JP2016132628A - Antibody that reacts with foot-and-mouth disease virus - Google Patents
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Abstract
Description
本発明は、口蹄疫ウイルスと反応する抗体に関する。より詳細には、口蹄疫ウイルスの血清型Ａと特異的に反応する抗体に関する。 The present invention relates to antibodies that react with foot-and-mouth disease virus. More specifically, it relates to an antibody that specifically reacts with serotype A of foot-and-mouth disease virus.
口蹄疫ウイルス（foot and mouth disease virus：ＦＭＤＶ）は、家畜の伝染病の１つである。口蹄疫ウイルスは、鯨偶蹄目（例えばウシ、ブタ、シカ、ヒツジ、ヤギなど）に属する動物を主な宿主としており、日本ではその感染疾患が家畜伝染病予防法において家畜伝染病（旧法定伝染病）に指定されている。口蹄疫ウイルスは、周囲環境において容易に不活性化しないため伝播性が高く、感染した家畜の生産性を著しく低下させ、感染した幼獣において高い致死率を示す。特に高い伝播性に起因して、口蹄疫ウイルスに感染した家畜は、感染が確認され次第、日本内では家畜伝染病予防法に基づいて殺処分に処される。また、口蹄疫ウイルスに感染した家畜が発見された地域、国家には家畜の移動制限が加えられるため、口蹄疫ウイルスは、畜産業に非常な経済的打撃を与え得る病原体として、世界的に認識されている。 Foot and mouth disease virus (FMDV) is one of the infectious diseases of livestock. The foot-and-mouth disease virus is mainly composed of animals belonging to the cetacean (for example, cattle, pigs, deer, sheep, goats, etc.). ) Is specified. The foot-and-mouth disease virus is not easily inactivated in the surrounding environment and is therefore highly transmissible, significantly reducing the productivity of infected livestock and showing a high mortality rate in infected cubs. Domestic animals infected with foot-and-mouth disease virus due to their particularly high spreadability are slaughtered in Japan as soon as infection is confirmed, based on the Act on Prevention of Livestock Infectious Diseases. In addition, since movement of livestock is restricted in areas and nations where livestock infected with foot-and-mouth disease virus is found, foot-and-mouth disease virus is recognized worldwide as a pathogen that can have a very economic impact on the livestock industry. Yes.
日本では口蹄疫ウイルスに感染した個体は、２０００年までおよそ１世紀にわたって確認されていなかった。しかし、２０００年および２０１０年に、口蹄疫ウイルスに感染した個体が確認された。このため、口蹄疫の流行に対する対処法を確立することの重要性が明らかに高まっている。上記対処法として最も重要なのは、感染の疑いのある家畜が、口蹄疫ウイルスに感染しているか否かを決定することである。 In Japan, individuals infected with foot-and-mouth disease virus have not been confirmed until 2000 for about a century. However, in 2000 and 2010, individuals infected with foot-and-mouth disease virus were identified. For this reason, the importance of establishing countermeasures against foot-and-mouth disease epidemics is clearly increasing. The most important countermeasure is to determine whether livestock suspected of being infected is infected with the foot-and-mouth disease virus.
一般的な決定方法としては、ＲＴ−ＰＣＲによって口蹄疫ウイルスの遺伝子を検出する方法である。この方法では、遺伝子の増幅が認められれば、陽性（口蹄疫ウイルスに感染している）と決定されるが、迅速かつ正確に決定するという点では、単独にＲＴ−ＰＣＲを利用する方法は好ましくない。そこで、他の抗原検出法（ＥＬＩＳＡまたはイムノクロマトグラフィーなど）を利用（併用）して口蹄疫ウイルスの検出する方法が検討されている（例えば、本発明者らによる非特許文献１）。 As a general determination method, a gene for foot-and-mouth disease virus is detected by RT-PCR. In this method, if gene amplification is observed, it is determined to be positive (infected with a foot-and-mouth disease virus). However, a method using RT-PCR alone is not preferable in terms of quick and accurate determination. . Therefore, methods for detecting foot-and-mouth disease virus using other antigen detection methods (such as ELISA or immunochromatography) (in combination) have been studied (for example, Non-Patent Document 1 by the present inventors).
口蹄疫ウイルスは、７つの血清型（Ｏ、Ａ、Ｃ、Ａｓｉａ１、ＳＡＴ１、ＳＡＴ２およびＳＡＴ３）に分類される。非特許文献１には、血清型Ａと特異的に反応する抗体が記載されている。 Foot-and-mouth disease viruses are classified into seven serotypes (O, A, C, Asia1, SAT1, SAT2, and SAT3). Non-Patent Document 1 describes an antibody that specifically reacts with serotype A.
本発明者らが調べたところ、非特許文献１に記載の血清型Ａと特異的に反応する抗体は、血清型Ａのウイルス株のいくつかに対する検出感度が非常に低いことが判明した。そのため、口蹄疫ウイルスの血清型Ａと反応する新たな抗口蹄疫ウイルス抗体の開発が望まれる。 When the present inventors investigated, the antibody which reacts specifically with the serotype A of the nonpatent literature 1 became clear [the detection sensitivity with respect to some of the serotype A virus strains] very low. Therefore, development of a new anti-foot-and-mouth disease virus antibody that reacts with serotype A of foot-and-mouth disease virus is desired.
本発明者らは、上記目的を達成するため鋭意研究を重ねた結果、口蹄疫ウイルスの血清型Ａのいくつかの株を特異的に認識することができる抗口蹄疫ウイルス抗体を産生するハイブリドーマの作製に成功し、本発明を完成するに至った。 As a result of intensive studies to achieve the above-mentioned object, the present inventors have produced a hybridoma that produces an anti-foot-and-mouth disease virus antibody that can specifically recognize several strains of serotype A of foot-and-mouth disease virus. The present invention has been completed successfully.
すなわち、本発明は、以下の（１）〜（１０）を提供する。
（１）口蹄疫ウイルスの血清型ＡのＡ／ＩＲＮ／１／２０１１株と反応し、口蹄疫ウイルスの血清型Ｏ、Ｃ、Ａｓｉａ１、ＳＡＴ１、ＳＡＴ２およびＳＡＴ３の何れとも反応しない、抗体。
（２）受託番号NITE P−01971で特定されるハイブリドーマによって産生される、上記（１）に記載の抗体。
（３）受託番号NITE P−01971で特定される抗体産生用ハイブリドーマ。
（４）口蹄疫ウイルスの血清型Ａと特異的に反応する、上記（１）または（２）に記載の抗体とは別の抗口蹄疫ウイルス抗体と、上記（１）または（２）に記載の抗体とを含む、組成物。
（５）上記（１）または（２）に記載の抗体を含む口蹄疫ウイルス検出キット。
（６）口蹄疫ウイルスの血清型Ａと特異的に反応する、上記（１）または（２）に記載の抗体とは別の抗口蹄疫ウイルス抗体をさらに含む、上記（５）に記載の口蹄疫ウイルス検出キット。
（７）口蹄疫ウイルスの血清型Ｏ、Ａ、Ｃ、Ａｓｉａ１、ＳＡＴ１、ＳＡＴ２およびＳＡＴ３の何れとも反応する抗口蹄疫ウイルス抗体をさらに含む、上記（５）または（６）に記載の口蹄疫ウイルス検出キット。
（８）上記（１）または（２）に記載の抗体を用いる口蹄疫ウイルスの検出方法。
（９）被験体から採取した試料と、上記（１）または（２）に記載の抗体とを接触させて、当該試料中の抗原と、当該抗体とを抗原抗体反応させる工程；および抗原抗体反応物を検出する工程を含む、上記（８）に記載の検出方法。
（１０）さらに、上記試料と、口蹄疫ウイルスの血清型Ａと特異的に反応する、上記（１）または（２）に記載の抗体とは別の抗口蹄疫ウイルス抗体とを接触させる、上記（９）に記載の検出方法。
That is, the present invention provides the following (1) to (10).
(1) An antibody that reacts with A / IRN / 1/2011 strain of serotype A of foot-and-mouth disease virus and does not react with any of serotypes O, C, Asia1, SAT1, SAT2, and SAT3 of foot-and-mouth disease virus.
(2) The antibody according to (1) above, which is produced by a hybridoma specified by accession number NITE P-01971.
(3) A hybridoma for antibody production identified by the accession number NITE P-01971.
(4) An anti-foot-and-mouth disease virus antibody different from the antibody according to (1) or (2), which specifically reacts with serotype A of the foot-and-mouth disease virus, and the antibody according to (1) or (2) And a composition comprising:
(5) A foot-and-mouth disease virus detection kit comprising the antibody according to (1) or (2) above.
(6) The foot-and-mouth disease virus detection according to (5), further comprising an anti-foot-and-mouth disease virus antibody different from the antibody according to (1) or (2) that specifically reacts with serotype A of the foot-and-mouth disease virus kit.
(7) The foot-and-mouth disease virus detection kit according to (5) or (6), further including an anti-foot-and-mouth disease virus antibody that reacts with any of serotypes O, A, C, Asia1, SAT1, SAT2, and SAT3 of foot-and-mouth disease virus.
(8) A method for detecting foot-and-mouth disease virus using the antibody according to (1) or (2) above.
(9) a step of bringing a sample collected from a subject into contact with the antibody described in (1) or (2) above, and causing an antigen-antibody reaction between the antigen in the sample and the antibody; and an antigen-antibody reaction The detection method according to (8) above, comprising a step of detecting an object.
(10) The above sample is further contacted with an anti-foot-and-mouth disease virus antibody different from the antibody according to (1) or (2), which specifically reacts with serotype A of the foot-and-mouth disease virus. ) Detection method.
本発明に係る抗体は、口蹄疫ウイルスの血清型ＡのＡ／ＩＲＮ／１／２０１１株を高感度で検出することができる。 The antibody according to the present invention can detect the A / IRN / 1/2011 strain of serotype A of foot-and-mouth disease virus with high sensitivity.
〔１．抗体〕
本発明に係る抗体は、口蹄疫ウイルスの血清型ＡのＡ／ＩＲＮ／１／２０１１株（文献：http://www.wrlfmd.org/fmd_genotyping/2011/WRLFMD-2011 00010%20A%20Iran%202011.pdf）と反応し、口蹄疫ウイルスの血清型Ｏ、Ｃ、Ａｓｉａ１、ＳＡＴ１、ＳＡＴ２およびＳＡＴ３の何れとも反応しない。なお、「反応する」とは、抗体が口蹄疫ウイルス（抗原）と抗原抗体反応することをいい、「結合する」または「認識する」と表現することもできる。
[1. antibody〕
The antibody according to the present invention is A / IRN / 1/20101 strain of foot-and-mouth disease virus serotype A (Reference: http://www.wrlfmd.org/fmd_genotyping/2011/WRLFMD-2011 00010% 20A% 20Iran% 202011. pdf) and does not react with any of the foot-and-mouth disease virus serotypes O, C, Asia1, SAT1, SAT2 and SAT3. “React” means that an antibody reacts with a foot-and-mouth disease virus (antigen) by an antigen-antibody reaction, and can also be expressed as “bind” or “recognize”.
本発明に係る抗体は、２価の抗体であってもよいし、１価の抗体であってもよい。また、本発明に係る抗体は、モノクローナル抗体であってもよいし、ポリクローナル抗体であってもよい。交差反応性を示すおそれの少なさという観点から、本発明に係る抗体はモノクローナル抗体であることが好ましい。 The antibody according to the present invention may be a divalent antibody or a monovalent antibody. Further, the antibody according to the present invention may be a monoclonal antibody or a polyclonal antibody. From the viewpoint of low risk of cross-reactivity, the antibody according to the present invention is preferably a monoclonal antibody.
本発明に係る抗体の一態様は、受託番号NITE P−01971で特定されるハイブリドーマによって産生されるモノクローナル抗体（「２Ａ１」と名付けた）である。２Ａ１抗体は、口蹄疫ウイルスの血清型ＡのＡ２２／ＩＲＱ／２４／６４株（文献：ARROWSMITH, A. E. M. (1975). Variation among strains of type A foot-and-mouth disease virus in the Eastern Mediterranean region 1964-1972. Journal of Hygiene 75, 387-397）およびＡ／ＩＲＮ／１／２０１１株を強く認識することができ、血清型Ａ以外の血清型とは反応しない。２Ａ１抗体は、受託番号NITE P−01971で特定されるハイブリドーマを、例えば、１０％胎児血清を含む培地中でまたは無血清培地中で、３７℃、５％ ＣＯ２の条件で培養し、産生された２Ａ１抗体を公知の方法に従って回収・精製することによって、製造することができる。 One embodiment of the antibody according to the present invention is a monoclonal antibody (named “2A1”) produced by a hybridoma identified by accession number NITE P-01971. The 2A1 antibody is A22 / IRQ / 24/64 strain of serotype A of foot-and-mouth disease virus (Reference: ARROWSMITH, AEM (1975). Variation among strains of type A foot-and-mouth disease virus in the Eastern Mediterranean region 1964-1972 Journal of Hygiene 75, 387-397) and A / IRN / 1/2011 strains can be strongly recognized and does not react with serotypes other than serotype A. The 2A1 antibody is produced by culturing the hybridoma specified by the accession number NITE P-01971, for example, in a medium containing 10% fetal serum or in a serum-free medium under conditions of 37 ° C. and 5% CO 2. In addition, the 2A1 antibody can be produced by recovery and purification according to a known method.
〔２．ハイブリドーマ〕
本発明はまた、受託番号NITE P−01971で特定される抗体産生用ハイブリドーマも提供する。本発明に係るハイブリドーマは、ウシから分離された口蹄疫ウイルスＡ／ＩＲＮ／１／２０１１株を抗原として、マウスに免疫し、免疫したマウスの脾臓細胞と、マウスのミエローマ細胞由来株とを融合させて作成されたハイブリドーマ（抗口蹄疫ウイルス血清型Ａ ｈｙｂｒｉｄｏｍａ ２Ａ１（受託番号NITE P−01971））である。ハイブリドーマ２Ａ１は、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（千葉県木更津市かずさ鎌足２丁目５番地８ １２２号室）に寄託されている。
[2. Hybridoma)
The present invention also provides a hybridoma for producing an antibody identified by accession number NITE P-01971. The hybridoma according to the present invention immunizes mice using the foot-and-mouth disease virus A / IRN / 1/2011 strain isolated from cattle as an antigen, and fuses the spleen cells of the immunized mice with the mouse myeloma cell-derived strain. It is a prepared hybridoma (anti-foot-and-mouth disease virus serotype A hybridoma 2A1 (Accession Number NITE P-01971)). The hybridoma 2A1 is deposited with the Patent Microorganism Depositary Center of the National Institute for Product Evaluation and Technology (Room No. 1222, Kazusa-Kamashita, 5-chome, Kisarazu-shi, Chiba).
〔３．組成物〕
本発明はさらに、口蹄疫ウイルスの血清型Ａと特異的に反応する（すなわち、他の血清型と反応しない）、本発明に係るに記載の抗体とは別の抗口蹄疫ウイルス抗体と、本発明に係る抗体とを含む、組成物を提供する。口蹄疫ウイルスの血清型Ａと特異的に反応する、本発明に係る抗体とは別の抗口蹄疫ウイルス抗体は、モノクローナル抗体であり得る。また、そのような別の抗口蹄疫ウイルス抗体は、Ａ／ＩＲＮ／１／２０１１株と反応しない抗体であり得る。Ａ／ＩＲＮ／１／２０１１株と反応しない抗体と本発明に係る抗体とを組み合わせることにより、網羅的に血清型Ａを検出することができる。また、そのような別の抗口蹄疫ウイルス抗体は、Ａ／ＴＡＩ／１０／２０１１株と反応する抗体であり得る。Ａ／ＴＡＩ／１０／２０１１株と反応する抗体と本発明に係る抗体とを組み合わせることにより、より網羅的に血清型Ａを検出することができる。
[3. Composition〕
The present invention further includes an anti-foot-and-mouth disease virus antibody that specifically reacts with serotype A of foot-and-mouth disease virus (that is, does not react with other serotypes) and is different from the antibody according to the present invention, and Compositions comprising such antibodies are provided. The anti-foot-and-mouth disease virus antibody different from the antibody according to the present invention that specifically reacts with serotype A of foot-and-mouth disease virus may be a monoclonal antibody. Further, such another anti-foot-and-mouth disease virus antibody may be an antibody that does not react with the A / IRN / 1/2011 strain. Serotype A can be comprehensively detected by combining an antibody that does not react with the A / IRN / 1/2011 strain and the antibody according to the present invention. In addition, such another anti-foot-and-mouth disease virus antibody may be an antibody that reacts with the A / TAI / 10/2011 strain. Serotype A can be detected more comprehensively by combining an antibody that reacts with the A / TAI / 10/2011 strain and the antibody according to the present invention.
そのような別の抗口蹄疫ウイルス抗体としては、例えば、非特許文献１に記載された１６Ｃ６抗体等が挙げられる。１６Ｃ６抗体は、血清型Ａに分類されるＡ／ＴＡＩ／１０／２０１１株およびＡ１５／ＴＡＩ／１／６０株を強く認識することができる一方で、Ａ／ＩＲＮ／１／２０１１株を認識することができない（後述の実施例を参照）。そのため、本発明に係る抗体と１６Ｃ６抗体とを組み合わせることによって、より網羅的に血清型Ａを検出することができる。 Examples of such another anti-foot-and-mouth disease virus antibody include the 16C6 antibody described in Non-Patent Document 1. The 16C6 antibody can strongly recognize A / TAI / 10/2011 and A15 / TAI / 1/60 strains classified as serotype A, while recognizing A / IRN / 1/2011 strain Cannot be performed (see the examples described later). Therefore, serotype A can be detected more comprehensively by combining the antibody according to the present invention and the 16C6 antibody.
本発明に係る組成物における本発明に係る抗体と本発明に係る抗体とは別の抗口蹄疫ウイルス抗体との比は、特に限定されないが、例えば、１：１とすることができる。 The ratio of the antibody according to the present invention and the anti-foot-and-mouth disease virus antibody different from the antibody according to the present invention in the composition according to the present invention is not particularly limited, but can be, for example, 1: 1.
〔４．口蹄疫ウイルス検出キット〕
本発明はさらに、本発明に係る抗体を含む口蹄疫ウイルス検出キットを提供する。本発明に係る口蹄疫ウイルス検出キットは、例えば、ＥＬＩＳＡ法、イムノクロマトグラフィー法、または免疫拡散測定法等を利用したキットであり得る。
[4. Foot-and-mouth disease virus detection kit]
The present invention further provides a foot-and-mouth disease virus detection kit comprising the antibody according to the present invention. The foot-and-mouth disease virus detection kit which concerns on this invention can be a kit using ELISA method, an immunochromatography method, or an immunodiffusion measuring method etc., for example.
本発明に係る口蹄疫ウイルス検出キットは、口蹄疫ウイルスの血清型Ａと特異的に反応する、本発明に係る抗体とは別の抗口蹄疫ウイルス抗体をさらに含んでいてもよい。そのような抗口蹄疫ウイルス抗体は、モノクローナル抗体であり得る。そのような抗口蹄疫ウイルス抗体については、上記〔３．組成物〕で説明したとおりである。 The foot-and-mouth disease virus detection kit according to the present invention may further include an anti-foot-and-mouth disease virus antibody different from the antibody according to the present invention that specifically reacts with serotype A of the foot-and-mouth disease virus. Such anti-foot-and-mouth disease virus antibodies can be monoclonal antibodies. Such anti-foot-and-mouth disease virus antibodies are described in [3. As described in [Composition].
また、本発明に係る口蹄疫ウイルス検出キットは、口蹄疫ウイルスの血清型Ｏ、Ａ、Ｃ、Ａｓｉａ１、ＳＡＴ１、ＳＡＴ２およびＳＡＴ３の何れとも反応する抗口蹄疫ウイルス抗体を含んでいてもよい。そのような抗口蹄疫ウイルス抗体は、モノクローナル抗体であり得る。そのような抗口蹄疫ウイルス抗体としては、例えば、非特許文献１に記載された１Ｈ５抗体、および後述の実施例に記載されている１６Ｄ６抗体等が挙げられる。本発明に係る抗体と７つ全ての血清型と反応する抗口蹄疫ウイルス抗体とを組み合わせる場合、ＥＬＩＳＡにおいては、７つ全ての血清型と反応する抗口蹄疫ウイルス抗体を捕捉抗体として用いることによって、血清型毎に抗体７種類を用意する必要がない。また、エピトープが競合しないという利点もある。また、同一血清型間でも抗原性が異なる、すなわちサブタイプあるいはトポタイプがヘテロの場合、保存性の高い領域を認識している７つ全ての血清型と反応する抗口蹄疫ウイルス抗体を用いることによって、検出漏れを低減することができる。 Further, the foot-and-mouth disease virus detection kit according to the present invention may contain an anti-foot-and-mouth disease virus antibody that reacts with any of serotypes O, A, C, Asia1, SAT1, SAT2, and SAT3 of foot-and-mouth disease virus. Such anti-foot-and-mouth disease virus antibodies can be monoclonal antibodies. Examples of such anti-foot-and-mouth disease virus antibodies include 1H5 antibody described in Non-Patent Document 1, and 16D6 antibody described in Examples described later. When combining the antibody according to the present invention with anti-foot-and-mouth disease virus antibodies that react with all seven serotypes, the ELISA uses anti-foot-and-mouth disease virus antibodies that react with all seven serotypes as a capture antibody. There is no need to prepare seven types of antibodies for each type. There is also an advantage that epitopes do not compete. In addition, by using anti-foot-and-mouth disease virus antibodies that react with all seven serotypes that recognize highly conserved regions when antigenicity is different even between the same serotypes, that is, when the subtype or topotype is heterozygous, Detection omission can be reduced.
また、本発明に係る口蹄疫ウイルス検出キットは、口蹄疫ウイルスの血清型Ｏ、Ｃ、Ａｓｉａ１、ＳＡＴ１、ＳＡＴ２およびＳＡＴ３の何れかと特異的に反応する抗口蹄疫ウイルス抗体を含んでいてもよい。そのような抗口蹄疫ウイルス抗体は、モノクローナル抗体であり得る。そのような抗口蹄疫ウイルス抗体としては、例えば、非特許文献１に記載された７０Ｃ４抗体（血清型Ｏ特異的）、および６５Ｈ６抗体（血清型Ｏ特異的）、ならびに、特開２０１３−４９６４５号に記載された１２Ｃ７抗体（血清型Ａｓｉａ１特異的）、および１３Ｆ１抗体（血清型Ｃ特異的）等が挙げられる。これらの抗体と反応性を比較することで、一度の検査で、被験体の検体中に含まれる口蹄疫ウイルスの血清型を判別することができる。 In addition, the foot-and-mouth disease virus detection kit according to the present invention may include an anti-foot-and-mouth disease virus antibody that specifically reacts with any of serotypes O, C, Asia1, SAT1, SAT2, and SAT3 of foot-and-mouth disease virus. Such anti-foot-and-mouth disease virus antibodies can be monoclonal antibodies. Examples of such anti-foot-and-mouth disease virus antibodies include, for example, the 70C4 antibody (serotype O specific) and 65H6 antibody (serotype O specific) described in Non-Patent Document 1, and JP2013-49645A Examples include 12C7 antibody (serotype Asia1-specific) and 13F1 antibody (serotype C-specific) described. By comparing the reactivity with these antibodies, the serotype of the foot-and-mouth disease virus contained in the specimen of the subject can be determined by a single test.
また、本発明に係る口蹄疫ウイルス検出キットは、口蹄疫ウイルスの血清型Ｏ、Ａ、Ｃ、Ａｓｉａ１、ＳＡＴ１、ＳＡＴ２およびＳＡＴ３の何れか２つ以上と特異的に反応する抗口蹄疫ウイルス抗体を含んでいてもよい。 In addition, the foot-and-mouth disease virus detection kit according to the present invention includes an anti-foot-and-mouth disease virus antibody that specifically reacts with any two or more of serotypes O, A, C, Asia1, SAT1, SAT2, and SAT3 of foot-and-mouth disease virus. Also good.
上述した本発明に係る抗体以外の抗体のうちの複数が、本発明に係る口蹄疫ウイルス検出キットに含まれていてもよい。また、本発明に係る抗体と本発明に係る抗体以外の抗体とは、混合されて使用されてもよいし、混合されずに使用されてもよい。混合して使用した場合であっても、阻害されずに、相補的に複数の血清型または株を網羅的に検出することができる。 A plurality of antibodies other than the antibodies according to the present invention described above may be included in the foot-and-mouth disease virus detection kit according to the present invention. Further, the antibody according to the present invention and the antibody other than the antibody according to the present invention may be used in a mixed manner or may be used without being mixed. Even when mixed and used, a plurality of serotypes or strains can be comprehensively detected without being inhibited.
また、本発明に係る抗体および／または上述の本発明に係る抗体以外の抗体は、標識に結合されていてもよい。標識としては、例えば、酵素、酵素基質、放射性同位元素、発光物質、蛍光物質、ビオチンおよび着色物質等が挙げられる。酵素の例としては、ペルオキシダーゼ、β−ガラクトシダーゼ、アルカリホスファターゼ、グルコースオキシダーゼ、アセチルコリンエステラーゼおよびグルコース−６−リン酸脱水素酵素等が挙げられる。これら酵素と抗体との結合は、マレイミド化合物およびＮ−ヒドロキシスクシンイミドエステル化合物等の架橋剤を用いる公知の方法により行うことができる。酵素基質としては、使用する酵素に応じて公知の物質を使用することができる。例えば、酵素としてペルオキシダーゼを使用する場合には、ＯＰＤ（オルトフェニレンジアミン）およびＴＭＢ（テトラメチルベンジジン）等を、また酵素としてアルカリホスファターゼを用いる場合には、ニトロブルーテトラゾリウム（ＮＢＴ）および５−ブロモ−４−クロロ−３−インドリルホスファターゼｐ−トルイジニル塩（ＢＣＩＰ）の混合基質等を用いることができる。 Further, the antibody according to the present invention and / or the antibody other than the antibody according to the present invention described above may be bound to a label. Examples of the label include enzymes, enzyme substrates, radioisotopes, luminescent substances, fluorescent substances, biotin, and colored substances. Examples of the enzyme include peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase and glucose-6-phosphate dehydrogenase. These enzymes and antibodies can be bound by a known method using a crosslinking agent such as a maleimide compound and an N-hydroxysuccinimide ester compound. As the enzyme substrate, a known substance can be used according to the enzyme to be used. For example, when peroxidase is used as an enzyme, OPD (orthophenylenediamine) and TMB (tetramethylbenzidine) are used. When alkaline phosphatase is used as an enzyme, nitroblue tetrazolium (NBT) and 5-bromo- A mixed substrate of 4-chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) or the like can be used.
放射性同位元素としては、１２５Ｉ、３Ｈまたは１４Ｃ等の通常のラジオイムノアッセイで用いられているものが挙げられる。抗体への放射標識は公知の方法を用いて行うことができる。蛍光色素としては、フルオレセインイソチオシアネート、テトラメチルローダミンイソチオシアネートおよびフィコエリスリン等の通常の蛍光抗体法に用いられるものが挙げられる。また、発光物質としては、イソルミノール、アクリジンエステルおよびルシゲニン等が挙げられる。この際、標識の方法は、公知の方法を用いることができる。また、着色物質としては、例えば、着色ラテックス粒子および金コロイド等が挙げられる。 Examples of the radioisotope include those used in usual radioimmunoassay such as 125 I, 3 H or 14 C. Radiolabeling of the antibody can be performed using a known method. Examples of the fluorescent dye include those used in usual fluorescent antibody methods such as fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate and phycoerythrin. Examples of the luminescent substance include isoluminol, acridine ester and lucigenin. At this time, a known method can be used as the labeling method. Examples of the coloring substance include colored latex particles and gold colloid.
また、本発明に係る抗体および／または上述の本発明に係る抗体以外の抗体は、基材に固定されていてもよい。この場合、基材も口蹄疫ウイルス検出キットの構成要素である。基材としては、ニトロセルロースメンブレン、シリカメンブレン、ガラスフィルター、ポリスチレン粒子、ポリスチレンプレート、またはシリカ磁性粒子等の公知のものを使用することができる。一例において、本発明に係る口蹄疫ウイルス検出キットは、基材に固定された本発明に係る抗体および標識された本発明に係る抗体以外の抗体を含んでいる。他の一例において、本発明に係る口蹄疫ウイルス検出キットは、基材に固定された本発明に係る抗体以外の抗体および標識された本発明に係る抗体を含んでいる。 In addition, the antibody according to the present invention and / or the antibody other than the antibody according to the present invention may be immobilized on a base material. In this case, the base material is also a component of the foot-and-mouth disease virus detection kit. As a base material, well-known things, such as a nitrocellulose membrane, a silica membrane, a glass filter, a polystyrene particle, a polystyrene plate, or a silica magnetic particle, can be used. In one example, the foot-and-mouth disease virus detection kit according to the present invention includes an antibody other than the antibody according to the present invention and the labeled antibody according to the present invention immobilized on a base material. In another example, a foot-and-mouth disease virus detection kit according to the present invention includes an antibody other than the antibody according to the present invention immobilized on a substrate and a labeled antibody according to the present invention.
本発明に係る口蹄疫ウイルス検出キットは、標識を検出するための試薬をさらに含んでいてもよい。また、本発明に係る口蹄疫ウイルス検出キットは、ＥＬＩＳＡ法、イムノクロマトグラフィー法、およびウエスタンブロット法等の免疫反応を行うために必要な部材（二次抗体、発色試薬、ブロッキング試薬等）、プレート（９６ウェルプレート等）およびチューブ等が含まれていてもよい。また、本発明に係る抗体を検出するための二次抗体および二次抗体に結合させた標識酵素の基質等を備えていてもよい。 The foot-and-mouth disease virus detection kit which concerns on this invention may further contain the reagent for detecting a label | marker. In addition, the foot-and-mouth disease virus detection kit according to the present invention is a member (secondary antibody, coloring reagent, blocking reagent, etc.) necessary for performing an immune reaction such as ELISA, immunochromatography, Western blotting, etc., plate (96 Well plate, etc.) and tubes may be included. Further, a secondary antibody for detecting the antibody according to the present invention, a label enzyme substrate bound to the secondary antibody, and the like may be provided.
また、本発明に係る検出キットを構成する成分を格納するための１つ以上の容器（例えば、バイアル、管、アンプルおよびビンなど）を備えていてもよい。また、本発明に係る検出キットにおいて使用する検出方法について詳細が記載された使用説明書をさらに備えていてもよい。当該検出方法としては、後述する〔５．口蹄疫ウイルスの検出方法〕で説明される検出方法が挙げられる。 Moreover, you may provide the 1 or more containers (for example, vial, tube, ampoule, bottle, etc.) for storing the component which comprises the detection kit which concerns on this invention. Moreover, you may further provide the use manual in which the detail was described about the detection method used in the detection kit which concerns on this invention. The detection method will be described later [5. The detection method demonstrated by the detection method of foot-and-mouth disease virus] is mentioned.
〔５．口蹄疫ウイルスの検出方法〕
本発明はさらに、本発明に係る抗体を用いる口蹄疫ウイルスの検出方法を提供する。被験体としては、ウシ、ブタ、シカ、ヒツジ、ヤギ、スイギュウ、イノシシ、およびカモシカ等が挙げられる。被験体から採取した試料としては、血液、水疱液、糞、尿、咽頭ぬぐい液、鼻腔ぬぐい液、鼻腔吸引液、水疱上皮乳剤、病変部拭い液、種々の組織・器官などが挙げられる。本発明に係る検出方法を用いて、試料中の口蹄疫ウイルスを検出することで、この試料が由来する被験体が口蹄疫ウイルスによる感染症に罹患しているか否かを迅速、容易かつ確実に診断できる。
[5. (Food and foot disease virus detection method)
The present invention further provides a method for detecting foot-and-mouth disease virus using the antibody according to the present invention. Examples of subjects include cattle, pigs, deer, sheep, goats, buffalos, wild boars, and antelopes. Samples collected from a subject include blood, blister fluid, feces, urine, throat swab, nasal swab, nasal aspirate, blister epithelium emulsion, lesion wipes, various tissues and organs, and the like. By detecting the foot-and-mouth disease virus in the sample using the detection method according to the present invention, it is possible to quickly and easily and reliably diagnose whether or not the subject from which the sample is derived is infected with the foot-and-mouth disease virus. .
本発明に係る検出方法は、例えば、ＥＬＩＳＡ法（例えば、サンドイッチＥＬＩＳＡ法等）、イムノクロマトグラフィー法、または免疫拡散測定法等に基づくことができる。 The detection method according to the present invention can be based on, for example, an ELISA method (for example, a sandwich ELISA method), an immunochromatography method, an immunodiffusion measurement method, or the like.
本発明に係る検出方法の一態様は、被験体から採取した試料と、本発明の抗体とを接触させて、当該試料中の抗原と、当該抗体とを抗原抗体反応させる工程；および抗原抗体反応物を検出する工程を含む。 One aspect of the detection method according to the present invention is a step of bringing a sample collected from a subject into contact with the antibody of the present invention, and causing an antigen-antibody reaction between the antigen in the sample and the antibody; and an antigen-antibody reaction Detecting an object.
抗原抗体反応させる工程では、試料と抗体とをインキュベートすればよい。抗原抗体反応物を検出する工程は、例えば、予め標識された上記抗体を検出する等の公知の方法を用いて行うことができる。 In the antigen-antibody reaction step, the sample and the antibody may be incubated. The step of detecting the antigen-antibody reaction product can be performed using a known method such as detecting the antibody labeled in advance.
本発明に係る検出方法の一態様では、さらに、試料と、口蹄疫ウイルスの血清型Ａと特異的に反応する、本発明の抗体とは別の抗口蹄疫ウイルス抗体とを接触させてもよい。そのような抗口蹄疫ウイルス抗体については、上記〔３．組成物〕で説明したとおりである。本発明に係る抗体と別の抗口蹄疫ウイルス抗体とは、混合されて使用されてもよいし、混合されずに使用されてもよい。混合して使用した場合であっても、阻害されずに、口蹄疫ウイルスの血清型Ａを網羅的に検出することができる。 In one embodiment of the detection method according to the present invention, the sample may be further contacted with an anti-foot-and-mouth disease virus antibody different from the antibody of the present invention that specifically reacts with serotype A of the foot-and-mouth disease virus. Such anti-foot-and-mouth disease virus antibodies are described in [3. As described in [Composition]. The antibody according to the present invention and another anti-foot-and-mouth disease virus antibody may be used as mixed or may be used without being mixed. Even when mixed and used, serotype A of foot-and-mouth disease virus can be comprehensively detected without being inhibited.
また、本発明に係る検出方法は、上記〔４．口蹄疫ウイルス検出キット〕に記載された、血清型Ａ以外の血清型と反応する抗口蹄疫ウイルス抗体と、試料とを接触させる工程を含んでいてもよい。 The detection method according to the present invention is the above [4. The step of contacting the sample with an anti-foot-and-mouth disease virus antibody that reacts with a serotype other than serotype A described in the foot-and-mouth disease virus detection kit] may be included.
以下に実施例を示し、本発明の実施の形態についてさらに詳しく説明する。もちろん、本発明は以下の実施例に限定されるものではなく、細部については様々な態様が可能であることはいうまでもない。さらに、本発明は上述した実施形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、それぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された文献の全てが参考として援用される。 Examples will be shown below, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention. Moreover, all the literatures described in this specification are used as reference.
〔モノクローナル抗体の作製〕
口蹄疫ウイルスＡ／ＩＲＮ／１／２０１１株をＩＢ−ＲＳ−２細胞に接種し、一晩回転培養した。回収した培養液を１５００Ｇで１０分間遠心した後、上清を回収した。上清を飽和硫酸アンモニウム溶液と等量混和し、一晩４℃で撹拌することによってウイルスを析出させた。析出させたウイルスを、５０００Ｇで３０分間遠沈し、上清を除去してから適量のＰＢＳに懸濁した。再び５０００Ｇで３０分間遠心し、上清を除去し、１ｍＬのＰＢＳに懸濁した。スクロース密度勾配（１５〜４５％）ウイルスの懸濁液を添加し、２００００ｒｐｍで２時間遠心を行い、目的のバンドを回収することによって、１４６Ｓの完全粒子を精製した。
[Production of monoclonal antibodies]
Foot-and-mouth disease virus A / IRN / 1/2011 strain was inoculated into IB-RS-2 cells and rotated overnight. The collected culture was centrifuged at 1500 G for 10 minutes, and the supernatant was collected. The supernatant was mixed with an equal volume of saturated ammonium sulfate solution and stirred overnight at 4 ° C. to precipitate the virus. The precipitated virus was spun down at 5000 G for 30 minutes, and the supernatant was removed and suspended in an appropriate amount of PBS. Again, centrifugation was performed at 5000 G for 30 minutes, and the supernatant was removed and suspended in 1 mL of PBS. A sucrose density gradient (15-45%) virus suspension was added, centrifuged at 20000 rpm for 2 hours, and the desired band was collected to purify 146S complete particles.
８〜１２週齢のメスのＢＡＬＢ／ｃマウスの腹腔内に、上述のように精製した口蹄疫ウイルスＡ／ＩＲＮ／１／２０１１株をそれぞれ接種した。初回免疫ではフロイントコンプリートアジュバント（ヤトロン社製）と精製したウイルス液との等量混合物、追加接種ではフロイントインコンプリートアジュバント（ヤトロン社製）と精製したウイルス液との等量混合物を、連結針を用いてミセル化させた。最終免疫後にマウスの脾臓から回収したリンパ球とマウスミエローマ細胞Ｐ３Ｕ１とをポリエチレングリコール４０００（メルク社製）を用いて融合させ、ＨＡＴ培地（０．１ｍＭのヒポキサンチン、０．４μＭのアミノプテリン、１６μＭのチミジン、２０％ＦＣＳ、および１０％ＢＭ Ｃｏｎｄｉｍｅｄ Ｈ１を含有するＲＰＭＩ−１６４０培地（ニッスイ））を用いて９６ウェルプレートにおいて培養した。２週間後からはＨＴ選択培地（０．１ｍＭのヒポキサンチン、１６μＭのチミジン、および２０％ＦＣＳを含有するＲＰＭＩ−１６４０培地（ニッスイ））を用いて培養した。 8 to 12-week-old female BALB / c mice were each inoculated with the foot-and-mouth disease virus A / IRN / 20201 strain purified as described above. For the first immunization, use an equivalent mixture of Freund's complete adjuvant (manufactured by Yatron) and purified virus solution, and for additional inoculation, use an equivalent mixture of Freund's complete adjuvant (manufactured by Yatron) and purified virus solution. To micelles. Lymphocytes recovered from the spleen of the mouse after the final immunization and mouse myeloma cell P3U1 were fused using polyethylene glycol 4000 (manufactured by Merck), and HAT medium (0.1 mM hypoxanthine, 0.4 μM aminopterin, 16 μM). Of RPMI-1640 medium (Nissui) containing thymidine, 20% FCS, and 10% BM Condimed H1. After 2 weeks, the cells were cultured using an HT selection medium (RPMI-1640 medium (Nissui) containing 0.1 mM hypoxanthine, 16 μM thymidine, and 20% FCS).
上述のように培養することによって、ハイブリドーマのコロニーを形成させ、ＥＬＩＳＡ、ウイルス中和試験および口蹄疫ウイルス感染細胞を用いた免疫染色によって目的のモノクローナル抗体を産生しているハイブリドーマをスクリーニングした。スクリーニングの詳細は以下の通りである。 By culturing as described above, colonies of hybridomas were formed, and hybridomas producing the desired monoclonal antibodies were screened by ELISA, virus neutralization test, and immunostaining using foot-and-mouth disease virus-infected cells. Details of the screening are as follows.
ＥＬＩＳＡでは、ウサギ抗口蹄疫ウイルス抗体を、固相に吸着させ、Ａ／ＩＲＮ／１／２０１１株ウイルスと反応させた。次いで、ウイルスを取り除いてからハイブリドーマの培養上清を加えて反応させた。そして、培養上清を取り除いてからＨＲＰ標識したマウス抗ＩｇＧ抗体を加えて反応させた。標識抗体を捨ててから発色基質と反応させて、吸光度を測定した。 In ELISA, a rabbit anti-foot-and-mouth disease virus antibody was adsorbed to a solid phase and reacted with the A / IRN / 1/2011 strain virus. Next, after removing the virus, the culture supernatant of the hybridoma was added and allowed to react. Then, after removing the culture supernatant, a mouse anti-IgG antibody labeled with HRP was added and reacted. After discarding the labeled antibody, it was reacted with a chromogenic substrate, and the absorbance was measured.
口蹄疫ウイルス感染細胞を用いた免疫染色では、複数のウェルを有しているプレートにＩＢ−ＳＲ−２細胞を播種した後に口蹄疫ウイルスを血清型に分けて接種した。感染後の適当な時間（感染細胞がはがれない程度）に培養液を捨て、冷却したアセトンを用いて感染細胞を固定した。スクリーニングの各タイムポイントにおいて使用するまで、各プレートを−８０℃に保存しておいた。つまり、スクリーニング対象のハイブリドーマの１つに対して４種類の血清型（Ｏ、Ａ、Ｃ、Ａｓｉａ１）に必要な分だけ感染細胞を準備した。保存しておいたプレートにスクリーニングするハイブリドーマの培養上清を加えてインキュベートした。上清を捨ててからＨＲＰ標識した抗マウスＩｇＧ抗体を加えて反応させた。標識抗体を捨ててから発色基質と反応させ、顕微鏡下において抗体の有無を判定した。 In immunostaining using foot-and-mouth disease virus-infected cells, IB-SR-2 cells were seeded on a plate having a plurality of wells, and then foot-and-mouth disease virus was inoculated into serotypes. The culture solution was discarded at an appropriate time after infection (so that the infected cells were not peeled off), and the infected cells were fixed using cooled acetone. Each plate was stored at −80 ° C. until used at each time point of screening. That is, infected cells were prepared in an amount necessary for four serotypes (O, A, C, Asia1) for one hybridoma to be screened. The culture supernatant of the hybridoma to be screened was added to the preserved plate and incubated. After discarding the supernatant, an anti-mouse IgG antibody labeled with HRP was added and reacted. The labeled antibody was discarded and reacted with a chromogenic substrate, and the presence or absence of the antibody was determined under a microscope.
培養上清に抗体が存在しているハイブリドーマをＥＬＩＳＡによって特定し、さらに、ＥＬＩＳＡによるスクリーニングの結果が非特異的な反応ではないことを確認するために、口蹄疫ウイルス感染細胞を用いた免疫染色によって再度スクリーニングした。 Hybridomas in which antibodies are present in the culture supernatant are identified by ELISA, and in order to confirm that the result of screening by ELISA is not a non-specific reaction, again by immunostaining with foot-and-mouth disease virus-infected cells. Screened.
スクリーニングによって選択したハイブリドーマを、標準的な方法に従ってクローニングすることによって、目的のモノクローナル抗体を安定して産生している１種類のハイブリドーマを確立した。このハイブリドーマを抗口蹄疫ウイルス血清型Ａ ｈｙｂｒｉｄｏｍａ ２Ａ１と名づけた。また、このハイブリドーマを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に寄託した（受託番号NITE P−01971）。このハイブリドーマによって産生されているモノクローナル抗体を、２Ａ１と名づけた。 By cloning the hybridoma selected by screening according to a standard method, one type of hybridoma stably producing the target monoclonal antibody was established. This hybridoma was named anti-foot-and-mouth disease virus serotype A hybridoma 2A1. In addition, this hybridoma was deposited with the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (Accession Number NITE P-01971). The monoclonal antibody produced by this hybridoma was named 2A1.
ウイルス中和試験では、感染用の細胞としてＩＢ−ＳＲ−２細胞を使用した。定法に従って、ハイブリドーマの培養上清もしくは増殖用の培地のみ、およびウイルス液の混合液をインキュベートした後に、細胞に接種した。感染後５日目に細胞変性効果を確認して、培養上清とのウイルス液の混合およびインキュベーションによって、ウイルスの増殖を抑制したか否かを評価した。 In the virus neutralization test, IB-SR-2 cells were used as cells for infection. In accordance with a conventional method, cells were inoculated after incubating a hybridoma culture supernatant or a growth medium alone and a mixture of virus solutions. On day 5 after the infection, the cytopathic effect was confirmed, and it was evaluated whether virus growth was suppressed by mixing and incubation of the virus solution with the culture supernatant.
CellTram/vario（エッペンドルフ社）を用いたマニピュレーション法により、クローニングしたモノクローナル抗体産生ハイブリドーマは、２０％ＦＣＳおよび１０％ＢＭ Ｃｏｎｄｉｍｅｄ Ｈ１（Ｒｏｃｈｅ社）を含むＲＰＭＩ培地を用いて培養した。 The cloned monoclonal antibody-producing hybridomas were cultured using RPMI medium containing 20% FCS and 10% BM Condimed H1 (Roche) by a manipulation method using CellTram / vario (Eppendorf).
なお、血清型Ａとして、Ａ／ＩＲＮ／１／２０１１株、Ａ２２／ＩＲＱ／２４／６４株、Ａ／ＴＡＩ／１０／２０１１株およびＡ１５／ＴＡＩ／１／６０株を用いた。血清型Ｏとして、Ｏ／Ｍａｎｉｓａ株を用いた。血清型Ａｓｉａ１として、Ａｓｉａ１ Ｓｈａｍｉｒ（ＩＳＲ ３／８９）株を用いた。血清型Ｃとして、Ｃ／ＰＨＩ／７／８４株を用いた。血清型ＳＡＴ１として、ＳＡＴ１／ＫＥＮ／１１７／２００９株を用いた。血清型ＳＡＴ２として、ＳＡＴ２／ＳＡＵ／６／２０００株を用いた。血清型ＳＡＴ３として、ＳＡＴ３／ＺＩＭ／３／８３株を用いた（http://www.wrlfmd.org/等を参照）。また、他のウイルスとの交差反応性を調べるために、豚水胞病ウイルス（swine vesicular disease virus；ＳＶＤＶ）を用いた。 As serotype A, A / IRN / 1/2011, A22 / IRQ / 24/64, A / TAI / 10/2011 and A15 / TAI / 1/60 were used. As serotype O, O / Manisa strain was used. As the serotype Asia1, the Asia1 Shamir (ISR 3/89) strain was used. As serotype C, C / PHI / 7/84 strain was used. As serotype SAT1, SAT1 / KEN / 117/2009 strain was used. As serotype SAT2, SAT2 / SAU / 6/2000 strain was used. As serotype SAT3, SAT3 / ZIM / 3/83 strain was used (see http://www.wrlfmd.org/ etc.). In addition, swine vesicular disease virus (SVDV) was used to examine cross-reactivity with other viruses.
結果を図１に示す。図１に示すように、２Ａ１抗体は、Ａ２２／ＩＲＱ／２４／６４株およびＡ／ＩＲＮ／１／２０１１株に対する反応性が高く、Ａ／ＴＡＩ／１０／２０１１株およびＡ１５／ＴＡＩ／１／６０株ならびに血清型Ａ以外の血清型に対する反応性が低かった。１Ｃ１抗体および３Ｈ２抗体（何れも上記スクリーニングで選択されなかったハイブリドーマから産生された抗体）は、Ａ２２／ＩＲＱ／２４／６４株およびＡ／ＩＲＮ／１／２０１１株に対する反応性が高かったが、血清型Ｏ、Ａｓｉａ１およびＣに対する反応性も高かった。一方、血清型Ａに対する公知の抗体１６Ｃ６は、Ａ２２／ＩＲＱ／２４／６４株およびＡ／ＩＲＮ／１／２０１１株に対する反応性が低く、Ａ／ＴＡＩ／１０／２０１１株およびＡ１５／ＴＡＩ／１／６０株に対する反応性は高かった。なお、上記スクリーニングで選択されなかったハイブリドーマ２Ｈ５はスクリーニングの途中で抗体産生能が脱落したが、２Ｈ５抗体は、Ａ２２／ＩＲＱ／２４／６４株およびＡ／ＩＲＮ／１／２０１１株に対する反応性が低く（＜０．３）、また、血清型Ａ型以外の型とは反応しなかった（図示せず）。 The results are shown in FIG. As shown in FIG. 1, the 2A1 antibody has high reactivity with the A22 / IRQ / 24/64 strain and the A / IRN / 1/2011 strain, and the A / TAI / 10/2011 strain and the A15 / TAI / 1/60 strain. The reactivity to serotypes other than the strain and serotype A was low. 1C1 antibody and 3H2 antibody (both antibodies produced from hybridomas not selected in the above screening) were highly reactive against A22 / IRQ / 24/64 strain and A / IRN / 1/2011 strain, but serum The reactivity to type O, Asia1 and C was also high. On the other hand, the known antibody 16C6 against serotype A has low reactivity to the A22 / IRQ / 24/64 strain and A / IRN / 1/2011 strain, and the A / TAI / 10/2011 strain and A15 / TAI / 1 / The reactivity against 60 strains was high. The hybridoma 2H5 that was not selected in the above screening lost its antibody-producing ability during screening, but the 2H5 antibody has low reactivity with the A22 / IRQ / 24/64 strain and the A / IRN / 1/2011 strain. (<0.3) and did not react with types other than serotype A (not shown).
このように、２Ａ１抗体は、１６Ｃ６抗体では検出感度が低い株に対する反応性が高い一方で、血清型Ａ以外の血清型とは反応しないという有用な特性を有している。この結果から、２Ａ１抗体は１６Ｃ６抗体では検出感度が低い株の検出を補強することができ、この２つの抗体を併用することにより、血清型Ａの検出に広く応用され得ることが期待された。 Thus, the 2A1 antibody has a useful property that it does not react with serotypes other than the serotype A, while the 16C6 antibody has high reactivity to strains with low detection sensitivity. From this result, it was expected that the 2A1 antibody can reinforce the detection of strains having low detection sensitivity with the 16C6 antibody, and that the two antibodies can be used together to detect serotype A widely.
〔モノクローナル抗体を用いたイムノクロマトグラフィー〕
（抗体の精製）
使用するモノクローナル抗体（２Ａ１、１６Ｃ６および１Ｈ５）をプロテインＧアフィニティーカラム（MAb Trap Kit; GE Healthcare 17-1128-01）にかけて、アッセイに使用するためのＩｇＧ画分のみを精製した。
[Immunochromatography using monoclonal antibodies]
(Purification of antibodies)
The monoclonal antibodies used (2A1, 16C6 and 1H5) were applied to a protein G affinity column (MAb Trap Kit; GE Healthcare 17-1128-01) to purify only the IgG fraction for use in the assay.
（抗体懸濁液における緩衝液の置換）
抗体の精製によって得られたＩｇＧ画分を含んでいる抗体懸濁液における緩衝液を、Amicon ultra Centrifugal filter units（Millipore社、商品コード：UFC805024）を用いて置換した。５ｍＭのリン酸バッファー（ＰＢ）（ｐＨ７．５）に置換したものを、２Ａ１抗体および１６Ｃ６抗体のそれぞれについて準備し、５ｍＭのＰＢ（ｐＨ８．０）に置換したものを１Ｈ５抗体について準備した。
(Replacement of buffer in antibody suspension)
The buffer in the antibody suspension containing the IgG fraction obtained by antibody purification was replaced with Amicon ultra Centrifugal filter units (Millipore, product code: UFC805024). Those substituted with 5 mM phosphate buffer (PB) (pH 7.5) were prepared for each of 2A1 antibody and 16C6 antibody, and those substituted with 5 mM PB (pH 8.0) were prepared for 1H5 antibody.
（抗体が塗布されているメンブレンの作製）
３％のエタノールを含んでいる５ｍＭのＰＢ（ｐＨ７．５）を用いて、２Ａ１抗体および１６Ｃ６抗体のそれぞれの濃度を約１５００μｇ／ｍＬに調整した。濃度を調整した２Ａ１抗体および１６Ｃ６抗体を、約１ｍｍ幅の線状として５ｍｍの間隔をあけて、イムノライナー２００（システムバイオティクス社）を用いてニトロセルロースメンブレン（Millipore社、HF135XSS）上に塗布した。上流から順に抗マウスＩｇＧコントロール抗体、血清型Ａに対するモノクローナル抗体（２Ａ１抗体単独、２Ａ１抗体と１６Ｃ６抗体との等量混合物、１６Ｃ６抗体単独）が並ぶように塗布した。また、抗体の濃度（混合物では合計）は１５００μｇ／ｍＬ、塗布抗体量は０．８μＬ／ｃｍとした。
(Preparation of antibody-coated membrane)
Using 5 mM PB (pH 7.5) containing 3% ethanol, the concentration of each of the 2A1 antibody and 16C6 antibody was adjusted to about 1500 μg / mL. Concentrated 2A1 antibody and 16C6 antibody were coated on a nitrocellulose membrane (Millipore, HF135XSS) using Immunoliner 200 (System Biotics) with a linear spacing of about 1 mm and a spacing of 5 mm. . An anti-mouse IgG control antibody and a monoclonal antibody against serotype A (2A1 antibody alone, an equal mixture of 2A1 antibody and 16C6 antibody, 16C6 antibody alone) were applied in order from the upstream. The antibody concentration (total for the mixture) was 1500 μg / mL, and the applied antibody amount was 0.8 μL / cm.
上記のメンブレンを５０℃で３０分間乾燥させた後、ブロッキングバッファー（０．５％のカゼインを含んでいる５０ｍＭのホウ酸バッファー（ｐＨ８．５））に室温で３０分間浸した。次いで、洗浄バッファー（０．５％のスクロースを含んでいる５０ｍＭのＴｒｉｓ−ＨＣｌ（ｐＨ７．４））に室温で３０分間浸した後、室温で風乾させた。 The membrane was dried at 50 ° C. for 30 minutes, and then immersed in blocking buffer (50 mM borate buffer (pH 8.5) containing 0.5% casein) at room temperature for 30 minutes. Subsequently, it was immersed in a washing buffer (50 mM Tris-HCl (pH 7.4) containing 0.5% sucrose) for 30 minutes at room temperature, and then air-dried at room temperature.
（検出用ストリップの組み立て）
バッキングシート（ARcare 7815 : Adhesives Research社）を用いて、乾燥させたメンブレンの上流側にサンプルパッド（セルロースファイバーメンブレン（Millipore社、CFSP223000））を貼り付け、下流側に吸収パッド（CF6（グラスファイバーとコットンとの混合物）、Whatman社）を貼り付けた。４０％以下の湿度の条件下で、シリカゲルが収められている密封容器内に検出用ストリップを保存した。
(Assembly of detection strip)
Using a backing sheet (ARcare 7815: Adhesives Research), a sample pad (cellulose fiber membrane (Millipore, CFSP223000)) is pasted on the upstream side of the dried membrane, and an absorbent pad (CF6 (glass fiber and A mixture with cotton), Whatman) was applied. The detection strip was stored in a sealed container containing silica gel under conditions of humidity of 40% or less.
（金コロイドによるモノクローナル抗体の標識化）
５ｍＭのＰＢ（ｐＨ８．０）を用いて１Ｈ５抗体の濃度を２０μｇ／１００μＬに調整した。金コロイド（直径４０〜５０ｎｍ）（ワインレッドケミカル社、WRGH2（ＯＤ５２０＝１２））を十分にソニケーションした後、１ｍＭのＫ２ＣＯ３を用いてｐＨ８．０に調整した。次にシリコンコーティングチューブ内において、１：８の割合で抗体液および金コロイド液を混合した。１％のウシ血清アルブミン（ＢＳＡ）および０．０５％のＰＥＧを加えて混和した後、１００００ｒｐｍ、室温で３０分間遠心した。遠心上清を除去し、ソニケーションした後、ＰＢＳに０．５％のＢＳＡおよび０．０５％ＰＥＧを加えた溶液（溶液Ａ）を用いて懸濁した。再び１００００ｒｐｍ、室温で３０分間遠心した後、遠心上清を除去し、ソニケーションによって懸濁させ、溶液Ａを用いてＯＤ５２０＝２．０に調整した。２０％のスクロースを含んでいる１５ｍＭのＴｒｉｓ−ＨＣｌ（ｐＨ８．２）を等量加えた後、金コロイド標識した抗体を含んでいる懸濁液を１００μＬずつ２ｍＬのチューブに分注した。各チューブの口をパラフィルムで覆ってからパラフィルムに数カ所の穴を空け、−８０℃で１時間凍結させた。真空乾燥機を用いて一晩真空乾燥させた後、パラフィルムを剥がし、チューブのキャップを閉めて４℃で保存した。
(Labeling monoclonal antibodies with colloidal gold)
The concentration of 1H5 antibody was adjusted to 20 μg / 100 μL using 5 mM PB (pH 8.0). Gold colloid (40-50 nm in diameter) (Wine Red Chemical Co., WRGH2 (OD 520 = 12)) was sufficiently sonicated, and adjusted to pH 8.0 using 1 mM K 2 CO 3 . Next, the antibody solution and the gold colloid solution were mixed at a ratio of 1: 8 in the silicon coating tube. 1% bovine serum albumin (BSA) and 0.05% PEG were added and mixed, and then centrifuged at 10,000 rpm at room temperature for 30 minutes. After removing the centrifugation supernatant and sonicating, it was suspended using a solution (solution A) in which 0.5% BSA and 0.05% PEG were added to PBS. After centrifugation again at 10000 rpm and room temperature for 30 minutes, the supernatant was removed, suspended by sonication, and adjusted to OD 520 = 2.0 using solution A. After adding an equal volume of 15 mM Tris-HCl (pH 8.2) containing 20% sucrose, 100 μL of the suspension containing colloidal gold labeled antibody was dispensed into 2 mL tubes. The mouth of each tube was covered with parafilm, and then several holes were made in the parafilm and frozen at -80 ° C for 1 hour. After vacuum drying overnight using a vacuum dryer, the parafilm was peeled off, the cap of the tube was closed and stored at 4 ° C.
（ラテラルフローアッセイの評価）
口蹄疫ウイルスの血清型Ａ（Ａ２２／ＩＲＱ／２４／６４株、Ａ／ＩＲＮ／１／２０１１株、Ａ／ＴＡＩ／１０／２０１１株およびＡ１５／ＴＡＩ／１／６０株）の量をＰＢＳで１０倍、１００倍および４００倍に希釈した。次に、凍結乾燥させた金コロイド標識１Ｈ５抗体を１００μＬの各ウイルス希釈液で溶解した後、ストリップを漬けて、１０分間反応させた。イムノクロマトリーダー（浜松ホトニクス社）を用いてｍＡＢＳを測定した。
(Evaluation of lateral flow assay)
10 times the amount of foot-and-mouth disease virus serotype A (A22 / IRQ / 24/64, A / IRN / 1/2011, A / TAI / 10/2011 and A15 / TAI / 1/60) in PBS Diluted 100-fold and 400-fold. Next, the lyophilized gold colloid-labeled 1H5 antibody was dissolved in 100 μL of each virus dilution, and then stripped and allowed to react for 10 minutes. MABS was measured using an immunochromatographic reader (Hamamatsu Photonics).
結果を図２に示す。図２に示すとおり、２Ａ１抗体と１６Ｃ６抗体とを組み合わせると、Ａ２２／ＩＲＱ／２４／６４株、Ａ／ＩＲＮ／１／２０１１株、Ａ／ＴＡＩ／１０／２０１１株およびＡ１５／ＴＡＩ／１／６０株の何れにおいても、高感度で検出することができた。 The results are shown in FIG. As shown in FIG. 2, when the 2A1 antibody and the 16C6 antibody are combined, the A22 / IRQ / 24/64 strain, the A / IRN / 1/2011 strain, the A / TAI / 10/2011 strain, and the A15 / TAI / 1/60 strain are combined. It was possible to detect with high sensitivity in any of the strains.
〔参考：７つの血清型全てと反応する抗体の取得〕
（モノクローナル抗体の作製）
口蹄疫ウイルスＡ２２／ＩＲＱ／２４／６４株をＩＢ−ＲＳ−２細胞に接種し、一晩回転培養した。回収した培養液を１５００Ｇで１０分間遠心した後、上清を回収した。上清を飽和硫酸アンモニウム溶液と等量混和し、一晩４℃で撹拌することによってウイルスを析出させた。析出させたウイルスを、５０００Ｇで３０分間遠沈し、上清を除去してから適量のＰＢＳに懸濁した。再び５０００Ｇで３０分間遠心し、上清を除去し、１ｍＬのＰＢＳに懸濁した。スクロース密度勾配（１５〜４５％）ウイルスの懸濁液を添加し、２００００ｒｐｍで２時間遠心を行い、目的のバンドを回収することによって、１４６Ｓの完全粒子を精製した。
[Reference: Acquisition of antibodies that react with all seven serotypes]
(Production of monoclonal antibodies)
IB-RS-2 cells were inoculated with foot-and-mouth disease virus A22 / IRQ / 24/64 strain, and rotated and cultured overnight. The collected culture was centrifuged at 1500 G for 10 minutes, and the supernatant was collected. The supernatant was mixed with an equal volume of saturated ammonium sulfate solution and stirred overnight at 4 ° C. to precipitate the virus. The precipitated virus was spun down at 5000 G for 30 minutes, and the supernatant was removed and suspended in an appropriate amount of PBS. Again, centrifugation was performed at 5000 G for 30 minutes, and the supernatant was removed and suspended in 1 mL of PBS. A sucrose density gradient (15-45%) virus suspension was added, centrifuged at 20000 rpm for 2 hours, and the desired band was collected to purify 146S complete particles.
８〜１２週齢のメスのＢＡＬＢ／ｃマウスの腹腔内に、上述のように精製した口蹄疫ウイルスＡ２２／ＩＲＱ／２４／６４株の完全粒子を接種した。初回免疫ではフロイントコンプリートアジュバント（ヤトロン社製）と精製したウイルス液との等量混合物、追加接種ではフロイントインコンプリートアジュバント（ヤトロン社製）と精製したウイルス液との等量混合物を、連結針を用いてミセル化させた。最終免疫後にマウスの脾臓細胞とマウスミエローマ細胞Ｐ３Ｕ１とをポリエチレングリコール４０００（メルク社製）を用いて融合させ、ＨＡＴ培地（０．１ｍＭのヒポキサンチン、０．４μＭのアミノプテリン、１６μＭのチミジン、および２０％ＦＣＳを含有するＲＰＭＩ−１６４０培地（ニッスイ））を用いて９６ウェルプレートにおいて培養した。２週間後からはＨＴ選択培地（０．１ｍＭのヒポキサンチン、１６μＭのチミジン、および２０％ＦＣＳを含有するＲＰＭＩ−１６４０培地（ニッスイ））を用いて培養した。 8-12 week old female BALB / c mice were inoculated intraperitoneally with complete particles of the foot-and-mouth disease virus A22 / IRQ / 24/64 strain purified as described above. For the first immunization, use an equivalent mixture of Freund's complete adjuvant (manufactured by Yatron) and purified virus solution, and for additional inoculation, use an equivalent mixture of Freund's complete adjuvant (manufactured by Yatron) and purified virus solution. To micelles. After final immunization, mouse spleen cells and mouse myeloma cells P3U1 were fused using polyethylene glycol 4000 (Merck), and HAT medium (0.1 mM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine, and Cultured in 96-well plates using RPMI-1640 medium (Nissui) containing 20% FCS. After 2 weeks, the cells were cultured using an HT selection medium (RPMI-1640 medium (Nissui) containing 0.1 mM hypoxanthine, 16 μM thymidine, and 20% FCS).
上述のように培養することによって、ハイブリドーマのコロニーを形成させ、ＥＬＩＳＡおよび口蹄疫ウイルス感染細胞を用いた免疫染色によって目的のモノクローナル抗体を産生しているハイブリドーマをスクリーニングした。スクリーニングの詳細は以下の通りである。 By culturing as described above, hybridoma colonies were formed, and hybridomas producing the desired monoclonal antibody were screened by ELISA and immunostaining using foot-and-mouth disease virus-infected cells. Details of the screening are as follows.
ＥＬＩＳＡでは、ウサギ抗口蹄疫ウイルス抗体を、固相に吸着させ、Ａ２２／ＩＲＱ／２４／６４株の完全粒子と反応させた。次いで、ウイルスを取り除いてからハイブリドーマの培養上清を加えて反応させた。そして、培養上清を取り除いてからＨＲＰ標識したマウス抗ＩｇＧ抗体を加えて反応させた。標識抗体を捨ててから発色基質と反応させて、吸光度を測定した。 In ELISA, a rabbit anti-foot-and-mouth disease virus antibody was adsorbed to a solid phase and reacted with complete particles of A22 / IRQ / 24/64 strain. Next, after removing the virus, the culture supernatant of the hybridoma was added and allowed to react. Then, after removing the culture supernatant, a mouse anti-IgG antibody labeled with HRP was added and reacted. After discarding the labeled antibody, it was reacted with a chromogenic substrate, and the absorbance was measured.
口蹄疫ウイルス感染細胞を用いた免疫染色では、複数のウェルを有しているプレートにＩＢ−ＳＲ−２細胞を播種した後に口蹄疫ウイルスを血清型に分けて接種した。感染後の適当な時間（感染細胞がはがれない程度）に培養液を捨て、冷却したアセトンを用いて感染細胞を固定した。スクリーニングの各タイムポイントにおいて使用するまで、各プレートを−８０℃に保存しておいた。つまり、スクリーニング対象のハイブリドーマの１つに対して７種類の血清型に必要な分だけ感染細胞を準備した。保存しておいたプレートにスクリーニングするハイブリドーマの培養上清を加えてインキュベートした。上清を捨ててからＨＲＰ標識した抗マウスＩｇＧ抗体を加えて反応させた。標識抗体を捨ててから発色基質と反応させ、顕微鏡下において抗体の有無を判定した。 In immunostaining using foot-and-mouth disease virus-infected cells, IB-SR-2 cells were seeded on a plate having a plurality of wells, and then foot-and-mouth disease virus was inoculated into serotypes. The culture solution was discarded at an appropriate time after infection (so that the infected cells were not peeled off), and the infected cells were fixed using cooled acetone. Each plate was stored at −80 ° C. until used at each time point of screening. In other words, as many infected cells as necessary for seven serotypes were prepared for one of the hybridomas to be screened. The culture supernatant of the hybridoma to be screened was added to the preserved plate and incubated. After discarding the supernatant, an anti-mouse IgG antibody labeled with HRP was added and reacted. The labeled antibody was discarded and reacted with a chromogenic substrate, and the presence or absence of the antibody was determined under a microscope.
培養上清に抗体が存在しているハイブリドーマをＥＬＩＳＡによって特定し、さらに、ＥＬＩＳＡによるスクリーニングの結果が非特異的な反応ではないことを確認するために、口蹄疫ウイルス感染細胞を用いた免疫染色によって再度スクリーニングした。 Hybridomas in which antibodies are present in the culture supernatant are identified by ELISA, and in order to confirm that the result of screening by ELISA is not a non-specific reaction, again by immunostaining with foot-and-mouth disease virus-infected cells. Screened.
スクリーニングによって選択したハイブリドーマを、標準的な方法に従ってクローニングすることによって、目的のモノクローナル抗体を安定して産生している１種類のハイブリドーマを確立した。このハイブリドーマを抗口蹄疫ウイルス ｈｙｂｒｉｄｏｍａ １６Ｄ６と名づけた。また、このハイブリドーマを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に寄託した（受託番号NITE P−01972）。このハイブリドーマによって産生されているモノクローナル抗体（７種類全ての血清型の口蹄疫ウイルスと反応するモノクローナル抗体）を、１６Ｄ６と名づけた。 By cloning the hybridoma selected by screening according to a standard method, one type of hybridoma stably producing the target monoclonal antibody was established. This hybridoma was named anti-foot-and-mouth disease virus hybridoma 16D6. The hybridoma was deposited with the Patent Microorganism Depositary Center for Product Evaluation Technology, an independent administrative agency (Accession Number NITE P-01972). The monoclonal antibody produced by this hybridoma (monoclonal antibody that reacts with foot-and-mouth disease virus of all seven serotypes) was named 16D6.
CellTram/vario（エッペンドルフ社）を用いたマニピュレーション法により、クローニングしたモノクローナル抗体産生ハイブリドーマは、BriClone Hybridoma Cloning Medium（キューイーディーバイオサイエンス社）を用いて培養した。 The monoclonal antibody-producing hybridoma cloned by the manipulation method using CellTram / vario (Eppendorf) was cultured using BriClone Hybridoma Cloning Medium (QD Bioscience).
（モノクローナル抗体の性能比較のための間接サンドイッチＥＬＩＳＡ）
モノクローナル抗体１６Ｄ６の性能を確かめるために、間接サンドイッチＥＬＩＳＡを行った。比較として、７種類全ての血清型の口蹄疫ウイルスと反応する既知のモノクローナル抗体１Ｈ５を用いた。１Ｈ５抗体については、非特許文献１を参照すればよい。
(Indirect sandwich ELISA for monoclonal antibody performance comparison)
To confirm the performance of monoclonal antibody 16D6, an indirect sandwich ELISA was performed. For comparison, a known monoclonal antibody 1H5 that reacts with foot-and-mouth disease virus of all seven serotypes was used. For the 1H5 antibody, Non-Patent Document 1 may be referred to.
捕捉抗体として各血清型の抗口蹄疫ウイルスウサギ抗体を０．０５Ｍの炭酸バッファー（ｐＨ９．６）で×８００に希釈し、５０μＬずつ９６ウェルプレート（Immulon II HB Thermo）に４℃で一晩固相化した。３００μＬ／ｗｅｌｌの０．００２Ｍ ＰＢＳで３回洗浄した後、口蹄疫ウイルス（抗原）を含むサンプルを５０μＬずつウェルに加え、１００ｒｐｍで振とうしながら３７℃で１時間反応させた。このとき、陰性検体として０．０１ＭのＰＢＳ（ｐＨ７．４）に０．０５%（ｖ／ｖ）のＴｗｅｅｎ２０を加えたＡ液を使用した。同様に洗浄した後、Ａ液に５％のスキムミルクを加えＢ液とし、３７℃で３０分間ブロッキングした。Ｂ液を捨てた後、洗浄せずに、Ａ液で１μｇ〜０．００００６４μｇの５倍階段希釈を行ったモノクローナル抗体１Ｈ５および１６Ｄ６をそれぞれ加えた。３７℃で１時間反応させた後、Ｂ液で×３０００に希釈したペルオキシダーゼ標識抗マウスイムノグロブリンヤギ抗体を５０μＬずつ加え、室温で４５分間反応させた。８回洗浄した後、発色基質ＴＭＢを５０μＬずつ加え、暗所にて室温で１５分間反応させた。５０μＬの１．２５Ｍ 硫酸で反応を停止させ、ＥＬＩＳＡプレートリーダーを用いて４５０ｎｍの波長を測定した。 As a capture antibody, anti-foot-and-mouth disease virus rabbit antibody of each serotype was diluted to × 800 with 0.05M carbonate buffer (pH 9.6), and 50 μL each was immobilized on a 96-well plate (Immulon II HB Thermo) at 4 ° C. overnight. Turned into. After washing three times with 300 μL / well of 0.002M PBS, 50 μL of the sample containing foot-and-mouth disease virus (antigen) was added to each well and reacted at 37 ° C. for 1 hour while shaking at 100 rpm. At this time, as a negative sample, solution A in which 0.05% (v / v) Tween 20 was added to 0.01 M PBS (pH 7.4) was used. After washing in the same manner, 5% skim milk was added to solution A to prepare solution B, which was blocked at 37 ° C. for 30 minutes. After discarding the B solution, monoclonal antibodies 1H5 and 16D6, each of which was 5-fold serially diluted with the A solution from 1 μg to 0.000064 μg, were added without washing. After reacting at 37 ° C. for 1 hour, 50 μL of peroxidase-labeled anti-mouse immunoglobulin goat antibody diluted to 3000 × B solution was added and reacted at room temperature for 45 minutes. After washing 8 times, 50 μL of chromogenic substrate TMB was added and reacted at room temperature for 15 minutes in the dark. The reaction was stopped with 50 μL of 1.25 M sulfuric acid and a wavelength of 450 nm was measured using an ELISA plate reader.
なお、血清型ＯとしてＯ／ＪＰＮ／２０００株、血清型ＡとしてＡ／ＩＲＮ／１／２０１１株およびＡ２２／ＩＲＱ／２４／６４株、血清型Ａｓｉａ１としてＡｓｉａ１ Ｓｈａｍｉｒ（ＩＳＲ ３／８９）株、血清型ＣとしてＣ／ＰＨＩ／７／８４株、血清型ＳＡＴ１としてＳＡＴ１／ＫＥＮ／１１７／２００９株、血清型ＳＡＴ２としてＳＡＴ２／ＳＡＵ／６／２０００株、血清型ＳＡＴ３としてＳＡＴ３／ＺＩＭ／３／８３株を用いた。 In addition, O / JPN / 2000 strain as serotype O, A / IRN / 1/2011 strain and A22 / IRQ / 24/64 strain as serotype A, Asia1 Shamir (ISR 3/89) strain as serotype Asia1, and serum C / PHI / 7/84 strain as type C, SAT1 / KEN / 117/2009 strain as serotype SAT1, SAT2 / SAU / 6/2000 strain as serotype SAT2, SAT3 / ZIM / 3/83 strain as serotype SAT3 Was used.
結果を図３に示す。図３に示すように、１６Ｄ６抗体では８種類の株のうち７種類の株において１Ｈ５抗体よりも反応性が高かった。特に、１Ｈ５抗体では反応性が比較的低いＡ／ＩＲＮ／１／２０１１株、ＳＡＴ１／ＫＥＮ／１１７／２００９株、ＳＡＴ２／ＳＡＵ／６／２０００株、およびＳＡＴ３／ＺＩＭ／３／８３株において、顕著に反応性が高くなっている。このように、１６Ｄ６抗体は１Ｈ５抗体と比較して非常に優れた反応性を示すことがわかった。 The results are shown in FIG. As shown in FIG. 3, the 16D6 antibody was more reactive than the 1H5 antibody in 7 out of 8 strains. In particular, the A / IRN / 1/2011 strain, SAT1 / KEN / 117/2009 strain, SAT2 / SAU / 6/2000 strain, and SAT3 / ZIM / 3/83 strain, which have relatively low reactivity with the 1H5 antibody, are prominent. The reactivity is high. Thus, it was found that the 16D6 antibody exhibits a very excellent reactivity as compared with the 1H5 antibody.
本発明は、口蹄疫ウイルスの検出に利用することができる。 The present invention can be used for detection of foot-and-mouth disease virus.
NITE P−01971
NITE P−01972
NITE P-01971
NITE P-01972
Claims (10)
抗原抗体反応物を検出する工程を含む、請求項８に記載の検出方法。 A step of contacting a sample collected from a subject with the antibody according to claim 1 or 2 to cause an antigen-antibody reaction between the antigen in the sample and the antibody; and a step of detecting an antigen-antibody reaction product. The detection method according to claim 8, comprising:
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| CN111172118A (en) * | 2020-03-25 | 2020-05-19 | 新疆畜牧科学院兽医研究所（新疆畜牧科学院动物临床医学研究中心） | anti-A-type foot-and-mouth disease antigen monoclonal antibody hybridoma cell strain, anti-A-type foot-and-mouth disease antigen monoclonal antibody and application thereof |
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Non-Patent Citations (3)
| Title |
|---|
| FAO WORLD REFERENCE LABORATORY FOR FOOT-AND-MOUTH DISEASE (WRLFMD) GENOTYPING REPORT, vol. [Retrieved on 2018 Sep 20], JPN6018038278, 24 May 2011 (2011-05-24), pages WRLFMD Ref No: IRN/1/2011項 * |
| MORIOKA K. ET AL., PLOS ONE, vol. Vol.9 Issue 4(2014), JPN6018038274, pages e94143 * |
| 森岡 一樹, 農林水産技術研究ジャーナル, vol. Vol.35 No.5(2012), JPN6018038271, pages 22 - 25 * |
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| CN111172118A (en) * | 2020-03-25 | 2020-05-19 | 新疆畜牧科学院兽医研究所（新疆畜牧科学院动物临床医学研究中心） | anti-A-type foot-and-mouth disease antigen monoclonal antibody hybridoma cell strain, anti-A-type foot-and-mouth disease antigen monoclonal antibody and application thereof |
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