JP2004231535A - Reagent for immunoassay to detect autoantibody against p53 gene product - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reagent excellent in sensitivity and specificity for immunoassay to detect an autoantibody against a p53 gene product. <P>SOLUTION: The reagent for immunoassay to detect the autoantibody against the p53 gene product comprises a polypeptide comprising as an essential constituting unit an amino acid sequence containing 8-50 amino acids such as Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００４８】
２．合成ポリペプチドへのビオチンによる修飾
合成ポリペプチチドＰ１の１重量部とビオチン残基導入試薬（商品名［Ｂｉｏｔｉｎ Ｓｕｌｆｏ−ＯＳｕ］、株式会社同仁化学研究所）１重量部を炭酸水素ナトリウム緩衝液（ｐＨ７．５）１０００重量部に溶解し、３０℃で２時間、静置反応した。反応溶液をＣ１８−逆相クロマトグラフィーで精製し、ビオチン結合ポリペプチドＰ１（ＰＢ−１）を作成した。Ｐ２〜Ｐ１１についても同様にしてビオチン結合ポリペプチド（ＰＢ−２〜ＰＢ−１１）を作成した。
【００４９】
＜実施例１＞
１．合成ポリペプチド結合ビーズの作成
（１）アビジン結合ビーズの作成
ストレプトアビジン（ロシュダイアグノティクス株式会社より購入）をｐＨ９の０．１Ｍ炭酸緩衝液に２０μｇ／ｍｌの濃度で溶解した。この溶液２０ｍｌに、直径３．２ｍｍのポリスチレンビーズ（イムノケミカル社製）１０００個を加え、２〜１０℃で４８時間静置させて、ポリスチレンビーズにストレプトアビジンを物理吸着させた。その後、溶液をアスピレーターで吸引除去し、２０ｍＬの０．１重量％牛血清アルブミン含有リン酸緩衝液（ｐＨ７．２）でビーズを２回洗浄し、アビジン結合ビーズを調製した。このアビジン結合ビーズを再度５０ｍＬの０．１％牛血清アルブミン含有リン酸緩衝液に浸漬し、浸漬状態で冷蔵（２〜１０℃）保存した。
【００５０】
（２）ｐ５３ポリペプチド結合ビーズ（ＰＢビーズ）の作成
浸漬液をアスピレーターで除いたアビジン結合ビーズ１０００個を、合成ポリペプチドＰＢ１を２μｇ／ｍＬの濃度で含むリン酸緩衝水溶液（０．０２Ｍ，ｐＨ７．０）２０ｍＬに加え、４〜１０℃で２４時間反応（２時間に１回転倒攪拌）した。その後、緩衝水溶液をアスピレーターで吸引除去し、２０ｍＬの０．１重量％牛血清アルブミン含有リン酸緩衝液（ｐＨ７．２）でビーズを２回洗浄した後、１０重量％のショ糖を含むリン酸緩衝液（ｐＨ７．２）に３０分間浸漬後、リン酸緩衝液をアスピレーターで除き、ビーズをろ紙上に撒き室温（約２５℃）で風乾し、合成ポリペプチド結合ビーズ（ＰＢ１ビーズ）を調整し、乾燥剤（シリカゲル）を入れた密閉容器中で冷蔵保存した。
【００５１】
２．免疫反応用緩衝液の作成
０．０２Ｍのリン酸緩衝液（ｐＨ８．０）に、カゼインを３ｇ／Ｌ及び塩化ナトリウムを８．５ｇ／Ｌの濃度になるように添加し、免疫反応用緩衝液を作成した。使用時まで冷蔵保存した。
【００５２】
３．ペルオキシダーゼ標識抗ヒトイムノグロブリン抗体の作成
抗ヒトイムノグロブリンポリクローナル抗体（ダコジャパン（株）製）及び西洋ワサビ由来ペルオキシダーゼ（東洋紡（株）製）を用い、文献［エス・ヨシタケ、エム・イマガワ、イー・イシカワ、エトール；ジェイ．バイオケム，Ｖｏｌ．９２（１９８２）１４１３−１４２４］に記載の方法でペルオキシダーゼ標識抗β２−マイクログロブリンポリクローナル抗体を調製し、冷凍（−３０℃）保存した。
【００５３】
４．酵素標識抗体液の作成
上記で作成した免疫反応緩衝液及びペルオキシダーゼ標識抗ヒトイムノグロブリン抗体を用いて次の通り酵素標識抗体液を作成した。すなわち、免疫反応用緩衝液１００ｍＬにペルオキシダーゼ標識抗ヒトイムノグロブリン抗体を蛋白量で１００μｇ添加し、攪拌混合し、これを酵素標識抗体液とした。
【００５４】
５．過酸化水素液の調製
２００μｌの３５％過酸化水素水を脱イオン水１リットルに溶解し、過酸化水素水とした。使用するまで冷蔵保存した。
【００５５】
６．基質液の調製
ルミノール（東京化成製）０．１８ｇ及び４−（シアノメチルチオ）フェノール０．１ｇ（三新化学製）を０．１Ｍ（モル／Ｌ）、ｐＨ８．５のトリス／塩酸緩衝液１リットルに溶解した。使用するまで遮光、冷蔵保存した。
【００５６】
＜実施例２〜１８＞
１．合成ポリペプチド結合ビーズの作成
実施例１と同様にして、表２及び３に示した実施例２〜１８の合成ポリペプチド結合ビーズを調整し、乾燥剤（シリカゲル）を入れた密閉容器中で冷蔵保存した。
尚、複数の合成ポリペプチドを組み合わせて使用する場合（実施例１２〜１８）、各合成ポリペプチドの量は同じとし、総量が２μｇ／ｍＬになるようにした。
【００５７】
また、免疫反応用緩衝液、ペルオキシダーゼ標識抗ヒトイムノグロブリン抗体、酵素標識抗体液、過酸化水素液及び基質液は、実施例１で作成したものを用いた。
【００５８】
＜比較例１＞ＧＳＴ融合ｐ５３蛋白質の作製
特開平９−２２９９３３号公報に記載の方法に準じて、４種類の蛋白質、すなわち、ｐ５３遺伝子のＮ末端からのアミノ酸配列が１−１１２のペプチド、１０８−３１０ペプチド、３０６−３９３ペプチド、１−３９３ペプチドとＧＳＴ（グルタチオンＳトランスフェラーゼ）との各融合蛋白質を以下の通り作製した。
【００５９】
１．ｐ５３遺伝子の調製
ヒト線維芽細胞ＷＩ−３８のＳＶ４０トランスフォーマントであるＷＩ−３８ＶＡ１３よりＲＮＡを抽出した。抽出は、ＡＧＰＣ法を用いて、次のようにして行った。
【００６０】
（ＡＧＰＣ法試薬の調製）
（１）１Ｍ クエン酸ナトリウム（ｐＨ７．０）の調製
クエン酸三ナトリウムの２９．４ｇを蒸留水８０ｍｌに溶解した後、クエン酸を加えて、ｐＨ７．０にあわせた後、水を加えて１００ｍｌとした。そして、オートクレーブ（１２０℃、２０分）してから使用した。
（２）Ｄ液の調製
ＧＴＣ（グアニジウムチオシアネート）２３６．３ｇ（４Ｍ）、ザルコシル ２．５ｇ（０．８重量％）、１Ｍ クエン酸ナトリウム緩衝液（ｐＨ７．０） １２．５ｍｌ（２５ｍＭ）及び蒸留水２５０ｍｌを８５℃で加温溶解した後、室温に戻し、水で４９６ｍｌにあわせた。細孔径０．４５μｍボトルトップフィルターで濾過して保存した。使用時にこの保存溶液５０ｍｌに対して２−メルカプトエタノール３６０μｌ（０．１Ｍ）を加え、Ｄ液とした。
（３）２Ｍ 酢酸ナトリウム（ｐＨ４．０）の調製
酢酸ナトリウム トリハイドレート ２７．２ｇを蒸留水１０ｍｌに溶解させた後、酢酸を用いてｐＨ４．０にあわせ、蒸留水を加えて１００ｍｌとした。細孔径０．４５μｍボトルトップフィルターで濾過して使用した。
【００６１】
（ＡＧＰＣ法の操作）
（１）チューブにＤ液０．５ｍｌを加え、ＷＩ−３８ＶＡ１３細胞（大日本製薬株式会社より購入）を分散し、細胞を破壊した。
（２）さらに、２Ｍ 酢酸ナトリウム ５０μｌ、フェノール ０．５ｍｌ及びクロロホルム／イソアミルアルコール（体積比４９／ｌ） １００μｌを順次加え、１種類いれるごとに、チューブを２〜３回振り、混ぜた。
（３）１０秒間激しく混ぜた後、１５分間氷冷した。
（４）遠心加速度１０，０００Ｇで１０℃、２０分間遠心した後、下部に分離した水層を、ＤＮＡを含む中間層が混入しないように、別のチューブに分取した。
（５）分取した水層に、イソプロパノール０．５ｍｌを加え、−２０℃で１時間冷やした。次に、１０分間遠心（１０，０００Ｇ、０℃）し、ＲＮＡを沈殿させた。
（６）沈殿したＲＮＡを０．５ｍｌのＤ液に再び溶解し、イソプロパノール０．５ｍｌを加え、−２０℃で１時間置く。
（７）遠心してＲＮＡを沈殿させ、沈殿を８０重量％エタノール水溶液１ｍＬで洗い、乾燥させた後、水に溶解する。
【００６２】
このようにして抽出したｍＲＮＡから、次の反応条件にて、ＲｅｖｅｒｓｅＴｒａｎｓｃｒｉｐｔａｓｅ（ＲＴ）反応によりｃＤＮＡを作成した。ＲＴ反応は、ＧＩＢＣＯ／ＢＲＬのＲｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔａｓｅ（商品名 Ｓｕｐｅｒｓｃｒｉｐｔ）を使用した。反応組成は次のとおりである。５０ｍＭ トリス（ヒドロキシメチル）アミノメタン／塩酸緩衝液（ｐＨ８．３）、７５ｍＭ塩化カリウム水溶液、３ｍＭ塩化マグネシウム水溶液、１０ｍＭ ｄＮＴＰ（デオキシリボヌクレオシド ５’−トリフォスフェート混合物）、７．５μｇ／ｍｌのランダムノナマー（シグマ社から購入）、ＲＮａｓｉｎ（プロメガ社、ＲＮａｓｅインヒビター）を１０００Ｕ／ｍｌ、ＲＮａｓｅＨ−（リバース トランスクリプターゼ）を２００，０００Ｕ／ｍｌ。
【００６３】
２．組み換え遺伝子の作成及び発現
作成したｃＤＮＡを制限酵素ＢａｍＨＩの認識配列を５´末端に有するＰＣＲｐｒｉｍｅｒにて増幅後、ＢａｍＨＩにて消化し、ｐＧＥＸ−５Ｘ−２ベクターのＢａｍＨＩ部位に組み込んだ。シークエンス法にてインサートが正しく組み込まれていることを確認した後、大腸菌ＢＬ２１（ＤＥ３），ＬｙｓＳ（Ｎｏｖａｇｅｎ社）にトランスフォーム後、０．１ｍＭのイソプロピルチオベータガラクトシド（ＩＰＴＧ）にて３７℃、２時間あるいは２５℃、６時間誘導をかけた。大腸菌の細胞ライゼイトより目的の融合蛋白質をグルタチオンセファロースを用いたアフィニティークロマトグラフィー法にて抽出、精製し、ＧＳＴ融合ｐ５３蛋白質（ＧＳＴ１［アミノ酸配列１−１１２を含む］、ＧＳＴ２［アミノ酸配列１０８−３１０を含む］、ＧＳＴ３［アミノ酸配列３０６−３９３を含む］、ＧＳＴ４［アミノ酸配列１−３９３を含む］を得た。
【００６４】
１，ＧＳＴ融合ｐ５３蛋白質結合ビーズ（ＧＳＴビーズ）の作成
実施例１のアビジン結合ビーズの作成に準じて作成した。すなわち、ＧＳＴ融合ｐ５３蛋白質ＧＳＴ１をｐＨ９の０．１Ｍ炭酸緩衝液に２０μｇ／ｍｌの濃度で溶解した。直径３．２ｍｍのポリスチレンビーズ（イムノケミカル社製）１０００個をこの溶液２０ｍｌに加え、２〜１０℃で４８時間静置させて、ポリスチレンビーズにＧＳＴ１を物理吸着させた。その後、溶液をアスピレーターで吸引除去し、２０ｍＬの０．１重量％牛血清アルブミン含有リン酸緩衝液（ｐＨ７．２）でビーズを２回洗浄した後、２０ｍＬの０．１重量％牛血清アルブミン含有リン酸緩衝液（ｐＨ７．２）に浸漬して２４時間冷蔵で放置した。浸漬液をアスピレーターで吸引除去し、２０ｍＬの０．１重量％牛血清アルブミン含有リン酸緩衝液（ｐＨ７．２）でビーズを２回洗浄した後、１０重量％のショ糖を含むリン酸緩衝液に浸漬後、液をアスピレーターで除き、ビーズをろ紙上に撒き室温で風乾し、ＧＳＴ１結合ビーズ（ＧＳＴ１ビーズ）とし、乾燥剤（シリカゲル）を入れた密閉容器中で冷蔵保存した。
【００６５】
＜比較例２〜５＞
１．ＧＳＴ融合ｐ５３蛋白質結合ビーズの作成
比較例１と同様にして、表３に示した比較例２〜５のＧＳＴ融合ｐ５３蛋白質結合ビーズを調整し、乾燥剤（シリカゲル）を入れた密閉容器中で冷蔵保存した。
尚、複数のＧＳＴ融合ｐ５３蛋白質を組み合わせて使用する場合（比較例５）、各ＧＳＴの量は同じとし、総量が２０μｇ／ｍＬになるようにした。
２．その他試薬の作成
免疫反応用緩衝液、酵素標識抗体液、過酸化水素液及び基質液、は実施例１で作成したものを用いた。
【００６６】
＜実施例１９＞
健常人プール血清及び癌患者血清を測定した例である。
１．検体
当社ボランティア５０名から採取した血清を各０．２ｍＬずつ混合し、健常人プール血清１０ｍＬを作成し、又、癌患者から得た血清（患者１〜３）を各１０ｍＬを用意し検体として用いた。
【００６７】
２．免疫反応操作
１２×７５ｍｍ試験管中に、免疫測定用緩衝液３００μＬ、検体（健常人プール血清又は癌患者血清）１０μＬ、及びＰＢビーズ又ＧＳＴビーズ１個を加え、３７℃で、１０分間反応させた。反応液をアスピレータで除去した後、生理食塩水２ｍＬを加てビーズを洗浄し、洗浄液をアスピレーターで除去した。さらに生理食塩水２ｍＬを加え同様に洗浄した。次に、酵素標識抗体液３００μＬを、洗浄後のビーズに加え３７℃、１０分反応させた。反応液をアスピレーターで除去し、生理食塩水２ｍＬを加えビーズを洗浄し、洗浄液をアスピレーターで除去した。さらに生理食塩水２ｍＬを加え同様に２回洗浄した。洗浄後のビーズについて、酵素活性の測定を行った。
【００６８】
３．酵素活性測定操作
洗浄後のビーズが入った試験管（１２×７５ｍｍ）をアロカ社製ルミネッセンスリーダーＢＬＲ−２０１型のサンプルホルダーにセットし、基質液２００μＬ及び過酸化水素水２００μＬを加え化学発光反応を開始した。発光反応開始４０秒後から１０秒間の発光量を積算計測し、これを酵素活性を示す発光量とした。なお、表２及び３中の数値は、上段が発光量を示し、下段は健常人プール血清の発光量を１．０としたときの各発光量の相対値である。
【００６９】
【表２】

【表３】

【００７０】
４．測定結果
結果を表２及び３に示した。実施例では比較例に比べ、健常人プール血清の発光量が低く、発光量比（表中下段の数値で、各測定発光量を健常人プール血清の測定発光量で除した値）が大きいことが判る。又、患者血清では個別のＰＢに対して反応性が低い場合があっても、複数のＰＢを組み合わせることにより感度が向上することが判る。
【００７１】
＜実施例２０＞
複数の健常人血清を測定して基準値を求め、カットオフ値を設定し、癌患者１０名からの血清を測定し、ｐ５３遺伝子産物に対する自己抗体の有無を判断した例である。
【００７２】
１．検体の測定
実施例１８で調整した合成ポリペプチド結合ビーズ（固相化ＰＢ１，ＰＢ２，ＰＢ１０，ＰＢ１１，ＰＢ３，ＰＢ４，ＰＢ５，ＰＢ６，ＰＢ７，ＰＢ８，ＰＢ９）及び比較例５で作成したＧＳＴ融合ｐ５３蛋白質結合ビーズ（固相化ＧＳＴ１，ＧＳＴ２，ＧＳＴ３）を用いて、実施例１９の方法で、健常人ボランティア１２０名からの血清検体及び実施例１９の健常人プール血清を測定した。
健常人ボランティア１２０名の測定値（発光量）の分布を図１（実施例１８）及び２（比較例５）に示した。図１の通り実施例１８の合成ポリペプチド結合ビーズでは発光量が低く、分布の範囲も狭いが、比較例５のＧＳＴ融合ｐ５３蛋白質結合ビーズ（図２）では発光量が高く、分布の範囲も広いことが判った。なお、図の横軸は発光量（ｃｐｓ）であり、点は検体の分布（１点が１検体に対応）を示す。
【００７３】
２．カットオフ値の設定
健常人の測定値（発光量）を統計処理ソフト「ＳＴＡＴＦＬＥＸ ｖ．４．１」（アーテック社製）を用いて分布型を解析した結果、実施例１８の合成ポリペプチド結合ビーズについての発光量分布は、歪度１．４９、尖度４．９０であるのに対して、比較例５のＧＳＴ融合ｐ５３蛋白質結合ビーズについての発光量分布は、歪度１．９８、尖度７．２４であった。なお、いずれの分布も正規分布していなかった。ノンパラメトリック法で９５％の信頼区間を求めたところ、実施例１８の合成ポリペプチド結合ビーズについての基準値は、発光量１２４５〜９３９７であったのに対して、比較例５のＧＳＴ融合ｐ５３蛋白質結合ビーズについての基準値は、発光量３７７１〜１７５３０であった。ここで上限発光量の１．５倍の発光量をカットオフ値と設定すると、実施例１８について発光量１４０９５、比較例５について発光量２６２９４であった。この時、実施例１８についての健常人プール血清の発光量は２４０６であり、カットオフ値の１／５．８６であり、比較例５についての健常人プール血清の発光量は６２４６であり、カットオフ値の１／４．２１であった。従って、健常人プール血清を、陰性コントロールとして測定した場合、実施例１８の合成ポリペプチド結合ビーズではカットオフ値＝「健常人プール血清測定発光量」×５．８６と設定され、一方、比較例５のＧＳＴ融合ｐ５３蛋白質結合ビーズではカットオフ値＝「健常人プール血清測定発光量」×４．２１と設定された。
【００７４】
３．検体の測定
表４記載の癌患者１０名（患者４〜１３）から採取した血清検体及び陰性コントロールとして実施例１９で作成した健常人プール血清を測定した。測定は、実施例１８の合成ポリペプチド結合ビーズ、及び比較例５のＧＳＴ融合ｐ５３蛋白質結合ビーズを用いて実施例１９と同様にして行った。
４．判定結果
健常人プール血清を陰性コントロールとし、実施例２０で設定し係数でカットオフ値（「健常人プール血清測定発光量」×係数）を求めた。カットオフ値以上（カットオフ比１．０以上）の測定値を自己抗体陽性、カットオフ値未満（カットオフ比１．０未満）の測定値を自己抗体陰性と判断した。結果を表４に示した。
【００７５】
【表４】

表４から、比較例５のＧＳＴ融合ｐ５３蛋白質結合ビーズではカットオフ値が高く、相対的に癌患者の発光量が低くなる（カットオフ比が小さくなる）ため、陰性と判定され頻度が高くなった。これは、健常人検体の測定値が、非特異吸着等の原因で、その分布が広いことに起因している。
これに対して、実施例１８の合成ポリペプチド結合ビーズではカットオフ値と癌患者検体の発光量差が大きく（カットオフ比が大きく）、自己抗体陰性と判断される頻度が小さかった。
【００７６】
【発明の効果】
本発明のｐ５３遺伝子産物に対する自己抗体を検出するための免疫測定用試薬は、非特異的反応が少なく、感度及び特異性に極めて優れている。すなわち、自己抗体を保有しない健常人からの検体の測定値と自己抗体を保有する癌患者からの検体の測定値との差が充分に大きく（高感度）、また自己抗体を保有しない健常人の検体に対して非特異的反応が極めて少ないので高精度の測定ができる。従って、本発明の測定試薬、測定試薬キット及び測定法によると、癌の極めて正確な診断が可能となる。
【００７７】
【図面の簡単な説明】
【図１】実施例１８の免疫測定用試薬を用いて、健常人ボランティア１２０名の測定値（発光量）の分布を示したグラフである（実施例２０）。
【図２】比較例５の免疫測定用試薬を用いて、健常人ボランティア１２０名の測定値（発光量）の分布を示したグラフである（実施例２０）。
【００７８】
【配列表】

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an immunoassay reagent for detecting an autoantibody to a p53 gene product, an immunoassay reagent kit, and an immunoassay method for an anti-p53 gene product autoantibody.
[0002]
[Prior art]
The p53 gene is a tumor suppressor gene that encodes a nuclear phosphorylated protein consisting of 393 amino acids (hereinafter, a product of the p53 gene), and is frequently deleted and point-mutated in various human cancers (non-p53). It is known that the expression of a mutant p53 gene product increases and accumulates in cancer cells because it has a very long half-life (Non-Patent Document 2). On the other hand, in cancer patients such as breast cancer and lung cancer, the appearance of autoantibodies that react with the p53 gene product in blood has been observed by Western blotting, immunoprecipitation, and the like (Non-patent Document 3). A method for measuring an autoantibody against a p53 gene product using a carrier on which the whole p53 gene product (393 amino acids) or a fragment (about 100 amino acids) is immobilized is known (for example, Patent Document 1).
[0003]
[Non-Patent Document 1] Science, 250, p. 1233, 1990
[Non-Patent Document 2] Oncogene, 6,1699, 1991
[Non-Patent Document 3] International Journal of Cancer (Int. J. Cancer), 30, p.403, 1982
[Patent Document 1]
JP-A-9-229933
[0004]
[Problems to be solved by the invention]
In the conventional method for measuring an autoantibody to the p53 gene product, the difference between the measured value of a sample from a healthy person without the autoantibody and the measured value of a sample from a cancer patient having the autoantibody is small (low sensitivity). In addition, a non-specific reaction sometimes resulted in an erroneous measurement value even in a sample of a healthy person having no autoantibody.
Accordingly, an object of the present invention is to provide an immunoassay reagent for detecting an autoantibody against a p53 gene product, which is excellent in sensitivity and specificity.
[0005]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the present inventors have found that a smaller peptide fragment of the p53 gene product (that is, a peptide consisting of a specific amino acid sequence in the p53 gene product) specifically binds to an autoantibody. The inventors have found that they are combined, and have completed the present invention. That is, the present invention relates to an immunoassay reagent for detecting an autoantibody to a p53 gene product, which is a Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trop Lys Leu Prognosis. Val Leu Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr (1), Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro LeuSer Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg (2), Ser Gly Thr Ala ysSerValThrCysThrSerProAlaLeuAsnLysMetPheCys (3), LeuArgValGluTyRalThArpAshPrAgAsPrAgThrThrPrAgAs Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val His Val Cys Ala Cys Pro Gly Arg (5), Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pr Gly Ser Thr Lys Arg Ala Leu Pro Asn (6), or Lys Asp Ala Gln Ala Gly LysGlu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly GlnSer Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp (7), characterized in that it comprises a polypeptide comprising at least one kind of amino acid sequence contained in Gly Pro Asp Ser Asp (7) and having 8 to 50 amino acids as an essential constituent unit. This is an immunoassay reagent.
[0006]
Also, the present invention is an immunoassay reagent for detecting an autoantibody against a p53 gene product, which is a Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr He Ser Asp Leu Trp Lys Leu Pro Lu Glu Pro Glu. Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr (1) is at least one kind of amino acid sequence, and has 8 to 50 amino acids. A polypeptide as a structural unit,
At least included in the Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp (7) An immunoassay reagent comprising: one kind of amino acid sequence; and a polypeptide having an amino acid sequence having 8 to 50 amino acids as a constituent unit.
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
Examples of the polypeptide contained in the immunoassay reagent of the present invention include a p53 gene product {Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr Seru Lup Rup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Lup Leu Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala Pro Thr Pro Ala Ala Pro Ala Pro Ala Pro er Trp Pro Leu Ser Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala ro Pro Gln His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val His Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn Leu rg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly ro Asp Ser Asp (62)} is a polypeptide comprising as essential constituent units a specific amino acid sequence contained in. The specific amino acid sequence is at least one amino acid sequence contained in the amino acid sequence (1), (2), (3), (4), (5), (6) or (7), and Is an amino acid sequence having 8 to 50 amino acids.
[0008]
The number of amino acids in the specific amino acid sequence is preferably 8 to 50, more preferably 8 to 30, and particularly preferably 8 to 20, from the viewpoint of antigenicity and preparation.
[0009]
The specific amino acid sequence includes the amino acid sequence (A) contained in the amino acid sequence (1), the amino acid sequence (B) contained in the amino acid sequence (2), the amino acid sequence (C) contained in the amino acid sequence (3), Amino acid sequence (D) contained in sequence (4), amino acid sequence (E) contained in amino acid sequence (5), amino acid sequence (F) contained in amino acid sequence (6) and amino acid sequence (7) Amino acid sequence (G) is included.
As the amino acid sequence (A) contained in the amino acid sequence (1), Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Lu Glu As Met Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr (1), Ser Asp Pro Ser Val GluPro LeuPr SerG (8), GlNpL Asn Asn Val Leu Ser Pro Leu (10), Pro Ser Gln Ala Met Asp Asp Leu Met Leu (11), Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr (12), Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu ThPhr Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu Ser Pro Leu (14), Leu Pro Glu Asn Asn Val Leu Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu (15), Pro Ser Gln Ala MetAsp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr 16), Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu (17), and Asp Asp Leu Met Leu Ser Pro Asp Asp Ile (18), and the like.
Of these, the amino acid sequences (17) and (18) are preferred.
[00010]
The amino acid sequence (B) contained in the amino acid sequence (2) includes Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser Gln Lys Thr Tyr Gly Gly Gly Gly G Thr Pro Ala Ala Pro Ala Pro AlaPro Ser (19), Trp Pro Leu Ser Ser Ser Val Pro Ser Gln (20), Lys Thr Tyr Gln Gary Gly Gly Aly Pyr Gyr Pyr Gyr Pyr Gyr Pyr Gyr Pyr Gyr Pyr Gyr Pyr Gyr Pyr Gyr Pyr Gyr Pyr Gyr Pyr Gyr Pyr Gyr Pyr Gyr Pyr Gyr Pyr Gyr Pyr Gyr Pyr Gyr Pry Gyr Pry Gyr Pry Gyr Pry Gyr Pry Gyr Pry Gly Ser Trp Pro Leu Ser Ser (22), Ser Trp Pro Leu Ser Ser Val Pro Ser Gln ys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg (23), Thr Pro Ala AlaPro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser (24), and Pro SerGinTryNyGySy .
Of these, the amino acid sequences (24) and (25) are preferred.
[0011]
Examples of the amino acid sequence (C) contained in the amino acid sequence (3) include Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys MetThrThrThyThrThyThrThyThrThyThrThrThyThrThyThrThrThrThyThrThyThrThrThyThrThrThyThrThrThrThyThrThrThyThrThrThyThrThrThyThrThyThrThyThrThyThrThyThrThyThrThyThrThyThrThyThrThrThyThrThyThrThyThrThyThrThyThrThyThrThyThrThyThrThyThrThyThrThyThrThyThrThyThrThyThrThyThrThyThrThrThyThrThyThrThyThrThyThrThrThy (26), Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys (27), Ser Gly Thr Ala Lys Ser Val Thr Thr Thr Ser TherThrSerPro (28), Thr CyrThrThrThr ), And Ala Lys Ser Val Thr Thr Cys Thr Tyr (30).
Of these, the amino acid sequence (30) is preferred.
[0012]
As the amino acid sequence (D) contained in the amino acid sequence (4), Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn Thr Phe Arg His Ser Val Val Progal Tyr (4), LeuArpGalGalGalGal (31), Thr Phe Arg His Ser Val Val Val Pro Tyr (32), Leu Arg Val Glu Tyr Leu Asp Asp Arg Asn Thr Phe Arg (33), Asp Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr (34 ), And Asp Arg Asn Thr Phe Arg His Ser Val Val Val (35).
Of these, the amino acid sequence (35) is preferred.
[0013]
The amino acid sequence (E) contained in the amino acid sequence (5) may be Tyr Met Cys Asn Ser Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu ulle Gle Sul Gleu L Phe Glu ValHis Val Cys Ala Cys Pro Gly Arg (5), Tyr Met Cys Asn Ser Ser Cys Met Gly Gly (36), Met Asn Arle Glei Leu G Asn Leu Leu (38), Gly Arg Asn Ser Phe Glu Val His Val Cys Ala C ysProGlyArg (39), TyrMetCysAsnSerSysCysMetGlyGlyMetAsnArgArgProg (40), AsnArgArgArsElePlEleLeThrIleThrIleThrIleThr Leu Leu Gly Arg Asn Ser Phe Glu (42), Ser Phe Glu ValHis Val Cys AlaCys Pro Gly Arg (43), AsnSerSerIsRyGryGlyGlyGlyGly And Asn Ser Phe Glu Val His Val Val Cys Ala Cys Pro (45) And the like.
Of these, the amino acid sequences (44) and (45) are preferred.
[0014]
As the amino acid sequence (F) contained in the amino acid sequence (6), Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr Lys Arg AluGuAluGuGuG Lys Lys Gly Glu Pro His (46), His Glu Leu Pro Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn (47), Glu Glu Asy LuGlyGlyGlyGlyGlyGlyGly Pro Gly (49), Pro Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn (50), and Gly Glu Pro His His Glu Leu Pro Pro Gly Ser (51) and the like.
Of these, the amino acid sequence (51) is preferred.
[0015]
The amino acid sequence (G) contained in the amino acid sequence (7) may be Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Alg Ala His Ser Ser His His Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Phe Lys Thr Glu Gly Pro Asp Ser Asp (7), Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly (52), Gly Ser Lys LyGyS LyGyS SerGySry His (54), Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp ( 5), Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala (56), Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Glys Lys Lys Glys Lys Gly Met Phe (58), Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp (59), Gly Lys Glu Pro Gly Gly Ser Lys Lys LyS Lys Lys Lys Lys Lys Lys 61) and the like.
Of these, the amino acid sequences (60) and (61) are preferred.
[0016]
Among these specific amino acid sequences, the amino acid sequence (A) and the amino acid sequence (G) are preferable from the viewpoint of reactivity (specificity and sensitivity) with the autoantibody, and more preferably the amino acid sequences (1) and (G). 7), (13), (16), (17), (18), (52), (53), (54) (55), (56), (58), (60) and (61), Particularly preferred are the amino acid sequences (17), (18), (60) and (61).
[0017]
The polypeptide only needs to contain at least one of these specific amino acid sequences per polypeptide molecule, and may contain two or more thereof at the same time. Further, one polypeptide molecule may contain a repeating sequence (polymer) of these specific amino acid sequences.
The polypeptide includes a polypeptide comprising at least one selected from the group consisting of the amino acid sequences (1) to (61), a polypeptide comprising a repeating sequence (polymer) thereof, and the like.
Among these, a polypeptide comprising at least one selected from the group consisting of the amino acid sequences (1) to (61) is preferable, and more preferably a polypeptide comprising any one of the amino acid sequences (1) to (61) Is a polypeptide.
[0018]
The polypeptide may be composed of only these specific amino acid sequences, and further, at the N-terminal and / or C-terminal, biotin, thioglycolic acid, and / or amino acids used as a linker (cysteine, lysine, tyrosine, glutamic acid and And the like may be included. Further, the N-terminal may be acetylated with acetic anhydride, N-acetylimidazole, or the like, and the C-terminal may be water-soluble carbodiimide (eg, 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride), isoxazolium (For example, N-methyl-5-phenylisoxazolium fluoroborate) or the like. Preferably, the polypeptide is modified with biotin or thioglycolic acid, and more preferably, it is modified with biotin from the viewpoint of simplicity in binding the polypeptide to a water-insoluble carrier. Also, these modifications can be used in combination.
When an amino acid is contained at the N-terminus and / or the C-terminus, the number of amino acids is preferably 1 to 5, more preferably 1 to 3, and particularly preferably 1 to 2, per N-terminus or C-terminus.
When biotin and / or thioglycolic acid is contained at the N-terminal and / or C-terminal, the number of biotin or thioglycolic acid is preferably 1 to 5, more preferably 1 to 3, particularly preferably 1 to 2, per polypeptide. is there.
[0019]
The number of amino acids constituting the polypeptide is preferably 10 or more, more preferably 11 or more, particularly preferably 13 or more, and most preferably 15 or more, and preferably 50 or less, more preferably 40 or less, and particularly preferably. Is 30 or less, most preferably 25 or less.
[0020]
The polypeptide can be prepared by a conventionally known gene recombination method or peptide synthesis method.
The genetic recombination method has the advantage that a polypeptide having a relatively large number of amino acids can be easily prepared.However, the presence of a host-derived narrow protein and the production of the polypeptide as a fusion protein make it possible to use a recombinant method. There is a disadvantage that non-specific reactions are likely to occur. Therefore, of these methods, the peptide synthesis method is preferred.
As a gene recombination method, after introducing SV40 virus, E1 adenovirus, E6 papilloma virus, etc. into cells such as human fibroblasts, cDNA was prepared from mRNA extracted from transformants and amplified by PCR. Cleavage with a restriction enzyme, the fragment is incorporated into a vector, a host such as Escherichia coli is transformed with the obtained expression vector, induction treatment is performed if necessary, the host is cultured, and the target fusion protein is obtained from the cell lysate. Is extracted and purified. In this way, a fusion protein containing a polypeptide can be prepared (for example, Biochemical Experiment Method 45, “Recombinant Protein Production Method”, Gakkai Shuppan Center, 2001). Alternatively, a polypeptide can be produced in the same manner by synthesizing a gene sequence corresponding to the target polypeptide from the p53 gene sequence with a DNA synthesizer and incorporating the gene sequence into a vector.
[0021]
Although the peptide synthesis method can be achieved in a solution or on a solid support, a solid phase synthesis method using a solid support is preferable, and a solid phase synthesis method using an automatic peptide synthesizer is more preferable. Polypeptide synthesis methods generally use activated amino acids protected with t-butyloxycarbonyl (BOC group) or 9-fluorenylmethoxy-carbonyl (Fmoc group). In addition, specific synthetic procedures, types of side-chain protection, cleavage methods, and the like are described in, for example, Solid Phase Peptide Synthesis Second Edition, Peace Chemical Company, 1984 (Stewart and Young, “Solid Phase Peptide Synthesis”, Second Edition). Edition, Pierce Chemical Company, 1984), and Solid Phase Peptide Synthesis, IR, 1989 (Atherton and Sheppard, "Solid Phase Peptide Synthesis", IRL Press, 1989).
[0022]
It is sufficient that the immunoassay reagent of the present invention contains at least one kind of such a polypeptide. However, the presence of two or more kinds of polypeptides indicates the reactivity (specificity and sensitivity) with autoantibodies. ) Is preferred in that it is higher. More preferably, it comprises a polypeptide comprising the amino acid sequence (A) and a polypeptide comprising the amino acid sequence (G), and particularly preferably a polypeptide comprising the amino acid sequence (A) and a polypeptide comprising the amino acid sequence (G). In addition to the peptide, a polypeptide containing at least one amino acid sequence of the amino acid sequences (B) to (F), and most preferably a polypeptide containing any one of the amino acid sequences (A) to (G), It is to include each one.
[0023]
Examples of preferable combinations of polypeptides for the immunoassay reagent of the present invention include, for example, the following combinations. The numbers in parentheses correspond to the amino acid sequence numbers, and represent the polypeptide consisting of the amino acid sequence. That is, (1) + (7) represents a combination of a polypeptide consisting of the amino acid sequence (1) and a polypeptide consisting of the amino acid sequence (7).
(1) + (7), (17) + (60), (18) + (61), (17) + (61), (17) + (18) + (60) + (61), (1) ) + (7) + (2), (1) + (7) + (24) + (25), (1) + (7) + (3), (1) + (7) + (30), (1) + (7) + (4), (1) + (7) + (35), (1) + (7) + (5), (1) + (7) + (44) + (45) ), (1) + (7) + (6), (1) + (7) + (51), (1) + (7) + (2) + (3), (1) + (7) + (2) + (4), (1) + (7) + (2) + (5), (1) + (7) + (2) + (6), (1) + (7) + (3) ) + (4), (1) + (7) + (3) + (5), (1) + (7) + (3) + (6), (1) + (7) + (4) + (5), (1) + (7) + (4) + 6), (1) + (7) + (5) + (6), (1) + (7) + (2) + (3) + (4), (1) + (7) + (2) + (3) + (5), (1) + (7) + (2) + (3) + (6), (1) + (7) + (3) + (4) + (5), ( 1) + (7) + (3) + (4) + (6), (1) + (7) + (4) + (5) + (6), (1) + (7) + (2) + (4) + (5), (1) + (7) + (2) + (4) + (6), (1) + (7) + (2) + (5) + (6), ( 1) + (7) + (3) + (5) + (6), (1) + (7) + (2) + (3) + (4) + (5), (1) + (7) + (2) + (3) + (4) + (6), (1) + (7) + (3) + (4) + (5) + (6), (1) + (7) + ( 2) + (4) + (5) + (6), (1) + (7) + (2) + (3) + ( ) + (6), (1) + (7) + (2) + (3) + (4) + (5) + (6), (17) + (18) + (60) + (61) + (2) + (3), (17) + (18) + (60) + (61) + (2) + (4), (17) + (18) + (60) + (61) + (2) ) + (5), (17) + (18) + (60) + (61) + (2) + (6), (17) + (18) + (60) + (61) + (3) + (4), (17) + (18) + (60) + (61) + (3) + (5), (17) + (18) + (60) + (61) + (3) + (6) ), (17) + (18) + (60) + (61) + (4) + (5), (17) + (18) + (60) + (61) + (4) + (6), (17) + (18) + (60) + (61) + (5) + (6), (17) + (18) + (60) + (61) + (2) + (3) + (4), (17) + (18) + (60) + (61) + (2) + (3) + (5), (17) + (18) + ( 60) + (61) + (2) + (3) + (6), (17) + (18) + (60) + (61) + (3) + (4) + (5), (17) + (18) + (60) + (61) + (3) + (4) + (6), (17) + (18) + (60) + (61) + (4) + (5) + ( 6), (17) + (18) + (60) + (61) + (2) + (4) + (5), (17) + (18) + (60) + (61) + (2) + (4) + (6), (17) + (18) + (60) + (61) + (2) + (5) + (6), (17) + (18) + (60) + ( 61) + (3) + (5) + (6), (17) + (18) + (60) + (61) + (2) + (3) + (4) + 5), (17) + (18) + (60) + (61) + (2) + (3) + (4) + (6), (17) + (18) + (60) + (61) + (3) + (4) + (5) + (6), (17) + (18) + (60) + (61) + (2) + (4) + (5) + (6), ( 17) + (18) + (60) + (61) + (2) + (3) + (5) + (6), (17) + (18) + (60) + (61) + (2) + (3) + (4) + (5) + (6), (17) + (18) + (60) + (61) + (24) + (25) + (30), (17) + ( 18) + (60) + (61) + (24) + (25) + (35), (17) + (18) + (60) + (61) + (24) + (25) + (44) + (45), (17) + (18) + (60) + (61) + (24) + (25) + (51), 17) + (18) + (60) + (61) + (30) + (35), (17) + (18) + (60) + (61) + (30) + (44) + (45) , (17) + (18) + (60) + (61) + (30) + (51), (17) + (18) + (60) + (61) + (35) + (44) + ( 45), (17) + (18) + (60) + (61) + (35) + (51), (17) + (18) + (60) + (61) + (44) + (45) + (51), (17) + (18) + (60) + (61) + (24) + (25) + (30) + (35), (17) + (18) + (60) + ( 61) + (24) + (25) + (30) + (44) + (45), (17) + (18) + (60) + (61) + (24) + (25) + (30) + (51), (17) + (18) + (60) + (61) + (30) + (35) + (44) + (45), (17) + (18) + (60) + (61) + (30) + (35) + (51), ( 17) + (18) + (60) + (61) + (35) + (44) + (45) + (51), (17) + (18) + (60) + (61) + (24) + (25) + (35) + (44) + (45), (17) + (18) + (60) + (61) + (24) + (25) + (35) + (51), ( 17) + (18) + (60) + (61) + (24) + (25) + (44) + (45) + (51), (17) + (18) + (60) + (61) + (30) + (44) + (45) + (51), (17) + (18) + (60) + (61) + (24) + (25) + (30) + (35) + ( 44) + (45), (17) + (18) + (6 ) + (61) + (24) + (25) + (30) + (35) + (51), (17) + (18) + (60) + (61) + (30) + (35) + (44) + (45) + (51), (17) + (18) + (60) + (61) + (24) + (25) + (35) + (44) + (45) + (51) ), (17) + (18) + (60) + (61) + (24) + (25) + (30) + (44) + (45) + (51), (17) + (18) + (60) + (61) + (24) + (25) + (30) + (35) + (44) + (45) + (51)
Among these, (1) + (7) + (2) + (3) + (4) + (5) + (6), (17) + (18) + (60) + (61) + (2) ) + (3) + (5) + (6) and (17) + (18) + (60) + (61) + (24) + (25) + (30) + (35) + (44) + (45) + (51), more preferably (17) + (18) + (60) + (61) + (24) + (25) + (30) + (35) + (44) + (45) + (51).
[0024]
The reagent for immunoassay of the present invention is not limited in the form and composition of the reagent as long as it contains the above-mentioned polypeptide. For example, the polypeptide is combined with a water-insoluble carrier and / or a labeled compound to form the reagent. May be included, or may be included alone in the polypeptide. That is, the reagent of the present invention can be applied to a homogeneous immunoassay such as a nephelometric method, a latex agglutination method, and a turbidimetric method, and a heterogeneous immunoassay using a water-insoluble carrier. Among them, a reagent for heterogeneous immunoassay is preferable in terms of measurement sensitivity. That is, the immunoassay reagent of the present invention is preferably used by binding the polypeptide to a water-insoluble carrier.
[0025]
As the water-insoluble carrier, carriers described in JP-A-2-205774 can be used, and inorganic and organic substances can be used. For example, cellulose, polystyrene, polypropylene, polyolefin, polyurethane, nitrocellulose, cellulose acetate, polyester, Examples include epoxy resin, phenolic resin, silk, fibroin, lignin, hemicellulose, chitin, ebonite, rubber, glass, quartz, silicon, and ceramics. Among them, polystyrene, glass, quartz and silicon are preferred, and polystyrene and glass are more preferred, and glass is particularly preferred.
[0026]
The shape of the water-insoluble carrier can be freely determined according to the purpose of use, such as true spherical or disk-shaped beads, plate-like or rod-like sticks, test tubes, and nonwoven fabric or filter strips (strip-like strips), Fine particles and the like. Of these, beads and microparticles are preferred, and more preferably spherical beads.
[0027]
The size of the water-insoluble carrier can be freely determined according to the purpose of use, but is usually a size that can be put into a reaction vessel having an inner diameter of about 4 to 10 mm and a depth of about 10 to 20 mm (when the water-insoluble carrier is a test tube). ). In the case of true spherical beads, the diameter (mm) is preferably 1 to 10, more preferably 2 to 8, and particularly preferably 3 to 7. In the case of disc-shaped beads, the diameter (mm) is preferably from 1 to 10, more preferably from 2 to 8, particularly preferably from 3 to 7, and the thickness (mm) is preferably from 0.1 to 5, more preferably. Is from 0.2 to 2, particularly preferably from 0.3 to 1. In the case of a stick, the length (mm) is preferably 2 to 10, more preferably 3 to 8, and particularly preferably 4 to 7. Also, the cross-sectional area of the stick (mm ² ) Is preferably 1 to 25, more preferably 2 to 16, particularly preferably 3 to 9. The cross-sectional area means a cross-sectional area of a cut portion when cut perpendicular to the long axis direction. In the case of a test tube, the length (mm) is preferably 5 to 100, more preferably 8 to 80, and particularly preferably 10 to 20. Further, the inner diameter (mm) of the test tube is preferably 5 to 20, more preferably 6 to 16, and particularly preferably 8 to 12. In the case of a strip, the length (mm) is preferably 5 to 100, more preferably 10 to 80, and particularly preferably 10 to 50. Further, the width (mm) of the strip is preferably 1 to 20, more preferably 2 to 16, and particularly preferably 3 to 10. The thickness (mm) is preferably from 0.1 to 2, more preferably from 0.1 to 0.5. The average pore size (μm) of the nonwoven fabric or the filter is preferably from 0.1 to 10, more preferably from 0.3 to 5. In the case of fine particles, the average particle diameter (μm) is preferably from 0.01 to 200, more preferably from 0.1 to 50, particularly preferably from 0.2 to 10. The average particle diameter can be measured by transmission electron microscopy, alignment measurement using an optical microscope, or the like.
[0028]
The polypeptide can be bound to the water-insoluble carrier by a conventionally known method such as a method of chemically binding or a method of physical adsorption. As a method of chemically bonding, a functional group such as an amino group and / or a sulfhydryl group introduced on the surface of a water-insoluble carrier and a functional group such as an amino group and / or a sulfhydryl group of a polypeptide are used as a binder (glutaryl). Crosslinking with aldehyde, succinaldehyde, m-maleimidobenzoyl-N-hydrosuccinimide ester, o-phenylenebismaleimide, and the like (US Pat. Nos. 4,280,992 and 365,2761). As a method by physical adsorption, when the water-insoluble carrier is polystyrene, a method of immersing the water-insoluble carrier in a 0.001 to 0.04% (W / V) carbonate buffer aqueous solution (pH 9.0) of the polypeptide for an appropriate time ( BioSim Biofizs Acta, Vol. 251, 427, 1971). This method can be applied to the case where the carrier is a substance other than polystyrene, such as polypropylene, silicon, glass, and cellulose. The polypeptide can also be indirectly bound to a water-insoluble carrier using a specific binding substance (for example, antigen-antibody, avidin-biotin, lectin-sugar chain, complementary gene chain, etc.). For example, a polypeptide can be bound to a water-insoluble carrier by modifying the polypeptide with biotin and reacting with a water-insoluble carrier bound to avidin. As avidin, egg white-derived avidin and streptavidin can be used, and streptavidin is preferable. Among these specific binding substances, antigen-antibody, avidin-biotin, and lectin-sugar chains are, for example, complementary to those described in [Biochemical Experiment 11, “Enzyme Immunoassay”, Tokyo Chemical Dojinsha, 1989]. Examples of the gene include a complementary gene described in JP-A-6-186232, for example, a combination of polydeoxyadenylic acid and polythymidylic acid.
[0029]
Among these, a method of chemically binding and a method of using a specific binding substance are preferable, and a method of using a specific binding substance is more preferable. Among the methods using a specific binding substance, this is a method utilizing avidin-biotin bond.
[0030]
The polypeptide may be dissolved in a buffer or the like conventionally used for an immunoassay. As the buffer, for example, a phosphate buffer and a Good's buffer can be used, and may contain a protein, a salt, and / or a surfactant. Proteins include albumin (bovine serum albumin, rabbit serum albumin, mouse serum albumin, ovalbumin, conalbumin, lactalbumin, etc.), antibodies (antibodies that have no binding to polypeptides such as normal rabbit IgG and normal mouse IgG) and gelatin And the like. Examples of the salt include sodium chloride, potassium chloride and lithium bromide. Examples of the surfactant include nonionic surfactants such as sorbitan lauric acid monoester ethylene oxide adduct (trade name: Tween 20, Tween 40, ICI America). The water-insoluble carrier to which the polypeptide is bound may be in a state of being immersed in the above-mentioned buffer solution. However, it is preferable that the entire water-insoluble carrier containing the polypeptide is coated with saccharides and proteins and then dried. Examples of the coating / drying method include a method of dipping in a solution containing a saccharide and a protein and then drying (JP-A-09-318628 and JP-B-5-41946).
[0031]
The immunoassay reagent kit of the present invention preferably contains a reagent containing an anti-human immunoglobulin antibody together with an immunoassay reagent containing a polypeptide.
As the anti-human immunoglobulin antibody, an antibody prepared by a conventionally known method can be used. For example, it can be prepared by immunizing a suitable animal (for example, mouse, rabbit, pig, goat, horse, etc.) with human immunoglobulin, and purifying the antibody from the obtained antiserum by salting out, an ion exchange column or the like. The anti-human immunoglobulin antibody may be a polyclonal antibody or a monoclonal antibody. ² , Fab ′ and the like. The human immunoglobulin includes all immunoglobulins such as IgG, IgA, and IgM, but is usually IgG and IgM, and is mainly IgG.
[0032]
The anti-human immunoglobulin antibody is preferably labeled with a labeling compound. Conventionally known compounds can be used as the labeling compound, and radioisotopes, fluorescent substances, luminescent substances, enzymes and the like are used. As an isotope, ¹²⁵ I, etc., the fluorescent substance includes a europium complex and the like, the luminescent substance includes N-methylacridium ester, and the like, and the enzyme includes horseradish peroxidase, alkaline phosphatase, β-galactosidase, and the like. Of these labeled compounds, enzymes are preferred, more preferably horseradish peroxidase, alkaline phosphatase and β-galactosidase, particularly preferably horseradish peroxidase and alkaline phosphatase.
[0033]
As a method of labeling a labeled compound with an anti-human immunoglobulin antibody, conventionally known methods and the like can be applied, and “Seizoku Chemistry Experiment Course 5 Immunobiochemical Experiment Method” (edited by The Japanese Biochemical Society, Tokyo Chemical Dojin, 1986, 102 To 112, and the following methods (1) to (4) can be applied.
(1) A method in which, when the labeled compound is an isotope, radioactive iodine is introduced into a tyrosine residue of an anti-hyaluronic acid antibody or a ligand using chloramine T as an oxidizing agent.
(2) When the labeling compound is a fluorescent substance, a method of reacting fluorescein isothiocyanate with an anti-hyaluronic acid antibody or a ligand in a buffer to bind to the lysine residue of the anti-hyaluronic acid antibody or the ligand.
(3) When the labeling substance is a luminescent substance, a trade name “Acridinium Derivative-I” (manufactured by Dojindo Laboratories) is reacted with an anti-hyaluronic acid antibody or a ligand in a buffer, and the anti-hyaluronic acid antibody or ligand is reacted. A method of binding to an amino group of a ligand.
(4) When the labeling substance is an enzyme, a method in which an amino group of the enzyme and a thiol group of an anti-hyaluronic acid antibody or a ligand are bound with a di-crosslinking reagent such as N-succinimidyl-6-maleide hexanoate. .
[0034]
The reagent kit for immunoassay of the present invention is not limited in the form of the reagent and the configuration of the reagent kit, as long as the reagent for immunoassay of the present invention is included. For example, the polypeptide may be a water-insoluble carrier and / or labeled It may be contained in the reagent kit in a form bound to the compound, or may be contained alone in the polypeptide. That is, the reagent kit of the present invention can be applied to a homogeneous immunoassay such as a nephelometric method, a latex agglutination method, and a turbidimetric method, as well as a heterogeneous immunoassay using an insoluble carrier. Among them, a reagent kit for heterogeneous sandwich immunoassay containing a labeled compound and containing an anti-human immunoglobulin antibody is most preferable in terms of measurement sensitivity. That is, when the peptide is reacted with the specimen, only the autoantibody against the polypeptide specifically binds to the peptide. Thus, when an anti-human immunoglobulin antibody is added, the antibody binds to the autoantibody to form an immune complex "polypeptide-autoantibody-anti-human immunoglobulin antibody". By quantifying the amount of the anti-human immunoglobulin antibody in the immune complex, the amount of the autoantibody can be quantified. The amount of the anti-human immunoglobulin antibody can be quantified by measuring the amount of the labeled compound.
[0035]
The measurement of the labeled compound can be performed by a conventionally known method or the like depending on the type of the labeled compound. When the labeling compound is a fluorescent substance, for example, the amount of fluorescence generated by irradiation with excitation light having an appropriate wavelength is quantified using a photomultiplier. When the labeling compound is a chemiluminescent substance, for example, in the case of acridinium ester, the amount of luminescence generated by adding an alkaline solution is quantified by a photomultiplier.
[0036]
When the labeled compound is an enzyme, the enzyme activity can be measured as an absorbance (absorbance measurement method), a fluorescence amount (fluorescence measurement method) or a luminescence amount (chemiluminescence measurement method) by reacting an appropriate substrate. For example, when the enzyme is peroxidase, the substrate is 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium (ABTS) (absorbance measurement method) and luminol / peroxide (chemiluminescence). Quantity measurement method) can be selected. When the enzyme is alkaline phosphatase, p-nitrophenyl phosphate (absorbance measurement method), 4-methylumbelliferyl phosphate (4-MUP) (fluorescence measurement method) and 3- (2′-spiroadamantane) are used as substrates. ) -4-methoxy-4- (3 ″ phosphoryloxy) phenyl-1,2-dioxetane disodium (AMPPD) (chemiluminescence assay) and the like. The absorbance is determined by a spectrophotometer, and the amount of fluorescence and the amount of chemiluminescence are determined by a photomultiplier. Of these, a chemiluminescence assay is preferable (ie, a method for quantifying autoantibodies using the reagent kit of the present invention is a chemiluminescent enzyme immunoassay), and more preferably peroxidase and luminol / peroxidase. This is a method for measuring the amount of chemiluminescence based on a combination of a substance and a combination of alkaline phosphatase and AMPPD.
[0037]
Luminol includes luminol, isoluminol, N-aminohexyl-N-ethylisoluminol (AHEI), N-aminobutyl-N-ethylisoluminol (ABEI), and metal salts thereof. As these metal salts, alkali metal (such as sodium and potassium) salts and alkaline earth metal (such as calcium and magnesium) salts can be used. Of these, luminol and a metal salt of luminol are preferred, more preferably a metal salt of luminol, and particularly preferably a sodium salt of luminol. As the peroxide, either an inorganic peroxide or an organic peroxide can be used. Inorganic peroxides include hydrogen peroxide, sodium perborate, potassium perborate, peroxide, carbon dioxide, dicarbonate, hypochlorous acid, potassium hypochlorite, chlorite, chlorine Acids, sodium chlorate, perchloric acid, perbronic acid, peroxosulfuric acid, peroxophosphoric acid, and the like. Examples of the organic peroxide include peracetic acid, perpropionic acid, dimethyl sulfoxide (DMSO), triethylamine oxide, methyldiethylamine oxide, and phthaloyl peroxide. Among these, from the viewpoint of storage stability and the like, inorganic peroxides are preferable, and hydrogen peroxide is more preferable.
[0038]
The reagent kit for immunoassay of the present invention includes, in addition to a reagent containing a polypeptide (reagent for immunoassay of the present invention) and a reagent containing anti-human immunoglobulin, a buffer for reaction and a buffer for B / F separation And a control sample. As the reaction buffer and the B / F separation buffer, buffers and the like conventionally used for immunoassays can be used, and phosphate buffers and Goods containing proteins, salts and / or surfactants and the like can be used. Can be used. Proteins include albumin (bovine serum albumin, rabbit serum albumin, mouse serum albumin, ovalbumin, conalbumin, lactalbumin, etc.), antibodies (antibodies that do not bind to p53 polypeptides such as normal rabbit IgG and normal mouse IgG) and Gelatin and the like can be mentioned. Examples of the salt include sodium chloride, potassium chloride and lithium bromide. Examples of the surfactant include nonionic surfactants such as sorbitan lauric acid monoester ethylene oxide adduct (trade name: Tween 20, Tween 40, ICI America).
[0039]
The control sample is used as a comparison sample for determining whether an autoantibody against the p53 gene product is present in the sample, and is usually a sample containing no autoantibody (negative control) and a sample containing the autoantibody (positive control). ) Is prepared. As a negative control, a buffer containing the same protein as the buffer for the immunoreaction, a human pool serum having no autoantibody against the p53 gene product, and the like can be used. As a positive control, a human pool serum containing an autoantibody to the p53 gene product, a buffer containing a protein similar to the buffer for immunoreaction, etc. to which a serum containing an autoantibody to the p53 gene product is added, and the like can be used. .
[0040]
The sample to be measured by the immunoassay reagent kit of the present invention is not particularly limited as long as it is a human-derived body fluid, and includes, for example, blood, urine, saliva, lymph, bile, gastric juice, pancreatic juice, etc. A homogenate extract of the obtained tissue can also be used. Of these, blood and urine are preferred, and blood (including whole blood, serum, plasma, etc.) is more preferred.
[0041]
Specific examples (steps 1 to 6) of a method for measuring an autoantibody against a p53 gene product using the reagent kit of the present invention (sandwich measurement method) are shown below.
Step 1. The sample is reacted with the water-insoluble carrier to which the polypeptide is bound to obtain a reaction mixture (including complex 1).
Step 2. The unreacted material is removed from the reaction mixture of Step 1 (B / F separation) to obtain a complex 1.
Step 3. The complex 1 obtained in step 2 is reacted with an anti-human immunoglobulin antibody labeled with a labeling compound to obtain a reaction mixture (including complex 2).
Step 4. Step 3 Unreacted substances are removed from the reaction mixture (B / F separation) to obtain a complex 2.
Step 5. The amount of the labeled compound of the complex 2 is measured.
Step 6. The presence or absence of an autoantibody is determined by comparing the amount of the labeled compound with a negative control and / or a positive control.
[0042]
In the step of determining the presence or absence of an autoantibody to the p53 gene product in the sample (step 6 in the above example), the amount of the anti-human immunoglobulin antibody (the amount of the labeled compound labeled with the anti-human immunoglobulin) in the blood as the sample Measured) and a reference value set from the amount of anti-human immunoglobulin (measured as the amount of labeled compound labeled with anti-human immunoglobulin) in the blood of a plurality of healthy persons. It is preferable to determine the presence or absence.
The method of setting the reference value is described in, for example, "Clinical Laboratory Drug Guide 1999-2000, p64-71, Bunkodo (1999)", "Clinical Laboratory Data Book 1997-1998, p8-13, Medical Shoin (1997)". Can be done in the manner described. That is, a plurality of healthy individuals (at least 50 or more, preferably 120 or more) are measured, and the amount of the labeled compound is determined (in the case of a chemiluminescent enzyme immunoassay, the luminescence is obtained). Statistical processing of the amount of labeled compounds ｛Parametric method, non-parametric method, etc. [The parametric method is used when the population shows a normal distribution (power transformation is possible). The non-parametric method is used when the population does not have a normal distribution. used. Then, a range including 95% of the sample group is set as a reference range, the upper limit is set as an upper reference value, and the lower limit is set as a lower reference value. Therefore, a sample exceeding the upper reference value has a high possibility of having an autoantibody, but may have a probability of 5% even in a healthy person. In order to prevent this, it is usual to multiply the upper limit reference value by a coefficient and / or set an added cutoff value. That is, a sample exceeding the cutoff value is determined as having an autoantibody. The cut-off value is set in the relationship between the upper limit reference value and the lower limit value of the distribution of the specimen having the autoantibody to the p53 gene product, and is usually about 1.5 to 3 times the upper limit reference value. .
[0043]
The negative control and the positive control described above were prepared so that the set cutoff value could be easily reproduced. For example, it is possible to set such that the cutoff value is three times the measured value (the amount of the labeled compound) of the negative control, or the average value of the negative control and the positive control is the cutoff value.
[0044]
【Example】
Hereinafter, the present invention will be further described with reference to Examples, but the present invention is not limited thereto.
[0045]
<Synthesis example 1>
1. Synthesis of polypeptide
Pepsin K polyamide-Keselguar resin functionalized with ethylenediamine and coupled with an acid labile linker {4- (2 ', 4'-dimethyloxyphenyl-Fmoc-aminomethyl) phenoxyacetic acid}. The polypeptides shown in Table 1 were synthesized by a chemical synthesis method described in Tetrahedron Letter (Rink, tetrahedron Lett. (1987) 28: 3787) using Milligen, Novato, CA as a solid phase.
[0046]
(1) The side chain w-amino group was protected by Boc, and the α-amino group was protected by Fmoc.
(2) The guanidino group of arginine was protected by a 2,2,5,7,8-pentamethylchroman-6-sulfonyl group.
(3) The imidazolyl group of histidine was protected with a Boc or trityl group, and the sulfhydryl group of cysteine was protected with a trityl group.
(4) Except in the case of arginine which is coupled by forming a hydroxybenzotriazole ester using diisopropylcarbodiimide, the elongation reaction of the peptide is performed by activating the carboxyl group of the amino acid to be added to form O-pentafluorophenyl. This was performed by coupling the esters and the α-amino group of the peptide.
{Circle around (5)} The N-terminals of all the peptides were N-acetylated with acetic anhydride.
{Circle around (6)} All the syntheses were carried out using a Milligen 9050 Pepsynthesizer (Novato, CA) (continuous flow method).
{Circle around (7)} The solid phase and the polypeptide were cleaved with trifluoroacetic acid in the presence of a collection agent (anisole and 1,2-ethanedithiol), and extracted with diethyl ether to obtain a crude synthetic polypeptide.
{Circle around (8)} Purification of these crudely synthesized polypeptides by a C18-reverse phase chromatography column (YMC-Hydrorosphere C18 20 × 150, manufactured by YM Co., Ltd.) Eluent: A: water / TFA (volume ratio: 100 / 0.1) And B: gradient of acetonitrile / TFA (volume ratio: 100 / 0.1) (0 to 40% by volume of B), flow rate of 1.0 mL / mL, temperature of 37 ° C., and detection using a wavelength of 220 nm. Thereafter, the resultant was freeze-dried to obtain synthetic polypeptides P1 to P11 in Table 1.
[0047]
[Table 1]

[0048]
2. Modification of synthetic polypeptides with biotin
1 part by weight of the synthetic polypeptide P1 and 1 part by weight of a biotin residue introduction reagent (trade name [Biotin Sulfo-OSu], Dojin Chemical Laboratories) are dissolved in 1000 parts by weight of a sodium hydrogen carbonate buffer (pH 7.5). Then, the reaction was allowed to stand at 30 ° C. for 2 hours. The reaction solution was purified by C18-reverse phase chromatography to produce biotin-binding polypeptide P1 (PB-1). Biotin-binding polypeptides (PB-2 to PB-11) were similarly prepared for P2 to P11.
[0049]
<Example 1>
1. Preparation of synthetic polypeptide binding beads
(1) Preparation of avidin-bound beads
Streptavidin (purchased from Roche Diagnostics Co., Ltd.) was dissolved in 0.1 M carbonate buffer at pH 9 at a concentration of 20 μg / ml. To 20 ml of this solution, 1,000 polystyrene beads having a diameter of 3.2 mm (manufactured by Immunochemical Co., Ltd.) were added and allowed to stand at 2 to 10 ° C. for 48 hours to physically adsorb streptavidin to the polystyrene beads. Thereafter, the solution was removed by suction with an aspirator, and the beads were washed twice with 20 mL of a phosphate buffer solution (pH 7.2) containing 0.1% by weight of bovine serum albumin to prepare avidin-bound beads. The avidin-bound beads were immersed again in 50 mL of a phosphate buffer containing 0.1% bovine serum albumin, and refrigerated (2 to 10 ° C.) in the immersed state.
[0050]
(2) Preparation of p53 polypeptide binding beads (PB beads)
1000 avidin-bound beads from which the immersion liquid has been removed with an aspirator are added to 20 mL of a phosphate buffer aqueous solution (0.02 M, pH 7.0) containing 2 μg / mL of the synthetic polypeptide PB1, and added at 4 to 10 ° C. for 24 hours. The reaction was performed (inverting and stirring once every two hours). Thereafter, the buffer aqueous solution was removed by suction using an aspirator, and the beads were washed twice with 20 mL of a phosphate buffer solution (pH 7.2) containing 0.1% by weight of bovine serum albumin. After immersion in a buffer solution (pH 7.2) for 30 minutes, the phosphate buffer solution was removed with an aspirator, the beads were spread on filter paper, and air-dried at room temperature (about 25 ° C.) to prepare synthetic polypeptide-bound beads (PB1 beads). And refrigerated in a closed container containing a desiccant (silica gel).
[0051]
2. Preparation of buffer for immune reaction
Casein and sodium chloride were added to a 0.02 M phosphate buffer (pH 8.0) at a concentration of 3 g / L and sodium chloride at a concentration of 8.5 g / L to prepare an immune reaction buffer. Stored refrigerated until use.
[0052]
3. Preparation of peroxidase-labeled anti-human immunoglobulin antibody
Using an anti-human immunoglobulin polyclonal antibody (manufactured by Dako Japan Co., Ltd.) and horseradish-derived peroxidase (manufactured by Toyobo Co., Ltd.), literatures [S. Yoshitake, M. Imagawa, E. Ishikawa, Ethol; J. Biochem, Vol. 92 (1982) 1413-1424], and a peroxidase-labeled anti-β2-microglobulin polyclonal antibody was prepared and stored frozen (−30 ° C.).
[0053]
4. Preparation of enzyme-labeled antibody solution
An enzyme-labeled antibody solution was prepared as follows using the immune reaction buffer solution prepared above and a peroxidase-labeled anti-human immunoglobulin antibody. That is, peroxidase-labeled anti-human immunoglobulin antibody (100 μg in protein amount) was added to 100 mL of the immune reaction buffer, and the mixture was stirred and mixed to obtain an enzyme-labeled antibody solution.
[0054]
5. Preparation of hydrogen peroxide solution
200 μl of 35% hydrogen peroxide solution was dissolved in 1 liter of deionized water to obtain a hydrogen peroxide solution. Stored refrigerated until use.
[0055]
6. Preparation of substrate solution
0.18 g of luminol (manufactured by Tokyo Kasei) and 0.1 g of 4- (cyanomethylthio) phenol (manufactured by Sanshin Chemical) were dissolved in 1 liter of a 0.1 M (mol / L), pH 8.5 Tris / HCl buffer solution. . Stored in the dark and refrigerated until use.
[0056]
<Examples 2 to 18>
1. Preparation of synthetic polypeptide binding beads
In the same manner as in Example 1, the synthetic polypeptide-bound beads of Examples 2 to 18 shown in Tables 2 and 3 were prepared, and refrigerated in a closed container containing a desiccant (silica gel).
When a plurality of synthetic polypeptides were used in combination (Examples 12 to 18), the amount of each synthetic polypeptide was the same, and the total amount was 2 μg / mL.
[0057]
In addition, the buffers prepared in Example 1 were used for the immunoreaction buffer, peroxidase-labeled anti-human immunoglobulin antibody, enzyme-labeled antibody solution, hydrogen peroxide solution, and substrate solution.
[0058]
<Comparative Example 1> Preparation of GST-fused p53 protein
According to the method described in JP-A-9-229933, four kinds of proteins, namely, a peptide having an amino acid sequence of 1-112 from the N-terminal of the p53 gene, a peptide of 108 to 310, a peptide of 306-393, Each fusion protein of the 393 peptide and GST (glutathione S transferase) was prepared as follows.
[0059]
1. Preparation of p53 gene
RNA was extracted from WI-38VA13, an SV40 transformant of human fibroblast WI-38. The extraction was performed as follows using the AGPC method.
[0060]
(Preparation of AGPC method reagent)
(1) Preparation of 1M sodium citrate (pH 7.0)
After dissolving 29.4 g of trisodium citrate in 80 ml of distilled water, citric acid was added to adjust the pH to 7.0, and then water was added to make 100 ml. And it used after autoclaving (120 degreeC, 20 minutes).
(2) Preparation of solution D
236.3 g (4 M) of GTC (guanidium thiocyanate), 2.5 g (0.8% by weight) of sarkosyl, 12.5 ml (25 mM) of 1 M sodium citrate buffer (pH 7.0) and 250 ml of distilled water at 85 ° C. After heating and dissolving with, the mixture was returned to room temperature and adjusted to 496 ml with water. The solution was filtered through a 0.45 μm bottle top filter and stored. At the time of use, 360 μl (0.1 M) of 2-mercaptoethanol was added to 50 ml of this stock solution to prepare solution D.
(3) Preparation of 2M sodium acetate (pH 4.0)
After dissolving 27.2 g of sodium acetate trihydrate in 10 ml of distilled water, the pH was adjusted to 4.0 with acetic acid, and distilled water was added to make 100 ml. It was used after being filtered with a 0.45 μm bottle top filter.
[0061]
(Operation of AGPC method)
(1) 0.5 ml of solution D was added to a tube, and WI-38VA13 cells (purchased from Dainippon Pharmaceutical Co., Ltd.) were dispersed to destroy the cells.
(2) Further, 50 μl of 2M sodium acetate, 0.5 ml of phenol and 100 μl of chloroform / isoamyl alcohol (volume ratio 49 / l) were sequentially added, and the tube was shaken two or three times for each type, and mixed.
(3) After mixing vigorously for 10 seconds, the mixture was ice-cooled for 15 minutes.
(4) After centrifugation at 10,000 G for 20 minutes at a centrifugal acceleration of 10,000 G, the aqueous layer separated at the bottom was separated into another tube so that an intermediate layer containing DNA was not mixed.
(5) 0.5 ml of isopropanol was added to the separated aqueous layer, and the mixture was cooled at -20 ° C for 1 hour. Next, it was centrifuged (10,000 G, 0 ° C.) for 10 minutes to precipitate RNA.
(6) The precipitated RNA is redissolved in 0.5 ml of solution D, 0.5 ml of isopropanol is added, and the mixture is left at -20 ° C for 1 hour.
(7) The RNA is precipitated by centrifugation, and the precipitate is washed with 1 mL of an 80% by weight aqueous ethanol solution, dried, and then dissolved in water.
[0062]
From the mRNA thus extracted, cDNA was prepared by a reverse transcriptase (RT) reaction under the following reaction conditions. For the RT reaction, GIBCO / BRL Reverse Transcriptase (trade name: Superscript) was used. The reaction composition is as follows. 50 mM Tris (hydroxymethyl) aminomethane / hydrochloric acid buffer (pH 8.3), 75 mM aqueous potassium chloride, 3 mM aqueous magnesium chloride, 10 mM dNTP (deoxyribonucleoside 5′-triphosphate mixture), 7.5 μg / ml random nona Mer (purchased from Sigma), RNasin (Promega, RNase inhibitor) at 1000 U / ml, and RNaseH- (reverse transcriptase) at 200,000 U / ml.
[0063]
2. Creation and expression of recombinant genes
The prepared cDNA was amplified with a PCR primer having a recognition sequence for the restriction enzyme BamHI at the 5 'end, and then digested with BamHI and inserted into the BamHI site of the pGEX-5X-2 vector. After confirming that the insert was correctly incorporated by a sequencing method, the transformant was transformed into Escherichia coli BL21 (DE3) and LysS (Novagen), and then incubated at 37 ° C with 0.1 mM isopropylthiobetagalactoside (IPTG) for 2 hours. Induction was performed for 6 hours at 25 ° C. The target fusion protein was extracted and purified from the cell lysate of E. coli by affinity chromatography using glutathione sepharose, and GST-fused p53 protein (GST1 [including amino acid sequence 1-112], GST2 [ GST3 [including amino acid sequence 306-393] and GST4 [including amino acid sequence 1-393].
[0064]
1. Preparation of GST-fused p53 protein-bound beads (GST beads)
It was prepared according to the preparation of the avidin-bound beads of Example 1. That is, GST-fused p53 protein GST1 was dissolved in 0.1 M carbonate buffer at pH 9 at a concentration of 20 μg / ml. 1,000 polystyrene beads having a diameter of 3.2 mm (manufactured by Immunochemical Co., Ltd.) were added to 20 ml of this solution, and allowed to stand at 2 to 10 ° C. for 48 hours to physically adsorb GST1 to the polystyrene beads. Thereafter, the solution was removed by suction with an aspirator, and the beads were washed twice with 20 mL of a phosphate buffer solution (pH 7.2) containing 0.1% by weight of bovine serum albumin, followed by 20 mL of containing 0.1% by weight of bovine serum albumin. It was immersed in a phosphate buffer (pH 7.2) and left refrigerated for 24 hours. The immersion liquid was removed by suction with an aspirator, and the beads were washed twice with 20 mL of a phosphate buffer solution (pH 7.2) containing 0.1% by weight of bovine serum albumin, and then a phosphate buffer solution containing 10% by weight of sucrose. After immersion, the liquid was removed with an aspirator, the beads were spread on filter paper, air-dried at room temperature to obtain GST1-bound beads (GST1 beads), and refrigerated in a sealed container containing a desiccant (silica gel).
[0065]
<Comparative Examples 2 to 5>
1. Preparation of GST-fused p53 protein-bound beads
In the same manner as in Comparative Example 1, the GST-fused p53 protein-bound beads of Comparative Examples 2 to 5 shown in Table 3 were prepared, and were refrigerated and stored in a sealed container containing a desiccant (silica gel).
When a plurality of GST-fused p53 proteins were used in combination (Comparative Example 5), the amount of each GST was the same, and the total amount was 20 μg / mL.
2. Creation of other reagents
The immune reaction buffer, enzyme-labeled antibody solution, hydrogen peroxide solution and substrate solution used were those prepared in Example 1.
[0066]
<Example 19>
This is an example in which the serum of a healthy person and the serum of a cancer patient were measured.
1. Specimen
Serum collected from 50 of our volunteers was mixed with 0.2 mL each to prepare 10 mL of healthy human pool serum, and 10 mL of each serum (patients 1 to 3) obtained from cancer patients was used as a sample. .
[0067]
2. Immune reaction manipulation
In a 12 × 75 mm test tube, 300 μL of an immunoassay buffer, 10 μL of a specimen (pool serum of a healthy person or serum of a cancer patient), and one PB bead or GST bead were added, and reacted at 37 ° C. for 10 minutes. After removing the reaction solution with an aspirator, 2 mL of physiological saline was added to wash the beads, and the washing solution was removed with an aspirator. Further, 2 mL of physiological saline was added and washed in the same manner. Next, 300 μL of the enzyme-labeled antibody solution was added to the washed beads, and reacted at 37 ° C. for 10 minutes. The reaction solution was removed with an aspirator, 2 mL of physiological saline was added to wash the beads, and the washing solution was removed with an aspirator. Further, 2 mL of physiological saline was added, and the mixture was washed twice in the same manner. The enzyme activity of the washed beads was measured.
[0068]
3. Enzyme activity measurement procedure
A test tube (12 × 75 mm) containing the washed beads was set in a sample holder of a luminescence reader BLR-201 manufactured by Aloka, and 200 μL of a substrate solution and 200 μL of a hydrogen peroxide solution were added to start a chemiluminescence reaction. The luminescence amount for 10 seconds from 40 seconds after the start of the luminescence reaction was integrated and measured, and this was defined as the luminescence amount showing the enzyme activity. In the numerical values in Tables 2 and 3, the upper row shows the luminescence level, and the lower row shows the relative value of each luminescence level when the luminescence level of the pool serum of healthy volunteers is set to 1.0.
[0069]
[Table 2]

[Table 3]

[0070]
4. Measurement result
The results are shown in Tables 2 and 3. In the example, compared with the comparative example, the luminescence of the pool serum of healthy volunteers is low, and the luminescence ratio (the value in the lower part of the table divided by the measured luminescence of the pool serum of healthy volunteers) is large. I understand. In addition, even if the patient serum has low reactivity to individual PBs, the sensitivity is improved by combining a plurality of PBs.
[0071]
<Example 20>
This is an example in which a plurality of healthy human sera were measured to obtain a reference value, a cutoff value was set, sera from 10 cancer patients were measured, and the presence or absence of an autoantibody to the p53 gene product was determined.
[0072]
1. Sample measurement
Synthetic polypeptide-bound beads prepared in Example 18 (immobilized PB1, PB2, PB10, PB11, PB3, PB4, PB5, PB6, PB7, PB8, PB9) and GST-fused p53 protein-bound beads prepared in Comparative Example 5 Using (immobilized GST1, GST2, and GST3), serum samples from 120 healthy volunteers and the pool serum of healthy subjects of Example 19 were measured by the method of Example 19.
1 (Example 18) and 2 (Comparative Example 5) show distributions of measured values (light emission amounts) of 120 healthy volunteers. As shown in FIG. 1, the synthetic polypeptide-bound beads of Example 18 have low luminescence and a narrow range of distribution, whereas the GST-fused p53 protein-bound beads of Comparative Example 5 (FIG. 2) have high luminescence and range of distribution. It turned out to be wide. The horizontal axis in the figure is the light emission amount (cps), and the points indicate the distribution of the specimen (one point corresponds to one specimen).
[0073]
2. Setting the cutoff value
As a result of analyzing the measured value (light emission amount) of a healthy person using the statistical processing software “STATFLEX v.4.1” (manufactured by Artec), the light emission amount distribution of the synthetic polypeptide-bound beads of Example 18 was obtained. Has a skewness of 1.49 and a kurtosis of 4.90, whereas the luminescence amount distribution of the GST-fused p53 protein-bound beads of Comparative Example 5 has a skewness of 1.98 and a kurtosis of 7.24. Was. Note that none of the distributions was normally distributed. When a 95% confidence interval was determined by a non-parametric method, the reference value for the synthetic polypeptide-bound beads of Example 18 was 1245 to 9397, while the GST-fused p53 protein of Comparative Example 5 was The reference value for the bound beads was a luminescence of 3771 to 17530. Here, when the light emission amount 1.5 times the upper limit light emission amount is set as the cutoff value, the light emission amount was 14095 in Example 18, and the light emission amount was 26294 in Comparative Example 5. At this time, the luminescence of the pool serum of healthy subjects for Example 18 was 2406, which was 1 / 5.86 of the cutoff value, and the luminescence of the pool serum of healthy subjects for Comparative Example 5 was 6246. It was 1 / 4.21 of the off value. Therefore, when the pool serum of healthy subjects was measured as a negative control, the cut-off value of the synthetic polypeptide-conjugated beads of Example 18 was set to “the measured luminescence of the pool serum of healthy volunteers” × 5.86, while the comparative example In the case of the GST-fused p53 protein-bound beads of No. 5, the cutoff value was set to “measured luminescence of pooled serum in healthy volunteers” × 4.21.
[0074]
3. Sample measurement
Serum samples collected from 10 cancer patients (patients 4 to 13) shown in Table 4 and pooled sera of healthy individuals prepared in Example 19 as a negative control were measured. The measurement was performed in the same manner as in Example 19 using the synthetic polypeptide-bound beads of Example 18 and the GST-fused p53 protein-bound beads of Comparative Example 5.
4. judgment result
Using the pooled serum of a healthy subject as a negative control, the cutoff value (“the measured amount of luminescence of the pooled serum of a healthy subject” × the coefficient) was determined by the coefficient set in Example 20. A measurement value equal to or higher than the cutoff value (cutoff ratio of 1.0 or more) was determined to be positive for autoantibodies, and a measurement value lower than the cutoff value (less than 1.0 for cutoff ratio) was determined to be negative for autoantibodies. The results are shown in Table 4.
[0075]
[Table 4]

From Table 4, it can be seen that the GST-fused p53 protein-bound beads of Comparative Example 5 have a high cut-off value, and the luminescence of cancer patients is relatively low (the cut-off ratio is low). Was. This is due to the fact that the measured values of healthy human samples have a wide distribution due to non-specific adsorption or the like.
In contrast, in the synthetic polypeptide-conjugated beads of Example 18, the difference between the cutoff value and the luminescence amount of the cancer patient specimen was large (the cutoff ratio was large), and the frequency of autoantibody negative judgment was low.
[0076]
【The invention's effect】
The immunoassay reagent for detecting an autoantibody to the p53 gene product of the present invention has few nonspecific reactions and is extremely excellent in sensitivity and specificity. That is, the difference between the measured value of a sample from a healthy person without an autoantibody and the measured value of a sample from a cancer patient with an autoantibody is sufficiently large (high sensitivity), and the difference between a healthy person without an autoantibody and Since there is very little non-specific reaction to the sample, highly accurate measurement can be performed. Therefore, according to the measuring reagent, the measuring reagent kit and the measuring method of the present invention, extremely accurate diagnosis of cancer can be made.
[0077]
[Brief description of the drawings]
FIG. 1 is a graph showing the distribution of measured values (light emission amount) of 120 healthy volunteers using the immunoassay reagent of Example 18 (Example 20).
FIG. 2 is a graph showing the distribution of measured values (light emission amount) of 120 healthy volunteers using the immunoassay reagent of Comparative Example 5 (Example 20).
[0078]
[Sequence list]

Claims (11)

An immunoassay reagent for detecting an autoantibody to a p53 gene product, comprising: Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr The Ser Asp Leu Trp Lys Leu Pro Lu A Lu Pro Lu G Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr (1), Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg ( ), Ser Gly ThrAla Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met Phe Cys (3), Leu Argh Val Glu Tyr LeuArpAg Met Cys Asn Ser Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val His Val Cys Ala Cys Pro Gly Arg (5), Glu Glu A n Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr Lys Arg Ala Leu Pro Asn (6), or Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Gly Gly Pro Asp Ser Asp (7) has at least one kind of amino acid sequence and has 8 to 50 amino acids. An immunoassay test comprising a polypeptide as an essential constituent unit. . An immunoassay reagent for detecting an autoantibody to a p53 gene product, comprising: Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr The Ser Asp Leu Trp Lys Leu Pro Lu A Lu Pro Lu G Ser Gln Ala Met Asp Leu Met Leu Ser Pro Asp Ile Glu Gln Trp Phe Thr (1) An amino acid sequence comprising at least one kind of amino acid sequence and having 8 to 50 amino acids. A polypeptide serving as a unit,
At least included in the Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp (7) A reagent comprising one kind of amino acid sequence and having an amino acid sequence having 8 to 50 amino acids as a constitutional unit. Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg (2), SerGly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala Leu Asn Lys Met PheCys (3), Leu Arg Val Glu Tyr Leu Asp Arg AsnThrPhe Arg His SerValValProSyGrSySynGySrSyGrSySyGrSyGySyGySyGySyGySyGySyGySySyGySyGySyGySyGySyGySyGySyGySyGySyGySyMgPyCy (3) e Ile Thr Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val His Val Cys Ala Cys Pro Gly Arg (5), or Glu Glu Asn Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser The polypeptide further comprising at least one kind of amino acid sequence contained in Thr Lys Arg Ala Leu Pro Asn (6) and having an amino acid sequence having 8 to 20 amino acids as a constitutional unit. The reagent according to 1. The reagent according to any one of claims 1 to 3, wherein the number of amino acids constituting the polypeptide is 10 to 50. The reagent according to any one of claims 1 to 4, wherein the polypeptide is bound to a water-insoluble carrier. An immunoassay reagent kit comprising the reagent according to any one of claims 1 to 5 and an anti-human immunoglobulin antibody. 7. The reagent kit according to claim 6, wherein the anti-human immunoglobulin antibody is labeled with a labeling compound. The reagent kit according to claim 7, wherein the labeling compound is an enzyme. A p53 gene comprising a step of applying the reagent kit according to any one of claims 6 to 8 to a sandwich method and quantifying an anti-human immunoglobulin antibody to determine the presence or absence of an autoantibody to a p53 gene product in the sample. An immunoassay method for autoantibodies to the product. The immunoassay according to claim 9, which is performed by a chemiluminescent enzyme immunoassay. In the step of determining the presence or absence of an autoantibody to the p53 gene product in the sample, using blood as a sample, the amount of anti-human immunoglobulin antibody against this sample,
The immunoassay method according to claim 9 or 10, which comprises comparing a reference value set based on the amount of anti-human immunoglobulin in blood of a plurality of healthy persons.

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