JP4726031B2 - Immunoassay method for X-type phospholipase A2 - Google Patents

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ｈＧＧＸ−Ｓは、コザック配列およびＭｌｕＩ認識部位を有する。また、ｈＧＸ−ＡＳは、ＮｏｔＩ認識部位を有する。
ＰＣＲは、９４度で３０秒、５０度で５０秒、７２度で２分の条件で３５サイクル行った。ＰＣＲ増幅断片は、ＭｌｕＩ及びＮｏｔＩで切断し、ｐＢＳ−ＳＫ（−）に挿入した。配列を確認後、カルボキシル基末端に６個のＨｉｓ残基を付加したＸ型ｓＰＬＡ_２−ＨｉｓＴａｇ体は、ｈＧＸ−Ｓプライマー及びｈＧＸ−Ｈ６ＡＳプライマー（５’−ｔｃａａｇｔｇｃｇｇｃｃｇｃｔｃａａｔｇｇｔｇａｔｇｇｔｇａｔｇａｔｇｇｔｃａｃａｃｔｔｇｇｇｃｇａｇｔｃｃｇｇｃｔ−３’：配列番号７）を用い、ＰＣＲにより構築した。ＰＣＲ増幅断片は、ＮｏｔＩ及びＸｈｏＩで切断し、Ｘ型ｓＰＬＡ_２プラスミドの対応する部位で置換した。ＰＣＲで増幅した領域の配列を確認した後、そのｃＤＮＡを哺乳類細胞用発現ベクターのＳＲ−αプロモーター下流に挿入した。
実施例２ ヒトＸ型ｓＰＬＡ _２ −ＨｉｓＴａｇ組換え蛋白質の発現、精製及び性状解析
Ｘ型ｓＰＬＡ_２−ＨｉｓＴａｇ ｃＤＮＡを含んだプラスミド２０μｇとともに、０．５μｇのｈｙｇｒｏｍｙｃｉｎ Ｂ抵抗遺伝子をＳＶ−４０で形質転換したヒト胎児腎臓由来２９３細胞（２９３Ｔ細胞）にＬｉｐｏｆｅｃｔＡＭＩＮＥ試薬（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｉｎｃ．）によりトランスフェクトした。Ｘ型ｓＰＬＡ_２−ＨｉｓＴａｇを安定的に発現する２９３Ｔクローンをｈｙｇｒｏｍｙｃｉｎ Ｂに対する抵抗性を基に選択することにより得た。組換え蛋白質の発現は、発色性ＰＬＡ_２活性測定法により検出した。本２９３Ｔクローンを１０％仔ウシ胎児血清含有培地で培養し、Ｘ型ｓＰＬＡ_２−ＨｉｓＴａｇ発現タンパクを含んだ培養上清を調製した。本培養上清を、１０ｍｍｏｌ／Ｌイミダゾール、０．５ｍｏｌ／Ｌ塩化ナトリウムを含む２０ｍｍｏｌ／Ｌリン酸ナトリウム緩衝液（ｐＨ７．４）で平衡化したＮｉ^２＋−ｃｈａｒｇｅｄ ｃｈｅｌａｔｉｎｇ Ｓｅｐｈａｒｏｓｅ ｆａｓｔ ｆｌｏｗカラム（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）に添加した。その後、本カラムに結合したＸ型ｓＰＬＡ_２−ＨｉｓＴａｇタンパクを、５００ｍｍｏｌ／Ｌイミダゾール、０．５ｍｏｌ／Ｌ塩化ナトリウムを含む２０ｍｍｏｌ／Ｌリン酸ナトリウム緩衝液（ｐＨ７．４）により溶出した。
得られたＸ型ｓＰＬＡ_２−ＨｉｓＴａｇタンパクを含む画分を、１ｍｍｏｌ／Ｌフッ化フェニルメチルスルホン酸を含む２０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ緩衝液にて透析し、ＨｉＴｒａｐ Ｑカラム（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）に添加した。結合したタンパクを、０から０．５ｍｏｌ／Ｌまでの塩化ナトリウムの濃度勾配で溶出し、Ｘ型ｓＰＬＡ_２−ＨｉｓＴａｇタンパクを含む画分を、次に逆相ＨＰＬＣカラム（Ｃｏｓｍｏｓｉｌ、５Ｃ１８ ３００ＡＲ、４．６×１５０ｍｍ、ナカライテスク社）に添加した。０．０５％トリフルオロ酢酸中で、２０から９５％までのアセトニトリルの濃度勾配で溶出し、得られた画分について発色性ＰＬＡ_２活性測定を行った結果、Ｘ型ｓＰＬＡ_２−ＨｉｓＴａｇは４３％のアセトニトリルで溶出されていた。溶出された各画分について、１５−２５％のアクリルアミド濃度勾配ゲル（第一化学）を用いて、ＳＤＳ−ポリアクリルアミドゲル電気泳動（ＳＤＳ−ＰＡＧＥ）により解析した。別の実験として、精製したＸ型ｓＰＬＡ_２−ＨｉｓＴａｇタンパクを、０．２ユニットのＮ−グリコシダーゼＦ（Ｅ−５００３；Ｏｘｆｏｒｄ ＧｌｙｃｏＳｙｓｔｅｍ）、５％ ２−メルカプトエタノール、３％ ノニデットＰ−４０を含む０．５％ＳＤＳ中にて３７℃、１８時間処理した後、ＳＤＳ−ＰＡＧＥを行い、既報に従ってＩｍｍｏｂｉｌｉｎ−Ｐ膜（Ｍｉｌｌｉｐｏｒｅ Ｃｏ．，）へ電気的にブロットした（Ｈａｎａｓａｋｉ，Ｋ．ｅｔ ａｌ．，Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２７０，７５４３−７５５０（１９９５））。ブロットされたタンパクをクーマシー ブリリアント ブルーで染色した後、Ｘ型ｓＰＬＡ_２−ＨｉｓＴａｇタンパクに相当するバンドを切り取り、そのＮ末端のアミノ酸配列を、Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ Ｐｒｏｃｉｓｅ Ｓｅｑｕｅｎｃｅｒにより解析した。
以上より得られたＣ末端にＨｉｓ残基を付加したＸ型ｓＰＬＡ_２の精製タンパク標品は、ＰＬＡ_２活性を有していた。ＳＤＳ−ＰＡＧＥ解析の結果、ほとんどが１６−ｋＤａの分子量を持つ分子種であったが、一部が１４−ｋＤａの分子量を示した。Ｎ−グリコシダーゼＦ（糖タンパク質からＮ−結合型糖鎖を遊離させる酵素）で処理した結果、１６−ｋＤａの分子種が消失し、すべて１４−ｋＤａの単一の分子種に集約した。この１４−ｋＤａタンパク質のＮ末端アミノ酸配列の解析を行なった結果、ヒトＸ型ｓＰＬＡ_２の活性型の配列（配列番号３記載のアミノ酸配列中、１位のＧｌｙから８位のＴｈｒまでのアミノ酸配列）と一致した。Ｘ型ｓＰＬＡ_２の配列中には、７１位にＮ−結合型糖鎖付加可能な部位が存在する。従って、これらの知見は、２９３Ｔ細胞において、ｃ末端にＨｉｓ残基を付加したＸ型ｓＰＬＡ_２のほとんどは、Ｎ−結合型糖鎖が付加された活性型の糖タンパク質として発現していることを示している。
実施例３ 抗Ｘ型ｓＰＬＡ _２ 抗体の調製ならびにその性状解析
精製したヒトＸ型ｓＰＬＡ_２−ＨｉｓＴａｇタンパクでウサギを免疫した後、抗血清を調製し、そのＩｇＧ画分をＨｉＴｒａｐプロテインＧ−カラム（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）を用いて精製した。抗血清及び精製した抗体の特異性と力価は、以下のＥＬＩＳＡ法により測定した。精製したヒトＩＢ型、ＩＩＡ型ならびにＸ型ｓＰＬＡ_２−ＨｉｓＴａｇタンパク（各１００ｎｇ）をコートした９６ウェルプレート（Ｎｕｎｃ、Ｉｍｍｕｎｏ ＭａｘｉＳｏｒｐ プレート）を調製し、ブロック・エース溶液（大日本製薬）で非特異的吸着をブロック後、抗ヒトＸ型ｓＰＬＡ_２−ＨｉｓＴａｇ抗体、抗ヒトＩＢ型ｓＰＬＡ_２抗体（生化学）及び抗ヒトＩＩＡ型ｓＰＬＡ_２血清（Ｃａｙｍａｎ Ｃｈｅｍｉｃａｌ Ｃｏ．）を加えて２時間反応させた。０．０５％Ｔｗｅｅｎ２０を含むＰＢＳ（Ｐｈｏｓｐｈａｔｅ−ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ，ｐＨ７．４）で洗浄後、さらにペロキシダーゼ結合型ヒツジ抗ウサギＩｇＧ抗体（Ｉｍｍｕｎｏｔｅｃｈ）と反応させ、最終的に０．５ｍｇ／ｍＬのｏ−フェニレンジアミンと０．０１％（ｖ／ｖ）のＨ_２Ｏ_２を含む５０ｍｍｏｌ／Ｌクエン酸ナトリウム緩衝液（ｐＨ５．３）を添加して発色させた。発色反応を２ｍｏｌ／Ｌ硫酸を添加して終了させ、発色強度を４９２ｎｍの波長で測定した。別の試験では、プレートをＸ型ｓＰＬＡ_２−ＨｉｓＴａｇあるいはＡｘｌ−ＨｉｓＴａｇ蛋白質でコートし、前述の方法で解析した。
ＰＬＡ_２活性に対する抗Ｘ型ｓＰＬＡ_２抗体の阻害作用は、発光性アッセイにより行った。すなわち、精製した各ｓＰＬＡ_２蛋白質（５０ｎｇ）、あるいはＩＩＤ型、Ｖ型ｓＰＬＡ_２を発現するＣＨＯ細胞の培養上清を抗Ｘ型ｓＰＬＡ_２抗体とともに２時間反応させ、その後ＰＬＡ_２活性を前述のとおり測定した。抗Ｘ型ｓＰＬＡ_２抗体の阻害作用は、それぞれのｓＰＬＡ_２反応が３７℃でほぼ最大に到達する条件下で試験した。
このようにして得た抗Ｘ型ｓＰＬＡ_２抗体は、ＨｉｓＴａｇ付加したヒトＸ型ｓＰＬＡ_２は認識するものの、ＩＢ型やＩＩＡ型ｓＰＬＡ_２とは反応しなかった。また、調製した抗体は、Ｘ型ｓＰＬＡ_２とは関係のないＨｉｓＴａｇ付加タンパク質とは反応しなかった。このことから、ＨｉｓＴａｇ付加部分は、抗Ｘ型ｓＰＬＡ_２抗体のエピトープではないことが明らかとなった。さらに、抗Ｘ型ｓＰＬＡ_２抗体による前処理によって、ＨｉｓＴａｇ付加したヒトＸ型ｓＰＬＡ_２の酵素活性が濃度依存的に抑制され、１００μｇ／ｍＬの濃度では、他の５種類のｓＰＬＡ_２活性には影響は認められず、Ｘ型ｓＰＬＡ_２の酵素活性のみが約９０％阻害された。以上の結果から、調製した抗Ｘ型ｓＰＬＡ_２抗体は、ヒトＸ型ｓＰＬＡ_２のタンパク部分に対し特異的であることが証明された。
実施例４ 組換えＸ型ｓＰＬＡ _２ 蛋白質の発現、精製および性状解析
Ｘ型ｓＰＬＡ_２発現プラスミドを用いて、本酵素を安定発現させたＣＨＯ細胞を前述のとおり作製した。このＣＨＯ細胞を、１０％仔ウシ胎児血清含有ダルベッコ修飾イーグル培地で培養し、細胞がコンフエントになった段階で、血清を含まないＰＭ−１０００培地（栄研、日本）に変え、細胞をさらに３日間培養した。得られた培養上清を、抗Ｘ型ｓＰＬＡ_２抗体を結合させたアフィニティーカラムに添加した。本カラムは、抗Ｘ型ｓＰＬＡ_２ ＩｇＧ（３０ｍｇ）を、常法に従ってＨｉＴｒａｐ Ｎ−ヒドロキシサクシンイミド活性化カラム（５ｍＬ；Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）に結合させて調製した。カラムをリン酸ナトリウム緩衝液（ｐＨ７．４）で洗浄した後、結合した蛋白質を０．１ｍｏｌ／Ｌグリシン−ＨＣｌ（ｐＨ１．７）で溶出した。この溶出画分を、前述の逆相ＨＰＬＣカラムに添加し、０．１％トリフルオロ酢酸中、２０から４０％までのアセトニトリルの濃度勾配で分離した。３つの大きな画分を得たので、回収し、乾燥させ、０．３ｍｏｌ／Ｌ塩化ナトリウムを含む５０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．４）に溶解した。
以上の方法により、ヒトＸ型ｓＰＬＡ_２の組換えタンパク質を精製した。抗体カラムにて得られた酸溶出画分のＳＤＳ−ＰＡＧＥ解析の結果、１５〜１８−ｋＤａにわたる幅広い分子量を示す分子種と、１４−ｋＤａ及び１３−ｋＤａに単一のバンドを示す２種の分子種の存在が認められた。Ｎ−グリコシダーゼＦ処置により、２つの分子種（１４−ｋＤａと１３−ｋＤａ）に集約し、それらのＮ末端アミノ酸配列解析の結果、過半数を占める１３−ｋＤａの蛋白質（ＧＩＬＥＬＡＧＴ：配列番号８）はヒトＸ型ｓＰＬＡ_２の推定成熟体（配列番号３に記載のアミノ酸配列中、１位のＧｌｙから１２３位のＡｓｐからなるポリペプチド）であり、一方、少数の１４−ｋＤａの蛋白質は、活性Ｘ型ｓＰＬＡ_２のＮ末端に１１残基からなるペプチド（ＥＡＳＲＩＬＲＶＨＲＲ：配列番号９）が付加したプロ型（配列番号３に記載のアミノ酸配列中、−１１位のＧｌｕから１２３位のＡｓｐからなるポリペプチド）であることが判明した。したがって、１５〜１８−ｋＤａにわたって幅広い分子量を示す分子種は、これらプロ型と活性型Ｘ型ｓＰＬＡ_２にＮ−結合型糖鎖が付加されたＸ型ｓＰＬＡ_２と考えられた。
逆相ＨＰＬＣによりさらに分離された３つの画分についてもＮ−グリコシダーゼＦ処理後にＳＤＳ−ＰＡＧＥで解析した結果、画分１は糖鎖非結合型の１３−ｋＤａ活性型のＸ型ｓＰＬＡ_２とＮ−結合型糖鎖が付加されたプロ型と活性型Ｘ型ｓＰＬＡ_２の混合物であり、画分２はＮ−結合型糖鎖付加された活性型のＸ型ｓＰＬＡ_２蛋白質であり、画分３は糖鎖非結合型の１３−ｋＤａの活性型蛋白質であることが明らかとなった。
実施例５ ヒトＸ型ｓＰＬＡ _２ の基質特異性の解析
精製したヒトＸ型ｓＰＬＡ_２各精製標品の基質特異性について、ｓｎ−１位にパルミチン酸を有し、ｓｎ−２位に種々の異なる脂肪酸を持つ極性基の異なる１２種類のリン脂質を市販購入し、それらを基質として試験した。各ｓＰＬＡ_２タンパク質（ＩＢ型、ＩＩＡ型ｓＰＬＡ_２及びヒトＸ型ｓＰＬＡ_２のＨＰＬＣ３画分）の酵素活性について、１ｍｍｏｌ／Ｌの各リン脂質と３ｍｍｏｌ／Ｌのデオキシコール酸から成る混合ミセル条件下で測定した。その結果、ＨＰＬＣ画分１（プロ型と活性型Ｘ型ｓＰＬＡ_２の混合物）は、活性型のみから成る画分２又は画分３に比べて３０〜４０％のＰＬＡ_２活性しか示さなかった。この減少は、画分１に含まれる１４−ｋＤａのプロ型酵素の存在に起因するものと考えられ、プロ型Ｘ型ｓＰＬＡ_２が活性型に比べて酵素活性が弱いことを示唆している。一方、糖鎖非結合型の活性型から成る画分３の活性は、Ｎ−結合型糖鎖を持つ活性型酵素である画分２の活性とほぼ同じ活性を示したことから、Ｘ型ｓＰＬＡ_２に結合するＮ−結合型糖鎖はＰＬＡ_２活性の発現には本質的に影響していないと考えられた。用いた１２種類のリン脂質基質の中で、Ｘ型ｓＰＬＡ_２の活性型（画分２及び３）は、ｓｎ−２位にアラキドン酸を保持するホスファチジルコリンに対して最も高い活性を示した。これに対して、ＩＢ型ならびにＩＩＡ型ｓＰＬＡ_２は、ｓｎ−２位にオレイン酸を保持するホスファチジルグリセロールに対して最も高い活性を示した。
本発明者らは、組換えヒトＸ型ｓＰＬＡ_２タンパク質の精製の過程で、活性型Ｘ型ｓＰＬＡ_２蛋白質のＮ末端に１１個のアミノ酸からなるプロペプチドが付加されたプロ型Ｘ型ｓＰＬＡ_２の存在を明らかにし、さらに基質特異性解析により、プロ型酵素が活性型に比べて酵素活性が非常に低い可能性を示した。さらに、シグナル配列の切断部位を予測するためのＳｉｇｎａｌＰコンピューター解析（Ｎｉｅｌｓｅｎら、Ｐｒｏｔｅｉｎ Ｅｎｇ．，１２，３−９（１９９９））を用いて、シグナルペプチダーゼにより最も切断されやすい部位が活性蛋白質のＮ末端より前の−１１と−１２番目の間であることを示すことにより、１１アミノ酸残基がプロペプチドであることを証明した。したがって、Ｘ型ｓＰＬＡ_２の最大酵素活性の発現には、サブチリジン様あるいはトリプシン様のプロテアーゼによるプロ型からのプロペプチドの切断が必須であることが示されるとともに、本プロペプチドの産生量を測定することにより、対として生じる活性型Ｘ型ｓＰＬＡ_２の産生量を予測することが可能となった。
実施例６ 抗Ｘ型ｓＰＬＡ _２ 抗体を用いたヒト大腸癌組織の免疫組織化学的解析
健常成人大腸組織ならびに大腸癌患者由来大腸癌組織の標本は、Ｂｉｏｃｈａｉｎ Ｉｎｃ．（Ｓａｎ Ｌｅａｎｄｒｏ，ＣＡ）より購入した。組織切片のスライドは、パラフィンワックスを取り除き、０．３％Ｈ_２Ｏ_２を含むメタノールに３０分間反応させ内因性ペルオキシターゼを除去した後、５％の正常ヤギ血清で２０分間処置した。その後、０．１％牛血清アルブミンを含むＰＢＳ中で、抗Ｘ型ｓＰＬＡ_２抗体（６μｇ／ｍＬ）と１４時間、４℃で反応させた。ＰＢＳで洗浄後、ビオチンを結合したヤギ抗ウサギＩｇＧ抗体と３０分間反応させ、ペルオキシダーゼ含有アビジン−ビオチン複合体試薬（Ｖｅｃｔｏｒ Ｌａｂｏｒａｔｏｒｉｅｓ）で処置した。洗浄後、２００μｇ／ｍＬのジアミノベンジジンと０．００６％Ｈ_２Ｏ_２を含む５０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）緩衝液中で１０分間反応させることによりペルオキシダーゼ活性を呈色し組織標本中のＸ型ｓＰＬＡ_２を可視化した。また、０．４％ヘマトキシリン水溶液との反応により、細胞核を対比染色した。Ｘ型ｓＰＬＡ_２陽性シグナルは、濃い茶色のジアミノベンジデンの沈着として検出した。Ｘ型ｓＰＬＡ_２シグナルの中和実験は、抗体をスライドに添加する前に、精製したＸ型ｓＰＬＡ_２蛋白質（６０μｇ／ｍＬ）と２時間反応させることにより行なった。
その結果、Ｘ型ｓＰＬＡ_２の陽性シグナルは、正常大腸組織にはほとんど検出されず、大腸腺癌組織中の癌細胞に強く検出された。これらのシグナルは、Ｘ型ｓＰＬＡ_２蛋白質の添加で消失したことからＸ型ｓＰＬＡ_２に特異的であることが確認された。また、これら陽性シグナルは、非免疫のウサギから調製したＩｇＧでは認められなかった。以上の結果から、大腸癌ではＸ型ｓＰＬＡ_２蛋白質の発現が顕著に高発現していることが示唆された。
実施例７ 抗Ｘ型ｓＰＬＡ _２ 抗体を用いたヒト肺組織の免疫組織化学的解析
健常成人肺組織ならびに肺癌患者由来肺組織の標本は、Ｂｉｏｃｈａｉｎ Ｉｎｃ．（Ｓａｎ Ｌｅａｎｄｒｏ，ＣＡ）より購入した。組織切片のスライドは、パラフィンワックスを取り除き、０．３％Ｈ_２Ｏ_２を含むメタノールに３０分間反応させ内因性ペルオキシターゼを除去した後、５％の正常ヤギ血清で２０分間処置した。その後、０．１％牛血清アルブミンを含むＰＢＳ中で、抗Ｘ型ｓＰＬＡ_２抗体（６μｇ／ｍＬ）と１４時間、４℃で反応させた。ＰＢＳで洗浄後、ビオチンを結合したヤギ抗ウサギＩｇＧ抗体と３０分間反応させ、ペルオキシダーゼ含有アビジン−ビオチン複合体試薬（Ｖｅｃｔｏｒ Ｌａｂｏｒａｔｏｒｉｅｓ）で処置した。洗浄後、２００μｇ／ｍＬのジアミノベンジジンと０．００６％Ｈ_２Ｏ_２を含む５０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）緩衝液中で１０分間反応させることによりペルオキシダーゼ活性を呈色し組織標本中のＸ型ｓＰＬＡ_２を可視化した。また、０．４％ヘマトキシリン水溶液との反応により、細胞核を対比染色した。Ｘ型ｓＰＬＡ_２陽性シグナルは、濃い茶色のジアミノベンジデンの沈着として検出した。Ｘ型ｓＰＬＡ_２シグナルの中和実験は、抗体をスライドに添加する前に、精製したＸ型ｓＰＬＡ_２蛋白質（６０μｇ／ｍＬ）と２時間反応させることにより行なった。
その結果、Ｘ型ｓＰＬＡ_２の陽性シグナルは、正常肺組織ではＩＩ型肺胞上皮細胞において低レベルながら検出されたが、肺癌患者由来肺組織においては、癌細胞に強く検出された。これらのシグナルは、Ｘ型ｓＰＬＡ_２蛋白質の添加で消失したことからＸ型ｓＰＬＡ_２に特異的であることが確認された。また、これら陽性シグナルは、非免疫のウサギから調製したＩｇＧでは認められなかった。以上の結果から、肺癌ではＸ型ｓＰＬＡ_２蛋白質の発現が顕著に高発現していることが示唆された。
実施例８ 抗Ｘ型ｓＰＬＡ _２ 抗体を用いたヒト肝臓組織の免疫組織化学的解析
健常成人肝臓組織ならびに肝臓癌患者由来肺組織の標本は、Ｂｉｏｃｈａｉｎ Ｉｎｃ．（Ｓａｎ Ｌｅａｎｄｒｏ，ＣＡ）より購入した。組織切片のスライドは、パラフィンワックスを取り除き、０．３％Ｈ_２Ｏ_２を含むメタノールに３０分間反応させ内因性ペルオキシターゼを除去した後、５％の正常ヤギ血清で２０分間処置した。その後、０．１％牛血清アルブミンを含むＰＢＳ中で、抗Ｘ型ｓＰＬＡ_２抗体（６μｇ／ｍＬ）と１４時間、４℃で反応させた。ＰＢＳで洗浄後、ビオチンを結合したヤギ抗ウサギＩｇＧ抗体と３０分間反応させ、ペルオキシダーゼ含有アビジン−ビオチン複合体試薬（Ｖｅｃｔｏｒ Ｌａｂｏｒａｔｏｒｉｅｓ）で処置した。洗浄後、２００μｇ／ｍＬのジアミノベンジジンと０．００６％Ｈ_２Ｏ_２を含む５０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）緩衝液中で１０分間反応させることによりペルオキシダーゼ活性を呈色し組織標本中のＸ型ｓＰＬＡ_２を可視化した。また、０．４％ヘマトキシリン水溶液との反応により、細胞核を対比染色した。Ｘ型ｓＰＬＡ_２陽性シグナルは、濃い茶色のジアミノベンジデンの沈着として検出した。Ｘ型ｓＰＬＡ_２シグナルの中和実験は、抗体をスライドに添加する前に、精製したＸ型ｓＰＬＡ_２蛋白質（６０μｇ／ｍＬ）と２時間反応させることにより行なった。
その結果、Ｘ型ｓＰＬＡ_２の陽性シグナルは、正常肝臓組織では肝小葉およびＫｕｐｆｆｅｒ星細胞において低レベルながら検出されたが、肝臓癌患者由来肝臓組織においては、癌細胞に強く検出された。これらのシグナルは、Ｘ型ｓＰＬＡ_２蛋白質の添加で消失したことからＸ型ｓＰＬＡ_２に特異的であることが確認された。また、これら陽性シグナルは、非免疫のウサギから調製したＩｇＧでは認められなかった。以上の結果から、肝臓癌ではＸ型ｓＰＬＡ_２蛋白質の発現が顕著に高発現していることが示唆された。
実施例９ 抗Ｘ型ｓＰＬＡ _２ 抗体を用いたヒト胃組織の免疫組織化学的解析
健常成人胃組織ならびに胃癌患者由来肺組織の標本は、Ｂｉｏｃｈａｉｎ Ｉｎｃ．（Ｓａｎ Ｌｅａｎｄｒｏ，ＣＡ）より購入した。組織切片のスライドは、パラフィンワックスを取り除き、０．３％Ｈ_２Ｏ_２を含むメタノールに３０分間反応させ内因性ペルオキシターゼを除去した後、５％の正常ヤギ血清で２０分間処置した。その後、０．１％牛血清アルブミンを含むＰＢＳ中で、抗Ｘ型ｓＰＬＡ_２抗体（６μｇ／ｍＬ）と１４時間、４℃で反応させた。ＰＢＳで洗浄後、ビオチンを結合したヤギ抗ウサギＩｇＧ抗体と３０分間反応させ、ペルオキシダーゼ含有アビジン−ビオチン複合体試薬（Ｖｅｃｔｏｒ Ｌａｂｏｒａｔｏｒｉｅｓ）で処置した。洗浄後、２００μｇ／ｍＬのジアミノベンジジンと０．００６％Ｈ_２Ｏ_２を含む５０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）緩衝液中で１０分間反応させることによりペルオキシダーゼ活性を呈色し組織標本中のＸ型ｓＰＬＡ_２を可視化した。また、０．４％ヘマトキシリン水溶液との反応により、細胞核を対比染色した。Ｘ型ｓＰＬＡ_２陽性シグナルは、濃い茶色のジアミノベンジデンの沈着として検出した。Ｘ型ｓＰＬＡ_２シグナルの中和実験は、抗体をスライドに添加する前に、精製したＸ型ｓＰＬＡ_２蛋白質（６０μｇ／ｍＬ）と２時間反応させることにより行なった。
その結果、Ｘ型ｓＰＬＡ_２の陽性シグナルは、正常胃組織ではほとんど検出されず、胃癌患者由来胃組織においては、癌細胞に強く検出された。これらのシグナルは、Ｘ型ｓＰＬＡ_２蛋白質の添加で消失したことからＸ型ｓＰＬＡ_２に特異的であることが確認された。また、これら陽性シグナルは、非免疫のウサギから調製したＩｇＧでは認められなかった。以上の結果から、胃癌ではＸ型ｓＰＬＡ_２蛋白質の発現が顕著に高発現していることが示唆された。
実施例１０ 抗Ｘ型ｓＰＬＡ _２ 抗体を用いたヒト腎臓組織の免疫組織化学的解析
健常成人腎臓組織ならびに腎臓癌患者由来肺組織の標本は、Ｂｉｏｃｈａｉｎ Ｉｎｃ．（Ｓａｎ Ｌｅａｎｄｒｏ，ＣＡ）より購入した。組織切片のスライドは、パラフィンワックスを取り除き、０．３％Ｈ_２Ｏ_２を含むメタノールに３０分間反応させ内因性ペルオキシターゼを除去した後、５％の正常ヤギ血清で２０分間処置した。その後、０．１％牛血清アルブミンを含むＰＢＳ中で、抗Ｘ型ｓＰＬＡ_２抗体（６μｇ／ｍＬ）と１４時間、４℃で反応させた。ＰＢＳで洗浄後、ビオチンを結合したヤギ抗ウサギＩｇＧ抗体と３０分間反応させ、ペルオキシダーゼ含有アビジン−ビオチン複合体試薬（Ｖｅｃｔｏｒ Ｌａｂｏｒａｔｏｒｉｅｓ）で処置した。洗浄後、２００μｇ／ｍＬのジアミノベンジジンと０．００６％Ｈ_２Ｏ_２を含む５０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）緩衝液中で１０分間反応させることによりペルオキシダーゼ活性を呈色し組織標本中のＸ型ｓＰＬＡ_２を可視化した。また、０．４％ヘマトキシリン水溶液との反応により、細胞核を対比染色した。Ｘ型ｓＰＬＡ_２陽性シグナルは、濃い茶色のジアミノベンジデンの沈着として検出した。Ｘ型ｓＰＬＡ_２シグナルの中和実験は、抗体をスライドに添加する前に、精製したＸ型ｓＰＬＡ_２蛋白質（６０μｇ／ｍＬ）と２時間反応させることにより行なった。
その結果、Ｘ型ｓＰＬＡ_２の陽性シグナルは、正常腎臓組織では糸球体メサンギウム細胞において低レベルながら検出されたが、腎臓癌患者由来腎臓組織においては、癌細胞に強く検出された。これらのシグナルは、Ｘ型ｓＰＬＡ_２蛋白質の添加で消失したことからＸ型ｓＰＬＡ_２に特異的であることが確認された。また、これら陽性シグナルは、非免疫のウサギから調製したＩｇＧでは認められなかった。以上の結果から、腎臓癌ではＸ型ｓＰＬＡ_２蛋白質の発現が顕著に高発現していることが示唆された。
実施例１１ 抗Ｘ型ｓＰＬＡ _２ 抗体を用いたヒト胆嚢組織の免疫組織化学的解析
健常成人胆嚢組織ならびに胆嚢癌患者由来肺組織の標本は、Ｂｉｏｃｈａｉｎ Ｉｎｃ．（Ｓａｎ Ｌｅａｎｄｒｏ，ＣＡ）より購入した。組織切片のスライドは、パラフィンワックスを取り除き、０．３％Ｈ_２Ｏ_２を含むメタノールに３０分間反応させ内因性ペルオキシターゼを除去した後、５％の正常ヤギ血清で２０分間処置した。その後、０．１％牛血清アルブミンを含むＰＢＳ中で、抗Ｘ型ｓＰＬＡ_２抗体（６μｇ／ｍＬ）と１４時間、４℃で反応させた。ＰＢＳで洗浄後、ビオチンを結合したヤギ抗ウサギＩｇＧ抗体と３０分間反応させ、ペルオキシダーゼ含有アビジン−ビオチン複合体試薬（Ｖｅｃｔｏｒ Ｌａｂｏｒａｔｏｒｉｅｓ）で処置した。洗浄後、２００μｇ／ｍＬのジアミノベンジジンと０．００６％Ｈ_２Ｏ_２を含む５０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）緩衝液中で１０分間反応させることによりペルオキシダーゼ活性を呈色し組織標本中のＸ型ｓＰＬＡ_２を可視化した。また、０．４％ヘマトキシリン水溶液との反応により、細胞核を対比染色した。Ｘ型ｓＰＬＡ_２陽性シグナルは、濃い茶色のジアミノベンジデンの沈着として検出した。Ｘ型ｓＰＬＡ_２シグナルの中和実験は、抗体をスライドに添加する前に、精製したＸ型ｓＰＬＡ_２蛋白質（６０μｇ／ｍＬ）と２時間反応させることにより行なった。
その結果、Ｘ型ｓＰＬＡ_２の陽性シグナルは、正常胆嚢組織ではほとんど検出されず、胆嚢癌患者由来胆嚢組織においては、癌細胞に強く検出された。これらのシグナルは、Ｘ型ｓＰＬＡ_２蛋白質の添加で消失したことからＸ型ｓＰＬＡ_２に特異的であることが確認された。また、これら陽性シグナルは、非免疫のウサギから調製したＩｇＧでは認められなかった。以上の結果から、胆嚢癌ではＸ型ｓＰＬＡ_２蛋白質の発現が顕著に高発現していることが示唆された。
実施例１２ 抗Ｘ型ｓＰＬＡ _２ 抗体を用いたヒト前立腺組織の免疫組織化学的解析
健常成人前立腺組織ならびに前立腺癌患者由来前立腺組織の標本は、Ｂｉｏｃｈａｉｎ Ｉｎｃ．（Ｓａｎ Ｌｅａｎｄｒｏ，ＣＡ）より購入した。組織切片のスライドは、パラフィンワックスを取り除き、０．３％Ｈ_２Ｏ_２を含むメタノールに３０分間反応させ内因性ペルオキシターゼを除去した後、５％の正常ヤギ血清で２０分間処置した。その後、０．１％牛血清アルブミンを含むＰＢＳ中で、抗Ｘ型ｓＰＬＡ_２抗体（６μｇ／ｍＬ）と１４時間、４℃で反応させた。ＰＢＳで洗浄後、ビオチンを結合したヤギ抗ウサギＩｇＧ抗体と３０分間反応させ、ペルオキシダーゼ含有アビジン−ビオチン複合体試薬（Ｖｅｃｔｏｒ Ｌａｂｏｒａｔｏｒｉｅｓ）で処置した。洗浄後、２００μｇ／ｍＬのジアミノベンジジンと０．００６％Ｈ_２Ｏ_２を含む５０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）緩衝液中で１０分間反応させることによりペルオキシダーゼ活性を呈色し組織標本中のＸ型ｓＰＬＡ_２を可視化した。また、０．４％ヘマトキシリン水溶液との反応により、細胞核を対比染色した。Ｘ型ｓＰＬＡ_２陽性シグナルは、濃い茶色のジアミノベンジデンの沈着として検出した。Ｘ型ｓＰＬＡ_２シグナルの中和実験は、抗体をスライドに添加する前に、精製したＸ型ｓＰＬＡ_２蛋白質（６０μｇ／ｍＬ）と２時間反応させることにより行なった。
その結果、Ｘ型ｓＰＬＡ_２の陽性シグナルは、正常前立腺組織ではほとんど検出されず、前立腺癌患者由来前立腺組織においては、癌細胞に強く検出された。これらのシグナルは、Ｘ型ｓＰＬＡ_２蛋白質の添加で消失したことからＸ型ｓＰＬＡ_２に特異的であることが確認された。また、これら陽性シグナルは、非免疫のウサギから調製したＩｇＧでは認められなかった。以上の結果から、前立腺癌ではＸ型ｓＰＬＡ_２蛋白質の発現が顕著に高発現していることが示唆された。
実施例１３ 抗Ｘ型ｓＰＬＡ _２ 抗体を用いたヒト膵臓組織の免疫組織化学的解析
健常成人膵臓組織ならびに膵臓癌患者由来膵臓組織の標本は、Ｂｉｏｃｈａｉｎ Ｉｎｃ．（Ｓａｎ Ｌｅａｎｄｒｏ，ＣＡ）より購入した。組織切片のスライドは、パラフィンワックスを取り除き、０．３％Ｈ_２Ｏ_２を含むメタノールに３０分間反応させ内因性ペルオキシターゼを除去した後、５％の正常ヤギ血清で２０分間処置した。その後、０．１％牛血清アルブミンを含むＰＢＳ中で、抗Ｘ型ｓＰＬＡ_２抗体（６μｇ／ｍＬ）と１４時間、４℃で反応させた。ＰＢＳで洗浄後、ビオチンを結合したヤギ抗ウサギＩｇＧ抗体と３０分間反応させ、ペルオキシダーゼ含有アビジン−ビオチン複合体試薬（Ｖｅｃｔｏｒ Ｌａｂｏｒａｔｏｒｉｅｓ）で処置した。洗浄後、２００μｇ／ｍＬのジアミノベンジジンと０．００６％Ｈ_２Ｏ_２を含む５０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）緩衝液中で１０分間反応させることによりペルオキシダーゼ活性を呈色し組織標本中のＸ型ｓＰＬＡ_２を可視化した。また、０．４％ヘマトキシリン水溶液との反応により、細胞核を対比染色した。Ｘ型ｓＰＬＡ_２陽性シグナルは、濃い茶色のジアミノベンジデンの沈着として検出した。Ｘ型ｓＰＬＡ_２シグナルの中和実験は、抗体をスライドに添加する前に、精製したＸ型ｓＰＬＡ_２蛋白質（６０μｇ／ｍＬ）と２時間反応させることにより行なった。
その結果、Ｘ型ｓＰＬＡ_２の陽性シグナルは、正常膵臓組織ではほとんど検出されず、膵臓癌患者由来膵臓組織においては、癌細胞に強く検出された。これらのシグナルは、Ｘ型ｓＰＬＡ_２蛋白質の添加で消失したことからＸ型ｓＰＬＡ_２に特異的であることが確認された。また、これら陽性シグナルは、非免疫のウサギから調製したＩｇＧでは認められなかった。以上の結果から、膵臓癌ではＸ型ｓＰＬＡ_２蛋白質の発現が顕著に高発現していることが示唆された。
実施例１４ 抗Ｘ型ｓＰＬＡ _２ 抗体を用いたヒト大脳組織の免疫組織化学的解析
健常成人大脳組織ならびにアルツハーマ患者由来大脳組織の標本は、Ｂｉｏｃｈａｉｎ Ｉｎｃ．（Ｓａｎ Ｌｅａｎｄｒｏ，ＣＡ）より購入した。組織切片のスライドは、パラフィンワックスを取り除き、０．３％Ｈ_２Ｏ_２を含むメタノールに３０分間反応させ内因性ペルオキシターゼを除去した後、５％の正常ヤギ血清で２０分間処置した。その後、０．１％牛血清アルブミンを含むＰＢＳ中で、抗Ｘ型ｓＰＬＡ_２抗体（６μｇ／ｍＬ）と１４時間、４℃で反応させた。ＰＢＳで洗浄後、ビオチンを結合したヤギ抗ウサギＩｇＧ抗体と３０分間反応させ、ペルオキシダーゼ含有アビジン−ビオチン複合体試薬（Ｖｅｃｔｏｒ Ｌａｂｏｒａｔｏｒｉｅｓ）で処置した。洗浄後、２００μｇ／ｍｌのジアミノベンジジンと０．００６％Ｈ_２Ｏ_２を含む５０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）緩衝液中で１０分間反応させることによりペルオキシダーゼ活性を呈色し組織標本中のＸ型ｓＰＬＡ_２を可視化した。また、０．４％ヘマトキシリン水溶液との反応により、細胞核を対比染色した。Ｘ型ｓＰＬＡ_２陽性シグナルは、濃い茶色のジアミノベンジデンの沈着として検出した。Ｘ型ｓＰＬＡ_２シグナルの中和実験は、抗体をスライドに添加する前に、精製したＸ型ｓＰＬＡ_２蛋白質（６０μｇ／ｍＬ）と２時間反応させることにより行なった。
その結果、Ｘ型ｓＰＬＡ_２の陽性シグナルは、正常大脳組織にはほとんど検出されず、アルツハイマー患者由来大脳組織中の神経細胞群の一部、特に老人班（ｓｅｎｉｌｅｐｌａｑｕｅ）や神経原繊維変化部位（ｎｅｕｒｏｆｉｂｒｉｌｌａｒｙ ｔａｎｇｌｅ ｒｅｇｉｏｎｓ）に強く検出された。これらのシグナルは、Ｘ型ｓＰＬＡ_２蛋白質の添加で消失したことからＸ型ｓＰＬＡ_２に特異的であることが確認された。また、これら陽性シグナルは、非免疫のウサギから調製したＩｇＧでは認められなかった。以上の結果から、アルツハイマー患者大脳ではＸ型ｓＰＬＡ_２蛋白質の発現が顕著に高発現していることが示唆された。
実施例１５ 抗Ｘ型ｓＰＬＡ _２ 抗体を用いたヒト肝硬変組織の免疫組織化学的解析
健常成人肝臓組織ならびに肝硬変患者由来肝臓組織の標本は、Ｂｉｏｃｈａｉｎ Ｉｎｃ．（Ｓａｎ Ｌｅａｎｄｒｏ，ＣＡ）より購入した。組織切片のスライドは、パラフィンワックスを取り除き、０．３％Ｈ_２Ｏ_２を含むメタノールに３０分間反応させ内因性ペルオキシターゼを除去した後、５％の正常ヤギ血清で２０分間処置した。その後、０．１％牛血清アルブミンを含むＰＢＳ中で、抗Ｘ型ｓＰＬＡ_２抗体（６μｇ／ｍＬ）と１４時間、４℃で反応させた。ＰＢＳで洗浄後、ビオチンを結合したヤギ抗ウサギＩｇＧ抗体と３０分間反応させ、ペルオキシダーゼ含有アビジン−ビオチン複合体試薬（Ｖｅｃｔｏｒ Ｌａｂｏｒａｔｏｒｉｅｓ）で処置した。洗浄後、２００μｇ／ｍｌのジアミノベンジジンと０．００６％Ｈ_２Ｏ_２を含む５０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）緩衝液中で１０分間反応させることによりペルオキシダーゼ活性を呈色し組織標本中のＸ型ｓＰＬＡ_２を可視化した。また、０．４％ヘマトキシリン水溶液との反応により、細胞核を対比染色した。Ｘ型ｓＰＬＡ_２陽性シグナルは、濃い茶色のジアミノベンジデンの沈着として検出した。Ｘ型ｓＰＬＡ_２シグナルの中和実験は、抗体をスライドに添加する前に、精製したＸ型ｓＰＬＡ_２蛋白質（６０μｇ／ｍＬ）と２時間反応させることにより行なった。
その結果、Ｘ型ｓＰＬＡ_２の陽性シグナルは、正常肝臓組織では肝小葉およびＫｕｐｆｆｅｒ星細胞において低レベルながら検出されたが、肝硬変患者由来肝臓組織では、偽小葉において顕著な発現増強が認められた。これらのシグナルは、Ｘ型ｓＰＬＡ_２蛋白質の添加で消失したことからＸ型ｓＰＬＡ_２に特異的であることが確認された。また、これら陽性シグナルは、非免疫のウサギから調製したＩｇＧでは認められなかった。以上の結果から、肝硬変組織ではＸ型ｓＰＬＡ_２蛋白質の発現が顕著に高発現していることが示唆された。
実施例１６ マウスＸ型ｓＰＬＡ _２ をコードする遺伝子のクローニング
Ｘ型ｓＰＬＡ_２のマウスホモローグを単離するために、ヒトＸ型ｓＰＬＡ_２配列に基づき２種類のプライマーを設計し、マウス胸腺ｃＤＮＡを鋳型としてＰＣＲを行なった。

ＰＣＲは、９２℃で１分、５８℃で１分、７２℃で１分の条件で４０サイクル行なった。
次に、マウス脾臓ｍａｒａｔｈｏｎ−ｒｅａｄｙ ｃＤＮＡ（Ｃｌｏｎｔｅｃｈ）を用いて、ｒａｐｉｄ ａｍｐｌｉｆｉｃａｔｉｏｎ ｏｆ ｔｈｅ ｃＤＮＡ ｅｎｄｓ（ＲＡＣＥ）法により３’−、５’−末端のクローニングを行い、最終的に以下のプライマーを用いて、マウスＸ型ｓＰＬＡ_２ｃＤＮＡのコード領域を、マウス脾臓ｃＤＮＡを鋳型としてＰＣＲにより得た。

マウスＸ型ｓＰＬＡ_２は、Ｖａｌｅｎｔｉｎら（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２７４，４４，３１１９５−３１２０２（１９９９））が報告するように、１５１個のアミノ酸から成り（配列番号１４）、ヒトＸ型ｓＰＬＡ_２に対して７０．９％の相同性を有することが推定された。マウスＸ型ｓＰＬＡ_２は、ヒトＸ型ｓＰＬＡ_２と同様にＮ末端に２８アミノ酸から成るプレプロペプチドを有し、Ｃ末端に延長構造を有する。しかしながら、マウスＸ型ｓＰＬＡ_２は、ヒトＸ型ｓＰＬＡ_２とは異なり、Ｎ−結合型糖鎖付加部位は有していなかった。
実施例１７ マウスプロ型及び活性型Ｘ型ｓＰＬＡ _２ の酵素活性
マウスＸ型ｓＰＬＡ_２の発現及び精製は、実施例４に記載の方法と同様にして行なった。
精製したプロ型及び活性型Ｘ型ｓＰＬＡ_２の酵素活性は、１−ｐａｌｍｉｔｏｙｌ−２−ｌｉｎｏｌｅｏｙｌ−ｐｈｏｓｐｈａｔｉｄｙｌｃｈｏｌｉｎｅ（ＰＬＰＣ）を基質として測定した。図２に示すように、プロ型Ｘ型ｓＰＬＡ_２は、活性型に比べてわずか８％の活性しか有していなかった。プロペプチドのＣ末端にはＡｒｇが存在していることから、トリプシン様エンドプロテアーゼが切断に関与している可能性が推察された。そこで、プロ型Ｘ型ｓＰＬＡ_２をトリプシンで消化後、ＳＤＳ−ＰＡＧＥにて解析した結果、トリプシン処理によって活性型への変換が確認され、それに対応して、ＰＬＡ_２活性が約５倍にまで上昇した（図２）。以上の解析から、エンドプロテアーゼによるプロ型からのプロペプチド部分の切断除去が、Ｘ型ｓＰＬＡ_２の最大活性発現には不可欠であることが示唆された。
実施例１８ 抗Ｘ型ｓＰＬＡ _２ プロペプチド抗体の調製ならびにその測定系
Ｘ型ｓＰＬＡ_２プロペプチドは本発明により教示されたアミノ酸配列情報に基づいてペプチド合成機により合成した。得られたプロペプチドは牛サイログロブリンと結合させ免疫原とし、ウサギを免疫し抗血清を調製した。ＩｇＧ画分はＨｉＴｒａｐプロテインＧ−カラム（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）を用いて精製した。抗血清及び精製した抗体の特異性と力価は、以下のＥＬＩＳＡ法により測定した。牛血清アルブミンと結合させたＸ型ｓＰＬＡ_２プロペプチドをコートした９６ウェルプレート（Ｎｕｎｃ、Ｉｍｍｕｎｏ ＭａｘｉＳｏｒｐプレート）を調製し、１％牛血清アルブミン溶液で非特異的吸着をブロック後、抗ヒトＸ型ｓＰＬＡ_２プロペプチド抗体を加えて反応させた。０．０５％Ｔｗｅｅｎ２０を含むＰＢＳ（Ｐｈｏｓｐｈａｔｅ−ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ，ｐＨ７．４）で洗浄後、さらにペルオキシダーゼ結合型ヤギ抗ウサギＩｇＧ抗体（Ｃａｐｐｅｌ）と反応させ、ＴＢＭｐｌｕｓ（ＤＡＫＯ）を添加して発色させた。発色反応を０．５ｍｏｌ／Ｌ硫酸を添加して終了させ、発色強度を４５０ｎｍの波長で測定した。
このようにして得た抗Ｘ型ｓＰＬＡ_２プロペプチド抗体は、実施例３に示したＥＬＩＳＡ系においてプロ型Ｘ型ｓＰＬＡ_２や活性型Ｘ型ｓＰＬＡ_２とは反応せず、プロペプチドのみを認識するものであった。この抗血清を用い、以下のＥＬＩＳＡ系第１法によりプロペプチドを測定した。プロペプチドをＥＺ−Ｌｉｎｋ Ｓｕｌｆｏ−ＮＨＳ−ＬＣ−ＬＣ−Ｂｉｏｔｉｎ（Ｐｉｅｒｃｅ）を反応させ、逆相ＨＰＬＣにより分離し、ビオチン化プロペプチドを調製した。アビジンをコートした９６ウェルプレートを調製し、１％牛血清アルブミン溶液で非特異的吸着をブロック後、ビオチン化プロペプチドを加えて２時間反応させた。０．０５％Ｔｗｅｅｎ２０を含むＰＢＳ（Ｐｈｏｓｐｈａｔｅ−ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ，ｐＨ７．４）で洗浄後、抗Ｘ型ｓＰＬＡ_２プロペプチド抗体と測定するべき検体または標準溶液を同時に添加し、１８時間反応させた。洗浄後、さらにペルオキシダーゼ結合型ヤギ抗ウサギＩｇＧ抗体（Ｃａｐｐｅｌ）と反応させ、洗浄後ＴＢＭｐｌｕｓ（ＤＡＫＯ）を添加して発色させた。発色反応を１Ｎ硫酸を添加して終了させ、発色強度を４５０ｎｍの波長で測定した。０ｎｇ／ｍＬの標準溶液で得られる吸光度Ｂ_０に対する各濃度の標準溶液で得られる吸光度Ｂの百分率Ｂ／Ｂ_０％を求めて標準曲線（図３）を作成し、それを用いて未知検体で得られる吸光度から求めたＢ／Ｂ_０％から未知検体中に含まれるプロペプチド濃度を算出した。また、以下のＥＬＩＳＡ系第２法によってもプロペプチドを測定した。牛血清アルブミンと結合させたＸ型ｓＰＬＡ_２プロペプチドをコートした９６ウェルプレートを調製し、１％牛血清アルブミン溶液で非特異的吸着をブロック後、抗Ｘ型ｓＰＬＡ_２プロペプチド抗体と測定するべき検体または標準溶液を同時に添加し、１８時間反応させた。洗浄後、第１法と同様に発色反応を行い、未知検体中に含まれるプロペプチド濃度を算出した。
実施例１９ 抗Ｘ型ｓＰＬＡ _２ プロペプチド抗体を用いたヒト大腸癌組織の免疫組織化学的解析
本試験には、抗ヒトＸ型ｓＰＬＡ２プロペプチド抗体を用いた。健常成人の大腸組織ならびに大腸癌患者由来大腸癌組織の標本は、Ｂｉｏｃｈａｉｎ Ｉｎｃ．（Ｓａｎ Ｌｅａｎｄｒｏ，ＣＡ）より購入した。組織切片のスライドは、パラフィンワックスを取り除き、０．３％Ｈ_２Ｏ_２を含むメタノールに３０分間反応させ内因性ペルオキシターゼを除去した後、５％の正常ヤギ血清で２０分間処置した。その後、０．１％牛血清アルブミンを含むＰＢＳ水溶液中で、抗ヒトＸ型ｓＰＬＡ_２（ウサギ）プロペプチド抗体（２４μｇ／ｍＬ）と１４時間、４℃で反応させた。０．１％Ｐｏｌｙｏｘｙｅｔｈｙｌｅｎｅ（２０）ｓｏｒｂｉｔａｎ ｍｏｎｏｌａｕｒａｔｅ（Ｔｗｅｅｎ２０；Ｗａｋｏ Ｐｕｒｅ Ｃｈｅｍｉｃａｌ Ｉｎｄｕｓｔｒｉｅｓ，Ｌｔｄ．Ｏｓａｋａ）含有のＰＢＳ水溶液で洗浄後、ビオチンを結合したヤギ抗ウサギＩｇＧ抗体と３０分間反応させ、ペルオキシダーゼ含有のストレプトアビジン試薬（ＤＡＫＯ Ｃｏｒｐｏｒａｔｉｏｎ，Ｃａｒｐｉｎｔｅｒｉａ，ＣＡ）を１５分間処置した。ＰＢＳ水溶液で洗浄した後、ビオチン標識タイラマイド溶液（ＤＡＫＯ Ｃｏｒｐｏｒａｔｉｏｎ，Ｃａｒｐｉｎｔｅｒｉａ，ＣＡ）を１５分間反応させ、引き続き０．１％Ｔｗｅｅｎ２０含有のＰＢＳ水溶液で充分洗浄した。さらにペルオキシターゼ標識ストレプトアビジン溶液を１５分間処理し、同様に０．１％Ｔｗｅｅｎ２０含有のＰＢＳ水溶液で充分に洗浄した。洗浄後、２００μｇ／ｍＬのジアミノベンジジンと０．００６％Ｈ_２Ｏ_２を含む５０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）緩衝液中で１０分間反応させることによりペルオキシダーゼ活性を呈色し、組織標本中のＸ型ｓＰＬＡ_２プロペプチドを可視化した。また、０．４％ヘマトキシリン水溶液との反応により、細胞核を対比染色した。Ｘ型ｓＰＬＡ_２プロペプチド陽性シグナルは、ジアミノベンジデンの茶褐色の沈着として検出した。プロペプチド陽性シグナルの吸収中和実験は、抗体をスライドに添加する前に、プロペプチド結合型牛血清アルブミン（３００μｇ／ｍＬ）と２時間反応させることにより行った。
その結果、Ｘ型ｓＰＬＡ_２プロペプチド陽性シグナルは、正常大腸組織にはほとんど検出されず、大腸腺癌組織中の癌細胞に強く検出された。これらのシグナルは、組織への抗体処理前のプロペプチド結合型牛血清アルブミンの添加で消失したことからＸ型ｓＰＬＡ_２プロペプチドに特異的であることが確認された。また、これら陽性シグナルは、非免疫のウサギから調製したＩｇＧでは認められなかった。以上の結果から、大腸癌ではプロ型Ｘ型ｓＰＬＡ_２から活性化に伴って遊離されたプロペプチド量が顕著に増加していることが示唆された。
実施例２０ ヒト活性型Ｘ型ｓＰＬＡ _２ に対するマウスモノクローナル抗体の調製
本発明によって教示されたアミノ酸配列情報に基づいて、ヒト活性型Ｘ型ｓＰＬＡ_２のＮ末端配列を含むペプチド（ＧＩＬＥＬ ＡＧＧＧＣ：配列番号１６）を合成し、それをＫＬＨに結合させたものを免疫原としてマウスを免疫した。抗体価の上昇は、以下のＰＥＧ法により測定した。チューブにマウス抗血漿および放射性沃素標識したＸ型ｓＰＬＡ_２蛋白質を添加し、４度で１８時間以上反応させた。反応後、１％牛ガンマーグロブリン溶液および２５％ＰＥＧ６０００溶液を添加し、遠心分離により抗体と結合して沈澱した放射活性をガンマーカウンターで測定した。
最も抗体価の上昇したマウスから脾細胞を調製し、マウスミエローマ細胞とＰＥＧ１５００の存在下で細胞融合を行った。生成した抗体産生ハイブリドーマは、以下に示すＲＩＡ法で検出した。９６ウエルプレートにヤギ抗マウスＩｇＧ抗体を固相し、ブロックエース（大日本製薬）で非特異的吸着をブロック後、ハイブリドーマ培養上精を反応させ含まれるモノクローナル抗体を捕捉した。洗浄後、放射性沃素標識したＸ型ｓＰＬＡ_２蛋白質を反応させ、洗浄後残存した放射活性を測定した。検出したハイブリドーマを限界希釈法によるクローニングを繰り返し、活性型Ｘ型ＰＬＡ_２に反応するマウスモノクローナル抗体産生ハイブリドーマ株３Ｃ１０を樹立した。該ハイブリドーマは、２０００年１２月１２日にＦＥＲＭ ＢＰ−７３９３（受託番号）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（日本国茨城県つくば市東１丁目１番地１ 中央第６）に受託されている。
３Ｃ１０抗体の特異性を上述のＰＥＧ法において検討した結果、Ｘ型ｓＰＬＡ_２プロペプチドおよび配列番号３記載のアミノ酸配列中−１１位から１０位のアミノ酸配列を含むペプチドを添加した場合には結合する放射活性の減少は認められず、活性型を含むＸ型ｓＰＬＡ_２蛋白質を添加した場合にのみ結合の阻害が認められたことから、本抗体が活性型Ｘ型ｓＰＬＡ_２を特異的に認識している事が示された（図４）。
実施例２１ Ｘ型ｓＰＬＡ _２ プロペプチドに対するマウスモノクローナル抗体の調製
本発明によって教示されたアミノ酸配列情報に基づいて、Ｘ型ｓＰＬＡ_２プロペプチドを含むペプチド（ＥＡＳＲＩ ＬＲＶＨＲ ＲＣ：配列番号１７）を合成し、それをＫＬＨに結合させたものを免疫原とし、マウスを免疫した。抗体価の上昇は以下のＥＬＩＳＡ法により測定した。９６ウエルプレートにヤギ抗マウスＩｇＧ抗体を固相し、ブロックエース（大日本製薬）で非特異的吸着をブロック後、マウス抗血漿を反応させ含まれる抗体を捕捉した。洗浄後、ビオチン化した配列番号３記載のアミノ酸配列中−１１位から１０位のアミノ酸配列を含むペプチドを反応させた。洗浄後、さらにペルオキシダーゼ標識ストレプトアビジン（Ｐｉｅｒｃｅ）と反応させ、最終的にＤＡＫＯ ＴＭＢ Ｏｎｅ−Ｓｔｅｐ Ｓｕｂｓｔｒａｔｅ Ｓｙｓｔｅｍ（ＤＡＫＯ）を添加して発色させ、発色強度を４５０ｎｍの波長で測定した。
最も抗体価の上昇したマウスから脾細胞を調製し、マウスミエローマ細胞とＰＥＧ１５００の存在下で細胞融合を行った。生成した抗体産生ハイブリドーマは上述のＥＬＩＳＡ法で検出した。検出したハイブリドーマを限界希釈法によるクローニングを繰り返し、Ｘ型ｓＰＬＡ_２プロペプチドに反応するマウスモノクローナル抗体産生ハイブリドーマ株ＨＰＸ−６Ｅ１を樹立した。該ハイブリドーマは、２００１年４月１７日にＦＥＲＭ ＢＰ−７５４７（受託番号）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（日本国茨城県つくば市東１丁目１番地１ 中央第６）に受託されている。
ＨＰＸ−６Ｅ１抗体の特異性を上述のＥＬＩＳＡ法において検討した結果、活性型を含むＸ型ｓＰＬＡ_２蛋白質を添加した場合には吸光度の減少は認められず、Ｘ型ｓＰＬＡ_２プロペプチドを添加した場合にのみ結合の阻害が認められ、本抗体がＸ型ｓＰＬＡ_２プロペプチドを特異的に認識している事が示された（図５）。
実施例２２ プロ型、活性型ヒトＸ型ｓＰＬＡ _２ の精製
前述のごとく確立された活性型Ｘ型ｓＰＬＡ_２特異モノクローナル抗体３Ｃ１０産生ハイブリドーマを大量培養後、ヌードマウス腹腔内に移植し、腹水を作製した。得られた腹水をＨｉＴｒａｐ Ｐｒｏｔｅｉｎ Ｇ カラム（Ａｍｅｒｃｈａｍ Ｐｈａｒｍａｃｉａ）に添加し、洗浄後１０ｍＭ ｃｉｔｒａｔｅ ｂｕｆｆｅｒ（ｐＨ３．０）で溶出し、精製抗体画分を得た。
Ｘ型ｓＰＬＡ_２プロペプチド特異モノクローナル抗体ＨＰＸ−６Ｅ１については、無血清培地で大量培養を行い、その培養上清をＨｉＴｒａｐ Ｐｒｏｔｅｉｎ Ｇ カラムに直接添加し、上記条件にて溶出することにより精製抗体を得た。
得られたそれぞれの精製抗体について、ＨｉＴｒａｐ ＮＨＳ−Ａｃｔｉｖａｔｅｄ（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ）に固相化し、３Ｃ１０抗体架橋カラム及びＨＰＸ−６Ｅ１抗体架橋カラムを作製した。
これらの３Ｃ１０抗体カラム及びＨＰＸ−６Ｅ１抗体カラムを用いて、活性型Ｘ型ｓＰＬＡ_２及びプロ型Ｘ型ｓＰＬＡ_２を単離した。本発明実施例４記載のＣＨＯ細胞発現組換えＸ型ｓＰＬＡ_２蛋白質を３Ｃ１０抗体カラムあるいはＨＰＸ−６Ｅ１抗体カラムに添加し、洗浄後、０．３Ｍ ＮａＣｌを含む０．１Ｍ リン酸緩衝液（ｐＨ１２）で溶出した。溶出した画分をＳＤＳ−ＰＡＧＥで分析した結果、３Ｃ１０抗体カラム吸着画分には活性型が、またＨＰＸ−６Ｅ１抗体カラム吸着画分にはプロ型が回収された。
３Ｃ１０抗体カラム吸着画分には微量のプロ型が、またＨＰＸ−６Ｅ１抗体カラム吸着画分には微量の活性型蛋白質が混入していたが、電気泳動像の画像解析により、その比率はそれぞれ４．０％および８．７％であった。
実施例２３ 活性型Ｘ型ｓＰＬＡ _２ 特異的ＥＬＩＳＡ系の確立
本発明実施例３記載の抗Ｘ型ｓＰＬＡ_２抗体から製したＩｇＧ画分を９６ウエルプレートに固相し、ブロックエース（大日本製薬）で非特異的吸着をブロック後、抗体カラムを用いて精製した活性型Ｘ型ｓＰＬＡ_２またはプロ型Ｘ型ｓＰＬＡ_２を含む溶液を反応させ、溶液中に含まれるＸ型ｓＰＬＡ_２を捕捉した。洗浄後、ビオチン化した３Ｃ１０抗体を反応させた。洗浄後さらにペルオキシダーゼ標識ストレプトアビジン（Ｐｉｅｒｃｅ）と反応させ、最終的にＤＡＫＯ ＴＭＢ Ｏｎｅ−Ｓｔｅｐ Ｓｕｂｓｔｒａｔｅ Ｓｙｓｔｅｍ（ＤＡＫＯ）を添加して発色させ、発色強度を４５０ｎｍの波長で測定した。
その結果、検体として活性型Ｘ型ｓＰＬＡ_２を用いた場合には強い発色が認められ、得られる吸光度は活性型Ｘ型ｓＰＬＡ_２濃度に比例した。しかし、プロ型Ｘ型ｓＰＬＡ_２を用いた場合には、活性型Ｘ型ｓＰＬＡ_２を抗原とした場合に比べて得られる反応性は微弱で、約５％の交叉反応性として検出されたが、前述のようにプロ型画分にはわずかに活性型の混在が認められる（８．７％）ことから、本ＥＬＩＳＡ系の活性型Ｘ型ＰＬＡ_２に対する特異性はきわめて高いと判断できる（図６）。
実施例２４ プロ型ｓＰＬＡ _２ 特異的ＥＬＩＳＡ系の確立
ＨＰＸ−６Ｅ１精製抗体を９６ウエルプレートに固相し、ブロックエース（大日本製薬）で非特異的吸着をブロック後、検体として、さまざまな濃度の活性型Ｘ型ｓＰＬＡ_２またはプロ型Ｘ型ｓＰＬＡ_２を含む溶液を反応させ、溶液中に含まれるｓＰＬＡ_２を捕捉した。洗浄後、ビオチン化した抗Ｘ型ｓＰＬＡ_２抗体ＩｇＧ画分を反応させた。洗浄後さらにペルオキシダーゼ標識ストレプトアビジン（Ｐｉｅｒｃｅ）と反応させ、最終的にＤＡＫＯ ＴＭＢ Ｏｎｅ−Ｓｔｅｐ Ｓｕｂｓｔｒａｔｅ Ｓｙｓｔｅｍ（ＤＡＫＯ）を添加して発色させ、発色強度を４５０ｎｍの波長で測定した。
その結果、検体としてプロ型Ｘ型ｓＰＬＡ_２を用いた場合には強い発色が認められ、得られる吸光度はプロ型Ｘ型ｓＰＬＡ_２濃度に比例した。活性型Ｘ型ｓＰＬＡ_２を用いた場合には、プロ型Ｘ型ｓＰＬＡ_２を抗原とした場合に比べて得られる反応性は微弱で、約４％の交叉反応性として検出されたが、活性型画分にはわずかにプロ型の混在が認められる（４．０％）ことから、本ＥＬＩＳＡ系のプロ型Ｘ型ｓＰＬＡ_２に対する特異性はきわめて高いと判断できる（図７）。
実施例２５ 血清中Ｘ型ｓＰＬＡ _２ 検出の検討
モノクローナル抗体を用いた活性型及びプロ型Ｘ型ｓＰＬＡ_２特異的ＥＬＩＳＡ系と、ポリクローナル抗体を用いた型非特異的Ｘ型ｓＰＬＡ_２測定系を用いて、正常者血清及び大腸癌患者血清中のＸ型ｓＰＬＡ_２濃度を測定した。
正常者血清５例のＸ型ｓＰＬＡ_２濃度の平均値を１としたときの大腸癌患者血清６例中のＸ型ｓＰＬＡ_２濃度は図８に示すとおりであり、活性型Ｘ型ｓＰＬＡ_２特異的測定系では大腸癌患者群の中に高値を示す検体を検出することが出来た。これに対して、プロ型Ｘ型ｓＰＬＡ_２特異的測定系及び型非特異的測定系においては患者群における高値検体を検出することが出来なかった。この成績は、本発明において作製した活性型Ｘ型ｓＰＬＡ_２特異抗体を用いた活性型Ｘ型ｓＰＬＡ_２特異的測定系が、大腸癌の診断において臨床上有用である可能性を示している（図８）。
産業上の利用可能性
本発明によれば、Ｘ型ｓＰＬＡ_２の一部を特異的に認識する抗体；該抗体を用いた活性型Ｘ型ｓＰＬＡ_２の測定方法；該抗体を含む診断用キットなどが提供される。
本発明の測定方法を用いることで、Ｘ型ｓＰＬＡ_２が特異的に関与する疾病の特定が可能となるとともに、Ｘ型ｓＰＬＡ_２に起因する特定疾患、特に大腸癌、肺癌、肝臓癌、胃癌、腎臓癌、胆嚢癌、前立腺癌、膵臓癌、精巣癌、卵巣癌、皮膚癌、アルツハイマー病、肝硬変の診断用キットなどが提供される。
【配列表】

【図面の簡単な説明】
図１は、プレプロ型Ｘ型ｓＰＬＡ_２、プロ型Ｘ型ｓＰＬＡ_２及び活性型Ｘ型ｓＰＬＡ_２の関係を示す図である。
図２は、マウス活性型Ｘ型ｓＰＬＡ_２、マウスプロ型Ｘ型ｓＰＬＡ_２およびトリプシン消化したプロ型Ｘ型ｓＰＬＡ_２の酵素活性を示す図である。図中、Ｍ、ＰおよびＰ＋Ｔは、それぞれ活性型、プロ型およびトリプシンにて消化されたプロ型Ｘ型ｓＰＬＡ_２を意味する。
図３は、本発明の抗Ｘ型ｓＰＬＡ_２プロペプチド抗体を用いたＥＬＩＳＡ法における標準曲線を示す。
図４は、本発明の活性型Ｘ型ｓＰＬＡ_２モノクローナル抗体（３Ｃ１０）の特異性を示す図である。
図５は、本発明のプロペプチドモノクローナル抗体（ＨＰＸ−６Ｅ１）の特異性を示す図である。
図６は、本発明の活性型Ｘ型ｓＰＬＡ_２モノクローナル抗体（３Ｃ１０）を用いたＥＬＩＳＡ系の標準曲線性および特異性を示す図である。
図７は、本発明のプロペプチドモノクローナル抗体（ＨＰＸ−６Ｅ１）を用いたＥＬＩＳＡ系の標準曲線性および特異性を示す図である。
図８は、各種測定系を用いた大腸癌患者由来サンプルの測定結果を示す図である。 Technical field
The present invention relates to X-type phospholipase A₂(X-type sPLA₂An antibody that specifically recognizes a part of the active X type sPLA using the antibody₂A diagnostic kit comprising the antibody.
Background art
Phospholipase A₂(PLA₂EC 3.1.1.4) is a generic name for phospholipid degrading enzymes that hydrolyze 2-acyl ester bonds of 3-sn-phosphoglycerides. PLA₂Is involved in the digestion of phospholipids in food and the formation and metabolism of biological membrane phospholipids, as well as the initiation of the arachidonic acid cascade leading to the production of lipid mediators such as prostaglandins, leukotrienes, platelet activating factor (PAF), and lysophospholipids Functions as an enzyme. Various PLAs in mammals₂The existence of calcium ions (Ca²⁺) Secreted PLA based on requirements and substrate specificity₂, Cytoplasmic PLA₂, Ca²⁺Independent PLA₂As well as four families of PAF-acetylhydrolases (Balsinde et al., J. Biol. Chem. 272, 16069-16072 (1997)).
Of these, secretory PLA₂(SPLA₂) Family is a low molecular weight (13,000-15,000) PLA secreted extracellularly₂It is a molecular group. In humans, six types of IB, IIA, IID, IIE, V, and X are known. Each molecular species has 12 to 16 Cys residues in its structure, forms an intramolecular disulfide bond, and preserves an active center site composed of His-Asp residues. In addition, common Ca²⁺It has a binding region, and the enzyme activity is expressed in the order of mM Ca.²⁺(Tischfield et al., J. Biol. Chem. 272, 17247-17250 (1997), Cupillard et al., J. Biol. Chem. 272, 15745-15752 (1997), Ishizaki et al., J. Biol. Chem. 274. , 24973-24979 (1999), and Suzuki et al., J. Biol. Chem. 275, 5785-5793 (2000)).
Human X-type sPLA₂SPLA from the DNA database₂Based on related sequences, it was cloned from fetal lung tissue (Cupillard, et al., J. Biol. Chem., 272, 15745-15752 (1997)). Human X-type sPLA₂The gene of other sPLA₂It is located on chromosome 16 unlike.
Human X-type sPLA₂Protein is sPLA₂It is the most acidic (pI = 5.3) in the family and contains a site capable of N-linked sugar chain binding. This sPLA₂Has 16 cysteine residues in the molecule and is type IB sPLA₂And IIA / IID / IIE sPLA₂All of the characteristic intramolecular disulfide bridges present in each. Furthermore, IIA / IID / IIE type sPLA₂Have a unique terminal extension structure of the carboxyl group. By Northern analysis, expression of X-type sPLA2 mRNA (1.5 kilobase) was detected in human spleen and thymus. Therefore, X-type sPLA₂Although the details regarding the physiological and pathological functions of these are unknown, the possibility that this enzyme is involved in the regulation of immune system and inflammatory pathology is presumed from the localization of the expression site.
X type sPLA₂Of active X form sPLA consisting of 123 amino acids₂And the presence of a prepropeptide (SEQ ID NO: 1; Cupillard, et al., J. Biol. Chem., 272, 15745-15752 (1997)). However, as pointed out by Cupillard et al., The cleavage site by the signal peptidase on the prepropeptide, that is, the signal peptide and the propeptide was not specified.
Disclosure of the invention
The object of the present invention is to provide an X-type sPLA₂An antibody that specifically recognizes a part of the protein; active X-type sPLA using the antibody₂A diagnostic kit containing the antibody.
We have human X-type sPLA₂In order to elucidate the physiological functions of the sPLA,₂Shows that there are two types of molecular species, an inactive pro type having a propeptide sequence at the N-terminus and an active type that is generated by cleaving the propeptide portion. The amino acid sequence of the propeptide portion to be identified was identified. Further, the amount of the propeptide or the active X-type sPLA₂Activated X-type sPLA by measuring its own amount₂A measuring method for specifically detecting the concentration was found and the present invention was completed.
That is, the present invention
(1) an antibody that specifically recognizes part of a polypeptide consisting of amino acids from Glu at position -11 to Asp at position 123 in the amino acid sequence set forth in SEQ ID NO: 3;
(2) The antibody according to (1) above, which specifically recognizes a polypeptide consisting of amino acids from Glu at position -11 to Arg at position -1 in the amino acid sequence described in SEQ ID NO: 3;
(3) The antibody according to (1), which specifically recognizes a polypeptide consisting of amino acids from Gly at position 1 to Asp at position 123 in the amino acid sequence described in SEQ ID NO: 3;
(4) The antibody according to any one of (1) to (3) above, which is a polyclonal antibody;
(5) The antibody according to any one of (1) to (3) above, which is a monoclonal antibody;
(6) The monoclonal antibody according to (3), which is produced by a hybridoma whose accession number is FERM BP-7393;
(7) PLA comprising the antibody according to (3) or (6) above₂Drugs for related diseases;
(8) a polypeptide comprising amino acids from Glu at position -11 to Arg at position -1 in the amino acid sequence set forth in SEQ ID NO: 3;
(9) a polypeptide comprising an amino acid from Gly at position 1 to Asp at position 123 in the amino acid sequence set forth in SEQ ID NO: 3;
(10) A standard antigen comprising the polypeptide according to (8) or (9) above;
(11) The method for measuring a polypeptide according to (8) or (9) above, wherein the antibody according to any one of (1) to (6) is used;
(12) Active X-type sPLA₂Measuring method according to (11) above, characterized in that
(13) The kit for measuring a polypeptide according to (8) or (9) above, comprising the antibody according to any one of (1) to (6) above;
(14) Active X-type sPLA₂Measuring kit according to (13) above, characterized in that
(15) A cancer diagnostic kit comprising a reagent capable of detecting the polypeptide according to (8) or (9) above;
(16) The cancer diagnostic kit according to (15), wherein the reagent is the antibody according to any one of (1) to (6) above;
(17) The cancer according to (15) or (16) above, wherein the cancer is colon cancer, lung cancer, liver cancer, stomach cancer, kidney cancer, gallbladder cancer, prostate cancer, pancreatic cancer, testicular cancer, ovarian cancer or skin cancer. Cancer diagnostic kit;
(18) A kit for diagnosing Alzheimer's disease comprising a reagent capable of detecting the polypeptide according to (8) or (9) above;
(19) The diagnostic kit for Alzheimer's disease according to (18) above, wherein the reagent is the antibody according to any one of (1) to (6) above;
(20) A kit for diagnosing liver cirrhosis, comprising a reagent capable of detecting the polypeptide according to (8) or (9) above;
(21) The kit for diagnosing cirrhosis according to claim 20, wherein the reagent is the antibody according to any one of (1) to (6) above;
(22) A screening method for cancer, Alzheimer's disease or cirrhosis characterized by detecting the polypeptide according to (8) or (9) above; and
(23) The screening method according to the above (22), wherein the cancer is colon cancer, lung cancer, liver cancer, stomach cancer, kidney cancer, gallbladder cancer, prostate cancer, pancreatic cancer, testicular cancer, ovarian cancer or skin cancer.
X type sPLA₂Depending on the degree of maturity, pre-pro type X sPLA₂, Pro type X type sPLA₂And activated X-type sPLA₂(Mature type X sPLA₂There are three types.
Immediately after translation from mRNA, X-type sPLA₂Is produced as a prepro form with a signal peptide. Pre-pro type X type sPLA₂Means a polypeptide consisting of amino acids from Met at position −32 to Asp at position 123 in the amino acid sequence set forth in SEQ ID NO: 3. Among them, the signal peptide is a polypeptide consisting of amino acids from Met at the −32 position to Gly at the −12 position in the amino acid sequence shown in SEQ ID NO: 3.
Professional type X type sPLA₂Occurs when the signal peptide is cleaved from the prepro form. Professional type X type sPLA₂Means a polypeptide consisting of amino acids from Glu at position -11 to Asp at position 123 in the amino acid sequence set forth in SEQ ID NO: 3.
Furthermore, professional type X type sPLA₂Is cleaved between Arg at position -1 and Gly at position 1 in the amino acid sequence shown in SEQ ID NO: 3, and a propeptide (from Glu at position -11 to -1 in the amino acid sequence shown in SEQ ID NO: 3). Polypeptide consisting of 11 amino acids up to Arg) and active X-type sPLA₂(Polypeptide consisting of amino acids from Gly at position 1 to Asp at position 123 in the amino acid sequence shown in SEQ ID NO: 3). In vivo, this active X-type sPLA₂Is phospholipase A₂It is thought that it has the function as.
In the present invention, a fragment consisting of a signal peptide and a propeptide may be referred to as a prepropeptide. The prepropeptide means a polypeptide consisting of amino acids from Met at position −32 to Arg at position −1 in the amino acid sequence shown in SEQ ID NO: 3 (FIG. 1).
In the present invention, “a part of a polypeptide comprising an amino acid sequence from Glu at position −11 to Asp at position 123 in the amino acid sequence shown in SEQ ID NO: 3” means pro-type X-type sPLA₂Means a part of Preferably, propeptide or activated X-type sPLA₂Means. In addition, “the polypeptide described in (6) or (7) above” means a propeptide or an activated X-type sPLA₂Means.
The present invention relates to active X-type sPLA₂Is intended to be measured by immunological means.
In the process of activation, pro-type X-type sPLA₂To activated X-type sPLA₂A propeptide is produced as a pair. We have activated X-type sPLA₂Or activated X-type sPLA in the process of activation₂Specific amount of propeptide produced in an equimolar amount with active X-type sPLA₂The measurement method was completed.
In the present invention, “an antibody that specifically recognizes a polypeptide consisting of amino acids from Glu at position -11 to Arg at position −1 in the amino acid sequence set forth in SEQ ID NO: 3” specifically recognizes a propeptide. Antibody. “Recognize propeptide specifically” means that it reacts with propeptide but is active type X sPLA₂It means that the cross-reactivity with is 5% or less. In addition, “an antibody that specifically recognizes a polypeptide comprising an amino acid from Gly at position 1 to Asp at position 123 in the amino acid sequence described in SEQ ID NO: 3” refers to active X-type sPLA₂Means an antibody that specifically recognizes. "Activated X-type sPLA₂Specifically recognizes "active X-type sPLA"₂Reacts with propeptide and pro-type X-sPLA₂It means that the cross-reactivity with is 5% or less. Such an antibody may be either a polyclonal antibody or a monoclonal antibody. For example, activated X-type sPLA₂As a monoclonal antibody specifically recognizing, it was entrusted to the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (1st, 1st East, 1st Street, Tsukuba City, Ibaraki, Japan) on December 12, 2000. And a monoclonal antibody produced by hybridoma 3C10 (accession number: FERM BP-7393). In addition, propeptide or active X-type sPLA used to examine the specificity of these antibodies₂Can be used as a “standard antigen” to generate a standard curve.
By using the antibody of the present invention, activated X-type sPLA₂It becomes possible to measure only.
Secreted PLA₂Is involved in the release of fatty acids (eg, arachidonic acid) from phospholipids present in cell membranes and lipoproteins. The release of excess fatty acids as well as the production of various lipid mediators by their metabolism causes diseases such as septic shock, adult dyspnea syndrome, pancreatitis, trauma, bronchial asthma, allergic rhinitis, rheumatoid arthritis and the like. “PLA” of the present invention₂“Related diseases” means these diseases. Active X-type sPLA of the present invention₂Antibodies against are useful in the treatment of these diseases. Further, by using the measurement method of the present invention, active X-type sPLA₂It is thought that the diagnosis of these diseases caused by the disease becomes possible. Therefore, the present invention is “active X-type sPLA”₂Measurement method ".
Furthermore, the present inventors have analyzed by immunohistochemical analysis, colorectal cancer tissues of colon cancer patients, lung cancer tissues of lung cancer patients, liver tissues of liver cancer patients, stomach tissues of stomach cancer patients, kidney tissues of kidney cancer patients, gallbladder Cancer patient gallbladder tissue, prostate cancer patient prostate tissue, pancreatic cancer patient pancreatic tissue, testicular cancer patient testis tissue, ovarian cancer patient ovarian tissue, skin cancer patient skin tissue, Alzheimer's disease cerebral tissue and cirrhosis patient X-type sPLA compared with those in normal human tissues₂Was confirmed to be remarkably highly expressed. Therefore, sPLA₂X-type sPLA using the phospholipid which is a substrate of the present invention or the antibody of the present invention as a “reagent”₂By confirming the activity or expression, it is possible to diagnose cancer, Alzheimer's disease or cirrhosis. The present invention provides a “cancer diagnostic kit”, “Alzheimer's disease diagnostic kit” or “cirrhosis diagnostic kit”. Each diagnostic kit includes at least a reagent capable of detecting the polypeptide of the present invention. As a reagent, sPLA₂Examples thereof include phospholipids that are substrates of the present invention or antibodies of the present invention. Examples of the detection method include precipitation reaction method, ELISA method, RIA method, Western blotting method and the like. For example, the antibody is reacted with an appropriately diluted patient serum on a microplate on which the antibody is solid-phased, washed, and then reacted with an anti-rabbit IgG antibody labeled with a secondary antibody peroxidase, and then reacted. The polypeptide of the present invention can be detected by adding a peroxidase substrate to develop color and measuring the absorbance at 450 nm.
When the detection reagent is an antibody, the kit of the present invention is measured from various reagents such as a coloring reagent, a reaction stopping reagent, a standard antigen reagent, and a sample pretreatment reagent in addition to the antibody of the present invention. An appropriate reagent according to the method can be appropriately selected and attached to the kit of the present invention.
Further, by using the diagnostic kit of the present invention, cancer, particularly colon cancer, lung cancer, liver cancer, stomach cancer, kidney cancer, gallbladder cancer, prostate cancer, pancreatic cancer, testicular cancer, ovarian cancer or skin cancer, Alzheimer's disease or Mass screening for the early detection of cirrhosis is possible. Specifically, by using a biological sample of a subject, for example, blood as a sample, and using the diagnostic kit of the present invention, an activated X-type sPLA₂The present invention also provides a “screening method” for pre-onset diagnosis of cancer, Alzheimer's disease, or cirrhosis in which A is involved.
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention mainly relates to an activated X-type sPLA using a propeptide.₂Related to measurement.
The propeptide preparation step, antibody preparation step, and immunoassay method using the antibody will be described below. In this specification, unless otherwise specified, gene recombination techniques known in the art, production techniques for recombinant proteins in animal cells, insect cells, yeast and E. coli, methods for separating and purifying expressed proteins, and analysis methods And immunological techniques are employed.
X type sPLA ₂ Of DNA encoding
X type sPLA₂Can be easily produced and obtained by general genetic engineering techniques based on the sequence information taught by the present invention (see Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989), etc.). . Specifically, X-type sPLA₂A cDNA library is prepared according to a conventional method from an appropriate source from which X is expressed, and X-type sPLA is prepared from the library.₂(Proc. Natl. Acad. Sci., USA., 78, 6613 (1981); Science, 22, 778 ( 1983)). The origin of cDNA is X-type sPLA₂Examples include various cells, tissues, and cultured cells derived therefrom. Separation of total RNA from these, separation and purification of mRNA, acquisition of cDNA, and cloning thereof can all be performed according to conventional methods. In addition, cDNA libraries are commercially available. In the present invention, these cDNA libraries, for example, various cDNA libraries commercially available from Clontech can also be used.
X type sPLA₂The method of screening the DNA from a cDNA library is not particularly limited, and can be performed according to a usual method. Specifically, for example, for a protein produced by cDNA, a method of selecting a corresponding cDNA clone by immunoscreening using the protein-specific antibody, or a probe that selectively binds to a target DNA sequence is used. Examples thereof include plaque hybridization, colony hybridization, and combinations thereof. As a probe used here, X-type sPLA₂In general, the DNA and the like chemically synthesized based on the information on the nucleotide sequence of the DNA of the present invention can be generally exemplified, but the DNA of the present invention and fragments already obtained can also be used satisfactorily. In addition, sense primers and antisense primers set based on the base sequence information of the DNA of the present invention can also be used as screening probes.
X-type sPLA₂The DNA encoding can be obtained by conventional chemical methods such as the phosphoric acid triester method (Narang et al., Meth. Enzymol., 68, 90-108 (1979)) or the phosphoric acid diester method (Brown et al., Meth. Enzymol., 68, 109-151 (1979)).
X type sPLA ₂ Protein preparation
(1) Recombinant X-type sPLA₂Protein expression
X type sPLA₂Protein is X-type sPLA₂In accordance with the base sequence information of Science, Science, 224, 1431 (1984); Biochem. Biophys. Res. Comm., 130, 692 (1985); Proc. Natl. Acad. Sci., USA. 80, 5990 (1983), etc.) can be obtained as a recombinant protein. More specifically, a gene encoding a desired protein is incorporated into an appropriate vector. This vector is introduced into a host cell to produce a transformant. A recombinant protein can be obtained by culturing the transformant.
Here, both eukaryotes and prokaryotes can be used as host cells. The eukaryotic cells include vertebrate and yeast cells. Examples of vertebrate cells include COS cells (Cell, 23, 175 (1981)) which are monkey cells and Chinese hamster ovary. Cells are often used.
As an expression vector, a vector having a promoter located upstream of a gene to be normally expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence, etc. can be used, and this further has an origin of replication if necessary. May be. Examples of the expression vector include pSV2dhfr (Mol. Cell. Biol., 1,854 (1981)) carrying the SV40 early promoter. As eukaryotic microorganisms, yeast is generally used, and among them, Saccharomyces yeasts can be used. As the yeast expression vector, for example, pAM82 (Proc. Natl. Acad. Sci., USA., 80, 1 (1983)) having a promoter for the acid phosphatase gene can be used.
As a prokaryotic host, Escherichia coli and Bacillus subtilis are commonly used. When these are used as hosts, for example, a plasmid vector that can be replicated in the host bacterium is used, and a promoter and an SD sequence are further upstream of the gene so that a desired gene can be expressed in the vector, and further, necessary for starting protein synthesis It is preferable to use an expression plasmid provided with a proper start codon. Examples of the host include E. coli. E. coli K12 strain is used. In general, pBR322 and its improved vector are often used as the vector, but not limited thereto, and various known strains and vectors can also be used. As the promoter, for example, trp promoter, lpp promoter, lac promoter, PL / PR promoter and the like can be used.
Various general methods can be employed as a method for introducing a desired recombinant DNA into a host cell and a transformation method using the method. Moreover, the obtained transformant can be cultured according to a conventional method, and a desired protein is produced by the culture. As the medium used for the culture, various media commonly used depending on the host cell can be appropriately selected and used, and the culture can also be performed under conditions suitable for the host cell. For example, the human X-type sPLA of the present invention downstream of the pSVL SV40 late promoter₂A transformant was prepared by introducing a vector containing the gene into monkey-derived cell COS-7, and this transformant was cultured at 37 ° C. for 3 days in the presence of 5% CO 2 to obtain recombinant human X type. sPLA₂Protein can be produced.
Proteins are separated and separated by various separation operations (Biochemistry, 25 (25), 8274 (1986); Eur. J. Biochem., 163, 313 (1987), etc.) utilizing their physical properties and chemical properties. It can be purified. Such methods include salting-out, centrifugation, osmotic shock, ultrasonic crushing, ultrafiltration, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography and other various liquid chromatographies. Examples thereof include graphy, dialysis, and combinations thereof.
Purified X-type sPLA₂Is a professional type X type sPLA₂Active X-type sPLA by cleaving with an appropriate protease (eg, subtilisin-like endoprotease, trypsin, etc.)₂Can be obtained.
X-type sPLA₂Protein or X-type sPLA₂Protein fragments (eg, propeptides) can also be synthesized by a peptide synthesizer based on the amino acid sequence information taught by the present invention.
Preparation of antibodies against the protein of the present invention
An antibody against the protein of the present invention is produced by the following method.
(1) Preparation of polyclonal antibody
X type sPLA₂Based on the amino acid sequence of the above, a synthetic peptide synthesized with an ordinary peptide synthesizer or an X-type sPLA₂Proteins produced by bacteria, yeasts, insect cells, animal cells and the like transformed with a vector that expresses are purified by conventional protein chemical methods and used as immunogens. This immunogen was used to refer to Antibodies; A Laboratory Manual, Lane, H. et al. D. In accordance with the method described in et al., Cold Spring Harbor Laboratory Press, New York 1989, etc., a polyclonal antibody that specifically recognizes a protein as an antigen is easily prepared and purified by immunizing the animal by an appropriate method. be able to. An animal such as a mouse, rat, hamster, or rabbit may be immunized and a polyclonal antibody derived from the serum may be prepared. (2) Monoclonal antibody
After removing lymphocytes from the spleen or lymph nodes of mice or rats immunized with the aforementioned immunogen and fusing with myeloma cells, hybridomas were prepared according to the method of Kohler and Milstein (Nature, 256, 495-497 (1975)). Monoclonal antibodies can be produced from the hybridoma. For example, the propeptide or activated X-type sPLA is obtained by the following steps₂Monoclonal antibodies against can be obtained:
(A) immunization of mice with proteins,
(B) removal of the spleen from the immunized mouse and separation of the spleen cells,
(C) Fusion of the isolated spleen cells and mouse myeloma cells in the presence of a fusion promoter (eg, polyethylene glycol) by the method described in Kohler et al.
(D) culture of hybridoma cells obtained in a selective medium in which unfused myeloma cells do not grow;
(E) selection of hybridoma cells producing a desired antibody by a method such as enzyme-linked immunosorbent assay (ELISA) or Western blot, and cloning by a limiting dilution method,
(F) The hybridoma cells producing the monoclonal antibody are cultured, and the monoclonal antibody is harvested.
Active X-type sPLA ₂ Measuring method
In the present invention, a propeptide or an activated X-type sPLA₂Active X-type sPLA₂Protein measurements can be made. The measurement method of the present invention comprises the amount of antigen (propeptide or activated X-type sPLA in the solution to be measured).₂The amount of the antibody, antigen or antibody-antigen complex corresponding to) is detected by chemical or physical means, and this is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. Any measurement method may be used. For example, nephrometry, competition method, immunometric method and sandwich method can be used.
In immobilizing an antigen or antibody, a chemical bond usually used for immobilizing a protein or an enzyme can be used. As the carrier, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass is used.
As the labeling agent, a radioisotope, an enzyme, or a fluorescent substance is used. As a radioisotope,¹²⁵I,³H,¹⁴C or the like is used. As the enzyme, peroxidase, β-galactosidase, β-glucosidase, alkaline phosphatase and the like are used.
Example
The invention is further illustrated by the following examples.
Example 1 Human X-type sPLA ₂ Preparation of expression vector
Human X-type sPLA₂The cDNA encoding was obtained by PCR using human placenta cDNA as a template. The following primers were used for PCR.

hGGX-S has a Kozak sequence and a MluI recognition site. HGX-AS has a NotI recognition site.
PCR was performed for 35 cycles under the conditions of 94 ° for 30 seconds, 50 ° for 50 seconds, and 72 ° for 2 minutes. The PCR amplified fragment was cut with MluI and NotI and inserted into pBS-SK (−). X-type sPLA with 6 His residues added to the end of the carboxyl group after confirming the sequence₂-HisTag body is expressed as hGX-S primer and hGX-H6AS primer (5'-tcaaggtgggccgctcaatggtgatggtgatgIt was constructed by PCR using gtcaactttgggcgagtccggct-3 ': SEQ ID NO: 7). The PCR amplified fragment was cleaved with NotI and XhoI, and X-type sPLA₂Substitution at the corresponding site in the plasmid. After confirming the sequence of the region amplified by PCR, the cDNA was inserted downstream of the SR-α promoter of the expression vector for mammalian cells.
Example 2 Human X-type sPLA ₂ -Expression, purification and characterization of HisTag recombinant protein
X type sPLA₂-Human fetal kidney-derived 293 cells (293T cells) transformed with SV-40 with 0.5 μg of hygromycin B resistance gene together with 20 μg of the plasmid containing HisTag cDNA were transfected with LipofectAMINE reagent (Life Technologies, Inc.). . X type sPLA₂-A 293T clone that stably expresses HisTag was obtained by selecting on the basis of resistance to hygromycin B. Recombinant protein is expressed in chromogenic PLA₂Detected by activity assay. This 293T clone was cultured in a medium containing 10% fetal calf serum, and X-type sPLA₂-A culture supernatant containing HisTag expressed protein was prepared. The main culture supernatant was equilibrated with 20 mmol / L sodium phosphate buffer (pH 7.4) containing 10 mmol / L imidazole and 0.5 mol / L sodium chloride.²⁺-Added to a charged chelating Sepharose fast flow column (Amersham Pharmacia Biotech). Then, X-type sPLA bound to this column₂-HisTag protein was eluted with 20 mmol / L sodium phosphate buffer (pH 7.4) containing 500 mmol / L imidazole and 0.5 mol / L sodium chloride.
Obtained X-type sPLA₂-The fraction containing HisTag protein was dialyzed against 20 mmol / L Tris-HCl buffer containing 1 mmol / L fluorinated phenylmethylsulfonic acid and added to a HiTrap Q column (Amersham Pharmacia Biotech). The bound protein is eluted with a sodium chloride concentration gradient from 0 to 0.5 mol / L, and X-type sPLA₂The fraction containing the HisTag protein was then added to a reverse phase HPLC column (Cosmosil, 5C18 300AR, 4.6 × 150 mm, Nacalai Tesque). Elute with a gradient of acetonitrile from 20 to 95% in 0.05% trifluoroacetic acid,₂As a result of activity measurement, X-type sPLA₂-HisTag was eluted with 43% acetonitrile. Each eluted fraction was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 15-25% acrylamide gradient gel (Daiichi Kagaku). In another experiment, purified X-type sPLA₂-HisTag protein was added at 37 ° C. in 0.5% SDS containing 0.2 units of N-glycosidase F (E-5003; Oxford GlycoSystem), 5% 2-mercaptoethanol, 3% nonidet P-40. After time treatment, SDS-PAGE was performed and electroblotted to Immobilin-P membrane (Millipore Co.,) according to previous reports (Hanazaki, K. et al., J. Biol. Chem., 270, 7543-7550). (1995)). Blotted protein was stained with Coomassie Brilliant Blue, then X-type sPLA₂-A band corresponding to the HisTag protein was cut out, and the amino acid sequence at the N-terminal thereof was analyzed by Applied Biosystems Sequence Sequencer.
X-type sPLA with a His residue added to the C-terminus obtained from the above₂The purified protein preparation is PLA₂Had activity. As a result of SDS-PAGE analysis, most were molecular species having a molecular weight of 16-kDa, but some showed a molecular weight of 14-kDa. As a result of treatment with N-glycosidase F (an enzyme that liberates N-linked sugar chains from glycoproteins), the 16-kDa molecular species disappeared, and all were aggregated into a single 14-kDa molecular species. As a result of analysis of the N-terminal amino acid sequence of this 14-kDa protein, human X-type sPLA₂(The amino acid sequence from Gly at position 1 to Thr at position 8 in the amino acid sequence described in SEQ ID NO: 3). X type sPLA₂In the sequence, there is a site where N-linked sugar chain can be added at position 71. Therefore, these findings indicate that X-type sPLA with a His residue added to the c-terminus in 293T cells₂Most of them indicate that they are expressed as active glycoproteins to which N-linked sugar chains are added.
Example 3 Anti-X type sPLA ₂ Preparation and characterization of antibodies
Purified human X-type sPLA₂-After immunizing rabbits with HisTag protein, antiserum was prepared and the IgG fraction was purified using HiTrap protein G-column (Amersham Pharmacia Biotech). Specificity and titer of the antiserum and the purified antibody were measured by the following ELISA method. Purified human IB, IIA and X sPLA₂-Prepare 96-well plate (Nunc, Immuno MaxiSorp plate) coated with HisTag protein (100ng each), block non-specific adsorption with Block Ace solution (Dainippon Pharmaceutical), then anti-human X-type sPLA₂-HisTag antibody, anti-human type IB sPLA₂Antibodies (biochemistry) and anti-human type IIA sPLA₂Serum (Cayman Chemical Co.) was added and allowed to react for 2 hours. After washing with PBS containing 0.05% Tween 20 (Phosphate-buffered saline, pH 7.4), it was further reacted with a peroxidase-conjugated sheep anti-rabbit IgG antibody (Immunotech), and finally 0.5 mg / mL o- Phenylenediamine and 0.01% (v / v) H₂O₂50 mmol / L sodium citrate buffer solution (pH 5.3) containing was added for color development. The color reaction was terminated by adding 2 mol / L sulfuric acid, and the color intensity was measured at a wavelength of 492 nm. In another test, the plate is X-type sPLA₂-Coated with HisTag or Axl-HisTag protein and analyzed by the method described above.
PLA₂Anti-X type sPLA for activity₂The inhibitory action of the antibody was performed by a luminescence assay. That is, each purified sPLA₂Protein (50 ng) or IID type, V type sPLA₂The culture supernatant of CHO cells expressing₂React with antibody for 2 hours, then PLA₂Activity was measured as described above. Anti-X type sPLA₂The inhibitory action of the antibody is₂The reaction was tested under conditions where it reached a maximum at 37 ° C.
The anti-X type sPLA thus obtained₂The antibody is a human X-type sPLA with HisTag added.₂IB type and IIA type sPLA₂Did not react. The prepared antibody is X-type sPLA.₂It did not react with the HisTag addition protein which has nothing to do with. From this, the HisTag addition part is anti-X type sPLA.₂It became clear that it was not an epitope of an antibody. Furthermore, anti-X type sPLA₂Human Tag sPLA with HisTag added by pretreatment with antibody₂The enzyme activity of sPLA was suppressed in a concentration-dependent manner, and at the concentration of 100 μg / mL, the other five types of sPLA₂No effect on activity, X-type sPLA₂Only 90% of the enzyme activity was inhibited. From the above results, the prepared anti-X type sPLA₂The antibody is human X-type sPLA₂It was proved to be specific for the protein part of.
Example 4 Recombinant X-type sPLA ₂ Protein expression, purification and characterization
X type sPLA₂Using the expression plasmid, CHO cells stably expressing this enzyme were prepared as described above. The CHO cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, and when the cells became confluent, the cells were changed to PM-1000 medium (Eiken, Japan) containing no serum, and the cells were further treated with 3 Cultured for days. The obtained culture supernatant was treated with anti-X type sPLA.₂The antibody was added to an affinity column to which antibody was bound. This column is an anti-X type sPLA₂  IgG (30 mg) was prepared by binding to a HiTrap N-hydroxysuccinimide activation column (5 mL; Amersham Pharmacia Biotech) according to conventional methods. The column was washed with sodium phosphate buffer (pH 7.4), and the bound protein was eluted with 0.1 mol / L glycine-HCl (pH 1.7). This eluted fraction was added to the aforementioned reverse phase HPLC column and separated with a gradient of acetonitrile from 20 to 40% in 0.1% trifluoroacetic acid. Three large fractions were obtained, collected, dried and dissolved in 50 mmol / L Tris-HCl (pH 7.4) containing 0.3 mol / L sodium chloride.
By the above method, human X-type sPLA₂Of the recombinant protein was purified. As a result of SDS-PAGE analysis of the acid-eluted fraction obtained from the antibody column, there are two types of molecular species showing a wide molecular weight ranging from 15 to 18-kDa, and two types showing a single band at 14-kDa and 13-kDa. The presence of molecular species was observed. By N-glycosidase F treatment, it is aggregated into two molecular species (14-kDa and 13-kDa), and as a result of N-terminal amino acid sequence analysis, a 13-kDa protein (GILELAGT: SEQ ID NO: 8) occupies the majority. Human X-type sPLA₂(A polypeptide consisting of Asp at position 1 to Gly in position 1 in the amino acid sequence of SEQ ID NO: 3), while a small number of 14-kDa proteins are active X-type sPLA₂Is a pro type (polypeptide consisting of Glu at position -11 to Asp at position 123 in the amino acid sequence described in SEQ ID NO: 3) to which a peptide consisting of 11 residues (EASRILRVHRR: SEQ ID NO: 9) is added at the N-terminus of It has been found. Therefore, molecular species exhibiting a wide molecular weight over 15-18-kDa are pro-type and active X-type sPLA.₂X-sPLA with N-linked sugar chain added to₂It was considered.
Three fractions further separated by reversed-phase HPLC were analyzed by SDS-PAGE after N-glycosidase F treatment. As a result, fraction 1 was a non-glycosylated 13-kDa active X-type sPLA.₂And N-linked sugar chain added pro-type and active X-type sPLA₂Fraction 2 is an active X-type sPLA with an N-linked glycosylation₂It was revealed that Fraction 3 is a 13-kDa active protein of non-glycan type.
Example 5 Human X-type sPLA ₂ Analysis of substrate specificity
Purified human X-type sPLA₂Regarding the substrate specificity of each purified sample, 12 types of phospholipids having different polar groups having palmitic acid at the sn-1 position and various different fatty acids at the sn-2 position were purchased commercially, and they were used as substrates. Tested. Each sPLA₂Protein (type IB, type IIA sPLA₂And human X-type sPLA₂The enzyme activity of HPLC 3 fractions) was measured under mixed micelle conditions consisting of 1 mmol / L of each phospholipid and 3 mmol / L of deoxycholic acid. As a result, HPLC fraction 1 (pro type and active X type sPLA₂30 to 40% PLA compared to fraction 2 or fraction 3 consisting only of the active form)₂Only showed activity. This decrease is thought to be due to the presence of the 14-kDa pro-type enzyme contained in fraction 1, and the pro-type X-type sPLA₂Suggests that the enzyme activity is weaker than the active form. On the other hand, since the activity of fraction 3 consisting of the non-glycosylated active form showed almost the same activity as that of fraction 2 which is an active enzyme having an N-linked sugar chain, X-type sPLA₂N-linked sugar chain that binds to PLA₂It was considered that the expression of activity was not essentially affected. Among the 12 types of phospholipid substrates used, X-type sPLA₂Active forms (fractions 2 and 3) showed the highest activity against phosphatidylcholine carrying arachidonic acid at the sn-2 position. In contrast, type IB and type IIA sPLA₂Showed the highest activity against phosphatidylglycerols carrying oleic acid at the sn-2 position.
We have recombinant human type X sPLA₂In the process of protein purification, active X-type sPLA₂Pro-type X-type sPLA with a propeptide consisting of 11 amino acids added to the N-terminus of the protein₂In addition, the substrate specificity analysis revealed that the pro-type enzyme had a much lower enzyme activity than the active type. Furthermore, using SignalP computer analysis (Nielsen et al., Protein Eng., 12, 3-9 (1999)) to predict the cleavage site of the signal sequence, the site most easily cleaved by signal peptidase is the N-terminus of the active protein. It was proved that 11 amino acid residues are propeptides by showing that it is between -11 and -12 before. Therefore, X-type sPLA₂It is shown that the cleavage of the propeptide from the pro form by a subtilidine-like or trypsin-like protease is essential for the expression of the maximum enzyme activity of, and it occurs as a pair by measuring the production amount of this propeptide Active X-type sPLA₂It became possible to predict the production amount.
Example 6 Anti-X-type sPLA ₂ Immunohistochemical analysis of human colon cancer tissues using antibodies
Samples of healthy adult colorectal tissue and colorectal cancer tissue derived from colorectal cancer patients are available from Biochain Inc. (San Leandro, CA). Tissue section slides are freed of paraffin wax and 0.3% H₂O₂After removing endogenous peroxidase by reacting with methanol containing for 30 minutes, it was treated with 5% normal goat serum for 20 minutes. Thereafter, anti-X sPLA in PBS containing 0.1% bovine serum albumin₂The mixture was reacted with the antibody (6 μg / mL) for 14 hours at 4 ° C. After washing with PBS, it was reacted with a biotin-conjugated goat anti-rabbit IgG antibody for 30 minutes and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 μg / mL diaminobenzidine and 0.006% H₂O₂X-type sPLA in a tissue specimen colored with peroxidase activity by reacting for 10 minutes in a 50 mmol / L Tris-HCl (pH 7.6) buffer solution containing₂Was visualized. Moreover, the cell nucleus was counterstained by reaction with 0.4% hematoxylin aqueous solution. X type sPLA₂Positive signals were detected as dark brown diaminobenzidene deposits. X type sPLA₂Signal neutralization experiments were performed using purified X-type sPLA prior to addition of antibody to the slide.₂The reaction was performed by reacting with protein (60 μg / mL) for 2 hours.
As a result, X-type sPLA₂The positive signal was hardly detected in normal colon tissue, but was strongly detected in cancer cells in colorectal adenocarcinoma tissue. These signals are X-type sPLA₂X-type sPLA disappeared due to the addition of protein₂It was confirmed to be specific. Also, these positive signals were not observed with IgG prepared from non-immunized rabbits. Based on the above results, X-type sPLA in colorectal cancer₂It was suggested that the protein expression was remarkably high.
Example 7 Anti-X type sPLA ₂ Immunohistochemical analysis of human lung tissue using antibodies
Samples of healthy adult lung tissue as well as lung tissue from lung cancer patients were obtained from Biochain Inc. (San Leandro, CA). Tissue section slides are freed of paraffin wax and 0.3% H₂O₂After removing endogenous peroxidase by reacting with methanol containing for 30 minutes, it was treated with 5% normal goat serum for 20 minutes. Thereafter, anti-X sPLA in PBS containing 0.1% bovine serum albumin₂The mixture was reacted with the antibody (6 μg / mL) for 14 hours at 4 ° C. After washing with PBS, it was reacted with a biotin-conjugated goat anti-rabbit IgG antibody for 30 minutes and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 μg / mL diaminobenzidine and 0.006% H₂O₂X-type sPLA in a tissue specimen colored with peroxidase activity by reacting for 10 minutes in a 50 mmol / L Tris-HCl (pH 7.6) buffer solution containing₂Was visualized. Moreover, the cell nucleus was counterstained by reaction with 0.4% hematoxylin aqueous solution. X type sPLA₂Positive signals were detected as dark brown diaminobenzidene deposits. X type sPLA₂Signal neutralization experiments were performed using purified X-type sPLA prior to addition of antibody to the slide.₂The reaction was performed by reacting with protein (60 μg / mL) for 2 hours.
As a result, X-type sPLA₂The positive signal was detected at a low level in type II alveolar epithelial cells in normal lung tissue, but was strongly detected in cancer cells in lung tissue derived from lung cancer patients. These signals are X-type sPLA₂X-type sPLA disappeared due to the addition of protein₂It was confirmed to be specific. Also, these positive signals were not observed with IgG prepared from non-immunized rabbits. Based on the above results, X-type sPLA in lung cancer₂It was suggested that the protein expression was remarkably high.
Example 8 Anti-X type sPLA ₂ Immunohistochemical analysis of human liver tissue using antibodies
Samples of healthy adult liver tissue as well as lung tissue from liver cancer patients were obtained from Biochain Inc. (San Leandro, CA). Tissue section slides are freed of paraffin wax and 0.3% H₂O₂After removing endogenous peroxidase by reacting with methanol containing for 30 minutes, it was treated with 5% normal goat serum for 20 minutes. Thereafter, anti-X sPLA in PBS containing 0.1% bovine serum albumin₂The mixture was reacted with the antibody (6 μg / mL) for 14 hours at 4 ° C. After washing with PBS, it was reacted with a biotin-conjugated goat anti-rabbit IgG antibody for 30 minutes and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 μg / mL diaminobenzidine and 0.006% H₂O₂X-type sPLA in a tissue specimen colored with peroxidase activity by reacting for 10 minutes in a 50 mmol / L Tris-HCl (pH 7.6) buffer solution containing₂Was visualized. Moreover, the cell nucleus was counterstained by reaction with 0.4% hematoxylin aqueous solution. X type sPLA₂Positive signals were detected as dark brown diaminobenzidene deposits. X type sPLA₂Signal neutralization experiments were performed using purified X-type sPLA prior to addition of antibody to the slide.₂The reaction was performed by reacting with protein (60 μg / mL) for 2 hours.
As a result, X-type sPLA₂A positive signal was detected at a low level in liver lobule and Kupffer stellate cells in normal liver tissue, but was strongly detected in cancer cells in liver tissue derived from liver cancer patients. These signals are X-type sPLA₂X-type sPLA disappeared due to the addition of protein₂It was confirmed to be specific. Also, these positive signals were not observed with IgG prepared from non-immunized rabbits. From the above results, X-type sPLA was found in liver cancer.₂It was suggested that the protein expression was remarkably high.
Example 9 Anti-X type sPLA ₂ Immunohistochemical analysis of human gastric tissue using antibodies
Samples of healthy adult gastric tissue and lung tissue from gastric cancer patients were obtained from Biochain Inc. (San Leandro, CA). Tissue section slides are freed of paraffin wax and 0.3% H₂O₂After removing endogenous peroxidase by reacting with methanol containing for 30 minutes, it was treated with 5% normal goat serum for 20 minutes. Thereafter, anti-X sPLA in PBS containing 0.1% bovine serum albumin₂The mixture was reacted with the antibody (6 μg / mL) for 14 hours at 4 ° C. After washing with PBS, it was reacted with a biotin-conjugated goat anti-rabbit IgG antibody for 30 minutes and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 μg / mL diaminobenzidine and 0.006% H₂O₂X-type sPLA in a tissue specimen colored with peroxidase activity by reacting for 10 minutes in a 50 mmol / L Tris-HCl (pH 7.6) buffer solution containing₂Was visualized. Moreover, the cell nucleus was counterstained by reaction with 0.4% hematoxylin aqueous solution. X type sPLA₂Positive signals were detected as dark brown diaminobenzidene deposits. X type sPLA₂Signal neutralization experiments were performed using purified X-type sPLA prior to addition of antibody to the slide.₂The reaction was performed by reacting with protein (60 μg / mL) for 2 hours.
As a result, X-type sPLA₂The positive signal was hardly detected in normal gastric tissue, and strongly detected in cancer cells in gastric cancer patient-derived gastric tissue. These signals are X-type sPLA₂X-type sPLA disappeared due to the addition of protein₂It was confirmed to be specific. Also, these positive signals were not observed with IgG prepared from non-immunized rabbits. From the above results, X-type sPLA is found in gastric cancer₂It was suggested that the protein expression was remarkably high.
Example 10 Anti-X sPLA ₂ Immunohistochemical analysis of human kidney tissue using antibodies
Samples of healthy adult kidney tissue as well as lung tissue from kidney cancer patients were obtained from Biochain Inc. (San Leandro, CA). Tissue section slides are freed of paraffin wax and 0.3% H₂O₂After removing endogenous peroxidase by reacting with methanol containing for 30 minutes, it was treated with 5% normal goat serum for 20 minutes. Thereafter, anti-X sPLA in PBS containing 0.1% bovine serum albumin₂The mixture was reacted with the antibody (6 μg / mL) for 14 hours at 4 ° C. After washing with PBS, it was reacted with a biotin-conjugated goat anti-rabbit IgG antibody for 30 minutes and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 μg / mL diaminobenzidine and 0.006% H₂O₂X-type sPLA in a tissue specimen colored with peroxidase activity by reacting for 10 minutes in a 50 mmol / L Tris-HCl (pH 7.6) buffer solution containing₂Was visualized. Moreover, the cell nucleus was counterstained by reaction with 0.4% hematoxylin aqueous solution. X type sPLA₂Positive signals were detected as dark brown diaminobenzidene deposits. X type sPLA₂Signal neutralization experiments were performed using purified X-type sPLA prior to addition of antibody to the slide.₂The reaction was performed by reacting with protein (60 μg / mL) for 2 hours.
As a result, X-type sPLA₂The positive signal was detected at a low level in glomerular mesangial cells in normal kidney tissue, but was strongly detected in cancer cells in kidney tissue derived from kidney cancer patients. These signals are X-type sPLA₂X-type sPLA disappeared due to the addition of protein₂It was confirmed to be specific. Also, these positive signals were not observed with IgG prepared from non-immunized rabbits. Based on the above results, X-type sPLA was found in kidney cancer.₂It was suggested that the protein expression was remarkably high.
Example 11 Anti-X type sPLA ₂ Immunohistochemical analysis of human gallbladder tissue using antibodies
Specimens of healthy adult gallbladder tissue and lung tissue from patients with gallbladder cancer were obtained from Biochain Inc. (San Leandro, CA). Tissue section slides are freed of paraffin wax and 0.3% H₂O₂After removing endogenous peroxidase by reacting with methanol containing for 30 minutes, it was treated with 5% normal goat serum for 20 minutes. Thereafter, anti-X sPLA in PBS containing 0.1% bovine serum albumin₂The mixture was reacted with the antibody (6 μg / mL) for 14 hours at 4 ° C. After washing with PBS, it was reacted with a biotin-conjugated goat anti-rabbit IgG antibody for 30 minutes and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 μg / mL diaminobenzidine and 0.006% H₂O₂X-type sPLA in a tissue specimen colored with peroxidase activity by reacting for 10 minutes in a 50 mmol / L Tris-HCl (pH 7.6) buffer solution containing₂Was visualized. Moreover, the cell nucleus was counterstained by reaction with 0.4% hematoxylin aqueous solution. X type sPLA₂Positive signals were detected as dark brown diaminobenzidene deposits. X type sPLA₂Signal neutralization experiments were performed using purified X-type sPLA prior to addition of antibody to the slide.₂The reaction was performed by reacting with protein (60 μg / mL) for 2 hours.
As a result, X-type sPLA₂The positive signal was hardly detected in normal gallbladder tissue, and was strongly detected in cancer cells in gallbladder cancer-derived gallbladder tissue. These signals are X-type sPLA₂X-type sPLA disappeared due to the addition of protein₂It was confirmed to be specific. Also, these positive signals were not observed with IgG prepared from non-immunized rabbits. From the above results, X-type sPLA was found in gallbladder cancer₂It was suggested that the protein expression was remarkably high.
Example 12 Anti-X type sPLA ₂ Immunohistochemical analysis of human prostate tissue using antibodies
Specimens of healthy adult prostate tissue as well as prostate tissue from prostate cancer patients are available from Biochain Inc. (San Leandro, CA). Tissue section slides are freed of paraffin wax and 0.3% H₂O₂After removing endogenous peroxidase by reacting with methanol containing for 30 minutes, it was treated with 5% normal goat serum for 20 minutes. Thereafter, anti-X sPLA in PBS containing 0.1% bovine serum albumin₂The mixture was reacted with the antibody (6 μg / mL) for 14 hours at 4 ° C. After washing with PBS, it was reacted with a biotin-conjugated goat anti-rabbit IgG antibody for 30 minutes and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 μg / mL diaminobenzidine and 0.006% H₂O₂X-type sPLA in a tissue specimen colored with peroxidase activity by reacting for 10 minutes in a 50 mmol / L Tris-HCl (pH 7.6) buffer solution containing₂Was visualized. Moreover, the cell nucleus was counterstained by reaction with 0.4% hematoxylin aqueous solution. X type sPLA₂Positive signals were detected as dark brown diaminobenzidene deposits. X type sPLA₂Signal neutralization experiments were performed using purified X-type sPLA prior to addition of antibody to the slide.₂The reaction was performed by reacting with protein (60 μg / mL) for 2 hours.
As a result, X-type sPLA₂The positive signal was hardly detected in normal prostate tissue, and was strongly detected in cancer cells in prostate cancer patient-derived prostate tissue. These signals are X-type sPLA₂X-type sPLA disappeared due to the addition of protein₂It was confirmed to be specific. Also, these positive signals were not observed with IgG prepared from non-immunized rabbits. From the above results, X-type sPLA is found in prostate cancer₂It was suggested that the protein expression was remarkably high.
Example 13 Anti-X type sPLA ₂ Immunohistochemical analysis of human pancreatic tissue using antibodies
Samples of healthy adult pancreatic tissue as well as pancreatic tissue from patients with pancreatic cancer were obtained from Biochain Inc. (San Leandro, CA). Tissue section slides are freed of paraffin wax and 0.3% H₂O₂After removing endogenous peroxidase by reacting with methanol containing for 30 minutes, it was treated with 5% normal goat serum for 20 minutes. Thereafter, anti-X sPLA in PBS containing 0.1% bovine serum albumin₂The mixture was reacted with the antibody (6 μg / mL) for 14 hours at 4 ° C. After washing with PBS, it was reacted with a biotin-conjugated goat anti-rabbit IgG antibody for 30 minutes and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 μg / mL diaminobenzidine and 0.006% H₂O₂X-type sPLA in a tissue specimen colored with peroxidase activity by reacting for 10 minutes in a 50 mmol / L Tris-HCl (pH 7.6) buffer solution containing₂Was visualized. Moreover, the cell nucleus was counterstained by reaction with 0.4% hematoxylin aqueous solution. X type sPLA₂Positive signals were detected as dark brown diaminobenzidene deposits. X type sPLA₂Signal neutralization experiments were performed using purified X-type sPLA prior to addition of antibody to the slide.₂The reaction was performed by reacting with protein (60 μg / mL) for 2 hours.
As a result, X-type sPLA₂The positive signal was hardly detected in normal pancreatic tissue, and was strongly detected in cancer cells in pancreatic cancer patient-derived pancreatic tissue. These signals are X-type sPLA₂X-type sPLA disappeared due to the addition of protein₂It was confirmed to be specific. Also, these positive signals were not observed with IgG prepared from non-immunized rabbits. Based on the above results, X-type sPLA in pancreatic cancer₂It was suggested that the protein expression was remarkably high.
Example 14 Anti-X-type sPLA ₂ Immunohistochemical analysis of human cerebral tissue using antibodies
Specimens of healthy adult cerebral tissue and cerebral tissue from Alzhama patients are available from Biochain Inc. (San Leandro, CA). Tissue section slides are freed of paraffin wax and 0.3% H₂O₂After removing endogenous peroxidase by reacting with methanol containing for 30 minutes, it was treated with 5% normal goat serum for 20 minutes. Thereafter, anti-X sPLA in PBS containing 0.1% bovine serum albumin₂The mixture was reacted with the antibody (6 μg / mL) for 14 hours at 4 ° C. After washing with PBS, it was reacted with a biotin-conjugated goat anti-rabbit IgG antibody for 30 minutes and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 μg / ml diaminobenzidine and 0.006% H₂O₂X-type sPLA in a tissue specimen colored with peroxidase activity by reacting for 10 minutes in a 50 mmol / L Tris-HCl (pH 7.6) buffer solution containing₂Was visualized. Moreover, the cell nucleus was counterstained by reaction with 0.4% hematoxylin aqueous solution. X type sPLA₂Positive signals were detected as dark brown diaminobenzidene deposits. X type sPLA₂Signal neutralization experiments were performed using purified X-type sPLA prior to addition of antibody to the slide.₂The reaction was performed by reacting with protein (60 μg / mL) for 2 hours.
As a result, X-type sPLA₂The positive signal is hardly detected in normal cerebral tissue, but is strongly detected in a part of nerve cells in cerebral tissue derived from Alzheimer's patients, particularly in senior citizens and neurofibrillar tangential regions. It was. These signals are X-type sPLA₂X-type sPLA disappeared due to the addition of protein₂It was confirmed to be specific. Also, these positive signals were not observed with IgG prepared from non-immunized rabbits. From the above results, X-type sPLA was observed in the Alzheimer cerebrum.₂It was suggested that the protein expression was remarkably high.
Example 15 Anti-X type sPLA ₂ Immunohistochemical analysis of human cirrhosis tissue using antibodies
Samples of healthy adult liver tissue as well as liver tissue from cirrhosis patients were obtained from Biochain Inc. (San Leandro, CA). Tissue section slides are freed of paraffin wax and 0.3% H₂O₂After removing endogenous peroxidase by reacting with methanol containing for 30 minutes, it was treated with 5% normal goat serum for 20 minutes. Thereafter, anti-X sPLA in PBS containing 0.1% bovine serum albumin₂The mixture was reacted with the antibody (6 μg / mL) for 14 hours at 4 ° C. After washing with PBS, it was reacted with a biotin-conjugated goat anti-rabbit IgG antibody for 30 minutes and treated with a peroxidase-containing avidin-biotin complex reagent (Vector Laboratories). After washing, 200 μg / ml diaminobenzidine and 0.006% H₂O₂X-type sPLA in a tissue specimen colored with peroxidase activity by reacting for 10 minutes in a 50 mmol / L Tris-HCl (pH 7.6) buffer solution containing₂Was visualized. Moreover, the cell nucleus was counterstained by reaction with 0.4% hematoxylin aqueous solution. X type sPLA₂Positive signals were detected as dark brown diaminobenzidene deposits. X type sPLA₂Signal neutralization experiments were performed using purified X-type sPLA prior to addition of antibody to the slide.₂The reaction was performed by reacting with protein (60 μg / mL) for 2 hours.
As a result, X-type sPLA₂The positive signal was detected at a low level in liver lobule and Kupffer stellate cells in normal liver tissue, but in liver tissue derived from cirrhosis patients, marked expression enhancement was observed in pseudolobule. These signals are X-type sPLA₂X-type sPLA disappeared due to the addition of protein₂It was confirmed to be specific. Also, these positive signals were not observed with IgG prepared from non-immunized rabbits. From the above results, X-type sPLA in cirrhotic tissues₂It was suggested that the protein expression was remarkably high.
Example 16 Mouse X-type sPLA ₂ Of the gene coding for
X type sPLA₂Human X-type sPLA for the isolation of mouse homologues₂Two types of primers were designed based on the sequence, and PCR was performed using mouse thymus cDNA as a template.

PCR was performed for 40 cycles under the conditions of 92 ° C. for 1 minute, 58 ° C. for 1 minute, and 72 ° C. for 1 minute.
Next, the mouse spleen marathon-ready cDNA (Clontech) was used to clone the 3′-, 5′-ends by the rapid amplification of the cDNA ends (RACE) method, and finally using the following primers: Mouse X type sPLA₂The coding region of cDNA was obtained by PCR using mouse spleen cDNA as a template.

Mouse X type sPLA₂Consists of 151 amino acids (SEQ ID NO: 14), as reported by Valentin et al. (J. Biol. Chem., 274, 44, 31195-3202 (1999)) and human X-type sPLA₂Was estimated to have 70.9% homology. Mouse X type sPLA₂Is human X-type sPLA₂Like the above, it has a prepropeptide consisting of 28 amino acids at the N-terminus and an extended structure at the C-terminus. However, mouse X type sPLA₂Is human X-type sPLA₂In contrast, it did not have an N-linked glycosylation site.
Example 17 Mouse Pro-type and Active X-type sPLA ₂ Enzyme activity
Mouse X type sPLA₂Was expressed and purified in the same manner as described in Example 4.
Purified pro-type and active X-type sPLA₂The enzyme activity of was measured using 1-palmitoyyl-2-linoleoyl-phosphophatideylcholine (PLPC) as a substrate. As shown in FIG. 2, the professional X-type sPLA₂Had only 8% activity compared to the active form. Since Arg is present at the C-terminus of the propeptide, it was speculated that trypsin-like endoprotease may be involved in the cleavage. Therefore, professional type X type sPLA₂As a result of digestion with trypsin and analysis by SDS-PAGE, conversion to the active form was confirmed by trypsin treatment.₂The activity increased to about 5 times (FIG. 2). From the above analysis, it was found that the propeptide portion was cleaved and removed from the pro-type by endoprotease.₂It was suggested that it is indispensable for the expression of maximum activity.
Example 18 Anti-X sPLA ₂ Preparation of propeptide antibody and its measurement system
X type sPLA₂Propeptides were synthesized by a peptide synthesizer based on the amino acid sequence information taught by the present invention. The obtained propeptide was combined with bovine thyroglobulin as an immunogen, and rabbits were immunized to prepare antiserum. The IgG fraction was purified using HiTrap protein G-column (Amersham Pharmacia Biotech). Specificity and titer of the antiserum and the purified antibody were measured by the following ELISA method. X-type sPLA combined with bovine serum albumin₂A 96-well plate (Nunc, Immuno MaxiSorp plate) coated with propeptide was prepared, and after blocking nonspecific adsorption with 1% bovine serum albumin solution, anti-human X type sPLA₂Propeptide antibody was added and reacted. After washing with PBS containing 0.05% Tween 20 (Phosphate-buffered saline, pH 7.4), it was further reacted with a peroxidase-conjugated goat anti-rabbit IgG antibody (Cappel), and TBMplus (DAKO) was added for color development. The coloring reaction was terminated by adding 0.5 mol / L sulfuric acid, and the coloring intensity was measured at a wavelength of 450 nm.
The anti-X type sPLA thus obtained₂The propeptide antibody is a pro type X sPLA in the ELISA system shown in Example 3.₂And activated X-type sPLA₂And only the propeptide was recognized. Using this antiserum, propeptide was measured by the following ELISA system first method. Propeptides were reacted with EZ-Link Sulfo-NHS-LC-LC-Biotin (Pierce) and separated by reverse phase HPLC to prepare biotinylated propeptides. A 96-well plate coated with avidin was prepared, non-specific adsorption was blocked with 1% bovine serum albumin solution, biotinylated propeptide was added and allowed to react for 2 hours. Anti-X-type sPLA after washing with PBS (Phosphate-buffered saline, pH 7.4) containing 0.05% Tween20₂The propeptide antibody and the sample or standard solution to be measured were added simultaneously and allowed to react for 18 hours. After washing, it was further reacted with a peroxidase-conjugated goat anti-rabbit IgG antibody (Cappel), and after washing, TBMplus (DAKO) was added to cause color development. The color reaction was terminated by adding 1N sulfuric acid, and the color intensity was measured at a wavelength of 450 nm. Absorbance B obtained with 0 ng / mL standard solution₀The percentage B / B of absorbance B obtained with the standard solution of each concentration with respect to₀% Is calculated and a standard curve (FIG. 3) is prepared, and B / B obtained from the absorbance obtained from an unknown sample using the standard curve.₀The propeptide concentration contained in the unknown sample was calculated from%. Propeptides were also measured by the following second ELISA method. X-type sPLA combined with bovine serum albumin₂After preparing a 96-well plate coated with propeptide and blocking nonspecific adsorption with 1% bovine serum albumin solution, anti-X type sPLA₂The propeptide antibody and the sample or standard solution to be measured were added simultaneously and allowed to react for 18 hours. After washing, a color development reaction was performed in the same manner as in the first method, and the propeptide concentration contained in the unknown sample was calculated.
Example 19 Anti-X type sPLA ₂ Immunohistochemical analysis of human colon cancer tissue using propeptide antibody
In this test, an anti-human X-type sPLA2 propeptide antibody was used. Samples of colon tissue from healthy adult colon cancer and colon cancer patients were obtained from Biochain Inc. (San Leandro, CA). Tissue section slides are freed of paraffin wax and 0.3% H₂O₂After removing endogenous peroxidase by reacting with methanol containing for 30 minutes, it was treated with 5% normal goat serum for 20 minutes. Then, anti-human X-type sPLA in an aqueous PBS solution containing 0.1% bovine serum albumin₂(Rabbit) It was reacted with a propeptide antibody (24 μg / mL) for 14 hours at 4 ° C. After washing with a PBS aqueous solution containing 0.1% Polyoxyethylene (20) sorbitan monolaurate (Tween 20; Wako Pure Chemical Industries, Ltd. Osaka) for 30 minutes, a biotin-conjugated goat anti-rabbit IgG antibody was reacted for 30 minutes. Reagent (DAKO Corporation, Carpinteria, CA) was treated for 15 minutes. After washing with an aqueous PBS solution, a biotin-labeled tyramide solution (DAKO Corporation, Carpinteria, Calif.) Was reacted for 15 minutes, and then thoroughly washed with an aqueous PBS solution containing 0.1% Tween 20. Further, the peroxidase-labeled streptavidin solution was treated for 15 minutes and similarly washed thoroughly with a PBS aqueous solution containing 0.1% Tween20. After washing, 200 μg / mL diaminobenzidine and 0.006% H₂O₂Peroxidase activity by reacting for 10 minutes in a 50 mmol / L Tris-HCl (pH 7.6) buffer solution containing X-type sPLA in a tissue specimen₂Propeptide was visualized. Moreover, the cell nucleus was counterstained by reaction with 0.4% hematoxylin aqueous solution. X type sPLA₂A propeptide positive signal was detected as a brown deposit of diaminobenzidene. The absorption neutralization experiment of the propeptide positive signal was performed by reacting with propeptide-bound bovine serum albumin (300 μg / mL) for 2 hours before adding the antibody to the slide.
As a result, X-type sPLA₂Propeptide positive signal was hardly detected in normal colon tissue, but was strongly detected in cancer cells in colorectal adenocarcinoma tissue. Since these signals disappeared by addition of propeptide-bound bovine serum albumin before antibody treatment to the tissue, X-type sPLA₂It was confirmed to be specific for the propeptide. Also, these positive signals were not observed with IgG prepared from non-immunized rabbits. From the above results, pro-type X-type sPLA in colorectal cancer₂From these results, it was suggested that the amount of propeptide released with the activation increased remarkably.
Example 20 Human Active Type X sPLA ₂ Of mouse monoclonal antibodies against
Based on the amino acid sequence information taught by the present invention, human active X-type sPLA₂A peptide containing the N-terminal sequence (GILEL AGGGC: SEQ ID NO: 16) was synthesized, and the mouse was immunized with the peptide bound to KLH as an immunogen. The increase in antibody titer was measured by the following PEG method. X-type sPLA labeled with mouse anti-plasma and radioiodine in tubes₂Protein was added and reacted at 4 degrees for 18 hours or longer. After the reaction, 1% bovine gamma globulin solution and 25% PEG6000 solution were added, and the radioactivity precipitated by binding to the antibody by centrifugation was measured with a gamma counter.
Spleen cells were prepared from the mouse with the highest antibody titer, and cell fusion was performed with mouse myeloma cells in the presence of PEG1500. The produced antibody-producing hybridoma was detected by the RIA method shown below. A goat anti-mouse IgG antibody was solid-phased on a 96-well plate, non-specific adsorption was blocked with Block Ace (Dainippon Pharmaceutical Co., Ltd.), and then the hybridoma culture supernatant was reacted to capture the contained monoclonal antibody. X-type sPLA labeled with radioiodine after washing₂The protein was reacted and the radioactivity remaining after washing was measured. Cloning of the detected hybridoma by limiting dilution is repeated, and active X-type PLA₂A mouse monoclonal antibody-producing hybridoma strain 3C10 that reacts with the above was established. The hybridoma was commissioned on December 12, 2000 as FERM BP-7393 (trust number) at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan). Has been.
As a result of examining the specificity of the 3C10 antibody in the above PEG method, X-type sPLA₂When a propeptide and a peptide containing the amino acid sequence from position -11 to position 10 in the amino acid sequence shown in SEQ ID NO: 3 were added, no decrease in the binding radioactivity was observed, and X-type sPLA containing the active form₂Since the inhibition of binding was observed only when the protein was added, the antibody was activated X-type sPLA₂Was specifically recognized (FIG. 4).
Example 21 X-type sPLA ₂ Preparation of mouse monoclonal antibody against propeptide
Based on the amino acid sequence information taught by the present invention, X-type sPLA₂A peptide containing a propeptide (EASRI LRVHR RC: SEQ ID NO: 17) was synthesized and conjugated to KLH as an immunogen, and mice were immunized. The increase in antibody titer was measured by the following ELISA method. A goat anti-mouse IgG antibody was immobilized on a 96-well plate, non-specific adsorption was blocked with Block Ace (Dainippon Pharmaceutical Co., Ltd.), and mouse anti-plasma was reacted to capture the contained antibody. After washing, a biotinylated peptide containing the amino acid sequence at positions -11 to 10 in the amino acid sequence described in SEQ ID NO: 3 was reacted. After washing, it was further reacted with peroxidase-labeled streptavidin (Pierce), finally added with DAKO TMB One-Step Substrate System (DAKO) to develop color, and the color intensity was measured at a wavelength of 450 nm.
Spleen cells were prepared from the mouse with the highest antibody titer, and cell fusion was performed with mouse myeloma cells in the presence of PEG1500. The produced antibody-producing hybridoma was detected by the above-described ELISA method. Cloning of the detected hybridoma by limiting dilution was repeated, and X-type sPLA₂A mouse monoclonal antibody-producing hybridoma strain HPX-6E1 that reacts with propeptide was established. The hybridoma was commissioned on April 17, 2001 as FERM BP-7547 (trusted number) to the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan). Has been.
As a result of examining the specificity of the HPX-6E1 antibody in the above-mentioned ELISA method, X-type sPLA containing the active form₂When protein is added, no decrease in absorbance is observed, and X-type sPLA₂Inhibition of binding was observed only when a propeptide was added, and this antibody was converted to X-type sPLA.₂It was shown that the propeptide was specifically recognized (FIG. 5).
Example 22 Pro-type and active human X-type sPLA ₂ Purification
Active X-type sPLA established as described above₂The specific monoclonal antibody 3C10-producing hybridoma was cultured in large quantities and then transplanted into the abdominal cavity of nude mice to produce ascites. The obtained ascites was added to a HiTrap Protein G column (Amercham Pharmacia), washed and eluted with a 10 mM citrate buffer (pH 3.0) to obtain a purified antibody fraction.
X type sPLA₂The propeptide-specific monoclonal antibody HPX-6E1 was cultured in a large amount in a serum-free medium, and the culture supernatant was directly added to a HiTrap Protein G column and eluted under the above conditions to obtain a purified antibody.
About each obtained purified antibody, it solid-phased on HiTrap NHS-Activated (Amersham Pharmacia), and produced the 3C10 antibody bridge | crosslinking column and the HPX-6E1 antibody bridge | crosslinking column.
Using these 3C10 antibody column and HPX-6E1 antibody column, activated X-type sPLA₂And professional type X type sPLA₂Was isolated. CHO cell expression recombinant X type sPLA described in Example 4 of the present invention₂The protein was added to the 3C10 antibody column or HPX-6E1 antibody column, washed, and then eluted with 0.1 M phosphate buffer (pH 12) containing 0.3 M NaCl. As a result of analyzing the eluted fraction by SDS-PAGE, the active form was recovered in the 3C10 antibody column adsorption fraction, and the pro form was recovered in the HPX-6E1 antibody column adsorption fraction.
The 3C10 antibody column adsorbed fraction contained a small amount of pro-type, and the HPX-6E1 antibody column adsorbed fraction contained a small amount of active protein. According to the image analysis of the electrophoresis image, the ratio was 4 respectively. 0.0% and 8.7%.
Example 23 Active X-type sPLA ₂ Establishment of specific ELISA system
Anti-X sPLA described in Example 3 of the present invention₂The IgG fraction produced from the antibody is solid-phased in a 96-well plate, non-specific adsorption is blocked with Block Ace (Dainippon Pharmaceutical), and then purified using an antibody column.₂Or professional type X type sPLA₂X-type sPLA contained in the solution₂Captured. After washing, biotinylated 3C10 antibody was reacted. After washing, it was further reacted with peroxidase-labeled streptavidin (Pierce), finally added with DAKO TMB One-Step Substrate System (DAKO) to develop color, and the color intensity was measured at a wavelength of 450 nm.
As a result, active X-type sPLA was used as a specimen.₂Strong color development was observed, and the absorbance obtained was the active X-type sPLA₂Proportional to concentration. However, professional type X type sPLA₂Is used, the activated X-type sPLA₂The reactivity obtained was weak compared to the case of using as an antigen, and was detected as a cross-reactivity of about 5%. However, as described above, the active fraction was slightly mixed (8). .7%) from this ELISA type active PLA₂It can be judged that the specificity for is extremely high (FIG. 6).
Example 24 Pro-type sPLA ₂ Establishment of specific ELISA system
HPX-6E1 purified antibody is solid-phased on a 96-well plate, non-specific adsorption is blocked with Block Ace (Dainippon Pharmaceutical Co., Ltd.), and various concentrations of activated X-type sPLA are used as samples.₂Or professional type X type sPLA₂SPLA contained in the solution₂Captured. After washing, biotinylated anti-X type sPLA₂The antibody IgG fraction was reacted. After washing, it was further reacted with peroxidase-labeled streptavidin (Pierce), finally added with DAKO TMB One-Step Substrate System (DAKO) to develop color, and the color intensity was measured at a wavelength of 450 nm.
As a result, the pro-type X-type sPLA as a specimen₂When using, strong color development was observed, and the absorbance obtained was pro-type X-type sPLA₂Proportional to concentration. Active X-type sPLA₂Is used, professional type X type sPLA₂The reactivity obtained was weak compared to the case of using an antigen as an antigen, and was detected as a cross-reactivity of about 4%, but a slightly mixed pro-type was observed in the active fraction (4.0%). Therefore, this ELISA-based professional X-type sPLA₂It can be judged that the specificity for is extremely high (FIG. 7).
Example 25 Serum X-type sPLA ₂ Detection considerations
Active and pro-type X-type sPLA using monoclonal antibodies₂Nonspecific X-type sPLA using specific ELISA system and polyclonal antibody₂X-type sPLA in normal serum and colorectal cancer patient serum using measurement system₂Concentration was measured.
X-type sPLA in 5 normal serums₂X-type sPLA in 6 sera of colorectal cancer patients when the average concentration is 1.₂The concentration is as shown in FIG. 8, and the active X-type sPLA₂The specific measurement system was able to detect specimens showing high values in the colorectal cancer patient group. In contrast, the professional type X type sPLA₂In the specific measurement system and the type non-specific measurement system, high-value samples in the patient group could not be detected. This result shows that the activated X-type sPLA produced in the present invention₂Active X-type sPLA using specific antibodies₂The specific measurement system has a possibility of being clinically useful in the diagnosis of colorectal cancer (FIG. 8).
Industrial applicability
According to the present invention, X-type sPLA₂An antibody that specifically recognizes a part of the protein; active X-type sPLA using the antibody₂A diagnostic kit containing the antibody is provided.
By using the measurement method of the present invention, X-type sPLA₂Is possible to identify diseases that are specifically involved, and X-type sPLA₂Kits for diagnosis of specific diseases caused by cancer, especially colon cancer, lung cancer, liver cancer, stomach cancer, kidney cancer, gallbladder cancer, prostate cancer, pancreatic cancer, testicular cancer, ovarian cancer, skin cancer, Alzheimer's disease, cirrhosis, etc. The
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows a pre-pro type X type sPLA₂, Pro type X type sPLA₂And activated X-type sPLA₂It is a figure which shows the relationship.
FIG. 2 shows mouse activated X-type sPLA₂, Mouse pro type X type sPLA₂And trypsin digested pro type X sPLA₂It is a figure which shows the enzyme activity. In the figure, M, P and P + T are pro-form X-type sPLA digested with active form, pro-form and trypsin, respectively.₂Means.
FIG. 3 shows the anti-X type sPLA of the present invention.₂The standard curve in ELISA method using a propeptide antibody is shown.
FIG. 4 shows the active X-type sPLA of the present invention.₂It is a figure which shows the specificity of a monoclonal antibody (3C10).
FIG. 5 shows the specificity of the propeptide monoclonal antibody (HPX-6E1) of the present invention.
FIG. 6 shows the activated X-type sPLA of the present invention.₂It is a figure which shows the standard curve property and specificity of ELISA system using a monoclonal antibody (3C10).
FIG. 7 is a diagram showing the standard curvilinearity and specificity of an ELISA system using the propeptide monoclonal antibody (HPX-6E1) of the present invention.
FIG. 8 is a diagram showing measurement results of samples derived from colorectal cancer patients using various measurement systems.

Claims (8)

A monoclonal antibody that specifically recognizes a polypeptide consisting of amino acids from Glu at position -11 to Arg at position -1 in the amino acid sequence described in SEQ ID NO: 3, which is produced by a hybridoma having a deposit number of FERM BP-7547. A monoclonal antibody that specifically recognizes a polypeptide consisting of amino acids from Gly at position 1 to Asp at position 123 in the amino acid sequence described in SEQ ID NO: 3, which is produced by a hybridoma having an accession number of FERM BP-7393 . In the amino acid sequence described in SEQ ID NO: 3, a polypeptide consisting of amino acids from Glu at position -11 to Arg at position -1 or a polypeptide consisting of amino acids from Gly at position 1 to Asp at position 123 is specifically recognized. A kit for diagnosing cancer , comprising an antibody . The cancer diagnostic kit according to claim 3 , wherein the cancer is colon cancer, lung cancer, liver cancer, stomach cancer, kidney cancer, gallbladder cancer, prostate cancer, pancreatic cancer, testicular cancer, ovarian cancer, or skin cancer. In the amino acid sequence described in SEQ ID NO: 3, a polypeptide consisting of amino acids from Glu at position -11 to Arg at position -1 or a polypeptide consisting of amino acids from Gly at position 1 to Asp at position 123 is specifically recognized. A diagnostic kit for Alzheimer's disease comprising an antibody . In the amino acid sequence described in SEQ ID NO: 3, a polypeptide consisting of amino acids from Glu at position -11 to Arg at position -1 or a polypeptide consisting of amino acids from Gly at position 1 to Asp at position 123 is specifically recognized. A kit for diagnosing liver cirrhosis , comprising an antibody . A polypeptide comprising an amino acid from Glu at position -11 to Arg at -1 position or a polypeptide comprising amino acid from Gly at position 1 to Asp at position 123 in the amino acid sequence described in SEQ ID NO: 3 And screening method for cancer, Alzheimer's disease or cirrhosis. The screening method according to claim 7 , wherein the cancer is colon cancer, lung cancer, liver cancer, stomach cancer, kidney cancer, gallbladder cancer, prostate cancer, pancreatic cancer, testicular cancer, ovarian cancer, or skin cancer.

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