JP2009077658A - Method for quickly detecting campylobacter in feces - Google Patents
Method for quickly detecting campylobacter in feces Download PDFInfo
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- JP2009077658A JP2009077658A JP2007249616A JP2007249616A JP2009077658A JP 2009077658 A JP2009077658 A JP 2009077658A JP 2007249616 A JP2007249616 A JP 2007249616A JP 2007249616 A JP2007249616 A JP 2007249616A JP 2009077658 A JP2009077658 A JP 2009077658A
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Abstract
Description
本発明は、食中毒菌の迅速検出方法、詳細には、便中のカンピロバクターの迅速検出方法に関する。 The present invention relates to a rapid detection method for food poisoning bacteria, and more particularly to a rapid detection method for Campylobacter in feces.
カンピロバクター腸炎は、カンピロバクター（Campylobacter）に汚染された食品を摂食することにより引き起こされる細菌性食中毒の１つであり、その発生件数は、わが国で発生している細菌性食中毒の中で最も多い。現在、カンピロバクター腸炎の診断は、細菌培養法により患者便からカンピロバクターを検出することにより実施されているが、それは煩雑な操作と最低２日の培養期間を必要とする。 Campylobacter enteritis is one of the bacterial food poisonings caused by eating food contaminated with Campylobacter, and the incidence is the largest among the bacterial food poisonings occurring in Japan. Currently, the diagnosis of Campylobacter enteritis is carried out by detecting Campylobacter from the patient's stool by a bacterial culture method, which requires a complicated operation and a culture period of at least 2 days.
カンピロバクター腸炎の２大原因菌はカンピロバクター・ジェジュニ（Campylobacter jejuni）およびカンピロバクター・コリ（Campylobacter coli）であるが、患者から分離されるカンピロバクターの９５〜９９％はC. jejuniであり、C. coliは数％に過ぎない。従って、C. jejuniがカンピロバクター腸炎の主要原因菌であり、これはLiorの血清型別システムにより数十種類の血清型に分類される。他のカンピロバクター菌種では、カンピロバクター・ラリ（Campylobacter lari）及びカンピロバクター・アプサリエンシス（Campylobacter upsaliensis）が、その発生は非常に稀であるが、ヒト腸炎との関連が報告されている。従って、検出率や診断精度を高めるためには、カンピロバクター属に対して特異性が高く、しかもこれらのヒト腸炎の原因となるカンピロバクターに属する広範な菌種（広範なカンピロバクター血清型）を検出できる方法が必要とされている。 Campylobacter enteritis has two major causative organisms: Campylobacter jejuni and Campylobacter coli, but 95-99% of Campylobacter isolates from patients are C. jejuni, and C. coli is a number It is only%. Therefore, C. jejuni is the main causative agent of Campylobacter enteritis, which is classified into several tens of serotypes by Lior's serotyping system. In other Campylobacter species, Campylobacter lari and Campylobacter upsaliensis are very rare but have been reported to be associated with human enteritis. Therefore, in order to increase the detection rate and diagnostic accuracy, it is highly specific for Campylobacter and can detect a wide range of bacterial species (a wide range of Campylobacter serotypes) belonging to Campylobacter that cause these human enteritis. Is needed.
上記のような問題を解決するために、これまでに様々な検出方法が開発されてきたが、カンピロバクター属に対して特異性が高く、カンピロバクターに属する広範な菌種（広範なカンピロバクター血清型）を検出でき、しかも迅速に検出を行うことのできる方法はなかった。例えば、本発明と同様にモノクローナル抗体を用いてカンピロバクターを検出しようとする試みもいくつかあったが（非特許文献１〜３参照）、これらの方法に用いられる抗体は、カンピロバクター以外の菌にも反応してしまう、C. jejuniの一部の血清型には反応しない、１種類ではサンドイッチ検出系を構築できない等の問題がある。また、ＥＬＩＳＡ法を用いる方法もあるが（非特許文献４参照）、迅速性および簡便性の点で満足とはいえない。
便中のカンピロバクターを迅速かつ高精度、かつ高特異性でもって検出する方法、そのためのキット等を提供することが本発明の課題である。 It is an object of the present invention to provide a method for detecting Campylobacter in stool quickly, with high accuracy and high specificity, a kit for that purpose, and the like.
本発明者らは上記事情に鑑みて鋭意研究を重ね、カンピロバクター腸炎の主要原因菌であるC. jejuniの菌体表面タンパク質に対するマウスモノクローナル抗体を新たに作出し、それを用いることにより患者便抽出液中のカンピロバクター抗原を１５分以内に簡便に検出することが可能であること、この抗体がカンピロバクター特異的であり、しかもカンピロバクター属の広範な種（広範な血清型）をカバーするものであること等を見出し、本発明を完成するに至った。 In light of the above circumstances, the present inventors have made extensive studies and newly created a mouse monoclonal antibody against the cell surface protein of C. jejuni, the main causative bacterium of Campylobacter enteritis, and by using it, a patient stool extract is used. Campylobacter antigen can be easily detected within 15 minutes, and this antibody is specific to Campylobacter and covers a wide range of Campylobacter species (wide serotypes) As a result, the present invention has been completed.
すなわち、本発明は：
（１）モノクローナル抗体４Ｂ４と便試料とを接触させることを特徴とする、便中のカンピロバクターの検出方法；
（２）（ａ）検出可能な標識を付したモノクローナル抗体４Ｂ４を便試料と反応させて複合体を形成させること、次いで、
（ｂ）（ａ）で得られた複合体を、固相上に固定化されたモノクローナル抗体４Ｂ４と反応させて、形成されたさらなる複合体を該標識を用いて検出すること
を特徴とする（１）記載の方法；
（３）工程（ｂ）がイムノクロマトグラフィー法またはＥＬＩＳＡ法を用いて行われる（２）記載の方法；
（４）モノクローナル抗体４Ｂ４と便試料とを接触させる前に、便試料を界面活性剤で処理する工程を含む（１）ないし（３）のいずれかに記載の方法；
（５）モノクローナル抗体４Ｂ４を必須構成成分として含む、便中のカンピロバクターを検出するためのキット；
（６）イムノクロマトグラフィー法またはＥＬＩＳＡ法を用いるものである（５）記載のキット；
（７）便試料がヒトから得られたものである（１）ないし（４）のいずれかに記載の方法、あるいは（５）または（６）記載のキット；
（８）独立行政法人 産業技術総合研究所 特許生物寄託センターに寄託され、受領番号ＦＥＲＭ ＡＰ−２１３６８を付与されたハイブリドーマ；
（９）（８）記載のハイブリドーマにより産生されるモノクローナル抗体４Ｂ４
を提供するものである。
That is, the present invention provides:
(1) A method for detecting Campylobacter in feces, which comprises contacting monoclonal antibody 4B4 with a fecal sample;
(2) (a) reacting monoclonal antibody 4B4 with a detectable label with a stool sample to form a complex;
(B) The complex obtained in (a) is reacted with monoclonal antibody 4B4 immobilized on a solid phase, and the further complex formed is detected using the label ( 1) The method according to the description;
(3) The method according to (2), wherein step (b) is performed using an immunochromatography method or an ELISA method;
(4) The method according to any one of (1) to (3), comprising a step of treating the stool sample with a surfactant before contacting the monoclonal antibody 4B4 with the stool sample;
(5) A kit for detecting Campylobacter in feces, comprising monoclonal antibody 4B4 as an essential component;
(6) The kit according to (5), which uses an immunochromatography method or an ELISA method;
(7) The method according to any one of (1) to (4), or the kit according to (5) or (6), wherein the stool sample is obtained from a human;
(8) A hybridoma deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, and given the receipt number FERM AP-21368;
(9) Monoclonal antibody 4B4 produced by the hybridoma according to (8)
Is to provide.
本発明によれば、便中のカンピロバクター属の様々な菌種（広範な血清型）を迅速かつ高精度、かつ高特異性で検出することができるので、本発明は、カンピロバクター食中毒の診断薬や診断キットの製造分野において利用可能である。本発明の検出方法は、一度に多くの検体を処理するハイスループット分析にも適したものである。 According to the present invention, various bacterial species of the genus Campylobacter (a wide range of serotypes) in stool can be detected quickly, with high accuracy, and with high specificity. Therefore, the present invention provides a diagnostic agent for Campylobacter food poisoning, It can be used in the field of manufacturing diagnostic kits. The detection method of the present invention is also suitable for high-throughput analysis in which many samples are processed at once.
本発明は、１の態様において、モノクローナル抗体４Ｂ４と便試料とを反応させることを特徴とする、便中のカンピロバクターの検出方法に関するものである。本発明において新たに取得されたモノクローナル抗体４Ｂ４は、カンピロバクター菌体表面タンパク質に特異的に結合する。しかも、カンピロバクター属の多くの菌種（広範な血清型）の菌体表面タンパク質に結合する。したがって、モノクローナル抗体４Ｂ４を用いれば、カンピロバクターに属する多くの種の細菌（広範な血清型）を特異的に検出することが可能である。そのうえ、モノクローナル抗体４Ｂ４は、後述のごとく、それのみを用いてイムノクロマトグラフィー（本明細書において「イムノクロマト」と略称することがある）やサンドイッチＥＬＩＳＡのようなサンドイッチ検出を行うことができる。その理由は、モノクローナル抗体４Ｂ４がカンピロバクター菌体表面タンパク質の２つまたはそれ以上の部位に結合するためと考えられる。 In one aspect, the present invention relates to a method for detecting Campylobacter in feces, which comprises reacting monoclonal antibody 4B4 with a fecal sample. The monoclonal antibody 4B4 newly obtained in the present invention specifically binds to Campylobacter cell surface protein. In addition, it binds to cell surface proteins of many species of Campylobacter species (wide serotype). Therefore, by using the monoclonal antibody 4B4, it is possible to specifically detect many types of bacteria (wide serotype) belonging to Campylobacter. Moreover, as described later, the monoclonal antibody 4B4 can be used alone to perform sandwich detection such as immunochromatography (sometimes abbreviated as “immunochromatography” in this specification) or sandwich ELISA. The reason is considered that the monoclonal antibody 4B4 binds to two or more sites of the Campylobacter cell surface protein.
モノクローナル抗体４Ｂ４と便試料との反応とは、モノクローナル抗体４Ｂ４と便試料中に存在するカンピロバクター属細菌の表面タンパク質とが特異的に反応し、結合することを意味する。 The reaction between the monoclonal antibody 4B4 and the stool sample means that the monoclonal antibody 4B4 and the surface protein of Campylobacter bacterium present in the stool sample specifically react and bind to each other.
本発明において分析される試料としては、動物、特にヒトの糞便が挙げられる。 Samples to be analyzed in the present invention include animals, particularly human feces.
モノクローナル抗体４Ｂ４は、本発明により得られたマウスハイブリドーマＣＪ−４Ｂ４によって産生される抗体である。マウスハイブリドーマＣＪ−４Ｂ４は、２００７年９月１９日に、日本国茨城県つくば市東１丁目１番地１中央第６の独立行政法人 産業技術総合研究所 特許生物寄託センターに寄託され、受領番号ＦＥＲＭ ＡＰ−２１３６８が付与された。上記ハイブリドーマおよびモノクローナル抗体４Ｂ４は本発明に包含される。 Monoclonal antibody 4B4 is an antibody produced by murine hybridoma CJ-4B4 obtained according to the present invention. Mouse hybridoma CJ-4B4 was deposited on September 19, 2007 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi 1-chome, Tsukuba, Ibaraki, Japan. -21368. The hybridoma and monoclonal antibody 4B4 are included in the present invention.
本発明は、もう１つの態様において、（ａ）検出可能な標識を付したモノクローナル抗体４Ｂ４を試料と反応させて複合体を形成させること、次いで、（ｂ）（ａ）で得られた複合体を、固相上に固定化されたモノクローナル抗体４Ｂ４と反応させて、形成されたさらなる複合体を該標識を用いて検出することを特徴とする、カンピロバクターの検出方法に関するものである。前述のように、モノクローナル抗体４Ｂ４は、それのみを用いることでイムノクロマトやサンドイッチＥＬＩＳＡ（通常、これらの手法は２種類以上の抗体を使用する必要があるが、それに適した優れた抗体を２種類以上作出することは容易ではない）などのサンドイッチ検出を行うことができるので、検出系の開発を容易にする。 In another embodiment, the present invention provides (a) reacting a monoclonal antibody 4B4 with a detectable label with a sample to form a complex, and then (b) the complex obtained in (a). Is reacted with monoclonal antibody 4B4 immobilized on a solid phase, and a further complex formed is detected using the label, which relates to a method for detecting Campylobacter. As described above, monoclonal antibody 4B4 can be used alone for immunochromatography or sandwich ELISA (usually, these methods require the use of two or more types of antibodies, but two or more types of excellent antibodies suitable for them are used. (It is not easy to create) and the like can be detected, which facilitates the development of a detection system.
先ず、検出可能な標識を付したモノクローナル抗体４Ｂ４と試料とを反応させて複合体を形成させる。この工程において、試料中のカンピロバクター菌体表面タンパク質あるいはカンピロバクターから遊離した表面タンパク質とモノクローナル抗体４Ｂ４とが反応して結合し、複合体が形成される。検出可能な標識としては、金コロイド、酵素（例えば、ペルオキシダーゼ、アルカリホスファターゼ、ルシフェラーゼ）、蛍光基（例えば、ＦＩＴＣ）、発光基（例えば、アクリジニウムなど）、放射性同位元素（例えば、３２Ｐ、３５Ｓ、３Ｈ）などが挙げられるが、これらに限らない。モノクローナル抗体４Ｂ４と標識の結合方法は当業者に公知であり、例えば、共有結合を介して抗体に標識を結合させることができる。当業者は、標識の種類や検出方法に応じて結合方法を適宜選択して用いることができる。標識の検出についても肉眼観察、分光学的処方による検出、放射能カウントなど、様々であり、やはり当業者が適宜検出方法を選択することができる。 First, a monoclonal antibody 4B4 with a detectable label is reacted with a sample to form a complex. In this step, the Campylobacter cell surface protein in the sample or the surface protein released from Campylobacter reacts and binds to the monoclonal antibody 4B4 to form a complex. Detectable labels include colloidal gold, enzymes (eg, peroxidase, alkaline phosphatase, luciferase), fluorescent groups (eg, FITC), luminescent groups (eg, acridinium), radioisotopes (eg, 32 P, 35 S). , 3 H) and the like. A method for binding the monoclonal antibody 4B4 and the label is known to those skilled in the art. For example, the label can be bound to the antibody via a covalent bond. A person skilled in the art can appropriately select and use a binding method according to the type of label and the detection method. There are various label detection methods such as visual observation, detection by spectroscopic prescription, and radioactivity count, and those skilled in the art can appropriately select a detection method.
次に、得られた複合体と固相に固定化されたモノクローナル抗体４Ｂ４とを反応させる。上記複合体が存在する場合には、固相に固定化されたモノクローナル抗体と上記複合体中のカンピロバクター表面タンパク質とが反応してさらなる複合体が形成される。モノクローナル抗体４Ｂ４の固相への固定化方法は公知であり、検出手段・方法に応じて適宜選択されうる。典型的な固定化方法としては、例えば、物理的吸着法、化学的結合法など挙げられる。上記さらなる複合体の検出方法としては、例えばイムノクロマト法やサンドイッチＥＬＩＳＡ法などが一般的であり、本発明においても使用可能である。また、ラテックス粒子にモノクローマル抗体４Ｂ４を結合させて、これを試料と混合し、顕微鏡観察により凝集の有無を調べることも可能である。本発明における検出方法としては、イムノクロマト法が迅速性の点から好ましいが、これらの方法に限られず、当業者は様々な方法を適用することができる。 Next, the obtained complex is reacted with the monoclonal antibody 4B4 immobilized on the solid phase. When the complex is present, the monoclonal antibody immobilized on the solid phase reacts with the Campylobacter surface protein in the complex to form a further complex. The method for immobilizing the monoclonal antibody 4B4 on the solid phase is known and can be appropriately selected depending on the detection means and method. Examples of typical immobilization methods include physical adsorption methods and chemical bonding methods. As a method for detecting the further complex, for example, an immunochromatography method or a sandwich ELISA method is generally used and can also be used in the present invention. It is also possible to bind the monoclonal antibody 4B4 to latex particles, mix this with a sample, and examine the presence or absence of aggregation by microscopic observation. As a detection method in the present invention, immunochromatography is preferable from the viewpoint of rapidity, but is not limited to these methods, and those skilled in the art can apply various methods.
本発明のカンピロバクターの検出方法において、便試料を界面活性剤にて処理してから、モノクローナル抗体４Ｂ４と接触させてもよい。モノクローナル抗体４Ｂ４と反応するカンピロバクターの菌体表面タンパク質は、界面活性剤処理により容易に菌体から遊離される性質を有する。使用されうる界面活性剤としては特に制限はないが、モノクローナル抗体４Ｂ４により認識されなくなるほどカンピロバクターの菌体表面タンパク質の構造を変化させるものは好ましくない。界面活性剤としては陰イオン系界面活性剤（脂肪酸塩、アルファスルホ脂肪酸エステルナトリウムなど）、陽イオン系界面活性剤（アルキルトリメチルアンモニウム塩、ジアルキルジメチルアンモニウム塩など）、両性イオン界面活性剤（アルキルアミノ脂肪酸塩、アルキルベタイン、アルキルアミンオキシドなど）、非イオン系界面活性剤（ショ糖脂肪酸エステルソルビタン脂肪酸エステル、ポリオキシエチレンソルビタン脂肪酸エステル、脂肪酸アルカノールアミドなど）があり、いずれの種類の界面活性剤でも使用可能である。多種多様な界面活性剤が市販されている。例えば、商品名ノニデット、トライトンＸ、プルロニック、ツインなどの商品名で市販されているものがある。使用される界面活性剤の濃度は、界面活性剤の種類によって異なるが、当業者であれば容易に決定しうる。なお、界面活性剤を有効成分とするタンパク質抽出剤を本発明に使用することもできる。本明細書において、界面活性剤を有効成分とするタンパク質抽出剤を「界面活性剤」に含めることとする。界面活性剤を有効成分とするタンパク質抽出剤は多くの種類があり、市販されている。界面活性剤を有効成分とする市販のタンパク質抽出剤としてはB-PER Bacterial Protein Extraction Reagent（PIERCE, Rockford, IL）、B-PER II Bacterial Protein Extraction Reagent（PIERCE）、BugBuster Protein Extraction Reagent（EMD Chemicals, San Diego, CA）などが例示される。 In the Campylobacter detection method of the present invention, the stool sample may be treated with a surfactant and then contacted with the monoclonal antibody 4B4. The cell surface protein of Campylobacter that reacts with the monoclonal antibody 4B4 has a property that it can be easily released from the cell by treatment with a surfactant. There are no particular limitations on the surfactant that can be used, but those that change the structure of the cell surface protein of Campylobacter so that it is not recognized by the monoclonal antibody 4B4 are not preferred. Surfactants include anionic surfactants (fatty acid salts, sodium alphasulfo fatty acid ester, etc.), cationic surfactants (alkyltrimethylammonium salts, dialkyldimethylammonium salts, etc.), zwitterionic surfactants (alkylamino) Fatty acid salts, alkylbetaines, alkylamine oxides, etc.), nonionic surfactants (sucrose fatty acid ester sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, fatty acid alkanolamide, etc.), any type of surfactant It can be used. A wide variety of surfactants are commercially available. For example, there are those marketed under trade names such as trade names Nonidet, Triton X, Pluronic, and Twin. The concentration of the surfactant used varies depending on the type of the surfactant, but can be easily determined by those skilled in the art. In addition, the protein extractant which uses surfactant as an active ingredient can also be used for this invention. In the present specification, a protein extractant containing a surfactant as an active ingredient is included in the “surfactant”. There are many types of protein extractants containing surfactants as active ingredients and are commercially available. Commercial protein extractants with surfactants as active ingredients include B-PER Bacterial Protein Extraction Reagent (PIERCE, Rockford, IL), B-PER II Bacterial Protein Extraction Reagent (PIERCE), BugBuster Protein Extraction Reagent (EMD Chemicals, San Diego, CA).
便中のカンピロバクターを免疫学的分析方法を用いて検出する場合、水または適当な緩衝液で希釈していわゆる乳剤状態とし、遠心分離を行うことにより滓を除去してからカンピロバクター検出手順に供するのが望ましいが、遠心分離によって大部分の菌体も滓とともに除去されてしまい、検出感度が低下するという問題がある。しかし、モノクローナル抗体４Ｂ４と反応するカンピロバクターの菌体表面タンパク質は、界面活性剤処理により容易に菌体から遊離される性質を有するので、界面活性剤処理後に遠心分離を行うことにより、菌体が滓とともに除去されても表面タンパク質は上清中に残り、上清を以後の検出操作に用いることで検出感度の向上を図ることができる。 When detecting Campylobacter in stool using an immunological analysis method, dilute it with water or a suitable buffer to form a so-called emulsion, and remove the sputum by centrifugation and use it for the Campylobacter detection procedure. However, there is a problem that most of the cells are removed together with the sputum by centrifugation and the detection sensitivity is lowered. However, since the cell surface protein of Campylobacter that reacts with the monoclonal antibody 4B4 has a property that it can be easily released from the cell by the surfactant treatment, the cell is removed by centrifugation after the surfactant treatment. Even if it is removed together, the surface protein remains in the supernatant, and the detection sensitivity can be improved by using the supernatant for the subsequent detection operation.
界面活性剤を用いる代わりに、上記の乳剤状態の試料をサンプルパッドなどのフィルターに適用し、滓を除去することもできる。この場合、界面活性剤は使用しても、使用しなくてもよい（表面タンパク質が菌体に付いたままであっても、イムノクロマト法や他の方法にて検出可能な場合がある）。 Instead of using a surfactant, the above emulsion sample can be applied to a filter such as a sample pad to remove wrinkles. In this case, the surfactant may or may not be used (even if the surface protein remains attached to the microbial cells, it may be detectable by immunochromatography or other methods).
これらの前処理方法はどのような試料にも適用可能であるが、試料がヒトの便である場合に特に効果的である。 These pretreatment methods can be applied to any sample, but are particularly effective when the sample is human feces.
本発明は、もう１つの態様において、上で説明した本発明の方法を用いてカンピロバクターを検出するためのキットに関するものである。本発明のキットはモノクローナル抗体４Ｂ４を必須構成成分として含むものである。本発明のキットは、例えば、イムノクロマト用のパーツ、試料処理のための界面活性剤、あるいはサンプルパッドなどを含んでいてもよい。本発明を構成する成分はこれらのものには限定されない。本発明のキットはハイスループット検査用であってもよい。通常、キットには取扱説明書が添付される。 In another aspect, the present invention relates to a kit for detecting Campylobacter using the method of the present invention described above. The kit of the present invention contains monoclonal antibody 4B4 as an essential component. The kit of the present invention may contain, for example, parts for immunochromatography, a surfactant for sample processing, or a sample pad. The components constituting the present invention are not limited to these. The kit of the present invention may be for high-throughput testing. Usually, an instruction manual is attached to the kit.
以下に実施例を示して本発明をより詳細かつ具体的に説明するが、実施例はあくまでも例示説明のためのものであって、本発明の範囲を限定するものではない。 The present invention will be described in more detail and specifically with reference to the following examples. However, the examples are for illustrative purposes only and do not limit the scope of the present invention.
４Ｂ４モノクローナル抗体の作製
（Ｉ）菌体抽出抗原の調製
下記手順により細菌を培養し、抗原を抽出した。
（１）寒天培地シャーレ１枚分の培養細菌をリン酸緩衝食塩水（ＰＢＳ）１ｍｌに懸濁した。
（２）１５０００ｒｐｍで５℃、５分間遠心した。
（３）上清を捨てた後、沈渣をＰＢＳ １ｍｌに再度懸濁した。
（４）１５０００ｒｐｍで５℃、５分間遠心した。
（５）上清を捨てた後、沈渣をB-PER II Bacterial Protein Extraction Reagent（PIERCE, Rockford, IL）１ｍｌに懸濁した。
（６）懸濁液を室温で１５分間攪拌した。
（７）１５０００ｒｐｍで５℃、５分間遠心し、その上清を菌体抽出抗原とした。
Production of 4B4 monoclonal antibody
(I) Preparation of cell-extracted antigen Bacteria were cultured according to the following procedure to extract the antigen .
(1) The cultured bacteria for one agar medium petri dish were suspended in 1 ml of phosphate buffered saline (PBS).
(2) Centrifugation was performed at 15000 rpm at 5 ° C. for 5 minutes.
(3) After discarding the supernatant, the sediment was resuspended in 1 ml of PBS.
(4) The mixture was centrifuged at 15000 rpm at 5 ° C. for 5 minutes.
(5) After discarding the supernatant, the sediment was suspended in 1 ml of B-PER II Bacterial Protein Extraction Reagent (PIERCE, Rockford, IL).
(6) The suspension was stirred at room temperature for 15 minutes.
(7) Centrifugation was performed at 15000 rpm at 5 ° C. for 5 minutes, and the supernatant was used as a cell-extracted antigen.
（ＩＩ）マウスの免疫
下記手順によりカンピロバクター抽出抗原を用いてマウスを免疫した。
（１）C. jejuni（Ｌｉｏｒ血清型７）の臨床分離株を使って調製した菌体抽出抗原（蛋白濃度１．２ｍｇ／ｍｌ）とFreund's complete adjuvantの等容量混合乳濁液を５匹のＢＡＬＢ／ｃマウス（ＳＰＦ−ｇｒａｄｅ，雌、８週齢）のそれぞれの腹腔内に０．１ｍｌ接種した。
（２）３、５、７及び１１週間後、同じ菌体抽出抗原とFreund's incomplete adjuvant等容量混合乳濁液をそれぞれのマウスの腹腔内に０．１ｍｌ追加免疫した。
（３）最後の追加免疫から２週間後、それぞれのマウスの眼底静脈叢から採血し、血清抗体の力価を、酵素免疫測定法（ＥＬＩＳＡ、詳細は（ＩＩＩ）に後記）により測定し、高度免疫されたマウスを選抜した。
（４）細胞融合３日前に、最も高いＥＬＩＳＡ抗体価を示した免疫マウスの腹腔内に、菌体抽出抗原のみを０．５ｍｌ（０．６ｍｇ）を最終免疫した。
(II) Immunization of mice Mice were immunized with Campylobacter extracted antigen by the following procedure.
(1) Equal volume mixed emulsion of bacterial extract antigen (protein concentration 1.2 mg / ml) and Freund's complete adjuvant prepared using a clinical isolate of C. jejuni (Lior serotype 7) 0.1 ml was inoculated into the abdominal cavity of each / c mouse (SPF-grade, female, 8 weeks old).
(2) After 3, 5, 7 and 11 weeks, 0.1 ml of the same bacterial cell-extracted antigen and Freund's incomplete adjuvant equal volume mixed emulsion was boosted intraperitoneally into each mouse.
(3) Two weeks after the last booster, blood was collected from the fundus venous plexus of each mouse, and the serum antibody titer was measured by enzyme immunoassay (ELISA, details are described later in (III)). Immunized mice were selected.
(4) Three days before cell fusion, 0.5 ml (0.6 mg) of the bacterial cell-extracted antigen alone was finally immunized into the peritoneal cavity of the immunized mouse that showed the highest ELISA antibody titer.
（ＩＩＩ）ＥＬＩＳＡによる免疫マウスの血清抗体価の測定
上で得られたマウスの血清抗体価を、以下の手順により測定した。
（１）免疫抗原（C. jejuni（Ｌｉｏｒ血清型７）の臨床分離株を使って調製した菌体抽出抗原）を０．０５Ｍ炭酸バッファー ｐＨ９．６で０．０１ｍｇ／ｍｌに希釈して、マイクロタイタープレートのウエルに０．１ｍｌ分注し、４℃で一夜放置した。
（２）抗原液をプレートから除去した後、ブロッキング液（２０％馬血清を含むＰＢＳ）を０．１５ｍｌ加えて室温１時間反応させた。
（３）ブロッキング液を除去した後、洗浄液（０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳ）で３回洗浄した。
（４）血清試料の１０倍階段希釈液（マウス血清を、２０％馬血清と０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳで希釈したもの）を０．１ｍｌ加えて、室温１時間反応させた。
（５）洗浄液で４回洗浄後、ペルオキシダーゼ標識抗マウスＩｇＧを２０％馬血清と０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳで１０００倍に希釈したものを０．１ｍｌ加えて、室温１時間反応させた。
（６）洗浄液で４回洗浄後、酵素基質（1-Step Ultra TMB-ELISA:PIERCE）を０．１ｍｌ加えて、室温１０分間反応させた後、反応停止液（１Ｍ硫酸）を０．１ｍｌ加えてから、吸光度の自動計測装置を用いて４５０ｎｍにおける吸光度を計測した。
(III) Measurement of serum antibody titer of immunized mouse by ELISA The serum antibody titer of the mouse obtained above was measured by the following procedure.
(1) An immune antigen (a cell extract antigen prepared using a clinical isolate of C. jejuni (Lior serotype 7)) is diluted to 0.01 mg / ml with 0.05 M carbonate buffer pH 9.6 to obtain micro 0.1 ml was dispensed into the well of the titer plate and left at 4 ° C. overnight.
(2) After removing the antigen solution from the plate, 0.15 ml of blocking solution (PBS containing 20% horse serum) was added and reacted at room temperature for 1 hour.
(3) After removing the blocking solution, the plate was washed 3 times with a washing solution (PBS containing 0.05% Tween 20).
(4) A 10-fold serial dilution of a serum sample (mouse serum diluted with 20% horse serum and PBS containing 0.05% Tween 20) was added in an amount of 0.1 ml and allowed to react for 1 hour at room temperature.
(5) After washing 4 times with the washing solution, 0.1 ml of peroxidase-labeled anti-mouse IgG diluted 1000 times with PBS containing 20% horse serum and 0.05% Tween 20 was added and allowed to react at room temperature for 1 hour.
(6) After washing 4 times with the washing solution, 0.1 ml of enzyme substrate (1-Step Ultra TMB-ELISA: PIERCE) is added and reacted at room temperature for 10 minutes, and then 0.1 ml of reaction stop solution (1M sulfuric acid) is added. Then, the absorbance at 450 nm was measured using an automatic absorbance measuring apparatus.
（ＩＶ）細胞融合
下記手順に従って、免疫マウスの脾臓細胞とミエローマ細胞を融合させた。
（１）最終免疫から３日後、マウスから脾臓を取り出し、Pre-Fusion Medium（StemCell Technology, Vancouver, BC, Canada）を入れたシャーレに置き、洗浄した。
（２）洗浄後の脾臓をPre-Fusion Mediumの入った別のシャーレに移し、ピンセットでホモジナイズした。
（３）脾臓のホモジナイズ液をナイロンメッシュで濾過し、均一な細胞懸濁液を得た。
（４）マウスリンパ球比重分離液（日本抗体研究所）を使って赤血球を除去した後、Pre-Fusion Mediumで３回遠心洗浄した（１６００ｒｐｍ，６分×３回）。
（５）３回洗浄後に、Fusion Medium（StemCell Technologies）２５ｍｌに懸濁し、それをＰＢＳで１０倍希釈したものの細胞数を測定した。（１匹の免疫マウスより、約１×１０８個の脾臓細胞が得られた）
（６）脾臓細胞懸濁液（約１×１０８個の脾臓細胞をFusion Mediumの２５ｍｌに懸濁したもの）に２×１０７個のミエローマ細胞（Ｐ３−Ｘ６３−Ａｇ８．Ｕ１）を加えた（細胞比；脾臓細胞：ミエローマ細胞＝５：１）後、遠心した（１６００ｒｐｍ，６分）。
（７）遠心後、血清成分を完全に除去するために、予め３７℃に保温したFusion Medium ２５ｍｌで３回遠心洗浄した（１６００ｒｐｍ，６分）。
（８）細胞沈渣をFusion Medium １０ｍｌに浮遊させ、１０ｍｌ丸底チューブに移して１６００ｒｐｍ，６分遠心した。
（９）遠心後、上清を完全に除去した。
（１０）細胞をタッピングで十分にほぐした後、１ｍｌの４０％ＰＥＧ溶液（４０ｇのＰＥＧ−４０００を５０ｍｌのＰＢＳに溶解後、１０ｍｌのＤＭＳＯと１ｍｌのポリ−Ｌ−アルギニン塩酸塩水溶液（１ｍｇ／ｍｌ）を加えて、最後に１Ｎ ＨＣｌを加えてｐＨを約７．８に調製したもの）をピペットの先端で細胞を緩やかに攪拌しながら１分間かけて添加した。
（１１）更に１分間、細胞を攪拌した。
（１２）Fusion Medium １ｍｌをピペットの先端で細胞を緩やかに攪拌しながら１分間かけて添加した。
（１３）Fusion Medium １ｍｌをピペットの先端で細胞を緩やかに攪拌しながら１分間かけて添加した。
（１４）Fusion Medium ７ｍｌをピペットの先端で細胞を緩やかに攪拌しながら（１ｍｌずつ７回に分けて）３分間かけて添加した。
（１５）１０００ｒｐｍ，５分遠心し上清を捨てた。
（１６）細胞をタッピングで優しくほぐした後、Pre-Fusion Medium １０ｍｌに懸濁し１０００ｒｐｍ，５分遠心した。
（１７）１０００ｒｐｍ，５分遠心し上清を捨てた。
（１８）細胞をタッピングで優しくほぐした後、Hybridoma Recovery Medium（StemCell Technologies）１０ｍｌに懸濁する。
（１９）懸濁液１０ｍｌをHybridoma Recovery Mediumが４０ｍｌ入った中フラスコに移した。
（２０）３７℃で１８時間培養した。
(IV) Cell fusion According to the following procedure, spleen cells and myeloma cells of immunized mice were fused.
(1) Three days after the final immunization, the spleen was removed from the mouse, placed in a petri dish containing Pre-Fusion Medium (StemCell Technology, Vancouver, BC, Canada), and washed.
(2) The washed spleen was transferred to another petri dish containing Pre-Fusion Medium and homogenized with tweezers.
(3) The homogenized spleen solution was filtered through a nylon mesh to obtain a uniform cell suspension.
(4) After removing erythrocytes using mouse lymphocyte specific gravity separation liquid (Nippon Antibody Laboratories), it was washed with Pre-Fusion Medium three times (1600 rpm, 6 minutes × 3 times).
(5) After washing three times, the cells were suspended in 25 ml of Fusion Medium (StemCell Technologies) and diluted 10-fold with PBS to measure the number of cells. (About 1 × 10 8 spleen cells were obtained from one immunized mouse)
(6) 2 × 10 7 myeloma cells (P3-X63-Ag8.U1) were added to a spleen cell suspension (approximately 1 × 10 8 spleen cells suspended in 25 ml of Fusion Medium). (Cell ratio; spleen cells: myeloma cells = 5: 1) and then centrifuged (1600 rpm, 6 minutes).
(7) After centrifugation, in order to completely remove serum components, centrifugal washing was performed three times with 25 ml of Fusion Medium previously kept at 37 ° C. (1600 rpm, 6 minutes).
(8) The cell sediment was suspended in 10 ml of Fusion Medium, transferred to a 10 ml round bottom tube, and centrifuged at 1600 rpm for 6 minutes.
(9) After centrifugation, the supernatant was completely removed.
(10) After sufficiently loosening the cells by tapping, 1 ml of 40% PEG solution (40 g of PEG-4000 dissolved in 50 ml of PBS, 10 ml of DMSO and 1 ml of poly-L-arginine hydrochloride aqueous solution (1 mg / ml) and finally 1N HCl added to adjust the pH to about 7.8) was added over 1 minute while gently stirring the cells with the tip of the pipette.
(11) The cells were further stirred for 1 minute.
(12) 1 ml of Fusion Medium was added over 1 minute while gently stirring the cells with the tip of a pipette.
(13) 1 ml of Fusion Medium was added over 1 minute while gently stirring the cells with the tip of a pipette.
(14) 7 ml of Fusion Medium was added over 3 minutes while gently stirring the cells with the tip of the pipette (divided into 7 portions of 1 ml each).
(15) Centrifuge at 1000 rpm for 5 minutes and discard the supernatant.
(16) The cells were gently loosened by tapping, then suspended in 10 ml of Pre-Fusion Medium and centrifuged at 1000 rpm for 5 minutes.
(17) The solution was centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded.
(18) The cells are gently loosened by tapping and then suspended in 10 ml of Hybridoma Recovery Medium (StemCell Technologies).
(19) 10 ml of the suspension was transferred to a medium flask containing 40 ml of Hybridoma Recovery Medium.
(20) The cells were cultured at 37 ° C. for 18 hours.
（Ｖ）選択培養とクローニング
上で得られた融合細胞を、下記手順にてり選択培養し、クローニングを行った。
（１）３７℃で１８時間培養した融合細胞懸濁液（５０ｍｌ）を１６００ｒｐｍ，６分遠心した。
（２）細胞沈渣をタッピングで優しくほぐした後、Hybridoma Recovery Mediumの１０ｍｌに懸濁した。
（３）懸濁液１０ｍｌをHybridoma Selection Medium（ヒポキサンチン・チミジン・アミノプテリン添加軟寒天培地、StemCell Technologies）が９０ｍｌ入ったボトルに移し、なるべく泡立たないようにして、ボトルを数回逆さに反転させながら中身を完全に混ぜ合わせた。
（４）ボトルの蓋を完全に閉めて、３７℃に１５分間静置した。
（５）２５枚の６０ｍｍペトリデイッシュのそれぞれに４ｍｌずつ分注した。
（６）３７℃、５％ＣＯ２で１０〜１３日間培養した。
(V) Selection culture and cloning The fused cells obtained by cloning were subjected to selection culture and cloning according to the following procedure.
(1) A fusion cell suspension (50 ml) cultured at 37 ° C. for 18 hours was centrifuged at 1600 rpm for 6 minutes.
(2) The cell sediment was gently loosened by tapping and then suspended in 10 ml of Hybridoma Recovery Medium.
(3) Transfer 10 ml of the suspension to a bottle containing 90 ml of Hybridoma Selection Medium (soft agar medium containing hypoxanthine / thymidine / aminopterin, StemCell Technologies), and invert the bottle several times to avoid bubbles as much as possible. While mixing the contents completely.
(4) The bottle lid was completely closed, and the bottle was allowed to stand at 37 ° C. for 15 minutes.
(5) 4 ml was dispensed to each of 25 60 mm Petri dishes.
(6) The cells were cultured at 37 ° C. and 5% CO 2 for 10 to 13 days.
（ＶＩ）コロニーの採取と陽性クローンの検出
上で得られた融合細胞のコロニーを採取し、ＥＬＩＳＡにて陽性クローンを検出し、再クローニングした。手順は下記のとおり。
（１）培養開始後１０〜１４日の間に、各ペトリデイッシュから融合細胞コロニーを採取した。
（２）２０μｌにセットした２０μｌピペットマンを使って融合細胞コロニーを採取し、Hybridoma Groth Medium（ヒポキサンチン・チミジン添加培地、StemCell Technologies）が２００μｌ／ウエル入った９６ウエルマイクロプレートのウエルに移した。
（３）最終的に、融合細胞を培養した２５枚のペトリデイッシュから、計１７１４個の融合細胞コロニーを採取し、Hybridoma Growth Mediumが２００μｌ／ウエル入った９６ウエルマイクロプレートの各ウエルにそれぞれ移した。
（４）３７℃、５％ ＣＯ２で４〜６日間培養した。
（５）培養後、各培養上清に含まれる免疫抗原に対する抗体価をＥＬＩＳＡ（詳細は（ＶＩＩ）に後記）で測定した。
（６）ＥＬＩＳＡで陽性であった５５ウエルの細胞懸濁液のそれぞれをHybridoma Growth Mediumが１ｍｌ／ウエル入った２４ウエルプレートの各ウエルに移し３７℃、５％ ＣＯ２で３〜４日間培養した。
（７）培養後、各培養上清に含まれる抗体の各種菌由来の菌体抽出抗原に対する反応性をＥＬＩＳＡ（詳細は（８）に後記）を用いて検討した。
（８）ＥＬＩＳＡの結果、培養上清がC. jejuni及びC. coliから調製された菌体抽出抗原には陽性反応を示したが、その他の菌種から調製された菌体抽出抗原には反応を示さなかった１２ウエルについて、その中の融合細胞（ハイブリドーマ）を再クローニングした。
(VI) Collecting colonies and detecting positive clones Colonies of the fused cells obtained were collected, positive clones were detected by ELISA, and recloned. The procedure is as follows.
(1) Between 10 and 14 days after the start of culture, a fused cell colony was collected from each petri dish.
(2) A fused cell colony was collected using a 20 μl pipetman set to 20 μl, and transferred to a well of a 96-well microplate containing 200 μl / well of Hybridoma Groth Medium (medium supplemented with hypoxanthine / thymidine, StemCell Technologies).
(3) Finally, a total of 1714 fused cell colonies were collected from 25 Petri dishes in which the fused cells were cultured, and transferred to each well of a 96-well microplate containing 200 μl / well of Hybridoma Growth Medium. did.
(4) The cells were cultured at 37 ° C. and 5% CO 2 for 4 to 6 days.
(5) After the culture, the antibody titer against the immunizing antigen contained in each culture supernatant was measured by ELISA (details are described later in (VII)).
(6) Each of the 55-well cell suspensions that were positive by ELISA was transferred to each well of a 24-well plate containing 1 ml / well of Hybridoma Growth Medium and cultured at 37 ° C., 5% CO 2 for 3-4 days. .
(7) After the culture, the reactivity of the antibody contained in each culture supernatant to the cell-derived antigen extracted from various bacteria was examined using ELISA (details are described later in (8)).
(8) As a result of ELISA, the culture supernatant showed a positive reaction to cell extract antigens prepared from C. jejuni and C. coli, but reacted to cell extract antigens prepared from other species. For the 12 wells that did not show any, the fusion cells (hybridomas) therein were recloned.
（ＶＩＩ）ＥＬＩＳＡによる培養上清中の抗体価の測定
上で得られたハイブリドーマ培養上清中の抗体価を、下記手順にて測定した。
（１）免疫抗原（C. jejuni（Ｌｉｏｒ血清型７）の臨床分離株を使って調製した菌体抽出抗原）を０．０５Ｍ炭酸バッファー ｐＨ９．６で０．０１ｍｇ／ｍｌに希釈して、マイクロタイタープレートのウエルに０．１ｍｌ分注し、４℃で一夜放置した。
（２）抗原液をプレートから除去した後、ブロッキング液（２０％馬血清を含むＰＢＳ）を０．１５ｍｌ加えて室温１時間反応させた。
（３）ブロッキング液を除去した後、洗浄液（０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳ）で３回洗浄した。
（４）培養上清の１０倍希釈液（マウス血清を、２０％馬血清と０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳで希釈したもの）を０．１ｍｌ加えて、室温１時間反応させた。
（５）洗浄液で４回洗浄後、ペルオキシダーゼ標識抗マウスＩｇＧを２０％馬血清と０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳで１０００倍に希釈したものを０．１ｍｌ加えて、室温１時間反応させた。
（６）洗浄液で４回洗浄後、酵素基質（1-Step Ultra TMB-ELISA:PIERCE）を０．１ｍｌ加えて、室温１０分間反応させた後、反応停止液（１Ｍ硫酸）を０．１ｍｌ加えてから、吸光度の自動計測装置を用いて４５０ｎｍにおける吸光度を計測した。
(VII) Measurement of antibody titer in culture supernatant by ELISA The antibody titer in the hybridoma culture supernatant obtained above was measured by the following procedure.
(1) An immune antigen (a cell extract antigen prepared using a clinical isolate of C. jejuni (Lior serotype 7)) is diluted to 0.01 mg / ml with 0.05 M carbonate buffer pH 9.6 to obtain micro 0.1 ml was dispensed into the well of the titer plate and left at 4 ° C. overnight.
(2) After removing the antigen solution from the plate, 0.15 ml of blocking solution (PBS containing 20% horse serum) was added and reacted at room temperature for 1 hour.
(3) After removing the blocking solution, the plate was washed 3 times with a washing solution (PBS containing 0.05% Tween 20).
(4) 0.1 ml of 10-fold diluted culture supernatant (mouse serum diluted with PBS containing 20% horse serum and 0.05% Tween 20) was added and allowed to react at room temperature for 1 hour.
(5) After washing 4 times with the washing solution, 0.1 ml of peroxidase-labeled anti-mouse IgG diluted 1000 times with PBS containing 20% horse serum and 0.05% Tween 20 was added and allowed to react at room temperature for 1 hour.
(6) After washing 4 times with the washing solution, 0.1 ml of enzyme substrate (1-Step Ultra TMB-ELISA: PIERCE) is added and reacted at room temperature for 10 minutes, and then 0.1 ml of reaction stop solution (1M sulfuric acid) is added. Then, the absorbance at 450 nm was measured using an automatic absorbance measuring apparatus.
（ＶＩＩＩ） ＥＬＩＳＡによる培養上清中の抗体の各種菌由来の菌体抽出抗原に対する反応性の検討
上記ハイブリドーマ培養上清中の抗体の各種細菌由来の抗原に対する反応性を、下記手順にて調べた。
（１）C. jejuni ３株（Ｌｉｏｒ血清型２，７及び９）、C. coli １株、Aeromonas hydrophira、Enterobacter aerogenes、Proteus vulgaris、Pseudomonas aeruginosa、Salmonella enteritidis及びVibrio cholera各１株からそれぞれ菌体抽出抗原を調製した。それぞれの菌体抽出抗原を０．０５Ｍ炭酸バッファー ｐＨ９．６で０．０１ｍｇ／ｍｌに希釈して、マイクロタイタープレートのウエルに０．１ｍｌ分注し、４℃で一夜放置した。
（２）抗原液をプレートから除去した後、ブロッキング液（２０％馬血清を含むＰＢＳ）を０．１５ｍｌ加えて室温１時間反応させた。
（３）ブロッキング液を除去した後、洗浄液（０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳ）で３回洗浄した。
（４）培養上清の１０倍希釈液（マウス血清を、２０％馬血清と０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳで希釈したもの）を０．１ｍｌ加えて、室温１時間反応させた。
（５）洗浄液で４回洗浄後、ペルオキシダーゼ標識抗マウスＩｇＧを２０％馬血清と０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳで１０００倍に希釈したものを０．１ｍｌ加えて、室温１時間反応させた。
（６）洗浄液で４回洗浄後、酵素基質（1-Step Ultra TMB-ELISA:PIERCE）を０．１ｍｌ加えて、室温１０分間反応させた後、反応停止液（１Ｍ硫酸）を０．１ｍｌ加えてから、吸光度の自動計測装置を用いて４５０ｎｍにおける吸光度を計測した。
(VIII) Examination of reactivity of antibody in culture supernatant to various cell-derived antigens by ELISA by ELISA The reactivity of antibody in hybridoma culture supernatant to antigens from various bacteria was examined by the following procedure. .
(1) Cell extracts from 3 strains of C. jejuni (Lior serotypes 2, 7 and 9), 1 strain of C. coli, Aeromonas hydrophira, Enterobacter aerogenes, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella enteritidis and Vibrio cholera Antigen was prepared. Each bacterial cell extract antigen was diluted to 0.01 mg / ml with 0.05 M carbonate buffer pH 9.6, dispensed 0.1 ml into the well of a microtiter plate, and left overnight at 4 ° C.
(2) After removing the antigen solution from the plate, 0.15 ml of blocking solution (PBS containing 20% horse serum) was added and reacted at room temperature for 1 hour.
(3) After removing the blocking solution, the plate was washed 3 times with a washing solution (PBS containing 0.05% Tween 20).
(4) 0.1 ml of 10-fold diluted culture supernatant (mouse serum diluted with PBS containing 20% horse serum and 0.05% Tween 20) was added and allowed to react at room temperature for 1 hour.
(5) After washing 4 times with the washing solution, 0.1 ml of peroxidase-labeled anti-mouse IgG diluted 1000 times with PBS containing 20% horse serum and 0.05% Tween 20 was added and allowed to react at room temperature for 1 hour.
(6) After washing 4 times with the washing solution, 0.1 ml of enzyme substrate (1-Step Ultra TMB-ELISA: PIERCE) is added and reacted at room temperature for 10 minutes, and then 0.1 ml of reaction stop solution (1M sulfuric acid) is added. Then, the absorbance at 450 nm was measured using an automatic absorbance measuring apparatus.
（ＩＸ）再クローニング
上記ＥＬＩＳＡにて選抜した１２個のクローンにつて、培養規模を拡大して培養を行った。手順は下記のとおり。
（１）ＥＬＩＳＡ（詳細は（ＶＩＩＩ）に前記）の結果により選抜された１２ウエル中のそれぞれのハイブリドーマクローンについて、細胞数を測定し、Pre-Fusion Mediumを使って、細胞濃度３００個／ｍｌの細胞懸濁液を調整した。
（２）その０．５ｍｌ（細胞数１５０個）をHybridoma Selection Medium ９．５ｍｌと混合し、３７℃、５％ ＣＯ２に１５分間静置した。
（３）それを２枚の６０ｍｍペトリデイッシュに４ｍｌ／ペトリデイッシュで泡立たないようにして分注し、残りは捨てた。
（４）３７℃、５％ ＣＯ２で１０〜１４日間培養した。
（５）１０〜１４日間後、各ペトリデイッシュのコロニーを肉眼で観察した。
（６）２０μｌにセットした２０μｌピペットマンを使って各コロニーを採取し、Hybridoma Growth Mediumが２００ｕｌ／ウエル入った９６ウエルマイクロプレートの各ウエルに移す。コロニーは、１クローン（ペトリデイッシュ２枚）につき大きいものから順に計２４個採取した。
（７）３７℃、５％ ＣＯ２で３〜４日間培養した。
（８）上清を採取し、ＥＬＩＳＡ（詳細は（７）に前記）で抗体価を測定した。
（９）培養上清のＥＬＩＳＡ抗体価高く、かつ細胞の増殖状態の良いものを、１クローンにつき１ウエル選抜し、Hybridoma Growth Mediumが１ｍｌ／ウエル入った２４ウエルプレートのウエルに移し３７℃、５％ ＣＯ２で３〜４日間培養した。
（１０）培養後、適当量のHybridoma Growth Mediumに懸濁（細胞濃度１〜２×１０５個／ｍｌに調整）して培養規模を拡大し、３７℃、５％ ＣＯ２で２日間培養した。
（１１）培養後、適当量のHybridoma Growth Mediumに懸濁（細胞濃度１〜２×１０５個／ｍｌに調整）して培養規模を拡大し、３７℃、５％ ＣＯ２で２日間培養した。
（１２）培養後、Hybridoma Expantion Medium（StemCell Technologies）／Hybridoma Growth Mediumの５０％混合液の適当量に懸濁（細胞濃度１〜２×１０５個／ｍｌに調整）して、３７℃、５％ ＣＯ２で２日間培養した。
（１３）Hybridoma Expantion Mediumを用いて順次、培養規模を拡大し、適当な時期に、細胞を凍結保存した。
（注）Hybridoma Expantion Mediumは、２０％ ＦＣＳ、２ｍＭ グルタミン、０．０５ｍＭ ２−メルカプトエタノール、トランスフェリン ０．００６ｍｇ／ｍｌ、インスリン ０．０２ｍｇ／ｍｌ及び抗生物質（ペニシリン １００単位／ｍｌ、ストレプトマイシン ０．１ｍｇ／ｍｌ、アンフォテリシンＢ ０．０００２５ｍｇ／ｍｌ）を含むイスコフ培地（Iscove's Modified Dulbecco's Medium）でも代用可能である。
(IX) Recloning The 12 clones selected by the above ELISA were cultured at an expanded culture scale. The procedure is as follows.
(1) The number of cells was measured for each hybridoma clone in 12 wells selected based on the result of ELISA (details are described in (VIII) above), and the cell concentration was 300 cells / ml using Pre-Fusion Medium. The cell suspension was adjusted.
(2) 0.5 ml (150 cells) was mixed with 9.5 ml of Hybridoma Selection Medium, and allowed to stand at 37 ° C., 5% CO 2 for 15 minutes.
(3) It was dispensed into two 60 mm Petri dishes without foaming at 4 ml / petri dish, and the rest was discarded.
(4) The cells were cultured at 37 ° C. and 5% CO 2 for 10 to 14 days.
(5) After 10 to 14 days, colonies of each petri dish were observed with the naked eye.
(6) Collect each colony using a 20 μl pipetman set to 20 μl and transfer it to each well of a 96-well microplate containing 200 ul / well of Hybridoma Growth Medium. A total of 24 colonies were collected in order from the largest for each clone (2 petri dishes).
(7) The cells were cultured at 37 ° C. and 5% CO 2 for 3 to 4 days.
(8) The supernatant was collected, and the antibody titer was measured by ELISA (for details, see (7) above).
(9) A culture supernatant with a high ELISA antibody titer and a good cell growth state was selected for each well, and transferred to a well of a 24-well plate containing 1 ml / well of Hybridoma Growth Medium at 37 ° C., 5 ° C. and cultured for 3-4 days in% CO 2.
(10) After culturing, the suspension was suspended in an appropriate amount of Hybridoma Growth Medium (adjusted to a cell concentration of 1-2 × 10 5 cells / ml) to expand the culture scale, and cultured at 37 ° C., 5% CO 2 for 2 days. .
(11) After culturing, the suspension was suspended in an appropriate amount of Hybridoma Growth Medium (cell concentration adjusted to 1-2 × 10 5 cells / ml) to expand the culture scale, and cultured at 37 ° C., 5% CO 2 for 2 days. .
(12) After culturing, the suspension is suspended in an appropriate amount of a 50% mixed solution of Hybridoma Expantion Medium (StemCell Technologies) / Hybridoma Growth Medium (adjusted to a cell concentration of 1 to 2 × 10 5 cells / ml) at 37 ° C., 5 Cultivated in% CO 2 for 2 days.
(13) The culture scale was sequentially expanded using Hybridoma Expantion Medium, and the cells were stored frozen at an appropriate time.
(Note) Hybridoma Expantion Medium is 20% FCS, 2 mM glutamine, 0.05 mM 2-mercaptoethanol, transferrin 0.006 mg / ml, insulin 0.02 mg / ml and antibiotics (
（Ｘ）モノクローナル抗体の大量調製
下記手順により、再クローニング後の１２クローンを大量培養して、モノクローナル抗体を得た。
（１）再クローニング後の１２クローンのそれぞれを、Hybridoma Expantion Mediumを用いて大量培養し、Fusion Mediumで２回洗浄して細胞浮遊液（２×１０７個／ｍｌ）を調製した。
（２）２４匹の８週齢の雄性ＢＡＬＢ／ｃマウスにプリスタン（２，６，１０，１４−テトラメチルペンタデカン，Aldrich Chemicals）０．５ｍｌを腹腔内注射し、一週間後更に０．５ｍｌを腹腔内注射した。
（３）約３日後に前記の細胞浮遊液を１ｍｌ腹腔内注射した（１クローンにつき、２匹のマウスに注射した）。
（４）約７〜１２日間で腹水が貯留して腹部が肥大したので、注射器により腹水採取した。こうしてマウス１匹当り５〜１０ｍｌの腹水が得られた。
（５）得られた腹水を冷凍遠心分離（４℃、３０００ｒｐｍ、１０分）して上清を集め、この上清に等容量の飽和硫酸アンモニア溶液（アンモニア水でｐＨ７．４に調製）を滴下して塩析を行った。
（６）高速遠心分離（４℃、１４，０００ｒｐｍ：１０，０００ｘＧ，３０分）して上清を除去し、塩析沈殿物を適当量のＰＢＳ（−）に溶解した。
(X) Large-scale preparation of monoclonal antibody By the following procedure, 12 clones after recloning were mass-cultured to obtain a monoclonal antibody.
(1) Each of the 12 clones after recloning was cultured in a large amount using Hybridoma Expantion Medium, washed twice with Fusion Medium to prepare a cell suspension (2 × 10 7 cells / ml).
(2) 24 male 8-week-old BALB / c mice were injected intraperitoneally with 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane, Aldrich Chemicals), and another week, 0.5 ml Intraperitoneal injection.
(3) After about 3 days, 1 ml of the cell suspension was injected intraperitoneally (two mice were injected per clone).
(4) Since ascites accumulated and the abdomen enlarged in about 7 to 12 days, ascites was collected with a syringe. In this way, 5 to 10 ml of ascites was obtained per mouse.
(5) The obtained ascites was frozen and centrifuged (4 ° C., 3000 rpm, 10 minutes) to collect the supernatant, and an equal volume of saturated ammonia sulfate solution (adjusted to pH 7.4 with aqueous ammonia) was dropped into this supernatant. And salting out.
(6) The supernatant was removed by high-speed centrifugation (4 ° C., 14,000 rpm: 10,000 × G, 30 minutes), and the salted out precipitate was dissolved in an appropriate amount of PBS (−).
（ＸＩ）大量調製したモノクローナル抗体の精製
上で大量調製されたモノクローナル抗体を、下記手順により精製した。
（１）塩析した抗体試料は、Protein A-Agaroseカラムクロマトグラフィーにより精製を行った。
（２）Protein A-Agarose充填カラム（ゲル５ｍｌ：Bio-Rad Laboratories, Hercules, CA）を５ベット容量の結合緩衝液（ｐＨ９．０：BioRad）で洗浄した。
（３）タンパク濃度約１０ｍｇ／ｍｌの抗体試料を前記結合緩衝液と等容量の割合で混合し、その５ｍｌを前記のカラムにアプライした。
（４）１５ベット容量の結合緩衝液によりカラムを洗浄した後、５ベット容量の溶出緩衝液（ｐＨ３．０：BioRad）によりマウスＩｇＧ画分を溶出した。溶出液のｐＨを中性に中和する目的で、溶出液１ｍｌ当り０．３ｍｌの１Ｍ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ９．０）を予め受器に入れておいた。
（５）精製抗体画分は分子篩（分子量：３０，０００）を用いる限外濾過により濃縮した後、ＰＤ−１０カラム（アマシャム）を用いるゲル濾過により溶媒をＰＢＳに置換した。
（６）得られた精製抗体溶液のタンパク濃度を５ｍｇ／ｍｌに調製した後、凍結保存した。
(XI) Purification of a large amount of monoclonal antibody A large amount of monoclonal antibody was purified by the following procedure.
(1) The salted out antibody sample was purified by Protein A-Agarose column chromatography.
(2) A Protein A-Agarose packed column (gel 5 ml: Bio-Rad Laboratories, Hercules, CA) was washed with 5 bed volumes of binding buffer (pH 9.0: BioRad).
(3) An antibody sample having a protein concentration of about 10 mg / ml was mixed with the binding buffer at an equal volume ratio, and 5 ml thereof was applied to the column.
(4) After washing the column with 15 bed volumes of binding buffer, the mouse IgG fraction was eluted with 5 bed volumes of elution buffer (pH 3.0: BioRad). In order to neutralize the pH of the eluate to neutral, 0.3 ml of 1M Tris-HCl buffer (pH 9.0) per 1 ml of eluate was previously placed in a receiver.
(5) The purified antibody fraction was concentrated by ultrafiltration using a molecular sieve (molecular weight: 30,000), and the solvent was replaced with PBS by gel filtration using a PD-10 column (Amersham).
(6) The protein concentration of the obtained purified antibody solution was adjusted to 5 mg / ml and then stored frozen.
（ＸＩＩ）イムノクロマト法に使用可能なモノクローナル抗体の選定
上で大量調製したモノクローナル抗体からイムノクロマト法に使用可能な抗体を選別し、そのアイソタイプを決定した。手順は下記のとおり。
（１）大量調製した１２種類のモノクローナル抗体のそれぞれを用いて、１２種類の抗体固相化メンブレン（作製法は後記）と１２種類の金コロイド標識抗体（作製法は後記）を作製した。
（２）１４４通りの抗体固相化メンブレンと金コロイド標識抗体の組み合わせのそれぞれについて、C. jejuni (Ｌｉｏｒ血清型７)の菌体抽出抗原を検出対象にしてイムノクロマト法を実施した。
（３）その結果、１４４通りの組み合わせの中で、１通りのみ（抗体固相化メンブレンも金コロイド標識抗体も、共に同じ抗体の組み合わせ）が陽性反応を示した。この抗体を４Ｂ４抗体と命名した。４Ｂ４抗体をカンピロバクターの迅速検出法に使用することにした。
(XII) Selection of monoclonal antibodies that can be used for immunochromatography The antibodies that can be used for immunochromatography were selected from the monoclonal antibodies that were prepared in large quantities, and their isotypes were determined. The procedure is as follows.
(1) Using each of the 12 types of monoclonal antibodies prepared in large quantities, 12 types of antibody-immobilized membranes (production method will be described later) and 12 types of gold colloid-labeled antibodies (production method will be described later) were prepared.
(2) For each of 144 combinations of antibody-immobilized membranes and colloidal gold-labeled antibodies, immunochromatography was performed using C. jejuni (Lior serotype 7) cell extract antigens as detection targets.
(3) As a result, of 144 combinations, only one (both antibody-immobilized membrane and colloidal gold labeled antibody were the same antibody combination) showed a positive reaction. This antibody was named 4B4 antibody. We decided to use the 4B4 antibody for rapid detection of Campylobacter.
この実験により、モノクローナル抗体４Ｂ４は、イムノクロマト法などのサンドイッチ法に、４Ｂ４抗体１種のみで使用可能であることがわかった。通常、イムノクロマト法などのサンドイッチ法には２種類以上の抗体を必要とするのが、それに適した優れた抗体を２種類以上作出することは容易ではない。従って、この４Ｂ４の性質は、イムノクロマト法などのサンドイッチ法を利用した菌の検出系の開発作業を容易なものとする。 From this experiment, it was found that the monoclonal antibody 4B4 can be used in a sandwich method such as an immunochromatography method with only one type of 4B4 antibody. Usually, sandwich methods such as immunochromatography require two or more types of antibodies, but it is not easy to produce two or more types of excellent antibodies suitable for them. Therefore, this 4B4 property facilitates the development of a bacteria detection system using a sandwich method such as an immunochromatography method.
上記実験で得られた、モノクローナル抗体４Ｂ４を産生するハイブリドーマ（ＣＪ−４Ｂ４）は、茨城県つくば市東１−１−１ つくばセンター 中央第６の、独立行政法人産業技術総合研究所 特許生物寄託センターに、２００７年９月１９日に受領され、受領番号ＦＥＲＭ ＡＰ−２１３６８を付与された。 The hybridoma (CJ-4B4) that produces the monoclonal antibody 4B4 obtained in the above experiment has been established at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, Tsukuba Center, 1-1-1, Tsukuba Center, Tsukuba City, Ibaraki Prefecture. , Received on September 19, 2007 and has been given the receipt number FERM AP-21368.
（ＸＩＩＩ）４Ｂ４抗体のアイソタイプの決定
（１）４Ｂ４抗体のアイソタイプは、モノクローナル抗体アイソタイピングキット（IsoStrip; Roche Diagnostics, Indianapolis）を使用した。
（２）その結果、４Ｂ４抗体のアイソタイプはＩｇＧ１（ｋ）サブクラスであった。
(XIII) Determination of isotype of 4B4 antibody (1) The isotype of 4B4 antibody was a monoclonal antibody isotyping kit (IsoStrip; Roche Diagnostics, Indianapolis).
(2) As a result, the isotype of the 4B4 antibody was IgG1 (k) subclass.
４Ｂ４モノクローナル抗体の認識抗原の同定
（Ｉ）４Ｂ４抗体が認識する抗原のＥＬＩＳＡによる解析
４Ｂ４モノクローナル抗体がどのような抗原を認識するのかを、下記手順にて調べた。
（１）４Ｂ４抗体が認識する抗原が、菌体表面に存在するかどうかを、C. jejuni（Ｌｉｏｒ血清型７）の生菌体を固相化抗原として用いたＥＬＩＳＡ法により、以下の手順で調べた。
（２）生菌体を０．０５Ｍ炭酸バッファー ｐＨ９．６で約１×１０７個／ｍｌに希釈して、マイクロタイタープレートのウエルに０．１ｍｌ分注し、４℃で一夜放置した。
（３）抗原液をプレートから除去した後、ブロッキング液（２０％馬血清を含むＰＢＳ）を０．１５ｍｌ加えて室温１時間反応させた。
（４）ブロッキング液を除去した後、洗浄液（０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳ）で３回洗浄した。
（５）４Ｂ４抗体希釈液（精製４Ｂ４抗体を、２０％馬血清と０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳで０．０１ｍｇ／ｍｌに希釈したもの）を０．１ｍｌ加えて、室温１時間反応させた。
（６）洗浄液で４回洗浄後、ペルオキシダーゼ標識抗マウスＩｇＧを２０％馬血清と０．０５％ Ｔｗｅｅｎ２０を含むＰＢＳで１０００倍に希釈したものを０．１ｍｌ加えて、室温１時間反応させた。
（７）洗浄液で４回洗浄後、酵素基質（1-Step Ultra TMB-ELISA:PIERCE）を０．１ｍｌ加えて、室温１０分間反応させた後、反応停止液（１Ｍ硫酸）を０．１ｍｌ加えてから、吸光度の自動計測装置を用いて４５０ｎｍにおける吸光度を計測した。
（８）４Ｂ４抗体希釈液を添加したウエルは、高い吸光度（２．８１１）を示したので、４Ｂ４抗体は、菌体の表面に存在する抗原に結合することがわかった。
Identification of recognition antigen of 4B4 monoclonal antibody
(I) Analysis of antigen recognized by 4B4 antibody by ELISA The following procedure examined what kind of antigen the 4B4 monoclonal antibody recognizes.
(1) Whether or not the antigen recognized by the 4B4 antibody is present on the surface of the microbial cell is determined by the following procedure using an ELISA method using a living microbial cell of C. jejuni (Lior serotype 7) as an immobilized antigen. Examined.
(2) The viable cells were diluted to about 1 × 10 7 cells / ml with 0.05 M carbonate buffer pH 9.6, dispensed 0.1 ml into the wells of the microtiter plate, and left at 4 ° C. overnight.
(3) After removing the antigen solution from the plate, 0.15 ml of blocking solution (PBS containing 20% horse serum) was added and reacted at room temperature for 1 hour.
(4) After removing the blocking solution, the plate was washed 3 times with a washing solution (PBS containing 0.05% Tween 20).
(5) 0.1 ml of 4B4 antibody dilution (purified 4B4 antibody diluted to 0.01 mg / ml with PBS containing 20% horse serum and 0.05% Tween 20) was added and allowed to react for 1 hour at room temperature. .
(6) After washing 4 times with the washing solution, 0.1 ml of peroxidase-labeled anti-mouse IgG diluted 1000-fold with PBS containing 20% horse serum and 0.05% Tween 20 was added and allowed to react at room temperature for 1 hour.
(7) After washing 4 times with the washing solution, 0.1 ml of enzyme substrate (1-Step Ultra TMB-ELISA: PIERCE) is added and reacted at room temperature for 10 minutes, and then 0.1 ml of reaction stop solution (1M sulfuric acid) is added. Then, the absorbance at 450 nm was measured using an automatic absorbance measuring apparatus.
(8) The well to which the 4B4 antibody diluted solution was added showed high absorbance (2.811), and thus it was found that the 4B4 antibody binds to the antigen present on the surface of the cells.
（ＩＩ）４Ｂ４抗体が認識する抗原のイムノブロッテイングによる解析
下記手順により、４Ｂ４抗体が認識する菌体抗原の分子量を調べた。
（１）免疫抗原（C. jejuni（Ｌｉｏｒ血清型７）の臨床分離株を使って調製した菌体抽出抗原）にＳＤＳ−ＰＡＧＥサンプルバッファー（６２．５ｍＭ Ｔｒｉｓ−ＨＣｌ ｐＨ６．８，２％ ＳＤＳ，１％ ２−メルカプトエタノール，１０％グリセロール，０．０１％ブロモフェノールブルー）を４倍量加えたものを１００℃３分間加熱して、ＳＤＳ−ＰＡＧＥサンプルとした。
（２）上記サンプルについて、２枚のゲル（１５％分離ゲル）を使ってＳＤＳ−ＰＡＧＥを実施し、１枚をＰＶＤＦメンブレンに転写し、残りの１枚をＣｏｏｍａｓｓｉｅ染色した。
（３）転写後のメンブレンは０．１％ Ｔｗｅｅｎ２０を含むトリス塩酸緩衝食塩水（タカラバイオ）で洗浄後、ブロックエース（雪印）を使ってブロッキングした。
（４）洗浄後のメンブレンに、４Ｂ４抗体希釈液（精製４Ｂ４抗体を、０．１％ Ｔｗｅｅｎ２０を含むトリス塩酸緩衝食塩水で０．０２ｍｇ／ｍｌに希釈したもの）で室温１時間反応させた。
（５）洗浄後のメンブレンに、ペルオキシダーゼ標識抗マウスＩｇＧを０．１％ Ｔｗｅｅｎ２０を含むトリス塩酸緩衝食塩水で４０００倍に希釈したものを、室温１時間反応させた。
（６）洗浄後のメンブレンに、酵素基質（TMB Membrane Peroxidase Substrate：フナコシ）を、室温５分間反応させた。
（７）メンブレンを大量の蒸留水で洗浄後、出現したバンドの位置を分子量マーカー（BioRad）及びＣｏｏｍａｓｓｉｅ染色したゲルと比較した。その結果、４Ｂ４抗体は、分子量約１５キロダルトンの菌体タンパク質を認識することが示された（図１参照）。
(II) Analysis by immunoblotting of the antigen recognized by the 4B4 antibody The molecular weight of the cell antigen recognized by the 4B4 antibody was examined by the following procedure.
(1) SDS-PAGE sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, immunization antigen (bacteria extracted antigen prepared using clinical isolate of C. jejuni (Lior serotype 7)) A 4% amount of 1% 2-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue) was heated at 100 ° C. for 3 minutes to obtain an SDS-PAGE sample.
(2) SDS-PAGE was performed on the above sample using two gels (15% separation gel), one was transferred to a PVDF membrane, and the remaining one was Coomassie stained.
(3) The membrane after transfer was washed with Tris-HCl buffered saline (Takara Bio) containing 0.1% Tween 20, and then blocked using Block Ace (snow mark).
(4) The washed membrane was reacted with a 4B4 antibody diluent (purified 4B4 antibody diluted to 0.02 mg / ml with Tris-HCl buffered saline containing 0.1% Tween 20) at room temperature for 1 hour.
(5) The membrane after washing was diluted 4000 times with Tris-HCl buffered saline containing 0.1% Tween 20 and reacted for 1 hour at room temperature.
(6) The washed membrane was reacted with an enzyme substrate (TMB Membrane Peroxidase Substrate: Funakoshi) for 5 minutes at room temperature.
(7) After the membrane was washed with a large amount of distilled water, the position of the appearing band was compared with the molecular weight marker (BioRad) and the gel stained with Coomassie. As a result, the 4B4 antibody was shown to recognize bacterial proteins having a molecular weight of about 15 kilodaltons (see FIG. 1).
モノクローナル抗体４Ｂ４を用いたイムノクロマト法の感度の検討
下記手順に従って、本発明のモノクローナル抗体４Ｂ４を用いたイムノクロマト法の感度を調べた。
（Ｉ）被検菌原液の調整及びそこに含まれる菌数の測定
（１）患者から分離される頻度の高い９種類の血清型（Ｌｉｏｒ血清型１，２，４，７，１１，２７，２８，３６及び５０）を含む１８種類の血清型から成る１８菌株と血清型別不能な５株、計２３株のC. jejuniを用いた。
（２）血液寒天培地シャーレ１枚分の培養菌をリン酸緩衝食塩水（ＰＢＳ）１ｍｌに懸濁し、被検菌原液とした。
（３）被検菌原液を、ＰＢＳを用いて１０段階希釈した。
（４）菌原液に含まれる菌数を算出するため、各段階希釈液の０．０１ｍｌを血液寒天培地に接種し、培養後のコロニーをカウントした。
（５）カウントしたコロニー数から菌原液に含まれる菌数を算出した。
Examination of sensitivity of immunochromatography method using monoclonal antibody 4B4 The sensitivity of immunochromatography method using monoclonal antibody 4B4 of the present invention was examined according to the following procedure.
(I) Preparation of test stock solution and measurement of the number of bacteria contained therein (1) Nine types of serotypes (Lior serotypes 1, 2, 4, 7, 11, 27, A total of 23 strains of C. jejuni were used, including 18 strains comprising 18 serotypes including 28, 36 and 50) and 5 strains that could not be serotyped.
(2) A culture medium for one blood agar medium Petri dish was suspended in 1 ml of phosphate buffered saline (PBS) to prepare a test bacterial stock solution.
(3) The test bacteria stock solution was diluted in 10 steps using PBS.
(4) In order to calculate the number of bacteria contained in the bacterial stock solution, 0.01 ml of each serial dilution was inoculated on a blood agar medium, and the colonies after culture were counted.
(5) The number of bacteria contained in the bacterial stock solution was calculated from the counted number of colonies.
（ＩＩ）イムノクロマト法（迅速検出法）の反応
（１）被検菌原液及びその段階希釈液に、B-PER II Bacterial Protein Extraction Reagent（PIRCE, Rockford, IL）を等量添加した。
（２）ボルテックスで１分間攪拌した。
（３）１５０００ｒｐｍで５℃、５分間遠心し、その上清を被検液とした。
（４）９６ウエルマイクロプレートのウエル内で、被検液０．０３ｍｌと金コロイド標識４Ｂ４抗体希釈液（−２０℃保存した金コロイド標識４Ｂ４抗体溶液を１．０％ ＢＳＡ、２％ ＴｒｉｔｏｎＸ１００を含む０．１Ｍ トリス塩酸緩衝液 ｐＨ１０．５で１０倍希釈したもの）０．０３ｍｌを混合した。
（５）そのウエル内の混合液の中に、抗体固相化メンブレンの先端を浸漬した。
（６）１５分後、明瞭なバンドが認められた場合、陽性とした。
（７）陽性反応が認められた最終希釈菌液に含まれる菌数（最小検出菌数）を、菌原液に含まれる菌数を最終希釈液の希釈倍数で割ることにより算出した。
(2) The mixture was stirred for 1 minute by vortexing.
(3) The mixture was centrifuged at 15000 rpm at 5 ° C. for 5 minutes, and the supernatant was used as a test solution.
(4) In a well of a 96-well microplate, 0.03 ml of a test solution and a diluted gold colloid-labeled 4B4 antibody solution (containing a colloidal gold-labeled 4B4 antibody solution stored at −20 ° C. containing 1.0% BSA and 2% Triton X100) 0.03 ml of 0.1M Tris-HCl buffer (diluted 10 times with pH 10.5).
(5) The tip of the antibody-immobilized membrane was immersed in the mixed solution in the well.
(6) If a clear band was observed after 15 minutes, it was determined as positive.
(7) The number of bacteria (minimum number of detected bacteria) contained in the final diluted bacterial solution in which a positive reaction was observed was calculated by dividing the number of bacteria contained in the bacterial stock solution by the dilution factor of the final diluted solution.
この実験により、モノクローナル抗体４Ｂ４を用いたイムノクロマト法において試験したすべての血清型を検出できることがわかった。モノクローナル抗体４Ｂ４を用いたイムノクロマト法の検出感度は、菌の血清型により異なるが、１．８〜１０４〜８．２ｘ１０５ＣＦＵ／ｍｌの範囲であった。 This experiment showed that all serotypes tested in the immunochromatography method using monoclonal antibody 4B4 could be detected. The detection sensitivity of the immunochromatography method using the monoclonal antibody 4B4 was in the range of 1.8 to 10 4 to 8.2 × 10 5 CFU / ml, although it varied depending on the serotype of the bacteria.
モノクローナル抗体４Ｂ４を用いたイムノクロマト法の特異性の検討
本発明のモノクローナル抗体４Ｂ４を用いたイムノクロマト法の特異性について、下記手順により調べた。
（Ｉ）被検菌原液の調整及びそこに含まれる菌数の測定
（１）カンピロバクター属の５菌種（計８株）とカンピロバクター属以外の２６菌種（計２６株）を用いた。
（２）寒天培地シャーレ１枚分の培養菌をリン酸緩衝食塩水（ＰＢＳ）１ｍｌに懸濁し、被検菌原液とした。
（３）被検菌原液を、ＰＢＳを用いて段階希釈した。
（４）菌原液に含まれる菌数を算出するため、各段階希釈液の０．０１ｍｌを寒天培地に接種し、培養後のコロニーをカウントした。
（５）カウントしたコロニー数から菌原液に含まれる菌数を算出した。
Examination of specificity of immunochromatography method using monoclonal antibody 4B4 The specificity of immunochromatography method using monoclonal antibody 4B4 of the present invention was examined by the following procedure.
(I) Preparation of test stock solution and measurement of the number of bacteria contained therein (1) Five species of Campylobacter (total 8 strains) and 26 species of species other than Campylobacter (total 26 strains) were used.
(2) A culture medium for one agar medium Petri dish was suspended in 1 ml of phosphate buffered saline (PBS) to prepare a test bacterial stock solution.
(3) The test bacteria stock solution was serially diluted with PBS.
(4) In order to calculate the number of bacteria contained in the bacterial stock solution, 0.01 ml of each serial dilution was inoculated on the agar medium, and the colonies after culture were counted.
(5) The number of bacteria contained in the bacterial stock solution was calculated from the counted number of colonies.
（ＩＩ）イムノクロマト法（迅速検出法）の反応
（１）被検菌原液及びその段階希釈液に、B-PER II Bacterial Protein Extraction Reagent（PIRCE, Rockford, IL）を等量添加した。
（２）ボルテックスで１分間攪拌した。
（３）１５０００ｒｐｍで５℃、５分間遠心し、その上清を被検液とした。
（４）９６ウエルマイクロプレートのウエル内で、被検液０．０３ｍｌと金コロイド標識４Ｂ４抗体希釈液（−２０℃保存した金コロイド標識４Ｂ４抗体溶液を１．０％ ＢＳＡ、２％ ＴｒｉｔｏｎＸ１００を含む０．１Ｍ トリス塩酸緩衝液 ｐＨ １０．５で１０倍希釈したもの）０．０３ｍｌを混合した。
（注）カンピロバクター属以外の２６菌種については、菌原液から調製された被検液でのみイムノクロマト法を実施した。
（５）そのウエル内の混合液の中に、抗体固相化メンブレンの先端を浸漬した。
（６）１５分後、明瞭なバンドが認められた場合、陽性とした。
（７）陽性反応が認められた最終希釈菌液に含まれる菌数（最小検出菌数）を、菌原液に含まれる菌数を最終希釈液の希釈倍数で割ることにより算出した。
(2) The mixture was stirred for 1 minute by vortexing.
(3) The mixture was centrifuged at 15000 rpm at 5 ° C. for 5 minutes, and the supernatant was used as a test solution.
(4) In a well of a 96-well microplate, 0.03 ml of a test solution and a diluted gold colloid-labeled 4B4 antibody solution (containing a colloidal gold-labeled 4B4 antibody solution stored at −20 ° C. containing 1.0% BSA and 2% Triton X100) 0.03 ml of 0.1 M Tris-HCl buffer (diluted 10-fold with pH 10.5).
(Note) For 26 bacterial species other than Campylobacter, immunochromatography was carried out only with the test solution prepared from the bacterial stock solution.
(5) The tip of the antibody-immobilized membrane was immersed in the mixed solution in the well.
(6) If a clear band was observed after 15 minutes, it was determined as positive.
(7) The number of bacteria (minimum number of detected bacteria) contained in the final diluted bacterial solution in which a positive reaction was observed was calculated by dividing the number of bacteria contained in the bacterial stock solution by the dilution factor of the final diluted solution.
モノクローナル抗体４Ｂ４抗体は、試験したカンピロバクター属の５菌種のうち３菌種と反応したが、カンピロバクター属以外の細菌とは反応しなかった。表１〜表３の結果をあわせると、モノクローナル抗体４Ｂ４を用いた場合、カンピロバクター腸炎患者から分離される頻度が非常に高いC. jejuniのみならず、患者から分離される頻度はC. jejuniほど高くないがカンピロバクター腸炎の原因菌であるC. coliについてもC. jejuniと同程度の検出感度で検出できることがわかった。また、その発生は希であるが、ヒト腸炎との関連が報告されているC. lariおよびC. upsaliensisについても、C. jejuni、と同程度の検出感度で検出できることもわかった。したがって、モノクローナル抗体４Ｂ４はカンピロバクター属の細菌に対して特異性が高く、またヒト腸炎の原因となる広範なカンピロバクター属の菌種（広範な血清型）を検出できるので、カンピロバクター腸炎の診断を、漏れなく正確に行うのに好適なものであることがわかった。 The monoclonal antibody 4B4 antibody reacted with 3 of the 5 species of Campylobacter tested, but did not react with bacteria other than Campylobacter. When the results of Tables 1 to 3 are combined, when the monoclonal antibody 4B4 is used, not only C. jejuni, which is very frequently separated from patients with Campylobacter enteritis, but also as frequently as C. jejuni. However, it was found that C. coli, the causative agent of Campylobacter enteritis, can be detected with the same detection sensitivity as C. jejuni. It was also found that the occurrence of C. lari and C. upsaliensis, which is rarely observed but related to human enterocolitis, can be detected with the same detection sensitivity as C. jejuni. Therefore, monoclonal antibody 4B4 is highly specific for Campylobacter bacteria and can detect a wide range of Campylobacter species (wide serotypes) that cause human enteritis. It was found to be suitable for performing accurately.
なお、本明細書に記載した実施例における寒天培地上での細菌の培養は以下のようにして行った。
C. jejuni, C. coli, C. lariは、血液寒天培地(Blood Agar Base No.2 [Oxoid Ltd., Basingstoke, United Kingdom] に無菌馬脱繊血を７％の割合で加えたもの)上において、４２℃、微好気下(アネロパック微好気、三菱ガス化学製を使用)で培養した。Campylobacter fetus subsp. fetus, Campylobacter fetus subsp. venerealis, C. upsaliensisi, Arcobacter butzleri, Arcobacter skirrowii, Helicobacter pyloriは、血液寒天培地上において、３７℃、微好気下で培養した。Arcobacter cryaerophilusは、血液寒天培地上において、３０℃で、微好気下で培養した。その他の菌種は、brain heart infusion寒天培地(brain heart infusion[Becton Dickinson Microbiology Systems, Sparks, MD.]にBacto agar[Becton Dickinson]を１．５％の割合で加えたもの)上において、３７℃で培養した。他の細菌の培養については常法により行った。
In addition, the culture | cultivation of the bacteria on the agar medium in the Example described in this specification was performed as follows.
C. jejuni, C. coli and C. lari are on blood agar medium (Blood Agar Base No.2 [Oxoid Ltd., Basingstoke, United Kingdom] plus 7% sterile horse defibrinated blood) Cultivated at 42 ° C. under slight aerobic condition (anaeropack slightly aerobic, using Mitsubishi Gas Chemical Co., Ltd.). Campylobacter fetus subsp. Fetus, Campylobacter fetus subsp. Venerealis, C. upsaliensisi, Arcobacter butzleri, Arcobacter skirrowii, Helicobacter pylori were cultured on a blood agar medium at 37 ° C. under slight aerobic condition. Arcobacter cryaerophilus was cultured on a blood agar medium at 30 ° C. under microaerobic conditions. Other bacterial species are 37 ° C on brain heart infusion agar medium (brain heart infusion [Becton Dickinson Microbiology Systems, Sparks, MD.] With Bacto agar [Becton Dickinson] added at a rate of 1.5%) In culture. The culture of other bacteria was performed by a conventional method.
ヒト糞便からのカンピロバクターの検出
先ず、タンパク質抽出剤（B-PER II Bacterial Protein Extraction Reagent（PIERCE）、50％）を含有する水に患者大便試料を懸濁して乳剤を得た。この操作により、試料中にカンピロバクターが存在する場合には、カンピロバクター菌体表面の４Ｂ４抗原タンパク質が遊離される。乳剤を遠心分離して菌体を沈降させて上清を得た。上清中に４Ｂ４抗原タンパク質の大部分が存在する。上清を本発明のモノクローナル抗体４Ｂ４（金コロイド標識したもの）と反応させて複合体を形成させた。このようにして得られた複合体を含む上清を、常法によりイムノクロマトにかけた。イムノクロマトの固相にも４Ｂ４抗体をバンド状に固定化しておいた（物理的吸着法により固定化）。約１５分置くと、上記複合体と固相の４Ｂ４抗体との間でさらなる複合体が形成され、上記標識により検出可能となったバンドが出現した。このバンドの出現により試料中にカンピロバクターが存在すること（陽性であること）がわかった。この実験の手順の概略を図２に示す。
Detection of Campylobacter from Human Feces First, a patient stool sample was suspended in water containing a protein extractant (B-PER II Bacterial Protein Extraction Reagent (PIERCE), 50%) to obtain an emulsion. By this operation, when Campylobacter is present in the sample, 4B4 antigen protein on the surface of Campylobacter is released. The emulsion was centrifuged to settle the cells, and a supernatant was obtained. The majority of 4B4 antigen protein is present in the supernatant. The supernatant was reacted with the monoclonal antibody 4B4 of the present invention (labeled with colloidal gold) to form a complex. The supernatant containing the complex thus obtained was subjected to immunochromatography by a conventional method. The 4B4 antibody was also immobilized in a band shape on the immunochromatographic solid phase (immobilized by physical adsorption). After about 15 minutes, a further complex was formed between the complex and the solid phase 4B4 antibody, and a band that was detectable by the label appeared. The appearance of this band showed that Campylobacter was present in the sample (positive). An outline of the procedure of this experiment is shown in FIG.
本発明によれば、このような簡単な操作手順により、短時間のうちに便中のカンピロバクターの有無を判定することができる。便を採取してから乳剤の調製、遠心分離、４Ｂ４抗体との混合、イムノクロマトを含む一連の操作を行っても、通常であれば３０分以内に終了し、従来の便からのカンピロバクター検出方法が最低でも２日間を要することをからすれば、大変な時間短縮となる。 According to the present invention, the presence or absence of Campylobacter in the stool can be determined in a short time by such a simple operation procedure. Even if a series of operations including preparation of emulsion, centrifugation, mixing with 4B4 antibody, and immunochromatography are performed after the stool is collected, it is usually completed within 30 minutes, and conventional methods for detecting Campylobacter from stool are available. If it takes at least two days, it will save a lot of time.
実施例で説明した実験において用いたその他の実験手法は当業者に公知のものであるか、公知の手法を当業者が容易に変更して行いうるものである。 Other experimental methods used in the experiments described in the examples are known to those skilled in the art or can be easily modified by those skilled in the art.
本発明は、便中のカンピロバクターの迅速検出方法を提供するので、カンピロバクター検査薬、検査キット等の分野において利用可能である。 Since the present invention provides a rapid detection method of Campylobacter in feces, it can be used in the fields of Campylobacter test agents, test kits and the like.
Claims (9)
（ｂ）（ａ）で得られた複合体を、固相上に固定化されたモノクローナル抗体４Ｂ４と反応させて、形成されたさらなる複合体を該標識を用いて検出すること
を特徴とする請求項１記載の方法。 (A) reacting monoclonal antibody 4B4 with a detectable label with a stool sample to form a complex;
(B) The complex obtained in (a) is reacted with a monoclonal antibody 4B4 immobilized on a solid phase, and the further complex formed is detected using the label. Item 2. The method according to Item 1.
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| WO2018181263A1 (en) | 2017-03-27 | 2018-10-04 | 日本ハム株式会社 | Substance that prevents antigen-antibody reaction inhibition by body fluid |
| EP3461841A1 (en) | 2017-10-02 | 2019-04-03 | Certest Biotec, S.L. | Antibodies and test devices for the detection of bacteria of the genus campylobacter |
| WO2019068733A1 (en) | 2017-10-02 | 2019-04-11 | Certest Biotec, S.L. | Antibodies and test devices for the detection of bacteria of the genus campylobacter |
| JP2020536117A (en) * | 2017-10-02 | 2020-12-10 | セルテスト・ビオテク，ソシエダッド・リミターダ | Antibodies and test devices for the detection of Campylobacter bacteria |
| US11312762B2 (en) | 2017-10-02 | 2022-04-26 | Certest Biotec, S.L. | Antibodies and test devices for the detection of bacteria of the genus Campylobacter |
| JP7212682B2 (en) | 2017-10-02 | 2023-01-25 | セルテスト・ビオテク，ソシエダッド・リミターダ | Antibodies and test devices for the detection of Campylobacter bacteria |
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