JP2016211853A - 微生物抗原の回収法 - Google Patents
微生物抗原の回収法 Download PDFInfo
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- JP2016211853A JP2016211853A JP2015092218A JP2015092218A JP2016211853A JP 2016211853 A JP2016211853 A JP 2016211853A JP 2015092218 A JP2015092218 A JP 2015092218A JP 2015092218 A JP2015092218 A JP 2015092218A JP 2016211853 A JP2016211853 A JP 2016211853A
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Abstract
【解決手段】微生物を含む検体を微生物を通過させない孔径を有する濾過膜に通し、前記検体中の微生物を濾過膜上に捕捉し、微生物を捕捉した濾過膜に微生物の膜を破壊し得る微生物破壊試薬を通すことにより捕捉した微生物を濾過膜上で破壊し、ろ液中に抗原を回収することを含む、微生物の抗原を回収する方法。
【選択図】なし
Description
[1] 微生物を含む検体を微生物を通過させない孔径を有する濾過膜に通し、前記検体中の微生物を濾過膜上に捕捉し、微生物を捕捉した濾過膜に微生物の膜を破壊し得る微生物破壊試薬を通すことにより捕捉した微生物を濾過膜上で破壊し、ろ液中に抗原を回収することを含む、微生物の抗原を回収する方法。
[2] 微生物が細菌であることを特徴とする[1]の微生物の抗原を回収する方法。
[3] 微生物破壊試薬が界面活性剤、アルカリ性溶液、又は界面活性剤とアルカリ性溶液の混合物であることを特徴とする[1]の微生物の抗原を回収する方法。
[4] アルカリ性溶液のpHが、11以上であることを特徴とする、[3]の微生物の抗原を回収する方法。
[5] アルカリ性溶液が水酸化ナトリウム溶液である、[3]の微生物の抗原を回収する方法。
[6] [1]〜[5]のいずれかの方法で回収した微生物の抗原を免疫学的測定法で測定することを含む、微生物の検出方法。
[7] 免疫学的測定法がイムノクロマト法である、[6]の微生物の検出方法。
1.微生物の抗原の回収
本発明の方法により抗原を回収することができる微生物は何ら限定されるものではなく、細菌、藻類、原生生物、酵母やカビなどの真菌、粘菌等の真核生物が含まれる。これらの中でもヒトや非ヒト動物の感染症を引き起こす病原体微生物が望ましい。例えば、メチシリン耐性黄色ブドウ球菌(MRSA)を含む黄色ブドウ球菌、病原大腸菌を含む大腸菌、サルモネラ菌、緑膿菌、コレラ菌、赤痢菌、炭疽菌、結核菌、ボツリヌス菌、破傷風菌、連鎖球菌、カンピロバクター、ウェルシュ菌、腸炎ビブリオ菌、クラミジア・トラコマティス、溶連菌、百日咳菌、ヘリコバクター・ピロリ、レプトスピラ、トレポネーマ・パリダム、ボレリア、等の細菌、カンジダ、白癬菌、アスペルギルス等が挙げられる。
回収した抗原の測定はいかなる方法で行うこともできるが、好ましくは測定しようとする抗原に特異的な抗体を用いて、抗原抗体反応を利用した免疫学的測定法により測定する。免疫学的測定法として、免疫染色法(蛍光抗体法、酵素抗体法、重金属標識抗体法、放射性同位元素標識抗体法を含む)、電気泳動法による分離と蛍光、酵素、放射性同位元素などによる検出方法とを組み合わせた方法(ウエスタンブロット法、蛍光二次元電気泳動法を含む)、酵素免疫測定吸着法(ELISA)、ドット・ブロッティング法、ラテックス凝集法(LA:Latex Agglutination-Turbidimetric Immunoassay)、イムノクロマト法等が挙げられる。好ましくは、イムノクロマト法又はELISA法により測定する。
(1)抗体感作ラテックス粒子の調製および乾燥化
抗PBP2’モノクローナル抗体を常法によりペプシンで処理しF(ab’)2を得た。これを粒子径0.4μmのラテックス粒子に感作し結合させ、ポリスチレン不織布に噴霧した。次いで減圧装置内で1時間減圧乾燥し、乾燥ラテックス抗体パッドとした。使用時には4mm間隔で裁断し、標識試薬部位2として用いた。
ラテックス感作に用いた抗PBP2’モノクローナル抗体とは認識部位を異にする第二の抗PBP2’モノクローナル抗体を常法によりペプシンで処理しF(ab’)2を得た。これを0.075%CHAPSを含むクエン酸緩衝液(pH6)で希釈し、ニトロセルロースメンブレン(固相支持体5)に塗布し、充分に乾燥した(捕捉試薬部位3)。対照用試薬としてAnti-Mouse IgGsを同様にニトロセルロースメンブレンに塗布し、充分に乾燥した(対照部位4)。
イムノクロマト法用試験片の構造を図1に示す。
検体浮遊用液として、0.1M水酸化ナトリウムと1.0% Triton X-100を混合した水溶液を調製した。
R2試薬(ろ液調整液)として、0.6Mトリス塩酸塩水溶液(pH6.0±0.5)を調製した。
上記の検体浮遊溶液、R1試薬及びR2試薬を併せてPBP2’抽出試薬と呼ぶ。
濾過膜のサイズの検討
血液寒天培地にて培養したMRSAを生理食塩水に浮遊させ、1.0×108個に調整したものを検体とした。
1)検体1:検体浮遊液1になるように混合し、全量2mLとした。
2)全量2mLを濾過膜(材質:セルロースアセテート、孔径:0.45μm、直径:25mm)でろ過した。
3)2)の濾過膜にR1試薬を1000μLろ過し、濾過膜内に液を残し5分静置した。
4)3)の濾過膜にR2試薬を400μLろ過し、ろ液を回収した。
5)実施例1で作製したPBP2’試験片での検体供給部位に滴下し、10分後に判定した。判定結果を表1に示す。「++」は比較的強い陽性を示し、「+++」は強い陽性を示す。
濾過膜の材質の検討
血液寒天培地にて培養したMRSAを生理食塩水に浮遊し、1.0×108個に調整したものを検体とした。
1)検体1:検体浮遊液1になるように混合し、全量2mLとした。
2)全量2mLを濾過膜(材質:セルロースアセテート、孔径:0.45μm、直径:25mm)でろ過した。
3)2)の濾過膜にR1試薬を1000μLろ過し、濾過膜内に液を残し5分静置した。
4)3)の濾過膜にR2試薬を400μLろ過し、ろ液を回収した。
5)実施例1で作製したPBP2’試験片での検体供給部位に滴下し、10分後に判定した。判定結果を表2に示す。「++」は比較的強い陽性を示し、「+++」は強い陽性を示す。
MCE: セルロース混合エステル
PES: ポリエーテルスルフォン
hydrophilic polyethersulfone: 親水性ポリエーテルスルホン
GHP: 親水性ポリプロピレン
NYL:ナイロン
CA:セルロースアセテート
GxF:多層グラスファイバー製プレフィルター
GF: ホウケイ酸グラスファイバー製プレフィルター
PTFE: ポリテトラフルオロエチレン
界面活性剤の検討
血液寒天培地にて培養したMRSAを生理食塩水に浮遊し、1.0×108個に調整したものを検体とした。
1)検体1:検体浮遊液1になるように混合し、全量2mLとした。
2)全量2mLを濾過膜(材質:セルロースアセテート、孔径:0.45μm、直径:25mm)でろ過した。
3)2)の濾過膜にR1試薬を1000μLろ過し、濾過膜内に液を残し5分静置した。
4)3)の濾過膜にR2試薬を400μLろ過し、ろ液を回収した。
5)実施例1で作製したPBP2’試験片での検体供給部位に滴下し、10分後に判定した。判定結果を表3に示す。「++」は比較的強い陽性を示し、「+++」は強い陽性を示す。
従来技術との比較実験
本発明の濾過膜法と従来技術である遠心法と再現性について比較した。
検体はMRSA菌株を血液培地(馬脱繊維血1:液体培地4)で希釈した。
1)検体1:検体浮遊液3になるように混合し、全量2mLとした。
2)7000g、5min、室温で遠心した。
3)上清を捨て、R1試薬を200μL加えた。
4)R2試薬を100μL添加し、混合した。
5)実施例1で作製したPBP2’試験片での検体供給部位に滴下し、10分後に判定した。
1)検体1:検体浮遊液1になるように混合し、全量2mLとした。
2)全量2mLを濾過膜(材質:セルロースアセテート、孔径:0.45μm、直径:25mm)でろ過した。
3)2)の濾過膜にR1試薬を1000μLろ過し、濾過膜内に液を残し5分静置した。
4)3)の濾過膜にR2試薬を400μLろ過し、ろ液を回収した。
5)実施例1で作製したPBP2’試験片での検体供給部位に滴下し、10分後に判定した。
2 標識試薬部位
3 捕捉試薬(キャプチャー抗体)部位
4 対照(コントロール)部位
5 固相支持体(ニトロセルロース膜)
6 吸収部位(アブソーベントパッド)
7 トップラミネートまたはハウジング
Claims (7)
- 微生物を含む検体を微生物を通過させない孔径を有する濾過膜に通し、前記検体中の微生物を濾過膜上に捕捉し、微生物を捕捉した濾過膜に微生物の膜を破壊し得る微生物破壊試薬を通すことにより捕捉した微生物を濾過膜上で破壊し、ろ液中に抗原を回収することを含む、微生物の抗原を回収する方法。
- 微生物が細菌であることを特徴とする請求項1記載の微生物の抗原を回収する方法。
- 微生物破壊試薬が界面活性剤、アルカリ性溶液、又は界面活性剤とアルカリ性溶液の混合物であることを特徴とする請求項1記載の微生物の抗原を回収する方法。
- アルカリ性溶液のpHが、11以上であることを特徴とする、請求項3記載の微生物の抗原を回収する方法。
- アルカリ性溶液が水酸化ナトリウム溶液である、請求項3記載の微生物の抗原を回収する方法。
- 請求項1〜5のいずれか1項に記載の方法で回収した微生物の抗原を免疫学的測定法で測定することを含む、微生物の検出方法。
- 免疫学的測定法がイムノクロマト法である、請求項6記載の微生物の検出方法。
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JP7313830B2 (ja) | 2019-01-29 | 2023-07-25 | デンカ株式会社 | 検体の展開を制御し得る、糖鎖抗原を抽出し測定するためのイムノクロマト試験キット |
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KR102561659B1 (ko) | 2023-07-28 |
JP6688561B2 (ja) | 2020-04-28 |
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CN107533059A (zh) | 2018-01-02 |
ES2894842T3 (es) | 2022-02-16 |
US10436783B2 (en) | 2019-10-08 |
KR20230117629A (ko) | 2023-08-08 |
EP3290920A4 (en) | 2018-11-07 |
US20180143195A1 (en) | 2018-05-24 |
KR20170140252A (ko) | 2017-12-20 |
EP3290920A1 (en) | 2018-03-07 |
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EP3290920B1 (en) | 2021-09-01 |
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