JP5331285B2 - Antibody for detection of Mycoplasma pneumonia - Google Patents
Antibody for detection of Mycoplasma pneumonia Download PDFInfo
- Publication number
- JP5331285B2 JP5331285B2 JP2001558014A JP2001558014A JP5331285B2 JP 5331285 B2 JP5331285 B2 JP 5331285B2 JP 2001558014 A JP2001558014 A JP 2001558014A JP 2001558014 A JP2001558014 A JP 2001558014A JP 5331285 B2 JP5331285 B2 JP 5331285B2
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- Prior art keywords
- antibody
- protein
- mycoplasma pneumoniae
- ribosomal protein
- bacteria
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Description
技術分野
本発明は、一般的な肺炎の原因微生物であるマイコプラズマ・ニューモニア（Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅ）に属する微生物の検出に有用な抗体、該微生物の検出方法、該微生物の検出用試薬キット、及び該微生物検出用抗体の製造方法に関する。
本発明は、医薬品業界、特にＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅによって惹起される非定型肺炎の診断に重要であり、医薬品の産業上有用である。
本発明は、検体、例えば、のど綿棒、組織サンプル、体液、実験用の溶液および培養物から採取された検体中に含まれる微生物、Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅを検出するのに有用である。
背景技術
微生物感染症の診断は、通常感染部位などでの原因菌の検出か、血清、体液中の原因菌に対する抗体の検出により確定される。特に、この診断は原因菌の検出が患者への迅速な治療を可能にする意味で重要である。
感染症原因菌の検出は、一般に、原因菌の分離培養を経て、その生理学的、生化学的あるいは構造的な特性に基づきこれを同定する培養同定法、原因菌の遺伝子をＰｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ（ＰＣＲ）法または特異的核酸ハイブリダイゼーションにより増幅させて、これを検出する遺伝子的診断法および抗体と原因菌の抗原マーカーとの特異的反応を利用して原因菌を検出する免疫学的方法に分類される。
しかしながら、培養同定法または遺伝子的診断法を用いる場合には、結果を得るのに長時間を要する。従って、原因菌を短時間にしかも高感度で検出することができ、迅速かつ適切な患者の治療につながる免疫学的方法による診断が汎用されている。
従来免疫学的方法による感染症原因菌の検出には、菌種によって様々なマーカー抗原と抗体の組み合わせが使われている。
Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅは肺炎、気管支炎および咽頭炎の一般的な原因菌である。小さな真菌様のグラム陰性菌であり、選択的にヒトの体内に侵入し、病気を引き起す。一般社会で集団的に発症する肺炎の約１５−２０％、および限定された集団内の肺炎の５０％までがＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅによって引き起されると言われている（Ｒａｇｎａｒ Ｎｏｒｒｂｙ １９９９）。
この菌は非常に微小の培養し難い微生物であり、濃厚培地中で小さなコロニーを形成する。栄養冨化寒天培地中でのＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅの成長は非常に緩慢であり、培地中に菌体を同定するまでに少なくとも２１日間を要する（Ｇｒａｎａｔｏ，Ｐｏｅ，ｅｔ ａｌ．１９８０）。ヒトの体内に起源を有するいくつかのマイコプラズマ種の微生物は同様の生化学的反応を起こす。一方、細胞壁がないため、明るい顕微鏡下での観察を一層困難なものとする（Ｋｎｕｄｓｏｎ ａｎｄ ＭａｃＬｅｏｄ １９７９）。従って、迅速に病原菌として検出する診断法としては、グラム染色法と培養法などを適用することができない。
Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅ検出の遺伝子配列に用いられるＤＮＡハイブリダイゼーション分析プローブは、Ｈｙｍａｎら、Ｂｕｃｋら及びＢｅｒｎｅｔら、（Ｈｙｍａｎ，Ｙｏｇｅｖ ｅｔ ａｌ．１９８７；Ｂｅｒｎｅｔ，Ｇａｒｒｅｔ ｅｔ ａｌ．１９９３；Ｂｕｃｋ，Ｏ′Ｈａｒａ ｅｔ ａｌ．１９９３）等によって報告されている。Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅリボソーマルＲＮＡ（ｒＲＮＡ）検出に用いられるプローブは、Ｔｉｌｔｏｎら（Ｔｉｌｔｏｎ，Ｄｉａｓ ｅｔ ａｌ．１９８０）、Ｙｏｇｅｖら（Ｙｏｇｅｖ，Ｈａｌａｃｈｍｉ ｅｔ ａｌ．１９８８）、Ｇｏｂｅｌら（Ｇｏｂｅｌ，Ｇｅｉｓｅｒ ｅｔ ａｌ．１９８７）、ＺｉｖｉｎとＭｏｎａｈａｎ（ＥＰＯ ３０５１４５）及びＧｏｂｅｌａｎｄとＳｔａｎｂｒｉｄｇｅ（ＥＰＯ２５０６６２）等に記載されている。Ｋａｉら（Ｋａｉ，Ｋａｍｉｙａ ｅｔ ａ１．１９８７）及びＪｅｎｓｅｎら（Ｊｅｎｓｅｎ，Ｓｏｎｄｅｒｇａｒｄ−Ａｎｄｅｒｓｅｎ ｅｔ ａｌ．１９９３）等はＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅの１６Ｓ ｒＲＮＡ配列のプライマーを記載している。ここに述べられた全てのプローブはＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＤＮＡまたはＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅとＭｙｃｏｐｌａｓｍａ ｇｅｎｉｔａｌｉｕｍのＤＮＡに対して設計されている。これらのプローブは比較的感度が高いので、ごく最近利用可能となった技術、例えば、ＰＣＲ法を用いてこれらを捕捉する。ＰＣＲ法は非常に感度が高く、しかもハイブリダイゼーションよりも短時間で行うことができる。しかしながら、培養Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅ陰性の無症状の人や、病気が治癒した後のキャリアの人に陽性を示す、擬陽性の問題がしばしば存在する。
特開昭６３−２９８号公報は、約４３キロダルトンのＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅの膜抗原蛋白質に対するモノクローナル抗体を用いたウェスタンブロット法をベースとする免疫検出法が記載されている。しかしながら、上記抗体および当該抗体を用いる検出法は、特異性が低くＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅに対する種の特異的な診断のためには不十分であるという問題を有する。さらに、この診断法によると結果がでるまでに少なくとも５時間かかるという問題もある（Ｍａｄｓｅｎ ｅｔ ａｌ．１９８８）
発明の開示
本発明は、上記のような課題を解決するためになされたものである。すなわち、本発明は、Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅに属する微生物を特異的、かつ高感度に迅速に検出する方法、その検出に用いる検出用抗体、検出用試薬キットを提供することを課題とする。さらに、本発明は、その検出に用いる検出用抗体の製造方法を提供することを課題とする。
本発明者等は、全ての微生物において同一の機能が保存されている蛋白質を有用な抗原蛋白質として見出した。通常、このような蛋白質の構造変化はきわめて少ないと予想される。しかし驚くべきことに、該蛋白質に対する抗体は、微生物の種あるいは属特異的であり、該蛋白質に対する抗体は、微生物の種あるいは属特異的な識別に用いることが可能な多様性を持つとともに、対象となる微生物についてはその全ての血清型を検出しうるものであることが見出されたのである。さらに詳しくは、該蛋白質はアミノ酸配列の変化を経て、同一種間では全く構造が同じであり、種が異なる場合は構造の変化を伴なう種特異性を示す有用な蛋白質である。
本発明者らは全ての微生物細胞に同一機能の分子として存在し、しかもそのアミノ酸構造が微生物間である程度の相違点をもつ細胞内分子、特にリボソーム蛋白質の一種であるＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質に着目した。Ｒｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質は分子量約１３キロダルトンの蛋白質であり、蛋白質合成に必須のリボソーム蛋白質として存在することが知られている。特にＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅを含むいくつかの微生物ではＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質の全アミノ酸配列が解析されている。
本発明者らはこの分子が微生物間で類似しているにもかかわらず、その一部に各微生物固有の構造部分を持つことに着目し、このＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質に対する抗体を利用することで様々な微生物、細菌の種特異的でかつ全ての同一菌種内の血清型について検出が可能であることを見いだした。
本発明者等は、Ｍｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅの該蛋白質に特異的な抗体が得られること、および該抗体を用いることによりＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅの特異的な検出が可能であることを見出し、本発明を完成した。
本発明により、Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質に対して特異的なモノクローナル抗体が見出され開発された。この抗体は新規であり、従来公知のいかなる抗体とも異なり、上記蛋白質と特異的に反応する性質を有する。
配列表において配列番号１及び２はＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２遺伝子のＤＮＡ配列及び対応するアミノ酸配列である（ＮＣＢＩ ｄａｔａｂａｓｅ ａｃｃｅｓｓｉｏｎ ＃ ＮＣ ０００９１２）。なお、配列表に記載されたアミノ酸配列の左端および右端はそれぞれアミノ基末端（以下、Ｎ末端）およびカルボキシル基末端（以下、Ｃ末端）であり、また塩基配列の左端および右端はそれぞれ５′末端および３′末端である。クロスマッチテストのアミノ酸配列のアミノ酸は１文字略字で表している。また、クロスマッチテストにおける「＋」の表記は、異なるアミノ酸であるが疎水性などの性質が類縁のアミノ酸であること、「」のブランクは性質も含めて全く異なるアミノ酸であることを示す。また、本発明で述べられる遺伝子操作の一連の分子生物学的な実験は通常の実験書の記載方法によって行うことができる。前記の通常の実験書としては、例えばＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ，Ａ ｌａｂｏｒａｔｏｒｙ ｍａｎｕａｌ，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ，Ｓｈａｍｂｒｏｏｋ，Ｊ．ら（１９８９）を挙げることができる。
本発明において抗体は、ポリクローナル抗体またはモノクローナル抗体を指し、上記Ｒｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質の全長あるいはその部分ペプチドを用いて作成することができる。抗体を作成するためのペプチドの長さは特に限定されないがＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質に対する抗体の場合、この蛋白質を特徴づけられる長さがあれば良く、好ましくは５アミノ酸以上、特に好ましくは８アミノ酸以上のペプチドを用いれば良い。このペプチドあるいは全長蛋白質をそのまま、またはＫＬＨ（ｋｅｙｈｏｌｅ−ｌｉｍｐｅｔ ｈｅｍｏｃｙａｎｉｎ）やＢＳＡ（ｂｏｖｉｎｅ ｓｅｒｕｍ ａｌｂｕｍｉｎ）といったキャリア蛋白質と架橋した後必要に応じてアジュバントとともに動物へ接種せしめ、その血清を回収することでＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質を認識する抗体（ポリクローナル抗体）を含む抗血清を得ることができる。また抗血清より抗体を精製して使用することもできる。接種する動物としてはヒツジ、ウマ、ヤギ、ウサギ、マウス、ラット等であり、特にポリクローナル抗体作製にはヒツジ、ウサギなどが好ましい。また、ハイブリドーマ細胞を作製する公知の方法によりモノクローナル抗体を得ることも可能であるが、この場合はマウスが好ましい。
また該蛋白質の全長または５残基以上、望ましくは８残基以上のアミノ酸配列をグルタチオンＳ−トランスフェラーゼ（ＧＳＴ）などとフュージョン蛋白質としたものを精製して、または未精製のまま、抗原として用いることもできる。
成書（Ａｎｔｉｂｏｄｉｅｓ；Ａ ｌａｂｏｒａｔｏｒｙ ｍａｎｕａｌ，Ｅ．Ｈａｒｌｏｗ ｅｔ ａｌ．，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ）に示された各種の方法ならびに遺伝子クローニング法などにより分離されたイムノグロブリン遺伝子を用いて培養した細胞に発現させた遺伝子組み換え抗体によっても作製することができる。
本発明のマーカー抗原として用いることができるＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２に対する抗体は、以下の方法あるいはその他の類似の方法によって取得することができるがこれらの方法に限定されるものではない。
ａ）Ｒｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質の遺伝子配列およびアミノ酸配列が既知の微生物については、他の微生物における該蛋白質のアミノ酸配列との類似性の少ない領域についてペプチド断片を合成し、それを免疫原としてポリクローナル抗体、あるいはモノクローナル抗体を作製することにより目的の抗体を取得することができる。
また、既知の該遺伝子の両端部位におけるＤＮＡ配列をプローブとしたＰＣＲ法による遺伝子増幅、相同部分配列を鋳型プローブとしたハイブリダイゼーション法など通常の遺伝子操作手法を用いることにより該遺伝子の全長配列を取得することができる。
その後他の蛋白質遺伝子とのフュージョン遺伝子などを構築し、大腸菌等を宿主として公知の遺伝子導入手法により宿主内に該当フュージョン遺伝子を挿入し大量に発現させた後にフュージョン蛋白質として用いた蛋白質に対する抗体アフィニティカラム法などにより発現蛋白質を精製することにより目的とする蛋白質抗原を取得することができる。この場合Ｒｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質の全長蛋白質が抗原となるため微生物間で保存されているアミノ酸部分に対する抗体を取得しても本発明の目的に合致しない。従って、本法によって取得した抗原に対しては公知の手法によりモノクローナル抗体を産生するハイブリドーマを取得し、該当する微生物とのみ反応する抗体を産生するクローンを選択することにより目的の抗体を取得することができる。
ｂ）Ｒｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質のアミノ酸配列が未知の微生物については１つにはＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質のアミノ酸配列が菌種間で５０〜６０％相同であることにより、そのアミノ酸配列の相同部分の配列を基にしてＰＣＲ法による特定配列部分の遺伝子増幅や相同部分配列を鋳型プローブとしたハイブリダイゼーション法など通常の遺伝子操作手法を用いることにより該蛋白質遺伝子を容易に取得することができる。
その後他の蛋白質遺伝子とのフュージョン遺伝子などを構築し、大腸菌等を宿主として公知の遺伝子導入手法により宿主内に該当フュージョン遺伝子を挿入し大量に発現させた後にフュージョン蛋白質として用いた蛋白質に対する抗体アフィニティカラム法などにより発現蛋白質を精製することにより目的とする蛋白質抗原を取得することができる。この場合Ｒｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質の全長蛋白質が抗原となるため微生物間で保存されているアミノ酸部分に対する抗体を取得しても本発明の目的に合致しない。従って、本法によって取得した抗原に対しては公知の手法によりモノクローナル抗体を産生するハイブリドーマを取得し、該当する微生物とのみ反応する抗体を産生するクローンを選択することにより目的の抗体を取得することができる。
ｃ）あるいはＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質のアミノ酸配列が未知な場合の別な方法として、既知のＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質のアミノ酸配列のうち微生物間で保存されている共通配列部分に相当する５〜３０アミノ酸の合成ペプチドを作製し、そのペプチド配列に対し公知の方法でポリクローナル抗体あるいはモノクローナル抗体を作製する。該抗体を用いたアフィニティカラムクロマトによって目的の微生物細胞破砕液を精製することにより高度に精製されたＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質を取得することができる。蛋白質の精製度が不足している場合は公知の精製手法であるイオン交換クロマトグラフィー、疎水クロマトグラフィー、ゲル濾過などの手法により精製したのち作製した抗体によるウェスタンブロットなどの方法によりＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質の溶出画分を同定し精製蛋白質を得ることができる。得られた精製Ｒｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質抗原を基にして公知の方法によりハイブリドーマを取得し、目的の微生物に特異的に反応するハイブリドーマを選択することにより目的の抗体を取得することができる。
上記の方法ａ）、ｂ）およびｃ）によって得られた種々の微生物に特異的な本発明の抗体は種々の免疫学的分析法に用いることにより、目的とする微生物に特異的な各種の診断試薬およびキットを提供することができる。例えば、この抗体は、公知の測定手法であるポリスチレンラテックス粒子上に該抗体を吸着させた凝集反応、マイクロタイタープレート中で行う公知技術であるＥＬＩＳＡ法、既存のイムノクロマト法、着色粒子もしくは発色能を有する粒子、または酵素もしくは蛍光体でラベルされた該抗体とともに捕捉（ｃａｐｔｕｒｅ）抗体で被覆した磁気微粒子などを用いるサンドイッチアッセイなど既知の全ての免疫測定手法に利用できる。
抗体を用いる微生物診断方法とは、ポリスチレンラテックス粒子上に該抗体を吸着させた凝集反応、マイクロタイタープレート中で行う公知技術であるＥＬＩＳＡ法、既存のイムノクロマト法、着色粒子もしくは発色能を有する粒子、または酵素もしくは蛍光体でラベルされた該抗体とともに捕捉抗体で被覆した磁気微粒子などを用いるサンドイッチアッセイなど既知の全ての免疫測定手法を利用する診断方法を意味する。
また、特に抗体を用いる有用な微生物診断方法として特表平７−５０９５６５号公報に記載されているシリコン、窒化珪素などにより形成された光学薄膜上で抗体反応をおこない光干渉原理等により検出するいわゆるオプティカルイムノアッセイ（ＯＩＡ，Ｏｐｔｉｃａｌ Ｉｍｍｕｎｏａｓｓａｙ）などが高感度な診断方法として有用である。
また該検出方法において必要となる微生物からの細胞内マーカー抗原の抽出方法としては、ＴｒｉｔｏｎＸ−１００、Ｔｗｅｅｎ−２０をはじめとする種々の界面活性剤を用いた抽出試薬による処理法、適当なプロテアーゼなどの酵素を用いる酵素処理法、物理的方法による微生物細胞の破砕をはじめ既知の細胞構造の破砕手法が用いられる。界面活性剤等の組み合わせにより微生物ごとに試薬による最適な抽出条件を設定することが望ましい。
また、本発明における、抗体を用いる微生物検出用試薬キットとは、当該検出方法を用いた検出用試薬キットに相当する。
Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質のアミノ酸配列及びＤＮＡ配列を配列表に示す。従って、この微生物の場合はＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質のアミノ酸配列を、配列表に「クロスマッチ」と記した類似した微生物の同種の蛋白質と比較することが可能である。相同性の低いセグメントのペプチドを合成し、これに対するポリクローナルあるいはモノクローナル抗体を作成することは、微生物に対する特異性を有するものの選択を省略することを可能とする。
特にポリクローナル抗体の場合、免疫した動物の抗血清をＰｒｏｔｅｉｎ Ａカラム等で精製しＩｇＧ面分を取得したのち、さらに動物の免疫に用いた合成ペプチドによるアフィニティ精製を実施することが望ましい。
更に、当該微生物のＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質のＤＮＡ配列からＮ末端とＣ末端の配列に基づいてＰＣＲプライマーが作成された。
このＰＣＲプライマーの相同性を利用して、ゲノムＤＮＡを用いてＰＣＲ法によりＤＮＡ断片を増殖させ、これを抽出し、常法によりＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２遺伝子の断片を取得することができる。Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２遺伝子の全長はこれらの断片のＤＮＡ配列情報を分析することにより知ることができる。
取得したＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２遺伝子は、例えばＧＳＴなどとフュージョン蛋白質遺伝子を構成し、適当な発現用プラスミドを用いて発現ベクターを構築後、大腸菌等を形質転換して該蛋白質を大量発現させうる。形質転換した大腸菌を適当量培養し、菌体破砕液をＧＳＴを用いたアフィニティカラムで精製することにより、Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質とＧＳＴのフュージョン蛋白質が得られる。
この蛋白質をそのまま、あるいはＧＳＴ部分を切断後、抗原蛋白質として公知の手法により、複数のハイブリドーマクローンを確立し、Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅ菌体あるいは菌体破砕液またはＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質に特異的な反応を示す抗体を選択することにより目的の特異的モノクローナル抗体を取得することも可能である。
本発明に基づき作製された抗体は、公知の測定手法であるポリスチレンラテックス粒子上に該抗体を吸着させた凝集反応、マイクロタイタープレート中で行う公知技術であるＥＬＩＳＡ法、既存のイムノクロマト法、着色粒子もしくは発色能を有する粒子、または酵素もしくは蛍光体でラベルされた該抗体とともに捕捉抗体で被覆した磁気微粒子などを用いるサンドイッチアッセイなど既知の全ての免疫測定手法に利用できる。
また本発明に基づき作製された抗体は全ての免疫測定手法において当該抗原蛋白質を固相あるいは液相中で捕獲するいわゆる捕捉抗体として機能しうると同時にパーオキシダーゼやアルカリフォスファターゼなどの酵素を公知の方法により修飾していわゆる酵素標識抗体とすることにより、検出用抗体としても機能しうる。
発明を実施するための最良の形態
以下の例は本発明を具体的に説明するためのものであって本発明について何らその範囲を限定するものではない。 TECHNICAL FIELD The present invention relates to an antibody useful for detecting a microorganism belonging to Mycoplasma pneumoniae , which is a common causative microorganism of pneumonia, a method for detecting the microorganism, a reagent kit for detecting the microorganism, and the microorganism detection The present invention relates to an antibody production method.
The present invention is important for the diagnosis of atypical pneumonia caused by the pharmaceutical industry, particularly Mycoplasma pneumoniae , and is useful in the pharmaceutical industry.
The present invention is useful for detecting microorganisms, Mycoplasma pneumoniae , contained in specimens collected from specimens such as throat swabs, tissue samples, body fluids, laboratory solutions and cultures.
BACKGROUND ART Diagnosis of microbial infection is usually confirmed by detecting causative bacteria at the site of infection or detecting antibodies against causative bacteria in serum and body fluids. In particular, this diagnosis is important in the sense that detection of the causative bacteria enables rapid treatment of the patient.
Infectious disease causative bacteria are generally detected through culture of causative bacteria, and a culture identification method for identifying the causal bacteria based on their physiological, biochemical or structural characteristics. Polymerase chain reaction (PCR) ) Method or genetic diagnostic method to detect and amplify by specific nucleic acid hybridization and immunological method to detect causative bacteria using specific reaction between antibody and antigenic marker of causative bacteria The
However, when using a culture identification method or a genetic diagnosis method, it takes a long time to obtain a result. Therefore, diagnosis by an immunological method that can detect causative bacteria in a short time and with high sensitivity and leads to rapid and appropriate treatment of patients is widely used.
Conventional combinations of marker antigens and antibodies have been used to detect infection-causing bacteria by immunological methods, depending on the species.
Mycoplasma pneumoniae is a common cause of pneumonia, bronchitis and pharyngitis. A small fungal-like gram-negative bacterium that selectively invades the human body and causes disease. It is said that about 15-20% of pneumonia that develops collectively in the general public, and up to 50% of pneumonia within a limited population, is caused by Mycoplasma pneumoniae (Ragnar Norrby 1999).
This bacterium is a very small microorganism that is difficult to cultivate, and forms a small colony in a concentrated medium. Growth of Mycoplasma pneumoniae in nutrient hatching agar is very slow and takes at least 21 days to identify cells in the medium (Granato, Poe, et al. 1980). Some mycoplasma species microorganisms that originate in the human body undergo similar biochemical reactions. On the other hand, the absence of cell walls makes observation under a bright microscope more difficult (Knudson and MacLeod 1979). Therefore, the Gram staining method and the culture method cannot be applied as a diagnostic method for rapidly detecting a pathogenic bacterium.
DNA hybridization analysis probes used for gene sequences for detection of Mycoplasma pneumoniae are Hyman et al., Buck et al. And Bernet et al. (Hyman, Yogev et al. 1987; Bernet, Garret et al. 1993; Buck, O'Hara et al. 1993). Probes used for Mycoplasma pneumoniae ribosomal RNA (rRNA) detection are Tilton et al. (Tilton, Dias et al. 1980), Yogev et al. (Yogev, Halachmi et al. 1988), Gobel et al. (Gobel, Geer 7). Zivin and Monahan (EPO 305145) and Gobeland and Stanbridge (EPO 250662). Kai et al. (Kai, Kamiya et al. 1.987) and Jensen et al. (Jensen, Sondergard-Andersen et al. 1993) et al. Describe primers for the 16S rRNA sequence of Mycoplasma pneumoniae . All probes described herein is designed for DNA of DNA or Mycoplasma pneumoniae and Mycoplasma genitalium in Mycoplasma pneumoniae. Since these probes are relatively sensitive, they are captured using techniques that have only recently become available, such as PCR. The PCR method is very sensitive and can be performed in a shorter time than hybridization. However, there are often false-positive problems that are positive for asymptomatic people who are negative for cultured Mycoplasma pneumoniae or for carriers after the disease has healed.
JP-A-63-298 describes an immunodetection method based on a Western blot method using a monoclonal antibody against a membrane antigen protein of about 43 kilodaltons of Mycoplasma pneumoniae . However, the above-described antibodies and detection methods using such antibodies have the problem of low specificity and are insufficient for specific diagnosis of species against Mycoplasma pneumoniae . Furthermore, there is a problem that it takes at least 5 hours to obtain a result according to this diagnostic method (Madsen et al. 1988).
DISCLOSURE OF THE INVENTION The present invention has been made to solve the above problems. That is, an object of the present invention is to provide a method for quickly and specifically detecting a microorganism belonging to Mycoplasma pneumoniae with high sensitivity, a detection antibody used for the detection, and a detection reagent kit. Furthermore, this invention makes it a subject to provide the manufacturing method of the antibody for a detection used for the detection.
The present inventors have found a protein having the same function in all microorganisms as a useful antigen protein. Usually, such protein structural changes are expected to be extremely small. Surprisingly, however, the antibody against the protein is microbial species or genus-specific, and the antibody against the protein has diversity that can be used for microbial species or genus-specific identification, It has been found that all the serotypes of the microorganisms that can be detected can be detected. More specifically, the protein is a useful protein that exhibits a species specificity that undergoes a change in amino acid sequence, has the same structure between the same species, and is accompanied by a change in structure when the species are different.
The present inventors are present in all microbial cells as molecules having the same function, and in addition to intracellular molecules having amino acid structures that differ to some extent among microorganisms, particularly Ribosomal Protein L7 / L12 protein, which is a kind of ribosomal protein. Pay attention. Ribosomal Protein L7 / L12 protein is a protein having a molecular weight of about 13 kilodaltons, and is known to exist as a ribosomal protein essential for protein synthesis. In particular, the entire amino acid sequence of the Ribosomal Protein L7 / L12 protein has been analyzed in several microorganisms including Mycoplasma pneumoniae .
The present inventors paid attention to the fact that although this molecule is similar between microorganisms, some of them have a structure part unique to each microorganism, and an antibody against the Ribosomal Protein L7 / L12 protein of this Mycoplasma pneumoniae is used. It was found that serotypes in various microorganisms and bacteria species-specific and within the same bacterial species can be detected by using them.
The present inventors have found that an antibody specific for the protein of Mcoplasma pneumoniae can be obtained and that Mycoplasma pneumoniae can be specifically detected by using the antibody, and have completed the present invention.
According to the present invention, a monoclonal antibody specific to Mycoplasma pneumoniae Ribosomal Protein L7 / L12 protein was found and developed. This antibody is novel and has a property of specifically reacting with the above protein unlike any conventionally known antibody.
In the sequence listing, SEQ ID NOs: 1 and 2 are the DNA sequence and corresponding amino acid sequence of the Ribosomal Protein L7 / L12 gene of Mycoplasma pneumoniae (NCBI database accession # NC 000912). The left end and right end of the amino acid sequences described in the sequence listing are the amino group end (hereinafter referred to as N-terminal) and the carboxyl group end (hereinafter referred to as C-terminal), respectively, and the left end and right end of the base sequence are each 5 ′ end And the 3 'end. Amino acids in the amino acid sequence of the cross match test are represented by single letter abbreviations. In addition, the notation “+” in the cross-match test indicates that the amino acid is a different amino acid but has similar properties such as hydrophobicity, and the blank “” is a completely different amino acid including the property. In addition, a series of molecular biological experiments of genetic manipulation described in the present invention can be performed by a method described in a normal experiment document. Examples of the above-mentioned normal experiment documents include Molecular Cloning, A laboratory manual, Cold Spring Harbor Laboratory Press, Shambrook, J. et al. (1989).
In the present invention, the antibody refers to a polyclonal antibody or a monoclonal antibody, and can be prepared using the full length of the above Ribosomal Protein L7 / L12 protein or a partial peptide thereof. The length of the peptide for producing the antibody is not particularly limited, but in the case of an antibody against the Ribosomal Protein L7 / L12 protein, it is sufficient that the protein has a characteristic length, preferably 5 amino acids or more, particularly preferably 8 amino acids. The above peptides may be used. This peptide or full-length protein is directly or after cross-linking with a carrier protein such as KLH (keyhole-limpet hemocyanin) or BSA (bovine serum albumin) and then inoculated to an animal together with an adjuvant as necessary, and then the serum is collected to recover the ribosomal protein. An antiserum containing an antibody (polyclonal antibody) that recognizes the L7 / L12 protein can be obtained. Further, the antibody can be purified from the antiserum and used. The animals to be inoculated include sheep, horses, goats, rabbits, mice, rats and the like, and sheep, rabbits and the like are particularly preferable for producing polyclonal antibodies. A monoclonal antibody can also be obtained by a known method for producing a hybridoma cell. In this case, a mouse is preferred.
Further, the full-length protein or an amino acid sequence of 5 or more residues, preferably 8 or more residues, which is a fusion protein with glutathione S-transferase (GST) or the like is purified or used as an antigen without purification. You can also.
It is expressed in cells cultured using immunoglobulin genes isolated by various methods and gene cloning methods shown in the literature (Antibodies; A laboratory manual, E. Harlow et al., Cold Spring Harbor Laboratory Press). It can also be produced by using a recombinant antibody.
An antibody against Ribosomal Protein L7 / L12 that can be used as the marker antigen of the present invention can be obtained by the following method or other similar methods, but is not limited to these methods.
a) For microorganisms whose gene sequence and amino acid sequence of Ribosomal Protein L7 / L12 protein are known, a peptide fragment is synthesized for a region having a low similarity to the amino acid sequence of the protein in other microorganisms, and a polyclonal antibody is used as an immunogen. The desired antibody can be obtained by preparing an antibody or a monoclonal antibody.
In addition, the full-length sequence of the gene can be obtained by using conventional gene manipulation techniques such as gene amplification by PCR using the DNA sequences at both ends of the known gene as probes and hybridization using homologous partial sequences as template probes. can do.
Then, construct a fusion gene with other protein genes, insert the corresponding fusion gene into the host by a known gene transfer method using Escherichia coli etc. as the host, express it in large quantities, and then antibody affinity column for the protein used as the fusion protein The target protein antigen can be obtained by purifying the expressed protein by a method or the like. In this case, since the full-length protein of Ribosomal Protein L7 / L12 protein serves as an antigen, obtaining an antibody against the amino acid portion conserved among microorganisms does not meet the purpose of the present invention. Therefore, for the antigen obtained by this method, obtain a hybridoma that produces a monoclonal antibody by a known technique, and obtain a target antibody by selecting a clone that produces an antibody that reacts only with the corresponding microorganism. Can do.
b) For microorganisms whose amino acid sequence of Ribosomal Protein L7 / L12 protein is unknown, one is that the amino acid sequence of Ribosomal Protein L7 / L12 protein is 50-60% homologous between the bacterial species, so the homology of the amino acid sequence The protein gene can be easily obtained by using normal gene manipulation techniques such as gene amplification of a specific sequence portion by PCR method based on the partial sequence and hybridization method using a homologous partial sequence as a template probe.
Then, construct a fusion gene with other protein genes, insert the corresponding fusion gene into the host by a known gene transfer method using Escherichia coli etc. as the host, express it in large quantities, and then antibody affinity column for the protein used as the fusion protein The target protein antigen can be obtained by purifying the expressed protein by a method or the like. In this case, since the full-length protein of Ribosomal Protein L7 / L12 protein serves as an antigen, obtaining an antibody against the amino acid portion conserved among microorganisms does not meet the purpose of the present invention. Therefore, for the antigen obtained by this method, obtain a hybridoma that produces a monoclonal antibody by a known technique, and obtain a target antibody by selecting a clone that produces an antibody that reacts only with the corresponding microorganism. Can do.
c) Alternatively, as another method when the amino acid sequence of the Ribosomal Protein L7 / L12 protein is unknown, the amino acid sequence of the Ribosomal Protein L7 / L12 protein, which corresponds to a common sequence portion conserved between microorganisms among the amino acid sequences of the known Ribosomal Protein L7 / L12 protein, A synthetic peptide of 30 amino acids is prepared, and a polyclonal antibody or a monoclonal antibody is prepared by a known method against the peptide sequence. A highly purified Ribosomal Protein L7 / L12 protein can be obtained by purifying the target microbial cell disruption solution by affinity column chromatography using the antibody. When the degree of protein purification is insufficient, Ribosomal Protein L7 / L12 is purified by a known purification technique such as ion exchange chromatography, hydrophobic chromatography, gel filtration, etc., and then by a method such as Western blotting using the prepared antibody. The purified protein can be obtained by identifying the eluted fraction of the protein. Based on the obtained purified Ribosomal Protein L7 / L12 protein antigen, a hybridoma is obtained by a known method, and a target antibody can be obtained by selecting a hybridoma that specifically reacts with the target microorganism.
The antibodies of the present invention specific for various microorganisms obtained by the above-mentioned methods a), b) and c) are used in various immunological analysis methods so that various diagnoses specific to the target microorganism can be obtained. Reagents and kits can be provided. For example, this antibody has an agglutination reaction in which the antibody is adsorbed onto polystyrene latex particles, which is a known measurement technique, an ELISA method that is a known technique performed in a microtiter plate, an existing immunochromatography method, colored particles, or coloring ability. The present invention can be used for all known immunoassay techniques such as sandwich assays using particles having magnetic particles coated with a capture antibody together with the antibody or the antibody labeled with an enzyme or a phosphor.
The microorganism diagnosis method using an antibody is an aggregation reaction in which the antibody is adsorbed on polystyrene latex particles, an ELISA method which is a known technique performed in a microtiter plate, an existing immunochromatography method, colored particles or particles having color developing ability, Alternatively, it means a diagnostic method using all known immunoassay techniques such as a sandwich assay using magnetic fine particles coated with a capture antibody together with the antibody labeled with an enzyme or a phosphor.
In addition, an antibody reaction is performed on an optical thin film formed of silicon, silicon nitride, or the like described in JP-A-7-509565 as a useful microorganism diagnosis method using an antibody in particular, so-called detection based on a light interference principle or the like. An optical immunoassay (OIA, Optical Immunoassay) is useful as a highly sensitive diagnostic method.
In addition, as a method for extracting intracellular marker antigens from microorganisms required in the detection method, treatment methods using extraction reagents using various surfactants such as Triton X-100 and Tween-20, suitable proteases, etc. Known cell structure disruption techniques are used, including enzyme treatment methods using these enzymes and disruption of microbial cells by physical methods. It is desirable to set optimum extraction conditions with reagents for each microorganism by combining surfactants and the like.
In the present invention, the reagent kit for detecting a microorganism using an antibody corresponds to a reagent kit for detecting using the detection method.
The amino acid sequence and DNA sequence of the Ribosomal Protein L7 / L12 protein of Mycoplasma pneumoniae are shown in the Sequence Listing. Therefore, in the case of this microorganism, it is possible to compare the amino acid sequence of the Ribosomal Protein L7 / L12 protein with the same type of protein of a similar microorganism whose sequence listing indicates “crossmatch”. By synthesizing a peptide having a segment with low homology and preparing a polyclonal or monoclonal antibody against the peptide, it is possible to omit selection of one having specificity for a microorganism.
In particular, in the case of a polyclonal antibody, it is desirable to purify the antiserum of the immunized animal using a Protein A column or the like to obtain an IgG surface portion, and then further carry out affinity purification using a synthetic peptide used for immunization of the animal.
Furthermore, PCR primers were prepared based on the N-terminal and C-terminal sequences from the DNA sequence of the Ribosomal Protein L7 / L12 protein of the microorganism.
Using this PCR primer homology, DNA fragments can be proliferated by PCR using genomic DNA, extracted, and fragments of Mycoplasma pneumoniae Ribosomal Protein L7 / L12 gene can be obtained by conventional methods. . The full length of the Ribosomal Protein L7 / L12 gene of Mycoplasma pneumoniae can be determined by analyzing the DNA sequence information of these fragments.
The obtained Mycoplasma pneumoniae Ribosomal Protein L7 / L12 gene constitutes a fusion protein gene with, for example, GST, etc., constructs an expression vector using an appropriate expression plasmid, and then transforms Escherichia coli to express the protein in large quantities. It can be made. The transformed Escherichia coli is cultured in an appropriate amount, and the cell disruption solution is purified with an affinity column using GST to obtain the Mycoplasma pneumoniae Ribosomal Protein L7 / L12 protein and the GST fusion protein.
A plurality of hybridoma clones are established by using a known method as an antigen protein after the protein is cut as it is or after cleavage of the GST portion, and is specific to Mycoplasma pneumoniae cells or cell disruption solution or Mycoplasma pneumoniae Ribosomal Protein L7 / L12 protein. It is also possible to obtain a specific monoclonal antibody of interest by selecting an antibody that exhibits an appropriate reaction.
The antibody prepared according to the present invention is a known measurement technique, such as an agglutination reaction in which the antibody is adsorbed onto polystyrene latex particles, an ELISA method that is a known technique performed in a microtiter plate, an existing immunochromatography method, a colored particle Alternatively, it can be used for all known immunoassay techniques such as a sandwich assay using particles having chromogenic ability, or magnetic fine particles coated with a capture antibody together with the antibody labeled with an enzyme or a phosphor.
In addition, the antibody prepared according to the present invention can function as a so-called capture antibody that captures the antigen protein in a solid phase or a liquid phase in all immunoassay methods, and at the same time, an enzyme such as peroxidase or alkaline phosphatase is a known method. It can also function as a detection antibody by modifying it with a so-called enzyme-labeled antibody.
BEST MODE FOR CARRYING OUT THE INVENTION The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅからのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２遺伝子のクローニングＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅ（ＡＴＣＣ１５５３１，ＡＴＣＣから分譲、購入）を、Ｍｙｃｏｐｌａｓｍａ Ｓｕｐｐｌｅｍｅｎｔ（ＤＩＦＣＯ，０８３６−６８−９）を加えたＰＰＬＯ寒天培地（ＤＩＦＣＯ，０４１２−１７−３）上に適当量植菌したのち、ＣＯ２インキュベーター中で３７℃、５％ＣＯ２条件で５時間培養した。生育したコロニーを最終的に５×１０９ＣＦＵ／ｍｌ前後になるようにＴＥ緩衝液（和光純薬工業製）に懸濁した。この懸濁液約１．５ｍｌを微量遠心チューブに移し取り１０，０００ｒｐｍで２分間遠心し、上澄み液を棄てた。沈殿部分を５６７μｌのＴＥ緩衝液に再懸濁した。さらに３０μｌの１０％ＳＤＳと３μｌの２０ｍｇ／ｍｌのＰｒｏｔｅｉｎａｓｅＫ溶液を加えて良く混合し、３７℃で１時間インキュベートした。懸濁液を５６℃で更に１時間インキュベートした。次に１０％のセチルトリメチルアンモニウムブロマイド／０．７Ｍ ＮａＣｌ溶液を８０μｌ加え、よく混合したのち６５℃で１０分間インキュベートした。同一体積の２４：１のクロロホルム−イソアミルアルコール混合液を７００μｌ加えよく攪拌した。
この溶液を微量遠心機で１２，０００ｒｐｍ、５分間、４℃で遠心処理したのち、水層画分を新しい微量遠心管に移した。そこに０．６倍量のイソプロパノールを加えチューブをよく振ってＤＮＡの沈殿を形成した。白いＤＮＡ沈殿をガラス棒ですくって１ｍｌの７０％エタノール（−２０℃に冷却したもの）が入った別の微量遠心管に移した。その後チューブを１０，０００ｒｐｍで５分間遠心し、上清を静かに除去した。１ｍｌの７０％エタノールを追加し、混合物を更に５分間遠心した。
再び上澄みを除去したのち沈殿を１００μｌのＴＥ緩衝液に溶解しＤＮＡ溶液を得た。このゲノムＤＮＡ溶液の濃度をＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ，Ａ ｌａｂｏｒａｔｏｒｙ ｍａｎｕａｌ，１９８９，Ｅｄｓ．Ｓｈａｍｂｒｏｏｋ，Ｊ．，Ｆｒｉｔｓｃｈ，Ｅ．Ｆ．，ａｎｄ Ｍａｎｉａｔｉｓ，Ｔ．，Ｃｏｌｄ Ｓｐｒｉｎｇ ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ ＰｒｅｓｓのＥ５，Ｓｐｅｃｔｒｏｐｈｏｔｏｍｅｔｒｉｃ Ｄｅｔｅｒｍｉｎａｔｉｏｎ ｏｆ ｔｈｅ Ａｍｏｕｎｔ ｏｆ ＤＮＡ ｏｒ ＲＮＡに従って定量した。
このゲノムＤＮＡのうち１０ｎｇを用いてＰＣＲ（ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ）を行った。ＰＣＲはＴａｑポリメラーゼ（宝酒造社製、コードＲ００１Ａ）を用いた。酵素に添付の緩衝液５μｌ、酵素に添付のｄＮＴＰ混合物４μｌおよび各２００ｐｍｏｌのオリゴヌクレオチド（配列表配列番号：３および４に示すもの）を酵素に加えた。全体の容量が５０μｌとなるように精製水を加えた。
この混合物を、ＴａＫａＲａ ＰＣＲ Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒ ４８０を用いて、９５℃１分、５０℃２分、７２℃３分を５サイクル行ったのち、９５℃１分、６０℃２分、７２℃３分を２５サイクル行った。このＰＣＲ生成物の一部を用いて、１．５％アガロースゲル中にて電気泳動を実施した。エチジウムブロマイド（日本ジーン社製）にて染色後、紫外線下で観察し、約４００ｂｐのｃＤＮＡが増幅されていることを確認した。制限酵素ＢａｍＨＩおよびＸｈｏＩを用いて切断後、１．５％アガロースゲル中にて電気泳動とエチジウムブロマイドによる染色を行った。ゲルから約４００ｂｐのバンドを切り取った。このバンドをＳｕｐｒｅｃ０１（宝酒造株式会社製）で精製し、一般的なベクターであるｐＧＥＸ−６Ｐ−１（Ｐｈａｒｍａｃｉａ社製）に挿入した。同ベクターは目的の遺伝子断片を適当な制限酵素サイトに組み込むことによりＧＳＴ蛋白質とのフュージョン蛋白質を発現しうる目的分子の発現ベクターとして機能することができる。
具体的にはベクターｐＧＥＸ−６Ｐ−１と先のＤＮＡとをそのモル比が１：３となるように混ぜ合わせて、Ｔ４ ＤＮＡリガーゼ（Ｉｎｖｉｔｒｏｇｅｎ社製）にてベクターにＤＮＡを組み込んだ。ＤＮＡを組み込んだベクターｐＧＥＸ−６Ｐ−１は大腸菌のワンショットコンピテントセルに遺伝子学的手法により導入し、ついで５０μｇ／ｍｌのアンピシリン（シグマ社）を含む半固体状の培養プレートであるＬＢ Ｌ−ブロス寒天（宝酒造株式会社製）に接種した。プレートを３７℃で１２時間インキュベートし、成長したコロニーを無差別に選択し、同じ濃度のアンピシリンを含むＬ−ブロス培養液に接種した。３７℃で８時間振とう培養・集菌後、Ｗｉｚａｒｄ Ｍｉｎｉｐｒｅｐを用い、添付の説明書に従ってプラスミドを分離した。プラスミドは制限酵素ＢａｍＨＩ／ＸｈｏＩにて切断処理した。約３７０ｂｐのＤＮＡを切断することによってＰＣＲ生成物の挿入を確認した。挿入されたＤＮＡの塩基配列を上記クローンを用いて決定した。
挿入ＤＮＡ断片の塩基配列の決定は、Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製の蛍光シークエンサーを用いて実施した。
シークエンスサンプルの調製はＰＲＩＳＭ，Ｒｅａｄｙ Ｒｅａｃｔｉｏｎ Ｄｙｅ Ｔｅｒｍｉｎａｔｏｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ Ｋｉｔ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製）を用いて行った。先ず、９．５μｌの反応液、４．０μｌの０．８ｐｍｏｌ／μｌのＴ７プロモータープライマー（Ｇｉｂｃｏ ＢＲＬ）、および６．５μｌの０．１６μｇ／μｌテンプレートＤＮＡを０．５ｍｌのマイクロチューブに加え、混合した。混合物を２層の１００μｌ鉱油で覆ったのち、２５サイクルＰＣＲ増幅処理を行った。ここで、１サイクルは、９６℃での３０秒間の処理、５５℃での１５秒間の処理、および６０℃での４分間の処理からなる。生成物を４℃で５分間保持した。反応終了後、８０μｌの無菌精製水を加え、攪拌した。生成物を遠心分離し、水層をフェノールークロロホルム混合液で３回抽出した。１０μｌの３Ｍ酢酸ナトリウムｐＨ５．２と３００μｌのエタノールを１００μｌの水層に加え、攪拌した。その後１４，０００ｒｐｍ、室温で１５分間遠心し、沈殿を回収した。沈殿を７５％エタノールで洗浄後、真空下に２分間静置して乾燥させ、シークエンス用サンプルとした。シークエンスサンプルは、４μｌの１０ｍＭのＥＤＴＡを含むホルムアミドに溶解して９０℃で２分間変性した。このものは氷中で冷却してシークエンスに供した。
無差別に選択した５個のクローンのうち２個はＰＣＲに用いたプローブと配列上の相同性を有していた。また、Ｒｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質の遺伝子配列と一致したＤＮＡ配列が明白であった。その構造遺伝子部分の全塩基配列及び対応するアミノ酸配列は配列表配列番号：１及び２に示すような配列であった。この遺伝子断片は、明らかにＭｉｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質の遺伝子をコードするものである。(Sale from ATCC15531, ATCC, purchase) Ribosomal Protein L7 / L12 gene cloning Mycoplasma pneumoniae from Mycoplasma pneumoniae and, Mycoplasma Supplement (DIFCO, 0836-68-9) to PPLO agar medium (DIFCO plus, 0412-17-3 ) After inoculating an appropriate amount above, the cells were cultured in a CO 2 incubator at 37 ° C. and 5% CO 2 for 5 hours. The grown colonies were suspended in TE buffer (manufactured by Wako Pure Chemical Industries, Ltd.) so that the final colony was around 5 × 10 9 CFU / ml. About 1.5 ml of this suspension was transferred to a microcentrifuge tube, centrifuged at 10,000 rpm for 2 minutes, and the supernatant was discarded. The pellet was resuspended in 567 μl TE buffer. Furthermore, 30 μl of 10% SDS and 3 μl of 20 mg / ml Proteinase K solution were added, mixed well, and incubated at 37 ° C. for 1 hour. The suspension was incubated for an additional hour at 56 ° C. Next, 80 μl of 10% cetyltrimethylammonium bromide / 0.7 M NaCl solution was added, mixed well, and then incubated at 65 ° C. for 10 minutes. 700 μl of the same volume of 24: 1 chloroform-isoamyl alcohol mixture was added and stirred well.
This solution was centrifuged at 12,000 rpm for 5 minutes at 4 ° C. in a microcentrifuge, and then the aqueous layer fraction was transferred to a new microcentrifuge tube. 0.6 times the amount of isopropanol was added thereto, and the tube was shaken well to form a DNA precipitate. The white DNA precipitate was scooped with a glass rod and transferred to another microcentrifuge tube containing 1 ml of 70% ethanol (cooled to -20 ° C). Thereafter, the tube was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was gently removed. 1 ml of 70% ethanol was added and the mixture was centrifuged for another 5 minutes.
After removing the supernatant again, the precipitate was dissolved in 100 μl of TE buffer to obtain a DNA solution. The concentration of this genomic DNA solution was measured according to Molecular Cloning, A laboratory manual, 1989, Eds. Shambrook, J. et al. , Fritsch, E .; F. , And Maniatis, T .; , Cold Spring Harbor Laboratory Press, E5, Spectrophotometric Determination of the Amount of DNA or RNA.
PCR (polymerase chain reaction) was performed using 10 ng of this genomic DNA. For PCR, Taq polymerase (Takara Shuzo Co., Ltd., code R001A) was used. 5 μl of buffer attached to the enzyme, 4 μl of dNTP mixture attached to the enzyme, and 200 pmol of oligonucleotides (shown in SEQ ID NOs: 3 and 4 in the sequence listing) were added to the enzyme. Purified water was added so that the total volume was 50 μl.
This mixture was subjected to 5 cycles of 95 ° C. for 1 minute, 50 ° C. for 2 minutes and 72 ° C. for 3 minutes using TaKaRa PCR Thermal Cycler 480, and then 95 ° C. for 1 minute, 60 ° C. for 2 minutes and 72 ° C. for 3 minutes for 25 minutes. Cycled. A portion of this PCR product was used for electrophoresis in a 1.5% agarose gel. After staining with ethidium bromide (manufactured by Nippon Gene Co., Ltd.), it was observed under ultraviolet light to confirm that about 400 bp cDNA was amplified. After digestion with restriction enzymes BamHI and XhoI, electrophoresis and staining with ethidium bromide were performed in a 1.5% agarose gel. A band of about 400 bp was cut from the gel. This band was purified with Suprec01 (Takara Shuzo Co., Ltd.) and inserted into a general vector pGEX-6P-1 (Pharmacia). This vector can function as an expression vector for a target molecule capable of expressing a fusion protein with a GST protein by incorporating the target gene fragment into an appropriate restriction enzyme site.
Specifically, the vector pGEX-6P-1 and the previous DNA were mixed so that the molar ratio was 1: 3, and the DNA was incorporated into the vector with T4 DNA ligase (manufactured by Invitrogen). The vector pGEX-6P-1 incorporating the DNA was introduced into a one-shot competent cell of Escherichia coli by a genetic method, and then a semi-solid culture plate LB L- containing 50 μg / ml ampicillin (Sigma). Broth agar (Takara Shuzo Co., Ltd.) was inoculated. Plates were incubated for 12 hours at 37 ° C. and grown colonies were selected indiscriminately and inoculated into L-broth cultures containing the same concentration of ampicillin. After shaking culture and collection at 37 ° C. for 8 hours, the plasmid was isolated using Wizard Miniprep according to the attached instructions. The plasmid was digested with restriction enzymes BamHI / XhoI. The insertion of the PCR product was confirmed by cleaving about 370 bp of DNA. The base sequence of the inserted DNA was determined using the above clone.
The nucleotide sequence of the inserted DNA fragment was determined using a fluorescent sequencer manufactured by Applied Biosystems.
Sequence samples were prepared using PRISM, Ready Reaction Dye Terminator Cycle Sequencing Kit (Applied Biosystems). First, add 9.5 μl reaction, 4.0 μl 0.8 pmol / μl T7 promoter primer (Gibco BRL), and 6.5 μl 0.16 μg / μl template DNA to a 0.5 ml microtube and mix did. The mixture was covered with two layers of 100 μl mineral oil followed by 25 cycles of PCR amplification. Here, one cycle consists of a treatment at 96 ° C. for 30 seconds, a treatment at 55 ° C. for 15 seconds, and a treatment at 60 ° C. for 4 minutes. The product was held at 4 ° C. for 5 minutes. After completion of the reaction, 80 μl of sterile purified water was added and stirred. The product was centrifuged and the aqueous layer was extracted three times with a phenol-chloroform mixture. 10 μl of 3M sodium acetate pH 5.2 and 300 μl of ethanol were added to 100 μl of the aqueous layer and stirred. Thereafter, the mixture was centrifuged at 14,000 rpm and room temperature for 15 minutes to collect the precipitate. The precipitate was washed with 75% ethanol, and left to stand for 2 minutes under vacuum to be dried to obtain a sample for sequencing. Sequence samples were dissolved in formamide containing 4 μl of 10 mM EDTA and denatured at 90 ° C. for 2 minutes. This was cooled in ice and subjected to sequencing.
Of the 5 clones selected indiscriminately, 2 had sequence homology with the probes used for PCR. In addition, a DNA sequence consistent with the gene sequence of Ribosomal Protein L7 / L12 protein was apparent. The entire base sequence of the structural gene portion and the corresponding amino acid sequence were those shown in SEQ ID NOs: 1 and 2. This gene fragment apparently encodes the gene for the Ribosomal Protein L7 / L12 protein of Micoplasma pneumoniae .
Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅからのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２遺伝子の大腸菌での大量発現と精製
発現ベクターを組み込んだ大腸菌をＬＢ培地中で５０ｍｌ、３７℃、一昼夜培養した。５００ｍｌの２倍濃度のＹＴ培地を３７℃で１時間加熱した。１晩培養した大腸菌液５０ｍｌを５００ｍｌの前述の培地に入れた。一時間後、５５０μｌの１００ｍＭイソプロピルβ−Ｄ（−）−チオガラクトピラノシド（ＩＰＴＧ）を導入し、４時間培養した。生成物を回収し、２５０ｍｌの遠心チューブに移し、７０００ｒｐｍで１９分間遠心した。上澄みを棄てて５０ｍＭ Ｔｒｉｓ緩衝液ｐＨ７．４、２５％ Ｓｕｃｒｏｓｅを含むＬｙｓｉｓ緩衝液２５ｍｌずつに溶解した。さらに１０％ ＮＰ−４０ １．２５ｍｌ、１Ｍ ＭｇＣｌ２ １２５μｌを加えてプラスチックチューブに移した。氷冷下、１分間の超音波処理を５回行った。その後、１２，０００ｒｐｍで１５分間遠心し、上清を回収した。
次に、ＰＢＳでコンディショニングしたグルタチオンアガロースカラムに前記の上澄み液を吸着させた。次に、２０ｍＭ Ｔｒｉｓ緩衝液ｐＨ７．４、４．２ｍＭ ＭｇＣｌ２、１ｍＭ ジチオスレイトール（ＤＴＴ）を含む洗浄液でカラムを２ベッドボリューム分洗浄した。５ｍＭのグルタチオンを含む５００ｍＭＴｒｉｓ緩衝液ｐＨ９．６中で溶離処理をした。溶出画分の蛋白質含有量をピグメント結合法（Ｂｒａｄｆｏｒｄ法；ＢｉｏＲａｄ Ｃｏ．）で決定し、主画分を取得した。
得られた精製ＧＳＴフュージョンＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質の純度は電気泳動法により確認したところ約７５％であり免疫源として充分な純度を確保できた。Mass expression and purification in Escherichia coli of Ribosomal Protein L7 / L12 gene from Mycoplasma pneumoniae E. coli incorporating the expression vector was cultured in LB medium at 50 ml, 37 ° C. overnight. 500 ml of double concentration YT medium was heated at 37 ° C. for 1 hour. 50 ml of the E. coli solution cultured overnight was placed in 500 ml of the aforementioned medium. One hour later, 550 μl of 100 mM isopropyl β-D (−)-thiogalactopyranoside (IPTG) was introduced and cultured for 4 hours. The product was collected and transferred to a 250 ml centrifuge tube and centrifuged at 7000 rpm for 19 minutes. The supernatant was discarded and dissolved in 25 ml of Lysis buffer containing 50 mM Tris buffer pH 7.4 and 25% sucrose. Further, 1.25 ml of 10% NP-40, 125 μl of 1M MgCl 2 was added and transferred to a plastic tube. The sonication for 1 minute was performed 5 times under ice cooling. Thereafter, the mixture was centrifuged at 12,000 rpm for 15 minutes, and the supernatant was collected.
Next, the supernatant was adsorbed onto a glutathione agarose column conditioned with PBS. Next, the column was washed for 2 bed volumes with a washing solution containing 20 mM Tris buffer pH 7.4, 4.2 mM MgCl 2 , 1 mM dithiothreitol (DTT). Elution was performed in 500 mM Tris buffer pH 9.6 containing 5 mM glutathione. The protein content of the eluted fraction was determined by the pigment binding method (Bradford method; BioRad Co.), and the main fraction was obtained.
The purity of the obtained purified GST fusion Ribosomal Protein L7 / L12 protein was about 75% as confirmed by electrophoresis, and sufficient purity as an immunogen could be secured.
Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質に対するモノクローナル抗体の作製
まずマウスの免疫についてはＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＧＳＴフュージョンＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質抗原１００μｇを２００μｌのＰＢＳに溶解後フロイントのコンプリートアジュバントを２００μｌ加え混合した。エマルジョン化したのち２００μｌを腹腔内に注射した。同じエマルジョン化抗原を２週間後、４週間後、および６週間後に腹腔内に注射した。２倍濃度のエマルジョン化抗原を１０週間後および１４週間後に腹腔内に注射した。最終の免疫化終了の３日後に脾臓を摘出し、細胞融合した。
無菌的に取り出したマウスの脾細胞１０８個に対し骨髄腫細胞２×１０７個をガラスチューブに取り良く混合したのち１５００ｒｐｍで５分間遠心し上澄みを棄て、その後細胞をよく混合した。
細胞融合に使用した骨髄腫細胞は、ＮＳ−１系の細胞株を用い１０％の牛胎児血清を含むＲＰＭＩ１６４０培地で培養し、細胞融合の２週間前から０．１３ｍＭのアザグアニン、０．５μｇ／ｍｌのＭＣ−２１０、１０％の牛胎児血清を含むＲＰＭＩ１６４０培地で１週間培養後、さらに１０％の牛胎児血清を含むＲＰＭＩ１６４０培地で１週間培養したものを用いた。
３７℃に保持したＲＰＭＩ１６４０培養液５０ｍｌを混合細胞試料に加え、１，５００ｒｐｍで遠心分離した。上澄み液を除去後、３７℃に保持した５０％ポリエチレングリコール１ｍｌを加え、１分間攪拌した。３７℃に保持したＲＰＭＩ１６４０培養液１０ｍｌを加え、混合液を殺菌したピペットで約５分間吸引・排出する事により激しく攪拌した。
５分間１，０００ｒｐｍで遠心分離し、上澄み液を除去したのち、細胞濃度が５×１０６／ｍｌとなるように３０ｍｌ ＨＡＴ培養液を加えた。この混合物を均一になるまで攪拌し、０．１ｍｌずつを９６穴の培養プレートに注ぎ、３７℃、７％の炭酸ガス雰囲気下で培養した。ＨＡＴ培地を、第１日、第１週、および第２週にそれぞれ０．１ｍｌずつ加え、ＥＬＩＳＡ法により所望の抗体を産生する細胞をスクリーニングした。
０．０５％のアジ化ソーダ含むＰＢＳ中に溶解したＧＳＴフュージョンＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質およびＧＳＴ蛋白質をそれぞれ１０μｇ／ｍｌ濃度で希釈した液を１００μｌずつ９６穴プレートに別々に分注し４℃で１晩吸着させた。
上澄み除去後、１％牛血清アルブミン溶液（ＰＢＳ中）２００μｌ添加し室温で１時間反応しブロッキングした。上澄み液を除去後、生成物を洗浄液（０．０２％Ｔｗｅｅｎ２０，ＰＢＳ）で洗浄した。これに融合細胞の培養液１００ｍｌを加え、室温にて２時間反応させた。上澄み液を除去し、沈殿を洗浄液で洗浄した。次いで、濃度５０ｎｇ／ｍｌのペルオキシダーゼでラベルしたｇｏａｔ ａｎｔｉ−ｍｏｕｓｅ ＩｇＧ抗体溶液１００μｌを加え、室温にて１時間反応させた。上澄み液を除去し、生成物を再び洗浄液で洗浄した。ＴＭＢ溶液（ＫＰＬ社製）を１００μｌずつ加え、混合物を室温にて２０分間反応させた。着色したところで１Ｎの硫酸１００μｌを加えて反応を停止し、４５０ｎｍの吸光度を測定した。
この結果、ＧＳＴフュージョンＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質にのみ反応しＧＳＴ蛋白質には反応しない陽性ウェルが見いだされＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質に対する抗体が含まれていることが判明した。
そこで陽性ウェル中の細胞をそれぞれ回収し２４穴プラスティックプレート中、ＨＡＴ培地で培養した。
培養した融合培地を細胞数が約２０個／ｍｌになるようにＨＴ培地で希釈し５０μｌを、ＨＴ培地に懸濁した６週齢のマウス胸腺細胞１０６個と９６穴培養プレート中で混合した。混合後、７％ＣＯ２条件下、３７℃で２週間培養した。
培養上澄み中の抗体活性を前述のＥＬＩＳＡ法にて同様に検定し、Ｒｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質との反応陽性の細胞を回収した。さらに、同様の希釈検定、クローニング操作を繰り返し、ハイブリドーマＭＰＲＢ−１〜５の計５クローンを取得した。For producing first immunization of mice of monoclonal antibodies against Ribosomal Protein L7 / L12 protein of Mycoplasma pneumoniae a GST fusion Ribosomal Protein L7 / L12 protein antigen 100μg of Mycoplasma pneumoniae were mixed complete adjuvant 200 [mu] l added after dissolution Freund of PBS 200 [mu] l. After emulsification, 200 μl was injected intraperitoneally. The same emulsified antigen was injected intraperitoneally after 2, 4 and 6 weeks. Double concentration of emulsified antigen was injected intraperitoneally after 10 and 14 weeks. Three days after the end of the final immunization, the spleen was removed and cell fused.
2 × 10 7 myeloma cells were collected from 10 8 spleen cells of aseptically removed mice in a glass tube, mixed well, centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and then the cells were mixed well.
The myeloma cells used for cell fusion were cultured in RPMI 1640 medium containing 10% fetal calf serum using NS-1 cell line, and 0.13 mM azaguanine, 0.5 μg / mg from 2 weeks before cell fusion. After culturing for 1 week in RPMI 1640 medium containing 10% fetal bovine serum in ml of MC-210, one further cultured in RPMI 1640 medium containing 10% fetal calf serum was used.
50 ml of RPMI 1640 culture maintained at 37 ° C. was added to the mixed cell sample and centrifuged at 1,500 rpm. After removing the supernatant, 1 ml of 50% polyethylene glycol maintained at 37 ° C. was added and stirred for 1 minute. 10 ml of RPMI 1640 culture solution maintained at 37 ° C. was added, and the mixture was agitated by aspirating and discharging with a sterilized pipette for about 5 minutes.
After centrifuging at 1,000 rpm for 5 minutes and removing the supernatant, 30 ml HAT culture solution was added so that the cell concentration became 5 × 10 6 / ml. The mixture was stirred until it was homogeneous, and 0.1 ml was poured into a 96-well culture plate and cultured at 37 ° C. in a 7% carbon dioxide atmosphere. HAT medium was added at 0.1 ml each on the first day, the first week, and the second week, and cells producing the desired antibody were screened by ELISA.
100 μl each of GST Fusion Ribosomal Protein L7 / L12 protein and GST protein dissolved in PBS containing 0.05% sodium azide at a concentration of 10 μg / ml was separately dispensed into a 96-well plate at 4 ° C. Adsorbed overnight.
After removing the supernatant, 200 μl of 1% bovine serum albumin solution (in PBS) was added and reacted at room temperature for 1 hour for blocking. After removing the supernatant, the product was washed with a washing solution (0.02% Tween 20, PBS). To this, 100 ml of a culture solution of the fused cells was added and reacted at room temperature for 2 hours. The supernatant was removed and the precipitate was washed with a washing solution. Subsequently, 100 μl of a goat anti-mouse IgG antibody solution labeled with a peroxidase at a concentration of 50 ng / ml was added and reacted at room temperature for 1 hour. The supernatant was removed and the product was washed again with the washing solution. 100 μl of TMB solution (manufactured by KPL) was added, and the mixture was allowed to react at room temperature for 20 minutes. When colored, 100 μl of 1N sulfuric acid was added to stop the reaction, and the absorbance at 450 nm was measured.
As a result, positive wells were found that reacted only with the GST fusion Ribosomal Protein L7 / L12 protein but not with the GST protein, and were found to contain antibodies against the Ribosomal Protein L7 / L12 protein.
Therefore, the cells in each positive well were collected and cultured in a HAT medium in a 24-well plastic plate.
The cultured fusion medium was diluted with HT medium so that the number of cells was about 20 cells / ml, and 50 μl was mixed with 10 6- week-old mouse thymocytes suspended in HT medium in a 96-well culture plate. . After mixing, the cells were cultured at 37 ° C. for 2 weeks under 7% CO 2 conditions.
The antibody activity in the culture supernatant was similarly assayed by the above-mentioned ELISA method, and cells positive for reaction with Ribosomal Protein L7 / L12 protein were recovered. Furthermore, the same dilution assay and cloning operation were repeated to obtain a total of 5 clones of hybridomas MPRB-1 to 5.
Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質を検出するモノクローナル抗体の選択
前述のようにして取得した陽性ハイブリドーマ細胞を用いて定法にしたがってモノクローナル抗体を生産回収した。
具体的には、ＲＰＭＩ１６４０培地（１０％ＦＣＳ入り）を用いて継代培養した細胞をあらかじめ２週間前に０．５ｍｌのプリスタンを腹腔内に注射したＢｌａｂ／Ｃマウスの腹腔内に５×１０６個（ＰＢＳ中）注射した。３週間後腹水を回収し、その遠心上澄みを取得した。
得られた抗体を含む溶液をＰｒｏｔｅｉｎ Ａカラム（５ｍｌ，Ｐｈａｒｍａｃｉａ製）に吸収させ、３倍量のＰＢＳで洗浄した。次いで、クエン酸緩衝液ｐＨ３で溶出した。抗体画分を回収し、各ハイブリドーマによって産生されたモノクローナル抗体を取得した。この５株のハイブリドーマ由来のモノクローナル抗体を用いてＥＬＩＳＡ法により評価した。
モノクローナル抗体の評価にはサンドウィッチ分析法を用いた。作成されたモノクローナル抗体をペルオキシダーゼに結合せしめることにより検出用の抗体として用いた。酵素標識はホースラディッシュパーオキシダーゼ（ＳｉｇｍａグレードＶＩ）を用い結合には試薬Ｓ−アセチルチオ酢酸Ｎ−ヒドロキシスクシンイミドを使用しＡｎａｌｙｔｉｃａｌ Ｂｉｏ−ｃｈｅｍｉｓｔｒｙ １３２（１９８３），６８−７３に述べられている方法に従って行った。ＥＬＩＳＡ反応においては市販の抗Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅポリクローナル抗体（Ｂｉｏｄｅｓｉｇｎ社、ウサギ）を１０μｇ／ｍｌ濃度で希釈した液を１００μｌずつ９６穴プレートに別々に分注し４℃で１晩吸着させた。
上澄み除去後、１％牛血清アルブミン溶液（ＰＢＳ中）２００μｌ添加し室温で１時間反応しブロッキングした。上澄み液を除去後、生成物を洗浄液（０．０２％Ｔｗｅｅｎ２０，ＰＢＳ）で洗浄した。これに各微生物の培養液に濃度０．３％となる量のＴｒｉｔｏｎ Ｘ−１００を加え常温で５分間抽出することにより得られた抗原溶液１００μｌを加え、室温で２時間反応させた。上澄み液を除去し、生成物を再び洗浄液で洗浄した。次いで、濃度５μｇ／ｍｌのペルオキシダーゼ標識抗Ｒｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質抗体溶液１００μｌを加え、室温にて１時間反応させた。上澄み液を除去し、生成物を再び洗浄液で洗浄した。ＴＭＢ溶液（ＫＰＬ社製）を１００μｌずつ加え、混合物を室温にて２０分間反応させた。着色したところで１Ｎの硫酸１００μｌを加えて反応を停止した。４５０ｎｍの吸光度を測定した。
酵素標識抗体としてハイブリドーマＭＰＲＢ−１由来のモノクローナル抗体を用いた場合、試験したＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅの全ての株を１０６個／ｍｌの感度の感度で検出すると同時に他のＨａｅｍｏｐｈｉｌｕｓ ｉｎｆｌｕｅｎｚａｅ，Ｋｌｅｂｓｉｅｌｌａ ｐｎｅｕｍｏｎｉａｅ，Ｃｈｌａｍｙｄｉａ ｐｎｅｕｍｏｎｉａｅおよびＮｅｉｓｓｅｒｉａ ｍｅｎｉｎｇｉｔｉｄｅｓ等の微生物について１０８個／ｍｌの高濃度でも反応性を示さずＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質に対するモノクローナル抗体を用いることでＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅ特異的な反応性をもつ抗体を取得したことが明確に確認できた。この抗体はＡＭＭＰ−１と名付けられた。表２はＡＭＭＰ−１を用いた結果のみを示す。別の微生物と交差反応を示す他の抗体を使用した結果はここで言及しない。
Specifically, intraperitoneally BLAB / C mice subcultured cells were pre 2 weeks pristane before 0.5ml were injected intraperitoneally with RPMI1640 medium (10% FCS-containing) 5 × 10 6 Injected (in PBS). After 3 weeks, ascites was collected and the supernatant was obtained.
The solution containing the obtained antibody was absorbed into a Protein A column (5 ml, manufactured by Pharmacia) and washed with 3 times the amount of PBS. Then, elution was performed with citrate buffer pH 3. Antibody fractions were collected to obtain monoclonal antibodies produced by each hybridoma. Evaluation was performed by ELISA using monoclonal antibodies derived from these five hybridomas.
A sandwich assay was used to evaluate the monoclonal antibody. The prepared monoclonal antibody was used as a detection antibody by binding to peroxidase. Enzyme labeling was performed according to the method described in Analytical Bio-chemistry 132 (1983), 68-73, using horseradish peroxidase (Sigma grade VI) and using the reagent S-acetylthioacetic acid N-hydroxysuccinimide for conjugation. . In an ELISA reaction, a solution obtained by diluting a commercially available anti- Mycoplasma pneumoniae polyclonal antibody (Biodesign, rabbit) at a concentration of 10 μg / ml was separately dispensed into 96-well plates in a 96-well plate and adsorbed overnight at 4 ° C.
After removing the supernatant, 200 μl of 1% bovine serum albumin solution (in PBS) was added and reacted at room temperature for 1 hour for blocking. After removing the supernatant, the product was washed with a washing solution (0.02% Tween 20, PBS). To this, 100 μl of an antigen solution obtained by adding Triton X-100 in an amount of 0.3% to the culture solution of each microorganism and extracting for 5 minutes at room temperature was added and reacted at room temperature for 2 hours. The supernatant was removed and the product was washed again with the washing solution. Next, 100 μl of a peroxidase-labeled anti-Ribosomal Protein L7 / L12 protein antibody solution at a concentration of 5 μg / ml was added and reacted at room temperature for 1 hour. The supernatant was removed and the product was washed again with the washing solution. 100 μl of TMB solution (manufactured by KPL) was added, and the mixture was allowed to react at room temperature for 20 minutes. When colored, 100 μl of 1N sulfuric acid was added to stop the reaction. Absorbance at 450 nm was measured.
When the monoclonal antibody derived from hybridoma MPRB-1 was used as the enzyme-labeled antibody, all the strains of Mycoplasma pneumoniae tested were detected with a sensitivity of 10 6 / ml and other Haemophilus influenzae , Klebsiella pneumoniae , Clebsiella pneumoniae, Neisseria microorganisms, etc. 10 8 cells / ml of the high concentration reactivity shown without Ribosomal protein L7 / L12 the use of monoclonal antibodies Mycoplasma pneumoniae-specific reactive antibodies clearly that was acquired with in to the protein at about meningitides It could be confirmed. This antibody was named AMMP-1. Table 2 shows only the results using AMMP-1. Results using other antibodies that cross-react with other microorganisms are not mentioned here.
Ｒｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質固定化アフィニティカラムを用いたＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅ Ｒｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質と特異的に反応するポリクローナル抗体の取得
実施例１に記載の方法により取得したＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅ Ｒｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質またはＴｒｉｔｏｎ Ｘ−１００処理した菌体の上清を抗原として使用した。１００μｇの抗原を含む生理食塩水約１．２ｍｌをフロイントアジュバント１．５ｍｌとともに乳化した。エマルジョンをＳＰＦ日本白色ウサギに皮下注射してウサギを免疫化した。２週間おきに５〜６回免疫し、抗体価を確認した。
抗体価の確認はＥＬＩＳＡ法により実施した。０．０５％のアジ化ソーダ含むＰＢＳ中に溶解したＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質を１０μｇ／ｍｌ濃度に希釈した液を１００μｌずつ９６穴プレートに分注し４℃で１晩吸着させた。上澄み除去後、１％牛血清アルブミン溶液（ＰＢＳ中）２００μｌ添加し室温で１時間反応しブロッキングした。上澄み液を除去後、生成物を洗浄液（０．０２％Ｔｗｅｅｎ２０，ＰＢＳ）で洗浄した。正常のウサギ血清および免疫化したウサギの抗血清を希釈して得られた溶液１００μｌを加え、室温にて２時間反応させた。上澄み液を除去し、生成物を再び洗浄液で洗浄した。次いで、濃度５０ｎｇ／ｍｌのペルオキシダーゼ標識抗ウサギＩｇＧ抗体溶液１００μｌを加え、室温にて１時間反応させた。上澄み液を除去し、生成物を再び洗浄液で洗浄した。ＯＰＤ溶液（Ｓｉｇｍａ社製）を１００μｌずつ加え、混合物を室温にて２０分間反応させた。着色したところで１Ｎの硫酸１００μｌを加えて反応を停止した。４９２ｎｍの吸光度を測定した。
抗体価上昇を確認後、大量採血を実施した。耳動脈から血液をガラス製遠心管に採取し、３７℃で１時間放置後、４℃で一晩静置した。その後３０００ｒｐｍ５分間遠心し、上清を回収した。得られた抗血清は４℃で保存した。
Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質を固定化したアフィニティカラムを調製した。ＨｉＴｒａｐ ＮＨＳ活性化カラム（１ｍｌ，Ｐｈａｒｍａｃｉａ社製）を用いた。カラムを１ｍＭ ＨＣｌで置換後直ちにＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質のＰＢＳ溶液（１ｍｇ／ｍｌ）を加えた。カラムを３０分間静置後、ブロッキング試薬を加え、ＰＢＳで平衡化した。
このＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質固定化アフィニティカラムを使用して、Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＴｒｉｔｏｎ Ｘ−１００処理した菌体の上清を抗原として得られた抗血清中のポリクローナル抗体の精製を行った。この抗血清をＰＢＳで５倍に希釈し、０．４５μｍのフィルターを通した後、流速０．５ｍｌ／ｍｉｎでＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質固定化カラムに吸着させた。その後０．１Ｍグリシシ緩衝液ｐＨ２．１でカラムから溶出し、直ちに１Ｍ Ｔｒｉｓ緩衝液ｐＨ９．０で中和した後、抗体価測定法と同様のＥＬＩＳＡ法により目的とする抗体の溶出画分を回収した。
このようにして得られたポリクローナル抗体は特表平７−５０９５６５号公報に記載されているＯＩＡ法により評価した。
精製した抗体はＯＩＡ法の捕捉抗体として使用した。また検出抗体としては実施例４に記載したＡＭＣＴ−１モノクローナル抗体をパーオキシダーゼで酵素標識したものを使用した。酵素標識はホースラディッシュパーオキシダーゼ（ＳｉｇｍａグレードＶＩ）を用い結合には試薬Ｓ−アセチルチオ酢酸Ｎ−ヒドロキシスクシンイミドを使用しＡｎａｌｙｔｉｃａｌ Ｂｉｏ−ｃｈｅｍｉｓｔｒｙ １３２（１９８３），６８−７３に述べられている方法に従って行った。
ＯＩＡ反応においては０．０５％アジ化ナトリウムを含むＰＢＳ中の精製ポリクローナル抗体を１０μｇ／ｍｌ濃度に０．１Ｍ ＨＥＰＥＳ緩衝液ｐＨ８．０で希釈した液を５０μｌずつシリコンウエハー上に添加し室温で３０分反応させた後、蒸留水で洗浄し、サッカロース及びアルカリ処理カゼインを含むコーティング溶液でコーティング後、使用した。
上記操作で得られた各微生物の培養液に濃度０．５％となる量のＴｒｉｔｏｎＸ−１００を加え常温で５分間抽出することにより得られた抗原溶液１５μｌを上記シリコンウエハーの上に添加し、室温で１０分間反応させた。次いで、２０μｇ／ｍｌペルオキシダーゼ標識化モノクローナル抗体を１５μｌ加え、１０分間反応させた。蒸留水で洗浄後、ＴＭＢ溶液（ＫＰＬ社製）を１５μｌずつ加え、混合物を室温にて５分間反応させた。生成物を蒸留水で洗浄し、酵素反応により生成した青色を肉眼で観察した。
この結果、表３に示すように、精製ポリクローナル抗体ＡＰＭＰ−１を捕捉抗体として用いることにより、Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅを１０８個／ｍｌの感度で検知できること、および他の微生物の反応性は検知できないことが明らかである。このようにしてＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅのＲｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質を固定化したアフィニティカラムにより、Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅに特異的に反応するポリクローナル抗体を取得したことを確認した。
本発明によると微生物の進化の過程で機能的に保持された細胞内分子に対する抗体を用いて特定の種の微生物を特異的に検出できるだけでなく、同一種内における全ての血清型の微生物を精度よく検出することができる。
このような抗体として微生物のリボソーム蛋白質、Ｒｉｂｏｓｏｍａｌ Ｐｒｏｔｅｉｎ Ｌ７／Ｌ１２蛋白質に対する抗体を用い、Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅの検出を精度良く行うことができる。また、このような抗体を構成要素とする微生物検出用試薬キットを用いることで、微生物の検出をより汎用的に精度良く行うことができる。
引用文献：
特許文献
＊米国特許Ｎｏ．５，５５２，２７９，Ｗｅｉｓｂｕｒｇ，ｅｔ ａｌ．，Ｓｅｐｔｅｍｂｅｒ３，１９９６「Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅおよびＭｙｃｏｐｌａｓｍａ ｇｅｎｉｔａｌｉｕｍ検出用核酸プローブ」
＊ＥＰＯ ３０５１４５，Ａｐｐｌｉｃａｔｉｏｎ Ｎｏ．８８３０７７９３．５ Ｚｉｖｉｎ ａｎｄ Ｍｏｎａｈａｎ．Ｍａｒｃｈ １，１９８９．Ｍｅｔｈｏｄｓ ａｎｄ ｐｒｏｂｅｓ ｆｏｒ ｄｅｔｅｃｔｉｎｇ ｎｕｃｌｅｉｃ ａｃｉｄ．
＊ＥＰＯ ２５０６６２，Ａｐｐｌｉｃａｔｉｏｎ Ｎｏ．８６３０４９１９，３ Ｇｏｂｅｌ ａｎｄ Ｓｔａｎｂｒｉｄｇｅ．Ｊａｎｕａｒｙ ７，１９８８，Ｄｅｔｅｃｔｉｏｎ ｏｆ Ｍｙｃｏｐｌａｓｍａ ｂｙ ＤＮＡ ｈｙｂｒｉｄｉｚａｔｉｏｎ．
＊特開昭６３（１９８８）−２９８
その他の特許文献
＊Ｂｅｒｎｅｔ，Ｃ．，Ｍ．Ｇａｒｒｅｔ，ｅｔ ａｌ．，（１９９３）「ポリメラーゼ連鎖反応を利用するＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅの検出」Ｊ Ｃｌｉｎ Ｍｉｃｒｏｂｉｏｌ ３１（４），１０１３−５
＊Ｂｕｃｋ，Ｇ．Ｅ．，Ｌ．Ｃ．Ｏ′Ｈａｒａ，ｅｔ ａｌ．，（１９９３）「ＤＮＡ増殖による疑似臨床試料中のＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅの迅速かつ高感度の検出法」Ｊ Ｃｌｉｎ Ｍｉｃｒｏｂｉｏｌ ３１（４），１０１３−５
＊Ｇｏｂｅｌ，Ｕ．Ｂ．，Ａ．Ｇｅｉｓｅｒ，ｅｔ ａｌ．，（１９８７）「Ｍｙｃｏｐｌａｓｍａ種間のリボソーマルＲＮＡ差別化可変領域に相補的なオリゴヌクレオチドプローブ」
＊Ｇｒａｎａｔｏ，Ｐ．Ａ．，Ｌ．Ｐｏｅ，ｅｔ ａｌ．，（１９８０）「Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅ成長における改良Ｎｅｗ Ｙｏｒｋ Ｃｉｔｙ培地の利用に関する報告書」Ｚｈ Ｍｉｋｒｏｂｉｏｌ Ｅｐｉｄｅｍｉｏｌ Ｉｍｍｕｎｏｂｉｏｌ（８），５０−３
＊Ｈｙｍａｎ，Ｈ．Ｃ．，Ｄ．Ｙｏｇｅｖ，ｅｔ ａｌ．，（１９８７）「Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅおよびＭｙｃｏｐｌａｓｍａ ｇｅｎｉｔａｌｉｕｍの検出・同定用ＤＮＡプローブ」
＊Ｊｅｎｓｅｎ，Ｊ．Ｓ．，Ｊ．Ｓｏｎｄｅｒｇａｒｄ Ａｎｄｅｒｓｅｎ，ｅｔ ａｌ．，（１９９３）「ポリメラーゼ連鎖反応を利用する疑似臨床サンプル中のＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅの検出」Ｊ Ｍｅｄ Ｍｉｃｒｏｂｉｏｌ ３８（３），１６６−７０
＊Ｋａｉ，Ｍ．，Ｓ．Ｋａｍｉｙａ，ｅｔ ａｌ．，（１９８７）「ポリメラーゼ連鎖反応を利用するＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅの迅速検出法」Ｊ Ｇｅｎ Ｍｉｃｒｏｂｉｏｌ １３３（Ｐｔ７），１９６９−７４
＊Ｋｎｕｄｓｏｎ，Ｄ．Ｌ．ａｎｄ Ｒ．ＭａｃＬｅｏｄ（１９７９）「Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅ ａｎｄ Ｍｙｃｏｐｌａｓｍａ ｓａｌｉｖａｒｉｕｍ−寒天中でのコロニー成長の電子顕微鏡写真」Ｓｃｉ Ｒｅｐ Ｒｅｓ Ｉｎｓｔ Ｔｏｈｏｋｕ Ｕｎｉｖ［Ｍｅｄ］２６（３−４），７１−９１
＊Ｍａｄｓｅｎ ＲＤ，Ｗｅｉｎｅｒ ＬＢ，ＭｃＭｉｌｌａｎ ＪＡ，Ｓａｅｅｄ ＪＡ，Ｎｏｒｔｈ ＪＡ，Ｃｏａｔｅｓ ＳＲ．（１９８８）．Ｄｉｒｅｃｔ ｄｅｔｅｃｔｉｏｎ ｏｆ Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅ ａｎｔｉｇｅｎ ｉｎ ｃｌｉｎｉｃａｌ ｓｐｅｃｉｍｅｎｓ ｂｙ ａ ｍｏｎｏｃｌｏｎａｌ ａｎｔｉｂｏｄｙ ｉｍｍｕｎｏｂｌｏｔ ａｓｓａｙ Ａｍ Ｊ Ｃｌｉｎ Ｐａｔｈｏｌ ８９（１）：９５−９
＊Ｒａｇｎａｒ Ｎｏｒｒｂｙ，Ｓ．（１９９９）「ノルディック諸国の代表的肺炎、その病因とフレロキサシンおよびドキシシクリンの比較臨床結果、Ｎｏｒｄｉｃ Ａｔｙｐｉｃａｌ Ｐｎｅｕｍｏｎｉａｅ Ｓｔｕｄｙ Ｇｒｏｕｐ」Ｊ Ｍｅｄ Ｍｉｃｒｏｂｉｏｌ ４８（１２），１１１５−２２
＊Ｔｉｌｔｏｎ，Ｒ．Ｃ．，Ｆ．Ｄｉａｓ，ｅｔ ａｌ．，（１９８０）「臨床試料中のＭｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅ検出用ＤＮＡプーロブと培養」Ｊ Ｃｌｉｎ Ｍｉｃｒｏｂｉｏｌ １２（６），７４８−５２
＊Ｙｏｇｅｖ，Ｄ．，Ｄ．Ｈａｌａｃｈｍｉ，ｅｔ ａｌ．（１９８８）「ｒＲＮＡ遺伝子プローブとゲノムＤＮＡ ｆｉｎｇｅｒｐｒｉｎｔｓによるＭｙｃｏｐｌａｓｍａ種およびＭｙｃｏｐｌａｓｍａ株（ｍｏｌｌｉｃｕｔｅｓ）の検出」Ｊ Ｃｌｉｎ Ｍｉｃｒｏｂｉｏｌ ２６（１１），２２６６−９
＊Ｃｈａｎ ＥＤ，Ｋａｌａｙａｎａｍｉｔ Ｔ，Ｌｙｎｃｈ ＤＡ，Ｔｕｄｅｒ Ｒ，Ａｒｎｄｔ Ｐ，ＷｉｎｎＲ．Ｓｃｈｗａｒｚ ＭＩ．「成人に重度の肺病気を引き起すＭｙｃｏｐｌａｓｍａ ｐｎｅｎｍｏｎｉａｅ関連細気管支炎−文献レビュー」Ｃｈｅｓｔ．，１９９９；１１５（４），１１８８−９４
＊ＮＣＢＩデータベース＃ＮＣ＃０００９１２．，Ｈｉｍｍｅｌｒｅｉｃｈ，Ｒ．，Ｈｉｌｂｅｌｔ，Ｈ．，Ｐｌａｇｅｎｓ，Ｈ．，Ｐｉｒｋｌ，Ｅ．，Ｌｉ，Ｂ．Ｃ．，ａｎｄ Ｈｅｒｒｍａｎｎ，Ｒ．
＊Ｃｉｍｏｌａｉ Ｎ．「Ｍｙｃｏｐｌａｓｍａ ｐｎｅｕｍｏｎｉａｅによる呼吸器感染症」Ｐｅｄｉａｔｒ Ｒｅｖ．，１９９８；１９（１０），３２７−３１
＊Ｈａｒｌｏｗ，Ｅ．，ａｎｄ Ｄ．Ｌａｎｅ（１９８８）「抗体、実験室マニュアル」Ｎｅｗ Ｙｏｒｋ，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ
＊Ｓｈａｍｂｒｏｏｋ，Ｊ．，Ｅ．Ｆ．Ｆｒｉｔｓｃｈ，ａｎｄ Ｔ．Ｍａｎｉａｔｉｓ，（１９８９）「モレキュラークローニング、実験室マニュアル、第２版」Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ ＰｒｅｓｓRibosomal Protein L7 / L12 protein Mycoplasma pneumoniae Ribosomal Protein L7 / L12 protein was obtained by the method described in the acquired first embodiment of Mycoplasma pneumoniae Ribosomal Protein L7 / L12 protein specifically reactive with a polyclonal antibody with immobilized affinity column or Triton X-100-treated cell supernatant was used as an antigen. About 1.2 ml of physiological saline containing 100 μg of antigen was emulsified with 1.5 ml of Freund's adjuvant. The emulsion was immunized subcutaneously into SPF Japanese white rabbits. Immunization was performed 5-6 times every 2 weeks, and the antibody titer was confirmed.
The antibody titer was confirmed by the ELISA method. 100 μl of a solution obtained by diluting Mycoplasma pneumoniae Ribosomal Protein L7 / L12 protein dissolved in PBS containing 0.05% sodium azide to a concentration of 10 μg / ml was adsorbed at 4 ° C. overnight at 4 ° C. . After removing the supernatant, 200 μl of 1% bovine serum albumin solution (in PBS) was added and reacted at room temperature for 1 hour for blocking. After removing the supernatant, the product was washed with a washing solution (0.02% Tween 20, PBS). 100 μl of a solution obtained by diluting normal rabbit serum and immunized rabbit antiserum was added and reacted at room temperature for 2 hours. The supernatant was removed and the product was washed again with the washing solution. Subsequently, 100 μl of a peroxidase-labeled anti-rabbit IgG antibody solution having a concentration of 50 ng / ml was added and reacted at room temperature for 1 hour. The supernatant was removed and the product was washed again with the washing solution. An OPD solution (manufactured by Sigma) was added by 100 μl each, and the mixture was reacted at room temperature for 20 minutes. When colored, 100 μl of 1N sulfuric acid was added to stop the reaction. Absorbance at 492 nm was measured.
After confirming an increase in antibody titer, a large amount of blood was collected. Blood was collected from the ear artery into a glass centrifuge tube, allowed to stand at 37 ° C. for 1 hour, and allowed to stand at 4 ° C. overnight. Thereafter, the mixture was centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected. The obtained antiserum was stored at 4 ° C.
An affinity column immobilizing the Ribosomal Protein L7 / L12 protein of Mycoplasma pneumoniae was prepared. A HiTrap NHS activation column (1 ml, manufactured by Pharmacia) was used. Immediately after replacement of the column with 1 mM HCl, a PBS solution of Ribosomal Protein L7 / L12 protein (1 mg / ml) was added. After allowing the column to stand for 30 minutes, a blocking reagent was added and equilibrated with PBS.
Using this Mycoplasma pneumoniae Ribosomal Protein L7 / L12 protein-immobilized affinity column, the polyclonal antibody in the antiserum obtained using the Mycoplasma pneumoniae Triton X-100-treated cell supernatant as an antigen was purified. . This antiserum was diluted 5-fold with PBS, passed through a 0.45 μm filter, and then adsorbed onto a Ribosomal Protein L7 / L12 protein-immobilized column of Mycoplasma pneumoniae at a flow rate of 0.5 ml / min. Then elute from the column with 0.1M glycisic buffer pH 2.1, immediately neutralize with 1M Tris buffer pH 9.0, and collect the elution fraction of the target antibody by the same ELISA method as the antibody titer measurement method did.
The polyclonal antibody thus obtained was evaluated by the OIA method described in JP-T-7-509565.
The purified antibody was used as a capture antibody for the OIA method. The detection antibody used was an AMCT-1 monoclonal antibody described in Example 4 enzyme-labeled with peroxidase. Enzyme labeling was performed according to the method described in Analytical Bio-chemistry 132 (1983), 68-73, using horseradish peroxidase (Sigma grade VI) and using the reagent S-acetylthioacetic acid N-hydroxysuccinimide for conjugation. .
In the OIA reaction, 50 μl of a purified polyclonal antibody in PBS containing 0.05% sodium azide diluted with 0.1 M HEPES buffer pH 8.0 to a concentration of 10 μg / ml was added to a silicon wafer at room temperature for 30 minutes. After reacting for a minute, it was washed with distilled water, used after coating with a coating solution containing saccharose and alkali-treated casein.
Add 15 μl of the antigen solution obtained by adding Triton X-100 in an amount of 0.5% to the culture solution of each microorganism obtained by the above operation and extracting it at room temperature for 5 minutes, on the silicon wafer, The reaction was allowed to proceed for 10 minutes at room temperature. Next, 15 μl of 20 μg / ml peroxidase-labeled monoclonal antibody was added and allowed to react for 10 minutes. After washing with distilled water, 15 μl of TMB solution (manufactured by KPL) was added, and the mixture was reacted at room temperature for 5 minutes. The product was washed with distilled water, and the blue color produced by the enzyme reaction was observed with the naked eye.
As a result, as shown in Table 3, by using the purified polyclonal antibody APMP-1 as a capture antibody, Mycoplasma pneumoniae can be detected with a sensitivity of 10 8 cells / ml, and the reactivity of other microorganisms cannot be detected. it is obvious. The Ribosomal Protein L7 / L12 affinity column where the protein immobilized in this way Mycoplasma pneumoniae, it was confirmed that the obtained polyclonal antibodies specifically reactive with Mycoplasma pneumoniae.
As such an antibody, an antibody against a ribosomal protein of a microorganism, Ribosomal Protein L7 / L12 protein can be used to accurately detect Mycoplasma pneumoniae . In addition, by using a microorganism detection reagent kit having such an antibody as a component, microorganisms can be detected more generally and accurately.
Cited references:
Patent Literature * US Patent No. 5,552,279, Weisburg, et al. , September 3, 1996 "Nucleic acid probe for detection of Mycoplasma pneumoniae and Mycoplasma genitalium "
* EPO 305145, Application No. 8830793.5 Zivin and Monahan. March 1, 1989. Methods and probes for detecting nuclear acid.
* EPO 250662, Application No. 8304919, 3 Gobel and Stanbridge. January 7, 1988, Detection of Mycoplasma by DNA hybridization.
* JP-A-63 (1988) -298
Other Patent Documents * Bernet, C.I. , M.M. Garret, et al. (1993) “Detection of Mycoplasma pneumoniae using polymerase chain reaction” J Clin Microbiol 31 (4), 1013-5.
* Buck, G. E. L. C. O'Hara, et al. , (1993) "Rapid and sensitive detection method of Mycoplasma pneumoniae in pseudo-clinical samples by DNA proliferation" J Clin Microbiol 31 (4), 1013-5
* Gobel, U. B. A. Geiser, et al. , (1987) "An oligonucleotide probe complementary to a ribosomal RNA differentiation variable region between Mycoplasma species".
* Granato, P.M. A. L. Poe, et al. , (1980) "A report on the use of improved New York City medium in growth of Mycoplasma pneumoniae " Zh Microbiol Epidemiol Immunobiol (8), 50-3.
* Hyman, H .; C. , D.C. Yogev, et al. , (1987) "DNA probe for detection and identification of Mycoplasma pneumoniae and Mycoplasma genitalium "
* Jensen, J.A. S. , J .; Sondergard Andersen, et al. (1993) "Detection of Mycoplasma pneumoniae in pseudoclinical samples using polymerase chain reaction" J Med Microbiol 38 (3), 166-70.
* Kai, M .; S. Kamiya, et al. (1987) "Rapid detection method of Mycoplasma pneumoniae using polymerase chain reaction" J Gen Microbiol 133 (Pt7), 1969-74.
* Knudson, D.M. L. and R.R. MacLeod (1979) " Mycoplasma pneumoniae and Mycoplasma salivarium- Electron micrograph of colony growth in agar" Sci Rep Res Inst Tokyo Univ [Med] 26 (3-4), 71-91
* Madsen RD, Weiner LB, McMillan JA, Seed JA, North JA, Coates SR. (1988). Direct detection of Mycoplasma pneumoniae antigen in clinical specifics by a monoclonal antigen immunoassay Am J-Clin Patol95 (95)
* Ragnar Norrby, S.A. (1999) "Representative pneumonia in Nordic countries, its etiology and comparative clinical results of fleroxacin and doxycycline, Nordic Atypical Pneumoniae Study Group" J Med Microbiol 48 (12), 1115-22.
* Tilton, R.A. C. , F.A. Dias, et al. (1980) "DNA prob and culture for detection of Mycoplasma pneumoniae in clinical samples" J Clin Microbiol 12 (6), 748-52.
* Yogev, D.M. , D.C. Halachmi, et al. (1988) "Detection of Mycoplasma species and Mycoplasma strains (mollicutes) with rRNA gene probes and genomic DNA fingerprints" J Clin Microbiol 26 (11), 2266-9.
* Chan ED, Kalayanamit T, Lynch DA, Tuder R, Arndt P, WinnR. Schwartz MI. “ Mycoplasma pnennoniae- associated bronchiolitis causing severe lung disease in adults-literature review” Chest. 1999; 115 (4), 1188-94.
* NCBI database # NC # 000912. , Himmelreich, R .; Hilbelt, H .; Plagens, H .; Pirkl, E .; Li, B .; C. , And Herrmann, R .;
* Cimolai N. "Respiratory infections caused by Mycoplasma pneumoniae " Pediatr Rev. 1998; 19 (10), 327-31.
* Harlow, E .; , And D.D. Lane (1988) "Antibodies, Laboratory Manual" New York, Cold Spring Harbor Laboratory Press.
* Shambrook, J. et al. , E.C. F. Fritsch, and T.R. Maniatis, (1989) "Molecular Cloning, Laboratory Manual, Second Edition" Cold Spring Harbor Laboratory Press.
Claims (4)
An antibody against bacterial ribosomal protein L7 / L12 that belong to Mycoplasma pneumoniae (Mycoplasma pneumoniae), reacts specifically with ribosomal protein L7 / L12 of the bacterium can be bacteria and identification of the bacteria other genera monoclonal bacteria detection method for, which comprises using the antibody to another bacteria identification of the bacteria other genera.
The method for detecting bacteria according to claim 1, wherein the antibody is a monoclonal antibody conjugated with an enzyme.
An antibody against bacterial ribosomal protein L7 / L12 that belong to Mycoplasma pneumoniae (Mycoplasma pneumoniae), reacts specifically with ribosomal protein L7 / L12 of the bacterium can be bacteria and identification of the bacteria other genera monoclonal which comprises using an antibody, bacteria and identify different bacteria detection reagent kit for the bacteria other genera.
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| JP2004201605A (en) * | 2002-12-26 | 2004-07-22 | Asahi Kasei Corp | Dna encoding ribosome l7/l12 protein of bacterium of genus legionella |
| KR100905066B1 (en) * | 2007-12-04 | 2009-06-30 | 주식회사 바이오랜드 | The recombinant proteins for the diagnosis of diseases infected from Mycoplasma pneumoniae and the diagnostic kits comprising the same |
| JP5458344B2 (en) * | 2008-01-30 | 2014-04-02 | 旭化成株式会社 | Antibody immobilization carrier |
| BR112012013328B1 (en) | 2009-12-04 | 2019-07-09 | Lsi Medience Corporation | METHODS TO DETECT MYCOPLASMA PNEUMONIAE OR MYCOPLASMA GENITALIUM |
| JP6150559B2 (en) * | 2013-02-28 | 2017-06-21 | 旭化成株式会社 | Detection method of Mycoplasma pneumonia |
| JP2016031353A (en) | 2014-07-30 | 2016-03-07 | 株式会社 富山研究所 | Mycoplasma pneumoniae detection reagent and use therefor |
| JP7402659B2 (en) * | 2019-11-01 | 2023-12-21 | 旭化成株式会社 | Antibodies for detecting Mycoplasma pneumoniae in a specimen, and methods, reagents, and kits for detecting Mycoplasma pneumoniae using such antibodies |
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| WO1996007910A1 (en) * | 1994-09-02 | 1996-03-14 | Meiji Milk Products Company Limited | Diagnostic drug for chlamydia infection |
| JPH0931097A (en) * | 1995-07-24 | 1997-02-04 | Yuka Medeiasu:Kk | Determination of chlamydia trachomatis antibody |
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| DE69131131T2 (en) * | 1990-09-11 | 1999-10-14 | Institute For Child Health Research | Cloning and sequencing of dermatophoid antigens (house dust mites) |
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| WO1996007910A1 (en) * | 1994-09-02 | 1996-03-14 | Meiji Milk Products Company Limited | Diagnostic drug for chlamydia infection |
| JPH0931097A (en) * | 1995-07-24 | 1997-02-04 | Yuka Medeiasu:Kk | Determination of chlamydia trachomatis antibody |
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| US10174103B2 (en) | 2013-08-23 | 2019-01-08 | Tauns Co., Ltd. | Mycoplasma pneumoniae immunological detection method and kit |
| US10550175B2 (en) | 2013-08-23 | 2020-02-04 | Tauns Co., Ltd. | Mycoplasma pneumoniae immunological detection method and kit |
| US10995135B2 (en) | 2013-08-23 | 2021-05-04 | Tauns Co., Ltd. | Mycoplasma pneumoniae immunological detection method and kit |
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| CA2398469A1 (en) | 2001-08-09 |
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