JP5604726B2 - Immunoassay for podoplanin - Google Patents
Immunoassay for podoplanin Download PDFInfo
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- JP5604726B2 JP5604726B2 JP2009160854A JP2009160854A JP5604726B2 JP 5604726 B2 JP5604726 B2 JP 5604726B2 JP 2009160854 A JP2009160854 A JP 2009160854A JP 2009160854 A JP2009160854 A JP 2009160854A JP 5604726 B2 JP5604726 B2 JP 5604726B2
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Description
本発明は、ポドプラニン（ｐｏｄｏｐｌａｎｉｎ）の測定方法に関するものである。更に詳しくは、体液中のポドプラニンを測定するポドプラニンの免疫測定方法に関するものである。 The present invention relates to a method for measuring podoplanin. More specifically, the present invention relates to an immunoassay method for podoplanin that measures podoplanin in body fluids.
肺癌の血液中のマーカーとしてガストリン放出ペプチド前駆体（ＰｒｏＧＲＰ）（例えば、特許文献１参照）、サイトケラチン１９フラグメント（Ｃｙｆｒａ）（例えば、特許文献２参照）等が知られている。近年、アスベスト暴露による悪性中皮腫の患者が急増する懸念が示唆されている。現在、悪性中皮腫の検査に使用できるマーカーとして、ポドプラニン（ｐｏｄｏｐｌａｎｉｎ）、メゾテリン（ｍｅｓｏｔｈｅｌｉｎ）及びカルレチニン（ｃａｌｒｅｔｉｎｉｎ）を代表として、いくつかのタンパク質が報告されているが、それらはいずれも細胞の免疫染色による中皮腫の鑑別マーカーとして使用されている（例えば、非特許文献１参照）。 Gastrin releasing peptide precursor (ProGRP) (see, for example, Patent Document 1), cytokeratin 19 fragment (Cyfra) (see, for example, Patent Document 2) and the like are known as markers in lung cancer blood. In recent years, there has been a concern that the number of patients with malignant mesothelioma due to asbestos exposure will increase rapidly. Currently, several proteins have been reported as representative markers that can be used for examination of malignant mesothelioma, such as podoplanin, mesothelin, and calretinin. It is used as a differentiation marker for mesothelioma by staining (see, for example, Non-Patent Document 1).
しかしながら、ＰｒｏＧＲＰ及びＣｙｆｒａ等の肺癌マーカーでは、検出できない肺癌が存在するという問題がある。また、メゾテリンは、悪性中皮腫の上皮型（中皮腫の内約７０％が該当）と肉腫型（中皮腫の内約３０％が該当）の２つの病理分類の内、上皮型にのみ発現しており、肉腫型では発現が無いため、すべての悪性中皮腫に対するマーカーとしては使用できないという問題がある。
本発明の課題は、肺癌及び悪性中皮腫の検査において従来の免疫染色で必須であった組織採取を必要とせず、患者の負担軽減を可能とし、肺癌及び悪性中皮腫の指標となるデータを収集する方法を提供することにある。
However, lung cancer markers such as ProGRP and Cyfra have a problem in that there are lung cancers that cannot be detected. In addition, mesothelin is classified into epithelial type in two pathological classifications of epithelial type of malignant mesothelioma (approx. 70% of mesothelioma) and sarcoma type (approx. 30% of mesothelioma). Since it is expressed only and not in the sarcoma type, there is a problem that it cannot be used as a marker for all malignant mesothelioma.
The problem of the present invention is that data collection that does not require tissue collection, which is essential for conventional immunostaining in the examination of lung cancer and malignant mesothelioma, can reduce the burden on the patient, and is an index of lung cancer and malignant mesothelioma It is to provide a way to collect.
本発明者らは上記課題を解決すべく鋭意研究を行った結果、ポドプラニンを高感度に測定する方法を見いだし、肺癌患者及び悪性中皮腫患者の血液等の体液中において従来存在が知られていなかったポドプラニンが存在しており、ポドプラニンを血液等の体液中の肺癌及び悪性中皮腫マーカーとして利用できること、及びポドプラニンは悪性中皮腫の上皮型と肉腫型のすべてに発現していることから、すべての悪性中皮腫マーカーとして利用できることを見出し、本発明に到達した。即ち、本発明は、肺癌又は中皮腫に対する指標としてポドプラニンを用いる方法であって、体液中の該ポドプラニンを測定することを特徴とし、細胞膜表面から外側に突出しているポドプラニンの細胞外領域を認識する抗体及びポドプラニンの細胞外領域以外を認識する抗体を含むモノクローナル抗体であって、該モノクローナル抗体を用いたポドプラニンの２抗体サンドイッチ定量的免疫測定法である。
As a result of diligent research to solve the above-mentioned problems, the present inventors have found a method for measuring podoplanin with high sensitivity, and it has been known to exist in body fluids such as blood of lung cancer patients and malignant mesothelioma patients. Podoplanin is present, and can be used as a marker for lung cancer and malignant mesothelioma in body fluids such as blood, and podoplanin is expressed in all epithelial and sarcoma types of malignant mesothelioma As a result, they have found that they can be used as all malignant mesothelioma markers, and have reached the present invention. That is, the present invention is a method of using podoplanin as an index for lung cancer or mesothelioma , characterized by measuring the podoplanin in body fluid, and recognizing the extracellular region of podoplanin protruding outward from the cell membrane surface This is a two-antibody sandwich quantitative immunoassay method for podoplanin using the monoclonal antibody, and a monoclonal antibody containing an antibody that recognizes other than the extracellular region of podoplanin.
本発明の測定方法により、肺癌及び悪性中皮腫の検査で、従来の免疫染色で必要であった組織採取が必要でなく、患者の負担を軽減できる。更に血液での検査が可能なため、肺癌及び悪性中皮腫のマススクリーニングが可能となる等、肺癌及び悪性中皮腫の検査に非常に有用である。また、ポドプラニンを定量的に測定できるため、肺癌及び中皮腫の治療効果等の判定に利用できるという効果がある。 According to the measurement method of the present invention, the examination of lung cancer and malignant mesothelioma does not require tissue collection, which is necessary for conventional immunostaining, and can reduce the burden on the patient. Furthermore, since it is possible to examine with blood, mass screening of lung cancer and malignant mesothelioma is possible, which is very useful for examination of lung cancer and malignant mesothelioma. In addition, since podoplanin can be measured quantitatively, there is an effect that it can be used to determine the therapeutic effect of lung cancer and mesothelioma.
ポドプラニンは、１６２アミノ酸からなる３８キロダルトンの１型膜貫通糖タンパク質として知られている。ポドプラニンは各腫の臓器及び細胞、例えば腎臓糸球体足細胞、骨格筋、胎盤、肺、心臓、筋線維芽細胞、リンパ管内皮及び中皮細胞等に存在していることが知られている。但し、血液等の体液中にポドプラニンが存在することはこれまで知られておらず、本発明により初めて明確にされたものである。
ポドプラニンはヒトを含む哺乳類動物に存在するが、本発明の方法により収集されたデータを疾患の検査に役立てる効果が大きいと言う観点からヒトのポドプラニンが好ましい。
Podoplanin is known as a 38 kilodalton type 1 transmembrane glycoprotein consisting of 162 amino acids. Podoplanin is known to be present in organs and cells of each tumor, such as kidney glomerular podocytes, skeletal muscle, placenta, lung, heart, myofibroblasts, lymphatic endothelium and mesothelial cells. However, the presence of podoplanin in body fluids such as blood has not been known so far and has been clarified for the first time by the present invention.
Podoplanin is present in mammals including humans, but human podoplanin is preferable from the viewpoint that the data collected by the method of the present invention has a large effect for use in disease tests.
本発明における体液としては、例えば、患者から分離採取した血液、漿液、腹水、胸水、リンパ液、尿、脳脊髄液、胸膜液、腹膜液、囲心腔液及び粘膜分泌物が挙げられる。これらの内、検体の採取のしやすさ及びポドプラニンの含有濃度の観点から血液、漿液、腹水、胸水及びリンパ液が好ましく、更に好ましいのは、血液、腹水、胸水及びリンパ液であり、特に好ましいのは血液及び胸水である。血液としては、全血、血清及び血漿が挙げられ、特に好ましくは血清及び血漿である。 Examples of the body fluid in the present invention include blood, serous fluid, ascites, pleural effusion, lymph fluid, urine, cerebrospinal fluid, pleural fluid, peritoneal fluid, pericardial fluid and mucosal secretions collected and collected from a patient. Among these, blood, serous fluid, ascites, pleural effusion, and lymph fluid are preferable from the viewpoint of easy sample collection and the concentration of podoplanin, and blood, ascites, pleural effusion, and lymph fluid are more preferable, and particularly preferable Blood and pleural effusion. Examples of blood include whole blood, serum and plasma, with serum and plasma being particularly preferred.
本発明における体液中のポドプラニンの測定は従来公知の高感度免疫測定法で実施できる。高感度免疫測定法としては、例えば、酵素免疫測定法、蛍光免疫測定法、発光免疫測定法及び放射性免疫測定法が挙げられる。これらの内、酵素免疫測定法、蛍光免疫測定法及び発光免疫測定法が好ましく、更に好ましいのは酵素免疫測定法、特に酵素の基質に化学発光物質を用いた化学発光酵素免疫測定法が好ましい。 The measurement of podoplanin in body fluids in the present invention can be carried out by a conventionally known highly sensitive immunoassay. Examples of the highly sensitive immunoassay include enzyme immunoassay, fluorescent immunoassay, luminescence immunoassay, and radioimmunoassay. Of these, enzyme immunoassay, fluorescence immunoassay, and luminescence immunoassay are preferred, and enzyme immunoassay, particularly chemiluminescence enzyme immunoassay using a chemiluminescent substance as the enzyme substrate is preferred.
免疫測定に用いるポドプラニンに対する抗体は、従来公知の方法で作製したポリクローナル抗体及びモノクローナル抗体が使用できるが、好ましい抗体は感度及び特異性（ポドプラニン類似タンパクとの交差反応性を示さない）の観点からポドプラニンに対するモノクローナル抗体である。例えばモノクローナル抗体は、細胞融合技術や遺伝子組替え技術等を利用した自体公知の方法〔例えばＥｕｒ．Ｊ．Ｉｍｍｕｎｏｌ，６５１１（１９７６）〕等によって産生されたものが使用できる。 As an antibody against podoplanin used for immunoassay, a polyclonal antibody and a monoclonal antibody prepared by a conventionally known method can be used, but preferred antibodies are podoplanin from the viewpoint of sensitivity and specificity (not showing cross-reactivity with podoplanin-like proteins). It is a monoclonal antibody against. For example, monoclonal antibodies can be produced by methods known per se using cell fusion technology, gene recombination technology, etc. [eg, Eur. J. et al. Immunol, 6511 (1976)] etc. can be used.
本発明において使用する抗体は、従来公知の高感度免疫測定に使用する抗体の場合と同様に、認識部位の異なる抗体を組み合わせて使用することが通常行われる。これは高感度免疫測定法の測定原理「２抗体サンドイッチ測定法」に基づくものである。即ち、測定対象である抗原の同一部位を認識する２つの抗体と抗原を反応させた場合、免疫複合体「抗体−抗原−抗体」を形成できず、免疫測定を実施できないことを回避するためである。 The antibodies used in the present invention are usually used in combination with antibodies having different recognition sites, as in the case of antibodies used for conventionally known highly sensitive immunoassays. This is based on the measurement principle of the high sensitivity immunoassay “2-antibody sandwich assay”. That is, in order to avoid the fact that an immune complex “antibody-antigen-antibody” cannot be formed when two antibodies recognizing the same site of the antigen to be measured are reacted with the antigen, and immunoassay cannot be performed. is there.
認識部位の異なる抗体は従来公知の方法で作製又は選択することができる。例えば、抗原の特定部位を遺伝子組み換えタンパク又は合成ペプチドとして調整し、これをマウス等に免役することで、抗原の特定部位に対する抗体（モノクローナル抗体が好ましい）が作製できる。また、モノクローナル抗体を選択する際に行うＥＬＩＳＡに使用する抗原を同様に調製することで、認識部位の異なる抗体を選択できる。更に、免疫染色、免疫組織化学染色及びフローサイトメトリー等での反応性を確認することでも認識部位の違う抗体を選ぶことができる。 Antibodies having different recognition sites can be prepared or selected by a conventionally known method. For example, by preparing a specific site of the antigen as a recombinant protein or synthetic peptide and immunizing it to a mouse or the like, an antibody (monoclonal antibody is preferred) against the specific site of the antigen can be produced. In addition, an antibody having a different recognition site can be selected by similarly preparing an antigen to be used for ELISA performed when selecting a monoclonal antibody. Furthermore, antibodies with different recognition sites can be selected by confirming reactivity by immunostaining, immunohistochemical staining, flow cytometry, and the like.
本発明において、使用する抗体の認識部位は免疫複合体「抗体−抗原(ポドプラニン）−抗体」を形成できる限り特に限定はないが、高感度の観点から、好ましくはポドプラニンの細胞外領域（細胞膜表面から外側に突出している部分）を認識する抗体と、ポドプラニンの細胞外領域以外を認識する抗体の組み合わせである。 In the present invention, the recognition site of the antibody to be used is not particularly limited as long as it can form an immune complex “antibody-antigen (podoplanin) -antibody”. A portion that protrudes outward from the region) and an antibody that recognizes other than the extracellular region of podoplanin.
本発明は、体液中のポドプラニンを測定することで、肺癌及び悪性中皮腫の指標となるデータの収集方法を提供するものであり、例えば多数の健常人から得た血液中のポドプラニンを測定して、その濃度分布から基準となる値を設定し、この基準値に対して、被検者から得た血液中のポドプラニン濃度を比較することにより、肺癌及び悪性中皮腫の検査に役立てることができる。 The present invention provides a method for collecting data serving as an indicator of lung cancer and malignant mesothelioma by measuring podoplanin in body fluids. For example, podoplanin in blood obtained from many healthy individuals is measured. By setting the reference value from the concentration distribution and comparing the podoplanin concentration in the blood obtained from the subject against this reference value, it can be useful for the examination of lung cancer and malignant mesothelioma. it can.
本発明において、肺癌としては、小細胞癌及び非小細胞癌等が挙げられ、非小細胞癌としては腺癌、扁平上皮癌、大細胞癌及び腺扁平上皮等が挙げられる。悪性中皮腫としては、胸膜中皮腫、腹膜中皮腫及び心膜中皮腫等が挙げられる。これらの内、特異性（疾患とポドプラニンの関連がより強い）の観点から好ましいのは悪性中皮腫であり、特に好ましいのは胸膜中皮腫及び腹膜中皮腫である。 In the present invention, examples of lung cancer include small cell carcinoma and non-small cell carcinoma, and examples of non-small cell carcinoma include adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and adenosquamous epithelium. Examples of malignant mesothelioma include pleural mesothelioma, peritoneal mesothelioma and pericardial mesothelioma. Among these, malignant mesothelioma is preferable from the viewpoint of specificity (a stronger association between the disease and podoplanin), and pleural mesothelioma and peritoneal mesothelioma are particularly preferable.
以下、実施例により、本発明を更に説明するが、本発明はこれに限定されるものではない。
[実施例1]
本実施例は、抗ポドプラニンモノクローナル抗体の作製と免疫測定に使用する抗体の選択例を示したものである。
＜抗ポドプラニンモノクローナル抗体の作製＞
１．免疫用抗原の作製
文献（Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｊｏｕｒｎａｌ ｏｆ Ｏｎｃｏｌｏｇｙ ３１：５０１−５０８，２００７）に記載の方法で、ヒトポドプラニン融合タンパク質を作製した。即ち、ポドプラニンの細胞外領域に相当するヒトポドプラニンｃＤＮＡの４８９塩基の断片（ＧｅｎＢａｎｋ ＡＢ１２７９５８のポジション１−４８９）を発現プラスミドｐＩｇｐｌｕｓに組み込み、ｐＩｇ−ｐｏｄｏｐｌａｎｉｎを作製した。このプラスミドはヒト免疫グロブリンのＦｃ領域のＣ末端に融合した形でヒトポドプラニンの細胞外領域タンパク質（ヒトポドプラニン融合タンパク質）を発現することができる。ｐＩｇ−ｐｏｄｏｐｌａｎｉｎを２９３T細胞にトランスフェクション試薬ＦｕＧＥＮＥ６（ロシュ社製）を用いて遺伝子導入した。この細胞をＦＣＳ（牛胎児血清）を含むＤＭＥＭ培地で５日培養し、培養液中にヒトポドプラニン融合タンパク質を分泌させた。培養液をプロテインAセファロースカラム（アマシャムファーマシアバイオテック社製）を用いてアフィニティークロマトグラフィーにより、ヒトポドプラニン融合タンパク質を精製した。このヒトポドプラニン融合タンパク質をマウスへの免疫用抗原とした。
EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to this.
[Example 1]
This example shows preparation of anti-podopranin monoclonal antibody and selection example of antibody used for immunoassay.
<Preparation of anti-podoplanin monoclonal antibody>
1. Production of antigen for immunization A human podoplanin fusion protein was produced by the method described in the literature (International Journal of Oncology 31: 501-508, 2007). Specifically, a 489 base fragment of human podoplanin cDNA corresponding to the extracellular region of podoplanin (position 1-489 of GenBank AB127958) was incorporated into the expression plasmid pIgplus to prepare pIg-podoplanin. This plasmid can express the human podoplanin extracellular region protein (human podoplanin fusion protein) in the form fused to the C-terminus of the Fc region of human immunoglobulin. pIg-podoplanin was introduced into 293T cells using the transfection reagent FuGENE6 (Roche). The cells were cultured for 5 days in a DMEM medium containing FCS (fetal calf serum), and human podoplanin fusion protein was secreted into the culture solution. The human podopranin fusion protein was purified by affinity chromatography using a protein A Sepharose column (manufactured by Amersham Pharmacia Biotech). This human podoplanin fusion protein was used as an antigen for immunization of mice.
２．モノクローナル抗体の作製
文献（Ｂｌｏｏｄ ９６：５４６−５５３，２０００）記載の方法でモノクローナル抗体を作製した。即ち、２０μｇの免疫用抗原をフロイントの完全アジュバント（Ｄｉｆｃｏ社製）で乳化し、この乳化液を８週齢のＢＡＬＢ／ｃマウスの皮下に投与した。その後、２０μｇの免疫用抗原をフロイントの不完全アジュバント（Ｄｉｆｃｏ社製）で乳化した乳化液を２週間間隔で３回追加免疫を行い、更に細胞融合3日前に尾静脈に５μｇの免疫用抗原を含む生理食塩水を注射した。マウス脾細胞を摘出し、マウス骨髄腫細胞（Ｐ３Ｕ１）と融合した後、ＨＡＴ培地によりハイブリドーマを選択培養した。
更にＥＬＩＳＡによって、免疫用抗原を認識し、ヒト免疫グロブリンのＦｃ領域を認識しない抗体を作るハイブリドーマを選択した。即ち、免疫用抗原を５０ｎｇ／ｍLの濃度で含む０．８５％塩化ナトリウム含有リン酸緩衝液（ＰＢＳ）を９６穴ＥＬＩＳＡプレート（ヌンク社製、Ｎｕｎｃ ｗｈｉｔｅプレート ｃａｔ．４３６１１０）の各穴に５０μＬずつ加え、４℃で１夜静置した。洗浄後、ブロックエース［雪印乳業（株）製］を用いてブロッキングし、抗原を固相化した９６穴ＥＬＩＳＡプレートを得た。この９６穴ＥＬＩＳＡプレートの各ウエルに、融合細胞の培養上清５０μLを添加し、室温で１時間反応後、洗浄し、ペルオキシダーゼ標識抗マウスＩｇＧ（ＨＬ鎖）を添加した。室温で１時間反応後洗浄し、基質液（ｏ−フェニレンジアミン）を各ウエルに５０μLづつ添加し、４９２ｎｍにおける吸光度（吸光度Ａ）を測定した。同様にヒト免疫グロブリンのＦｃ領域を固相化したプレートで吸光度（吸光度Ｂ）を測定した。吸光度Ａから吸光度Ｂを引いた値が大きい５クローンを選択した。
５種のハイブリドーマを更に限界希釈法により３回クローニングを行い、クローニング後のハイブリドーマを1週間前にプリスタン腹腔内注射済みのＢＡＬＢ／ｃマウスの腹腔内に注射し、１０日後に腹水を採取した。最終的に、腹水中のＩｇＧは硫安塩析後プロテインＡにて精製し、５種の抗ポドプラニンモノクローナル抗体（Ｎｏ．７Ｂ１０、Ｎｏ．５、Ｎｏ．７、Ｎｏ．３１及びＮｏ．３６）を得た。
2. Preparation of monoclonal antibody A monoclonal antibody was prepared by the method described in the literature (Blood 96: 546-553, 2000). That is, 20 μg of immunizing antigen was emulsified with Freund's complete adjuvant (Difco), and this emulsion was administered subcutaneously to 8-week-old BALB / c mice. Thereafter, an emulsion obtained by emulsifying 20 μg of immunizing antigen with Freund's incomplete adjuvant (Difco) was boosted three times at 2-week intervals, and 5 μg of immunizing antigen was added to the tail vein 3 days before cell fusion. The containing saline was injected. Mouse splenocytes were excised and fused with mouse myeloma cells (P3U1), and then hybridomas were selectively cultured in HAT medium.
Furthermore, a hybridoma that produces an antibody that recognizes an antigen for immunization and does not recognize the Fc region of human immunoglobulin was selected by ELISA. That is, a phosphate buffer solution (PBS) containing 0.85% sodium chloride containing an antigen for immunization at a concentration of 50 ng / mL, 50 μL in each well of a 96-well ELISA plate (Nunc white plate, Nunc white plate cat. 436110). In addition, the mixture was allowed to stand at 4 ° C. overnight. After washing, blocking was performed using Block Ace (manufactured by Snow Brand Milk Products Co., Ltd.) to obtain a 96-well ELISA plate on which the antigen was immobilized. To each well of the 96-well ELISA plate, 50 μL of the fusion cell culture supernatant was added, reacted for 1 hour at room temperature, washed, and peroxidase-labeled anti-mouse IgG (HL chain) was added. After the reaction at room temperature for 1 hour, the plate was washed, 50 μL of substrate solution (o-phenylenediamine) was added to each well, and the absorbance at 492 nm (absorbance A) was measured. Similarly, the absorbance (absorbance B) was measured using a plate on which the Fc region of human immunoglobulin was immobilized. Five clones having a large value obtained by subtracting the absorbance B from the absorbance A were selected.
Five hybridomas were further cloned three times by limiting dilution. The cloned hybridoma was injected intraperitoneally into BALB / c mice that had been injected intraperitoneally with pristane one week ago, and ascites was collected 10 days later. Finally, IgG in ascites was purified with protein A after salting out with ammonium sulfate, and 5 types of anti-podopranin monoclonal antibodies (No. 7B10, No. 5, No. 7, No. 31 and No. 36) were obtained. Got.
３．モノクローナル抗体の反応性確認
上記５種のモノクローナル抗体について文献（Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｊｏｕｒｎａｌ ｏｆ Ｏｎｃｏｌｏｇｙ ３１：５０１−５０８，２００７）記載の方法で、免疫蛍光染色及びフローサイトメトリー（ＦＡＣＳ）での反応性を確認した。使用した細胞は、
文献（Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｊｏｕｒｎａｌ ｏｆ Ｏｎｃｏｌｏｇｙ ３１：５０１−５０８，２００７）記載のｐｃＤＮＡ−ｐｏｄｏｐｌａｎｉｎ−Ｖ５（ヒトポドプラニン全体の遺伝子を含む発現プラスミド）をトランスフェクトしたＮＩＨ ３Ｔ３細胞、及び内在性のポドプラニンを有するヒトリンパ内皮細胞［ｌｙｍｐｈａｔｉｃ ｅｎｄｏｔｈｅｌｉａｌ ｃｅｌｌｓ（ＬＥＣｓ）］（ＡｎｇｉｏＢｉｏ社から購入）を用いた。
免疫蛍光染色は次のように行った、細胞を４％ＰＦＡ含有ＰＢＳを用いて氷上で１０分間固定した後、ＰＢＳで洗浄した。洗浄した細胞を１％ＢＳＡ含有ＰＢＳを用いて室温３０分間ブロッキングした。この細胞をモノクローナル抗体5μｇ／ｍＬの濃度で含むＰＢＳでインキュベートし、更にＰＢＳで１０００倍希釈したロバ抗マウスＡｌｅｘａ Ｆｌｕｐｒ ４８８抗体（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ社製）でインキュベートした後、細胞の蛍光を確認した。
フローサイトメトリー（ＦＡＣＳ）は、ＦＣＳ Ｃａｌｉｂｕｒ（Ｂｅｃｔｏｎ−Ｄｉｃｋｉｎｓｏｎ社製）で実施した。培養した細胞を０．２５％トリプシン−ＥＤＴＡ（Ｄｉｆｃｏ社製）で処理して集めて使用した。集めた細胞を、モノクローナル抗体（１０6個の細胞を含む液１ｍＬ当たり５μｇ）と抗ヒトＶＥ−Ｃａｈｈｅｒｉｎヤギ抗体（１０6個の細胞を含む液１ｍＬ当たり０．１μｇ）（Ｒ＆Ｄ Ｓｙｓｔｅｍｓ社製）で染色し、続いて１０００倍希釈したロバ抗マウスＡｌｅｘａ Ｆｌｕｐｒ ４８８抗体及び１０００倍希釈したウサギ抗ヤギＡｌｅｘａ Ｆｌｕｐｒ ６３３抗体（Ｉｎｖｉｔｒｏｇｅｎ社製）とインキュベートして、フローサイトメトリーにかけた。結果を表１に示した。
3. Confirmation of Reactivity of Monoclonal Antibodies The reactivity of the above five monoclonal antibodies was confirmed by immunofluorescence staining and flow cytometry (FACS) by the method described in the literature (International Journal of Oncology 31: 501-508, 2007). The cells used were
NIH 3T3 cells transfected with pcDNA-podoplanin-V5 (an expression plasmid containing the entire gene of human podoplanin) described in the literature (International Journal of Oncology 31: 501-508, 2007), and human lymphatic endothelial cells having endogenous podoplanin [ Lymphatic endothelial cells (LECs)] (purchased from AngioBio) were used.
Immunofluorescence staining was performed as follows. Cells were fixed on ice for 10 minutes using PBS containing 4% PFA, and then washed with PBS. The washed cells were blocked with PBS containing 1% BSA for 30 minutes at room temperature. The cells were incubated with PBS containing a monoclonal antibody at a concentration of 5 μg / mL, and further incubated with a donkey anti-mouse Alexa Flupr 488 antibody (manufactured by Molecular Probes) diluted 1000-fold with PBS, and then the fluorescence of the cells was confirmed.
Flow cytometry (FACS) was performed with FCS Calibur (Becton-Dickinson). The cultured cells were treated with 0.25% trypsin-EDTA (Difco) and collected for use. The collected cells were treated with a monoclonal antibody (5 μg per mL containing 10 6 cells) and an anti-human VE-Cahherin goat antibody (0.1 μg per mL containing 10 6 cells) (manufactured by R & D Systems). Staining was followed by incubation with donkey anti-mouse Alexa Flupr 488 antibody diluted 1000-fold and rabbit anti-goat Alexa Flupr 633 antibody diluted 1000-fold (Invitrogen) and subjected to flow cytometry. The results are shown in Table 1.
モノクローナル抗体Ｎｏ．７Ｂ１０は、いずれにも強い反応性（＋＋＋）を示した。モノクローナル抗体Ｎｏ．５は、ＮＩＨ ３Ｔ３細胞（ｐｃＤＮＡ−ｐｏｄｏｐｌａｎｉｎ−Ｖ５）への蛍光免疫染色で弱い反応性（＋）を示したが、そのほかには反応性を示さなかった。モノクローナル抗体Ｎｏ．７は、ＮＩＨ ３Ｔ３細胞（ｐｃＤＮＡ−ｐｏｄｏｐｌａｎｉｎ−Ｖ５）への免疫蛍光染色で弱い反応性（＋）及びＦＡＣＳでは反応性（＋＋）を示し、ＬＥＣｓへの蛍光免疫染色で反応性（＋＋）及びＦＡＣＳで弱い反応性（＋）を示した。モノクローナル抗体Ｎｏ．３１は、いずれにも反応性（＋＋）を示した。モノクローナル抗体Ｎｏ．３６は、ＮＩＨ ３Ｔ３細胞（ｐｃＤＮＡ−ｐｏｄｏｐｌａｎｉｎ−Ｖ５）への免疫蛍光染色で反応性（＋＋）及びＦＡＣＳでは弱い反応性（＋）を示し、ＬＥＣｓへの蛍光免疫染色及びＦＡＣＳで強い反応性（＋＋＋）を示した。
モノクローナル抗体Ｎｏ．５はＥＬＩＳＡでポドプラニン融合タンパク質に対して反応性を示すが、細胞表面に存在するポドプラニンへの反応性はＮＩＨ ３Ｔ３細胞（ｐｃＤＮＡ−ｐｏｄｏｐｌａｎｉｎ−Ｖ５）への蛍光免疫染色で弱い反応性（＋）を示した以外は認められなかったことから、モノクローナル抗体Ｎｏ．５は細胞膜から突出した領域ではなく、細胞外領域以外の領域を実質的に認識していると考えられる。
Monoclonal antibody no. 7B10 showed strong reactivity (++++). Monoclonal antibody no. No. 5 showed weak reactivity (+) in fluorescence immunostaining to NIH 3T3 cells (pcDNA-podoplanin-V5), but no other reactivity. Monoclonal antibody no. 7 shows weak reactivity (+) in immunofluorescence staining to NIH 3T3 cells (pcDNA-podoplanin-V5) and reactivity (++) in FACS, and reactivity (++) and FACS in fluorescence immunostaining to LECs. Showed weak reactivity (+). Monoclonal antibody no. All 31 showed reactivity (++). Monoclonal antibody no. 36 shows reactivity (++) in immunofluorescence staining to NIH 3T3 cells (pcDNA-podoplanin-V5) and weak reactivity (+) in FACS, and strong reactivity (++) in fluorescence immunostaining and FACS to LECs. )showed that.
Monoclonal antibody no. 5 shows reactivity to the podoplanin fusion protein by ELISA, but the reactivity to podoplanin existing on the cell surface is weakly reactive (+) by fluorescent immunostaining to NIH 3T3 cells (pcDNA-podoplanin-V5). Since nothing other than the above was observed, monoclonal antibody No. 5 is considered not to be a region protruding from the cell membrane but to substantially recognize a region other than the extracellular region.
＜ポドプラニン測定用モノクローナル抗体の選択＞
１．モノクローナル抗体固相化プレートの作製
抗ポドプラニンモノクローナル抗体Ｎｏ．５をＰＢＳで１０μｇ／ｍＬの濃度に希釈し、９６穴ＥＬＩＳＡAプレート（ヌンク社製、Ｎｕｎｃ Ｗｈｉｔｅプレート ｃａｔ．４３６１１０）の各穴に５０μLづつ加え、４℃で１夜静置した。洗浄後、ブロックエース［雪印乳業（株）製］を用いてブロッキングをし、モノクローナル抗体固相化プレートＮｏ．５を得た。抗ポドプラニンモノクローナル抗体Ｎｏ．７、抗ポドプラニンモノクローナル抗体Ｎｏ．３１、抗ポドプラニンモノクローナル抗体Ｎｏ．３６及び抗ポドプラニンモノクローナル抗体Ｎｏ．７Ｂ１０についても同様に操作し、モノクローナル抗体固相化プレートＮｏ．５、モノクローナル抗体固相化プレートＮｏ．７、モノクローナル抗体固相化プレートＮｏ．３１、モノクローナル抗体固相化プレートＮｏ．３６及びモノクローナル抗体固相化プレートＮｏ．７Ｂ１０を得た。
<Selection of monoclonal antibody for podoplanin measurement>
1. Preparation of monoclonal antibody solid-phase plate Anti-podoplanin monoclonal antibody No. 5 was diluted with PBS to a concentration of 10 μg / mL, 50 μL was added to each well of a 96-well ELISA plate (Nunc, Nunc White plate cat. 436110), and allowed to stand overnight at 4 ° C. After washing, blocking was performed using Block Ace (manufactured by Snow Brand Milk Products Co., Ltd.), and monoclonal antibody-immobilized plate No. 5 was obtained. Anti-podopranin monoclonal antibody No. 7, anti-podopranin monoclonal antibody No. 31, anti-podopranin monoclonal antibody no. 36 and anti-podoplanin monoclonal antibody No. The same operation was carried out for 7B10. 5. Monoclonal antibody immobilized plate No. 5 7. Monoclonal antibody immobilized plate No. 7 31. Monoclonal antibody-immobilized plate No. 31 36 and monoclonal antibody immobilized plate No. 7B10 was obtained.
２．ビオチン化モノクローナル抗体の作製
抗ポドプラニンモノクローナル抗体Ｎｏ．５ ０．１ｍｇとビオチニル−N−ヒドキシサクシニミドエステル０．０２ｍｇを混合し、０．１M炭酸水素ナトリウム（ｐＨ８．０）に溶解し、室温で４時間静置し、抗体のビオチン化を行った。更に反応液をＰＢＳ ２Ｌに対して４℃で一夜透析し、ビオチン化モノクローナル抗体Ｎｏ．５を得た。抗ポドプラニンモノクローナル抗体Ｎｏ．７、抗ポドプラニンモノクローナル抗体Ｎｏ．３１、抗ポドプラニンモノクローナル抗体Ｎｏ．３６及び抗ポドプラニンモノクローナル抗体Ｎｏ．７Ｂ１０についても同様に操作し、ビオチン化モノクローナル抗体Ｎｏ．７、ビオチン化モノクローナル抗体Ｎｏ．３１、ビオチン化モノクローナル抗体Ｎｏ．３６及びビオチン化モノクローナル抗体Ｎｏ．７Ｂ１０を得た。
2. Preparation of biotinylated monoclonal antibody Anti-podopranin monoclonal antibody No. 1 5 0.1 mg and biotinyl-N-hydroxysuccinimide ester 0.02 mg are mixed, dissolved in 0.1 M sodium bicarbonate (pH 8.0), and left at room temperature for 4 hours to biotinylate the antibody. went. Furthermore, the reaction solution was dialyzed overnight at 4 ° C. against 2 L of PBS, and biotinylated monoclonal antibody No. 5 was obtained. Anti-podopranin monoclonal antibody No. 7, anti-podopranin monoclonal antibody No. 31, anti-podopranin monoclonal antibody no. 36 and anti-podoplanin monoclonal antibody No. The same operation was performed for 7B10, and biotinylated monoclonal antibody No. 7. Biotinylated monoclonal antibody No. 31, biotinylated monoclonal antibody no. 36 and biotinylated monoclonal antibody no. 7B10 was obtained.
３．モノクローナル抗体固相化プレート／ビオチン化モノクローナル抗体の組み合わせ試験
抗原（ヒトポドプラニン融合タンパク質）を検体希釈液（1％ＢＳＡ含有ＰＢＳ）にて０〜１０００ｎｇ／ｍＬ（濃度ポイント：０、１０、１００、１０００ｎｇ／ｍＬ）の濃度に調整し、標準液を作製した。その標準液をモノクローナル抗体固相化プレートＮｏ．５の各穴に５０μLづつ添加し、室温で６０分間反応させた。洗浄後、ビオチン化モノクローナル抗体Ｎｏ．５を検体希釈液（1％ＢＳＡ含有ＰＢＳ）で０．２μｇ／ｍＬの濃度に希釈した液を５０μLづつ添加し、室温で６０分間反応させた。洗浄後、Ｓｔｒｅｐｔａｖｉｄｉｎ ＨＲＰ Ｃｏｎｊｕｇａｔｅを添加し、室温で３０分間反応させ、洗浄後、基質液（ｏ−フェニレンジアミン）を５０μLづつ添加し、室温で３０分間反応させた後、各ウエルに１Ｎ硫酸を５０μLづつ添加し、４９２ｎｍの吸光度を測定した。同様にして、モノクローナル抗体固相化プレートＮｏ．５、モノクローナル抗体固相化プレートＮｏ．７、モノクローナル抗体固相化プレートＮｏ．３１、モノクローナル抗体固相化プレートＮｏ．３６及びモノクローナル抗体固相化プレートＮｏ．７Ｂ１０から選んだ１種類とビオチン化モノクローナル抗体Ｎｏ．５、ビオチン化モノクローナル抗体Ｎｏ．７、ビオチン化モノクローナル抗体Ｎｏ．３１、ビオチン化モノクローナル抗体Ｎｏ．３６及びビオチン化モノクローナル抗体Ｎｏ．７Ｂ１０から選んだ1種類を組み合わせて測定を行い、測定吸光度を比較した。結果を表２に示した。
3. Monoclonal antibody-immobilized plate / biotinylated monoclonal
固相化プレートとビオチン化抗体に同一の抗体を使用した組み合わせでは、抗原濃度に応じた吸光度の上昇が認められず、ポドプラニン測定に使用できないことが判った。同様に、モノクローナル抗体Ｎｏ．７Ｂ１０、Ｎｏ．３１及びＮｏ．３６から２つの抗体を組み合わせ、モノクローナル抗体Ｎｏ．５とＮｏ．７の組み合わせも、抗原濃度に応じた吸光度の上昇が認められず、ポドプラニン測定に使用できないことが判った。
測定感度の点で好ましい組み合わせは、抗体Ｎｏ．５又はＮｏ．７と、Ｎｏ．７Ｂ１０、Ｎｏ．３１及びＮｏ．３６から選ばれる１つの抗体との組み合わせてあった。それらの組み合わせの中で、抗体Ｎｏ．５とＮｏ．７Ｂ１０の組み合わせ、及び抗体Ｎｏ．７とＮｏ．７Ｂ１０の組み合わせが優れており、特に抗体Ｎｏ．５とＮｏ．７Ｂ１０の組み合わせが最も測定感度が高い組み合わせであることが判った。
In the combination using the same antibody as the solid-phased plate and the biotinylated antibody, no increase in absorbance was observed according to the antigen concentration, indicating that it could not be used for podoplanin measurement. Similarly, monoclonal antibody no. 7B10, no. 31 and no. 36 to 2 antibodies were combined, and monoclonal antibody No. 5 and no. The combination of 7 was also found to be unable to be used for podoplanin measurement because no increase in absorbance was observed according to the antigen concentration.
A preferred combination in terms of measurement sensitivity is antibody No. 5 or No. 7 and no. 7B10, no. 31 and no. It was combined with one antibody selected from 36. Among these combinations, antibody no. 5 and no. 7B10 combination and antibody no. 7 and no. The combination of 7B10 is excellent. 5 and no. It was found that the combination of 7B10 was the combination with the highest measurement sensitivity.
[実施例２]
本実施例は、マイクロプレートを用いた化学発光酵素免疫測定用法によるポドプラニンの測定例である。
＜測定用試薬＞
１．モノクローナル抗体固相化プレート
実施例１で作製したモノクローナル抗体固相化プレートＮｏ．５及びモノクローナル抗体固相化プレートＮｏ．７を使用した。
２．ビオチン化モノクローナル抗体液
実施例１で作製したビオチン化モノクローナル抗体Ｎｏ．７Ｂ１０を検体希釈液（１％ＢＳＡ含有ＰＢＳ）で抗体濃度として０．２μｇ／ｍＬに希釈して用いた。
３．ストレプトアビジン結合アルカリフォスファターゼ液
Ｓｔｒｅｐｔａｖｉｄｉｎ Ａｌｋａｌｉｎｅ ｐｈｏｓｐｈａｔａｓｅ（ＭＡＢＴＥＣＨ社製、Ｃｏｄｅ３３１０−８）を検体希釈液（１％ＢＳＡ含有ＰＢＳ）で１０００倍希釈して用いた。
４．発光試薬
ルミホス５３０［和光純薬工業（株）製］をそのまま使用した。
５．標準液
実施例１と同様に標準液を調製した。但し、濃度範囲は０〜５００ｐｇ／ｍＬ（濃度ポイント：０、７．８、１５．６、３１．３、６２．５、１２５、５００ｐｇ／ｍＬ）とした。
[Example 2]
This example is a measurement example of podoplanin by a chemiluminescent enzyme immunoassay method using a microplate.
<Reagent for measurement>
1. Monoclonal antibody-immobilized plate Monoclonal antibody-immobilized plate No. 1 prepared in Example 1 was used. 5 and monoclonal antibody immobilized plate No. 5 7 was used.
2. Biotinylated monoclonal antibody solution Biotinylated monoclonal antibody No. 1 prepared in Example 1 7B10 was used by diluting it to 0.2 μg / mL as an antibody concentration with a specimen diluent (PBS containing 1% BSA).
3. Streptavidin-binding alkaline phosphatase solution Streptavidin Alkaline phosphatase (manufactured by MABTECH, Code 3310-8) was diluted 1000-fold with a sample diluent (PBS containing 1% BSA).
4). Luminfos 530 [manufactured by Wako Pure Chemical Industries, Ltd.] was used as it was.
5. Standard solution A standard solution was prepared in the same manner as in Example 1. However, the concentration range was 0 to 500 pg / mL (concentration points: 0, 7.8, 15.6, 31.3, 62.5, 125, 500 pg / mL).
＜測定操作＞
１．検量線の作製（抗体Ｎｏ．５とＮｏ．７Ｂ１０の組み合わせ）
モノクローナル抗体固相化プレートＮｏ．５のブロッキング液を捨て、検体希釈液にて上記各濃度の標準液を２倍希釈した液を５０μLづつ各ウエルに分注し、室温で６０分反応させた。ウエルを洗浄後、ビオチン化モノクローナル抗体液Ｎｏ．７Ｂ１０を５０μL分注し、室温で６０分反応させた。ウエルを洗浄後、ストレプトアビジン結合アルカリフォスファターゼ液を５０μL分注し、室温で３０分反応させた。ウエルを洗浄液で洗浄後、発光試薬５０μLを分注し、室温で３０分反応させた。反応後、Ｗａｌｌａｃ１４２０（パーキンエルマーライフサイエンス社製発光計測器）にて発光量を測定した。
２．検量線の作製（抗体Ｎｏ．７とＮｏ．７Ｂ１０の組み合わせ）
モノクローナル抗体固相化プレートＮｏ．５をモノクローナル抗体固相化プレートＮｏ．７に代えた以外は上記１．と同様にして測定した。
３．同時再現性の確認
濃度３１．３ｐｇ／ｍＬ及び１２５ｐｇ／ｍＬの標準液について上記検量線の作成における操作と同様にして発光量を各１０回測定した。
４．検出感度の設定
濃度０、７．８、１５．６及び３１．３ｐｇ／ｍＬの標準液について各１５回発光量を測定した。各濃度の測定発光の平均値及び標準偏差（ＳＤ）を算出し、測定発光量平均値±３ＳＤの範囲が、濃度０ｐｇ／ｍＬの標準液を使用した場合の測定発光量平均値±３ＳＤの範囲と重ならない最低の濃度を検出感度とした。
<Measurement operation>
1. Preparation of calibration curve (combination of antibodies No. 5 and No. 7B10)
Monoclonal antibody immobilized plate No. The blocking solution of No. 5 was discarded, and 50 μL of a solution obtained by diluting the standard solution of each concentration described above twice with a sample dilution solution was dispensed into each well and reacted at room temperature for 60 minutes. After washing the wells, biotinylated monoclonal antibody solution No. 50 μL of 7B10 was dispensed and reacted at room temperature for 60 minutes. After washing the wells, 50 μL of a streptavidin-conjugated alkaline phosphatase solution was dispensed and reacted at room temperature for 30 minutes. After washing the well with a washing solution, 50 μL of a luminescent reagent was dispensed and reacted at room temperature for 30 minutes. After the reaction, the amount of luminescence was measured with Wallac 1420 (a luminescence measuring instrument manufactured by Perkin Elmer Life Sciences).
2. Preparation of calibration curve (combination of antibodies No. 7 and No. 7B10)
Monoclonal antibody immobilized plate No. 5 is a monoclonal antibody-immobilized plate No. 5; 7 except for the above. Measured in the same manner as above.
3. Confirmation of simultaneous reproducibility The amount of luminescence was measured 10 times for each of the standard solutions having concentrations of 31.3 pg / mL and 125 pg / mL in the same manner as in the preparation of the calibration curve.
4). Setting of detection sensitivity The luminescence amount was measured 15 times for each of standard solutions having concentrations of 0, 7.8, 15.6 and 31.3 pg / mL. Calculate the average value and standard deviation (SD) of the measured luminescence at each concentration, and the range of the measured luminescence average value ± 3SD is the range of the measured luminescence average value ± 3SD when using a standard solution with a concentration of 0 pg / mL. The lowest concentration that does not overlap with the detection sensitivity was taken as the detection sensitivity.
＜測定結果＞
１．検量線
結果を図１に示した。抗体Ｎｏ．５とＮｏ．７Ｂ１０の組み合わせ、抗体Ｎｏ．７とＮｏ．７Ｂ１０の組み合わせとも、０〜５００ｐｇ／ｍＬの範囲でほぼ直線となる良好な検量線が得られた。但し、発光量は抗体Ｎｏ．７とＮｏ．７Ｂ１０の組み合わせに対して、抗体Ｎｏ．５とＮｏ．７Ｂ１０の組み合わせで約１．７倍の値を示した。
２．同時再現性
同一サンプルを１０回測定した場合の各測定値、平均値、標準偏差（ＳＤ）及び変動係数（ＣＶ）を表３に示した。抗体Ｎｏ．５とＮｏ．７Ｂ１０の組み合わせではＣＶ１０％以下と良好であった。抗体Ｎｏ．７とＮｏ．７Ｂ１０の組み合わせでは、低濃度サンプル（サンプル１：標準液のポドプラニン濃度３１．３ｐｇ／ｍＬ）でＣＶ１０％を超える結果となった。
３．検出感度試験
検出感度試験の結果を図２及び図３に示した。各濃度の標準液を１５回測定し±３ＳＤをバーで示した。抗体Ｎｏ．５とＮｏ．７Ｂ１０の組み合わせでは、０ｐｇ／ｍＬの３ＳＤと重ならない１５．６ｐｇ／ｍＬを検出感度とした。抗体Ｎｏ．７とＮｏ．７Ｂ１０の組み合わせでは、０ｐｇ／ｍＬの３ＳＤと重ならない３１．３ｐｇ／ｍＬを検出感度とした。
<Measurement results>
1. Calibration curve The results are shown in FIG. Antibody No. 5 and no. 7B10, antibody no. 7 and no. With the combination of 7B10, a good calibration curve almost linear in the range of 0 to 500 pg / mL was obtained. However, the amount of luminescence is antibody No. 7 and no. Antibody No. 7 against the combination of 7B10. 5 and no. The combination of 7B10 showed a value of about 1.7 times.
2. Simultaneous Reproducibility Table 3 shows each measured value, average value, standard deviation (SD), and coefficient of variation (CV) when the same sample was measured 10 times. Antibody No. 5 and no. The combination of 7B10 was as good as
3. Detection sensitivity test The results of the detection sensitivity test are shown in FIGS. The standard solution of each concentration was measured 15 times, and ± 3SD was indicated by a bar. Antibody No. 5 and no. In the combination of 7B10, 15.6 pg / mL not overlapping with 0 pg / mL 3SD was set as the detection sensitivity. Antibody No. 7 and no. In the combination of 7B10, 31.3 pg / mL which does not overlap with 0 pg / mL 3SD was set as the detection sensitivity.
[実施例３]
本実施例は、健常人から得た血清中のポドプラニンを測定した例である。
＜健常人血清中のポドプラニン測定＞
健常人（健康診断受診者２５７名、平均年齢３９．２才）から採取した血清中のポドプラニンを測定した。ポドプラニンの測定は実施例２に記載の測定操作の「１．検量線の作製（抗体Ｎｏ．５とＮｏ．７Ｂ１０の組み合わせ）」で標準液の代わりに血清検体を用いる以外は同様に操作して発光量を測定し、実施例２に記載の検量線を用いて算出した。
＜測定結果＞
健常人から得た血清中のポドプラニン濃度は、平均±標準偏差 ２３．７±１１．６ｐｇ／ｍＬ（最大値９６．６、中央値２１．３、最小値０）という結果であった。パラメトリック法により求めた健常人のポドプラニンの濃度範囲は８．０〜５２．３ｐｇ／ｍＬ（べき乗＝０．１、変換原点＝−０．７９、 P＝９５．０００％）であった。測定値の分布を図４に示した。
[Example 3]
In this example, podoplanin in serum obtained from a healthy person was measured.
<Measurement of podoplanin in serum of healthy subjects>
Podoplanin in serum collected from healthy individuals (257 health check-up examinees, average age 39.2 years) was measured. The measurement of podoplanin was carried out in the same manner except that a serum sample was used in place of the standard solution in “1. Preparation of calibration curve (combination of antibodies No. 5 and No. 7B10)” in the measurement procedure described in Example 2. The amount of luminescence was measured and calculated using the calibration curve described in Example 2.
<Measurement results>
The concentration of podoplanin in serum obtained from healthy subjects was the result of mean ± standard deviation 23.7 ± 11.6 pg / mL (maximum value 96.6, median value 21.3, minimum value 0). The concentration range of the healthy person's podoplanin obtained by the parametric method was 8.0 to 52.3 pg / mL (power = 0.1, conversion origin = −0.79, P = 95.000%). The distribution of measured values is shown in FIG.
[実施例４]
本実施例は、中皮腫患者から得た血清又は胸水中のポドプラニンを測定した例である。
＜中皮腫患者血清中のポドプラニン測定＞
中皮腫患者５名から採取した血清中のポドプラニン及び中皮腫患者３名から採取した胸水中のポドプラニンを測定した。ポドプラニンの濃度は、実施例２に記載の測定操作の「１．検量線の作製（抗体Ｎｏ．５とＮｏ．７Ｂ１０の組み合わせ）」で標準液の代わりに血清検体又は胸水検体を用いる以外は同様に操作して発光量を測定し、実施例２に記載の検量線を用いて算出した。
＜測定結果＞
中皮腫患者から得た血清又は胸水中のポドプラニン濃度を表４及び図５に示した。実施例３で得た健常人での濃度範囲上限５２．３ｐｇ／ｍＬを疾患の基準とすると、血清で陽性率６０％（３／５）、胸水で陽性率１００％（３／３）となり、中皮腫患者から得た血清又は胸水中のポドプラニン濃度は高いことが判った。
[Example 4]
In this example, podoplanin in serum or pleural effusion obtained from a mesothelioma patient was measured.
<Measurement of podoplanin in mesothelioma patient serum>
Podoplanin in serum collected from 5 patients with mesothelioma and podoplanin in pleural fluid collected from 3 patients with mesothelioma were measured. The concentration of podoplanin is the same except that a serum sample or pleural effusion sample is used instead of the standard solution in “1. Preparation of calibration curve (combination of antibodies No. 5 and No. 7B10)” in the measurement procedure described in Example 2. The amount of luminescence was measured by operating the above and calculated using the calibration curve described in Example 2.
<Measurement results>
The concentrations of podoplanin in serum or pleural effusion obtained from mesothelioma patients are shown in Table 4 and FIG. When the upper limit of the concentration range in healthy subjects obtained in Example 3 is 52.3 pg / mL, the positive rate is 60% (3/5) for serum and 100% (3/3) is positive for pleural effusion, Serum or pleural fluid podoplanin concentrations from mesothelioma patients were found to be high.
[実施例５]
本実施例は、中皮腫患者に手術及び化学療法を実施し、その前後で血清中のポドプラニンを測定した例である。
＜中皮腫患者治療前後の血清中ポドプラニン測定＞
中皮腫患者２名に手術及び化学療法を実施し、その前後で採取した血清中のポドプラニンを測定した。ポドプラニンの測定は実施例２記載の測定操作の「１．検量線の作製（抗体Ｎｏ．５とＮｏ．７Ｂ１０の組み合わせ）」で標準液の代わりに血清検体を用いる以外は同様操作して発光量を測定し、実施例２に記載の検量線を用いて算出した。尚、手術及び治療前の測定値は実施例４と同値である。
＜測定結果＞
結果を表５に示した。２例とも手術及び化学療法により血清中のポドプラニン濃度が低下した。血中ポドプラニン濃度は治療効果を反映していると考えられた。
[Example 5]
In this example, surgery and chemotherapy were performed on mesothelioma patients, and serum podoplanin was measured before and after that.
<Measurement of serum podoplanin before and after treatment of mesothelioma patients>
Two patients with mesothelioma were subjected to surgery and chemotherapy, and the serum podoplanin collected before and after that was measured. Podoplanin was measured in the same manner as in Example 2 except that a serum sample was used in place of the standard solution in “1. Preparation of calibration curve (combination of antibodies No. 5 and No. 7B10)”. Was calculated using the calibration curve described in Example 2. The measured values before surgery and treatment are the same as those in Example 4.
<Measurement results>
The results are shown in Table 5. In both cases, the serum podoplanin concentration decreased due to surgery and chemotherapy. The blood podoplanin concentration was considered to reflect the therapeutic effect.
[実施例６]
本実施例は全自動化学発光酵素免疫測定装置スフィアライト１８０［オリンパス（株）製］を用いてポドプラニンを高感度かつ短時間で測定し、各種の肺癌患者から得た手術等の治療前後に採取した血清中のポドプラニン濃度を測定した例である。
[Example 6]
In this example, podoplanin was measured with high sensitivity and in a short time using a fully automated chemiluminescent enzyme immunoassay device Spherelite 180 (Olympus Co., Ltd.) and collected before and after treatments obtained from various lung cancer patients. This is an example in which the concentration of podoplanin in the serum was measured.
＜試薬の調製＞
１．抗体ビーズの作製
１重量％γ−アミノプロピルトリエトキシシラン含有アセトン溶液２０ｍLの入った蓋付きポリエチレン瓶にガラスビーズ１０００個を加え、１時間、２５℃で反応させ、反応液をアスピレーターで吸引除去した。
次いで脱イオン水２０ｍＬを加えて蓋をし、ポリエチレン瓶をゆっくりと２回倒置攪拌した後、液をアスピレーターで吸引除去してガラスビーズを洗浄した。この洗浄操作を３回行った。
次いで、この洗浄後のガラスビーズ１０００個を２重量％グルタルアルデヒド含有水溶液２０ｍＬの入った蓋付きポリエチレン瓶に加え、２５℃で１時間反応させた。そして、脱イオン水２０ｍＬを加えて蓋をし、ポリエチレン瓶をゆっくりと２回倒置攪拌した後、液をアスピレーターで吸引除去してガラスビーズを洗浄した。この洗浄操作を３回行った。
更にこの洗浄後のガラスビーズ１０００個を抗ポドプラニンモノクローナル抗体Ｎｏ．７を２０μｇ／ｍＬの濃度で含む０．０２Ｍリン酸緩衝液（ｐＨ８．７）２０ｍＬの入った蓋付きポリエチレン瓶に加え、２５℃で１時間反応させた。
反応後、抗ポドプラニンモノクローナル抗体含有リン酸緩衝液を除去し、ガラスビーズを０．１重量％の牛血清アルブミン含有の０．０２Ｍリン酸緩衝液（ｐＨ７．２）２０ｍＬに８時間浸漬し、この液を除去した後、ビーズを２０重量％ショ糖含有０．０２Ｍリン酸緩衝液（ｐＨ７．２）２０ｍＬに浸漬した。２時間浸漬後、液を除去したビーズを濾紙上で風乾してビーズ表面をショ糖でコーティングし、抗体ビーズを得た。
<Preparation of reagents>
1. Preparation of
Next, 20 mL of deionized water was added, the cap was capped, the polyethylene bottle was slowly inverted and stirred twice, and the glass beads were washed by sucking and removing the liquid with an aspirator. This washing operation was performed three times.
Next, 1000 glass beads after washing were added to a polyethylene bottle with a lid containing 20 mL of an aqueous solution containing 2% by weight glutaraldehyde, and reacted at 25 ° C. for 1 hour. Then, 20 mL of deionized water was added, the cap was capped, the polyethylene bottle was slowly inverted and stirred twice, and then the liquid was sucked and removed with an aspirator to wash the glass beads. This washing operation was performed three times.
Further, 1000 glass beads after washing were treated with anti-podopranin monoclonal antibody No. 7 was added to a polyethylene bottle with a lid containing 20 mL of 0.02 M phosphate buffer (pH 8.7) containing 20 μg / mL and reacted at 25 ° C. for 1 hour.
After the reaction, the phosphate buffer containing anti-podoplanin monoclonal antibody was removed, and the glass beads were immersed in 20 mL of 0.02M phosphate buffer (pH 7.2) containing 0.1% by weight of bovine serum albumin for 8 hours. After removing this solution, the beads were immersed in 20 mL of 0.02 M phosphate buffer (pH 7.2) containing 20% by weight of sucrose. After immersion for 2 hours, the beads from which the liquid had been removed were air-dried on filter paper and the bead surface was coated with sucrose to obtain antibody beads.
２．免疫反応用緩衝液の作製
０．０５Ｍリン酸緩衝液（ｐＨ７．０）１００ｍＬに、牛血清アルブミン（ＢＳＡ）１ｇ、塩化ナトリウム０．８５ｇ及び界面活性剤としてのツイーン８０ ０．５ｇを添加し、免疫反応用緩衝液を作製した。
３．酵素標識抗体液の作製
抗ポドプラニンモノクローナル抗体Ｎｏ．７Ｂ１０及び西洋ワサビ由来ペルオキシダーゼを用い、文献（エス・ヨシタケ、エム・イマガワ、イー・イシカワ、エトール；ジェイ．バイオケム，Ｖｏｌ．９２，１９８２，１４１３−１４２４）に記載の方法でＰＯＤ標識抗ポドプラニン抗体を調製し、冷凍（−３０℃）保存した。
上記で作製したＰＯＤ標識抗ポドプラニン抗体を免疫反応用緩衝液で抗体濃度として５μｇ／ｍＬとなるように希釈し、酵素標識抗体液を作製した。
2. Preparation of buffer for immune reaction To 100 mL of 0.05 M phosphate buffer (pH 7.0), 1 g of bovine serum albumin (BSA), 0.85 g of sodium chloride and 0.5 g of
3. Preparation of enzyme-labeled antibody solution Anti-podoplanin monoclonal antibody No. 7B10 and horseradish-derived peroxidase were used, and a POD-labeled anti-podoplanin antibody was produced by the method described in the literature (S Yoshitake, M Imagawa, E Ishikawa, Etol; J. Biochem, Vol. 92, 1982, 1413-1424). Prepared and stored frozen (−30 ° C.).
The POD-labeled anti-podoplanin antibody prepared above was diluted with an immune reaction buffer to an antibody concentration of 5 μg / mL to prepare an enzyme-labeled antibody solution.
４．基質液の作製
ルミノールナトリウム［シグマアルドリッチジャパン（株）製］０．２８ｇ及び４−（シアノメチルチオ）フェノール［三新化学工業（株）製］０．１ｇを、グッド緩衝液（０．１ＭのＥＰＰＥＳ、ｐＨ８．５）１００ｍＬに溶解し、基質液を作製した。
５．過酸化水素液の調製
３５重量％過酸化水素水［和光純薬工業（株）製］を脱イオン水で１８００倍に希釈し、過酸化水素水とした。
６．測定用試薬の調製
測定用カートリッジ［オリンパス（株）製］に抗体ビーズ１個、免疫反応用緩衝液２００μL、酵素標識抗体液２４０μL、基質液１４０μL、過酸化水素液１４０μLを入れ、アルミシールで密閉し測定用試薬とした。測定まで２〜１０℃で冷蔵保存した。
７．標準液
実施例１と同様に標準液を調製した。但し濃度範囲は０〜５０００ｐｇ／ｍＬ（濃度ポイント ０，５０，２００，４００，８００，２０００，５０００ｐｇ／ｍＬ）とした。
4). Preparation of substrate solution 0.28 g of luminol sodium [manufactured by Sigma Aldrich Japan Co., Ltd.] and 0.1 g of 4- (cyanomethylthio) phenol [manufactured by Sanshin Chemical Industry Co., Ltd.] were added to Good buffer (0.1 M EPPES PH 8.5) was dissolved in 100 mL to prepare a substrate solution.
5. Preparation of
6). Preparation of reagent for measurement Place one antibody bead, 200 μL of immune reaction buffer, 240 μL of enzyme-labeled antibody solution, 140 μL of substrate solution, 140 μL of hydrogen peroxide solution in a measurement cartridge [Olympus Co., Ltd.], and seal with aluminum seal This was used as a measuring reagent. Refrigerated at 2-10 ° C. until measurement.
7). Standard solution A standard solution was prepared in the same manner as in Example 1. However, the concentration range was 0 to 5000 pg / mL (concentration points 0, 50, 200, 400, 800, 2000, 5000 pg / mL).
＜測定操作＞
全自動化学発光酵素免疫分析装置スフィアライト１８０［オリンパス（株）製］を用いて測定を実施した。本装置は検体の分注から測定値の出力まで自動的に行う。測定の概要は次の通りである。
測定用試薬及び検体を所定の位置にセットする。測定用試薬のアルミシールを開封し、抗体ビーズが１個入った反応槽に免疫反応用緩衝液８０μＬ及び検体６０μＬを加え、３７℃で１４分間免疫反応する。１回当たり５００μＬの洗浄液（リン酸緩衝液）で６回洗浄してＢ／Ｆ分離を行った後、酵素標識抗体液２４０μＬを加え、３７℃で１４分間免疫反応する。同様にＢ／Ｆ分離を行った後、基質液７０μＬ及び過酸化水素液７０μＬを加え、３７℃で１分間反応させた後、発光量を計測する。 予め測定した既知検体の発光量で検量線を作成し、検体の発光量から濃度を算出する。
<Measurement operation>
The measurement was carried out using a fully automatic chemiluminescent enzyme immunoanalyzer Spherelite 180 [Olympus Co., Ltd.]. This device automatically performs from sample dispensing to measurement value output. The outline of the measurement is as follows.
A measuring reagent and a sample are set at predetermined positions. The aluminum seal of the reagent for measurement is opened, and 80 μL of immune reaction buffer and 60 μL of specimen are added to a reaction tank containing one antibody bead, and immunoreaction is performed at 37 ° C. for 14 minutes. After washing with 500 μL of washing solution (phosphate buffer solution) 6 times and performing B / F separation, 240 μL of enzyme-labeled antibody solution is added and immunoreacted at 37 ° C. for 14 minutes. Similarly, after B / F separation, 70 μL of a substrate solution and 70 μL of a hydrogen peroxide solution are added and reacted at 37 ° C. for 1 minute, and then the amount of luminescence is measured. A calibration curve is created based on the luminescence amount of a known specimen measured in advance, and the concentration is calculated from the luminescence amount of the specimen.
＜検体＞
検体として、標準液（上記操作中に記載の既知検体として使用）及び肺癌患者２０名から手術等の治療前後に得た血清を用いた。肺癌患者２０名の内訳は、肺腺癌１２名、肺扁平上皮癌３名、肺神経内分泌大細胞癌２名及び肺大細胞癌３名である。
<Sample>
As samples, standard solutions (used as known samples described in the above operation) and serum obtained from 20 lung cancer patients before and after treatment such as surgery were used. The breakdown of the 20 lung cancer patients is 12 lung adenocarcinoma, 3 lung squamous cell carcinoma, 2 lung neuroendocrine large cell carcinoma and 3 lung large cell carcinoma.
＜結果＞
結果を図６に示した。治療前後の比較で５０％（１０／２０）の患者にポドプラニン濃度の低下が認められた。特に治療前のポドプラニン濃度が４０ｐｇ／ｍＬ以上であった１２名の患者では、８３％（１０／１２）と高率でポドプラニン濃度の低下が認められ、肺癌に関連したポドプラニンが存在することが確認された。
<Result>
The results are shown in FIG. A decrease in podoplanin concentration was observed in 50% (10/20) of patients before and after treatment. In particular, in 12 patients whose podoplanin concentration before treatment was 40 pg / mL or more, the podoplanin concentration decreased at a high rate of 83% (10/12), and it was confirmed that podoplanin related to lung cancer was present It was done.
本発明は臨床検査、特に体液中のポドプラニンを免疫測定して肺癌及び中皮腫を検査する臨床検査薬に有用である。 INDUSTRIAL APPLICABILITY The present invention is useful for clinical examinations, particularly clinical examination drugs for examining lung cancer and mesothelioma by immunoassay of podoplanin in body fluids.
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