JP3820519B2 - Sandwich measurement kits for detecting ciguatoxin CTX3C - Google Patents

Description

【００１４】
【化４】

【００１５】
Ｃ、タンパク質コンジュゲートの調製までを反応式２に示す。
化合物３，３’〜化合物４，４’の合成；
２０ｍＬナス型フラスコに化合物３，３’(4.7 mg, 8.5 μmol)、ｔ−ＢｕＯＨ(0.5 mL)、水 (125μL)、ＬｉＯＨ・Ｈ₂Ｏ(2.8 mg, 68 μmol) を加えて、室温で1時間撹拌した。ＫＨＳＯ₄(18.6 mg, 136 μmol) を加え、溶液のｐＨ（3-４程度）を確認した後、酢酸エチル (10 mL) で希釈した。無水硫酸マグネシウムで乾燥、ろ過、濃縮し、粗化合物４，４’を得た。これにＤＭＦ(200 μL)、Ｎ−ヒドロキシスクシンイミド (9.7 mg, 85μmol)、 ＥＤＣ・ＨＣｌ(8.1 mg, 43 μmol) を加えて、室温で１２時間攪拌した。反応溶液を酢酸エチル(10 ml)で希釈し、有機層を水で３回洗浄し、無水硫酸ナトリウムで乾燥、濃縮し、活性化エステルである化合物６，６’を得た。これにＤＭＦ(100 μL) を加えた溶液を作製し、コンジュゲートの調製に使用した。
【００１６】
ＫＬＨコンジュゲートの調製
ＫＬＨ(7.0 mg)のPBS buffer 溶液 (2.0 ml)に活性化エステルである化合物６，６’(約4.2 μmol) のＤＭＦ溶液(50 μL) を加えて１０分間撹拌した。一日静置した後、４℃で透析した。この後、１４、１９時間後にＰＢＳバッファー(700 mL) を交換し、２８時間後に透析膜からエッペンドルフチューブに移し換えて、−７８℃で保存した。
【００１７】
ＢＳＡコンジュゲートの調製
ＢＳＡ (7.0 mg) のＰＢＳバッファー溶液 (2.0 mL) に活性化エステルである化合物６，６’(約4.2 μmol) のＤＭＦ溶液(50 μL)を加えて１０分間撹拌した。一日静置した後、４℃で透析した。この後、１４、１９時間後にＰＢＳバッファー(700 mL) を交換し、２８時間後に透析膜からエッペンドルフチューブに移し換えて、−７８℃で保存した。
【００１８】
【化５】

【００１９】
（Ｐｒはキャリヤータンパク。ｎは１以上の整数）
【００２０】
ハプテン価の解析
透析して得た ＢＳＡコンジュゲートを、ＭＡＬＤＩ−ＴＯＦ−ＭＳで質量分析した。ＢＳＡコンジュゲートの平均分子量は約７１８００であった（ＢＳＡの分子量約６６４００）。ハプテンの分子量が (５４０) であるので、ＢＳＡコンジュゲートには平均１０個（化合物６．６’においてｎの平均値）のハプテンが連結されていることが分かった。
【００２１】
Ｄ、モノクローナル抗体調製；
前項で得たＩＪＫＬＭ−ＫＬＨ（100μg）にＲＩＢＩアジュバンド（ＲＩＢＩ Ｉｍｍｕｎｏｌ．Res. Inst.社製）を加え、良く攪拌してエマルジョンとしたのち、これをＢａｌｂ／ｃマウス（５匹）に二週間毎に三回、腹腔内投与した。初回免疫から３５日目にマウスの血清を採取し、ＩＪＫＬＭ−ＢＳＡ（式４）を用い、ＥＬＩＳＡ法で血清の抗体価を滴定した。
【００２２】
【化６】

【００２３】
E、ＥＬＩＳＡ法による抗体価の測定；
９６ウエルＥＬＩＳＡ用プレート（ファルコン社製、3910）の各ウエルに50 μＬの ＩＪＫＬＭ−ＢＳＡ溶液を入れ、室温で２時間放置後、４℃で一晩放置して、プレートにコンジュゲートを吸着させた。プレートをＰＢＳ−Ｔｗｅｅｎ〔PBS バッファー に５％ Ｔｗｅｅｎ ２０(和光純薬製 ポリオキシエチレン（２０）ソルビタンモノラウレート（Sorbitan Monolaurate）、ICI社、商標Tween 20 相当品 No. 167-11515、を含むもの〕で３回洗浄後、ＭＩＬＬＩ−Ｑ水で一度洗浄し、吸着しなかったコンジュゲートを除去した。ハイブリドーマ培養上清（もしくは抗血清、精製抗体溶液）を加え、室温で１時間放置後、PBS-Tweenと、ＭＩＬＬＩ−Ｑ水で順次洗浄した。50μＬの 酵素標識二次抗体（ヤギ抗マウスIgG−西洋わさびペルオキシダーゼ：ＨＲＰ）（ＺＹＭＥＤ社製、62-6520 1000倍希釈）を各ウエルに入れ、室温で１時間放置後、PBS-Tweenと、ＭＩＬＬＩ−Ｑ水で順次洗浄した。100μＬの基質溶液〔基質溶液の組成: １，２−フェニレンジアミン（1,2-phenylenediamine）4.0 mg、過酸化水素水 10 μＬ、0.1Ｍ クエン酸バッファー (pH 5.0) 10 ml〕を加え、５分間呈色反応を進行させた後、２規定 硫酸（50 μＬ）で反応を停止した。マイクロプレート吸光度測定装置（ＢＩＯ−ＲＡＤ, Benchmark 170-6850）を用いて、４５０ｎｍの吸光度を測定し、陽性のクローンを判別した。
【００２４】
血清中の抗体価の測定；
ＩＪＫＬＭ−ＢＳＡ溶液を吸着した９６ウエルＥＬＩＳＡ用プレートの最上段に、５０μＬのＰＢＳバッファーを入れた。２００倍に希釈したマウスの抗血清(50 μＬ) を最上段のウエル(Ａ１)に加え、この溶液を順次２倍希釈した（縦列Ａ１−Ａ８に４００倍から５１，２００倍の希釈系列を作製）。プレートを室温で１時間放置後、上述の方法で４５０ｎｍの吸光度を測定した。血清希釈率の対数（横軸）と吸光度をプロット（ＯＤ、縦軸）すると、シグモイド状の滴定曲線となり、血清濃度依存的に、血清中の抗体がＩＪＫＬＭ−ＢＳＡコンジュゲートと結合することが分かった。また、ＡＢＣ−ＢＳＡとはほとんど結合しないことを確認した。
【００２５】
５匹の中で、最も高い抗体価を示したマウスに、腹腔内にＩＪＫＬＭ−ＫＬＨ（100 μg）を追加免疫し、３日後に脾臓を摘出した。脾臓に付着している組織や臓器の断片をピンセットで取り除いた後、基本培地〔RPMI Medium 1640 (GIBCO 社製、一袋)、炭酸水素ナトリウム2 g, ペニシリン-ストレプトマイシン（GIBCO 社製）20 mg, 200 mM-グルタミン２０mLを蒸留水に溶かして１０００mＬとしてｐＨを７．２に調製したもの〕入りのシャーレに移し、ピンセットで脾臓内の細胞を浮遊させた。脾細胞浮遊液を濾過後、５０mL遠心管に移した。さらに、基本培地１５ｍＬを加え、良くピペッティングして濾過し、全量を３０ｍＬとした。８００回転／分で５分間、室温で遠心分離し、上清を除去、タッピングした。ＨＴ・ＢＣ培地［牛胎児血清（ＦＣＳ）200 ｍＬ, ＨＴ（コスモバイオ社製ＨＴ液（５０倍濃縮）２０ｍＬ,ＢＣ（Bioresearch island社製 BriClone）５０ｍＬ, 基本培地７３０ ｍＬを混合したもの）を３０ｍＬ加え、細胞を浮遊させた。
【００２６】
ハイブリドーマの調製；
冷凍庫から（−１３０℃）からミエローマ細胞〔Ｐ３Ｘ６３−Ａｇ８．５６３（大日本製薬社製）〕の凍結したチューブを取り出し、３７℃恒温槽中で速やかに解凍した。チューブをアルコール綿でよく消毒したのち、チューブ内の細胞浮遊液を基本培地３０ｍＬに移した。８００回転／分で５分間、室温で遠心分離し、上清を除去した。タッピング後、１０ＦＣＳ培地(基本培地に１０％のＦＣＳを添加)を１０ｍＬ加え、細胞を浮遊させ、５０ｍＬ培養フラスコに移した。フラスコの栓をゆるめ、炭酸ガス培養装置にいれた。１?２日毎に継代し、２５０ｍＬフラスコ２本分（９０-１００ｍＬ）にした。
【００２７】
マウスから取り出した脾細胞（２ｘ１０⁸個）とミエローマ細胞（5ｘ１０⁷個）を混合し、遠心（８００回転／分、５分間、室温）し、上清を除去、タッピングした。この後、ＥＣＦバッファー〔マンニトール 45.5 g, 10 mＭ 塩化カルシウム（10 mＬ）, 10 mＭ 塩化マグネシウム（10 mL）, 20 mＭ トリスバッファー〔トリス（ヒドロキシメチル）アミノメタン〕 ｐＨ７．２を蒸留水で溶かして、1000 mlにしたもの〕 を３０ｍＬを加え、遠心（８００回転／分、５分間、室温）、上清除去、タッピングする操作を２回繰り返した後、ＥＣＦバッファー(4.8 mＬ) を加えた。これを６ウエルプレート（ＳＵＭＩＲＯＮ製）に、１．２ｍＬずつ分注し、島津社製 ＳＳＨ−１０細胞融合装置を用い、以下の条件で細胞融合した〔電極間距離:1.0 mm; 交流周波数: 1 MHz; 交流初期印加電圧: 80 V; 交流初期印加時間: 10 s; パルス幅: 40 μｓ; パルス電圧: 920 V; パルス電界強度: 2.30 kV/cm; 交流２次印加電圧: 80 V; パルス印加間隔: 1.0 s; 印加パルス数: 1; パルス電圧変化: +0 V; 交流最終印加時間: 10 s; 交流電圧減衰率: 0%; 接触強化: off〕。
【００２８】
前記調製したハイブリドーマ細胞を、各ウエルにＨＡＴ培地（選択培地）〔基本培地110 ml, FCS 30 ml, BC 7.5 ml, HAT（コスモバイオ社製 HAT液（50 倍濃縮）を混合したもの）100μＬを加えた９６ウエルプレート１０枚に移した。２週間後、ハプテンとしたＩＪＫＬＭ環部に結合する抗体を産生するハイブリドーマをＩＪＫＬＭ−ＢＳＡを用い、ＥＬＩＳＡ法でスクリーニングした。陽性のウエルを選択し、２回クローニング後に、再度ＥＬＩＳＡ陽性となった抗体を産生するクローンを順次培養し、それぞれ約２００ mＬまで増殖させた。この結果、ＩＪＫＬＭ−ＫＬＭを免疫原として、３種のモノクローナル抗体、すなわち、IＭ（ＩからＭの環を持つことを意味する）−３Ｄ１１（受託番号ＦＥＲＭ Ｐ−１８７５０として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受託され、平成１５年２月１３日に前記原寄託よりブタペスト条約に基づく寄託への移管申請により受託番号ＦＥＲＭ ＰＢ−８２９３として受託されたハイブリドーマにより産生された抗体。抗体名を前記受託ハイブリドーマを識別する表示と同一にした。）、ＩＭ−２Ｃ７およびＩＭ−８Ｂ１２を調製することができた。得られたモノクローナル抗体の結合試験をＥＬＩＳＡ法で検討した。抗原として、シガトキシン類の部分構造、すなわちＡＢＣ、ＡＢＣＤ、Ａ＊ＢＣ(Ａ環部がシガトキシン型のものを意味する)、およびＩＪＫＬＭ環構造を連結したＢＳＡコンジュゲートを用いた。その結果、ＩＪＫＬＭ 環部親和性の高い抗体として、ＩｇＧ抗体ＩＭ−３Ｄ１１を選別した。ＩＪＫＬＭに対する解離定数Ｋｄは８．６ｎＭであった（測定法については後述する。）。
【００２９】
抗体の精製及びサブクラスの決定
上清を抗マウスＩｇＧ、ＩｇＭアフィニティーカラム（ＮＧＦ Industries, Ltd.社製）で精製した（結合用リン酸バッファー（pH 7.0）、溶出用バッファー（0.2 M Glycine-HCl, pH 2.5）。精製した抗体をＳＤＳ−ＰＡＧＥ（ドデシル硫酸ナトリウムーポリアクリルアミドゲル電気泳動）で分析し、＞９５％の純度であることを確認した。ＰＩＥＲＣＥ社製タイピングキット（37501）を用いて、それそれの抗体のサブクラスを決定した。
【００３０】
抗体の親和性解析（競争阻害実験）
次に、前記選別した３種のモノクローナル抗体を精製し、ハプテンとの解離定数(Ｋｄ)を求めた。ＥＬＩＳＡ用プレート（Ａ１からＡ１２ウエル）に順次２倍希釈した競合阻害剤（ＰＢＳ溶液 各30 μＬ）の溶液を調製した。これに抗体溶液(30 μＬ)を加え、室温で２時間放置した。抗体と阻害剤の混合溶液５０μＬを、ハプテン−ＢＳＡ溶液を吸着した９６ウエルＥＬＩＳＡ用プレート（抗体の親和性解析参照）に加え、室温で２０分間放置した。プレートを洗浄後、吸光度を測定し、滴定曲線を得た。Ｆｒｉｇｕｅｔらの方法［Journal of Immunological Method, 第77巻（1985年）, 第305 頁］を参考に、Ｋｌｏｔｚプロットして得られた直線の傾きから阻害剤のＫｄ値を求めた。この結果、IＭ−３Ｄ１１はＩＪＫＬＭ 環部（ＩＭ）（式５）にかなり高い親和性（Ｋｄ＝ 8.6 nM）を示すことが分かった。
【００３１】
【化７】

【００３２】
この結果をふまえ、上述の競争阻害の実験系を利用して毒本体ＣＴＸ３Ｃとの結合試験を検討した。ＩＭ−３Ｄ１１はＣＴＸ３Ｃに強く結合した（Ｋｄ＝１２２ｎＭ）。さらに、ＩＭ−３Ｄ１１については、構造の類似した海産ポリエーテル毒素との結合試験を検討したところ、オカダ酸や、マイトトキシンとはほとんど結合しなかった。赤潮毒ブレベトキシン類とは交差活性を検出したが、ＣＴＸ３Ｃとの親和性と比べると、３５０分の１程度（ＢＴＸＡ:Ｋｄ＝４３μＭ）の非常に弱いものであることが分かった。
【００３３】
３Ｄ１１抗体の酵素標識法〔3D11-ＨＲＰ（西洋わさびペルオキシダーゼ）；の合成〕（サンドイッチ法用）
３Ｄ１１抗体のＨＲＰ標識はPierce社のEZ-Link（商標名）Plus Activated Peroxidase and Kitを用い、Pierce社説明書に従って行った。３Ｄ１１抗体(1mg) のcarbonate-bicarbonate buffer溶液 (1 mL)に1mgのEZ-Link Plus Activated Peroxidaseの水溶液(100 μL)を加え、室温で1時間反応させた。10 μLのReductant solution（ＮａＢＨ₃ＣＮが主成分）を反応混合液へ加え、１５分間室温で反応させた。２０ mLのクエンチバッファー（Quench Buffer）を加え、得られた溶液を室温で１５分間静置した。反応液は１ＬＰＢＳで３回透析後、１ｍＬのグリセロールを加え、−２０℃で保存した。
標識体としては、周知のものを使用できる。
【００３４】
シガトキシンのＡ〜Ｅ（ＡＥと略称する場合もある）の環構造をもつ前記標識モノクローナル抗体と組み合わせるモノクローナル抗体の調製（この技術は、本出願と同日付けにて特許出願した）の説明；
１．合成ハプテンとなる前記式２（請求項３）のジアステレオマー混合物の合成。
下記の工程１により得られる。
【００３５】
【化８】

【００３６】
化合物７のＯＨをベンジル基（Ｂｎ）保護基で保護した化合物の合成方法は、M. Maruyama et al,Heterocycles,2001,54,93-99,に記載されている。化合物１は前記文献２に記載の化合物のＢｎをＤＤＱ（2,3-ジクロロ-5,6-ジシアノ-1,4-ベンゾキノン）を用いて脱保護することにより得られる。化合物１のアルコール(4.0 mg,6.0 μmol)をＴＨＦ（テトラヒドロフラン）(2 ｍＬ)に溶かし、室温でテトラブチルアンモニウムフルオリド (ＴＢＡＦ,１Ｍ ＴＨＦ溶液：１８μL,１８μmol)を加えた。３０分後、反応溶液を濃縮して、シリカゲルクロマトグラフィーで精製し、化合物８(2.5 mg, 5.9 μmol,９８％)を得た。
【００３７】
２０ｍＬナス型フラスコに化合物８(2.8 mg, 6.6 μmol),ＣＨ₂Ｃｌ₂(300 μL), （ＭｅＯ）₂ＣＨＣＨ₂ＣＯＯＭｅ(１０μl,７０ μmol), ＴｓＯＨ・Ｈ₂Ｏ(0.5 mg, 3 μmol) を加えた。室温で１．５時間攪拌した後、トルエン(1 ｍＬ) を加えて、ロータリーエバポレーターで減圧（120 mbar/hPa）し、１時間後に大気圧に戻した。シリカゲルカラムクロマトグラフィーで精製して、アセタール位に関してのジアステレオマー混合物 (式２の上の化合物：下の化合物＝２：１) を(1.9 mg, 3.8 μmol, 57%) 得た。式２の化合物の物性を表２に示す。
【００３８】
【表２】

【００３９】
式１及び式２のＲをＨにした化合物９は、式２の化合物をＬｉＯＨで処理して得られる。工程においてＬがスクシンイミドの化合物４は工程２で得られる。
【００４０】
【化９】

【００４１】
２０mLナシ型フラスコに式１及び式２の化合物(2.5 mg, 4.9 μmol)、ｔ−ＢｕＯＨ(0.5 mL)、水 (125 μL)、ＬｉＯＨ・Ｈ₂Ｏ(2.8 mg, 68 μmol) を加えて、室温で１時間撹拌した。ＫＨＳＯ₄(18.6 mg, 136 μmol) を加え、溶液のｐＨ（3-４程度）を確認した後、酢酸エチル (40 mL) で希釈した。無水硫酸マグネシウムで乾燥、ろ過、濃縮し、粗化合物８を得た。これにＤＭＦ(ＤＭＦ＝Ｎ，Ｎ−ジメチルホルムアミド、２００μL)、Ｎ−ヒドロキシスクシンイミド (5.6 mg, 49 μmol)、ＥＤＣ−ＨＣｌ (ＥＤＣ＝1-(3-ジメチルアミノプロピル)-3-エチルカルボジイミド、4.8 mg, 25 μmol) を加えて、室温で１２時間攪拌した。反応溶液を酢酸エチル(40 mL)で希釈し、有機層を水で３回洗浄し、無水硫酸ナトリウムで乾燥、濃縮し、活性化エステルである化合物９を得た。これにＤＭＦ(100 μL) を加えた溶液を作製し、コンジュゲートの調製に使用した。
【００４２】
２．タンパクコンジュケートの製造。
ＫＬＨコンジュゲートの調製；
ＫＬＨ(5.0 mg)のＰＢＳバッファー溶液 (1.0 mL)に活性化エステルである化合物９(約2.4 μmol) の ＤＭＦ溶液(50 μL) を加えて１０分間撹拌した。一日静置した後、４℃で透析した。この後、５．９時間後にＰＢＳバッファー(700 mL)を交換し、１２時間後に透析膜からエッペンドルフチューブに移し換えて、−７８℃で保存した。
【００４３】
ＢＳＡコンジュゲートの調製；
ＢＳＡ(7.0 ｍｇ) のＰＢＳバッファー溶液 (2.0 ｍＬ) に活性化エステルである化合物９(約2.4 μmol) のＤＭＦ溶液 (50 μL) を加えて１０分間撹拌した。一日静置した後、４℃で透析した。この後、５．９時間後にＰＢＳバッファー (700 ｍＬ) を交換し、１２時間後に透析膜からエッペンドルフチューブに移し換えて、−７８℃で保存した。
【００４４】
３．ハプテン価の解析
透析して得た ＢＳＡコンジュゲートを、ＭＡＬＤＩ−ＴＯＦ−ＭＳで質量分析した。ＢＳＡコンジュゲートの平均分子量は約７０２００であった（ＢＳＡの分子量約66400）。ハプテン部分の分子量が（４７６）であるので、ＢＳＡコンジュゲートには平均８個のハプテンが連結されていることが分かった。
【００４５】
４．モノクローナル抗体（非標識抗体として用いる）の作製；
前項で得たＡＢＣＤＥ−ＫＬＨ(100μg）にＲＩＢＩアジュバンド（RIBI Immunol. Res. Inst.社製）を加え、良く攪拌してエマルジョンとしたのち、これをＢａｌｂ／ｃマウス（５匹）に二週間毎に３回、腹腔内投与した。初回免疫から３９日目にマウスの血清を採取し、ＡＢＣＤＥ−ＢＳＡを用い、ＥＬＩＳＡ法で血清の抗体価を滴定した。
【００４６】
ＥＬＩＳＡ法；
９６ウエルＥＬＩＳＡ用プレート（ファルコン社製、3910）の各ウエルに５０ μＬの ABCDE−ＢＳＡ溶液を入れ、室温で２時間放置後、４℃で一晩放置して、プレートにコンジュゲートを吸着させた。プレートをＰＢＳ−Ｔｗｅｅｎ〔ＰＢＳバッファーに５％Ｔｗｅｅｎ ２０〔和光純薬製 ポリオキシエチレン（polyoxyethylene） (２０) ソルビタン モノラウレート（Sorbitan Monolaurate）ＩＣＩ社 商標Ｔｗｅｅｎ−２０ 相当品 No. 167-11515〕〕を含むもの〕で３回洗浄後、ＭＩＬＬＩ-Ｑ水（Millipore 社製の複数段階の樹脂やフィルターを用いて、精製水を脱イオン化もの）で一度洗浄し、吸着しなかったコンジュゲートを除去した。ハイブリドーマ培養上清（もしくは抗血清、精製抗体溶液）を加え、室温で１時間放置後、ＰＢＳ−Ｔｗｅｅｎと、ＭＩＬＬＩ-Ｑ水で順次洗浄した。５０μＬの 酵素標識二次抗体（ヤギ抗マウスＩｇＧ−西洋わさびペルオキシダーゼ）（ＺＹＭＥＤ社製、62-6520 1000倍希釈）を各ウエルに入れ、室温で１時間放置後、ＰＢＳ−Ｔｗｅｅｎと、ＭＩＬＬＩ-Ｑ水で順次洗浄した。１００μＬの基質溶液 〔基質溶液の組成: 1,2-phenylenediamine 4.0 mg、過酸化水素水 10 μＬ、０．１Ｍ クエン酸 バッファー(pH 5.0) 10 ml〕を加え、５分間呈色反応を進行させた後、２規定 硫酸（50 μl）で反応を停止した。マイクロプレート吸光度測定装置（ＢＩＯ−ＲＡＤ,Ｂｅｎｃｈｍａｒｋ 170-6850）を用いて、４５０ｎｍの吸光度を測定し、陽性のクローンを判別した。
【００４７】
血清中の抗体価の測定
ハプテン（ＡＢＣＤＥあるいはＩＪＫＬＭ）−ＢＳＡ溶液を吸着した９６ウエルＥＬＩＳＡ用プレートの最上段に、５０μＬのＰＢＳバッファーを入れた。２００倍に希釈したマウスの抗血清(５０μl) を最上段のウエル(Ａ１)に加え、この溶液を順次２倍希釈した（縦列Ａ１−Ａ８に４００倍から５１，０００倍の希釈系列を作製）。プレートを室温で１時間放置後、上述の方法で４５０ｎｍの吸光度を測定した（図２参照）。血清希釈率の対数と吸光度をプロットすると、血清濃度依存的に、血清中の抗体が ＡＢＣＤＥ−ＢＳＡコンジュゲートと結合することが分かった。
【００４８】
５．抗体価の高いマウスからの脾臓の摘出、培養
５匹の中で、最も高い抗体価を示したマウスに、腹腔内にＡＢＣＤＥ−ＫＬＨ（100 μg）を追加免疫し、３日後に脾臓を摘出した。脾臓に付着している組織や臓器の断片をピンセットで取り除いた後、基本培地〔ＲＰＭＩ Ｍｅｄｉｕｍ 1640 (GIBCO 社製、一袋、炭酸水素ナトリウム２g, ペニシリン-ストレプトマイシン（GIBCO 社製）20 mg, 200 mM-グルタミン２０ｍＬを蒸留水に溶かして1000ｍＬとしてｐＨを7.2に調製したもの〕入りのシャーレに移し、ピンセットで脾臓内の細胞を浮遊させた。脾細胞浮遊液を濾過後、５０ｍＬ遠心管に移した。さらに、基本培地１５ｍＬを加え、良くピペッティングして濾過し、全量を３０ｍＬとした。８００回転／分で５分間、室温で遠心分離し、上清を除去、タッピングした。ＨＴ・ＢＣ培地〔牛胎児血清（FCS）200 ml,ＨＴコスモバイオ社製ＨＴ液（５０倍濃縮）２０ｍＬ、ＢＣ（Bioresearch island社製 BriClone）５０ｍＬ,
基本培地 ７３０ｍＬを混合したもの〕を３０mＬ加え、細胞を浮遊させた。
【００４９】
冷凍庫から（−１３０℃）からミエローマ細胞Ｐ３Ｘ６３−Ａｇ８．６５３（大日本製薬社製）を凍結したチューブを取り出し、３７℃恒温槽中で速やかに解凍した。チューブをアルコール綿でよく消毒したのち、チューブ内の細胞浮遊液を基本培地３０ｍＬに移した。８００ｒｐｍ（回転／分）で５分間、室温で遠心分離し、上清を除去した。タッピング後、１０％ＦＣＳ(基本培地に１０％のFCSを添加)を１０ｍＬ加え、細胞を浮遊させ、５０ｍＬ培養フラスコに移した。フラスコの栓をゆるめ、炭酸ガス培養装置にいれた。１−２日毎に継代し、２５０ｍＬフラスコ２本分（９０−１００ｍＬ）にした。
【００５０】
マウスから取り出した脾細胞（２ｘ１０⁸個）とミエローマ細胞（5ｘ１０⁷個）を混合し、遠心（８００回転／分、５分間、室温）し、上清を除去、タッピングした。この後、ＥＣＦバッファー〔マンニトール 45.5 g, 10 mM 塩化カルシウム（10 ｍＬ）、１０ｍＭ塩化マグネシウム（１０ｍＬ）、２０ｍＭ ＴｒｉｓバッファーｐＨ7.2を蒸留水で溶かして、1000ｍＬにしたもの〕 を３０ｍＬを加え、遠心（８００回転／分、５分間、室温）、上清除去、タッピングする操作を２回繰り返した後、ＥＣＦバッファー(4.8ｍＬ) を加えた。これを６ウエルプレート（ＳＵＭＩＲＯＮ製）に、１．２ｍＬずつ分注し、島津社製ＳＳＨ−１０細胞融合装置を用い、以下の条件で細胞融合した〔［電極間距離:１．０ｍｍ; 交流周波数: 1 ＭＨｚ; 交流初期印加電圧: ８０Ｖ; 交流初期印加時間:１０秒（s）; パルス幅:４０（マイクロ秒（μｓ）; パルス電圧:９２０Ｖ; パルス電界強度:２．３０ｋＶ／ｃｍ; 交流２次印加電圧:８０Ｖ; パルス印加間隔:１．０秒;印加パルス数: １; パルス電圧変化:＋０Ｖ; 交流最終印加時間:１０秒; 交流電圧減衰率:０％; 接触強化:ｏｆｆ〕。
【００５１】
調製したハイブリドーマ細胞を、各ウエルにＨＡＴ培地 [HAT-RPMI 1640, 20% FCS, 5% Briclone (BioResearch Ireland社製)含有] １００μＬを加えた９６ウエルプレート１０枚に移した。２週間後、ハプテンとしたＡＢＣＤＥ環部に結合する抗体を産生するハイブリドーマ１０Ｃ９（受託番号ＦＥＲＭ Ｐ−１８７４９とて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受託された。受託日平成１４年３月５日。平成１５年２月１３日に前記原寄託よりブタペスト条約に基づく寄託への移管申請により受託番号ＦＥＲＭ ＰＢ−８２９２として受託された。）をＡＢＣＤＥ−ＢＳＡを用い、ＥＬＩＳＡ法でスクリーニングした。陽性のウエルを選択し、２ 回クローニング後に、再度ＥＬＩＳＡ陽性となった抗体を産生するモノクローンを順次培養し、それぞれ約２００ｍＬまで増殖させた。この結果、６種のモノクローナル抗体を調製することができた。得られたモノクローナル抗体の結合試験をＥＬＩＳＡ法で検討した。抗原として、シガトキシン類の部分構造を連結したＢＳＡコンジュゲートを用いた。その結果（表３）、ＡＢＣＤＥ−ＫＬＨ環部を免疫して得られたＩｇＧ抗体は、ＡＢＣＤＥ−ＢＳＡ（化学式ＡＥ−ＢＳＡ）へ強く結合し、IＪＫＬＭ−ＢＳＡ（化合物１０）とは結合しないことがわかった。
【００５２】
【化１０】

【００５３】
【表３】

【００５４】
【化１１】

【００５５】
サンドイッチ検定法
Ｃｏａｓｔｅｒ社ＥＬＩＳＡ Ｐｌａｔｅ（83590）に１０Ｃ９のＰＢＳ溶液 (4.3 μg/mL)を５０μＬ/ウエル入れ、４℃で一晩静置した。溶液を捨て、１％スキム ミルク入りＰＢＳを加えて(400 μL/ウエル)室温で１時間静置した。溶液を廃棄後、ＰＢＳ−Ｔｗｅｅｎで３回洗浄した後(200 μL/ウエル) 、ＣＴＸ３Ｃの希釈溶液を入れ(50 μL/ウエル)１時間静置した。溶液を廃棄後、ＰＢＳ−Ｔｗｅｅｎで３回洗浄した(200 μL/ウエル)。３Ｄ１１−ＨＲＰのＰＢＳ−Ｔｗｅｅｎ溶液( 1 μg/mL, 50 μL/ウエル)を加え、室温で１時間静置した。溶液を廃棄して、ＰＢＳ−Ｔｗｅｅｎで３回洗浄した(200 μL/ウエル)後、ＯＰＤ溶液(100 μL/ウエル、Sigma社FAST（商標名）ｏ−PHENYLENE DIAMINE DIHYDROCHLORIDE TABLET SETSを使用)を加えて、室温にて５−１０分発色させた。２規定硫酸水溶液(50μL/ウエル)を加えて反応を停止させ、吸光度(450ｎｍ)をBio-Rad社製Microplate Reader Benchmarkにて測定した。測定結果を図１に示す。
【００５６】
【発明の効果】
以上述べたように、本発明において設計した合成ハプテン(ＩＪＫＬＭ環部)、および合成ハプテン(ＡＢＣＤＥ環部)を用いて得られたモノクローナル抗体の組み合わせたサンドイッチＥＬＩＳＡ法を用いることにより、シガトキシンを高感度で検定できることを見出した。従来の、１種の抗体のサンプルへの結合を指標とするシガテラ毒検定キットにおいては、しばしば、陽性となっても夾雑物に結合し陽性となった擬陽性のケースを避けることができなかった。従来技術ではそのことの判別も不可能であったので、本発明の測定キットの提供は、社会生活の向上に資すること顕著である。被検出非タンパク化合物に対する２つの抗体を組み合わせたサンドイッチ式検定法は、本発明により初めて確立したものであり、特に、シガトキシンＣＴＸ３Ｃの特異性の高い検出方法を確立した点において優れた効果をもたらしたものである。
【００５７】
本明細書で使用の一部の略号表
BSA: bovine serum albumin
CTX: ciguatoxin
ELISA: enzyme linked immunosorbent assay
HRP: Horseradish Peroxidase (西洋わさびペルオキシダーゼ)
O.D.: Optical Density
OPD: o-Phenylene Diamine
PBS: Phosphate Buffered Saline
Bn: benzyl
DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
EDC；1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
FCS: fetal calf serum
Ig: Immunoglobulin
KLH: keyhole limpet hemocyanine
TBAF: tetra-n-butylammonium fluoride
Ts: p-tolenesulfonyl
THF: tetrahydrofuran
OVA: ovalbumin
【図面の簡単な説明】
【図１】 本発明のサンドイッチ法によるＣＴＸ３Ｃの検出特性
【図２】 １−５はシガトキシンのＡ〜Ｅ環部を含むＢＳＡとのコンジュゲートＡＥ−ＢＳＡに対する抗体価、（１）−（５）はシガトキシンのＩ〜Ｍ環部を含むＢＳＡとのコンジュゲートＩＭ−ＢＳＡに対する抗体価[0001]
BACKGROUND OF THE INVENTION
The present invention provides a plurality of monoclonals that specifically bind to the detected non-protein polymer compound obtained by using a plurality of synthetic haptens derived from partial structures in the molecule constituting the detected non-protein polymer compound. An unlabeled antibody, in particular a monoclonal antibody that specifically reacts with ciguatoxins, and a labeled compound, for example, a labeled monoclonal antibody that binds an enzyme labeling compound and a monoclonal antibody that is different from the monoclonal antibody that specifically reacts with ciguatoxins The present invention relates to a sandwich assay kit useful for the detection of ciguatoxins combined with a monoclonal antibody, particularly ciguatoxin CTX3C. Further, a hybridoma producing a monoclonal antibody that obtains the labeled monoclonal antibody, a synthetic hapten used for producing the hybridoma, and an immunizing compound used for producing the hybridoma obtained from the synthetic hapten and a carrier protein About.
[0002]
[Prior art]
Research to investigate sea venoms, especially ciguatoxins (CTX), using immunological techniques began around 1977 with the development of radioimmunoassay.
Only a very small amount of ciguatera venom is collected from nature (850 animals, 4t poison moray cucumber is only 0.35 mg of cigar toxin in the body of the poison), and production by culture is difficult, which is an obstacle to antibody preparation. The University of Hawaii, Hokama et al. Prepared a conjugate in which a valuable toxin body ciguatoxin (1 μg) was linked to human serum albumin by the carbodiimide method, and used this as an antigen to immunize mice and prepared monoclonal antibodies (Toxicon Vol. 15, (1977). Year), p. 317). This antibody binds to ciguatoxin but exhibits strong cross-activity with okadaic acid, and the difference in affinity is only about 5 times (Journal of Clinical Laboratory Analysis Vol. 6, (1992), p. 54). In addition, brevetoxin, mitotoxin, palytoxin and the like have been shown to show cross-activity (Journal of AOAC International Vol. 81, (1998), p. 727), but detailed data have not been published. Reagents, kits (Cigua Check ™), and the like have been developed to detect fish contaminated with ciguatoxins by immunological techniques using the antibody of Hokama et al.
[0003]
The present inventors chemically synthesized the left ABC ring part of ciguatoxin, and prepared three types of monoclonal antibodies using protein conjugates using this as a synthetic hapten. Only very weak affinity was shown (Synthesis (1999), page 1431). Other groups have also tried to immunize conjugates of synthetic haptens (JKLM ring) but have not yet prepared monoclonal antibodies [Toxicon 38 (2000) 669]. Under such circumstances, the present inventors further designed and synthesized a synthetic hapten containing the IJKLM ring, which is a partial structure at the right end of ciguatoxins, and immunized mice with the protein conjugate of this synthetic hapten. By a method comprising the step of Accession number FERM PB-8293 And successfully produced a monoclonal antibody highly specific for ciguatoxins using the hybridoma. The monoclonal antibody was added to 3D11 (Independent Administrative Agency, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, March 5, 2002). Accession number FERM P-18750. On February 13, 2003, the original deposit was accepted as the deposit number FERM PB-8293 by the transfer application to the deposit based on the Budapest Treaty. ) Named. The binding dissociation constant (Kd) of the monoclonal antibody for ciguatoxin CTX3C was 122 nM. In addition, when the cross-activity of the marine polyether toxin having a structure similar to ciguatoxin and the above antibody was measured, cross-activity with red tide toxin brevetoxins was confirmed, but this cross-activity was compared with the binding with ciguatoxins. It was found to be very weak, less than 1/350.
[0004]
In addition, as a synthetic hapten for the purpose of obtaining a monoclonal antibody of ciguatoxins, C of the ABC ring portion of ciguatoxin is used. ₁₆ A conjugate compound was synthesized from a derivative having a carboxylate linker introduced at the position and the carrier protein BSA or KLH, emulsified in RIBI adjuvant, and injected four times into three Balb / c mice at three-week intervals. The spleen is removed from the mouse, the spleen cells are fused with myeloma cells (P3X63-Ag8.653) to obtain the antibody, and the reaction specificity of the antibody against the synthetic hapten is evaluated [ Synthesis 1999, No. SI.1431-1436 ISSN 0039-7881,]. That is, one of the ring parts of synthetic ciguatoxins useful for producing monoclonal antibodies useful for investigating ciguatoxins (CTX) by immunochemical techniques using the partial structure of ciguatoxins, particularly ciguatoxin CTX3C. Synthetic haptens using parts have been studied.
[0005]
The present inventors have designed a synthetic hapten capable of obtaining a monoclonal antibody with further improved affinity for ciguatoxins, and the ABCDE ring part which is a partial structure of the left end of ciguatoxin CTX3C. A hybridoma 10C9 (Accessory No. FERM BP-8292), which is designed and synthesized as a hapten and containing a step of immunizing a mouse with a protein conjugate of this synthetic hapten. Date of trust of the center, March 5, 2002, trust number FERM P-18749. On February 13, 2003, the original deposit was accepted as the deposit number FERM PB-8292 by the transfer application to the deposit based on the Budapest Treaty. ) And succeeded in preparing a monoclonal antibody highly specific for ciguatoxins using the hybridoma.
[0006]
[Problems to be solved by the invention]
The object of the present invention is basically to provide a kit for detecting ciguatoxins by the sandwich method with improved detection characteristics by combining the two monoclonal antibodies developed by the present inventors.
Also useful for obtaining a hybridoma producing a monoclonal antibody for obtaining the kit, a synthetic hapten useful for obtaining the hybridoma, and a hybridoma producing the monoclonal antibody in which the synthetic hapten and a carrier protein are bound. Synthetic hapten-protein conjugates are provided.
In particular, the present inventors studied to obtain a useful sandwich method kit by binding a label useful for the sandwich method to one of the two monoclonal antibodies, and obtained a monoclonal hapten having a IJKLM ring constituting ciguatoxin CTX3C. It was discovered that a kit useful for the sandwich method can be obtained by binding an enzyme label to an antibody, and the above problems could be solved.
Further, in the above investigation, the detected compound specifically binds to the detected non-protein compound obtained by using a plurality of synthetic haptens derived from partial structures in the molecule constituting the detected compound. It was confirmed that by using a sandwich measurement kit in which a plurality of monoclonal antibodies were combined, it was possible to significantly reduce false positives, and the technical idea of the sandwich measurement kit was established.
[0007]
[Means for Solving the Problems]
Of the present invention The first is (1) according to claim 1 Obtained using the synthetic hapten of Formula 1 Accession Number FERM BP-8293 Monoclonal antibodies produced by the hybridoma 3D11 of Claim 1 Obtained using the synthetic hapten of formula 2 Accession Number FERM BP-8292 A sandwich assay kit for detecting ciguatoxins comprising a combination with a monoclonal antibody produced by hybridoma 10C9. Preferably, (2) A sandwich assay kit for detecting ciguatoxins, wherein one of the monoclonal antibodies to be combined is labeled, more preferably, 3) The sandwich measurement kit according to claim 1, wherein the labeling compound is an enzyme label. The sandwich measurement kit is characterized in that an unlabeled monoclonal antibody is adsorbed on a plate.
[0008]
Of the present invention The second is (4) according to claim 1 It is a novel compound represented by Formula 1. Of the present invention The third is (5) described in claim 1 Use as a synthetic hapten for obtaining a hybridoma that produces a monoclonal antibody that recognizes ciguatoxins of the novel compound represented by Formula 1.
[0009]
The fourth aspect of the present invention is (6) According to claim 6 A monoclonal antibody that specifically reacts with ciguatoxins obtained by immunization with a reaction product of a synthetic hapten of formula 3 and a carrier protein, preferably (7) The carrier protein in the reaction product of the synthetic hapten and the carrier protein is bovine serum albumin (BSA), keyhole limpet hemocyanine (KLM) or ovalbumin (egg albumin, OVA). Said monoclonal antibody.
[0010]
Of the present invention The fifth is (8) Deposited under the deposit number FERM BP-8293 It is a hybridoma that produces each of the monoclonal antibodies that specifically reacts with the ciguatoxins prepared.
[0011]
[Embodiments of the present invention]
The present invention will be described in more detail.
A. Detection of compounds present in trace amounts in the environment, food, etc., especially the establishment of a highly accurate detection method, is strongly required for safe social life. A combination of a plurality of monoclonal antibodies that specifically bind to the detected non-protein compound obtained by using a plurality of synthetic haptens derived from partial structures in the molecule constituting the detected non-protein compound of the present invention. Establishment of a sandwich measurement method for detecting non-protein compounds to be detected is an innovative technique in that it provides highly accurate detection.
B. Of the present invention Claim 1 Included in the synthetic hapten of Formula 1 Compounds 3, 3 ′ (described in Reaction Scheme 1) Is obtained by the following reaction formula 1. The starting compound 1 of Reaction Scheme 1 is known [Literature; Chem. Comm. 2001, 381-382. ]. Compound 2 was compounded in a 20 mL eggplant type flask with Compound 1 (7.8 mg, 11 μmol), ethyl acetate (EtOAc) (100 μL), methanol (300 μL), Pd (OH) ₂ (20% on carbon: 1.1 mg, 11 μmol) was added. The mixture was stirred at room temperature for 3 hours under a hydrogen atmosphere (using a thick balloon). Dilute with ethyl acetate and filter through celite. The filtrate was concentrated to obtain compound 2 (6.2 mg, 13 μmol) quantitatively.
[0012]
A mixture of compounds 3 and 3 ′ was added to compound 2 (4.2 mg, 9.0 μmol), CH in a 20 mL eggplant type flask. ₂ Cl ₂ (300 μL), (MeO) ₂ CHCH ₂ CO ₂ Me (25 μL, 180 μmol), TsOH · H ₂ O (0.5 mg, 3 μmol) was added. After stirring at room temperature for 1.5 hours, toluene (1 mL) was added, the pressure was reduced with a rotary evaporator (120 mbar / hPa), and the pressure was returned to atmospheric pressure after 1 hour. After diluting with ethyl acetate, the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution, water and saturated brine, dried over anhydrous magnesium sulfate, filtered and concentrated. Purification by silica gel column chromatography gave a diastereomeric mixture (3: 3 ′ = 3: 1) with respect to the acetal position (4.7 mg, 8.5 μmol, 94%). The physical properties of the compounds 3 and 3 ′ mixture are shown in Table 1.
[0013]
[Table 1]

[0014]
[Formula 4]

[0015]
C, the preparation of the protein conjugate is shown in Reaction Scheme 2.
Synthesis of compounds 3, 3 ′ to compounds 4, 4 ′;
In a 20 mL eggplant-shaped flask, compounds 3, 3 ′ (4.7 mg, 8.5 μmol), t-BuOH (0.5 mL), water (125 μL), LiOH · H ₂ O (2.8 mg, 68 μmol) was added, and the mixture was stirred at room temperature for 1 hour. KHSO _Four (18.6 mg, 136 μmol) was added, and the pH of the solution (about 3-4) was confirmed, followed by dilution with ethyl acetate (10 mL). Drying over anhydrous magnesium sulfate, filtration and concentration gave crude compounds 4, 4 ′. DMF (200 μL), N-hydroxysuccinimide (9.7 mg, 85 μmol) and EDC · HCl (8.1 mg, 43 μmol) were added thereto, and the mixture was stirred at room temperature for 12 hours. The reaction solution was diluted with ethyl acetate (10 ml), and the organic layer was washed three times with water, dried over anhydrous sodium sulfate, and concentrated to obtain activated esters 6 and 6 ′. A solution with DMF (100 μL) added thereto was prepared and used for the preparation of the conjugate.
[0016]
Preparation of KLH conjugate
To a PBS buffer solution (2.0 ml) of KLH (7.0 mg), a DMF solution (50 μL) of compound 6,6 ′ (about 4.2 μmol) as an activated ester was added and stirred for 10 minutes. After leaving still for one day, it dialyzed at 4 degreeC. Thereafter, the PBS buffer (700 mL) was changed after 14 and 19 hours, and after 28 hours, the dialysis membrane was transferred to an Eppendorf tube and stored at -78 ° C.
[0017]
Preparation of BSA conjugate
To a PBS buffer solution (2.0 mL) of BSA (7.0 mg), a DMF solution (50 μL) of compound 6,6 ′ (about 4.2 μmol) as an activated ester was added and stirred for 10 minutes. After leaving still for one day, it dialyzed at 4 degreeC. Thereafter, the PBS buffer (700 mL) was changed after 14 and 19 hours, and after 28 hours, the dialysis membrane was transferred to an Eppendorf tube and stored at -78 ° C.
[0018]
[Chemical formula 5]

[0019]
(Pr is a carrier protein, n is an integer of 1 or more)
[0020]
Analysis of hapten number
The BSA conjugate obtained by dialysis was subjected to mass spectrometry by MALDI-TOF-MS. The average molecular weight of the BSA conjugate was about 71800 (BSA molecular weight of about 66400). Since the molecular weight of the hapten was (540), it was found that an average of 10 haptens (average value of n in compound 6.6 ′) was linked to the BSA conjugate.
[0021]
D, monoclonal antibody preparation;
After adding RIBI adjuvant (manufactured by RIBI Immunol. Res. Inst.) To IJKLM-KLH (100 μg) obtained in the previous section and stirring well to make an emulsion, this was added to Balb / c mice (5 mice) for 2 weeks. Three intraperitoneal doses were given every time. On day 35 from the first immunization, mouse serum was collected, and the antibody titer of the serum was titrated by ELISA using IJKLM-BSA (Formula 4).
[0022]
[Chemical 6]

[0023]
E, measurement of antibody titer by ELISA method;
50 μL of IJKLM-BSA solution was placed in each well of a 96-well ELISA plate (Falcon, 3910), allowed to stand at room temperature for 2 hours, and then allowed to stand overnight at 4 ° C. to adsorb the conjugate to the plate. . Plate with PBS-Tween [PBS buffer containing 5% Tween 20 (polyoxyethylene (20) Sorbitan Monolaurate, manufactured by Wako Pure Chemical Industries, ICI, trademark Tween 20 equivalent No. 167-11515) After washing three times with MILLI-Q water, the conjugate that was not adsorbed was removed, and the hybridoma culture supernatant (or antiserum, purified antibody solution) was added, and the mixture was allowed to stand at room temperature for 1 hour. -Tween and sequentially washed with MILLI-Q water 50 μL of enzyme-labeled secondary antibody (goat anti-mouse IgG-horseradish peroxidase: HRP) (ZYMED, 62-6520 diluted 1000 times) was added to each well. After standing at room temperature for 1 hour, the plate was washed with PBS-Tween and MILLI-Q water in order 100 μL of substrate solution [composition of substrate solution: 1,2-phenylenediamine 4 0.04 mg, hydrogen peroxide solution 10 μL, 0.1 M citrate buffer (pH 5.0) 10 ml] was added and the color reaction was allowed to proceed for 5 minutes, and then the reaction was stopped with 2N sulfuric acid (50 μL). Using a microplate absorbance measurement apparatus (BIO-RAD, Benchmark 170-6850), the absorbance at 450 nm was measured to discriminate positive clones.
[0024]
Measurement of antibody titer in serum;
50 μL of PBS buffer was placed on the top of a 96-well ELISA plate adsorbed with IJKLM-BSA solution. Mouse antiserum (50 μL) diluted 200-fold was added to the uppermost well (A1), and this solution was serially diluted 2-fold (preparing 400 to 51,200-fold dilution series in columns A1-A8) ). After leaving the plate at room temperature for 1 hour, the absorbance at 450 nm was measured by the method described above. Plotting the logarithm of the serum dilution rate (horizontal axis) and absorbance (OD, vertical axis) gives a sigmoid titration curve, which shows that the antibody in the serum binds to the IJKLM-BSA conjugate depending on the serum concentration. It was. Moreover, it confirmed that it was hardly couple | bonded with ABC-BSA.
[0025]
Of the 5 mice, the mouse having the highest antibody titer was boosted with IJKLM-KLH (100 μg) intraperitoneally, and the spleen was removed 3 days later. After removing tissue and organ fragments attached to the spleen with tweezers, basic medium [RPMI Medium 1640 (GIBCO, one bag), sodium bicarbonate 2 g, penicillin-streptomycin (GIBCO) 20 mg, 20 mL of 200 mM glutamine was dissolved in distilled water and adjusted to 1000 mL with pH adjusted to 7.2]. The mixture was transferred to a petri dish and cells in the spleen were suspended with tweezers. The splenocyte suspension was filtered and transferred to a 50 mL centrifuge tube. Further, 15 mL of basic medium was added, and pipetting was performed well, followed by filtration to make the total volume 30 mL. Centrifugation was performed at 800 rpm for 5 minutes at room temperature, and the supernatant was removed and tapped. HT / BC medium [Fetal bovine serum (FCS) 200 mL, HT (Cosmo Bio HT solution (concentration 50 times) 20 mL, BC (Bioresearch island BriClone) 50 mL, basic medium 730 mL)] In addition, the cells were suspended.
[0026]
Preparation of hybridomas;
A frozen tube of myeloma cells [P3X63-Ag8.563 (manufactured by Dainippon Pharmaceutical Co., Ltd.)] was taken out from the freezer (−130 ° C.) and quickly thawed in a 37 ° C. constant temperature bath. After the tube was thoroughly disinfected with alcohol cotton, the cell suspension in the tube was transferred to 30 mL of basic medium. The supernatant was removed by centrifugation at 800 rpm for 5 minutes at room temperature. After tapping, 10 mL of 10FCS medium (10% FCS added to the basic medium) was added, the cells were suspended, and transferred to a 50 mL culture flask. The flask was loosened and placed in a carbon dioxide culture apparatus. It was subcultured every 1-2 days to make two 250 mL flasks (90-100 mL).
[0027]
Splenocytes removed from mice (2 × 10 ⁸ Cells and myeloma cells (5x10) ⁷ The mixture was mixed and centrifuged (800 rpm, 5 minutes, room temperature), and the supernatant was removed and tapped. After this, dissolve ECF buffer [mannitol 45.5 g, 10 mM calcium chloride (10 mL), 10 mM magnesium chloride (10 mL), 20 mM Tris buffer [Tris (hydroxymethyl) aminomethane] pH 7.2 with distilled water. , 1000 ml] was added, and centrifugation (800 rpm / min, 5 minutes, room temperature), supernatant removal, and tapping were repeated twice, and then ECF buffer (4.8 mL) was added. This was dispensed into a 6-well plate (manufactured by SUMIRON) in an amount of 1.2 mL, and cell fusion was performed under the following conditions using a Shimadzu SSH-10 cell fusion device [distance between electrodes: 1.0 mm; AC frequency: 1 MHz; AC initial applied voltage: 80 V; AC initial applied time: 10 s; Pulse width: 40 μs; Pulse voltage: 920 V; Pulse electric field strength: 2.30 kV / cm; AC secondary applied voltage: 80 V; Pulse applied Interval: 1.0 s; Number of applied pulses: 1; Pulse voltage change: +0 V; AC final application time: 10 s; AC voltage decay rate: 0%; Contact enhancement: off].
[0028]
100 μL of the prepared hybridoma cells are added to each well in a HAT medium (selection medium) (basic medium 110 ml, FCS 30 ml, BC 7.5 ml, HAT (mixed HAT solution (concentrated 50 times) by Cosmo Bio). Transferred to 10 additional 96-well plates. Two weeks later, a hybridoma that produces an antibody that binds to the IJKLM ring as a hapten was screened by ELISA using IJKLM-BSA. Positive wells were selected, and after cloning twice, clones producing the antibodies that became ELISA positive again were sequentially cultured and grown to about 200 mL each. As a result, using IJKLM-KLM as an immunogen, three types of monoclonal antibodies, namely, IM (meaning having a ring from I to M) -3D11 (independent administrative agency, National Institute of Advanced Industrial Science and Technology as accession number FERM P-18750) To the Patent Biological Depositary Center, Accepted under the accession number FERM PB-8293 by the transfer application from the original deposit to the deposit under the Budapest Treaty on February 13, 2003 Antibody produced by the hybridoma. The name of the antibody was the same as that used to identify the trust hybridoma. ), IM-2C7 and IM-8B12 could be prepared. The binding test of the obtained monoclonal antibody was examined by ELISA. As antigens, partial structures of ciguatoxins, that is, ABC, ABCD, A * BC (A ring means that of the ciguatoxin type), and BSA conjugate linked with IJKLM ring structure were used. As a result, IgG antibody IM-3D11 was selected as an antibody with high IJKLM ring affinity. The dissociation constant Kd for IJKLM was 8.6 nM (the measurement method will be described later).
[0029]
Antibody purification and subclass determination
The supernatant was purified by anti-mouse IgG, IgM affinity column (manufactured by NGF Industries, Ltd.) (binding phosphate buffer (pH 7.0), elution buffer (0.2 M Glycine-HCl, pH 2.5)). Was analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and was confirmed to be> 95% pure, using the PIERCE typing kit (37501) to determine the subclass of each antibody. Were determined.
[0030]
Antibody affinity analysis (competitive inhibition experiment)
Next, the three selected monoclonal antibodies were purified, and the dissociation constant (Kd) with the hapten was determined. A solution of competitive inhibitor (30 μL each in PBS solution) was prepared by sequentially diluting twice into ELISA plates (A1 to A12 wells). To this was added an antibody solution (30 μL) and left at room temperature for 2 hours. 50 μL of the antibody / inhibitor mixed solution was added to a 96-well ELISA plate adsorbed with a hapten-BSA solution (see antibody affinity analysis) and allowed to stand at room temperature for 20 minutes. After washing the plate, the absorbance was measured to obtain a titration curve. With reference to the method of Friguet et al. [Journal of Immunological Method, Vol. 77 (1985), p. 305], the Kd value of the inhibitor was determined from the slope of the straight line obtained by Klotz plotting. As a result, it was found that IM-3D11 exhibits a considerably high affinity (Kd = 8.6 nM) for the IJKLM ring part (IM) (Formula 5).
[0031]
[Chemical 7]

[0032]
Based on this result, a binding test with the poison main body CTX3C was examined using the above-described experimental system for inhibition of competition. IM-3D11 bound strongly to CTX3C (Kd = 122 nM). Furthermore, when IM-3D11 was examined for a binding test with a marine polyether toxin having a similar structure, it hardly bound to okadaic acid or mitotoxin. Cross-activity was detected with red tide venom brevetoxins, but it was found to be very weak, about 1/350 (BTXA: Kd = 43 μM), compared with the affinity with CTX3C.
[0033]
Enzyme labeling method of 3D11 antibody [synthesis of 3D11-HRP (horseradish peroxidase)] (for sandwich method)
HRP labeling of 3D11 antibody was performed using Pierce's EZ-Link (trade name) Plus Activated Peroxidase and Kit according to the Pierce instructions. To a carbonate-bicarbonate buffer solution (1 mL) of 3D11 antibody (1 mg), 1 mg of an aqueous solution of EZ-Link Plus Activated Peroxidase (100 μL) was added and reacted at room temperature for 1 hour. 10 μL Reductant solution (NaBH _Three CN was the main component) and added to the reaction mixture and allowed to react for 15 minutes at room temperature. 20 mL of quench buffer was added and the resulting solution was allowed to stand at room temperature for 15 minutes. The reaction solution was dialyzed 3 times with 1 L PBS, added with 1 mL of glycerol, and stored at -20 ° C.
A well-known thing can be used as a labeled body.
[0034]
Description of the preparation of a monoclonal antibody in combination with the labeled monoclonal antibody having the ring structure of ciguatoxins AE (sometimes abbreviated as AE) (this technology was patented on the same date as the present application);
1. Synthesis of the diastereomeric mixture of formula 2 (Claim 3) to be a synthetic hapten.
It is obtained by the following step 1.
[0035]
[Chemical 8]

[0036]
A method for synthesizing the compound 7 in which OH is protected with a benzyl (Bn) protecting group is described in M. Maruyama et al, Heterocycles, 2001, 54, 93-99. Compound 1 can be obtained by deprotecting Bn of the compound described in Document 2 using DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone). Compound 1 alcohol (4.0 mg, 6.0 μmol) was dissolved in THF (tetrahydrofuran) (2 mL), and tetrabutylammonium fluoride (TBAF, 1 M THF solution: 18 μL, 18 μmol) was added at room temperature. After 30 minutes, the reaction solution was concentrated and purified by silica gel chromatography to obtain Compound 8 (2.5 mg, 5.9 μmol, 98%).
[0037]
Compound 8 (2.8 mg, 6.6 μmol), CH in a 20 mL eggplant type flask ₂ Cl ₂ (300 μL), (MeO) ₂ CHCH ₂ COOMe (10 μl, 70 μmol), TsOH · H ₂ O (0.5 mg, 3 μmol) was added. After stirring at room temperature for 1.5 hours, toluene (1 mL) was added, and the pressure was reduced (120 mbar / hPa) with a rotary evaporator. After 1 hour, the pressure was returned to atmospheric pressure. Purification by silica gel column chromatography gave a mixture of diastereomers with respect to the acetal position (upper compound of formula 2: lower compound = 2: 1) (1.9 mg, 3.8 μmol, 57%). Table 2 shows the physical properties of the compound of Formula 2.
[0038]
[Table 2]

[0039]
Compound 9 in which R in Formula 1 and Formula 2 is H is obtained by treating the compound of Formula 2 with LiOH. In the step, compound 4 in which L is succinimide is obtained in step 2.
[0040]
[Chemical 9]

[0041]
In a 20 mL pear-shaped flask, compounds of formula 1 and formula 2 (2.5 mg, 4.9 μmol), t-BuOH (0.5 mL), water (125 μL), LiOH · H ₂ O (2.8 mg, 68 μmol) was added and stirred at room temperature for 1 hour. KHSO _Four (18.6 mg, 136 μmol) was added, and the pH of the solution (about 3-4) was confirmed, followed by dilution with ethyl acetate (40 mL). Drying over anhydrous magnesium sulfate, filtration and concentration gave crude compound 8. DMF (DMF = N, N-dimethylformamide, 200 μL), N-hydroxysuccinimide (5.6 mg, 49 μmol), EDC-HCl (EDC = 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, 4.8 mg, 25 μmol) was added, and the mixture was stirred at room temperature for 12 hours. The reaction solution was diluted with ethyl acetate (40 mL), and the organic layer was washed 3 times with water, dried over anhydrous sodium sulfate, and concentrated to obtain Compound 9 as an activated ester. A solution with DMF (100 μL) added thereto was prepared and used for the preparation of the conjugate.
[0042]
2. Production of protein conjugates.
Preparation of KLH conjugates;
To a PBS buffer solution (1.0 mL) of KLH (5.0 mg), a DMF solution (50 μL) of compound 9 (about 2.4 μmol) as an activated ester was added and stirred for 10 minutes. After leaving still for one day, it dialyzed at 4 degreeC. Thereafter, the PBS buffer (700 mL) was changed after 5.9 hours, and after 12 hours, the dialysis membrane was transferred to an Eppendorf tube and stored at -78 ° C.
[0043]
Preparation of a BSA conjugate;
To a PBS buffer solution (2.0 mL) of BSA (7.0 mg), a DMF solution (50 μL) of compound 9 (about 2.4 μmol) as an activated ester was added and stirred for 10 minutes. After leaving still for one day, it dialyzed at 4 degreeC. Thereafter, PBS buffer (700 mL) was changed after 5.9 hours, and after 12 hours, the dialysis membrane was transferred to an Eppendorf tube and stored at -78 ° C.
[0044]
3. Analysis of hapten number
The BSA conjugate obtained by dialysis was subjected to mass spectrometry by MALDI-TOF-MS. The average molecular weight of the BSA conjugate was about 70200 (BSA molecular weight of about 66400). Since the molecular weight of the hapten moiety was (476), it was found that an average of 8 haptens were linked to the BSA conjugate.
[0045]
4). Production of monoclonal antibodies (used as unlabeled antibodies);
After adding RIBI adjuvant (manufactured by RIBI Immunol. Res. Inst.) To ABCDE-KLH (100 μg) obtained in the previous section and stirring well to make an emulsion, this was added to Balb / c mice (5 mice) for 2 weeks. Three intraperitoneal doses were given. On day 39 after the first immunization, mouse serum was collected, and the antibody titer of the serum was titrated by ELISA using ABCDE-BSA.
[0046]
ELISA method;
50 μL of ABCDE-BSA solution was placed in each well of a 96-well ELISA plate (Falcon, 3910), allowed to stand at room temperature for 2 hours, and then allowed to stand overnight at 4 ° C. to adsorb the conjugate to the plate. . The plate was PBS-Tween [5% Tween 20 in PBS buffer [polyoxyethylene (20) Sorbitan Monolaurate manufactured by Wako Pure Chemicals Co., Ltd., trade name Tween-20 equivalent No. 167-11515]] The product was washed three times with MILLI-Q water (purified water was deionized using a multi-stage resin and filter manufactured by Millipore) to remove the non-adsorbed conjugate. . Hybridoma culture supernatant (or antiserum, purified antibody solution) was added, and the mixture was allowed to stand at room temperature for 1 hour, and then washed sequentially with PBS-Tween and MILLI-Q water. 50 μL of enzyme-labeled secondary antibody (goat anti-mouse IgG-horseradish peroxidase) (manufactured by ZYMED, 62-6520 diluted 1000 times) was placed in each well, allowed to stand at room temperature for 1 hour, PBS-Tween, and MILLI-Q. Washed sequentially with water. 100 μL of substrate solution [Composition of substrate solution: 4.0 mg of 1,2-phenylenediamine, 10 μL of hydrogen peroxide, 10 ml of 0.1 M citrate buffer (pH 5.0)] was allowed to proceed for 5 minutes. Then, the reaction was stopped with 2N sulfuric acid (50 μl). Using a microplate absorbance measurement apparatus (BIO-RAD, Benchmark 170-6850), absorbance at 450 nm was measured to discriminate positive clones.
[0047]
Measurement of antibody titer in serum
50 μL of PBS buffer was placed on the top of a 96-well ELISA plate adsorbed with a hapten (ABCDE or IJKLM) -BSA solution. Mouse antiserum (50 μl) diluted 200-fold was added to the uppermost well (A1), and this solution was diluted 2-fold sequentially (preparing 400-fold to 51,000-fold dilution series in columns A1-A8). . The plate was allowed to stand at room temperature for 1 hour, and then the absorbance at 450 nm was measured by the method described above (see FIG. 2). When the logarithm of the serum dilution ratio and the absorbance were plotted, it was found that antibodies in the serum bound to the ABCDE-BSA conjugate in a serum concentration-dependent manner.
[0048]
5). Extraction and culture of spleen from mice with high antibody titer
Among the 5 mice, the mouse showing the highest antibody titer was boosted with ABCDE-KLH (100 μg) intraperitoneally, and the spleen was removed 3 days later. After removing tissue and organ fragments adhering to the spleen with forceps, basic medium [RPMI Medium 1640 (GIBCO, 1 bag, sodium bicarbonate 2 g, penicillin-streptomycin (GIBCO) 20 mg, 200 mM -Dissolved 20 mL of glutamine in distilled water and adjusted to pH 7.2 with 1000 mL] transferred to a petri dish containing cells and suspended cells in the spleen with tweezers, filtered and transferred to 50 mL centrifuge tube Further, 15 mL of basic medium was added, pipetted well, and filtered to make a total volume of 30 mL, centrifuged at 800 rpm for 5 minutes at room temperature, and the supernatant was removed and tapped. Fetal calf serum (FCS) 200 ml, HT Cosmo Bio HT solution (concentrated 50 times) 20 mL, BC (Bioresearch island BriClone) 50 mL,
30 mL of a basic medium mixed with 730 mL] was added to suspend the cells.
[0049]
The tube which frozen myeloma cell P3X63-Ag8.653 (Dainippon Pharmaceutical Co., Ltd.) was taken out from the freezer (-130 degreeC), and it thawed | decompressed rapidly in a 37 degreeC thermostat. After the tube was thoroughly disinfected with alcohol cotton, the cell suspension in the tube was transferred to 30 mL of basic medium. Centrifugation was performed at 800 rpm (rotation / min) for 5 minutes at room temperature, and the supernatant was removed. After tapping, 10 mL of 10% FCS (10% FCS added to the basic medium) was added, the cells were suspended, and transferred to a 50 mL culture flask. The flask was loosened and placed in a carbon dioxide culture apparatus. It was subcultured every 1-2 days to make two 250 mL flasks (90-100 mL).
[0050]
Splenocytes removed from mice (2 × 10 ⁸ Cells and myeloma cells (5x10) ⁷ The mixture was mixed and centrifuged (800 rpm, 5 minutes, room temperature), and the supernatant was removed and tapped. Then, add 30 mL of ECF buffer (manitol 45.5 g, 10 mM calcium chloride (10 mL), 10 mM magnesium chloride (10 mL), 20 mM Tris buffer pH 7.2 dissolved in distilled water to 1000 mL), and centrifuge. (800 rotations / minute, 5 minutes, room temperature) After removing the supernatant and tapping twice, ECF buffer (4.8 mL) was added. This was dispensed 1.2 mL each into a 6-well plate (manufactured by SUMIRON), and cell fusion was performed under the following conditions using an SSH-10 cell fusion device manufactured by Shimadzu [[Distance between electrodes: 1.0 mm; AC frequency : 1 MHz; AC initial application voltage: 80 V; AC initial application time: 10 seconds (s); Pulse width: 40 (microseconds (μs); Pulse voltage: 920 V; Pulse electric field strength: 2.30 kV / cm; AC 2 Next applied voltage: 80 V; Pulse application interval: 1.0 seconds; Number of applied pulses: 1; Pulse voltage change: +0 V; AC final application time: 10 seconds; AC voltage decay rate: 0%; Contact enhancement: off].
[0051]
The prepared hybridoma cells were transferred to 10 wells of 96 well plates to which 100 μL of HAT medium [containing HAT-RPMI 1640, 20% FCS, 5% Briclone (manufactured by BioResearch Ireland)] was added to each well. Two weeks later, the hybridoma 10C9 (accession number FERM P-18749, which produces an antibody that binds to the ABCDE ring as a hapten, was deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. March 5th. On February 13, 2003, the original deposit was accepted as the deposit number FERM PB-8292 by the transfer application to the deposit based on the Budapest Treaty. ) Was screened by ELISA using ABCDE-BSA. Positive wells were selected, and after cloning twice, monoclonal clones producing the antibodies that became ELISA positive again were sequentially cultured and grown to about 200 mL each. As a result, 6 types of monoclonal antibodies could be prepared. The binding test of the obtained monoclonal antibody was examined by ELISA. As an antigen, a BSA conjugate linked with a partial structure of ciguatoxins was used. As a result (Table 3), the IgG antibody obtained by immunizing the ABCDE-KLH ring part strongly binds to ABCDE-BSA (chemical formula AE-BSA) and does not bind to IJKLM-BSA (compound 10). all right.
[0052]
[Chemical Formula 10]

[0053]
[Table 3]

[0054]
Embedded image

[0055]
Sandwich test
A 10 C9 PBS solution (4.3 μg / mL) was added to Coaster ELISA Plate (83590) at 50 μL / well and allowed to stand at 4 ° C. overnight. The solution was discarded, PBS containing 1% skim milk was added (400 μL / well), and the mixture was allowed to stand at room temperature for 1 hour. After discarding the solution, the plate was washed 3 times with PBS-Tween (200 μL / well), and then a diluted solution of CTX3C was added (50 μL / well) and allowed to stand for 1 hour. After discarding the solution, it was washed 3 times with PBS-Tween (200 μL / well). PBS-Tween solution of 3D11-HRP (1 μg / mL, 50 μL / well) was added, and the mixture was allowed to stand at room temperature for 1 hour. After discarding the solution and washing 3 times with PBS-Tween (200 μL / well), add OPD solution (100 μL / well, using Sigma FAST (trade name) o-PHENYLENE DIAMINE DIHYDROCHLORIDE TABLET SETS). The color was allowed to develop for 5-10 minutes at room temperature. A 2N aqueous sulfuric acid solution (50 μL / well) was added to stop the reaction, and the absorbance (450 nm) was measured with a Microplate Reader Benchmark manufactured by Bio-Rad. The measurement results are shown in FIG.
[0056]
【The invention's effect】
As described above, by using the sandwich ELISA method combining the synthetic hapten designed in the present invention (IJKLM ring part) and the monoclonal antibody obtained using the synthetic hapten (ABCDE ring part), cigartoxin is highly sensitive. It was found that it can be tested. In the conventional Sigatera venom assay kit using the binding of one kind of antibody to a sample as an index, it is often impossible to avoid a false positive case that becomes positive by binding to contaminants even if it becomes positive. Since it was impossible to determine this with the prior art, the provision of the measurement kit of the present invention is conspicuous to contribute to the improvement of social life. A sandwich assay method combining two antibodies against a non-protein compound to be detected was established for the first time by the present invention, and in particular, it had an excellent effect in establishing a highly specific detection method for ciguatoxin CTX3C. Is.
[0057]
Some abbreviation tables used in this specification
BSA: bovine serum albumin
CTX: ciguatoxin
ELISA: enzyme linked immunosorbent assay
HRP: Horseradish Peroxidase
OD: Optical Density
OPD: o-Phenylene Diamine
PBS: Phosphate Buffered Saline
Bn: benzyl
DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
EDC; 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride
FCS: fetal calf serum
Ig: Immunoglobulin
KLH: keyhole limpet hemocyanine
TBAF: tetra-n-butylammonium fluoride
Ts: p-tolenesulfonyl
THF:
OVA: ovalbumin
[Brief description of the drawings]
FIG. 1 Detection characteristics of CTX3C by the sandwich method of the present invention
FIG. 2 is a conjugate of BSA containing A to E ring parts of ciguatoxin against AE-BSA. (1)-(5) is a conjugate of BSA containing I to M ring parts of ciguatoxin. Antibody titer against gate IM-BSA

Claims (8)

Produced by hybridoma 10C9 of accession number FERM BP-8292 obtained using the hybridoma 3D11 obtained using the synthetic hapten of formula 1 and the monoclonal antibody produced by accession number FERM BP-8293 and the synthetic hapten of formula 2. The kit for detecting cigartoxin detection sandwich according to claim 2, comprising a combination with a monoclonal antibody.

R is H or a methyl group. The kit for detecting a ciguatoxin detection sandwich according to claim 1 , wherein one of the monoclonal antibodies to be combined is labeled. The sandwich measurement kit according to claim 2 , wherein the labeling compound is an enzyme label. A novel compound represented by the formula 1 according to claim 1 .  Use as a synthetic hapten for obtaining a hybridoma that produces a monoclonal antibody that recognizes ciguatoxins of the novel compound represented by the formula 1. A monoclonal antibody that specifically reacts with ciguatoxins obtained by immunization with a reaction product of a synthetic hapten represented by Formula 3 and a carrier protein.

In Formula 3, n is an integer of 1 or more.  The carrier protein in the reaction product of the synthetic hapten and the carrier protein is bovine serum albumin (BSA), keyhole limpet hemocyanine (KLM) or ovalbumin (egg albumin, OVA). Item 7. The monoclonal antibody according to Item 6. Hybridoma 3D11 producing a monoclonal antibody that specifically reacts with ciguatoxins deposited under accession number FERM PB-8293 .

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