JP5469783B1 - Drug resistant influenza virus detection kit - Google Patents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
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Abstract
本発明は、受託番号がＮＩＴＥ ＢＰ−０１７００であるハイブリドーマによって産生される、配列番号１のペプチドに対するモノクローナル抗体及びインフフエンザウイルスのリボ核タンパク質に対する抗体を含む、オセルタミビル耐性インフルエンザウイルス検出のためのキットに関する。 The present invention relates to a kit for detecting oseltamivir-resistant influenza virus comprising a monoclonal antibody against the peptide of SEQ ID NO: 1 and an antibody against INF nucleovirus ribonucleoprotein produced by a hybridoma whose accession number is NITE BP-01700. About.
Description
本発明は、薬剤耐性インフルエンザウイルス検出キット及びそれを用いる検出方法に関する。 The present invention relates to a drug resistant influenza virus detection kit and a detection method using the same.
インフルエンザは、インフルエンザウイルスによって引き起こされる極めて重要な疾患である。日本などの温帯では、季節性インフルエンザは冬季に毎年のように流行する。抗インフルエンザウイルス薬として、ザナミビルやオセルタミビル等が知られている。オセルタミビルは、非常に有効であり、かつ、経口剤であることから使用しやすことから、インフルエンザ治療に第一選択薬として用いられている。 Influenza is a vital disease caused by influenza viruses. In temperate zones such as Japan, seasonal influenza is prevalent every year in winter. Zanamivir, oseltamivir, etc. are known as anti-influenza virus drugs. Oseltamivir is very effective, and since it is an oral preparation, it is used as a first-line drug for the treatment of influenza.
しかし、オセルタミビルに耐性のインフルエンザウイルスが報告されている。この場合、オセルタミビルがインフルエンザの治療に無効であるばかりか、オセルタミビルのみを使用した場合には、インフルエンザをさらに悪化させ、重症化させてしまう可能性がある。このため、感染インフルエンザウイルスが、オセルタミビルに耐性であるか否かを、迅速かつ簡便に判定する技術が切望されている。 However, influenza viruses resistant to oseltamivir have been reported. In this case, oseltamivir is not only ineffective for the treatment of influenza, but if only oseltamivir is used, influenza may be further exacerbated and made serious. For this reason, a technique for quickly and simply determining whether or not an infectious influenza virus is resistant to oseltamivir is eagerly desired.
インフルエンザウイルスのオセルタミビル耐性出現のメカニズムは、ウイルスビリオンのノイラミニダーゼを構成するペプチドのアミノ酸配列の特定の部分の突然変異、すなわち、位置２７４のアミノ酸のヒスチジンからチロシンへの置換（Ｈｉｓ２７４Ｔｙｒ）であることが知られている。このため、ウイルスビリオンのノイラミニダーゼ遺伝子の塩基配列を増幅し、その塩基配列の解析から耐性の有無が判断されている。しかし、この方法は、労力と時間を要し、臨床的に応用することは極めて困難である。 It is known that the mechanism of emergence of oseltamivir resistance in influenza virus is a mutation in a specific part of the amino acid sequence of the peptide constituting the neuraminidase of the viral virion, that is, substitution of the amino acid at position 274 from histidine to tyrosine (His274Tyr). It has been. For this reason, the base sequence of the neuraminidase gene of a viral virion is amplified, and the presence or absence of resistance is judged from the analysis of the base sequence. However, this method requires labor and time and is extremely difficult to apply clinically.
最近、ＥＫＧＫＶＴＫＳＩＥＬＮＡＰＮＦＹＹＥＥ（配列番号１）からなるペプチドに対する抗体を用いる方法が報告されているが、実際に、臨床的に応用した場合の感度及び特異性は、報告されていない（特許文献１）。また、特許文献１には、ウイルスビリオンの薬剤耐性ノイラミニダーゼペプチドに対し特異的に反応するモノクローナル抗体と抗インフルエンザウイルスリボ核タンパク質抗体との組合わせについては、記載も示唆もされていない。 Recently, a method using an antibody against a peptide consisting of EKGKVTKSIELNAPNFYYEE (SEQ ID NO: 1) has been reported, but the sensitivity and specificity when actually applied clinically has not been reported (Patent Document 1). Patent Document 1 does not describe or suggest a combination of a monoclonal antibody that specifically reacts with a drug-resistant neuraminidase peptide of a viral virion and an anti-influenza virus ribonucleoprotein antibody.
本発明は、薬剤耐性インフルエンザ検出キット及びそれを用いる検出方法を提供することを課題とする。 An object of the present invention is to provide a drug resistant influenza detection kit and a detection method using the same.
本発明者は、予想外にも、ウイルスビリオンの薬剤耐性ノイラミニダーゼペプチドに対し特異的に反応するモノクローナル抗体と抗インフルエンザウイルスリボ核タンパク質抗体を組合わせることによって、高感度かつ特異的にオセルタミビル耐性ウイルスを検出することができることを初めて見出し、本発明を完成させた。 Unexpectedly, the present inventor has achieved a highly sensitive and specific oseltamivir resistant virus by combining a monoclonal antibody that reacts specifically with a drug resistant neuraminidase peptide of a viral virion and an anti-influenza virus ribonucleoprotein antibody. The present invention was completed by finding for the first time that it could be detected.
すなわち、本発明は、下記のとおりである。
［１］受託番号がＮＩＴＥ ＢＰ−０１７００であるハイブリドーマによって産生される、配列番号１のペプチドに対するモノクローナル抗体及びインフルエンザウイルスのリボ核タンパク質に対する抗体を含む、オセルタミビル耐性インフルエンザウイルス検出のためのキット、
［２］インフルエンザウイルスのリボ核タンパク質に対する抗体が、モノクローナル抗体である、［１］に記載のキット、
［３］キットが、ＥＬＩＳＡのためである、［２］に記載のキット、
［４］キットが、イムノクロマトグラフィーのためである、［２］に記載のキット。
［５］さらに、金コロイドを含む、［４］に記載のキット、
［６］さらに、白金コロイドを含む、［４］に記載のキット、
［７］さらに、パラジウムコロイドを含む、［４］に記載のキット、
［８］［１］〜［７］のいずれかに記載のキットを用いる、オセルタミビル耐性インフルエンザウイルスを検出するための方法。That is, the present invention is as follows.
[1] A kit for detecting oseltamivir-resistant influenza virus, comprising a monoclonal antibody against the peptide of SEQ ID NO: 1 and an antibody against influenza virus ribonucleoprotein, produced by a hybridoma whose accession number is NITE BP-01700,
[2] The kit according to [1], wherein the antibody against the ribonucleoprotein of influenza virus is a monoclonal antibody,
[3] The kit according to [2], wherein the kit is for ELISA,
[4] The kit according to [2], wherein the kit is for immunochromatography.
[5] The kit according to [4], further comprising gold colloid,
[6] The kit according to [4], further comprising platinum colloid,
[7] The kit according to [4], further comprising a palladium colloid,
[8] A method for detecting oseltamivir resistant influenza virus using the kit according to any one of [1] to [7].
本発明のハイブリドーマからモノクローナル抗体を取得する方法は、従来公知の任意の方法を用いることができる。たとえば、ハイブリドーマを所望数以上に増殖させた後、動物、たとえば、マウスの腹腔内に移植し、ハイブリドーマを腹水内で増殖させ、得られた腹水を精製することによって、目的のモノクローナル抗体を得ることができる。腹水の精製には、プロテインＧ、プロテインＡ等を用いたアフィニティークロマトグラフィーが公的に用いられる。また、抗原を固相化したアフィニティークロマトグラフィーを用いることもできる。さらには、イオン交換クロマトグラフィー、ゲルろ過クロマトグラフィー、硫安分画及び遠心分離等の手段を用いることもできる。 As a method for obtaining a monoclonal antibody from the hybridoma of the present invention, any conventionally known method can be used. For example, after a desired number of hybridomas are grown, transplanted into the abdominal cavity of an animal, for example, a mouse, the hybridoma is grown in ascites, and the resulting ascites is purified to obtain the desired monoclonal antibody. Can do. For purification of ascites, affinity chromatography using protein G, protein A or the like is publicly used. Alternatively, affinity chromatography in which an antigen is immobilized may be used. Furthermore, means such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used.
本発明のキットは、抗原抗体反応を利用した様々な測定方法に用いることができる。具体的には、蛍光免疫測定法（ＦＩＡ法）、酵素免疫測定法（ＥＩＡ法）、イムノクロマトグラフィーを例示することができる。好ましい測定法としては、ＥＩＡ法及びイムノクロマトグラフィーを挙げることができる。これらの方法によれば、高感度、迅速かつ簡便に検出することができる。ＥＩＡ法では、酵素標識したＩｇＧを用い、酵素反応に基づく発色をシグナルとして免疫複合体を検出する。ＥＩＡ法の中でも、サンドイッチＥＬＩＳＡが、特に好ましい。 The kit of the present invention can be used in various measurement methods utilizing antigen-antibody reaction. Specifically, fluorescent immunoassay (FIA method), enzyme immunoassay (EIA method), and immunochromatography can be exemplified. Preferable measurement methods include EIA method and immunochromatography. According to these methods, high sensitivity can be detected quickly and easily. In the EIA method, enzyme-labeled IgG is used, and an immune complex is detected using color development based on an enzyme reaction as a signal. Among the EIA methods, sandwich ELISA is particularly preferable.
イムノクロマトグラフィーの原理は、概略下記のとおりである。被分析物と結合可能な固定化試薬（抗体、抗原等）を含む少なくとも１つの反応部位を有するクロマトグラフィー担体を固定相として用いる。このクロマトグラフィー担体上で、分析対象物に結合可能な試薬によって修飾された検出用標識物が分散している分散液を移動させる。分析対象物と検出用標識物とが特異的に結合し、反応部位まで到達する。反応部位において、分析対象物と検出用標識物との複合体が固定化試薬に特異的に結合することにより、被分析液中に分析対象物が存在する場合にのみ、固定化試薬部に検出用標識物が濃縮される。これを目視または適当な機器を用いて被分析液中に被検出物が存在することを定性的に分析する手法である。
イムノクロマトグラフィーは、簡便性の点で特に優れた方法といえる。イムノクロマトグラフィーを利用した検出には、通常、テストストリップが用いられる。The principle of immunochromatography is roughly as follows. A chromatography carrier having at least one reaction site containing an immobilization reagent (antibody, antigen, etc.) that can bind to the analyte is used as the stationary phase. On this chromatographic carrier, the dispersion liquid in which the detection label modified by the reagent capable of binding to the analyte is dispersed is moved. The analyte and the detection label are specifically bound to reach the reaction site. Only when the analyte is present in the liquid to be analyzed, the complex of the analyte and the label for detection binds specifically to the immobilized reagent at the reaction site. The label is concentrated. This is a technique for qualitatively analyzing the presence of an object to be detected in a liquid to be analyzed by visual observation or using an appropriate instrument.
Immunochromatography can be said to be a particularly excellent method in terms of simplicity. For detection using immunochromatography, a test strip is usually used.
検出用抗体の標識には、従来公知の任意のものを用いることができる。なかでも、検出結果を肉眼で観察することができ、迅速かつ簡便な判定が可能になることから、呈色物質が好ましい。呈色物質としては、金属コロイド（金コロイド、白金コロイド、パラジウムコロイド）、顔料などで着色した合成ラテックス、天然ラテックスを例示することができる。 Any conventionally known label can be used for the detection antibody label. Among these, a color substance is preferable because the detection result can be observed with the naked eye and a quick and simple determination can be made. Examples of the coloring substance include metal colloid (gold colloid, platinum colloid, palladium colloid), synthetic latex colored with pigments, and natural latex.
ペプチドモノクローナル抗体の作製
配列番号１の合成ペプチド１ｍｇをＤＭＳＯ１０μｌ及びＰＢＳ１９０μｌに懸濁し、この懸濁液に卵白アルブミン（２ｍｇ／ｍｌ）２００μｌを加え、さらに２％グルタルアルデヒド８０μｌを加え、室温で６０分間反応させた。反応終了後に、１Ｍ Ｔｒｉｓ４０μｌを加えて反応を停止し、１Ｎ ＨＣｌ １０μｌを加えて中和し、免疫用ペプチド抗原を作製した。Preparation of Peptide Monoclonal Antibody 1 mg of the synthetic peptide of SEQ ID NO: 1 is suspended in DMSO 10 μl and PBS 190 μl, ovalbumin (2 mg / ml) 200 μl is added to this suspension, 2% glutaraldehyde 80 μl is added, and the reaction is performed at room temperature for 60 minutes. I let you. After completion of the reaction, 40 μl of 1M Tris was added to stop the reaction, and neutralized by adding 10 μl of 1N HCl to prepare a peptide antigen for immunization.
免疫には、マウスを用いた。得られた免疫用ペプチド抗原１５０μｌにＴｉｔｅｒ Ｍａｘ（登録商標）ｇｏｌｄ１５０μｌを加え、エマルションを調製した。このエマルションをマウス足底部に２０μｌ注射し、さらに２週間後に同量を注射した。１週間後に、免疫用ペプチド４０μｌを足底部に注射し、３日後に脾臓を摘出し、脾細胞をマウスミエローマ細胞（Ｐ３Ｕ１）と融合させた。常法にしたがって、融合細胞をクローニングし、ＥＬＩＳＡでスクリーニングし、５株のハイブリドーマを得た（ＲＮＡ−１〜５）。 Mice were used for immunization. 150 μl of Titer Max (registered trademark) gold was added to 150 μl of the obtained peptide antigen for immunization to prepare an emulsion. 20 μl of this emulsion was injected into the sole of the mouse, and the same amount was injected two weeks later. One week later, 40 μl of immunizing peptide was injected into the sole of the foot, and after 3 days, the spleen was removed and the spleen cells were fused with mouse myeloma cells (P3U1). According to a conventional method, the fused cells were cloned and screened by ELISA to obtain 5 hybridomas (RNA-1 to 5).
得られたハイブリドーマの中でＲＮＡ−３は、２０１３年８月３０日付けで独立行政法人製品評価技術基盤機構 特許微生物寄託センター（2920818 日本国千葉県木更津市かずさ鎌足２−５−８）に、受託番号ＮＩＴＥ ＢＰ−０１７００で寄託された。 Among the obtained hybridomas, RNA-3 was transferred to the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (2920818, Kazusa Kamashi 2-5-8, Kisarazu City, Chiba Prefecture, Japan) on August 30, 2013. And deposit number NITE BP-01700.
抗インフルエンザウイルスリボ核タンパク質抗体
抗インフルエンザウイルスリボ核タンパク質抗体は、市販のものを用いることができる。たとえば、イムノプローブ社のインフルエンザモノクローナル抗体 Ｎｏ．２を挙げることができる。Anti-influenza virus ribonucleoprotein antibody A commercially available anti-influenza virus ribonucleoprotein antibody can be used. For example, influenza monoclonal antibody no. 2 can be mentioned.
５株のハイブリドーマ ＲＮＡ−１〜５から得られたモノクローナル抗体ＲＮＡ１〜５（２ｍｇ／ｍｌ）１０μｌを、それぞれ、ニトロセルロースメンブラン（ミリポア）に直線状に塗布し、５０℃で３時間乾燥させた。 10 μl of monoclonal antibody RNA1-5 (2 mg / ml) obtained from 5 strains of hybridoma RNA-1-5 were each applied linearly to a nitrocellulose membrane (Millipore) and dried at 50 ° C. for 3 hours.
金コロイド（BB International、４０ｎｍ）１００μｌに、抗インフルエンザウイルスリボ核タンパク質抗体（インフルエンザモノクローナル抗体 Ｎｏ．２、４０μｇ／ｍｌ）１００μｌを混合し、室温で１０分間放置した。洗浄後、ブロックエース（ＤＳファーマバイオメディカル）で３０分間ブロッキングし、次いで洗浄し、１００μｌのＰＢＳ（ｐＨ７．２）に懸濁して、感作金コロイドを調製した。 100 μl of gold colloid (BB International, 40 nm) was mixed with 100 μl of anti-influenza virus ribonucleoprotein antibody (influenza monoclonal antibody No. 2, 40 μg / ml) and left at room temperature for 10 minutes. After washing, blocking with Block Ace (DS Pharma Biomedical) for 30 minutes, followed by washing and suspending in 100 μl PBS (pH 7.2) to prepare a sensitized gold colloid.
オセルタミビル耐性インフルエンザウイルスＡ／Ａｉｃｈｉ／１７３／０９（１０７／ｍｌ）と感作金コロイドを混合し、モノクローナル抗体塗沫ラインから約２ｃｍ離れたメンブラン上に滴下した。室温で１５分間放置した後に、モノクローナル抗体塗沫ライン上の金コロイドの発色の有無を肉眼で判定した。対照として、オセルタミビル感受性インフルエンザウイルスＡソ連型（Ｈ１Ｎ１）を用いた。Oseltamivir resistant influenza virus A / Aichi / 173/09 (10 7 / ml) was mixed with sensitized gold colloid and dropped onto a membrane about 2 cm away from the monoclonal antibody smear line. After leaving at room temperature for 15 minutes, the presence or absence of color development of colloidal gold on the monoclonal antibody smear line was judged with the naked eye. As a control, oseltamivir sensitive influenza virus A USSR type (H1N1) was used.
オセルタミビル耐性インフルエンザウイルスでは、ＲＮＡ１〜３で明瞭な発色が認められたが、ＲＮＡ４及び５では、発色が認めらなかった。一方、オセルタミビル感受性インフルエンザウイルスＡソ連型では、ＲＮＡ３及び４〜５では、発色が認めらなかったが、ＲＮＡ１及び２では、発色が認められた。結果として、ＲＮＡ３のみが、ＳＮＡ抗原（オセルタミビル感受性インフルエンザウイルスＡソ連型（Ｈ１Ｎ１））との非特異反応なしで、ＲＮＡ抗原（オセルタミビル耐性ウイルスＡ／Ａｉｃｈｉ／１７３／０９）を特異的に検出することができた。なお、供試したウイルス株は、名古屋大学医学系研究科分子病原細菌学山田景子博士から分与されたものである。ＲＮＡ１及び２は、ＲＮＡ抗原を特異的に検出することができたが、ＳＮＡ抗原との反応が認められた。ＲＮＡ４及び５は、ＲＮＡ抗原を検出することができなかった。 In the oseltamivir resistant influenza virus, clear color development was observed in RNA 1 to 3, but no color development was observed in RNA 4 and 5. On the other hand, in oseltamivir-sensitive influenza virus A Soviet type, no color development was observed in RNA 3 and 4-5, but color development was observed in RNA 1 and 2. As a result, only RNA3 can specifically detect RNA antigen (oseltamivir resistant virus A / Aichi / 173/09) without non-specific reaction with SNA antigen (oseltamivir sensitive influenza virus A solitary type (H1N1)) I was able to. The tested virus strains were distributed from Dr. Keiko Yamada, Molecular Pathogenic Bacteriology, Graduate School of Medicine, Nagoya University. RNA1 and 2 were able to specifically detect the RNA antigen, but a reaction with the SNA antigen was observed. RNAs 4 and 5 were unable to detect RNA antigens.
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| WO2011096302A1 (en) * | 2010-02-02 | 2011-08-11 | 国立大学法人名古屋大学 | Antibody specific to drug-resistant influenza virus and use of same |
| WO2012029694A1 (en) * | 2010-09-01 | 2012-03-08 | 富士レビオ株式会社 | Method for determining whether or not the amino acid in the 100th position of the influenza a virus nucleoprotein is arginine |
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| WO2011096302A1 (en) * | 2010-02-02 | 2011-08-11 | 国立大学法人名古屋大学 | Antibody specific to drug-resistant influenza virus and use of same |
| WO2012029694A1 (en) * | 2010-09-01 | 2012-03-08 | 富士レビオ株式会社 | Method for determining whether or not the amino acid in the 100th position of the influenza a virus nucleoprotein is arginine |
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