JP2008107339A - Method and kit for detecting allergen - Google Patents
Method and kit for detecting allergen Download PDFInfo
- Publication number
- JP2008107339A JP2008107339A JP2007256550A JP2007256550A JP2008107339A JP 2008107339 A JP2008107339 A JP 2008107339A JP 2007256550 A JP2007256550 A JP 2007256550A JP 2007256550 A JP2007256550 A JP 2007256550A JP 2008107339 A JP2008107339 A JP 2008107339A
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- Prior art keywords
- actinidine
- monoclonal antibody
- sds
- kiwi
- allergen
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
Description
本発明は、食品等の試料中に含まれるＳＤＳ未変性及びＳＤＳ変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体を用いるキウイアレルゲンの検出方法や、それに用いられるアレルゲンの検出用キットに関する。 The present invention relates to a kiwi allergen detection method using two or more monoclonal antibodies that recognize SDS-unmodified and SDS-modified actinidine contained in a sample such as food, and an allergen detection kit used therefor.
キウイはマタタビ科の植物で、サルナシ、マタタビが同属の植物となる。キウイは、ビタミンＣが豊富なフルーツとして世界中で食されている一方で、キウイは平成１３年３月２１日付けの厚生労働省からの通知「アレルギー物質を含む食品に関する表示について」（食企発第２号食監発第４６号）により、表示が推奨される２０品目の一つに指定されている。表示推奨品目の果物類の中でキウイは最も患者数が多く、口腔内アレルギー症候群（Oral Allergy Syndrome；OAS）や重篤なアレルギー症状を示すものとして知られている。キウイアレルギーの原因物質としては、アクチニジン（ＥＣ ３．４．２２．１４）が知られており、キウイに含まれる総たんぱく質の４０〜５０％を占め、分子量２７ｋＤａ、２２０残基のアミノ酸よりなるシステインプロテアーゼである。 Kiwi is a member of the family Tabata, and the plants belonging to the same genus are Sarnasashi and Matabi. While Kiwi is eaten all over the world as a fruit rich in vitamin C, Kiwi has received a notice from the Ministry of Health, Labor and Welfare on March 21, 2001 regarding “labeling about foods containing allergens” According to No. 2 Food Supervision No. 46), it is designated as one of 20 recommended items for display. Kiwi has the largest number of patients among the recommended fruits for labeling and is known to show oral allergy syndrome (OAS) and severe allergic symptoms. Actinidin (EC 3.4.22.14) is known as a causative agent of kiwi allergy, occupies 40 to 50% of the total protein contained in kiwi, has a molecular weight of 27 kDa, and consists of 220 amino acids. It is a protease.
従来、アレルゲンの検出する方法としては、例えば、アレルゲンに特異的に反応するイムノグロブリンを定量する方法（例えば、特許文献１参照）や、抗原抗体複合体を含有する検体中の該抗原抗体複合体を酸処理等により解離させ、必要に応じてアルカリを用いて中和処理を行った後、該検体中のアレルゲン特異的ＩｇＥ抗体を測定する方法（例えば、特許文献２参照）等が知られている。 Conventional methods for detecting allergens include, for example, a method for quantifying immunoglobulins that specifically react with allergens (see, for example, Patent Document 1), and antigen-antigen complexes in a specimen containing an antigen-antibody complex. Is known to be dissociated by acid treatment or the like, neutralized with alkali as necessary, and then allergen-specific IgE antibody in the sample is measured (for example, see Patent Document 2). Yes.
また、現在、乳、卵、小麦、そば、落花生の特定原材料を検出するための公定法として、加熱・非加熱複合抗原より得られるポリクローナル抗体を用いた免疫学的な検出方法（例えば、特許文献３参照；以下「市販公定法Ａ」という）、あるいは精製抗原より得られたポリクローナル抗体を用いた免疫学的な検出方法（以下「市販公定法Ｂ」という）が用いられている。これらは、特異的にアレルゲンを検出するために有効な方法であるが問題も多い。例えば、市販公定法Ａでは複合抗原を用いているため、何に対する抗体なのかが不明で、交差性が高く、例えば、イムノブロット法などによる抗原の同定ができず、また非特異反応が増える可能性がある。また、市販公定法Ｂでは、抗原が精製されているため抗体の特異性は明確であるものの、未変性の抗原を用いて作製された抗体を使用しているため、変性／未変性により抗体が結合する程度に違いがあるため、同じ添加量であっても、加熱前、加熱後での定量値が異なるという問題があった。 Currently, as an official method for detecting specific raw materials of milk, egg, wheat, buckwheat, and peanuts, an immunological detection method using a polyclonal antibody obtained from a combined heat / non-heat antigen (for example, patent literature) 3; hereinafter referred to as “commercial official method A”) or an immunological detection method using a polyclonal antibody obtained from a purified antigen (hereinafter referred to as “commercial official method B”). These are effective methods for specifically detecting allergens, but have many problems. For example, since the commercial official method A uses a complex antigen, it is unclear what the antibody is against, has high cross-ability, for example, the antigen cannot be identified by immunoblotting, etc., and non-specific reactions may increase. There is sex. In addition, in the commercial official method B, the specificity of the antibody is clear because the antigen is purified, but since the antibody produced using the unmodified antigen is used, the antibody is modified by denaturation / denaturation. Since there is a difference in the degree of binding, there is a problem that the quantitative values before and after heating are different even with the same addition amount.
本発明の課題は、キウイアレルゲンを含む食品等の被検試料から、ＳＤＳを用いて、キウイアレルゲンであるアクチニジンを抽出し、上記アレルゲンがＳＤＳ変性／ＳＤＳ未変性のいかなる状態にあっても検出できる高感度な免疫学的な検出方法及びそれに用いられる検出キット等を提供することにある。 An object of the present invention is to extract actinidine, which is a kiwi allergen, from a test sample such as a food containing a kiwi allergen using SDS, and detect the allergen in any state of SDS denaturation / SDS non-denaturation. An object is to provide a highly sensitive immunological detection method and a detection kit used therein.
本発明者らは、キウイのアレルゲンを検出する方法について鋭意検討し、ＳＤＳ未変性及びＳＤＳ変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体を用いると、これら特定原材料のＳＤＳ抽出液を用いてキウイアレルゲンを正確に検出することができることを見い出し、本発明を完成するに至った。 The present inventors diligently studied a method for detecting a kiwi allergen, and when using two or more monoclonal antibodies recognizing SDS-unmodified and SDS-modified actinidine, the SDS extract of these specific raw materials was used. Thus, the inventors have found that kiwi allergen can be accurately detected, and have completed the present invention.
すなわち本発明は（１）キウイアレルゲンであるアクチニジン含有又は未含有試料を、ＳＤＳ未変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体と、ＳＤＳ変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体とを用いて免疫反応に供することを特徴とするキウイアレルゲンの検出方法や、（２）キウイアレルゲンであるアクチニジン含有又は未含有試料として、ＳＤＳと２−メルカプトエタノールによる抽出液を用いることを特徴とする前記（１）記載のキウイアレルゲンの検出方法や、（３）ＳＤＳ未変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体が、抗アクチニジンモノクローナル抗体Ｎ−ＡＣＴ１と抗アクチニジンモノクローナル抗体Ｎ−ＡＣＴ２、又は、抗アクチニジンモノクローナル抗体Ｎ−ＡＣＴ３と抗アクチニジンモノクローナル抗体Ｎ−ＡＣＴ４であることを特徴とする前記（１）又は（２）記載のキウイアレルゲンの検出方法や、（４）ＳＤＳ変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体が、抗アクチニジンモノクローナル抗体Ｄ−ＡＣＴ１と抗アクチニジンモノクローナル抗体Ｄ−ＡＣＴ２、抗アクチニジンモノクローナル抗体Ｄ−ＡＣＴ４と抗アクチニジンモノクローナル抗体Ｄ−ＡＣＴ５、又は、抗アクチニジンモノクローナル抗体Ｎ−ＡＣＴ５と抗アクチニジンモノクローナル抗体Ｄ−ＡＣＴ３であることを特徴とする前記（１）又は（２）記載のキウイアレルゲンの検出方法に関する。 That is, the present invention comprises (1) a sample containing or not containing actinidine which is a kiwi allergen, two or more monoclonal antibodies recognizing SDS-native actinidine, and two or more types recognizing SDS-modified actinidine. A method for detecting a kiwi allergen characterized by subjecting it to an immune reaction using a monoclonal antibody, and (2) using an extract of SDS and 2-mercaptoethanol as a sample containing or not containing a kiwi allergen, actinidin The kiwi allergen detection method as described in (1) above, or (3) two or more monoclonal antibodies recognizing SDS native actinidin are anti-actinidine monoclonal antibody N-ACT1 and anti-actinidine monoclonal antibody N -ACT2 or anti-A The method for detecting a kiwi allergen according to the above (1) or (2), which is a tinidine monoclonal antibody N-ACT3 and an anti-actinidine monoclonal antibody N-ACT4, or (4) recognizing SDS-modified actinidine One or more types of monoclonal antibodies are anti-actinidine monoclonal antibody D-ACT1 and anti-actinidine monoclonal antibody D-ACT2, anti-actinidine monoclonal antibody D-ACT4 and anti-actinidine monoclonal antibody D-ACT5, or anti-actinidine monoclonal antibody N-ACT5. And a method for detecting a kiwi allergen according to (1) or (2) above, which is anti-actinidine monoclonal antibody D-ACT3.
また本発明は（５）ＳＤＳ未変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体と、ＳＤＳ変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体とを備えたことを特徴とするキウイアレルゲンの検出セットや、（６）ＳＤＳ未変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体が、抗アクチニジンモノクローナル抗体Ｎ−ＡＣＴ１と抗アクチニジンモノクローナル抗体Ｎ−ＡＣＴ２、又は、抗アクチニジンモノクローナル抗体Ｎ−ＡＣＴ３と抗アクチニジンモノクローナル抗体Ｎ−ＡＣＴ４であることを特徴とする前記（５）記載のキウイアレルゲンの検出セットや、（７）ＳＤＳ変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体が、抗アクチニジンモノクローナル抗体Ｄ−ＡＣＴ１と抗アクチニジンモノクローナル抗体Ｄ−ＡＣＴ２、抗アクチニジンモノクローナル抗体Ｄ−ＡＣＴ４と抗アクチニジンモノクローナル抗体Ｄ−ＡＣＴ５、又は、抗アクチニジンモノクローナル抗体Ｎ−ＡＣＴ５と抗アクチニジンモノクローナル抗体Ｄ−ＡＣＴ３であることを特徴とする前記（５）記載のキウイアレルゲンの検出セットに関する。 The present invention also includes (5) Kiwi, comprising two or more monoclonal antibodies recognizing SDS-unmodified actinidine and two or more monoclonal antibodies recognizing SDS-modified actinidine. An allergen detection set, or (6) two or more monoclonal antibodies recognizing SDS-native actinidin are anti-actinidine monoclonal antibody N-ACT1 and anti-actinidine monoclonal antibody N-ACT2, or anti-actinidine monoclonal antibody N -The detection set of kiwi allergen according to (5) above, which is ACT3 and anti-actinidine monoclonal antibody N-ACT4, and (7) two or more monoclonal antibodies that recognize SDS-modified actinidin, Anti-actinidinmo It is clonal antibody D-ACT1 and anti-actinidine monoclonal antibody D-ACT2, anti-actinidine monoclonal antibody D-ACT4 and anti-actinidine monoclonal antibody D-ACT5, or anti-actinidine monoclonal antibody N-ACT5 and anti-actinidine monoclonal antibody D-ACT3. The kiwi allergen detection set described in (5) above,
本発明によると、食品等に含まれるキウイアレルゲンについての免疫学的な検出方法において、キウイアレルゲンが、変性／未変性のいかなる状態にあっても、正確に定性かつ定量的に検出することができる。 According to the present invention, in the immunological detection method for kiwi allergen contained in foods and the like, the kiwi allergen can be accurately and qualitatively and quantitatively detected regardless of whether it is in a denatured / native state. .
本発明のキウイアレルゲンの検出方法としては、キウイアレルゲンであるアクチニジン含有又は未含有試料を、ＳＤＳ未変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体と、ＳＤＳ変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体とを用いて免疫反応に供する方法であれば特に制限されず、また、上記キウイアレルゲンであるアクチニジン含有又は未含有試料としては、ＳＤＳと２−メルカプトエタノールによる抽出液を用いることが好ましく、より具体的には、０．５％ＳＤＳ及び０．５％２−メルカプトエタノールを含む緩衝液による抽出液を用いることが好ましい。 As a method for detecting a kiwi allergen of the present invention, a sample containing or not containing actinidine, which is a kiwi allergen, two or more monoclonal antibodies recognizing SDS-native actinidine and two types recognizing SDS-modified actinidine In addition, the method is not particularly limited as long as it is a method for subjecting to an immune reaction using a monoclonal antibody higher than that, and an extract containing SDS and 2-mercaptoethanol is used as a sample containing or not containing actinidin, which is the kiwi allergen. More specifically, it is preferable to use an extract with a buffer containing 0.5% SDS and 0.5% 2-mercaptoethanol.
また、本発明のキウイアレルゲンの検出キットとしては、ＳＤＳ未変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体と、ＳＤＳ変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体とを備えたキットであれば特に制限されず、２種類のモノクローナル抗体の少なくとも一つが、イムノクロマト用に用いられる金コロイドで標識されたモノクローナル抗体であることが好ましい。 Moreover, the kiwi allergen detection kit of the present invention comprises two or more monoclonal antibodies recognizing SDS-unmodified actinidine and two or more monoclonal antibodies recognizing SDS-modified actinidine. The kit is not particularly limited, and at least one of the two types of monoclonal antibodies is preferably a monoclonal antibody labeled with a colloidal gold used for immunochromatography.
上記ＳＤＳ未変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体として、具体的には、ハイブリドーマ（ＦＥＲＭ ＡＰ−２１３７７）が産生する抗アクチニジンモノクローナル抗体Ｎ−ＡＣＴ１とハイブリドーマ（ＦＥＲＭ ＡＰ−２１３７８）が産生するモノクローナル抗体Ｎ−ＡＣＴ２との組み合わせ、ハイブリドーマ（ＦＥＲＭ Ｐ−２１０４６）が産生する抗アクチニジンモノクローナル抗体Ｎ−ＡＣＴ３とハイブリドーマ（ＦＥＲＭ Ｐ−２１０４７）が産生する抗アクチニジンモノクローナル抗体Ｎ−ＡＣＴ４との組み合わせ等を好適に例示することができ、また、ＳＤＳ変性のアクチニジンを認識する２種類又はそれ以上のモノクローナル抗体として、プリマハム株式会社基礎研究所（茨城県土浦市中向原６３５）にて所有するハイブリドーマが産生する抗アクチニジンモノクローナル抗体Ｄ−ＡＣＴ１と、ハイブリドーマ（ＦＥＲＭ Ｐ−２１０４５）が産生する抗アクチニジンモノクローナル抗体Ｄ−ＡＣＴ２との組み合わせ、ハイブリドーマ（ＦＥＲＭ ＡＰ−２１３７９）が産生する抗アクチニジンモノクローナル抗体Ｎ−ＡＣＴ５と、ハイブリドーマ（ＦＥＲＭ ＡＰ−２１３８０）が産生する抗アクチニジンモノクローナル抗体Ｄ−ＡＣＴ３との組み合わせ、ハイブリドーマ（ＦＥＲＭ ＡＰ−２１３８１）が産生する抗アクチニジンモノクローナル抗体Ｄ−ＡＣＴ４とハイブリドーマ（ＦＥＲＭ ＡＰ−２１３８２）が産生する抗アクチニジンモノクローナル抗体Ｄ−ＡＣＴ５との組み合わせ等を好適に例示することができる。抗アクチニジンモノクローナル抗体Ｎ−ＡＣＴ５とＤ−ＡＣＴ３との組み合わせは、１００℃・３０分、１２１℃・１５分の加熱変性したアクチニジンを認識することができる。 Specific examples of the two or more monoclonal antibodies that recognize SDS-native actinidine include anti-actinidine monoclonal antibody N-ACT1 and hybridoma (FERM AP-21378) produced by hybridoma (FERM AP-21377). Combination with monoclonal antibody N-ACT2 produced, combination of anti-actinidine monoclonal antibody N-ACT3 produced by hybridoma (FERM P-21046) and anti-actinidine monoclonal antibody N-ACT4 produced by hybridoma (FERM P-21047), etc. And two or more monoclonal antibodies recognizing SDS-modified actinidine, the Primaham Co., Ltd. Basic Research Institute (Takaura, Tsuchiura, Ibaraki) 635) is combined with the anti-actinidine monoclonal antibody D-ACT1 produced by the hybridoma owned by 635) and the anti-actinidine monoclonal antibody D-ACT2 produced by the hybridoma (FERM P-21045), and the hybridoma (FERM AP-21379) is produced. Combination of anti-actinidine monoclonal antibody N-ACT5 and anti-actinidine monoclonal antibody D-ACT3 produced by hybridoma (FERM AP-21380), anti-actinidine monoclonal antibody D-ACT4 produced by hybridoma (FERM AP-21281) and hybridoma ( A combination with the anti-actinidine monoclonal antibody D-ACT5 produced by FERM AP-21382) can be preferably exemplified. The combination of anti-actinidine monoclonal antibodies N-ACT5 and D-ACT3 can recognize heat-modified actinidine at 100 ° C. for 30 minutes and 121 ° C. for 15 minutes.
したがって、抗アクチニジンモノクローナル抗体である、Ｎ−ＡＣＴ１とＮ−ＡＣＴ２とＤ−ＡＣＴ１とＤ−ＡＣＴ２との組み合わせや、Ｎ−ＡＣＴ３とＮ−ＡＣＴ４とＤ−ＡＣＴ１とＤ−ＡＣＴ２との組み合わせや、Ｎ−ＡＣＴ１とＮ−ＡＣＴ２とＮ−ＡＣＴ５とＤ−ＡＣＴ３との組み合わせや、Ｎ−ＡＣＴ３とＮ−ＡＣＴ４とＮ−ＡＣＴ５とＤ−ＡＣＴ３との組み合わせや、Ｎ−ＡＣＴ１とＮ−ＡＣＴ２とＤ−ＡＣＴ４とＤ−ＡＣＴ５との組み合わせや、Ｎ−ＡＣＴ３とＮ−ＡＣＴ４とＤ−ＡＣＴ４とＤ−ＡＣＴ５との組み合わせや、Ｎ−ＡＣＴ１とＮ−ＡＣＴ２とＮ−ＡＣＴ５とＤ−ＡＣＴ３とＤ−ＡＣＴ４とＤ−ＡＣＴ５との組み合わせや、Ｎ−ＡＣＴ３とＮ−ＡＣＴ４とＮ−ＡＣＴ５とＤ−ＡＣＴ３とＤ−ＡＣＴ４とＤ−ＡＣＴ５との組み合わせを用いることで、特に有利にサンドイッチＥＬＩＳＡやイムノクロマトを行うことができ、ＳＤＳ未変性及びＳＤＳ変性のアクチニジンを定量することができる。 Therefore, a combination of anti-actinidine monoclonal antibodies N-ACT1, N-ACT2, D-ACT1, D-ACT2, N-ACT3, N-ACT4, D-ACT1, and D-ACT2, -A combination of ACT1, N-ACT2, N-ACT5 and D-ACT3, a combination of N-ACT3, N-ACT4, N-ACT5 and D-ACT3, N-ACT1, N-ACT2 and D-ACT4 , D-ACT5, N-ACT3, N-ACT4, D-ACT4, D-ACT5, N-ACT1, N-ACT2, N-ACT5, D-ACT3, D-ACT4, D -Combination with ACT5, N-ACT3, N-ACT4, N-ACT5, D-ACT3, D-ACT4 and D-ACT5 By using a combination of, particularly preferably it can perform sandwich ELISA and immunochromatography can be quantified actinidin of SDS native and SDS denaturation.
以上の本発明の免疫学的なキウイアレルゲンの検出方法は、未変性／変性のキウイアレルゲンを含む試料を、標識化した前記本件モノクローナル抗体と接触させ、あるいは標識化した抗体の存在下に前記本件モノクローナル抗体と接触させ、抗原抗体反応により標識化免疫複合体として捕捉する免疫反応段階と、生成した該免疫複合体をその分子中に存在する標識物質を用いて分離・測定する検出段階とからなり、かかる免疫反応段階における抗原抗体反応の方法も特に制限されず、例えば、以下の方法を例示することができる。 In the immunological kiwi allergen detection method of the present invention described above, a sample containing a native / denatured kiwi allergen is contacted with the labeled monoclonal antibody of the present invention or in the presence of the labeled antibody. It consists of an immune reaction stage that is brought into contact with a monoclonal antibody and captured as a labeled immune complex by an antigen-antibody reaction, and a detection stage in which the generated immune complex is separated and measured using a labeling substance present in the molecule. The method of antigen-antibody reaction in such an immune reaction stage is not particularly limited, and examples thereof include the following methods.
不溶性担体に結合した本件モノクローナル抗体に試料中のキウイアレルゲンを捕捉させた後に標識化抗ＩｇＧ抗体を反応させるサンドイッチ法や、不溶性担体に結合した本件モノクローナル抗体と異なるエピトープを認識する標識本件モノクローナル抗体（第二抗体）を用いるサンドイッチ二抗体法や、不溶性担体に結合した本件モノクローナル抗体に試料中のキウイアレルゲンを標識化抗原の存在下で反応させる競合法や、キウイアレルゲンを含有する試料にこれらと特異的に反応する磁気ビーズ結合標識本件モノクローナル抗体を作用させた後、磁力により分離した免疫複合体中の標識物質を検出する磁気ビーズ法や、キウイアレルゲンを含有する試料にこれらと特異的に反応する標識本件モノクローナル抗体を作用させて凝集沈殿させた後、遠心分離により分離した免疫複合体中の標識物質を検出する凝集沈殿法や、金コロイド等で標識された本件モノクローナル抗体とキウイアレルゲンであるアクチニジンが結合した抗原抗体複合体が試験ストリップ上を毛管現象等により移動する途中に、キウイアレルゲンと結合する本件モノクローナル抗体をあらかじめ固定しておき、抗原抗体複合体を捕捉させることで現れる着色ラインの有無によって定性分析するイムノクロマト法の他、二重免疫拡散法、放射免疫拡散法など公知の免疫測定法を利用することができるが、本件モノクローナル抗体として、それぞれ異なるエピトープを認識する２以上のモノクローナル抗体を用いる方法、例えば、食品中の未変性キウイアレルゲン及び／又は変性キウイアレルゲンが１００〜１０００ｐｐｂの濃度範囲においても定性的かつ定量的に分析しうる高感度の点でサンドイッチ二抗体法が、定性的には簡便性からイムノクロマト法が好ましい。 A sandwich method in which the monoclonal antibody bound to an insoluble carrier captures kiwi allergen in the sample and then reacted with a labeled anti-IgG antibody, or a labeled monoclonal antibody that recognizes an epitope different from the monoclonal antibody bound to an insoluble carrier ( Secondary antibody), a competitive method in which the monoclonal antibody bound to an insoluble carrier is reacted with the kiwi allergen in the sample in the presence of the labeled antigen, and specific to samples containing the kiwi allergen Reactively reacting magnetic bead binding label After the monoclonal antibody is acted on, it reacts specifically with the magnetic bead method for detecting the labeling substance in the immune complex separated by magnetic force or the sample containing kiwi allergen Labeled with the monoclonal antibody After that, an aggregation-precipitation method for detecting the labeled substance in the immune complex separated by centrifugation, or the antigen-antibody complex in which the present monoclonal antibody labeled with colloidal gold or the like and the actinidin kiwi allergen are bound on the test strip. In the middle of movement due to capillary action, etc., the monoclonal antibody that binds to the kiwi allergen is immobilized in advance, and in addition to the immunochromatography method that performs qualitative analysis based on the presence or absence of a colored line that appears by capturing the antigen-antibody complex, double immunization Although known immunoassay methods such as diffusion method and radioimmunodiffusion method can be used, a method using two or more monoclonal antibodies recognizing different epitopes as the present monoclonal antibody, for example, native kiwi allergen in food And / or 100 to 100 denatured kiwi allergen Sandwich double antibody method in qualitative and terms of high sensitivity which can quantitatively analyze even at ppb concentration range is qualitative immunochromatography from simplicity in preferred.
上記抗原抗体反応において用いられる不溶性担体としては、例えば、ポリスチレン、ポリエチレン、ポリプロピレン、ポリエステル、ポリアクリロニトリル、フッ素樹脂、架橋デキストラン、ポリサッカライド等の高分子化合物、その他、ガラス、金属、磁性粒子及びこれらの組み合わせ等を挙げることができ、また、不溶性担体の形状としては、例えば、トレイ状、球状、繊維状、棒状、盤状、容器状、セル、マイクロプレート、試験管、ラテックスビーズ状等の種々の形状で用いることができる。更に、これら不溶性担体への抗原又は抗体の固定化方法は特に限定されるものでなく、物理的吸着法、共有結合法、イオン結合法等を用いることができる。 Examples of insoluble carriers used in the antigen-antibody reaction include high molecular compounds such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, polysaccharides, glass, metal, magnetic particles, and the like. Examples of the shape of the insoluble carrier include various shapes such as a tray shape, a spherical shape, a fiber shape, a rod shape, a disk shape, a container shape, a cell, a microplate, a test tube, and a latex bead shape. Can be used in shape. Furthermore, the method for immobilizing the antigen or antibody on these insoluble carriers is not particularly limited, and a physical adsorption method, a covalent bond method, an ion bond method, or the like can be used.
本発明のアレルゲンの検出方法やアレルゲン検出用キットに用いられる本件モノクローナル抗体の免疫グロブリンのクラス及びタイプは特に制限されないが、本件モノクローナル抗体として、ＩｇＧクラス、タイプκの抗体が好適に用いられる。また、モノクローナル抗体の形態としては、全抗体又はＦ（ａｂ’）２、Ｆａｂ等の断片を用いることもできる。抗体の由来は特に限定されるものではないが、マウス、ラット、ヒト、兎、鶏等を挙げることができるが、作製の簡便性からマウスに由来するモノクローナル抗体が好適に用いられる。また、本件モノクローナル抗体は、未変性又はＳＤＳ変性のキウイアレルゲンであるアクチニジンで免疫した動物から採取した抗体産生細胞とミエローマ細胞との細胞融合により調製されるハイブリドーマを培地上で培養するか、又は動物腹腔内に投与して腹水内で増殖させた後、該培養物又は腹水から採取することにより製造することができる。 The class and type of immunoglobulin of the monoclonal antibody used in the allergen detection method and the allergen detection kit of the present invention are not particularly limited, but IgG class and type κ antibodies are preferably used as the monoclonal antibody. In addition, as a form of the monoclonal antibody, a whole antibody or a fragment such as F (ab ′) 2 or Fab can be used. The origin of the antibody is not particularly limited, and examples thereof include mice, rats, humans, rabbits, chickens and the like. Monoclonal antibodies derived from mice are preferably used because of the ease of production. In addition, the present monoclonal antibody is obtained by culturing a hybridoma prepared by cell fusion of antibody-producing cells and myeloma cells collected from an animal immunized with an adenidine, a native or SDS-denatured kiwi allergen, on a medium, or an animal It can be produced by being intraperitoneally administered and grown in ascites and then harvested from the culture or ascites.
本件モノクローナル抗体産生ハイブリドーマは、例えば、未変性又はＳＤＳ変性のキウイアレルゲンであるアクチニジンを用いてＢＡＬＢ／ｃマウスを免疫し、免疫されたマウスの抗体産生細胞とマウスミエローマ細胞とを、常法により細胞融合させ、免疫蛍光染色パターンによりスクリーニングすることにより、本件モノクローナル抗体産生ハイブリドーマを作出することができる。上記の抗体産生細胞としては、例えばＳＤＳ未変性及び／若しくはＳＤＳ変性のキウイアレルゲンであるアクチニジン又はこれを含有する組成物を投与して免疫した動物から得られる脾臓細胞、リンパ節細胞、Ｂ−リンパ球等を挙げることができる。免疫する動物としてはマウス、ラット、ウサギ、ウマ等が挙げられる。免疫は、例えば未変性又はＳＤＳ変性のキウイアレルゲンであるアクチニジンをそのまま又は適当なアジュバントと共に動物の皮下、筋肉内又は腹腔内に１〜２回／月、１〜６ケ月間投与することにより行なわれる。抗体産生細胞の分離は、最終免疫から２〜４日後に免疫動物から採取することにより行なわれる。ミエローマ細胞としては、マウス、ラット由来のもの等を使用することができる。抗体産生細胞とミエローマ細胞とは同種動物由来であることが好ましい。 The present monoclonal antibody-producing hybridoma is prepared by, for example, immunizing BALB / c mice with actinidin, a native or SDS-denatured kiwi allergen, and immunizing mouse antibody-producing cells and mouse myeloma cells by conventional methods. The present monoclonal antibody-producing hybridoma can be produced by fusing and screening by immunofluorescence staining pattern. Examples of the antibody-producing cells include spleen cells, lymph node cells, B-lymph obtained from an animal immunized with actinidin which is an SDS-denatured and / or SDS-denatured kiwi allergen or a composition containing the same. Spheres etc. can be mentioned. Examples of animals to be immunized include mice, rats, rabbits and horses. Immunization is performed, for example, by administering actinidin, which is a native or SDS-modified kiwi allergen, as it is or with an appropriate adjuvant, subcutaneously, intramuscularly or intraperitoneally, once or twice per month for 1 to 6 months. . Separation of antibody-producing cells is performed by collecting from the immunized animal 2 to 4 days after the final immunization. As myeloma cells, those derived from mice and rats can be used. The antibody-producing cells and the myeloma cells are preferably derived from the same species.
細胞融合は、例えばダルベッコ改変イーグル培地（ＤＭＥＭ）等の培地中で抗体産生細胞とミエローマ細胞とをポリエチレングリコール等の融合促進剤の存在下で混合することにより行なうことができる。細胞融合終了後、ＤＭＥＭ等で適当に希釈し、遠心分離し、沈殿をＨＡＴ培地等の選択培地に懸濁して培養することによりハイブリドーマを選択し、次いで、培養上清を用いて酵素抗体法により抗体産生ハイブリドーマを検索し、限界希釈法等によりクローニングを行ない、本件モノクローナル抗体を産生するハイブリドーマを得ることができる。また、未変性のキウイアレルゲンであるアクチニジンのみを用いて免疫した抗免疫動物から、有利に抗変性アクチニジンモノクローナル抗体を得ることができる場合もある。この場合、抗変性アクチニジンモノクローナル抗体等の本件モノクローナル抗体産生ハイブリドーマをスクリーニングしてもよいし、あるいは、固相状態でのＥＬＩＳＡで未変性のアクチニジンに対するモノクローナル抗体産生ハイブリドーマを選択し、この抗体産生ハイブリドーマが産生するモノクローナル抗体から液相状態で未変性のアクチニジンに対してのみ特異的に反応する本件モノクローナル抗体を得ることができる。前記のように、抗体産生ハイブリドーマを培地中又は生体内で培養しモノクローナル抗体を培養物から採取することができるが、培養物又は腹水からのモノクローナル抗体の分離・精製方法としては、タンパク質の精製に一般的に用いられる方法であればどのような方法でもよく、例えば、ＩｇＧ精製に通常使用される硫安分画法、陰イオン交換体又はプロテインＡ、Ｇ等のカラムによるクロマトグラフィーによって行なうことができる。 Cell fusion can be performed, for example, by mixing antibody-producing cells and myeloma cells in a medium such as Dulbecco's modified Eagle medium (DMEM) in the presence of a fusion promoter such as polyethylene glycol. After completion of cell fusion, the hybridoma is selected by diluting appropriately with DMEM, centrifuging, suspending the precipitate in a selective medium such as HAT medium and culturing, and then using the culture supernatant by the enzyme antibody method. An antibody-producing hybridoma can be searched and cloned by limiting dilution or the like to obtain a hybridoma that produces this monoclonal antibody. In some cases, an anti-denatured actinidine monoclonal antibody can be advantageously obtained from an anti-immunized animal immunized with only a native kiwi allergen, actinidin. In this case, the present monoclonal antibody-producing hybridoma such as an anti-denatured actinidine monoclonal antibody may be screened, or a monoclonal antibody-producing hybridoma against native actinidine is selected by ELISA in a solid phase, and this antibody-producing hybridoma is selected. The monoclonal antibody of the present invention that reacts specifically only with native actinidine in the liquid phase can be obtained from the monoclonal antibody produced. As described above, antibody-producing hybridomas can be cultured in a culture medium or in vivo, and monoclonal antibodies can be collected from the culture. As a method for separating and purifying monoclonal antibodies from the culture or ascites, protein purification can be performed. Any method can be used as long as it is a commonly used method. For example, it can be carried out by an ammonium sulfate fractionation method commonly used for IgG purification, chromatography using an anion exchanger or a column such as protein A or G. .
また、標識化抗体作製に用いられる標識物質としては、単独でまたは他の物質と反応することにより検出可能なシグナルをもたらすことができる標識物質であればよく、酵素、蛍光物質、化学発光物質、放射性物質、金コロイド等を使用するのができ、酵素としてはペルオキシダーゼ、アルカリホスファターゼ、β−Ｄ−ガラクトシダーゼ、グルコースオキシダーゼ、グルコース−６−ホスフェートデヒドロゲナーゼ、アルコール脱水素酵素、リンゴ酸脱水素酵素、ペニシリナーゼ、カタラーゼ、アポグルコースオキシダーゼ、ウレアーゼ、ルシフェラーゼ若しくはアセチルコリンエステラーゼ等を、蛍光物質としては、フルオレスセインイソチオシアネート、フィコビリタンパク、希土類金属キレート、ダンシルクロライド若しくはテトラメチルローダミンイソチオシアネート等を、発光物質としては、ルミノール類、ジオキセタン類、アクリジニウム塩類等を、放射性物質としては３Ｈ、１４Ｃ、１２５Ｉ若しくは１３１Ｉ等を例示することができる。標識物質が酵素である場合には、その活性を測定するために基質、必要により発色剤、蛍光剤、発光剤等が用いることができる。 In addition, the labeling substance used for the preparation of the labeled antibody may be any labeling substance that can provide a detectable signal either alone or by reacting with another substance, such as an enzyme, a fluorescent substance, a chemiluminescent substance, A radioactive substance, colloidal gold, etc. can be used, and as an enzyme, peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, Catalase, apoglucose oxidase, urease, luciferase, acetylcholinesterase, etc., and fluorescent substances include fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelate, dansyl chloride or tetra Chill isothiocyanate, etc. Examples of the luminescent substance include luminol compound, dioxetanes, acridinium salts, and the like, as the radioactive material can be exemplified 3 H, 14 C, 125 I or 131 I and the like. When the labeling substance is an enzyme, a substrate, and if necessary, a color former, a fluorescent agent, a luminescent agent and the like can be used for measuring the activity.
本発明のアレルゲン検出用キットには、有効成分としての本件モノクローナル抗体、好ましくはそれぞれ異なるエピトープを認識する２以上の本件モノクローナル抗体を含むが、これらは保存安定性の点から、溶液状態よりも凍結乾燥物として収容されていることが好ましく、検出用キットにはかかる本件モノクローナル抗体溶解する緩衝液や培養液の他、試料を調製するためのＳＤＳと２−メルカプトエタノールを含む緩衝液等を含んでいてもよい。また、より好ましい別の態様の本発明のアレルゲン検出用キットとしては、前記イムノクロマト法における試験ストリップを挙げることができる。この場合、異なるエピトープを認識する２種類のモノクローナル抗体の少なくとも一つを、イムノクロマト用に用いられる金コロイドで標識されたモノクローナル抗体とすることが好ましい。 The allergen detection kit of the present invention contains the present monoclonal antibody as an active ingredient, preferably two or more of the present monoclonal antibodies that recognize different epitopes, but these are more frozen than in solution from the viewpoint of storage stability. Preferably, the detection kit contains a buffer solution containing SDS and 2-mercaptoethanol for preparing a sample in addition to the buffer solution and culture solution for dissolving the monoclonal antibody. May be. In addition, the allergen detection kit of the present invention of another preferred embodiment may include a test strip in the immunochromatography method. In this case, it is preferable that at least one of two types of monoclonal antibodies recognizing different epitopes is a monoclonal antibody labeled with a colloidal gold used for immunochromatography.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
Ｉ．抗アクチニジンＭＡｂの作製
１．材料および方法
１）アクチニジンの精製
アクチニジンは、Pastorello et al .（Identification of actinidin as the major allergen of kiwi fruit. J Allergy Clin Immunol. 101; 531-7.1998）を一部改変し、精製した。すなわち、新鮮なキウイ果実（ヘイワード種、Actinidia deliciosa、以下キウイ）の皮をむき、最終濃度２％ ＰＶＰＰ、２μｇ／ｍＬ Ｅ−６４ を含む０．１ ｍｏｌ／Ｌリン酸カリウム緩衝液（ｐＨ７．０）と混合し（１：２ｗｔ／ｖｏｌ）、 ホモジナイズした。４℃で１２，０００ｇ×３０分の遠心分離後、上清を０．１ｍｏｌ／Ｌ リン酸カリウム緩衝液（ｐＨ７）に対し４８時間、４℃で透析した。透析後、４℃で３０００ｇ×３時間の遠心分離を行い、粗画分を得た。粗画分をRESOURCE Q カラムを用いた陰イオンクロマトグラフィで分画した。分離には２０ｍｍｏｌ／Ｌ Ｔｒｉｓ ＨＣｌ（ｐＨ７．５）を用い、０から０．５ｍｏｌ／Ｌ ＮａＣｌのグラジエントにより分画した。得られたアクチニジンを含む画分をさらにゲルろ過カラムにより精製後、蒸留水に対し透析し、凍結乾燥したものを精製未変性アクチニジン（以下Ｎ−ＡＣＴ）とした。一方、粗画分よりＰｒｅｐセル（日本バイオラッド）を用いたＳＤＳ−ポリアクリルアミド電気泳動法によりアクチニジンを精製後、蒸留水に対して透析し、凍結乾燥したものを精製変性アクチニジン（以下Ｄ−ＡＣＴ）とした。
I. Preparation of anti-actinidine MAb Materials and Methods 1) Purification of actinidine Actinidine was purified by partially modifying Pastorello et al. (Identification of actinidin as the major allergen of kiwi fruit. J Allergy Clin Immunol. 101; 531-7.1998). That is, a fresh kiwi fruit (Hayward seed, Actinidia deliciosa, hereinafter referred to as Kiwi) is peeled and 0.1 mol / L potassium phosphate buffer (pH 7.0) containing 2% PVPP and 2 μg / mL E-64 at a final concentration of 2% PVPP. (1: 2 wt / vol) and homogenized. After centrifugation at 12,000 g × 30 minutes at 4 ° C., the supernatant was dialyzed against 0.1 mol / L potassium phosphate buffer (pH 7) for 48 hours at 4 ° C. After dialysis, centrifugation was performed at 4 ° C. at 3000 g × 3 hours to obtain a crude fraction. The crude fraction was fractionated by anion chromatography using a RESOURCE Q column. For separation, 20 mmol / L Tris HCl (pH 7.5) was used, and fractionation was performed using a gradient of 0 to 0.5 mol / L NaCl. The obtained fraction containing actinidine was further purified by a gel filtration column, dialyzed against distilled water, and freeze-dried to obtain purified unmodified actinidine (hereinafter N-ACT). On the other hand, actinidine was purified from the crude fraction by SDS-polyacrylamide electrophoresis using a Prep cell (Nippon Bio-Rad), dialyzed against distilled water, and lyophilized to obtain purified modified actinidine (hereinafter D-ACT). ).
２）供試動物
６週齢のオスのＢＡＬＢ／ｃマウスを計１０尾供試した。
2) Test animals A total of 10 6-week-old male BALB / c mice were tested.
３）抗原溶液の作製
生理食塩水で０．１％のＮ−ＡＣＴあるいはＤ−ＡＣＴ溶液を作製し、１ｍＬ容エッペンドルフチューブに５００μＬずつ分注し、免疫に供するまで−２０℃で凍結保管した。
3) Preparation of antigen solution A 0.1% N-ACT or D-ACT solution was prepared with physiological saline, dispensed 500 μL each into a 1 mL Eppendorf tube, and stored frozen at −20 ° C. until immunization.
４）免疫
初回免疫には、complete Freund's adjuvant（Difco）を０．１％のＮ−ＡＣＴあるいはＤ−ＡＣＴが５００μＬ入ったエッペンドルフチューブに等量加え、ボルテックスミキサーにて攪拌して作製したエマルジョンを供試した。Ｎ−ＡＣＴに５尾、Ｄ−ＡＣＴに５尾を充て、各エマルジョンを１尾当たり１５０μＬ腹腔内に注射した。
4) Immunization For the initial immunization, an emulsion prepared by adding an equal amount of complete Freund's adjuvant (Difco) to an Eppendorf tube containing 500 μL of 0.1% N-ACT or D-ACT and stirring with a vortex mixer is provided. tried. Each of the emulsions was injected intraperitoneally at 150 μL per animal, with 5 N-ACT and 5 D-ACT.
追加免疫は、３週間の間隔で２回行った。免疫には、incomplete Freund's adjuvant（Difco）を０．１％のＮ−ＡＣＴあるいはＤ−ＡＣＴが５００μＬ入ったエッペンドルフチューブに等量加え、ボルテックスミキサーにて攪拌して作製したエマルジョンを供試した。このエマルジョンを１尾当たり１５０μＬ腹腔内に注射した。 Booster immunizations were performed twice at 3 week intervals. For immunization, an incomplete Freund's adjuvant (Difco) was added to an Eppendorf tube containing 500 μL of 0.1% N-ACT or D-ACT, and an emulsion prepared by stirring with a vortex mixer was used. This emulsion was injected intraperitoneally at 150 μL per tail.
５）血中抗体価の測定
初回あるいは追加免疫でＮ−ＡＣＴあるいはＤ−ＡＣＴを注射した１週間後に、各ＢＡＬＢ／ｃマウスの尾部静脈より採血を行った。採血した血液は室温に２時間放置後、遠心分離を行い、血清を得た。これらの血清の１０倍希釈段を作製し、非競合法ＥＬＩＳＡによりマウス血中の抗Ｎ−ＡＣＴあるいはＤ−ＡＣＴ抗体価を調べた。なお、二次抗体にはアルカリフォスファターゼ標識抗マウスＩｇＧ（Ｈ＋Ｌ）抗体を用いた。
5) Measurement of antibody titer in blood Blood was collected from the tail vein of each BALB / c mouse one week after the injection of N-ACT or D-ACT in the first or booster immunization. The collected blood was allowed to stand at room temperature for 2 hours and then centrifuged to obtain serum. Ten-fold dilutions of these sera were prepared, and anti-N-ACT or D-ACT antibody titers in mouse blood were examined by non-competitive ELISA. As the secondary antibody, an alkaline phosphatase-labeled anti-mouse IgG (H + L) antibody was used.
６）ハイブリドーマの作製
ハイブリドーマの作製は、Kolher and Milstein (1975)に従った。すなわち、十分に抗体価が上がったマウスに、０．１％Ｎ−ＡＣＴあるいはＤ−ＡＣＴ溶液１００μＬを尾部静脈より注射した。静脈注射から４日後、マウスより脾臓を無菌的に摘出した。脾臓を細切後、ＲＰＭＩ１６４０で洗浄して、滅菌ナイロンメッシュ（Cell Strainer, ７０ｍｍ,Becton Dickinson）を通し、脾臓細胞懸濁液を得た。１，０００ｒｐｍ×１０分の遠心分離により脾臓細胞を集め、再度ＲＰＭＩ１６４０で再懸濁し細胞数をカウントした。この脾臓細胞懸濁液とマウスミエローマ細胞懸濁液（P3X63Ag8.653）を細胞数が１０：１になるように混合し、再度１，０００ｒｐｍ×１０分の遠心分離を行い、ペレットを得た。このペレットに４５％ポリエチレングリコールを滴下し細胞融合を行った。細胞溶液にＲＰＭＩ１６４０を加え希釈後、遠心分離でペレットにした。このペレットに、ハイブリドーマ用培地（１０％牛胎児血清、４０ｍＭ ２−メルカプトエタノール（以下２-ＭＥ）、１００Ｕ／ｍＬペニシリン、１００μｇ／ｍＬストレプトマイシンを含むＲＰＭＩ１６４０培地）に１００μＭヒポキサンチン、０．４μＭアミノプテリン、１６μＭチミジンを含むＨＡＴ選択培地を加え、５×１０６cells/wellとなるように２４ウェルの細胞培養用プレート（Becton Dickinson）に分注し、５％ＣＯ２下で培養した。
6) Production of hybridoma Hybridoma was produced according to Kolher and Milstein (1975). That is, 100 μL of 0.1% N-ACT or D-ACT solution was injected into the mouse whose antibody titer was sufficiently increased from the tail vein. Four days after intravenous injection, the spleen was aseptically removed from the mouse. The spleen was minced, washed with RPMI 1640, and passed through a sterile nylon mesh (Cell Strainer, 70 mm, Becton Dickinson) to obtain a spleen cell suspension. Spleen cells were collected by centrifugation at 1,000 rpm × 10 minutes, resuspended in RPMI 1640 and counted. This spleen cell suspension and mouse myeloma cell suspension (P3X63Ag8.653) were mixed so that the cell number became 10: 1, and centrifuged again at 1,000 rpm × 10 minutes to obtain a pellet. Cell fusion was performed by dropping 45% polyethylene glycol into the pellet. RPMI1640 was added to the cell solution for dilution, and then pelleted by centrifugation. To this pellet, hybridoma medium (RPMI1640 medium containing 10% fetal bovine serum, 40 mM 2-mercaptoethanol (hereinafter 2-ME), 100 U / mL penicillin, 100 μg / mL streptomycin), 100 μM hypoxanthine, 0.4 μM aminopterin. Then, a HAT selection medium containing 16 μM thymidine was added, dispensed to a 24-well cell culture plate (Becton Dickinson) at 5 × 10 6 cells / well, and cultured under 5% CO 2 .
７）限界希釈法によるクローニング
細胞培養用プレートの各ウェルの培養上清について、ＥＬＩＳＡの一次抗体として供試し、抗Ｎ−ＡＣＴあるいはＤ−ＡＣＴ抗体を産生しているハイブリドーマの存在を調べた。ＥＬＩＳＡによりＮ−ＡＣＴあるいはＤ−ＡＣＴに対して陽性を示したウェルのハイブリドーマについて、０．９ｃｅｌｌ／ｗｅｌｌとなるように９６穴細胞培養用プレート（Becton Dickinson）に移し、限界希釈法によるクローニングを行った。なお、フィーダー細胞として、４週齢ＢＡＬＢ／ｃマウス胸腺細胞を５×１０６ｃｅｌｌｓ／ｗｅｌｌとなるように９６穴細胞培養用プレートの各ウェルに加えた。クローニングされたハイブリドーマの培養には、１０％牛胎児血清、４０ｍＭ ２-ＭＥ、１００Ｕ／ｍＬペニシリン、１００μｇ／ｍＬストレプトマイシンを含むＲＰＭＩ１６４０培地を用いた。
7) Cloning by limiting dilution The culture supernatant of each well of the cell culture plate was used as a primary antibody for ELISA to examine the presence of hybridomas producing anti-N-ACT or D-ACT antibodies. Hybridomas in wells that were positive for N-ACT or D-ACT by ELISA were transferred to a 96-well cell culture plate (Becton Dickinson) at 0.9 cell / well and cloned by limiting dilution. It was. As feeder cells, 4-week-old BALB / c mouse thymocytes were added to each well of a 96-well cell culture plate at 5 × 10 6 cells / well. RPMI1640 medium containing 10% fetal bovine serum, 40 mM 2-ME, 100 U / mL penicillin, 100 μg / mL streptomycin was used for the culture of the cloned hybridoma.
８）腹水の採取およびＭＡｂの精製
Jonesら（１９９０）に従い、まず、ＢＡＬＢ／ｃマウスにincomplete Freund's adjuvantを０．２ｍＬ腹腔内に注射した。１週間後、一尾当たり５×１０６ cellsのクローニングされたハイブリドーマを接種した。腹水貯留後、シリンジにより腹水を採取した。採種した腹水をProtein G カラム（アマシャム ファルマシア）により精製し、以下に供試した。
8) Ascites collection and MAb purification
According to Jones et al. (1990), BALB / c mice were first injected with 0.2 mL of incomplete Freund's adjuvant intraperitoneally. One week later, 5 × 10 6 cells of the cloned hybridoma were inoculated per fish. After ascites retention, ascites was collected with a syringe. The collected ascites was purified by Protein G column (Amersham Pharmacia) and used as follows.
９）ＭＡｂのクラス、サブクラスおよびタイプ
ＭＡｂのクラス及びサブクラスについては、Monoclonal mouseimmunoglobulinisotyping kit（Pharmingen）により、ＩｇＧ１、ＩｇＧ２ａ、ＩｇＧ２ｂ、ＩｇＧ３、ＩｇＭ、ＩｇＡ、ＩｇＬ（κ）及びＩｇＬ（γ）を決定した。
9) Class, subclass and type of MAb Regarding the class and subclass of MAb, IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, IgL (κ) and IgL (γ) were determined by Monoclonal mouse immunooglobulinisotyping kit (Pharmingen).
１０）ＭＡｂのビオチン化
精製したＭＡｂについて、サンドイッチＥＬＩＳＡに供試するため、それぞれビオチン化処理を行った。５０ｍＭ炭酸緩衝液（ｐＨ８．５）に２０ｍｇ／ｍＬとなるよう調製し、ＤＭＳＯに３ｍｇ／１００μｌで溶解したＮＨＳ−ビオチン溶液を１０μｌ加え、撹拌後、氷冷しながら２時間静置した。その後、２０ｍｇ／ｍＬとなるようにＰＢＳで置換した。
10) Biotinylation of MAb Each of the purified MAbs was subjected to biotinylation treatment for use in sandwich ELISA. 10 μl of NHS-biotin solution prepared in 50 mg carbonate buffer (pH 8.5) to 20 mg / mL, dissolved in DMSO at 3 mg / 100 μl was added, stirred and allowed to stand for 2 hours with ice cooling. Then, it substituted by PBS so that it might become 20 mg / mL.
２．結果および考察
１）各抗Ｎ−ＡＣＴ ＭＡｂおよび抗Ｄ−ＡＣＴ ＭＡｂの特異性とクラス、サブクラス、抗Ｎ−ＡＣＴ ＭＡｂ８種類、抗Ｄ−ＡＣＴ ＭＡｂ２０種類を得た。それぞれの特異性およびクラス・サブクラスおよびタイプを表１に示す。
2. Results and Discussion 1) Specificity, class, subclass, anti-N-ACT MAb, and 20 anti-D-ACT MAbs of each anti-N-ACT MAb and anti-D-ACT MAb were obtained. Each specificity and class / subclass and type is shown in Table 1.
２）組合せ条件
Ｎ−ＡＣＴを検出するためのＭＡｂあるいはＤ−ＡＣＴを検出するためのＭＡｂの組合せは、サンドイッチＥＬＩＳＡにより選出した。その結果、最も感度の高い組み合わせとして、Ｎ−ＡＣＴではキウイのみ検出できるＮ−ＡＣＴ１とＮ−ＡＣＴ２、近縁種であるサルナシも検出できるＮ−ＡＣＴ３とＮ−ＡＣＴ４を、またＤ−ＡＣＴではサルナシも検出できるＤ−ＡＣＴ１とＤ−ＡＣＴ２、Ｄ−ＡＣＴ４とＤ−ＡＣＴ５、１２１℃・１５分加熱したキウイでも検出できるＮ−ＡＣＴ３とＤ−ＡＣＴ３を選択した。
2) Combination conditions Combinations of MAbs for detecting N-ACT or MAbs for detecting D-ACT were selected by sandwich ELISA. As a result, the most sensitive combinations include N-ACT1 and N-ACT2, which can detect only kiwi in N-ACT, N-ACT3 and N-ACT4 which can also detect related species, such as Sarnashi, and D-ACT. D-ACT1 and D-ACT2, D-ACT4 and D-ACT5, and N-ACT3 and D-ACT3 capable of detecting even kiwi heated at 121 ° C. for 15 minutes were selected.
II．サンドイッチＥＬＩＳＡによるＮ−ＡＣＴあるいはＤ−ＡＣＴの検出
１．材料および方法
１）Ｎ−ＡＣＴ検出系の検討
〔１〕抽出後のアクチニジン検出率の変化
アクチニジンはプロテアーゼであることから、キウイあるいは被検食品から抽出後、自己消化する可能性が考えられるため、抽出液中での安定性を調べた。Ｉ．１．１）に従い、Ｅ−６４を加えずにキウイから抽出した粗画分を４℃、２５℃、３７℃に置き、６、１２、２４および４８時間後にサンプリングし−４０℃でＥＬＩＳＡに供するまで保管した。キウイのみ検出できるＮ−ＡＣＴ１とＮ−ＡＣＴ２、近縁種であるサルナシも検出できるＮ−ＡＣＴ３とＮ−ＡＣＴ４のサンドイッチＥＬＩＳＡにより残存するアクチニジンを測定した。
II. Detection of N-ACT or D-ACT by sandwich ELISA Materials and methods 1) Examination of N-ACT detection system [1] Change in actinidin detection rate after extraction Since actinidine is a protease, it may be extracted from kiwi or a test food and then self-digested. The stability in the extract was examined. I. According to 1.1), the crude fraction extracted from kiwi without adding E-64 is placed at 4 ° C., 25 ° C., 37 ° C., sampled after 6, 12, 24 and 48 hours and subjected to ELISA at −40 ° C. Stored. Remaining actinidine was measured by sandwich ELISA of N-ACT1 and N-ACT2, which can detect only kiwi, and N-ACT3 and N-ACT4, which can also detect the related species, Sarnashi.
〔２〕加熱温度による検出率の変化
加工食品は加工工程中で加熱処理を行われる場合があるため、アクチニジンの加熱による安定性を調べた。キウイ粗たんぱく質画分を、ＢＳＡを標準物質としてプロテインアッセイキット（日本バイオラッド）でたんぱく質濃度を測定後１，０００ｐｐｍに調製し、６３℃・３０分、８０℃・３０分、１００℃・３０分加熱した。冷却後、Ｎ−ＡＣＴ１とＮ−ＡＣＴ２、およびＮ−ＡＣＴ３とＮ−ＡＣＴ４のサンドイッチＥＬＩＳＡによりアクチニジンを測定した。
[2] Change in detection rate due to heating temperature Since processed foods may be heat-treated during the processing process, the stability of actinidine by heating was examined. Kiwi crude protein fraction was prepared to 1,000 ppm after measuring the protein concentration with a protein assay kit (Nippon Bio-Rad) using BSA as a standard substance, 63 ° C / 30 minutes, 80 ° C / 30 minutes, 100 ° C / 30 minutes. Heated. After cooling, actinidine was measured by sandwich ELISA of N-ACT1 and N-ACT2, and N-ACT3 and N-ACT4.
〔３〕他の食品への交差性
市販食品２４種類に対する交差性を調べた。各食品を、フードカッターで粉砕後、１ｇ量り、ＰＢＳＴを１９ｍＬ加えホモジナイズし、遠心分離後、フィルターろ過したろ液をさらにＰＢＳで２０倍に希釈し、サンプルとした。
[3] Crossability to other foods Crossability to 24 kinds of commercially available foods was examined. Each food was pulverized with a food cutter, weighed 1 g, homogenized with 19 mL of PBST, centrifuged, and the filtrate filtered by filtration was further diluted 20 times with PBS to prepare a sample.
２）Ｄ−ＡＣＴ検出系の検討
〔１〕抽出後のアクチニジン検出率の変化
特定原材料の通知法（食発第１１０６００１号、最終改正 平成１７年１０月１１日食安発第１０１１００２号）の抽出液によりキウイより抽出した粗たんぱく質を、４℃、２５℃、３７℃に置き、５、１０、２４および４８時間後にサンプリングし−４０℃でＥＬＩＳＡに供するまで保管した。Ｄ−ＡＣＴ１とＤ−ＡＣＴ２のサンドイッチＥＬＩＳＡにより残存するアクチニジンを測定した。
2) Examination of D-ACT detection system [1] Change in detection rate of actinidine after extraction Extraction of notification method for specific raw materials (Food No. 1106001, Final revision October 11, 2005 Food Safety No. 1011002) The crude protein extracted from kiwi with the liquid was placed at 4 ° C., 25 ° C., and 37 ° C., sampled after 5, 10, 24, and 48 hours, and stored at −40 ° C. until it was subjected to ELISA. Remaining actinidine was measured by sandwich ELISA of D-ACT1 and D-ACT2.
〔２〕加熱温度による検出率の変化
キウイ粗画分を、ＢＳＡを標準物質としてプロテインアッセイ（日本バイオラッド）で１０００ｐｐｍに調製後、６３℃・３０分、８０℃・３０分、１００℃・３０分、１２１℃・１５分加熱しサンプルとした。冷却後、未加熱のものを対照として、サンプルを特定原材料の通知法の抽出液で１００００倍に希釈後、１２時間振とう抽出し、Ｄ−ＡＣＴ１とＤ−ＡＣＴ２のサンドイッチＥＬＩＳＡによりアクチニジンを測定した。また、同様に、Ｎ−ＡＣＴ５とＤ−ＡＣＴ３、及びＤ−ＡＣＴ４とＤ−ＡＣＴ５の他、Ｎ−ＡＣＴ５とＤ−ＡＣＴ３とＤ−ＡＣＴ４とＤ−ＡＣＴ５の４種類を組み合わせたサンドイッチＥＬＩＳＡによりアクチニジンを測定した。
[2] Change in detection rate due to heating temperature After preparing Kiwi crude fraction to 1000 ppm by protein assay (Nippon Bio-Rad) using BSA as a standard substance, 63 ° C / 30 minutes, 80 ° C / 30 minutes, 100 ° C / 30 And heated at 121 ° C. for 15 minutes to obtain a sample. After cooling, using a non-heated sample as a control, the sample was diluted 10,000 times with the extract of the specific raw material notification method, extracted by shaking for 12 hours, and actinidine was measured by sandwich ELISA of D-ACT1 and D-ACT2. . Similarly, in addition to N-ACT5 and D-ACT3, and D-ACT4 and D-ACT5, N-ACT5, D-ACT3, D-ACT4 and D-ACT5 were combined in a sandwich ELISA that combined four types of actinidine. It was measured.
〔３〕他の食品への交差性
市販食品２４種類に対する交差性を調べた。各食品を、フードカッターで粉砕後、特定原材料の通知法に従い抽出液を加え１２時間震とう抽出し、遠心分離後、フィルターろ過したろ液をサンプルとした。
[3] Crossability to other foods Crossability to 24 kinds of commercially available foods was examined. After each food was pulverized with a food cutter, an extract was added according to the notification method for specific raw materials, shaken and extracted for 12 hours, centrifuged, and filtered to obtain a sample.
２．結果および考察
１）Ｎ−ＡＣＴ検出系の検討
〔１〕抽出後のアクチニジンの検出率の変化
抽出後のアクチニジンの検出率を図１ａ、１ｂに示した。４℃保管されたアクチニジンは４８時間後もＮ−ＡＣＴ１とＮ−ＡＣＴ２、およびＮ−ＡＣＴ３とＮ−ＡＣＴ４で９５％以上検出され、また、２５℃保管でも４８時間後で９０％検出された。一方、３７℃保管ではＮ−ＡＣＴ１とＮ−ＡＣＴ２で８０％、Ｎ−ＡＣＴ３とＮ−ＡＣＴ４で７２％検出された。これらの結果から、アクチニジンはプロテアーゼではあるものの自己消化は少なく、サンプル抽出後、低温に保管することで、検出率の低下は防げるものと考えられた。
2. Results and Discussion 1) Examination of N-ACT detection system [1] Change in detection rate of actinidine after extraction The detection rates of actinidine after extraction are shown in FIGS. 1a and 1b. Actinidin stored at 4 ° C. was detected in N-ACT1 and N-ACT2 and N-ACT3 and N-ACT4 in more than 95% after 48 hours, and 90% was detected after 48 hours in storage at 25 ° C. On the other hand, in storage at 37 ° C., 80% was detected with N-ACT1 and N-ACT2, and 72% was detected with N-ACT3 and N-ACT4. From these results, it was considered that actinidin is a protease, but has little autolysis, and it can be prevented that the detection rate is lowered by storing it at a low temperature after sample extraction.
〔２〕加熱温度による検出率の変化
加熱温度によるアクチニジンの検出率の変化を、Ｎ−ＡＣＴ１とＮ−ＡＣＴ２の結果を図２ａ、Ｎ−ＡＣＴ３とＮ−ＡＣＴ４の結果を２ｂに示した。いずれの組み合わせでも、６３℃・３０分で約６０％、８０℃・３０分で約２０％、１００℃・３０分で約３％にまでアクチニジンの検出率は低下した。このことから、Ｎ−ＡＣＴ１とＮ−ＡＣＴ２、及びＮ−ＡＣＴ３とＮ−ＡＣＴ４の組合せは未加熱のアクチニジンを特異的に検出するときに有利に用いることができ、加熱後のジュース、缶詰などに混入したキウイを検出するには適していないと考えられた。
[2] Change in Detection Rate with Heating Temperature The change in the detection rate of actinidine with heating temperature is shown in FIG. 2a, the result of N-ACT1 and N-ACT2, and the result of N-ACT3 and N-ACT4 is shown in 2b. In any combination, the actinidine detection rate decreased to about 60% at 63 ° C. for 30 minutes, about 20% at 80 ° C. for 30 minutes, and about 3% at 100 ° C. for 30 minutes. Therefore, the combination of N-ACT1 and N-ACT2, and N-ACT3 and N-ACT4 can be advantageously used when specifically detecting unheated actinidine, and can be used for juice, canned food, etc. after heating. It was considered unsuitable for detecting mixed kiwi.
〔３〕他の食品への交差性
Ｎ−ＡＣＴ１とＮ−ＡＣＴ２の標準曲線を図３ａ、およびＮ−ＡＣＴ３とＮ−ＡＣＴ４の標準曲線を図３ｂに示した。これらの標準曲線を用いて市販食品２４種に対する交差性を調べた。その結果、Ｎ−ＡＣＴ１とＮ−ＡＣＴ２、およびＮ−ＡＣＴ３とＮ−ＡＣＴ４はいずれも、他の食品への交差性を示さなかった。特に、アクチニジンはシステインプロテアーゼとしてパパイン、ブロメラインと相同性が認められているが、パパイア、パイナップルに対して交差性を示すことは無く、特異的にキウイを検出できる抗体の組み合わせであった。
[3] Crossability to other foods The standard curves of N-ACT1 and N-ACT2 are shown in FIG. 3a, and the standard curves of N-ACT3 and N-ACT4 are shown in FIG. 3b. Using these standard curves, crossover properties for 24 kinds of commercially available foods were examined. As a result, none of N-ACT1 and N-ACT2, and N-ACT3 and N-ACT4 showed crossover properties to other foods. In particular, actinidine was recognized as a cysteine protease having homology with papain and bromelain, but was not a cross-reactivity with papaya and pineapple, and was a combination of antibodies capable of specifically detecting kiwi.
２）Ｄ−ＡＣＴ検出系の検討
〔１〕抽出後のアクチニジンの安定性
抽出後のアクチニジンのＤ−ＡＣＴ１とＤ−ＡＣＴ２による検出率を図４に示した。ＳＤＳと２-ＭＥを含む通地法の抽出液で抽出後、各温度で保管されたアクチニジンは４８時間後の残存率は１００％であった。システインプロテアーゼであるアクチニジンは、２-ＭＥ存在下で活性化される（http://www1.ttv.ne.jp/~kiwi/actinidin-0.html）とされているが、試料の保管は４８時間程度であれば問題ないものと考えられた。
2) Examination of D-ACT detection system [1] Stability of actinidine after extraction The detection rate of actinidine after extraction by D-ACT1 and D-ACT2 is shown in FIG. Actinidin stored at each temperature after extraction with a conventional method extract containing SDS and 2-ME had a residual rate of 100% after 48 hours. Actinidin, a cysteine protease, is activated in the presence of 2-ME (http://www1.ttv.ne.jp/~kiwi/actinidin-0.html). It was thought that there was no problem if it was about time.
〔２〕加熱温度による検出率の変化
Ｄ−ＡＣＴ１とＤ−ＡＣＴ２を用いた加熱温度によるアクチニジンの検出率を図５に示した。６３℃・３０分、８０℃・３０分、１００℃・３０分で加熱したものは未加熱のものとほぼ同等の検出率となった。一方、１２１℃・１５分も未加熱に比較し、４０％程度検出可能であった。同様に、Ｄ−ＡＣＴ４とＤ−ＡＣＴ５とＮ−ＡＣＴ５とＤ−ＡＣＴ３を用いた加熱温度によるアクチニジンの検出率を図６に示した。Ｄ−ＡＣＴ４とＤ−ＡＣＴ５の組み合わせでは６３℃・３０分、８０℃・３０分、１００℃・３０分で１００％程度の検出となり、１２１℃・１５分では４０％の検出率であった。また、Ｎ−ＡＣＴ５とＤ−ＡＣＴ３の組み合わせでは６３℃・３０分で３００％、８０℃・３０分で４３０％、１００℃・３０分で５７０％、１２１℃・１５分では６１０％の検出となり、１２１℃・１５分のようなレトルト処理したアクチニジンを高く検出する組み合わせであった。さらに、Ｎ−ＡＣＴ５とＤ−ＡＣＴ３とＤ−ＡＣＴ４とＤ−ＡＣＴ５の４種類を組み合わせた場合、１２１℃・１５分で８５％程度の検出となり、４種類を組み合わせることによって、未加熱から１２１℃・１５分処理したアクチニジンまで検出できるものと考えられた。
[2] Change in Detection Rate by Heating Temperature FIG. 5 shows the detection rate of actinidine by the heating temperature using D-ACT1 and D-ACT2. Those heated at 63 ° C. for 30 minutes, 80 ° C. for 30 minutes, and 100 ° C. for 30 minutes had almost the same detection rate as that for the non-heated ones. On the other hand, about 40% was detectable at 121 ° C. for 15 minutes compared to unheated. Similarly, the detection rate of actinidine by the heating temperature using D-ACT4, D-ACT5, N-ACT5, and D-ACT3 is shown in FIG. In the combination of D-ACT4 and D-ACT5, detection was about 100% at 63 ° C./30 minutes, 80 ° C./30 minutes, 100 ° C./30 minutes, and 40% detection rate at 121 ° C./15 minutes. The combination of N-ACT5 and D-ACT3 is 300% at 63 ° C / 30 minutes, 430% at 80 ° C / 30 minutes, 570% at 100 ° C / 30 minutes, and 610% at 121 ° C / 15 minutes. , 121 ° C. for 15 minutes, a combination that highly detected actinidine treated with retort. Furthermore, when four types of N-ACT5, D-ACT3, D-ACT4, and D-ACT5 are combined, detection is about 85% in 121 ° C./15 minutes, and by combining the four types, it is 121 ° C. from unheated.・ It was thought that even actinidine treated for 15 minutes could be detected.
〔３〕他の食品への交差性
Ｄ−ＡＣＴ１とＤ−ＡＣＴ２の標準曲線を図７に示す。この標準曲線を用いて市販食品２４種に対する交差性を調べた。その結果、Ｄ−ＡＣＴ１とＤ−ＡＣＴ２は、他の食品への交差性を示さず、また、サルナシやアクチニジンの含有量が極めて低いゼスプリゴールドキウイ（Actinidia chinensis）も検出可能であった。
[3] Crossability to other foods Standard curves of D-ACT1 and D-ACT2 are shown in FIG. Using this standard curve, the crossability for 24 kinds of commercially available foods was examined. As a result, D-ACT1 and D-ACT2 did not show crossing properties to other foods, and zepsprigold kiwi (Actinidia chinensis) with a very low content of sarnashi and actinidin could be detected.
III．イムノクロマト法によるＮ−ＡＣＴあるいはＤ−ＡＣＴの検出
１．材料および方法
１）金コロイド標識及びコンジュゲートパッドの作製
２ｍＭホウ酸緩衝液（ｐＨ９．０）で１ｍｇ／ｍＬとなるようにＮ−ＡＣＴ２、Ｎ−ＡＣＴ４およびＤ−ＡＣＴ２のＭＡｂ溶液を調製した。あらかじめ０．２Ｍ炭酸カリウム溶液でｐＨ９．０に調製した金コロイド溶液（シグマ社製）５ｍＬにＭＡｂ溶液を５００μＬ加え室温で３０分間反応した後、１０％ＢＳＡ溶液を６２５μＬを加え、さらに１５分間反応させた。遠心分離を行い、１％ＢＳＡ溶液でＯＤ５２５＝１．０になるよう調製した。ガラスウール製コンジュゲートパッド（Ｓｃｈｌｅｉｃｈｅｒ＆Ｓｃｈｕｅｌｌ社製）に６８ｍＬ／ｃｍ２となるよう塗布し、乾燥させた。
III. 1. Detection of N-ACT or D-ACT by immunochromatography Materials and Methods 1) Preparation of Gold Colloid Label and Conjugate Pad MAb solutions of N-ACT2, N-ACT4 and D-ACT2 were prepared so as to be 1 mg / mL with 2 mM borate buffer (pH 9.0). 500 μL of MAb solution was added to 5 mL of a gold colloid solution (manufactured by Sigma) previously adjusted to pH 9.0 with 0.2 M potassium carbonate solution, reacted at room temperature for 30 minutes, added with 625 μL of 10% BSA solution, and further reacted for 15 minutes. I let you. Centrifugation was performed, and a 1% BSA solution was prepared so that OD525 = 1.0. It was applied to a glass wool conjugate pad (Schleicher & Schuell) at 68 mL / cm 2 and dried.
２）抗体固定化メンブレンの作製
ＰＢＳで４ｍｇ／ｍＬとなるようにＮ−ＡＣＴ１、Ｎ−ＡＣＴ３およびＤ−ＡＣＴ１のＭＡｂ溶液を調製し、ニトロセルロースメンブレンに直線状に塗布し乾燥させた。その後、１％ＢＳＡ、０．１％Ｔｗｅｅｎ２０を含むＰＢＳで３７℃、２時間ブロッキング後、ＰＢＳで洗浄し乾燥させた。
2) Preparation of antibody-immobilized membrane MAb solutions of N-ACT1, N-ACT3, and D-ACT1 were prepared so as to be 4 mg / mL with PBS, applied linearly to a nitrocellulose membrane, and dried. Then, after blocking with PBS containing 1% BSA and 0.1% Tween 20 at 37 ° C. for 2 hours, it was washed with PBS and dried.
３）イムノクロマトストリップの組立
上記で作製したコンジュゲートパッド、抗体固定化メンブレンに加えて、被検液スポット用のガラスウール製サンプルパッド、被検液吸収用のガラスウール製吸収パッドを別途用意し、サンプルパッド、コンジュゲートパッド、抗体固定化メンブレン、吸収パッドの順にそれぞれ貼り付け、Ｎ−ＡＣＴ１とＮ−ＡＣＴ２、Ｎ−ＡＣＴ３とＮ−ＡＣＴ４をＮ−ＡＣＴ検出用、またＤ−ＡＣＴ１とＤ−ＡＣＴ２をＤ−ＡＣＴ検出用として３組のイムノクロマトストリップを作製した。
3) Assembly of immunochromatographic strip In addition to the conjugate pad and antibody-immobilized membrane prepared above, a glass wool sample pad for the test liquid spot and a glass wool absorption pad for the test liquid absorption are prepared separately. A sample pad, a conjugate pad, an antibody-immobilized membrane, and an absorbent pad are attached in this order, and N-ACT1 and N-ACT2, N-ACT3 and N-ACT4 are for N-ACT detection, and D-ACT1 and D-ACT2 Three sets of immunochromatographic strips were prepared for D-ACT detection.
４）検出感度の検証
〔１〕Ｎ−ＡＣＴ溶液の調製
ＰＢＳによりキウイ粗たんぱく質画分を抽出し、ＢＳＡを標準物質としてプロテインアッセイキットでたんぱく質濃度を測定後、ＰＢＳで１００ｐｐｂ、１０ｐｐｂ、１ｐｐｂ、０ｐｐｂ溶液を調製し、６３℃・３０分、８０℃・３０分、１００℃・３０分加熱した。冷却後、未加熱を対照として、作製したＮ−ＡＣＴ１とＮ−ＡＣＴ２、Ｎ−ＡＣＴ３とＮ−ＡＣＴ４の２組のイムノクロマトキットに１００μＬずつ供試した。
4) Verification of detection sensitivity [1] Preparation of N-ACT solution Kiwi crude protein fraction was extracted with PBS, protein concentration was measured with a protein assay kit using BSA as a standard substance, and then 100 ppb, 10 ppb, 1 ppb, 0 ppb with PBS. Solutions were prepared and heated at 63 ° C. for 30 minutes, 80 ° C. for 30 minutes, and 100 ° C. for 30 minutes. After cooling, 100 μL each of the prepared N-ACT1 and N-ACT2 and N-ACT3 and N-ACT4 immunochromatography kits was used in an amount of 100 μL as a control.
〔２〕Ｄ−Ａｃｔ溶液の調製
ＰＢＳによりキウイ粗たんぱく質画分を抽出し、ＢＳＡを標準物質としてプロテインアッセイキットでたんぱく質濃度を測定後、２０００ｐｐｂ、２００ｐｐｂ、２０ｐｐｂ、０ｐｐｂ溶液を調製し、６３℃・３０分、８０℃・３０分、１００℃・３０分、１２１℃・１５分加熱した。冷却後、未加熱を対照として、サンプルに特定原材料の通知法の抽出液を２０倍量加え、１２時間震とう抽出を行い、Ｄ−ＡＣＴ１とＤ−ＡＣＴ２の組合せのイムノクロマトキットに１００μＬずつ供試した。
[2] Preparation of D-Act solution Extract the kiwi crude protein fraction with PBS, measure the protein concentration with a protein assay kit using BSA as a standard substance, and then prepare 2000 ppb, 200 ppb, 20 ppb, and 0 ppb solutions. Heated for 30 minutes, 80 ° C./30 minutes, 100 ° C./30 minutes, 121 ° C./15 minutes. After cooling, using unheated as a control, add 20 times the extract of the specified raw material notification method, shake extract for 12 hours, and use 100 μL each in an immunochromatography kit of D-ACT1 and D-ACT2 combination. did.
２．結果および考察
１）Ｎ−ＡＣＴ１とＮ−ＡＣＴ２のイムノクロマトキット
１ｐｐｂの濃度のキウイ粗たんぱく質を検出可能であった。また、各加熱温度の影響では、１００ｐｐｂでは、８０℃・３０分でわずかに検出したが１００℃・３０分は検出できず、ＥＬＩＳＡの結果と一致するものとなった。特異性に関しては、ＥＬＩＳＡと同様、キウイのみ検出するイムノクロマトキットであった。
2. Results and Discussion 1) Immunochromatography kit of N-ACT1 and N-ACT2 Kiwi crude protein having a concentration of 1 ppb was detectable. In addition, under the influence of each heating temperature, 100 ppb was detected slightly at 80 ° C. for 30 minutes, but 100 ° C. for 30 minutes could not be detected, which was consistent with the ELISA result. Regarding specificity, it was an immunochromatography kit that detects only kiwi as in ELISA.
２）Ｎ−ＡＣＴ３とＮ−ＡＣＴ４のイムノクロマトキット
１ｐｐｂの濃度のキウイ粗たんぱく質を検出可能であった。また、各加熱温度の影響では、１００ｐｐｂでは、８０℃・３０分でわずかに検出したが１００℃・３０分は検出できず、ＥＬＩＳＡの結果と一致するものとなった。特異性に関しては、ＥＬＩＳＡと同様、キウイのみならず、サルナシやアクチニジンをほとんど含まないゼスプリゴールドキウイなどキウイ類を幅広く検出できるイムノクロマトキットであった。
2) Immunochromatography kit for N-ACT3 and N-ACT4 Kiwi crude protein at a concentration of 1 ppb could be detected. In addition, under the influence of each heating temperature, 100 ppb was detected slightly at 80 ° C. for 30 minutes, but 100 ° C. for 30 minutes could not be detected, which was consistent with the ELISA result. Regarding the specificity, it was an immunochromatography kit capable of detecting not only kiwi but also kiwis such as zestprigold kiwi containing almost no monkey or actinidin as in ELISA.
３）Ｄ−ＡＣＴ１とＤ−ＡＣＴ２のイムノクロマトキット
低濃度のキウイ粗たんぱく質を検出可能であった。特異性に関しては、ＥＬＩＳＡと同様、キウイのみならず、サルナシやアクチニジンをほとんど含まないゼスプリゴールドキウイなどキウイ類を幅広く検出できるイムノクロマトキットであった。
3) D-ACT1 and D-ACT2 immunochromatography kit A low concentration of crude kiwi protein could be detected. Regarding the specificity, it was an immunochromatography kit capable of detecting not only kiwi but also kiwis such as zestprigold kiwi containing almost no monkey or actinidin as in ELISA.
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