JP6699823B2 - 子宮頸がんの試験方法およびそれに用いる試験試薬 - Google Patents
子宮頸がんの試験方法およびそれに用いる試験試薬 Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57411—Specifically defined cancers of cervix
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
- C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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Description
本発明の試験方法は、前述のように、子宮頸がんの罹患の可能性を試験する方法であって、子宮頸部から単離された生体試料について、MUCの発現を検出する工程を含むことを特徴とする。
本発明の試験試薬は、前述のように、子宮頸がんの試験試薬であり、MUCに対する結合物質を含むことを特徴とする。本発明の試験試薬は、前記本発明の子宮頸がんの試験方法に使用することができる。本発明の試験試薬は、前記結合物質を含むことが特徴であって、その他の条件や構成は、何ら制限されない。また、本発明の試験試薬は、特に示さない限り、前記本発明の試験方法における前記結合物質の記載を援用できる。
本発明の診断方法は、例えば、子宮頸がんの診断方法であり、子宮頸部から単離された生体試料について、MUCの発現を検出する工程を含むことを特徴とする。本発明の診断方法は、前記生体試料についてMUCの発現を検出することが特徴であり、その他の条件や工程は、何ら制限されない。本発明の診断方法は、前記本発明の試験方法における記載を援用でき、医師の行為を含んでもよい。
細胞診により子宮頸部上皮内腫瘍(CIN)が確認された患者30人から、子宮頸部上皮の生体組織を採取し、これらの検体について、MUC1の発現を確認した。
細胞診により子宮頸がんと診断された患者から、子宮頸部上皮の生体組織を採取し、液状細胞診(LBC)用の検体を調製し、前記検体について、MUC1の発現を確認した。
(1) 前記キットの容器内でLBC用の検体を混和し、ディスポーザブルのスポイトで、前記容器中のLBC細胞浮遊液を10ml採取してスピッツ管に移し、800G、2分間で遠心後、上清を除去した。
(2)前記スピッツ管内の沈渣に1%カーボワックス含有50%エタノール5mlを加え、前記沈渣を再浮遊させ、5分間静置後、800G、2分間で遠心し、さらに上清を除去した。
(3)前記スピッツ管内の沈渣を、前記(1)で使用したディスポーザブルピペットで吸い上げ、適量(0.5〜1ml)を、2枚のスライドガラスの下から1/3位の領域にすり合わせ、30分間、ふ卵器内で、完全に塗抹細胞を乾燥させた。
(4)局方エタノールを染色用バットに用意し、乾燥させた前記スライドを入れて、30分間以上固定した。
(5)固定完了後、蒸留水バットに前記乾燥スライドを5分間浸し、免疫染色用PBSバットに移した。この染色前処理を施した前記スライドを、免疫染色に使用した。
(a)Alexa Fluor 488で標識化した2次抗体
商品名 Alexa Fluor 488, Life Technologies社、goat anti−mouse IgG
(b)デキストランポリマーに抗マウスIgGとHRPが結合した2次抗体
商品名 EnVision(商標)+Kit, K4007 DAKO社、HRP(ペルオキシダーゼ)標識goat anti−mouse IgG
発色基質:DAB
(c)ビオチン標識した抗マウスIgGにAP(アルカリホスファターゼ)標識ストレプトアビジンを結合した2次抗体
商品名 ヒストファインSAB−APキット
発色基質:Fast Red
商品名 Vulcam Fast Red Chromogen Kit2、Biocare Medical社
(1) 前記スライドを軽くPBSに浸してから、細胞塗抹領域を避けて余分なPBSを拭き上げたのち、湿潤チャンバー内にスライドを並べ、前記スライドに、500倍に希釈した前記(a)、(b)または(c)の抗MUC1抗体を100μl滴下して、前記スライドにおける塗抹面全体にわたせた。そして、30分反応後、前記スライドをPBSで3回洗浄後、前記各発色系または蛍光系に応じて、下記(2a)、(2b)または(2c)の染色ステップを行った。
MUC1を抗原とする異なる抗MUC1抗体を用いて、子宮頸部上皮の検体について染色性の確認を行った。
子宮頸部上皮組織において、正常扁平上皮組織と子宮頸部上皮内病変との境界が、MUC1の局在によって判別できるかを確認した。
子宮頸部の内膜腺細胞(EC)におけるMUC1の染色性を確認した。
ベセスダシステムにより、HSIL(高度扁平上皮内病変)、ASC−US(意義不明異型扁平上皮細胞)、ASC−H(HSILを除外できない異型扁平上皮細胞)と判定された検体について、抗MUC1抗体によりMUC1の発現を確認した。
ベセスダシステムにより、NILM(上皮内病変または悪性病変を認めず)と判定された検体について、抗MUC1抗体によりMUC1の発現を確認した。
Claims (6)
- 子宮頸部から単離された子宮頸部上皮試料について、MUC1の発現を検出する工程を含み、前記MUC1の発現が、子宮頸がんの罹患の可能性を示す指標であることを特徴とする子宮頸がんの罹患の可能性を試験する試験方法。
- 前記検出工程が、MUC1に対する結合物質により、MUC1の発現を検出する工程である、請求項1記載の試験方法。
- 前記検出工程において、MUC1に結合した前記結合物質を検出することにより、間接的に、MUC1の発現を検出する、請求項2記載の試験方法。
- 前記MUC1と前記結合物質との結合によって直接的または間接的に生じるシグナルを検出することにより、MUC1の発現を検出する、請求項2または3記載の試験方法。
- 前記結合物質が、抗MUC1抗体である、請求項2から4のいずれか一項に記載の試験方法。
- 前記子宮頸部上皮試料が、子宮頸部上皮由来の細胞または組織である、請求項1から5のいずれか一項に記載の試験方法。
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US15/766,630 US20180299447A1 (en) | 2015-10-08 | 2016-06-30 | Cervical cancer testing method and test reagent used therefor |
PCT/JP2016/069468 WO2017061152A1 (ja) | 2015-10-08 | 2016-06-30 | 子宮頸がんの試験方法およびそれに用いる試験試薬 |
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