydes or an aldehyde donor compound which releases aldehydes when decomposed. Examples of compounds containing aldehydes include formaldehyde, glutaraldehyde, and glyoxal. Examples of aldehyde donor compounds include imidazolidinyl urea, benzyl hemiformal (phenylmethoxymethanol), 5-bromo-5-nitro-1,3-dioxane, and bronopol (2-bromo-2-nitropropane-1,3-diol). , Diazolidinyl urea, DMDM hydantoin (1,3-dimethylol-5,5-dimethylhydantoin), mesenamine (hexamethylenetetramine), quatanium-15 (mesenamine 3-chloroallylochloride), sodium hydroxymethylglycine, amines and amides Examples include methylol, hydroxymethyl derivatives, methylol, methamine, and paraformaldehyde. Above all, formaldehyde and paraformaldehyde can be said to be one of the most preferable aldehyde cross-linking agents in that they are effective in retaining proteins inside target cells and / or unnecessary cells and on the membrane surface while maintaining the hemolytic effect. The amount of the aldehyde cross-linking agent to be added may be appropriately determined in consideration of the properties of the protein used for detection of target cells and / or unnecessary cells contained in the blood sample. Formalin (formaldehyde aqueous solution) is used as the aldehyde cross-linking agent. In this case, the formaldehyde concentration in the blood sample may be between 0.01% (w / v) and 10% (w / v), and may be between 0.1% (w / v) and 4% (w / v). ), More preferably between 0.5% (w / v) and 2% (w / v).
本発明の溶血剤に含ませるＰＥＧの濃度は、アルデヒド架橋剤と同様、血液試料に含まれる標的細胞および／もしくは不要細胞の検出に用いるタンパク質の性質を考慮して適宜決定すればよいが、標的細胞および／もしくは不要細胞の検出を、当該細胞の膜表面タンパク質に対する蛍光標識抗体を用いて行なう場合、後述の実施例および比較例からＰＥＧ濃度が１．０％（ｗ／ｖ）以上とすると蛍光輝度が低下する傾向となるため、０．１％（ｗ／ｖ）以上１．０％（ｗ／ｖ）未満とすると好ましい。なおＰＥＧ濃度を０．１％（ｗ／ｖ）以上０．７％（ｗ／ｖ）以下の範囲とするとより好ましく、０．２％（ｗ／ｖ）以上０．５％（ｗ／ｖ）以下の範囲とするとさらに好ましい。また本発明の溶血剤に含ませるＰＥＧの分子量としては、平均分子量として６００以上であればよく、１０００以上１００万以下の範囲とすると好ましく、１０００以上１０万以下の範囲とするとさらに好ましい。 The concentration of PEG contained in the hemolytic agent of the present invention may be appropriately determined in consideration of the properties of the protein used for detecting target cells and / or unnecessary cells contained in the blood sample, as in the case of the aldehyde crosslinking agent. When the detection of cells and / or unnecessary cells is performed using a fluorescently labeled antibody against the membrane surface protein of the cells, when the PEG concentration is set to 1.0% (w / v) or more according to Examples and Comparative Examples described later, Since the luminance tends to decrease, it is preferable that the luminance be 0.1% (w / v) or more and less than 1.0% (w / v). The PEG concentration is more preferably in the range of 0.1% (w / v) to 0.7% (w / v), and more preferably in the range of 0.2% (w / v) to 0.5% (w / v). It is more preferable to set the following range. The molecular weight of PEG contained in the hemolytic agent of the present invention may be 600 or more in average molecular weight, preferably in the range of 1,000 to 1,000,000, and more preferably in the range of 1,000 to 100,000.
本発明の溶血剤に含ませるサポニンは、非イオン性界面活性剤であり、赤血球を含む細胞の細胞膜を溶解させることができる。本発明の溶血剤に含ませるサポニンの濃度は、溶血剤添加後の血液試料中に含まれるサポニンの終濃度として０．０１％（ｗ／ｖ）以上１％（ｗ／ｖ）以下の範囲とすると好ましく、０．１％（ｗ／ｖ）以上０．５％（ｗ／ｖ）以下の範囲とするとより好ましい。 Saponin contained in the hemolytic agent of the present invention is a nonionic surfactant and can lyse cell membranes of cells including erythrocytes. The concentration of saponin contained in the hemolytic agent of the present invention is in the range of 0.01% (w / v) to 1% (w / v) as the final concentration of saponin contained in the blood sample after the addition of the hemolytic agent. It is more preferable that the content be in the range of 0.1% (w / v) or more and 0.5% (w / v) or less.
溶血剤に含まれる各成分の重量濃度の比率としては、サポニンに対するポリエチレングリコールの比率が１以上５以下であると好ましく、１以上３以下であるとより好ましく、またアルデヒド架橋剤に対するポリエチレングリコールの比率が０．２以上１以下であると好ましく、０．２以上０．５以下であるとより好ましい。 本発明の溶血剤は血液試料を溶血処理させる際、当該試料に添加する物質のことをいう。したがって本発明の溶血剤を用いて血液試料を溶血処理させる際は、
本発明の溶血剤を構成する全ての物質を一度に血液試料に添加し溶血処理させてもよく、
本発明の溶血剤を構成する一部の物質を血液試料に添加後、前記構成する残りの物質を血液試料に添加し溶血処理させてもよい。
As the ratio of the weight concentration of each component contained in the hemolytic agent, the ratio of polyethylene glycol to saponin is preferably 1 or more and 5 or less, more preferably 1 or more and 3 or less, and the ratio of polyethylene glycol to aldehyde crosslinking agent. Is preferably from 0.2 to 1 and more preferably from 0.2 to 0.5. The hemolytic agent of the present invention refers to a substance to be added to a blood sample when the sample is subjected to hemolysis. Therefore, when performing hemolysis treatment of a blood sample using the hemolytic agent of the present invention,
All the substances constituting the hemolytic agent of the present invention may be added to a blood sample at a time and subjected to hemolysis,
After adding a part of the substance constituting the hemolytic agent of the present invention to the blood sample, the remaining substance constituting the above may be added to the blood sample and subjected to hemolysis.
本発明の溶血剤を血液試料に添加する前に、当該血液試料に安定化剤を添加し、血液試料を保存処理してもよい。当該保存処理を行なうことで、血液の長期保存安定性が向上するため、特に採血後から標的細胞の検出までに時間が要する場合、血液試料に安定化剤を添加するとよい。添加する安定化剤として特に限定はないが、標的細胞および／もしくは不要細胞の検出に用いる、当該細胞質内または当該細胞膜表面のタンパク質が漏出しにくい条件で行なうとよく、一態様として特開２０１７−０５８３２６号公報に記載の安定化剤があげられる。 Before adding the hemolytic agent of the present invention to a blood sample, a stabilizer may be added to the blood sample to preserve the blood sample. By performing the storage treatment, long-term storage stability of the blood is improved. Therefore, particularly when it takes time from the collection of blood to the detection of target cells, a stabilizer may be added to the blood sample. The stabilizer to be added is not particularly limited, and may be used under conditions where the detection of target cells and / or unnecessary cells does not easily leak proteins in the cytoplasm or on the cell membrane surface. Stabilizers described in JP-A-0558326.
本発明の溶血剤を用いて血液試料中に含まれる標的細胞を検出するには、本発明の溶血剤を前記血液試料に添加することで溶血処理後、当該処理液を遠心分離することで標的細胞を含むペレットを回収し、検出すればよい。なお前記ペレットを回収後、検出工程を実施する前に、前記ペレットを親水性高分子を結合したタンパク質に懸濁させる工程を追加すると、血液試料中に含まれる標的細胞を効率的に回収できる点で好ましい。親水性高分子は電荷を持たない親水性高分子であればよく、一例としてポリエチレングリコール、ポリビニルピロリドン、ポリビニルアルコール、ポリ（ヒドロキシアルキル）メタクリレート、ポリアクリルアミド、ホスホリルコリン基を側鎖に有するポリマー、多糖類、ポリペプチドがあげられる。タンパク質は水溶性を有していればよく、一例として血清由来タンパク質や血漿由来タンパク質などの血液由来タンパク質や乳由来タンパク質があげられ、さらに具体的な例として当業者が通常用いる血清由来タンパク質であるウシ血清アルブミン（ＢＳＡ）や、当業者が通常用いる乳由来タンパク質であるカゼインがあげられる。親水性高分子を結合したタンパク質は、親水性高分子とタンパク質とが一定の割合で結合したタンパク質であり、例えば、タンパク質と結合可能な官能基（例えば、Ｎ−ヒドロキシスクシンイミド基）を付与した親水性高分子とタンパク質とを一定のモル比で反応させることで得られる。なお親水性高分子とタンパク質との反応比は、タンパク質に対し親水性高分子を０．０１以上のモル比で反応させればよく、０．５以上のモル比で反応させればより好ましく、２以上のモル比で反応させると最も好ましい（タンパク質に対し親水性高分子を２以上のモル比で反応させると、血液由来タンパク質の場合は前記タンパク質に対し親水性高分子が実測モル比１以上で結合し、乳由来タンパク質の場合は前記タンパク質に対し親水性高分子が実測モル比０．２以上で結合する）。また前記ペレットを、マンニトール、グルコース、スクロースなどの糖を含む溶液に懸濁させると、標的細胞へのダメージが少なくなるため好ましく、前記糖を含む溶液に塩化カルシウムや塩化マグネシウムなどの電解質や、ＢＳＡやカゼイン等のタンパク質をさらに含んでもよい。添加する糖の濃度は等張液となる濃度とすればよく、糖としてマンニトールを用いる場合は終濃度で２５０ｍＭから３５０ｍＭの間とすればよい。 To detect target cells contained in a blood sample using the hemolytic agent of the present invention, the hemolytic treatment is performed by adding the hemolytic agent of the present invention to the blood sample, and then the target is centrifuged. A pellet containing cells may be collected and detected. If the step of suspending the pellet in a protein bound with a hydrophilic polymer is added after the collection of the pellet and before the detection step is performed, target cells contained in the blood sample can be efficiently collected. Is preferred. The hydrophilic polymer may be a hydrophilic polymer having no charge, and examples thereof include polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, poly (hydroxyalkyl) methacrylate, polyacrylamide, a polymer having a phosphorylcholine group in a side chain, and a polysaccharide. And polypeptides. The protein may be water-soluble, and examples include blood-derived proteins and milk-derived proteins such as serum-derived proteins and plasma-derived proteins, and more specific examples are serum-derived proteins commonly used by those skilled in the art. Examples include bovine serum albumin (BSA) and casein, a milk-derived protein commonly used by those skilled in the art. The protein to which a hydrophilic polymer is bound is a protein in which a hydrophilic polymer and a protein are bound at a certain ratio, for example, a hydrophilic group to which a functional group capable of binding to a protein (for example, N-hydroxysuccinimide group) is added. It is obtained by reacting a conductive polymer with a protein at a fixed molar ratio. The reaction ratio between the hydrophilic polymer and the protein may be such that the hydrophilic polymer is reacted with the protein at a molar ratio of 0.01 or more, more preferably at a molar ratio of 0.5 or more, Most preferably, the reaction is carried out at a molar ratio of 2 or more. (When a hydrophilic polymer is reacted with a protein at a molar ratio of 2 or more, in the case of a blood-derived protein, the hydrophilic polymer is actually measured at a molar ratio of 1 or more with respect to the protein. And in the case of a milk-derived protein, a hydrophilic polymer binds to the protein at a measured molar ratio of 0.2 or more). Suspension of the pellet in a solution containing sugars such as mannitol, glucose, and sucrose is preferable because damage to target cells is reduced, and an electrolyte such as calcium chloride or magnesium chloride or BSA is added to the solution containing the sugars. And protein such as casein. The concentration of the sugar to be added may be a concentration at which an isotonic solution is obtained, and when mannitol is used as the sugar, the final concentration may be between 250 mM and 350 mM.
本発明の溶血剤で血液試料を溶血処理し、当該処理液を遠心分離することで分離回収した標的細胞は、例えば、スライドに塗布したり、顕微鏡や光学検出器などで観察したり、フローサイトメトリーを用いて検出すればよい。なお顕微鏡や光学検出器などにより観察することで細胞を検出する場合、前記細胞を含む懸濁液を、前記細胞を保持可能な保持部を有した細胞保持手段に導入し、前記保持部に前記細胞を保持した後、顕微鏡や光学検出器などで観察するとよい。保持部の例として、前記細胞を収納可能な孔や、前記細胞を固定可能な材料（例えば、ポリ−Ｌ−リジン）で覆われた面があげられる。なお保持部の大きさを前記細胞を一つだけ保持可能な大きさとすると、特定細胞の採取および解析（形態学的分析、組織型分析、遺伝子分析など）が容易に行なえる点で好ましい。また細胞を保持部に保持させる際、誘電泳動力を用いると、保持部に細胞を効率的に保持させることができる点で好ましい。誘電泳動力を用いる場合、具体的には、交流電圧を印加することで誘電泳動を発生させ、保持部内へ細胞を導入すればよい。印加する交流電圧は、保持部内の細胞の充放電が周期的に繰り返される波形を有した交流電圧であると好ましく、周波数を１００ｋＨｚから３ＭＨｚの間とし、電界強度を１×１０５Ｖ／ｍから５×１０５Ｖ／ｍの間とすると特に好ましい（ＷＯ２０１１／１４９０３２号および特開２０１２−０１３５４９号公報参照）。 Target cells separated and recovered by subjecting a blood sample to hemolysis with the hemolytic agent of the present invention and centrifuging the treated solution are, for example, applied to slides, observed with a microscope or an optical detector, or analyzed by flow cytometry. What is necessary is just to detect using a metrology. When detecting cells by observing with a microscope or an optical detector or the like, the suspension containing the cells is introduced into a cell holding unit having a holding unit capable of holding the cells, and the holding unit is provided with the suspension. After holding the cells, it is preferable to observe them with a microscope or an optical detector. Examples of the holding portion include a hole capable of storing the cells and a surface covered with a material (eg, poly-L-lysine) capable of fixing the cells. In addition, it is preferable that the size of the holding portion is a size that can hold only one of the cells, because collection and analysis of specific cells (morphological analysis, tissue type analysis, gene analysis, and the like) can be easily performed. It is preferable to use dielectrophoretic force when holding the cells in the holding section, since the holding section can efficiently hold the cells. When the dielectrophoretic force is used, specifically, dielectrophoresis may be generated by applying an AC voltage, and cells may be introduced into the holding unit. The applied AC voltage is preferably an AC voltage having a waveform in which charging and discharging of the cells in the holding unit are periodically repeated. The frequency is set between 100 kHz and 3 MHz, and the electric field intensity is set at 1 × 10 5 V / m. It is particularly preferred to be between 5 × 10 5 V / m (see WO2011 / 149032 and JP-A-2012-013549).
本発明の溶血剤を用いて血液試料中に含まれる標的細胞を検出するには、例えば、本発明の溶血剤で溶血処理した血液試料を遠心分離して標的細胞を分離回収後、少なくとも当該標的細胞内で発現するタンパク質を利用して標的細胞を検出すればよい。標的細胞内で発現するタンパク質に特に限定はなく、一例として、標的細胞がＣＴＣなどの腫瘍細胞の場合、上皮系細胞の細胞質内タンパク質や細胞膜タンパク質をあげることができ、より具体的にはサイトケラチン（ＣＫ）やＥｐＣＡＭ（Ｅｐｉｔｈｅｌｉａｌ Ｃｅｌｌ Ａｄｈｅｓｉｏｎ Ｍｏｌｅｃｕｌｅ）が例示できる。なおＣＫにはＣＫ１からＣＫ２０まで２０種類のタンパク質が知られているが、そのいずれもが前記標的細胞の検出で利用可能な、標的細胞内で発現するタンパク質に含まれる。また、標的細胞でない細胞（不要細胞）も併せて検出することで標的細胞を検出してもよく、一例として血液試料中に含まれる腫瘍細胞を標的細胞として検出する場合、標的細胞以外の不要細胞である白血球を検出することで、標的細胞を検出してもよい。白血球を検出する方法の一例として、白血球細胞の膜タンパク質であるＣＤ４５を検出する方法があげられる。 In order to detect target cells contained in a blood sample using the hemolytic agent of the present invention, for example, centrifuging a blood sample hemolyzed with the hemolytic agent of the present invention to separate and collect the target cells, and then collecting at least the target cells The target cell may be detected using a protein expressed in the cell. The protein expressed in the target cell is not particularly limited. For example, when the target cell is a tumor cell such as CTC, a cytoplasmic protein of an epithelial cell or a cell membrane protein can be mentioned, and more specifically, cytokeratin. (CK) and EpCAM (Epithelial Cell Adhesion Molecular). Note that CK is known to include 20 types of proteins from CK1 to CK20, all of which are included in proteins expressed in target cells that can be used for detection of the target cells. Further, the target cells may be detected by simultaneously detecting cells (unnecessary cells) that are not target cells. For example, when detecting tumor cells contained in a blood sample as target cells, unnecessary cells other than the target cells may be detected. The target cells may be detected by detecting leukocytes that are as follows. One example of a method for detecting leukocytes is a method for detecting CD45, which is a membrane protein of leukocyte cells.
標的細胞内で発現するタンパク質の検出方法に特に限定はなく、当該タンパク質を直接呈色試薬や蛍光試薬で染色させて検出してもよいし、当該タンパク質に対する標識化抗体を用いて検出してもよいし、当該タンパク質の遺伝子を特異的に増幅して検出してもよい。中でも当該タンパク質に対する標識化抗体を用いて検出する方法は、当該タンパク質を簡便、高感度、かつ特異的に検出できる方法であり好ましい方法といえる。なお抗体に標識する物質も特に限定はなく、一例としてフルオレセインイソチオシアネート（ＦＩＴＣ）、Ａｌｅｘａ Ｆｌｕｏｒ（商品名）などの蛍光物質があげられる。 The method for detecting the protein expressed in the target cell is not particularly limited, and the protein may be directly detected by staining with a color reagent or a fluorescent reagent, or may be detected by using a labeled antibody against the protein. Alternatively, the protein gene may be specifically amplified and detected. Above all, a method of detecting the protein using a labeled antibody against the protein is a method capable of detecting the protein simply, with high sensitivity, and specifically, and can be said to be a preferable method. The substance to be labeled on the antibody is not particularly limited, and examples thereof include fluorescent substances such as fluorescein isothiocyanate (FITC) and Alexa Fluor (trade name).
以下、本発明の溶血剤を利用した、血液試料中に含まれる標的細胞の検出方法の一例として、血液試料中に含まれる腫瘍細胞（ＣＴＣ）を検出する方法を用いて詳細に説明するが、本発明は本説明の内容に限定されるものではない。 Hereinafter, as an example of a method for detecting a target cell contained in a blood sample using the hemolytic agent of the present invention, a method for detecting a tumor cell (CTC) contained in a blood sample will be described in detail. The present invention is not limited to the contents of the present description.
（１）がんの疑いのある患者から血液試料を採取する。なお血液試料を採取する際、クエン酸やエチレンジアミン四酢酸（ＥＤＴＡ）などのキレート剤に代表される抗凝固剤を添加すると好ましい。 (1) Collect a blood sample from a patient suspected of having cancer. When collecting a blood sample, it is preferable to add an anticoagulant represented by a chelating agent such as citric acid or ethylenediaminetetraacetic acid (EDTA).
（２）（１）で採取した血液試料に、ＣＴＣを安定化させるためのイミダゾリジニル尿素とＰＥＧとＥＤＴＡとを含む安定化剤を添加する。血液試料への保存剤の添加量は、血液試料１ｍＬあたり０．０１ｍＬから１０ｍＬの間であればよく、０．０４ｍＬから２ｍＬの間であればより好ましい。安定化剤を添加した血液試料は、室温で少なくとも５日間は安定に保存が可能である。 (2) A stabilizer containing imidazolidinyl urea, PEG and EDTA for stabilizing CTC is added to the blood sample collected in (1). The amount of the preservative added to the blood sample may be between 0.01 mL and 10 mL per 1 mL of the blood sample, and more preferably between 0.04 mL and 2 mL. The blood sample to which the stabilizer is added can be stably stored at room temperature for at least 5 days.
（３）安定化剤を添加した血液試料（血液保存試料）に含まれる赤血球を、本発明の溶血剤（ホルマリンとＰＥＧとサポニンとを少なくとも含む）を用いて破砕する（溶血処理する）。本操作により、分離回収したＣＴＣの観察が良好になる。なお溶血処理時間は３０分以内がよく、１０分以内であればより好ましい。 (3) Erythrocytes contained in a blood sample (blood preservation sample) to which a stabilizer has been added are crushed (haemolyzed) using the hemolytic agent of the present invention (containing at least formalin, PEG and saponin). This operation improves the observation of the separated and recovered CTC. The hemolysis time is preferably within 30 minutes, more preferably within 10 minutes.
（４）赤血球破砕処理（溶血処理）後、遠心分離することで血液成分を除去し、血液試料中に含まれるＣＴＣをペレット状にした後、適切な溶液を用いてＣＴＣを懸濁させる。なおＣＴＣを懸濁させる溶液に、親水性高分子を結合したタンパク質（例えば、ＰＥＧを結合したＢＳＡ）を含ませてもよい。当該タンパク質を含ませるとＣＴＣの回収効率が向上するため好ましい。親水性高分子を結合したタンパク質の濃度は、懸濁液でのタンパク質の終濃度として、０．０１％（ｗ／ｖ）以上２５％（ｗ／ｖ）以下の範囲であればよく、０．０２％（ｗ／ｖ）以上５％（ｗ／ｖ）以下の範囲であれば好ましく、０．０５％（ｗ／ｖ）以上２％（ｗ／ｖ）以下の範囲であればより好ましい。 (4) After the erythrocyte crushing treatment (hemolysis treatment), the blood component is removed by centrifugation, the CTC contained in the blood sample is pelletized, and the CTC is suspended using an appropriate solution. The solution in which the CTC is suspended may contain a protein having a hydrophilic polymer bound thereto (for example, PEG-bound BSA). It is preferable to include the protein because the recovery efficiency of CTC is improved. The concentration of the protein bound with the hydrophilic polymer may be in the range of 0.01% (w / v) to 25% (w / v) as the final concentration of the protein in the suspension. The range is preferably from 02% (w / v) to 5% (w / v), more preferably from 0.05% (w / v) to 2% (w / v).
（５）（４）で調製したＣＴＣを含む懸濁液を再度遠心分離し、ＣＴＣを含むペレットを回収する。なお必要に応じ、前記回収したペレットを親水性高分子を結合したタンパク質を含む溶液に再度懸濁させ、遠心分離する工程を追加してもよい。 (5) The suspension containing CTC prepared in (4) is centrifuged again to collect a pellet containing CTC. If necessary, a step of resuspending the recovered pellet in a solution containing a protein to which a hydrophilic polymer is bound and centrifuging the recovered pellet may be added.
（６）（５）で得られたＣＴＣを、例えばＷＯ２０１１／１４９０３２号に記載の装置を用いて保持部へ保持させた後、当該ＣＴＣに対し保存および膜透過処理を施す。保存処理剤としては、アルデヒド架橋剤（例えば、ホルムアルデヒド、ホルムアルデヒドドナー化合物（加水分解を受けることでホルムアルデヒドを放出可能な化合物）、グルタルアルデヒド）や、メタノール、エタノールなどのアルコール類や、重金属を含む溶液が例示できる。細胞膜透過処理剤としては、メタノール、エタノールなどのアルコール類や、サポニンなどの界面活性剤が例示できる。 (6) After holding the CTC obtained in (5) in a holding unit using, for example, an apparatus described in WO2011 / 149032, the CTC is subjected to storage and membrane permeation processing. Examples of preservatives include aldehyde cross-linking agents (eg, formaldehyde, formaldehyde donor compounds (compounds that can release formaldehyde by undergoing hydrolysis), glutaraldehyde), alcohols such as methanol and ethanol, and solutions containing heavy metals. Can be exemplified. Examples of the cell membrane permeation treating agent include alcohols such as methanol and ethanol, and surfactants such as saponin.
（７）抗体による非特異的な反応を防ぐため、保存および膜透過処理後のＣＴＣを保持した保持部に対しタンパク質によるブロッキング処理を施した後、蛍光基が修飾されたＣＴＣが発現するタンパク質に対する抗体や、細胞核を蛍光染色させる試薬を添加し、洗浄後、蛍光顕微鏡などで細胞の蛍光像を観察することで、ＣＴＣを検出する。ＣＴＣが発現するタンパク質に対する抗体としては、抗サイトケラチン抗体や抗ＥｐＣＡＭ抗体などを用いることができる。また不要細胞である白血球を検出することを目的に、抗ＣＤ４５抗体といった白血球を特異的に認識する抗体を用いてもよい。細胞核を蛍光染色させる試薬としては、４’，６−ｄｉａｍｉｄｉｎｏ−２−ｐｈｅｎｙｌｉｎｄｏｌｅ（ＤＡＰＩ）やＨｏｅｃｈｓｔ ３３３４２（商品名）などを用いることができる。 (7) In order to prevent non-specific reactions due to the antibody, after performing a blocking treatment with a protein on the holding portion holding the CTC after storage and membrane permeation treatment, the CTC with a fluorescent group-modified CTC is expressed. CTC is detected by adding an antibody or a reagent for fluorescently staining cell nuclei, washing, and observing a fluorescent image of the cells with a fluorescent microscope or the like. As an antibody against a protein expressed by CTC, an anti-cytokeratin antibody, an anti-EpCAM antibody, or the like can be used. For the purpose of detecting leukocytes, which are unnecessary cells, an antibody such as an anti-CD45 antibody that specifically recognizes leukocytes may be used. As a reagent for fluorescently staining the cell nucleus, 4 ', 6-diamidino-2-phenylindole (DAPI), Hoechst 33342 (trade name), or the like can be used.
本発明は、血液試料を溶血処理するための溶血剤であって、アルデヒド架橋剤とＰＥＧとサポニンとを少なくとも含むことを特徴としている。 The present invention relates to a hemolytic agent for hemolyzing a blood sample, which is characterized by containing at least an aldehyde crosslinking agent, PEG and saponin.
血液試料を溶血させる方法として従来用いられている、塩化アンモニウム溶液を用いた方法や、従来の界面活性剤を用いた溶血法では、細胞質内タンパク質や細胞膜タンパク質を精度高く検出することは困難であった。一方、本発明の溶血剤を用いることで、細胞質内タンパク質の検出精度が向上し、かつ従来の界面活性剤を用いた溶血剤での課題であった細胞膜タンパク質の漏出も抑制できたため、血液試料中に含まれる標的細胞の検出を、当該細胞質内および／もしくは当該細胞膜に存在するタンパク質に基づき、精度高く行なえる。 It has been difficult to accurately detect cytoplasmic proteins and cell membrane proteins by a method using an ammonium chloride solution, which has been conventionally used as a method for hemolyzing a blood sample, or a conventional hemolysis method using a surfactant. Was. On the other hand, by using the hemolytic agent of the present invention, the detection accuracy of cytoplasmic proteins was improved, and leakage of cell membrane proteins, which was a problem with a conventional hemolytic agent using a surfactant, could be suppressed. Detection of target cells contained therein can be performed with high accuracy based on proteins present in the cytoplasm and / or the cell membrane.
一例として本発明を、血液試料中に含まれる腫瘍細胞（ＣＴＣ）の検出に適用することで、ＣＴＣの有無の判断結果に対する信頼性が向上し、精度高くがんを検出できる。 For example, by applying the present invention to the detection of tumor cells (CTC) contained in a blood sample, the reliability of the determination result of the presence or absence of CTC is improved, and cancer can be detected with high accuracy.
以下、実施例、比較例および参考例を用いて本発明をさらに詳細に説明するが、本発明はこれら例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples, Comparative Examples, and Reference Examples, but the present invention is not limited to these examples.
実施例１
（１）アルデヒド架橋剤であるイミダゾリジニル尿素２．３ｇ、分子量６０００のポリエチレングリコール（ＰＥＧ）２．３ｇ、エチレンジアミン四酢酸（ＥＤＴＡ）３０ｍｇを、超純水で総量３０ｍＬになるよう溶解し、得られた溶液を安定化剤として用いた。
Example 1
(1) 2.3 g of imidazolidinyl urea as an aldehyde cross-linking agent, 2.3 g of polyethylene glycol (PEG) having a molecular weight of 6000, and 30 mg of ethylenediaminetetraacetic acid (EDTA) were dissolved in ultrapure water to a total volume of 30 mL. The solution was used as a stabilizer.
（２）インフォームドコンセントを得た健常人から血液をＥＤＴＡ−２Ｋ採血管（ＶＰ−ＤＫ０５０Ｋ、テルモ社製）に５ｍＬ採血後、前記採血した血液０．２ｍＬ、および（１）で調製した安定化剤０．０３ｍＬを添加し、得られた溶液を血液保存試料とした。 (2) Blood was collected from a healthy individual who obtained informed consent in an EDTA-2K blood collection tube (VP-DK050K, manufactured by Terumo Corporation) after 5 mL of blood was collected, 0.2 mL of the collected blood, and the stabilization prepared in (1). 0.03 mL of the agent was added, and the resulting solution was used as a blood preservation sample.
（３）血液保存試料を２５℃で１０分間放置した。 (3) The blood preservation sample was left at 25 ° C. for 10 minutes.
（４）放置後の血液保存試料全量に対して、以下の（ａ）から（ｆ）に示すいずれかの界面活性剤とアルデヒド架橋剤であるホルムアルデヒドとを含むＰＢＳ（Ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ）を添加した。なおホルムアルデヒドの終濃度は１％（ｗ／ｖ）であり、ＰＥＧの終濃度は０．２９％（ｗ／ｖ）である。
（ａ）サポニン（終濃度：０．２％（ｗ／ｖ））
（ｂ）Ｔｒｉｔｏｎ Ｘ−１００（商品名）（終濃度：０．２％（ｗ／ｖ））
（ｃ）ＣＨＡＰＳ（３−［（３−Ｃｈｏｌａｍｉｄｏｐｒｏｐｙｌ）ｄｉｍｅｔｈｙｌａｍｍｏｎｉｏ］ｐｒｏｐａｎｅｓｕｌｆｏｎａｔｅ）（終濃度：０．２％（ｗ／ｖ））
（ｄ）デオキシコール酸ナトリウム（終濃度：０．２％（ｗ／ｖ））
（ｅ）Ｔｗｅｅｎ ２０（商品名）（終濃度：０．２（ｗ／ｖ））
（ｆ）Ｔｗｅｅｎ ２０（商品名）（終濃度：１．２（ｗ／ｖ））
（５）界面活性剤添加後の血液保存試料を２５℃で１０分放置した。
(4) Phosphate buffered saline (PBS) containing any one of the following surfactants (a) to (f) and formaldehyde as an aldehyde crosslinker was added to the whole amount of the preserved blood sample. . The final concentration of formaldehyde is 1% (w / v), and the final concentration of PEG is 0.29% (w / v).
(A) Saponin (final concentration: 0.2% (w / v))
(B) Triton X-100 (trade name) (final concentration: 0.2% (w / v))
(C) CHAPS (3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate) (final concentration: 0.2% (w / v))
(D) Sodium deoxycholate (final concentration: 0.2% (w / v))
(E) Tween 20 (trade name) (final concentration: 0.2 (w / v))
(F) Tween 20 (trade name) (final concentration: 1.2 (w / v))
(5) The blood preservation sample after addition of the surfactant was left at 25 ° C. for 10 minutes.
（６）（５）で放置した試料の濁度を目視で観察することで、溶血能を評価した。具体的には、赤血球が破砕されている（溶血している）と前記試料の透過性が高くなる一方、溶血していないと濁度が高く透過性が低くなることを利用し、評価した。 (6) The hemolytic ability was evaluated by visually observing the turbidity of the sample left in (5). Specifically, the evaluation was performed using the fact that the permeability of the sample was increased when erythrocytes were crushed (has lysed), whereas the turbidity was high and the permeability was low when erythrocytes were not lysed.
溶血能の結果をまとめて表１に示す。界面活性剤として、サポニン、Ｔｒｉｔｏｎ Ｘ−１００およびデオキシコール酸ナトリウムを用いたときは、血液保存試料の透過性が上がり、溶血されていることが確認された。一方、界面活性剤として、ＣＨＡＰＳおよびＴｗｅｅｎ ２０を用いたときは、溶血が確認されなかった。非イオン性界面活性剤であるＴｗｅｅｎ ２０および両性界面活性剤であるＣＨＡＰＳでは溶血できないのに対して、非イオン性界面活性剤であるサポニンおよびＴｒｉｔｏｎ Ｘ−１００ならびに陰イオン性界面活性剤であるデオキシコール酸ナトリウムでは溶血できることから、血液試料の溶血能は、界面活性剤が有する電荷とは無関係であることがわかる。
Table 1 summarizes the results of the hemolytic ability. When saponin, Triton X-100 and sodium deoxycholate were used as the surfactant, the permeability of the blood preservation sample was increased, and it was confirmed that the sample was hemolyzed. On the other hand, when CHAPS and
実施例２
（１）一方の末端がメトキシ基であり、もう一方の末端がＮ−ヒドロオキシスクシンイミドエステル基である、分子量５０００のポリエチレングリコール（ｍＰＥＧ−ＮＨＳ）と、ウシ血清アルブミン（ＢＳＡ）（３００ｍｇ、０．３ｍｍｏｌ）とを、炭酸水素ナトリウム緩衝液（０．１Ｍ、１５ｍＬ）に溶解させ、当該溶液を２５℃近傍で３時間撹拌することでポリエチレングリコールを結合したＢＳＡ（ＰＥＧ−ＢＳＡ）を調製した。なお調製する際、ｍＰＥＧ−ＮＨＳとＢＳＡとのモル比（ｍＰＥＧ−ＮＨＳ／ＢＳＡ）を２となるようにした。調製後、分画分子量１００００の透析膜を用いて、純水への溶液置換を３日間行なった。
Example 2
(1) One end is a methoxy group and the other end is an N-hydroxysuccinimide ester group, a polyethylene glycol having a molecular weight of 5000 (mPEG-NHS), and bovine serum albumin (BSA) (300 mg, 0.1 mg / ml). 3 mmol) was dissolved in a sodium hydrogen carbonate buffer solution (0.1 M, 15 mL), and the solution was stirred at about 25 ° C. for 3 hours to prepare polyethylene glycol-bound BSA (PEG-BSA). In the preparation, the molar ratio between mPEG-NHS and BSA (mPEG-NHS / BSA) was adjusted to 2. After the preparation, the solution was replaced with pure water for 3 days using a dialysis membrane having a molecular weight cut off of 10,000.
（２）インフォームドコンセントを得た健常人から血液をＥＤＴＡ−２Ｋ採血管（ＶＰ−ＤＫ０５０Ｋ、テルモ社製）に５ｍＬ採血後、前記採血した血液０．２ｍＬ、および実施例１（１）で調製した安定化剤０．０３ｍＬを添加し、得られた溶液を血液保存試料とした。 (2) Blood was collected from a healthy individual who obtained informed consent in an EDTA-2K blood collection tube (VP-DK050K, manufactured by Terumo Corporation) after collecting 5 mL of the blood, and then prepared in 0.2 mL of the collected blood and Example 1 (1). 0.03 mL of the obtained stabilizer was added, and the obtained solution was used as a blood preservation sample.
（３）血液保存試料を２５℃で１０分間放置した。 (3) The blood preservation sample was left at 25 ° C. for 10 minutes.
（４）（３）で放置した試料全量に対して、界面活性剤であるサポニンとアルデヒド架橋剤であるホルムアルデヒドとＰＥＧとを含むＰＢＳを添加した。前記ＰＢＳを血液保存試料に添加した後の液量は０．８ｍＬであり、各成分の終濃度は、サポニンが０．２％（ｗ／ｖ）であり、ホルムアルデヒドが１％（ｗ／ｖ）であり、ＰＥＧが１％（ｗ／ｖ）である。
(4) PBS containing surfactant, saponin, formaldehyde, which is an aldehyde cross-linking agent, and PEG, was added to the entire amount of the sample left in (3). The solution volume after adding the PBS to the blood preservation sample was 0.8 mL, and the final concentration of each component was saponin 0.2% (w / v) and
（５）溶血剤添加後の血液試料を２５℃で５分放置し、３００×ｇで５分間、室温にて遠心分離し、上清を除去した。 (5) The blood sample after addition of the hemolytic agent was allowed to stand at 25 ° C. for 5 minutes, centrifuged at 300 × g for 5 minutes at room temperature, and the supernatant was removed.
（６）血液細胞を含むペレットを、（１）で調製したＰＥＧ−ＢＳＡ（ＢＳＡとして０．１％（ｗ／ｖ））および３００ｍＭマンニトールを含む溶液１ｍＬを添加して再懸濁し、３００×ｇで５分間、室温にて遠心分離した。上清を除去し、血液細胞を含むペレットを再度ＰＥＧ−ＢＳＡ（ＢＳＡとして０．１％（ｗ／ｖ））および３００ｍＭマンニトールを含む溶液１ｍＬで再懸濁した。本操作は、血液成分および溶血剤成分（サポニンおよびアルデヒド架橋剤）を除去するための操作である。 (6) The pellet containing blood cells is resuspended by adding 1 mL of a solution containing PEG-BSA (0.1% (w / v) as BSA) and 300 mM mannitol prepared in (1), and resuspended at 300 × g. For 5 minutes at room temperature. The supernatant was removed, and the pellet containing blood cells was resuspended again in 1 mL of a solution containing PEG-BSA (0.1% (w / v) as BSA) and 300 mM mannitol. This operation is an operation for removing a blood component and a hemolytic agent component (saponin and aldehyde crosslinking agent).
（７）（６）で上清を除去した血液細胞を含む懸濁液を図１および２に示す細胞保持装置１００に導入し、信号発生器５０から電極基板３１・３２に導線４０を介して交流電圧（１ＭＨｚ、２０Ｖｐ−ｐ）を１０分間印加することで前記装置が有する保持部６０に血液細胞７０を保持させた。本実施例で用いた細胞保持装置１００は、直径３０μｍの貫通孔１２ａを複数有した絶縁体１２と直径３０μｍの貫通孔１１ａを複数有した遮光性のクロム膜（遮光部材１１）と電極基板３１とを上から絶縁体１２−遮光部材１１−電極基板３１の順に密着して設け、さらに絶縁体１２の上面に試料の導入口２１、排出口２２および貫通部２３を有する厚さ１ｍｍのスペーサー２０を、スペーサー２０の上面に電極基板３２を、それぞれ密着して設けてなる装置である。なお貫通孔１１ａ／１２ａおよび電極基板３１により、直径３０μｍ、深さ４０μｍからなる細胞７０を保持可能な保持部６０が形成されている。
(7) The suspension containing the blood cells from which the supernatant has been removed in (6) is introduced into the
（８）（７）の条件で交流電圧を印加しながら、０．０１（ｗ／ｖ）％のポリ−Ｌ−リジンを含む３００ｍＭマンニトール水溶液を導入し、２分間静置後、前記交流電圧の印加を停止し、前記水溶液を吸引除去した。 (8) While applying an AC voltage under the conditions of (7), a 300 mM mannitol aqueous solution containing 0.01 (w / v)% poly-L-lysine was introduced, and allowed to stand for 2 minutes. The application was stopped, and the aqueous solution was removed by suction.
（９）５０％（ｖ／ｖ）エタノールと１％（ｗ／ｖ）ホルムアルデヒドを含む水溶液（以下、細胞膜透過試薬）を導入し、１０分間静置することで、細胞膜を透過させ、保持部に導入した細胞を標本化した。 (9) An aqueous solution containing 50% (v / v) ethanol and 1% (w / v) formaldehyde (hereinafter referred to as a cell membrane permeating reagent) is introduced, and allowed to stand for 10 minutes to allow the cell membrane to permeate and to be retained in the holding portion. The transfected cells were sampled.
（１０）細胞膜透過試薬を吸引除去し、ＰＢＳを導入することで、残留した細胞膜透過試薬を洗浄した。 (10) The cell membrane permeating reagent was removed by suction, and PBS was introduced to wash the remaining cell membrane permeating reagent.
（１１）細胞膜外のタンパク質と特異的に結合可能な蛍光標識された抗体と、細胞核を標識する蛍光試薬を含む水溶液（以下、標識試薬）を導入し、３０分間静置した。なお前記標識された抗体として、白血球表面に発現しているＣＤ４５に対する抗体を用いている。 (11) An aqueous solution (hereinafter, labeled reagent) containing a fluorescently labeled antibody capable of specifically binding to a protein outside the cell membrane and a fluorescent reagent for labeling cell nuclei was introduced, and allowed to stand for 30 minutes. Note that an antibody against CD45 expressed on the surface of leukocytes is used as the labeled antibody.
（１２）標識試薬を吸引除去し、ＰＢＳを導入することで、残留した標識試薬を除去した。 (12) The labeling reagent was removed by suction, and PBS was introduced to remove the remaining labeling reagent.
（１３）保持部６０に対して蛍光顕微鏡による蛍光観察を行なった。細胞核の蛍光が確認され、かつＣＤ４５抗体の蛍光が確認された細胞を白血球として、前記白血球へのＣＤ４５抗体の結合度合いを、蛍光輝度分布を基に評価した。
(13) The holding
比較例１
実施例２（４）で調製した溶液に含まれる界面活性剤として、終濃度０．２％（ｗ／ｖ）のサポニンに替えて、終濃度０．２％（ｗ／ｖ）のＴｒｉｔｏｎ Ｘ−１００（商品名）または終濃度０．２％（ｗ／ｖ）のデオキシコール酸ナトリウムを用いた他は、実施例２と同様な方法で、白血球へのＣＤ４５抗体の結合度合いを、蛍光輝度分布を基に評価した。
Comparative Example 1
As a surfactant contained in the solution prepared in Example 2 (4), instead of saponin having a final concentration of 0.2% (w / v), Triton X- having a final concentration of 0.2% (w / v) was used. 100 (trade name) or sodium deoxycholate at a final concentration of 0.2% (w / v), except that the degree of binding of the CD45 antibody to leukocytes was determined in the same manner as in Example 2, except for the fluorescent luminance distribution. The evaluation was based on
実施例２および比較例１での蛍光輝度分布の結果をまとめて図３に示す。界面活性剤としてサポニンを用いる（実施例２、図３（Ａ））ことで、Ｔｒｉｔｏｎ Ｘ−１００（ｚ図３（Ｂ））およびデオキシコール酸ナトリウム（図３（Ｃ））を用いた（比較例１）ときと比較し、ＣＤ４５蛍光輝度が大幅に上昇しており、白血球へのＣＤ４５抗体の結合度合いが向上していることがわかる。 FIG. 3 shows the results of the fluorescence luminance distribution in Example 2 and Comparative Example 1. By using saponin as a surfactant (Example 2, FIG. 3 (A)), Triton X-100 (z FIG. 3 (B)) and sodium deoxycholate (FIG. 3 (C)) were used (comparison). Compared to Example 1), the CD45 fluorescence luminance was significantly increased, indicating that the degree of binding of the CD45 antibody to leukocytes was improved.
具体的には、蛍光輝度が低い（蛍光輝度０．１１以下）細胞の割合が、デオキシコール酸ナトリウムを用いたときは全体の４３％であるのに対し、サポニンを用いたときは全体の６％と大幅に減少した。一方、蛍光輝度が高い（蛍光輝度が０．５１以上）細胞の割合は、Ｔｒｉｔｏｎ Ｘ−１００を用いたときは全体の５％であるのに対し、サポニンを用いたときは全体の１６％と大幅に上昇した。 Specifically, the percentage of cells having a low fluorescence luminance (fluorescence luminance of 0.11 or less) is 43% of the total when sodium deoxycholate is used, and 6% when the saponin is used. % Decreased significantly. On the other hand, the ratio of cells having high fluorescence luminance (fluorescence luminance is 0.51 or more) is 5% of the whole when using Triton X-100, and 16% of the whole when using saponin. Rose significantly.
参考例１
（１）インフォームドコンセントを得た健常人から血液をＥＤＴＡ−２Ｋ採血管（ＶＰ−ＤＫ０５０Ｋ、テルモ社製）に５ｍＬ採血した。
Reference Example 1
(1) Blood was collected from a healthy individual who obtained informed consent in an EDTA-2K blood collection tube (VP-DK050K, manufactured by Terumo Corporation) at a volume of 5 mL.
（２）採血した血液０．２ｍＬに対して、以下の（Ａ）から（Ｃ）に示すいずれかの溶液を添加した。なお（Ａ）または（Ｂ）の溶液を添加した場合、前記溶液を血液保存試料に添加した後の液量は０．６ｍＬである。
（Ａ）サポニン（終濃度０．２％（ｗ／ｖ））を含むＰＢＳ
（Ｂ）サポニン（終濃度０．２％（ｗ／ｖ））とホルムアルデヒド（終濃度１％（ｗ／ｖ））とを含むＰＢＳ
（Ｃ）０．９％（ｗ／ｖ）塩化アンモニウムと０．１％（ｗ／ｖ）炭酸水素カリウムと含む水溶液１０ｍＬ
（３）実施例２（５）から（１３）と同様な方法で、白血球へのＣＤ４５抗体の結合度合いを、蛍光輝度分布を基に評価した。
(2) One of the following solutions (A) to (C) was added to 0.2 mL of the collected blood. When the solution (A) or (B) is added, the volume of the solution after adding the solution to the blood preservation sample is 0.6 mL.
(A) PBS containing saponin (final concentration 0.2% (w / v))
(B) PBS containing saponin (final concentration 0.2% (w / v)) and formaldehyde (
(C) 10 mL of an aqueous solution containing 0.9% (w / v) ammonium chloride and 0.1% (w / v) potassium hydrogen carbonate
(3) In the same manner as in Examples 2 (5) to (13), the degree of binding of the CD45 antibody to leukocytes was evaluated based on the fluorescence luminance distribution.
参考例１での蛍光輝度分布の結果を図４に示す。血液試料の溶血を行なう溶血剤として、サポニンのみを用いた場合、塩化アンモニウムを含む溶血剤を用いた場合（最大輝度分布を示す細胞の蛍光輝度０．８２から１．００、図４（Ｃ））と比較して、ＣＤ４５抗体の結合による最大輝度分布を示す白血球の蛍光輝度が大幅に低下（最大輝度分布を示す細胞の蛍光輝度０．００から０．１１、図４（Ａ））しており、白血球へのＣＤ４５抗体の結合能が大幅に低下していることがわかる。この結果から、サポニンのみを溶血剤として用いた場合、細胞膜タンパク質であるＣＤ４５が細胞外に漏出し、ＣＤ４５抗体の結合性が低下したのに対して、塩化アンモニウムを含む溶血剤を用いた場合は、細胞膜に大きな損傷を与えることなく溶血できるため、ＣＤ４５抗体の結合性が低下せず、細胞膜タンパク質（ＣＤ４５）の染色能が高い結果となったことがわかる。 FIG. 4 shows the result of the fluorescence luminance distribution in Reference Example 1. When only a saponin is used as a hemolytic agent for hemolyzing a blood sample, and when a hemolytic agent containing ammonium chloride is used (fluorescent luminance of cells exhibiting the maximum luminance distribution from 0.82 to 1.00, FIG. 4C) ), The fluorescence luminance of leukocytes exhibiting the maximum luminance distribution due to the binding of the CD45 antibody is significantly reduced (the fluorescence luminance of the cells exhibiting the maximum luminance distribution is from 0.00 to 0.11, FIG. 4 (A)). Thus, it can be seen that the binding ability of the CD45 antibody to leukocytes is significantly reduced. From these results, it was found that when only saponin was used as a hemolytic agent, CD45, which is a cell membrane protein, leaked out of the cell and the binding of the CD45 antibody was reduced, whereas when a hemolytic agent containing ammonium chloride was used, It can be seen that hemolysis can be performed without causing significant damage to the cell membrane, so that the binding ability of the CD45 antibody does not decrease and the staining ability of the cell membrane protein (CD45) is high.
一方、血液試料の溶血を行なう溶血剤としてサポニンの他にホルマリンをＰＢＳに入れることで、サポニンのみをＰＢＳに加えた場合と比較し、ＣＤ４５抗体の結合による最大輝度分布を示す白血球の蛍光輝度が大幅に向上（最大輝度分布を示す細胞の蛍光輝度０．８２から１．００、図４（Ｂ））しており、白血球へのＣＤ４５抗体の結合能が大幅に向上していることがわかる。この結果から、サポニンの他にホルムアルデヒドを含むホルマリンを添加することでタンパク質固定能が向上し、細胞膜タンパク質の染色能が向上したことがわかる。 On the other hand, by adding formalin to PBS in addition to saponin as a hemolytic agent for hemolyzing a blood sample, compared to the case where only saponin was added to PBS, the fluorescence luminance of leukocytes showing the maximum luminance distribution due to the binding of the CD45 antibody was reduced. The fluorescence intensity of the cells exhibiting the maximum luminance distribution is significantly improved (from 0.82 to 1.00, FIG. 4B), indicating that the binding ability of the CD45 antibody to leukocytes is greatly improved. From these results, it can be seen that the addition of formalin containing formaldehyde in addition to saponin improved the protein fixing ability and the staining ability of cell membrane proteins.
参考例２
（１）ヒト非小細胞肺がん細胞（ＰＣ９）を、５％ＣＯ２環境下、１０％ＦＢＳを含むＤ−ＭＥＭ／Ｈａｍ’ｓ Ｆ−１２培地を用いて３７℃で２４時間から９６時間培養後、０．２５％トリプシン／１ｍＭ ＥＤＴＡを用いて培地から細胞を剥離した。
Reference example 2
(1) After culturing human non-small cell lung cancer cells (PC9) in D-MEM / Ham's F-12 medium containing 10% FBS in a 5% CO 2 environment at 37 ° C. for 24 to 96 hours The cells were detached from the medium using 0.25% trypsin / 1 mM EDTA.
（２）剥離したＰＣ９細胞懸濁液０．２ｍＬに対して、界面活性剤であるサポニンとＰＢＳとを含む溶血剤０．４ｍＬ、または０．９％（ｗ／ｖ）塩化アンモニウムと０．１％（ｗ／ｖ）炭酸水素カリウムと含む水溶液を１０ｍＬ添加した。界面活性剤を含む溶血剤をＰＣ９細胞懸濁液に添加した際のサポニンの終濃度は０．１％（ｗ／ｖ）となるよう溶血剤を調製した。 (2) To 0.2 mL of the detached PC9 cell suspension, 0.4 mL of a hemolytic agent containing saponin as a surfactant and PBS or 0.1% (w / v) ammonium chloride and 0.1% 10 mL of an aqueous solution containing% (w / v) potassium bicarbonate was added. The hemolytic agent was prepared such that the final concentration of saponin when the hemolytic agent containing a surfactant was added to the PC9 cell suspension was 0.1% (w / v).
（３）細胞として（２）で調製したＰＣ９細胞を用いた他は、実施例２（５）から（１０）と同様な方法で、細胞の膜透過処理を行なった。 (3) The cells were subjected to membrane permeation treatment in the same manner as in Examples 2 (5) to (10), except that the PC9 cells prepared in (2) were used as the cells.
（４）細胞質内のタンパク質と特異的に結合可能な蛍光標識された抗体と、細胞核を標識する蛍光試薬を含む水溶液（以下、標識試薬）を導入し、３０分間静置した。なお前記標識された抗体として、血中循環がん細胞（ＣＴＣ）の細胞質内で発現しているサイトケラチンに対する抗体を用いている。 (4) An aqueous solution (hereinafter, labeling reagent) containing a fluorescently labeled antibody capable of specifically binding to a protein in the cytoplasm and a fluorescent reagent for labeling cell nuclei was introduced, and allowed to stand for 30 minutes. As the labeled antibody, an antibody against cytokeratin expressed in the cytoplasm of circulating cancer cells (CTC) is used.
（５）標識試薬を吸引除去し、ＰＢＳを導入することで、残留した標識試薬を除去した。 (5) The labeling reagent was removed by suction, and the remaining labeling reagent was removed by introducing PBS.
（６）（５）で標識した細胞が保持された保持部６０に対して蛍光顕微鏡による蛍光観察を行なった。細胞核の蛍光が確認され、かつサイトケラチン抗体の蛍光が確認された細胞をＰＣ９細胞として、前記ＰＣ９細胞へのサイトケラチン抗体の結合度合いを、蛍光輝度分布を基に評価した。
(6) Fluorescence observation was performed with a fluorescence microscope on the holding
参考例２での蛍光輝度分布の結果を図５に示す。血液試料の溶血を、サポニンを含む溶血剤を用いて行なうことで、塩化アンモニウムを含む溶血剤を用いた場合（最大輝度分布を示す細胞の蛍光輝度０．０８から０．１５、図５（Ｂ））と比較し、サイトケラチン抗体の結合による最大輝度分布を示すＰＣ９細胞の蛍光輝度が大幅に向上（最大輝度分布を示す細胞の蛍光輝度０．７８から１．００、図５（Ａ））しており、ＰＣ９細胞へのサイトケラチン抗体の結合能が大幅に向上していることがわかる。 FIG. 5 shows the result of the fluorescence luminance distribution in Reference Example 2. The hemolysis of the blood sample is performed using a hemolytic agent containing saponin, so that a hemolytic agent containing ammonium chloride is used (the fluorescent luminance of the cell exhibiting the maximum luminance distribution is 0.08 to 0.15; FIG. )), The fluorescence brightness of PC9 cells showing the maximum brightness distribution due to the binding of the cytokeratin antibody is greatly improved (the fluorescence brightness of the cells showing the maximum brightness distribution from 0.78 to 1.00, FIG. 5 (A)). Thus, it can be seen that the binding ability of the cytokeratin antibody to PC9 cells has been significantly improved.
サポニンのみを溶血剤として用いた場合、細胞質内タンパク質を抗体等で染色する際は高い染色能を示す一方、細胞膜タンパク質を抗体等で染色する際は、細胞膜タンパク質が漏出してしまい染色能が低下してしまう。このことから、細胞質内および細胞膜タンパク質を共に高い染色能で染色するためには、サポニンとホルムアルデヒドとを少なくとも含む溶血剤を用いる必要があることがわかる。 When only saponin is used as a hemolytic agent, it shows high staining ability when staining cytoplasmic proteins with antibodies, etc., but when staining cell membrane proteins with antibodies, etc., cell membrane proteins leak out and staining ability is reduced Resulting in. This indicates that it is necessary to use a hemolytic agent containing at least saponin and formaldehyde in order to stain both cytoplasmic and cell membrane proteins with high staining ability.
実施例３
実施例２（４）で調製した溶液に含まれるＰＥＧの濃度を、０．２９％（ｗ／ｖ）、０．４４％（ｗ／ｖ）、０．６６％（ｗ／ｖ）のいずれかとした他は、実施例２と同様な方法で、白血球へのＣＤ４５抗体の結合度合いを、蛍光輝度分布を基に評価した。
Example 3
The concentration of PEG contained in the solution prepared in Example 2 (4) was set to 0.29% (w / v), 0.44% (w / v), or 0.66% (w / v). Other than the above, the degree of binding of the CD45 antibody to leukocytes was evaluated based on the fluorescence luminance distribution in the same manner as in Example 2.
比較例２
（１）アルデヒド架橋剤であるイミダゾリジニル尿素２．３ｇ、ＥＤＴＡ ３０ｍｇを、超純水で総量３０ｍＬになるよう溶解し、得られた溶液を安定化剤として用いた。
Comparative Example 2
(1) 2.3 g of imidazolidinyl urea, which is an aldehyde cross-linking agent, and 30 mg of EDTA were dissolved in ultrapure water to a total volume of 30 mL, and the obtained solution was used as a stabilizer.
（２）インフォームドコンセントを得た健常人から血液をＥＤＴＡ−２Ｋ採血管（ＶＰ−ＤＫ０５０Ｋ、テルモ社製）に５ｍＬ採血後、前記採血した血液０．２ｍＬ、および（１）で調製した安定化剤０．０３ｍＬを添加し、得られた溶液を血液保存試料とした。 (2) Blood was collected from a healthy individual who obtained informed consent in an EDTA-2K blood collection tube (VP-DK050K, manufactured by Terumo Corporation) after 5 mL of blood was collected, 0.2 mL of the collected blood, and the stabilization prepared in (1). 0.03 mL of the agent was added, and the resulting solution was used as a blood preservation sample.
（３）血液保存試料を２５℃で１０分間放置した。 (3) The blood preservation sample was left at 25 ° C. for 10 minutes.
（４）放置後の血液保存試料全量に対して、界面活性剤であるサポニンとアルデヒド架橋剤であるホルムアルデヒドとを含むＰＢＳを添加した。前記ＰＢＳを血液保存試料に添加した後の液量は０．８ｍＬであり、各成分の終濃度は、サポニンが０．２％（ｗ／ｖ）であり、ホルムアルデヒドが１％（ｗ／ｖ）である。
(4) PBS containing saponin as a surfactant and formaldehyde as an aldehyde cross-linking agent was added to the entire amount of the blood preservation sample after standing. The solution volume after adding the PBS to the blood preservation sample was 0.8 mL, and the final concentration of each component was saponin 0.2% (w / v) and
（５）実施例２（５）から（１３）と同様な方法で、白血球へのＣＤ４５抗体の結合度合いを、蛍光輝度分布を基に評価した。 (5) In the same manner as in Examples 2 (5) to (13), the degree of binding of the CD45 antibody to leukocytes was evaluated based on the fluorescence luminance distribution.
比較例３
実施例２（４）で血液保存試料に添加する溶液として、０．９％（ｗ／ｖ）塩化アンモニウムと０．１％（ｗ／ｖ）炭酸水素カリウムと含む水溶液１０ｍＬを用いた他は、実施例２と同様な方法で、白血球へのＣＤ４５抗体の結合度合いを、蛍光輝度分布を基に評価した。
Comparative Example 3
In Example 2 (4), 10 mL of an aqueous solution containing 0.9% (w / v) ammonium chloride and 0.1% (w / v) potassium hydrogen carbonate was used as a solution to be added to the blood preservation sample. In the same manner as in Example 2, the degree of binding of the CD45 antibody to leukocytes was evaluated based on the fluorescence luminance distribution.
実施例３ならびに比較例２および３での蛍光輝度分布の結果をまとめて図６に示す。アルデヒド架橋剤であるホルムアルデヒドとサポニンとＰＥＧとを含む溶血剤（図６（Ｂ）から（Ｅ））は、アルデヒド架橋剤であるホルムアルデヒドとサポニンとを含みＰＥＧを含まない溶血剤（図６（Ａ））や塩化アンモニウムを含む溶血剤（図６（Ｆ））と比較し、ＣＤ４５蛍光輝度が上昇しており、白血球へのＣＤ４５抗体の結合度合いが向上していることがわかる。 FIG. 6 shows the results of the fluorescent luminance distribution in Example 3 and Comparative Examples 2 and 3. The hemolytic agent containing formaldehyde, saponin and PEG as the aldehyde cross-linking agent (FIGS. 6B to 6E) is a hemolytic agent containing formaldehyde and saponin as the aldehyde cross-linking agent and containing no PEG (FIG. 6A )) And a hemolytic agent containing ammonium chloride (FIG. 6 (F)), indicating that the CD45 fluorescence luminance is increased and the degree of binding of the CD45 antibody to leukocytes is improved.
具体的には、蛍光輝度が低い（蛍光輝度０．１９以下）細胞の割合が、ホルムアルデヒドとサポニンとを含みＰＥＧを含まない溶血剤（図６（Ａ））を用いたときは全体の５０％であるのに対し、ホルムアルデヒドとサポニンとＰＥＧとを含む溶血剤を用いたときは全体の１０％（ＰＥＧ濃度０．２９％（ｗ／ｖ）、図６（Ｂ））から３７％（ＰＥＧ濃度１％（ｗ／ｖ）、図６（Ｅ））と大幅に減少している。 Specifically, the ratio of cells having a low fluorescence luminance (fluorescence luminance of 0.19 or less) is 50% of the total when a hemolytic agent containing formaldehyde and saponin and containing no PEG (FIG. 6 (A)) is used. On the other hand, when a hemolytic agent containing formaldehyde, saponin and PEG was used, 10% (PEG concentration 0.29% (w / v), FIG. 6 (B)) to 37% (PEG concentration) 1% (w / v) and FIG. 6 (E)).
また蛍光輝度が高い（蛍光輝度が０．８２以上）細胞の割合は、ホルムアルデヒドとサポニンとを含みＰＥＧを含まない溶血剤（図６（Ａ））ならびに塩化アンモニウムを含む溶血剤（図６（Ｆ））を用いたときは全体の８％であるのに対し、ホルムアルデヒドとサポニンとＰＥＧとを含む溶血剤を用いたときは、ＰＥＧ濃度が０．２９％（ｗ／ｖ）から０．６６％（ｗ／ｖ）の場合、全体の１１％から１４％と上昇した（図６（Ｂ）から（Ｄ））。一方ＰＥＧ濃度が１％（ｗ／ｖ）の場合（図６（Ｅ））、全体の８％と、ホルムアルデヒドとサポニンとを含みＰＥＧを含まない溶血剤や塩化アンモニウムを含む溶血剤を用いたときと同等であった。 The proportion of cells having high fluorescence luminance (fluorescence luminance of 0.82 or more) is determined by the ratio of the hemolytic agent containing formaldehyde and saponin and containing no PEG (FIG. 6 (A)) and the hemolytic agent containing ammonium chloride (FIG. 6 (F)). )) Is 8% of the total, whereas when a hemolytic agent containing formaldehyde, saponin and PEG is used, the PEG concentration is from 0.29% (w / v) to 0.66%. In the case of (w / v), it increased from 11% to 14% of the whole (FIGS. 6B to 6D). On the other hand, when the PEG concentration is 1% (w / v) (FIG. 6 (E)), when 8% of the whole and a hemolytic agent containing formaldehyde and saponin and containing no PEG or a hemolytic agent containing ammonium chloride are used. Was equivalent to
以上の結果から、アルデヒド架橋剤とＰＥＧとサポニンとを少なくとも含む溶血剤を用いることで、細胞膜タンパク質の漏出が抑制され、細胞膜タンパク質への抗体等の結合能が向上することがわかる。 From the above results, it can be seen that the use of the hemolytic agent containing at least the aldehyde cross-linking agent, PEG and saponin suppresses the leakage of cell membrane proteins and improves the binding ability of antibodies and the like to cell membrane proteins.
１００：細胞保持装置
１１：遮光部材
１２：絶縁体
１１ａ・１２ａ：貫通孔
２０：スペーサー
２１：導入口
２２：排出口
２３：貫通部
３１・３２：電極基板
４０：導線
５０：信号発生器
６０：保持部
７０：細胞
REFERENCE SIGNS LIST 100: cell holding device 11: light shielding member 12:
Claims (8)
前記血液試料を請求項３または４に記載の方法で溶血させる工程、および
溶血した血液試料中の標的細胞を光学的に検出する工程、を含む前記方法。 A method for detecting a target cell contained in a blood sample,
5. The method according to claim 3, further comprising the steps of lysing the blood sample by the method according to claim 3 or 4, and optically detecting target cells in the lysed blood sample.
前記血液試料を請求項３または４に記載の方法で溶血させる工程、および
溶血した血液試料中の標的細胞および／もしくは標的細胞以外の不要細胞を光学的に検出する工程、を含む前記方法。 A method for detecting a target cell contained in a blood sample,
The method comprising the steps of hemolyzing the blood sample by the method according to claim 3 or 4, and optically detecting target cells and / or unnecessary cells other than the target cells in the hemolyzed blood sample.
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| CN112014573B (en) * | 2020-08-25 | 2023-08-08 | 武汉生之源生物科技股份有限公司 | Preparation method of high-sensitivity assay kit for troponin I in human whole blood sample and kit |
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