JP6655302B2 - 被検対象の検出方法並びにそのための免疫測定器具及びモノクローナル抗体 - Google Patents
被検対象の検出方法並びにそのための免疫測定器具及びモノクローナル抗体 Download PDFInfo
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- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1253—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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Description
1.マイコプラズマ・ニューモニエ抗原の調製
マイコプラズマ・ニューモニエを培養し、その培養液を60℃、30分間の熱処理で不活化したものを用いた。
1.のマイコプラズマ不活化抗原をBALB/cマウスに免疫し、一定期間飼育したマウスから脾臓を摘出し、ケラーらの方法(Kohler et al., Nature, vol, 256, p495-497(1975))によりマウスミエローマ細胞(P3×63)と融合した。得られた融合細胞(ハイブリドーマ)を、37℃インキュベーター中で維持し、マイコプラズマ・ニューモニエP1抗原を固相したプレートとマイコプラズマ・ニューモニエP30抗原を固相したプレートを用いたELISAにより上清の抗体活性を確認しながら細胞の純化(単クローン化)を行った。
実施例1で得た各モノクローナル抗体とマイコプラズマ・ニューモニエとの反応性をウエスタンブロッティングで確認した。マイコプラズマ・ニューモニエはPPLO Brothで培養し、その培養液をサンプルとした。
1.抗マイコプラズマ・ニューモニエ抗体のニトロセルロースメンブレンへの固定化
実施例1で作製した抗体を1.0mg/mLになるように精製水で希釈した液及び抗マウスIgG抗体を準備し、PETフィルムで裏打ちされたニトロセルロースメンブレンのサンプルパッド側に抗体、吸収体側に抗マウスIgG抗体をそれぞれ線状に塗布した。その後、ニトロセルロースメンブレンを45℃、30分間乾燥させ、抗マイコプラズマ・ニューモニエ抗体固定化メンブレンを得た。本実施例において抗体固定化メンブレンと呼ぶ。
実施例1で作製した抗体を1.0mg/mLになるように精製水で希釈し、これに着色ポリスチレン粒子を0.1%になるように加え、攪拌後、カルボジイミドを1%になるように加え、さらに攪拌する。遠心操作により上清を除き、50mM Tris(pH9.0)、3%BSAに再浮遊し、抗マイコプラズマ・ニューモニエ抗体結合着色ポリスチレン粒子を得た。本実施例において、抗体固定化粒子と呼ぶ。
1で作製した抗体固定化メンブレンと他部材(バッキングシート、吸収帯、サンプルパッド)とを貼り合せて5mm幅に切断し、マイコプラズマ・ニューモニ試験片とした(図2参照)。これらを本実施例において、試験片と呼ぶ。
P1、P30及びP1とP30を測定する免疫測定器具の感度比較
実施例1で得られたモノクローナル抗体を表2の組み合わせで実施例3の手順によりP1試験片(比較例1)、P30試験片(比較例2)、P1−P30試験片(実施例4)の3種類作製した。
P1−P30試験片の使用抗体をP1−P30AとP1−P30Bの組み合わせで実施例4と同様に試験した。その結果、P1−P30Aのみの組み合わせ(実施例4)と比較して2〜4倍優れた感度を示した。
ロ 標識体部
ハ 検出領域
ニ サンプルパッド
ホ 吸収帯
へ バッキングシート
Claims (6)
- 被検対象の検出を可能とする2種類以上の抗原が存在する被検対象の検出方法であって、前記2種類以上の抗原を認識するモノクローナル抗体又はその抗原結合性断片を用いた免疫測定方法により行う、前記被検対象の検出方法であって、前記被検対象がマイコプラズマ・ニューモニエであり、前記2種類以上の抗原がP1タンパク質とP30タンパク質である、方法。
- 前記モノクローナル抗体又はその抗原結合性断片が標識または固相の少なくともいずれか一方に使用される、前記免疫測定方法がサンドイッチ法である請求項1記載の方法。
- 前記免疫測定方法がイムノクロマトグラフィー法である請求項2記載の方法。
- 請求項1記載の方法を行うための免疫測定器具であって、前記モノクローナル抗体又はその抗原結合性断片が標識または固相の少なくともいずれか一方に使用される免疫測定器具であり、前記被検対象がマイコプラズマ・ニューモニエであり、前記2種類以上の抗原がP1タンパク質とP30タンパク質である、免疫測定器具。
- イムノクロマト試験片である請求項4記載の免疫測定器具。
- マイコプラズマ・ニューモニエ由来のP1タンパク質とP30タンパク質に対して特異的に反応するモノクローナル抗体。
Priority Applications (7)
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JP2015110585A JP6655302B2 (ja) | 2015-05-29 | 2015-05-29 | 被検対象の検出方法並びにそのための免疫測定器具及びモノクローナル抗体 |
MYPI2017704569A MY194325A (en) | 2015-05-29 | 2016-05-27 | Test object detection method, and immunoassay instrument and monoclonal antibody for same |
EP16803232.4A EP3306319B1 (en) | 2015-05-29 | 2016-05-27 | Test object detection method, and immunoassay instrument and monoclonal antibody for same |
KR1020177037381A KR102569269B1 (ko) | 2015-05-29 | 2016-05-27 | 피검 대상의 검출 방법 및 그것을 위한 면역 측정 기구 및 모노클로널 항체 |
CN201680031403.XA CN107683412A (zh) | 2015-05-29 | 2016-05-27 | 被检对象的检测方法以及用于该方法的免疫测定仪器和单克隆抗体 |
PCT/JP2016/065683 WO2016194797A1 (ja) | 2015-05-29 | 2016-05-27 | 被検対象の検出方法並びにそのための免疫測定器具及びモノクローナル抗体 |
US15/577,542 US20180164316A1 (en) | 2015-05-29 | 2016-05-27 | Test object detection method, and immunoassay instrument and monoclonal antibody for same |
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CN111548423B (zh) * | 2020-06-04 | 2021-11-30 | 珠海丽珠试剂股份有限公司 | 肺炎支原体融合抗原及其制备方法和应用 |
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EP0327648B1 (en) * | 1986-12-15 | 1993-06-09 | MITSUI TOATSU CHEMICALS, Inc. | Human monoclonal antibody and drug for prophylaxis and treatment of infectious diseases comprising same as effective ingredient |
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WO2003012445A1 (en) * | 2001-07-30 | 2003-02-13 | Quest Diagnostics Investments Incorporated | Methods and kits for detecting ita in a biological sample |
JP4117563B2 (ja) * | 2003-02-03 | 2008-07-16 | 東洋紡績株式会社 | 皮膚糸状菌の検出方法、検出用試薬および抗原性賦活化方法 |
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US8323966B2 (en) * | 2009-06-25 | 2012-12-04 | Geron Corporation | Differentiated pluripotent stem cell progeny depleted of extraneous phenotypes |
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CN102928606B (zh) * | 2012-11-16 | 2015-10-07 | 武汉明德生物科技股份有限公司 | 多抗体标记的降钙素原快速检测试剂盒 |
CN103275196B (zh) * | 2013-06-24 | 2015-09-30 | 武汉市长立生物技术有限责任公司 | 一种肺炎支原体重组抗原及其制备方法和应用 |
EP3037822B1 (en) * | 2013-08-23 | 2019-10-09 | Tauns Co., Ltd. | Mycoplasma pneumoniae immunological detection method and kit |
CN104198703B (zh) * | 2014-08-18 | 2015-12-30 | 湖北工业大学 | 人肺炎支原体金标银染免疫层析检测试剂盒及其制备方法和应用 |
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MY194325A (en) | 2022-11-28 |
KR20180020992A (ko) | 2018-02-28 |
KR102569269B1 (ko) | 2023-08-22 |
EP3306319B1 (en) | 2020-03-25 |
EP3306319A1 (en) | 2018-04-11 |
JP2016223913A (ja) | 2016-12-28 |
CN107683412A (zh) | 2018-02-09 |
EP3306319A4 (en) | 2019-01-09 |
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