JP3850143B2 - Immune analysis elements - Google Patents

Description

【００８７】
この試薬層の表面に下記の被覆量になるように下記試薬含有水溶液を塗布し、ゼラチン層を膨潤させ、その上に50デニール相当のPET 紡績糸36ゲージ編みした厚さ約250μｍのトリコット編物布地をほぼ一様に軽く圧力をかけてラミネートして多孔性展開層を設けた。

【００８８】
次に、実施例１で得られたエンド型酵素反応特異的なカルボキシメチル化澱粉を用いて、下記の被覆量になるように基質を塗布、乾燥して基質層を設けてＣＲＰ分析用多層分析要素を調製した。
カルボキシメチル化澱粉 3.5 g/m²
マンニトール 3.0 g/m²
ＭＥＳ（2-モルホリノエタンスルホン酸） 2.0 g/m²
【００８９】
次いでこの分析要素を14mm×13mm四方のチップに裁断し、特開昭57-63452に記載のスライドの枠に収めて、本実施例のＣＲＰ分析用多層乾式スライド１とした。
【００９０】
比較例として、エンド型酵素反応特異的なカルボキシメチル化澱粉の代わりに、未処理のカルボキシメチル化澱粉（エキスプロタブ）を基質として用いた他は、実施例スライド１と全く同様な構成の比較用スライド２を作製した。
【００９１】
【実施例３】
50mMグリセロ燐酸緩衝溶液（pH7）の10μLを実施例２の乾式スライド１及び、比較例の乾式スライド２に点着した。この後、各スライドを37℃に保って、PET支持体側から中心波長650nmの反射光学濃度を経時的に測定した。点着後の反射光学濃度の時間変化を図４に示す。
【００９２】
未処理のカルボキシメチル化澱粉を非拡散性基質として使用した比較例のスライド２では、緩衝溶液点着後から経時的に反射光学濃度の増加が見られる（図中、-○-）。このことは、未処理カルボキシメチル化澱粉が、試薬層から移行したエキソ反応型酵素であるグルコアミラーゼ（低分子化酵素）により分解されて、グルコースを生成していることを示している。
【００９３】
一方、予めグルコアミラーゼ処理によりエンド型酵素反応特異的にしたカルボキシメチル化澱粉を使用した実施例２のスライド１では、反射光学濃度は一定値で増加することはなかった。（図４、-●-）。
【００９４】
【実施例４】
アミラーゼ標識抗ＣＲＰ−ＩｇＧ(0.1mg/mL）を含み、既知量のＣＲＰを含有する50mMグリセロ燐酸緩衝溶液（pH7）10μLを、実施例２のスライド１と、比較用スライド２に点着した。37℃に保って、中心波長650nmの可視光でＰＥＴ支持体側から各スライド１，２の反射光学濃度を測定した。点着から４分後および６分後の反射光学濃度の差（ΔＯＤ_6-4）を図５に示す。図５の検量線より、エンド型酵素反応特異的にしたカルボキシメチル化澱粉を使用した本発明の乾式免疫分析要素（図５，-●-）は、ＣＲＰの定量が精度良く行えることが明らかである。また比較例（図５、-○-）に比べ、ＣＲＰ濃度変化に対するΔＯＤ_6-4の変化が大きく、感度が上昇していた。
【００９５】
【実施例５】
実施例２と全く同様の手順、構成で、多層分析要素を作成し、その基質層兼展開層であるトリコット編物布地層に、さらにアミラーゼ標識ＣＲＰ−ＩｇＧを３mg/m²の被覆量となるようにしてエタノール溶液を塗布し含浸させ乾燥させてＣＲＰ分析用多層免疫スライド３（実施例５）を作成した。同様に、実施例２で作成した比較用スライド２の多層分析要素のトリコット編物布地層に、アミラーゼ標識ＣＲＰ−ＩｇＧを３mg/m²の被覆量となるようにしてエタノール溶液を塗布し含浸させ乾燥させて比較用のＣＲＰ分析用多層免疫スライド４を作成した。
【００９６】
【実施例６】
このスライド３及びスライド４に、各種濃度のＣＲＰを含有する50mMグリセロ燐酸緩衝溶液(pH7)を10μLを点着し、37℃に保って、支持体側から650nmの反射光学濃度を測定し、点着から４分後および６分後の反射光学濃度の差（ΔＯＤ_6-4）を求めた。図６の検量線に示すように、エンド型反応特異的カルボキシメチル化澱粉を使用した本発明の乾式免疫分析要素（図６、-●-）は、比較例（-○-）に比べ、より高感度にＣＲＰを定量分析できることが示された。
【００９７】
【実施例７】
実施例５のスライド３と比較用スライド４の再現性（ＣＶ）を比較検討した。スライド３及びスライド４に、ＣＲＰ含有ヒト標準血清４検体（Ｌ-1,Ｌ-2,Ｍ、Ｈ）を10μLを点着し、37℃に保って、支持体側から650nmの反射光学濃度を測定し、点着から４分後および６分後の反射光学濃度の差（ΔＯＤ_6-4）を求めた。実施例５のスライド３の場合の検量線と、比較用スライド４の場合の検量線を別途求めておき、得られた各検体のΔＯＤ_6-4からＣＲＰ濃度（測定値）を換算した。この測定を各検体について１０回行い、その平均値、標準偏差及びＣＶ値を求めた。結果を下記の表１，２に示す。
【００９８】
【表１】

【００９９】
【表２】

【０１００】
表１，２に示すように、従来の比較用スライド４では、測定値（濃度）のバラツキが大きいのに対して、本発明のエンド反応特異的基質を使用した実施例５のスライド３はバラツキが小さく、同時再現性に優れていた。特に低濃度域（検体Ｌ−１，Ｌ−２）と高濃度域（検体Ｈ）において、実施例スライド３のＣＶ値は比較用スライド４のそれに比べ１／３程度まで減少し、本発明による免疫分析要素は再現性に優れ精度が高いことが示された。
【０１０１】
【実施例８】
実施例５のスライド３と比較用スライド４の保存安定性を比較検討した。乾式分析要素は通常４℃程度で１ヶ月間程度安定であるが、ここでは加速試験として２５℃でスライドを保存し、スライド作製後１日、３日及び７日目の測定値をスライド作製直後（０日）の測定値と比較した。作製後各日におけるスライド３（実施例５）及びスライド４（比較例）にＣＲＰ含有標準試料ＣＰ１(1.4mg/dL)、 ＣＰ２(4.2mg/dL)、ＣＰ３(10.0mg/dL)をそれぞれ10μL点着し、37℃に保って、支持体側から650nmの反射光学濃度を測定し、点着から４分後および６分後の反射光学濃度の差（ΔＯＤ_6-4）を求めた。結果を下記の表３，４に示す。
【０１０２】
【表３】

【０１０３】
【表４】

【０１０４】
従来の未処理のカルボキシメチル化澱粉を基質としたスライド４（比較例）では、２５℃保存により日が経つにつれΔＯＤ_6-4の値は減少しており、３日目には５〜10％の減少が見られ、７日目には15〜20％程度の大きな減少が見られた。これに対し、本発明によるエンド型反応特異的基質としたカルボキシメチル化澱粉を使用したスライド３（実施例５）では、ΔＯＤ_6-4値の変動は３日目では２〜４％の減少とわずかであり、保存７日目でも３〜５％程度の減少しか認められなかった。このように本発明による免疫分析要素は保存安定性に優れていることが示された。
【０１０５】
【実施例９】
エンド型選択反応性基質の調製（その２）
カルボキシメチル化澱粉（エキスプロタブ、Edward Mendel Company Inc.製）453ｇに8960ｇの超純水を加え、約４時間攪拌して膨潤させた。これに1139ｇの0.5 N NaOH を加えて約１時間攪拌することによりアルカリ処理した。この後、酢酸32ｇを添加してpH７に戻した。これをＭＥＳ(2-モノホリノエタンスルホン酸)緩衝液でpH5.7に調整した後、80ｇのグルコアミラーゼ酵素液（東洋紡製、320 k Unit/Ｌ, 5mM MES, pH6.0）を添加し、37℃で12〜20時間反応させた。反応後、純水を加えて総量を140Ｌにしてから、この懸濁液をセラミック膜フィルター（日本ガイシ製、CEFILT-MF(登録商標)、モノリスタイプ、孔径2μm、膜面積 0.24m²）で濾過した。透過した水の電気伝導度が20μS/cm以下になるまで、このセラミック膜フィルターに純水を循環させて、可溶性成分を除去した。フィルターからＣＭ化澱粉残査を集め、純水を加えて100Ｌとした。その内の約3.6 L を7500rpm、10分間の遠心にかけ、残査を集めた。これにイソプロピルアルコール約18Ｌを加えて、沈殿してきた白色物質を減圧濾過で集めた。この遠心からイソプロピルアルコール沈殿までの操作を約２６回行い、これを集めて35℃で24時間乾燥した。以上の操作により、220ｇのエンド型酵素反応特異的なグルコアミラーゼ処理ＣＭ化澱粉（以下、ＧＡ処理ＣＭＳともいう）を得た。
【０１０６】
グルコアミラーゼ処理をしなかった他は全て上記と同じに処理したＣＭ化澱粉を比較例（グルコアミラーゼ未処理ＣＭ化澱粉：以下、未処理ＣＭＳともいう）とした。
【０１０７】
【実施例１０】
実施例９で得られたＧＡ処理ＣＭＳと未処理ＣＭＳのカルボキシメチル化度と、水に分散したときの膨潤度を調べた。カルボキシメチル化度は、全グルコース単位中、カルボキシメチル基で修飾されたグルコース単位の割合を¹³Ｃ−ＮＭＲで調べた。膨潤度は、各ＣＭＳの0.7％水溶液10mLを調製し、試験管に入れて、25℃、30分間静置した後に、沈降したＣＭＳの体積（mL）を膨潤度と定義した。
【０１０８】
下記の表５に示すように、グルコアミラーゼ処理したＣＭ化澱粉ではカルボキシメチル化度が増加しており、全構成グルコース単位中の２８％がカルボキシメチル基を有することが分かった。このことはグルコアミラーゼ処理により非還元性末端からカルボキシメチル化部位の直前までグルコシド結合が水解されたため、ＣＭ化澱粉中の非ＣＭ化グルコース単位の量が減少し、結果としてＣＭ化グルコース単位の割合が増加したことを示している。また親水性基であるカルボキシメチル基の導入率が高くなったことにより、水和されやすくなり膨潤度が高まったものと考えられる。
【０１０９】
【表５】

【０１１０】
【実施例１１】
ＧＡ処理ＣＭＳのグルコアミラーゼとの反応性
実施例９で得たグルコアミラーゼ処理ＣＭ化澱粉と、比較例のグルコアミラーゼ未処理ＣＭ化澱粉を、それぞれ５mM ＭＥＳ緩衝液(pH 6.0、0.5％ブロックエース（雪印乳業社製）、68μM CaCl₂含有）に分散し、0.2％(W/V)分散液とした。この0.2％ＣＭＳ分散液に70μLのグルコアミラーゼ溶液（東洋紡製、320 k Unit/Ｌ, 5mM MES, pH6.0）を加えて37℃、60分間、120rpmで振盪させながら反応させた。反応後、10％リン酸70μLを添加し、氷冷して反応を停止させた。反応液を12,000rpmで４分遠心して未反応のＣＭＳを沈降させた。生成グルコースを含んでいる遠心上清100μLを、下記組成の反応液Ａの３mLに添加し、37℃、30分間、120rpmで振盪して発色反応を行わせ、波長727nmの吸光度を測定した。検量線は、既知濃度のグルコース溶液100μLを反応液３mLに加え、同様の発色反応を行うことにより作成した。
【０１１１】

【０１１２】
測定は、ＧＡ処理ＣＭＳについて５回、未処理ＣＭＳについて２回行った。表６に示すように、グルコアミラーゼ処理をしていないＣＭ化澱粉では、１グラム当たり約33μgのグルコースを生成したのに対し、予めグルコアミラーゼ処理したＣＭ化澱粉では、平均0.1μg以下のグルコースしか生成しなかった。このことは、本発明のＧＡ処理ＣＭＳの非還元性末端グルコースの殆どが修飾されているか糖鎖分枝点にあり、エキソ反応型酵素であるグルコアミラーゼの基質とはならないことを示している。
【０１１３】
【表６】

【０１１４】
【実施例１２】
ＧＡ処理ＣＭＳのα−アミラーゼとの反応性（湿式法）
実施例９で得たグルコアミラーゼ処理ＣＭ化澱粉と、比較例のグルコアミラーゼ未処理ＣＭ化澱粉を、それぞれ５mM ＭＥＳ緩衝液(pH 6.0、0.5％ブロックエース（雪印乳業社製）、68μM CaCl₂含有）に分散し、0.2％(W/V)分散液とした。この0.2％ＣＭＳ分散液７mLに、各種濃度のα−アミラーゼ溶液70μLを加えて37℃、60分間、120rpmで振盪させながら反応させた。反応後、10％リン酸70μLを添加し、氷冷して反応を停止させた。反応液を12,000rpmで４分遠心して未反応のＣＭＳを沈降させた。生成オリゴ糖を含んでいる遠心上清100μLを、下記組成の反応液Ｂの３mLに添加し、37℃、30分間、120rpmで振盪して発色反応を行わせ、波長727nmの吸光度を測定した。既知濃度のオリゴ糖（マルトテトラオース）溶液100μLを反応液Ｂの３mLに加え、同様の発色反応を行うことにより作成した検量線を用いて、生成オリゴ糖に換算してグラフを作成した（図７）。
【０１１５】

【０１１６】
図７に示すように、グルコアミラーゼ処理したＣＭ化澱粉では、エンド活性型酵素であるα−アミラーゼに対する反応性（図中、検量線の傾き）は、未処理のＣＭ化澱粉に比べ約半分に減少していた。しかし、未処理ＣＭＳではバックグランド値が約1.3ppmであったのに対し、ＧＡ処理ＣＭＳではバックグランド値をほぼゼロにすることができた。
【０１１７】
α−アミラーゼ量がゼロであるのに未処理ＣＭＳからの生成オリゴ糖がゼロとはならないのは以下の理由によるものと思われる。図７縦軸はオリゴ糖濃度で表現しているが、この実施例の反応系では最終的に生成するグルコース量を定量している。α−アミラーゼと反応した後の各ＣＭＳは遠心により除去しているが、微量のＣＭＳ粒子は上清に残る。生成オリゴ糖として上清を採取したときに、この微量ＣＭＳが混入すると、ＣＭＳ粒子もその後発色反応液Ｂ中に含まれるグルコアミラーゼの攻撃を受けることになる。未処理ＣＭＳでは、末端グルコースが除かれていないので、グルコアミラーゼによって末端グルコースが水解され検出されることになる。この量は使用したα−アミラーゼ量に依存せずほぼ一定なので、図７でバックグランド値として検量線全体を上に押し上げることになる。
【０１１８】
これに対し、予めグルコアミラーゼ処理したＣＭ化澱粉では、エクソ活性型酵素であるグルコアミラーゼと反応する非修飾グルコース末端が存在しない。このため、たとえＣＭＳ粒子が発色反応系に混入してもバックグランド値を押し上げることがない。
【０１１９】
ＣＭ化澱粉のα−アミラーゼに対する反応性は、グルコアミラーゼ処理により低下するのは以下の理由によるものと思われる。α−アミラーゼはグルコース鎖長４分子以上の多糖類に対して反応性を示し、重合度の高い（鎖長の長い）ものほど反応性が高い。糖鎖中のグルコースがアトランダムにカルボキシメチル化されると、α−アミラーゼが高い反応性を示すグルコース分子連続部分が減少することになる。この結果、本来、澱粉はカルボキシメチル化することによりα−アミラーゼに対する反応性が低下する。さらにグルコアミラーゼ処理すると、ＣＭ化澱粉のそれぞれの枝鎖は非還元末端から分解されて除かれる。枝鎖の途中にＣＭ化部位があれば、その部位まで非還元末端からグルコース単位で水解・除去される。このため、グルコアミラーゼ処理ＣＭ化澱粉は一種の限界デキストリンとなり、α−アミラーゼが高い反応性を示すグルコース分子連続部分は見かけ上さらに少なくなる。図７はこのことを示している。
【０１２０】
このように、α−アミラーゼに対する反応性が低下しているグルコアミラーゼ処理ＣＭ化澱粉であるが、以下の実施例１３，１４に示すように乾式分析要素内の環境では、α−アミラーゼに対する反応性が増大している。
【０１２１】
【実施例１３】
ＧＡ処理ＣＭＳのα−アミラーゼとの反応性（乾式法）
実施例９で得たグルコアミラーゼ処理ＣＭ化澱粉を用いて、実施例５と全く同様の手順、構成で、多層分析要素を作製した。
【０１２２】
すなわち、実施例２で用いたカルボキシメチル化澱粉の代わりに実施例９で得たグルコアミラーゼ処理ＣＭ化澱粉を基質層に使用した以外は実施例２と全く同様な構成のスライドを作製し、その後実施例５と同様に、その基質層兼展開層であるトリコット編物布地層に、さらにアミラーゼ標識ＣＲＰ−ＩｇＧを３mg/m²の被覆量となるようにしてエタノール溶液を塗布し含浸させ乾燥させてＣＲＰ分析用多層免疫スライド５（実施例１３）を作製した。
【０１２３】
比較例として、グルコアミラーゼ処理カルボキシメチル化澱粉の代わりに、未処理のカルボキシメチル化澱粉を基質として用いた他は、実施例１３と全く同様な構成の比較用スライド６を作製した。
【０１２４】
【実施例１４】
このスライド５及びスライド６に、0, 1.4, 4.2, 10 mg/dLの各濃度のＣＲＰを含有すると50 mMグリセロ燐酸緩衝溶液(pH7)を10μLを点着し、37℃に保って、支持体側から625 nmの反射光学濃度を経時的に測定した。ＣＲＰ溶液点着後の反射光学密度の時間変化を図８に示す。
【０１２５】
0 mg/dLのＣＲＰ溶液を点着した場合、標識抗体中のα−アミラーゼは抗原（ＣＲＰ）との結合による立体障害を受けないから、非拡散性基質であるＣＭ化澱粉に対する水解活性は最大となる。このようにα−アミラーゼが最大活性を示すＣＲＰ濃度 0 mg/dLのときの活性は、ＧＡ処理ＣＭＳを使用したスライド５（実施例１４、図８右側）では、比較例（未処理ＣＭＳを使用したスライド６、第８図左側）に比べ、大きく上昇していた。反射光学密度の計時変化の傾きを指標にすると、グルコアミラーゼ処理したＣＭ化澱粉では、未処理のものに比べ約２倍程度までα−アミラーゼに対する反応性が増大していた。
【０１２６】
抗原であるＣＲＰ濃度を増加させるに従い、α−アミラーゼに対する立体障害が増大すると、α−アミラーゼに対する反応性の低下はＧＡ処理ＣＭＳを使用したスライド５（実施例１４、図８右側）の方が著しかった。このことは、乾式分析要素内でのα−アミラーゼのＣＭ化澱粉に対する反応性は、予めグルコアミラーゼ処理してエンド活性型酵素反応特異的にすることにより、ＣＲＰ濃度変化に対する反射光学密度の変化が大きく、感度が鋭敏になることを示している。
【０１２７】
最後に本発明に好ましい態様をまとめると、以下の通りである。
(1) 抗原と酵素標識抗体との反応、もしくは抗原と抗体と酵素標識抗原との反応、もしくは抗原と、抗原と高分子化合物との結合物と、酵素標識抗体との間の反応のいずれかの反応により生じた標識酵素活性の変化を測定することにより抗原の量を分析する免疫分析要素において、
前記標識酵素により拡散性物質を生成する非拡散性基質を含有する基質層と、前記拡散性物質をさらに低分子生成物にする低分子化酵素を含有する試薬層を備え、前記非拡散性基質が前記標識酵素とのみ反応し前記低分子化酵素とは反応しない基質であることを特徴とする免疫分析要素。
(2) 前記非拡散性基質が高分子多糖類であり、前記標識酵素がエンド活性型の糖質分解酵素であり、前記変換酵素がエキソ活性型の糖質分解酵素であることを特徴とする(1)記載の免疫分析要素。
(3) 前記標識酵素はエンド活性型糖質加水分解酵素であり、前記低分子化酵素はエクソ活性型糖質加水分解酵素であって、前記非拡散性基質は非還元末端グルコースが修飾されたエンド型選択反応性基質であることを特徴とする(1)記載の免疫分析要素。
(4) 前記エンド型選択反応性基質は、非還元末端グルコースがカルボキシメチル基で修飾されていることを特徴とする(3)記載の免疫分析要素。
(5) 前記標識酵素がα−アミラーゼであり、前記低分子化酵素がグルコアミラーゼ又はα−グルコシダーゼであり、前記非拡散性基質が非還元末端グルコース部位からカルボキシメチル修飾されたグルコース単位部位まで、又はグルコース鎖分岐部位まで、予めエクソ活性型糖質加水分解酵素によって限定分解されたカルボキシメチル化澱粉であることを特徴とする(3)記載の免疫分析要素。
(6) 前記試薬層は親水性ポリマーをバインダとして含有する層で形成されていることを特徴とする(1)記載の免疫分析要素。
(7) 前記基質層は多孔性媒体からなる多孔性層であることを特徴とする(1)記載の免疫分析要素。
(8) 前記酵素標識抗体が、前記基質層、又は基質層の上に積層された層に含有されていることを特徴とする(1)記載の免疫分析要素。
(9) 前記抗体が、前記基質層、又は基質層の上に積層された層に含有されていることを特徴とする(1)記載の免疫分析要素。
(10) 前記酵素標識抗原が、前記基質層、又は基質層の上に積層された層に含有されていることを特徴とする(1)記載の免疫分析要素。
(11) 前記抗体と、前記酵素標識抗原とが、前記基質層、又は基質層の上に積層された層に含有されていることを特徴とする請求項(1)記載の免疫分析要素。
(12) 抗原と高分子化合物との前記結合物と、前記酵素標識抗体とが、前記基質層、又は基質層の上に積層された層に含有されていることを特徴とする請求項(1)記載の免疫分析要素。
(13) 前記低分子生成物と反応して可視吸収を有する色素を生成する試薬組成物を、前記試薬層又は他の水浸透性層に含有していることを特徴とする(1)記載の免疫分析要素。
(14) 前記試薬組成物が、酸化により発色するロイコ色素を含む(13)記載の免疫分析要素。
(15) 前記試薬組成物が、ロイコ色素の水不溶性溶媒からなる溶液の水性液中への分散物を含む(14)記載の免疫分析要素。
(16) 前記試薬組成物が、ペルオキシダーゼ及びロイコ色素を含む(15)記載の免疫分析要素。
(17) 抗体と酵素標識抗原との反応、もしくは抗体と抗原と酵素標識抗体との反応により生じた標識酵素活性の変化を測定することにより抗体の量を分析する免疫分析要素において、
前記標識酵素により拡散性物質を生成する非拡散性基質を含有する基質層と、前記拡散性物質をさらに低分子生成物にする低分子化酵素を含有する試薬層を備え、前記非拡散性基質が前記標識酵素とのみ反応し前記低分子化酵素とは反応しない基質であることを特徴とする免疫分析要素。
(18) 前記非拡散性基質が高分子多糖類であり、前記標識酵素がエンド活性型の糖質分解酵素であり、前記変換酵素がエキソ活性型の糖質分解酵素であることを特徴とする(17)記載の免疫分析要素。
(19) 前記標識酵素はエンド活性型糖質加水分解酵素であり、前記低分子化酵素はエクソ活性型糖質加水分解酵素であって、前記非拡散性基質は非還元末端グルコースが修飾されたエンド型選択反応性基質であることを特徴とする(17)記載の免疫分析要素。
(20) 前記エンド型選択反応性基質は、非還元末端グルコースがカルボキシメチル基で修飾されていることを特徴とする(19)記載の免疫分析要素。
(21) 前記標識酵素がα−アミラーゼであり、前記低分子化酵素がグルコアミラーゼ又はα−グルコシダーゼであり、前記非拡散性基質が非還元末端グルコース部位からカルボキシメチル修飾されたグルコース単位部位まで、又はグルコース鎖分岐部位まで、予めエクソ活性型糖質加水分解酵素によって限定分解されたカルボキシメチル化澱粉であることを特徴とする(19)記載の免疫分析要素。
(22) 前記試薬層は親水性ポリマーをバインダとして含有する層で形成されていることを特徴とする(17)記載の免疫分析要素。
(23) 前記基質層は多孔性媒体からなる多孔性層であることを特徴とする(17)記載の免疫分析要素。
(24) 前記酵素標識抗体が、前記基質層、又は基質層の上に積層された層に含有されていることを特徴とする(17)記載の免疫分析要素。
(25) 前記抗体が、前記基質層、又は基質層の上に積層された層に含有されていることを特徴とする(17)記載の免疫分析要素。
(26) 前記酵素標識抗原が、前記基質層、又は基質層の上に積層された層に含有されていることを特徴とする(17)記載の免疫分析要素。
(27) 前記抗体と、前記酵素標識抗原とが、前記基質層、又は基質層の上に積層された層に含有されていることを特徴とする請求項(17)記載の免疫分析要素。
(28) 抗原と高分子化合物との前記結合物と、前記酵素標識抗体とが、前記基質層、又は基質層の上に積層された層に含有されていることを特徴とする請求項(17)記載の免疫分析要素。
(29) 前記低分子生成物と反応して可視吸収を有する色素を生成する試薬組成物を、前記試薬層又は他の水浸透性層に含有していることを特徴とする(17)記載の免疫分析要素。
(30) 前記試薬組成物が、酸化により発色するロイコ色素を含む(29)記載の免疫分析要素。
(31) 前記試薬組成物が、ロイコ色素の水不溶性溶媒からなる溶液の水性液中への分散物を含む(30)記載の免疫分析要素。
(32) 前記試薬組成物が、ペルオキシダーゼ及びロイコ色素を含む(29)記載の免疫分析要素。
【図面の簡単な説明】
【図１】本発明の免疫分析要素の一実施態様例の構成図である。
【図２】本発明の免疫分析要素の他の実施態様例の構成図である。
【図３】本発明の免疫分析要素のさらに他の実施態様例の構成図である。
【図４】実施例４の結果を示す図であり、実施例スライド１及び比較例スライド２の免疫分析要素に標識酵素非含有緩衝液を点着した場合の反射光学濃度の経時変化を示す図である。
【図５】実施例４の結果を示す図であり、実施例スライド１及び比較例スライド２の免疫分析要素の検量線を示す図である。
【図６】実施例６の結果を示す図であり、実施例スライド３及び比較例スライド４のＣＲＰ分析用免疫分析要素の検量線を示す図である。
【図７】実施例１２の結果を示す図であり、実施例９で得たグルコアミラーゼ処理ＣＭ化澱粉と、比較例のグルコアミラーゼ未処理ＣＭ化澱粉の、分散液中でのα−アミラーゼに対する反応性を調べたものである。
【図８】実施例１４の結果を示す図であり、実施例１３で作成した実施例スライド５と比較例スライド６に各種濃度のＣＲＰ溶液を点着した後の、反射光学濃度の経時変化を示す図である。
【符号の説明】
１０ 透光性支持体
１２ 試薬層
１４ 基質層
１６ 抗体（又は酵素標識抗原）を含有する水浸透性層
１８ 酵素標識抗原（又は抗体）を含有する水浸透性層
２０ 抗体と酵素標識抗原とを含有する水浸透性層[0001]
[Industrial application fields]
The present invention relates to a dry immunoassay element to which an enzyme immunoassay is applied. Specifically, in an immunoassay element comprising a substrate layer containing a non-diffusible substrate that generates a diffusible substance with a labeling enzyme, and a reagent layer containing a depolymerizing enzyme that further converts the diffusible substance into a low-molecular product, The present invention relates to an immunoassay element in which a non-diffusible substrate is a substrate that reacts only with a labeling enzyme and does not react with a low molecular weight enzyme.
[0002]
BACKGROUND OF THE INVENTION
Analysis of biological components, drugs, and the like contained in body fluids such as blood and urine is very useful for diagnosing pathological conditions and determining the course of treatment, and plays an important role in the field of clinical tests. As a method for analyzing such a trace component (ligand), there is an enzyme immunoassay method (enzyme immunoassay). Enzyme immunoassay methods include non-homogeneous systems that require B / F separation and homogeneous systems that do not require B / F separation. The homogeneous reaction is based on the fact that the enzyme activity of the labeled enzyme is subjected to some interference when the antibody and the antigen (ligand) are bound to each other, and utilizes the inhibitory action by the antigen-antibody binding. In general, the enzyme activity that occurs when an antigen is labeled with an enzyme and an antibody, which is a large molecule, binds to it, resulting in steric hindrance to the binding of the enzyme to the substrate or a change in the three-dimensional structure of the enzyme. Detect suppression of
[0003]
In contrast, when the antigen is a polymer, suppression of enzyme activity due to the antigen-antibody binding reaction can be detected even if the antibody is labeled with an enzyme.
[0004]
On the other hand, in clinical examinations in which a large number of specimen samples are handled and routineed, it is desired that analysis can be performed easily and quickly and automatic operation can be performed. 49-53888 (corresponding to US 3,992,158), JP 55-90859 (US 4,258,001), JP 55-164356 (US 4,292,272), JP 60-222769 (EP 0 162 302A), JP 59-77356 (EP 0 097 952A), JP 59-102388 (US 4,861,552), JP 61-501866 (US 4,459,358)).
[0005]
The following dry analysis elements are known that allow enzyme-labeled antibodies to undergo a homogeneous enzyme immunoreaction (JP-A-1-321360). this is
(A) Polymerized antigen (ligand / polymer compound conjugate),
(B) a water-insoluble polymer substrate,
(C) a conjugate of an antibody against a ligand and an enzyme against a substrate,
Are included in the same layer or separate layers of the multilayer analytical element. The antigen spotted and supplied to the analytical element competes with the polymerized antigen and binds to the antibody-enzyme conjugate. This antigen-antibody-enzyme complex reacts with a water-insoluble polymeric substrate to produce a soluble small molecule product. On the other hand, a polymer antigen-antibody-enzyme complex formed by binding to a polymerized antigen cannot exhibit enzyme activity for a polymer substrate. Therefore, as the amount of antigen in the sample increases, the enzyme reaction product increases. This product is transferred to a detection layer, and the amount of antigen in the sample is analyzed by measuring the optical density of the absorption given by the colored chemical group.
[0006]
The immunoassay element described in Japanese Patent No. 2576910 (corresponding to US Pat. No. 5,569,589) is a further improvement of this. This analytical element is provided with a reagent layer containing a low molecular weight enzyme that further reduces the molecular weight of the labeled enzyme degradation product, and the sensitivity is increased by detecting this low molecular weight product.
[0007]
When the measurement target is a high molecular antigen, the immunoassay element described in Japanese Patent No. 2576913 can be used. This analytic element is
(A) a water-insoluble polymer substrate,
(B) a conjugate of an antibody against a macromolecular antigen and an enzyme against a substrate;
Are included in the same layer or separate layers of the multilayer analytical element, and the labeled enzyme degradation product is further reduced in molecular weight as in the case of the immunological analytical element described in Japanese Patent No. 2576910 (corresponding to US Pat. No. 5,569,589). A reagent layer containing a depolymerizing enzyme is provided, and the sensitivity is increased by detecting the demolecularized product.
[0008]
In either case, the non-diffusible substrate will not migrate to the reagent layer. Moreover, it was thought that the low molecular-weight enzyme does not reverse from the reagent layer to the substrate layer, which is a layer above it. For this reason, the substrate specificity of the non-diffusible substrate is not so strict, and one that can react with either the low molecular weight enzyme or the labeling enzyme has been selected.
[0009]
However, in the analytical element spotted with the sample solution, there is a slight diffusion of soluble components from the lower reagent layer to the upper substrate layer. The inventors examined this and found that when a low molecular weight enzyme transferred from the reagent layer to the upstream substrate layer reacts with a non-diffusible substrate to produce a low molecular weight product, this cannot be ignored. Turned out to be. Such noise could reduce the accuracy of the analysis. In addition, if a polymer substrate held as a non-diffusible substrate in the substrate layer contains a small amount of a polymer having a low degree of polymerization or a particle having a small particle size, the upper reagent layer is changed to the lower reagent layer as the sample solution is supplied. There will be something that will shift to. The transferred trace amount of the polymer substrate reacts with the depolymerizing enzyme in the reagent layer, which also causes noise.
[0010]
Therefore, the present inventors prepared an immunoassay element using a substrate that reacts only with a labeling enzyme and does not react with a low molecular weight enzyme as a non-diffusible substrate, and examined its performance. It was found that not only the sensitivity increased, but also the reproducibility was excellent and the temporal stability (storability) was also excellent.
[0011]
OBJECT OF THE INVENTION
The present invention has been made on the basis of the above-described findings, and aims to provide an immunoassay element capable of performing highly accurate, reproducible and highly sensitive analysis and having excellent storage stability. To do.
[0012]
[Structure of the invention]
  Such an object of the present invention is to react a reaction between an antigen and an enzyme-labeled antibody, or a reaction between an antigen and an antibody and an enzyme-labeled antigen, or a conjugate of an antigen, an antigen and a polymer compound, and an enzyme-labeled antibody. A substrate containing a non-diffusible substrate that produces a diffusible substance with the labeled enzyme in an immunoassay element that analyzes the amount of antigen by measuring a change in the labeled enzyme activity caused by any of the reactions between And a reagent layer containing a low molecular weight enzyme that further converts the diffusible substance into a low molecular product,The labeling enzyme is α-amylase, and the depolymerizing enzyme is glucoamylase or α-glucosidase,Substrate in which the non-diffusible substrate reacts only with the labeling enzyme and does not react with the depolymerizing enzymeCarboxymethylated starch that has been limitedly degraded by exo-active carbohydrate hydrolase in advance from the non-reducing terminal glucose site to the carboxymethyl-modified glucose unit site or the glucose chain branch siteAchieved by an immunoassay element, characterized in that
[0013]
  When the test substance is an antibody, the object of the present invention is to measure the change in the labeled enzyme activity caused by the reaction between the antibody and the enzyme-labeled antigen or the reaction between the antibody, the antigen and the enzyme-labeled antibody. In the immunoassay element for analyzing the amount of the antibody, a substrate layer containing a non-diffusible substrate that generates a diffusible substance by the labeling enzyme, and a low molecular weight enzyme that further converts the diffusible substance into a low molecular product. Comprising a reagent layer containing,The labeling enzyme is α-amylase, and the depolymerizing enzyme is glucoamylase or α-glucosidase,Substrate in which the non-diffusible substrate reacts only with the labeling enzyme and does not react with the depolymerizing enzymeCarboxymethylated starch that has been limitedly degraded by exo-active carbohydrate hydrolase in advance from the non-reducing terminal glucose site to the carboxymethyl-modified glucose unit site or the glucose chain branch siteAchieved by an immunoassay element, characterized in that
[0014]
  That is, the analysis element of the present invention uses, as a non-diffusible substrate, a substrate that reacts only with an antigen (or antibody) labeling enzyme and does not react with a low molecular weight enzyme originally contained in the reagent layer.SubstrateIt is contained in the layer. As a result, even if the low molecular weight enzyme diffuses from the reagent layer to the upstream substrate layer after the sample solution is spotted, the non-diffusible substrate will not be decomposed by this low molecular weight enzyme, and noise Will decrease. Even if the non-diffusible substrate used contains an insoluble polymer substrate that has a low degree of polymerization or is not retained in the substrate layer due to its small particle size and moves to the reagent layer, Does not react with low molecular weight enzymes. By using such a non-diffusible substrate, the reproducibility of measured values and storage stability are also improved.
[0015]
When the antigen (or antibody) labeling enzyme is an endoactive carbohydrate hydrolase and the low molecular weight enzyme in the reagent layer is an exoactive carbohydrate hydrolase, the non-diffusible substrate in the substrate layer is In a preferred embodiment, the non-reducing terminal glucose is a modified endo-type reactive substrate.
[0016]
Alternatively, if the non-reducing terminal glucose is not modified, the non-reducing terminal glucose can be used as an endo-type selective reactive substrate if it is located at the branching point of the glucose sugar chain. Can do.
[0017]
  Endoselective reactive substrates include non-reducing terminal glucose sitesCarboxymethylLimited to the modified glucose unit site or glucose chain branch site in advance by exo-active carbohydrate hydrolaseCarboxymethylModified starch can be used. Limited decompositionCarboxymethylWhen the modified starch is used, the reactivity with the labeling enzyme can be further increased and the measurement sensitivity can be increased.
[0018]
  A high-molecular polysaccharide such as starch is used as a non-diffusible substrate, a glucose oligomer is generated from this high-molecular polysaccharide using an antibody or antigen labeling enzyme as an endoactive saccharide-degrading enzyme, and this oligomer is converted to glucose. (Disassemble)Low molecular weightIn a preferred embodiment, the enzyme is an exoactive glycolytic enzyme.
[0019]
DETAILED DESCRIPTION OF THE STRUCTURE OF THE INVENTION
Layer structure of immunoassay elements
FIG. 1 shows an embodiment of the immunoassay element of the present invention. In FIG. 1, reference numeral 10 denotes a light-transmitting support, on which a reagent layer 12 and a substrate layer 14 are laminated.
[0020]
The substrate layer 14 is composed of a water-permeable layer, and contains a non-diffusible substrate that is a substrate for an enzyme bound as a label to an antibody or an antigen.
[0021]
The reagent layer 12 is composed of a water-permeable layer, and contains a low molecular weight enzyme that converts the diffusible substance that has diffused and migrated from the substrate layer into a product with a lower molecular weight. The substrate layer 12 also contains a reagent composition for detecting this low molecular product.
[0022]
This basic configuration is the same regardless of whether the measurement target is a low molecular weight antigen, a high molecular weight antigen, or an antibody. However, when the measurement target is a low molecular antigen, a mixed solution in which a sample, a polymerized antigen (a conjugate of an antigen and a high molecular compound) and an enzyme-labeled antibody are mixed and subjected to a competitive reaction is used as a substrate layer. Analysis is performed by spotting or feeding 14. Alternatively, the analysis is performed by spotting or supplying a mixed solution obtained by mixing the specimen, the antibody, and the enzyme-labeled antigen and performing a competitive reaction onto the substrate layer 14. In any case, the diffusible material produced increases as the amount of the ligand (small molecule antigen) increases.
[0023]
On the other hand, when the measurement target is a high molecular weight antigen, only the specimen and the enzyme-labeled antibody are mixed to cause an antigen-antibody binding reaction, and the reaction solution is spotted or supplied to the substrate layer 14. In this case, the amount of diffusible material produced decreases as the amount of ligand (polymer antigen) increases.
[0024]
When the measurement target is an antibody, analysis is performed by spotting or supplying a mixed solution obtained by mixing and reacting the specimen and the enzyme-labeled antigen to the substrate layer 14. In this case, the diffusible substance produced decreases as the amount of the test antibody increases. Alternatively, the analysis is performed by spotting or supplying a mixed solution obtained by mixing a specimen, an antigen, and an enzyme-labeled antibody and performing a competitive reaction onto the substrate layer 14. In this case, the diffusible substance produced increases as the amount of the test antibody increases.
[0025]
Measurement target
The measurement object of the present invention is an antigen or antibody contained in a specimen. An antigen is a ligand having an antigenic determinant. The type of specimen is not limited, and examples thereof include blood (whole blood, plasma, serum) lymph and urine. If there are suspended substances such as blood cells, it is preferable to remove them beforehand. However, when an appropriate filtration layer is provided in the uppermost layer of the analysis element, it may be spotted and supplied to the analysis element as it is.
[0026]
  As long as the antigen (ligand) is antigenic and the antibody can be prepared, antigens from low molecular weight substances to high molecular weight substances can be analyzed by the analytical element of the present invention. Note that the presence of antigenicity means that it can react with the corresponding antibody, and the antigenic determinant (epitope). Even if there is no immunogenicity by itself, it can be a ligand (antigen) to be measured in the present invention as long as an antibody can be obtained by immunization as a hapten.
[0027]
Examples of low molecular weight antigens include, for example, drug derivatives such as digoxin, theophylline, phenobarbital, phenytoin, penicillin, amikacin (for example, a conjugate of a drug and a biological substance such as protein), prostaglandin, testosterone, progesterone, thyroxine And hormones.
[0028]
Examples of high molecular weight antigens include plasma proteins such as hormones derived from various endocrine glands, immunoglobulins, albumin, ferritin, HCG (human chorionic gonadotropin), C-reactive protein (hereinafter abbreviated as CRP), and HB. There are various antigens such as viruses such as antigens, bacteria, α-fetoprotein, carcinoembryonic antigen (CEA), and antigens present in blood and urine.
[0029]
In addition, when the ligand to be measured is an antigen having a high molecular weight that exhibits an interference (suppression) action on the enzyme activity when it is enzyme-labeled, the ligand and the antigenic determinant are added as described later. What is necessary is just to carry out enzyme labeling of the other low molecular weight substance made common and to make this into an enzyme labeled antigen. The enzyme-labeled antigen used here is a concept including not only an enzyme-labeled ligand (antigen) which is a test substance but also an enzyme-labeled substance having a common ligand and antigenic determinant. Not only analogs on the chemical structure, such as ligand derivatives, but also compounds that exhibit similar behavior to ligands in immune responsiveness to antibodies can be used as enzyme-labeled antigens by labeling them.
[0030]
As used herein, a high molecular weight antigen refers to a high molecular weight antigen that binds to an enzyme-labeled antibody and exhibits an interference (suppression) action on enzyme activity. For example, the molecular weight is 20,000 daltons or more, preferably about 50,000. It refers to antigens of Dalton or higher. On the other hand, a low molecular antigen as used herein refers to an antigen having a molecular weight that does not significantly affect enzyme activity even when bound to an enzyme-labeled antibody, for example, an antigen having a molecular weight of 20,000 daltons or less. However, these molecular weight values are only a guideline. The determination of whether a ligand is a low molecular weight antigen or a high molecular weight antigen is based on a competitive reaction with a conjugate (polymerized antigen) bound to a high molecular compound. It depends on whether you use it.
[0031]
Polymerized antigen
A polymerized antigen, that is, a conjugate of a ligand (antigen) and a polymer compound, binds to an antibody, thereby suppressing the activity of an enzyme that labels the antibody. It is used when the measurement target is a low molecular antigen, and is not used when the measurement target is a high molecular antigen. The molecular weight of the polymer compound is preferably a molecular weight of 50,000 daltons or more and water-soluble. Examples of such a polymer compound include proteins such as gelatin, hemocyanin and ferritin, and polyethylene glycol. It is sufficient for these to have the above-mentioned conditions in a state of being bound to a ligand. For example, even a relatively low molecular weight material such as bovine serum albumin is highly polymerized by self-polymerizing it into a multimer. It may be molecular.
[0032]
The binding method between the antigen (ligand) and the polymer compound can be determined in consideration of both functional groups. As the functional group, an amino group, a carboxyl group, a hydroxyl group, a thiol group, an imidazole group, a phenyl group, or the like can be used. For example, many methods for bonding amino groups are known, such as an isocyanate method, a glutaraldehyde method, a difluorobenzene method, and a benzoquinone method. As a method for bonding an amino group and a carboxyl group, a carbodiimide method, a Woodward reagent method and the like are known in addition to a method for converting a carboxyl group into a succinylimide ester, and periodate oxidation that crosslinks an amino group and a sugar chain. The law (Nakane method) is also applicable. In the case of using a thiol group, for example, a carboxyl group on one side is converted to a succinylimide ester, a cysteine is reacted with this to introduce a thiol group, and both are bonded using a thiol group-reactive divalent crosslinking agent. be able to. Examples of the method using a phenyl group include a diazotization method and an alkylation method. The binding method is not limited to these examples. For example, “Method in Immunology and Immunochemistry” Vol. 1, (CAWilliams, MWChase, Academic Press, 1967) or Ishikawa, Kawai, edited by Miyai, “Enzyme immunization” It can be used by appropriately selecting from methods described in a book such as “Measurement Method” (Medical School, issued in 1978). Needless to say, the binding ratio between the ligand and the polymer compound is not limited to 1: 1, and may be any ratio depending on the purpose. After the binding reaction, it is purified by gel filtration, ion exchange chromatography or the like, and if necessary, is dried by lyophilization or the like.
[0033]
Alternatively, the ligand itself may be polymerized to obtain a polymerized antigen. In this case, the polymerization method can be carried out in accordance with the above-described bonding method. For example, the polymerization may be performed with a divalent crosslinking agent such as carbodiimide or glutaraldehyde.
[0034]
In place of the ligand, a ligand derivative having a cross-reactivity with an antibody against the ligand may be bound to the polymer compound. The ligand derivative as used herein refers not only to a chemical structural analog but also to a substance that behaves similarly to a ligand in immunoreactivity. For example, when an antibody against theophylline, which is a ligand, also cross-reacts with caffeine, a caffeine derivative can also be used as a material for the polymerized antigen.
[0035]
If the ligand or ligand derivative does not have an appropriate functional group for binding to the polymer compound, an amino group, a carboxyl group or a thiol group may be introduced into these. In that case, it may be introduced via a spacer to facilitate the bonding with the polymer compound. For example, when the ligand is theophylline, 8-propylcarboxytheophylline having a carboxyl group introduced can be bound to the polymer compound.
[0036]
antibody
An antibody is used when a specimen containing a test antigen, an antibody, and an enzyme-labeled antigen are mixed and subjected to a competitive reaction. A specific antibody against the antigen to be tested is used. When an antigen (ligand) derivative is used as the enzyme-labeled antigen, one that reacts with an antigenic determinant common to the ligand and the ligand derivative is used. Although a polyclonal antibody obtained by a conventional method may be used, the sensitivity is further improved by using a monoclonal antibody. This antibody is also F (ab ')₂, Fab ′, Fab and the like.
[0037]
Enzyme labeled antigen
The enzyme-labeled antigen is used when a specimen containing a test antigen, an antibody, and an enzyme-labeled antigen are mixed and subjected to a competitive reaction. It is also used when a sample containing a test antibody is mixed with an enzyme-labeled antigen to cause a binding reaction and the amount of the test antibody is analyzed from the change in the activity of the labeled enzyme.
[0038]
When the test substance is an antigen (ligand), the enzyme-labeled antigen is a conjugate of a ligand (or a ligand-like substance having a common antigen determinant with this ligand) and an enzyme. When the ligand is a low molecular weight substance such as a drug, it may be directly bound to the enzyme. If the ligand is a large molecular weight substance that interferes with the enzyme activity when it is bound to the enzyme as it is, such as a protein, the protein may be fragmented and enzyme-labeled. It is only necessary that the protein having a low molecular weight by fragmentation has the same antigenic determinant as the non-fragmented protein (ie, ligand).
[0039]
The method for binding the ligand and the enzyme can be performed in the same manner as the method for binding the antigen (ligand) and the polymer compound.
[0040]
  Instead of the ligand, a ligand derivative having cross-reactivity with an antibody against the ligand may be bound to the enzyme. The ligand derivative as used herein refers not only to a chemical structure analog but also to a substance that behaves like a ligand in immunoreactivity. For example, if an antibody against the ligand theophylline also cross-reacts with caffeine, a derivative of caffeineAlso antigenIt can be used as a material.
[0041]
In addition, when there is no suitable functional group for binding with an enzyme in a ligand or a ligand derivative, you may introduce | transduce an amino group, a carboxyl group, or a thiol group to these. In that case, it may be introduced via a spacer to facilitate binding to the enzyme. For example, when the ligand is theophylline, 8-propylcarboxytheophylline into which a carboxyl group has been introduced can be bound to the enzyme.
[0042]
Enzyme-labeled antibody
The enzyme-labeled antibody is used when a specimen containing a test antibody, an antigen, and an enzyme-labeled antibody are mixed and subjected to a competitive reaction. It is also used when a sample containing a test antigen is mixed with an enzyme-labeled antibody to cause a binding reaction and the amount of the test antigen is analyzed from the change in the activity of the labeled enzyme. When the test antigen is a low molecular weight substance, it is used when a specimen containing the test antigen, a high molecular weight antigen, and an enzyme-labeled antibody are mixed and subjected to a competitive reaction.
[0043]
In a reaction system in which a competitive reaction between a test antibody, an antigen, and an enzyme-labeled antibody is performed, the enzyme-labeled antibody must be one that recognizes the same antigenic determinant on the same antigen that the test antibody recognizes. use.
[0044]
In a reaction system in which a competitive reaction is carried out between a test antigen, a polymerized antigen, and an enzyme-labeled antibody, the enzyme-labeled antibody is one that reacts with an antigenic determinant common to the antigen and the polymerized antigen. .
[0045]
  antibodyThe method for binding the enzyme to the enzyme can be performed in the same manner as the method for binding the antigen (ligand) to the polymer compound.
[0046]
Enzyme-Non-diffusible substrate-Decreasing enzyme
Enzyme bound as a label to an antigen or antibody decomposes a non-diffusible substrate consisting of a polymer, and generates a diffusible product that produces a low molecular product (glucose) by a low molecular weight enzyme. . The non-diffusible substrate is non-diffusible with respect to the aqueous specimen liquid and does not diffuse or migrate from the substrate layer 14 to the reagent layer 12 itself. The depolymerizing enzyme converts a diffusible product produced from a non-diffusible substrate by an enzyme bound as a label to an antigen (or antibody) into a further detectable low-molecular product. Contained in the reagent layer 12 of the element.
[0047]
In these combinations, the enzyme acts on a non-diffusible substrate to generate a diffusible substance, and this diffusible product can be easily detected by generating a low molecular weight product by the low molecular weight enzyme described later. You can choose from such combinations.
[0048]
enzyme
As such an enzyme, there is a degrading enzyme that generates a diffusible oligomer from a non-diffusible substrate made of a polymer, and examples thereof include a saccharide hydrolase. Examples of such a carbohydrate hydrolase include endo-active hydrolases such as α-amylase, β-amylase, and dextranase.
[0049]
  These enzymes are preferably unaffected by interfering factors present in any sample, and it is preferable that there is no competing enzyme of the same type in the sample. However, when an enzyme of the same type as the labeling enzyme is contained in the sample, this enzyme inhibitor may be used. The enzyme inhibitor only needs to have a greater degree of inhibiting the enzyme in the sample than inhibiting the activity of the labeling enzyme. Most preferably, the enzyme inhibitor completely inactivates the enzyme in the specimen, but does not inhibit the labeled enzyme at all. However, in practice, it is not necessary to simply increase the blank value at the time of measurement, and the enzyme activity in the specimen may be recovered after the measurement by deactivating the enzyme inhibitor. The enzyme inhibitor is an enzyme-labeled ligand.(Or antibody)Any enzyme may be used as long as it does not inhibit the enzyme, and the enzyme in the free state may be inhibited. As this enzyme inhibitor, a known enzyme inhibitor having the above-described specificity may be selected and used. Or you may make the antibody with respect to the enzyme which becomes a problem in a test substance, and may use this as an enzyme inhibitor.
[0050]
Non-diffusible substrate
Examples of substrates for the aforementioned α-amylase, β-amylase, dextranase and the like include carboxymethylated starch, starch, amylose, amylopectin and the like. However, in the present invention, a substrate that reacts only with the antigen (or antibody) labeling enzyme and does not react with the low molecular weight enzyme originally contained in the reagent layer is used as the non-diffusible substrate. As a result, even if the low molecular weight enzyme diffuses from the reagent layer to the upstream substrate layer after the sample solution is spotted, the non-diffusible substrate will not be decomposed by this low molecular weight enzyme, and noise Will decrease. In addition, even if an insoluble polymer substrate that has a low degree of polymerization or is not retained in the substrate layer and moves to the reagent layer is mixed in the non-diffusible substrate, the low molecular weight in the reagent layer Does not react with oxidase.
[0051]
  When using α-amylase as the labeling enzyme and glucoamylase or α-glucosidase described later as the depolymerizing enzyme, non-reducing terminal glucose is used.CarboxymethylInsoluble polysaccharides such as starch modified with a modifying group such as a group may be used. These modified substrates serve as α-amylase substrates, but not glucoamylase or α-glucosidase substrates. The non-reducing terminal glucose modifying group is preferably a non-reactive functional group that does not affect the activity of the labeling enzyme or the depolymerizing enzyme. In order to increase the reactivity in the dry analytical element, a hydrophilic modifying group is desirable. As such a modifying groupCarboxymethylAnd hydrophilic groups such as hydroxypropyl and hydroxyethyl groups. Alternatively, terminal glucose may be phosphate esterified, sulfate esterified, or nitrate esterified.
[0052]
Alternatively, this non-reducing terminal glucose is bound to an adjacent glucose unit in a binding mode other than an α-1,4 glucoside bond (eg, α-1,6 bond, α-1,3 bond), and glucose Any substance located at the branching point of the sugar chain can be used as the non-diffusible substrate of the present invention. An exo-active carbohydrate hydrolase such as glucoamylase is generally an enzyme that degrades the α-1,4 bond or α-1,6 bond of the linear part of the sugar chain from the non-reducing end to a glucose unit. The glucoside bond at the branch point (mainly α-1,6 bond, rarely α-1,3 bond) cannot be broken down. Therefore, even when the non-reducing terminal glucose is in a position branched from the sugar chain, it cannot be hydrolyzed.
[0053]
α-Amylase is an endo-active carbohydrate hydrolase that hydrolyzes α-1,4 glucoside bonds of sugar chains of 4 units or more of glucose, and is a substrate molecule with or without terminal glucose modification. Can be hydrolyzed within.
[0054]
  For example, in the glucose unit in the middle of the sugar chainCarboxymethylGroup introducedCarboxymethylFrom the non-reducing terminal glucose siteCarboxymethylBy subjecting the modified glucose unit site to limited degradation by exo-active carbohydrate hydrolase,CarboxymethylIt can be a substrate modified with a group. Examples of the exoactive carbohydrate hydrolase used here include glucoamylase and α-glucosidase.
[0055]
In this way, the carboxymethylated starch whose degree of carboxymethylation has been increased by limited decomposition of terminal glucose has increased reactivity with endo-active enzymes such as α-amylase within the dry analytical element. This is a new finding by the inventor of the present application, as will be described later in Examples. The details of why the enzyme activity increases by increasing the degree of carboxymethylation (introduction rate) of insoluble polysaccharides such as starch are unknown, but can be estimated as follows.
[0056]
  CarboxymethylThe group is a hydrophilic group, and by increasing the introduction rate of this functional group, the hydration property of the insoluble polysaccharide is increased, and it becomes easy to swell in an aqueous solvent. However, the reaction with the dry analytical element is performed by spotting a small amount of sample liquid (usually 100 μL or less), and the amount of water supplied is extremely small. The supplied water (sample solution) develops in the layer and immediately moves to the adjacent layer, so that the amount of water is extremely limited in the layer where the enzyme reaction takes place. In a layer with such a limited amount of water, the substrate swells sufficiently and cannot react sufficiently with the enzyme. However, if the degree of swelling of the substrate increases even slightly, in such a limiting environment, it becomes easy to expose the reaction site to the enzyme due to a microscopic structural change. This seems to increase the reactivity to the enzyme in the dry analytical element, leading to an increase in sensitivity.
[0057]
  On the other hand, when a substrate is reacted with an enzyme in an aqueous solution, the substrate is already sufficiently swollen to ensure reactivity with the enzyme. This is because, in an environment where there is a sufficient amount of water, the substrate can already swell enough to the extent necessary for the reaction.CarboxymethylIt seems that even if the degree of swelling is increased by increasing the degree of conversion and increasing the hydrophilicity, the reactivity to the enzyme is not further increased.
[0058]
Low molecular enzyme
This low molecular weight enzyme may be the same type of enzyme as the labeling enzyme. In this case, the labeling enzyme is an endo-active enzyme that cleaves from the inside of the molecule to produce an oligomer, and the depolymerizing enzyme acts from the end of the molecule to produce a monomer. It is preferable to have. For example, when the non-diffusible substrate is a polymer (eg, starch), a substance capable of decomposing a diffusible oligomer (eg, maltose) produced by a labeling enzyme into a monomer (eg, glucose) is used. Examples of such a low molecular weight enzyme include sugar hydrolase, and more specifically, α-amylase, β-amylase, dextranase, glucoamylase, α-glucosidase and the like.
[0059]
  As a non-diffusible substrate and labeling enzymeCarboxymethylWhen cellulose and cellulase are used, C1 enzyme can be used as a depolymerizing enzyme. Similarly, when galactan and galactanase are used, β-galactosidase can be used, and when RNA and ribonuclease are used, exoribonuclease can be used as the low molecular weight enzyme.
  Combinations of these labeling enzymes, non-diffusible substrates, and low molecular weight enzymes are known in the literature (eg, “Enzyme Handbook” (supervised by Bunji Maruo, Nobuo Tamiya, published by Asakura Shoten in 1982), “Handbook of Biochemistry” (Nobumasa Imura). , Other editions, published in Maruzen 1984).
[0060]
Low molecular product detection system
The low molecular product produced by the depolymerizing enzyme in the reagent layer can be optically detected by a known detection system reagent. As a method for detecting glucose finally produced by the low molecular weight enzyme, for example,
(1) As a method for detecting hydrogen peroxide produced by oxidizing glucose in the presence of glucose oxidase, for example,
(1-1) Ann.Clin.Biochem.,6, 24 (1964), J.Clin.Pathol.,twenty two, 246 (1969), using the Trinder reagent;
(1-2) A method using a Trinder reagent described in JP-A-49-50991 (corresponding US Pat. No. 3,886,045), US Pat. No. 3,992,158, JP-A-55-164356 (corresponding US Pat. No. 4,292,272);
(1-3) a method using a reagent containing a triaryl-substituted imidazole leuco dye described in JP-A-53-26188 (corresponding US Pat. No. 4,089,747), JP-A-58-45557, etc .;
(1-4) A reagent containing a diaryl-monoaralkyl-substituted imidazole leuco dye described in JP-A-59-193352 (corresponding European Patent Publication EP 0122641A), JP-A-60-224677 (corresponding US Pat. No. 4,665,023), etc. How to use;
(2) a method for detecting NADH produced in the presence of glucose dehydrogenase and NAD;
(3) a method for detecting glucose-6-phosphate produced in the presence of hexokinase;
For example, a known method can be used. Among these detection methods, a method of detecting hydrogen peroxide generated by oxidizing glucose in the presence of glucose oxidase using a peroxidase and a leuco dye is most desirable in terms of sensitivity.
[0061]
These detection reagents may be contained in the reagent layer 12 of the analysis element together with the depolymerizing enzyme, but are contained in another layer (for example, the second reagent layer or the detection layer) provided in the lower layer of the reagent layer 12. You may make it detect in this layer. In addition, when using a leuco pigment | dye, it is preferable on stability of the pigment | dye produced | generated to disperse this in a hydrophilic binder as a solution of a water immiscible solvent.
[0062]
Layer structure of analysis elements
The dry immunoassay element of the present invention can have a layer structure similar to that of various known dry analysis elements. In addition to the substrate layer and the reagent layer, the element may be a multilayer including a support, a spreading layer, a detection layer, a light shielding layer, an adhesive layer, a water absorbing layer, an undercoat layer, and other layers. Examples of such analytical elements include JP-A-49-53888 (corresponding US Pat. No. 3,992,158), JP-A-51-40191 (corresponding US Pat. No. 4,042,335), and JP-A-55-164356 (corresponding US Pat. No. 4,292,272). JP-A-61-4959 (corresponding to EPC published patent 0166365A).
[0063]
In the case of using a light-permeable water-impermeable support, the dry immunoassay element of the present invention can practically have the following configuration. However, the content of the present invention is not limited to this.
(1) One having a reagent layer on a support and a substrate layer thereon.
(2) One having a reagent layer, an adhesive layer, and a substrate layer in this order on a support.
(3) One having a detection layer, a reagent layer, and a substrate layer in this order on a support.
(4) One having a reagent layer, a light reflecting layer, and a substrate layer in this order on a support.
(5) A substrate having a detection layer, a reagent layer, a light reflection layer, and a substrate layer in this order on a support.
(6) One having a detection layer, a light reflection layer, a reagent layer, and a substrate layer in this order on a support.
(7) One having a second reagent layer, a light reflecting layer, a first reagent layer, and a substrate layer in this order on a support.
(8) A substrate having a detection layer, a second reagent layer, a light reflection layer, a first reagent layer, and a substrate layer in this order on a support.
[0064]
In the above (1) to (6), the reagent layer may be composed of a plurality of different layers. Further, the substrate layer may be an immune reaction layer containing a component capable of performing an immune reaction as described later. A water absorption layer may be provided between the support and the reagent layer or the detection layer. Moreover, you may provide a filtration layer between each layer. Further, a developing layer may be provided on the substrate layer, or the substrate layer may have a developing action and function as a developing layer.
[0065]
Substrate layer
The substrate layer 14 is composed of a water-permeable layer, and contains a non-diffusible substrate that is a substrate for an enzyme that labels an antigen or antibody. In order to ensure the water permeability of the substrate layer, it is preferable to use a porous layer made of a porous medium or a layer made of a hydrophilic polymer binder.
[0066]
The porous layer may be fibrous or non-fibrous. As the fibrous material, for example, filter paper, non-woven fabric, woven fabric (for example, plain woven fabric), knitted fabric (for example, tricot knitted fabric), glass fiber filter paper, and the like can be used. Non-fibrous materials include membrane filters composed of cellulose acetate described in JP-A-49-53888, etc .; JP-A-49-53888, JP-A-55-90859 (corresponding US Pat. No. 4,258,001), JP-A-58- 70163 (corresponding U.S. Pat. No. 4,486,537) or the like, and may be any of a continuous void-containing granular structure layer made of inorganic or organic fine particles. JP 61-4959 (corresponding European published EP 0166365A), JP 62-116258, JP 62-138756 (corresponding European published EP 0226465A), JP 62-138757 (corresponding European published EP 0226465A), special A laminate of a plurality of partially bonded porous layers described in, for example, Kokai 62-138758 (corresponding European publication EP 0226465A) is also suitable.
[0067]
The porous layer may be a spreading layer having a so-called metering action that spreads the liquid in an area approximately proportional to the amount of liquid supplied. Of these, a woven fabric, a knitted fabric and the like are preferable as the spreading layer. A woven fabric or the like may be subjected to glow discharge treatment as described in JP-A-57-66359. In the development layer, in order to adjust the development area, the development speed, etc., JP-A-60-222770 (corresponding to EP 0162301A), JP-A-62-219397 (corresponding German Patent Publication DE 37 17 913A), JP-A-63. -112999 (corresponding: DE 37 17 913A), JP-A 62-182652 (corresponding: DE 37 17 913A) may contain a hydrophilic polymer or a surfactant.
[0068]
For example, after impregnating or coating a substrate on paper, cloth, a porous membrane made of a polymer or the like in advance, on another water-permeable layer provided on the support, for example, a reagent layer, JP-A-55-164356 It is also a useful method to adhere by such a method. As another method, the porous layer may be adhered to another water-permeable layer (for example, a reagent layer) by the method described above, and then the composition containing the substrate may be applied to the porous layer. A known method can be used for impregnating or coating the porous layer. For coating, for example, dip coating, doctor coating, hopper coating, curtain coating, etc. are appropriately selected and used.
[0069]
The thickness of the substrate layer thus prepared is not particularly limited, but when it is provided as a coating layer, a range of about 1 μm to 50 μm, preferably 2 μm to 30 μm is appropriate. In the case of a method other than coating, such as lamination by lamination, the thickness can vary greatly in the range of several tens to several hundreds of μm.
[0070]
When the substrate layer is composed of a water-permeable layer composed of a hydrophilic polymer binder, examples of the hydrophilic polymer that can be used include the following. Gelatin and derivatives thereof (for example, phthalated gelatin), cellulose derivatives (for example, hydroxyethyl cellulose), agarose, sodium alginate, acrylamide copolymer, methacrylamide copolymer, acrylamide or copolymers of methacrylamide and various vinyl monomers Polyhydroxyethyl methacrylate, polyvinyl alcohol, polyvinylpyrrolidone, sodium polyacrylate, copolymers of acrylic acid and various vinyl monomers, and the like.
[0071]
Substrate layers composed of hydrophilic polymer binders are disclosed in JP-B 53-21677 (corresponding US Pat. No. 3,992,158), JP-A 55-164356 (corresponding US Pat. No. 4,292,272), JP-A 54-101398 (corresponding US patent). 4,132,528), JP-A-61-292063 (Chemical Abstracts,106210567y) and the like, by applying an aqueous solution or aqueous dispersion containing a substrate or other reagent composition and a hydrophilic polymer on another layer such as a support or a detection layer and drying the solution. Can be provided. The dry thickness of the substrate layer with a hydrophilic polymer binder is about 2 μm to about 50 μm, preferably about 4 μm to about 30 μm, and the coating amount is about 2 g / m.²~ About 50g / m², Preferably about 4 g / m²~ About 30g / m²Range.
[0072]
In addition to the non-diffusible substrate, the substrate layer has an enzyme activator, a coenzyme, a surfactant, for the purpose of improving various properties such as coating characteristics, diffusibility of the diffusible compound, reactivity, and storage stability. Various additives consisting of organic substances or inorganic substances can be added, such as pH buffer compositions, fine powders, antioxidants, and the like. Examples of buffering agents that can be contained in the substrate layer include the Chemical Society of Japan "Chemical Handbook Basics" (Tokyo, Maruzen Co., Ltd., 1966), pages 1312-1320, RMCDawson et al, "Data for Biochemical Research "Second Edition (Oxford at the Clarendon Press, 1969), pages 476-508," Biochemistry "Five, 467-477 (1966), `` Analytical Biochemistry ''104, 300-310 (1980). Buffers containing tris (hydroxymethyl) aminomethane (Tris) as specific examples of pH buffers; buffers containing phosphate; buffers containing borate; buffers containing citric acid or citrate; containing glycine There are good buffering agents such as a buffering agent; a buffering agent containing Bicine; a buffering agent containing HEPES; and a buffering agent containing MES (2-morpholinoethanesulfonic acid).
[0073]
Reagent layer
The reagent layer 12 contains a detection reagent composition for detecting a low molecular weight enzyme and, if necessary, a low molecular weight product generated by the low molecular weight enzyme. The reagent layer 12 is composed of a water-permeable layer, and is preferably a continuous layer made of a hydrophilic polymer binder among the water-permeable layers described in the description of the substrate layer. The hydrophilic polymer binder to be used is determined in consideration of the diffusible product produced in the substrate layer, the coloring reagent contained in the reagent layer, and the like.
[0074]
Support
As the support 10, any one of light-impermeable (opaque), light-translucent (translucent), and light-transmissive (transparent) can be used, but generally it is light-transmissive and water-impermeable. A sex support is preferred. Preferable materials for the light-transmissive water-impermeable support are polyethylene terephthalate and polystyrene. In order to firmly adhere the hydrophilic layer, an undercoat layer is usually provided or a hydrophilic treatment is performed.
[0075]
  Immune reaction layer
  The substrate layer 14 in FIG. 1 may contain not only a diffusible substrate but also molecular species necessary for an immune reaction. For example,
(1) When analyzing the amount of the test antigen by reacting the test antigen with the enzyme-labeled antibody, the enzyme-labeled antibody is used;
(2) When analyzing the amount of the test antigen by reacting the test antigen, the antibody and the enzyme-labeled antigen, the antibody and the enzyme-labeled antigen;
(3)Test antigenWhen the amount of the test antigen is analyzed by reacting the polymerized antigen with the enzyme-labeled antibody, the polymerized antigen and the enzyme-labeled antibody;
(4) When analyzing the amount of the test antibody by reacting the test antibody with the enzyme-labeled antigen, the enzyme-labeled antigen is used;
(5) When analyzing the amount of the test antibody by reacting the test antibody with the antigen and the enzyme-labeled antibody, the antigen and the enzyme-labeled antibody;
It is contained in the substrate layer. Thus, the substrate layer functions as an immune reaction layer that causes an immune reaction to be performed in the layer. In this case, a homogeneous enzyme immunoreaction can proceed within the element simply by spotting a specimen on the element.
[0076]
Alternatively, any of these may be contained in the substrate layer, and the remaining one may be contained in the water-permeable layer laminated on the upper layer of the substrate layer. Alternatively, a water-permeable layer composed of one or a plurality of layers may be provided on the substrate layer, and these may contain molecular species necessary for an immune reaction. The layer structure of these immune reaction layers can be arbitrarily determined according to the reaction mode of the immune reaction.
[0077]
For example, in the case of (2) above, the antibody and enzyme-labeled antigen may be separately contained in a plurality of layers other than the substrate layer. For example, as shown in FIG. 2, an immunoassay element is constructed by providing a water-permeable layer 16 containing an antibody on a substrate layer 14 and further providing a water-permeable layer 18 containing an enzyme-labeled antigen thereon. May be. In this case, the antigen (ligand) in the specimen diffuses and penetrates into the layer 16 together with the enzyme-labeled antigen in the layer 18. In the layer 16, the antigen and the enzyme-labeled antigen each bind to the antibody, and further move to the substrate layer 14.
[0078]
On the contrary, the immunoassay element may be constituted by containing an enzyme-labeled antigen in the water-permeable layer 16 and an antibody in the water-permeable layer 18 thereon. In this case, the antigen (ligand) in the specimen binds to the antibody in the layer 18 and moves to the layer 16. In the layer 16, the antibody that has not been bound to the antigen binds to the enzyme-labeled antigen and further moves to the substrate layer 14.
[0079]
Further, the antibody and the enzyme-labeled antigen may be contained together in one layer other than the substrate layer in a substantially dry state or substantially in the absence of water. For example, as shown in FIG. 3, a water permeable layer 20 containing an antibody and an enzyme-labeled antigen in a substantially dry state or substantially in the absence of water is provided on the substrate layer 14 to provide an immunoassay element. It may be configured. In this case, when a test solution in which water is a solvent is spotted and supplied to the layer 20, the ligand (antigen) in the specimen and the enzyme label in the water derived from the test solution in the layer 20 Each antigen binds to an antibody and moves to the substrate layer 14. In order to contain the antibody and the enzyme-labeled ligand together in a substantially dry state or in the absence of water in one layer, one or both of the antibody and the enzyme-labeled antigen are mixed with alcohol (eg, ethanol), etc. The water-permeable layer may be impregnated by dissolving or dispersing in a non-aqueous solvent.
[0080]
  When no immune reaction layer is provided, the analysis element of the present invention can also be used for analysis of an enzyme that degrades a non-diffusible substrate contained in the substrate layer 14. For example, as a non-diffusible substrateCarboxymethylWhen modified starch, starch, amylose, amylopectin or the like is used, it can be used for analysis of endo-active carbohydrate hydrolase such as α-amylase or β-amylase.
[0081]
Method for producing immunoassay element
The dry immunoassay element of the present invention can be prepared by known methods described in the aforementioned patent specifications. The analysis element of the present invention is cut into small pieces such as a square having a side of about 15 mm to about 30 mm or a circle of about the same size, and the like, and Japanese Patent Publication No. 57-28331 (corresponding US Pat. ), Japanese Unexamined Patent Publication No. 57-63452, Japanese Utility Model Publication No. 58-32350, Special Table No. 58-501144 (corresponding international publication: WO 83/00391), etc. From the viewpoint of packaging, transportation, storage, measurement operation and the like. Depending on the purpose of use, it can be used in the form of a long tape stored in a cassette or magazine, or a small piece can be attached to or stored in a card with an opening.
[0082]
Analysis method using immunoassay elements
The analysis element of the present invention can perform quantitative analysis of an antigen or antibody, which is a test substance in a liquid sample, by the same operation as described in the aforementioned patent specifications. For example, an aqueous liquid sample solution such as plasma, serum, urine or the like in the range of about 5 μL to about 30 μL, preferably 8 to 15 μL is spotted on the substrate layer 14. The spotted analytical element is incubated at a constant temperature in the range of about 20 ° C. to about 45 ° C., preferably at a constant temperature in the range of about 30 ° C. to about 40 ° C. for 1 to 10 minutes. The color development or discoloration in the element is reflected and photometrically measured from the side of the light-transmitting support, and the amount of ligand in the specimen can be determined based on the principle of colorimetric measurement using a calibration curve prepared in advance. Quantitative analysis can be performed with high accuracy by keeping the amount of liquid sample to be spotted, incubation time and temperature constant.
[0083]
Highly accurate quantitative analysis can be performed with extremely easy operations using the chemical analyzer described in JP-A-60-125543, JP-A-60-220862, JP-A-61-294367, JP-A-58-161867 (corresponding US Pat. No. 4,424,191). it can. Depending on the purpose and required accuracy, semi-quantitative measurement may be performed by visually judging the degree of color development.
[0084]
When the analytical element does not have an immune reaction layer, that is, when the molecular element necessary for the immune reaction system with the antigen or antibody as the test substance is not contained in the analytical element, an appropriate component outside the analytical element is used. After a necessary immune reaction is performed in a reaction solution, the test solution can be analyzed as a change in the labeled enzyme activity by spotting the reaction solution on the element. For example, when analyzing antigens, mix the aqueous sample solution with the solution containing the antibody and enzyme-labeled ligand to allow sufficient binding reaction before spotting on the element before spotting on the element. That's fine.
[0085]
[Example 1]
  Preparation of endo-type reactive substrate
  Carboxymethyl10 g of modified starch (Expprotab, Edward Mendel Company Inc.) was dispersed in 1 L of 0.1 M borate buffer (pH 10) and stirred for 1 hour. After adjusting the pH to 5.5 with hydrochloric acid and phosphoric acid, 1000 U of glucoamylase (Toyobo) was added and reacted at 37 ° C. for 16 hours. After the reaction, a high-speed centrifuge was applied at about 10,000 G, and the centrifugal purification was repeated until the electrical conductivity of the centrifugal upper layer liquid was 20 μS / cm or less to remove soluble components. While stirring the dispersion, 10 L of ethanol was added, and the precipitated white substance was collected by vacuum filtration. This was dried at 30 ° C for 10 hours, and 5.2 g (yield 52%) of endo-type enzyme reaction specificCarboxymethylA modified starch was obtained.
[0086]
[Example 2]
Creating analytical elements
A reagent solution containing a cross-linking agent is applied on a colorless and transparent polyethylene terephthalate (PET) sheet (support) having a thickness of 180 μm provided with a gelatin subbing layer so as to have the following coating amount, and dried to form a reagent layer. Provided.

[0087]
The following reagent-containing aqueous solution is applied to the surface of the reagent layer so as to have the following coating amount, the gelatin layer is swollen, and a PET spun yarn equivalent to 50 denier is knitted on 36 gauge and has a thickness of about 250 μm. Were laminated with light pressure almost uniformly to provide a porous spreading layer.

[0088]
  Next, endo-type enzyme reaction specific obtained in Example 1CarboxymethylUsing the modified starch, a substrate was applied so as to have the following coating amount and dried to provide a substrate layer to prepare a multilayer analysis element for CRP analysis.
    Carboxymethylated starch 3.5 g / m²
    Mannitol 3.0 g / m²
    MES (2-morpholinoethanesulfonic acid) 2.0 g / m²
[0089]
Next, this analytical element was cut into a 14 mm × 13 mm square chip and placed in a slide frame described in JP-A-57-63452 to obtain a multilayer dry slide 1 for CRP analysis of this example.
[0090]
  As a comparative example, endo-type enzyme reaction specificCarboxymethylUntreated instead of modified starchCarboxymethylA comparative slide 2 having the same structure as that of the example slide 1 was prepared except that the modified starch (expro tab) was used as a substrate.
[0091]
[Example 3]
10 μL of 50 mM glycerophosphate buffer solution (pH 7) was spotted on the dry slide 1 of Example 2 and the dry slide 2 of Comparative Example. Thereafter, each slide was kept at 37 ° C., and the reflection optical density at the central wavelength of 650 nm was measured over time from the PET support side. FIG. 4 shows the change over time in the reflection optical density after spotting.
[0092]
  RawCarboxymethylIn the slide 2 of the comparative example using the modified starch as the non-diffusible substrate, the reflection optical density increases with time after the buffer solution is spotted (-o- in the figure). This indicates that untreated carboxymethylated starch is decomposed by glucoamylase (low molecular enzyme), which is an exo-reactive enzyme transferred from the reagent layer, to produce glucose.
[0093]
  On the other hand, endo-type enzyme reaction was made specific by glucoamylase treatment in advance.CarboxymethylIn the slide 1 of Example 2 using the modified starch, the reflection optical density did not increase at a constant value. (Fig. 4,-●-).
[0094]
[Example 4]
  10 μL of 50 mM glycerophosphate buffer solution (pH 7) containing amylase-labeled anti-CRP-IgG (0.1 mg / mL) and containing a known amount of CRP was spotted on slide 1 of Example 2 and slide 2 for comparison. The reflection optical density of each of slides 1 and 2 was measured from the PET support side with visible light having a center wavelength of 650 nm while maintaining the temperature at 37 ° C. Difference in reflection optical density (ΔOD after 4 minutes and 6 minutes after spotting)_6-4) Is shown in FIG. Based on the calibration curve in Fig. 5, the endo-type enzyme reaction was made specific.CarboxymethylIt is clear that the dry immunoassay element of the present invention using the modified starch (FIG. 5,-●-) can accurately quantify CRP. In addition, compared to the comparative example (FIG. 5,-○-), ΔOD with respect to CRP concentration change_6-4The change of was large and the sensitivity increased.
[0095]
[Example 5]
A multilayer analytical element was prepared in exactly the same procedure and configuration as in Example 2, and amylase-labeled CRP-IgG was further added to 3 mg / m 2 on the tricot knitted fabric layer that was the substrate layer and the development layer.²A multi-layer immunoslide 3 for CRP analysis (Example 5) was prepared by applying an ethanol solution so as to have a coating amount of, impregnating and drying. Similarly, 3 mg / m of amylase-labeled CRP-IgG was added to the tricot knitted fabric layer of the multilayer analytical element of the comparative slide 2 prepared in Example 2.²A multilayer immunoslide 4 for CRP analysis for comparison was prepared by applying an ethanol solution so as to have a coating amount of, impregnating and drying.
[0096]
[Example 6]
  10 μL of 50 mM glycerophosphate buffer solution (pH 7) containing various concentrations of CRP is spotted on slide 3 and slide 4 and maintained at 37 ° C., and the reflection optical density at 650 nm is measured from the support side. Difference in reflection optical density after 4 minutes and 6 minutes from_6-4) As shown in the calibration curve of FIG.CarboxymethylIt was shown that the dry immunoassay element of the present invention using the modified starch (FIG. 6,-●-) can quantitatively analyze CRP with higher sensitivity than the comparative example (-○-).
[0097]
[Example 7]
The reproducibility (CV) of the slide 3 of Example 5 and the comparative slide 4 was compared and examined. Drop 10 μL of 4 CRP-containing human standard sera (L-1, L-2, M, H) on slide 3 and slide 4 and keep the temperature at 37 ° C to measure the reflection optical density at 650 nm from the support side. The difference in reflection optical density (ΔOD after 4 minutes and 6 minutes after spotting)_6-4) Separately, a calibration curve in the case of the slide 3 of Example 5 and a calibration curve in the case of the comparison slide 4 are obtained, and ΔOD of each obtained sample is obtained._6-4From this, the CRP concentration (measured value) was converted. This measurement was performed 10 times for each specimen, and the average value, standard deviation, and CV value were determined. The results are shown in Tables 1 and 2 below.
[0098]
[Table 1]

[0099]
[Table 2]

[0100]
As shown in Tables 1 and 2, the conventional comparison slide 4 has a large variation in the measured value (concentration), whereas the slide 3 of Example 5 using the endo-reaction specific substrate of the present invention varies. Was small and excellent in simultaneous reproducibility. In particular, in the low concentration range (specimen L-1, L-2) and the high concentration range (specimen H), the CV value of the example slide 3 is reduced to about ３ compared to that of the comparison slide 4, and according to the present invention. The immunoassay element was shown to be highly reproducible and highly accurate.
[0101]
[Example 8]
  The storage stability of the slide 3 of Example 5 and the comparative slide 4 was compared and examined. Dry analysiselementIs normally stable at about 4 ° C for about 1 month, but here, as an accelerated test, the slide was stored at 25 ° C, and the measured values on the 1st, 3rd, and 7th days after the slide were prepared were immediately ) And measured values. 10 μL each of CRP-containing standard samples CP1 (1.4 mg / dL), CP2 (4.2 mg / dL), and CP3 (10.0 mg / dL) on slide 3 (Example 5) and slide 4 (comparative example) on each day after preparation Spotted and kept at 37 ° C., the reflection optical density at 650 nm was measured from the support side, and the difference in reflection optical density (ΔOD after 4 and 6 minutes after spotting)_6-4) The results are shown in Tables 3 and 4 below.
[0102]
[Table 3]

[0103]
[Table 4]

[0104]
  Conventional unprocessedCarboxymethylIn slide 4 (comparative example) using modified starch as a substrate, ΔOD over time by storage at 25 ° C._6-4The value of was decreased, and a decrease of 5 to 10% was observed on the third day, and a large decrease of about 15 to 20% was observed on the seventh day. In contrast, the endo-type reaction specific substrate according to the present invention was used.CarboxymethylIn slide 3 (Example 5) using modified starch, ΔOD_6-4The fluctuation of the value was a slight decrease of 2 to 4% on the third day, and only a decrease of about 3 to 5% was observed even on the seventh day of storage. Thus, it was shown that the immunoassay element according to the present invention is excellent in storage stability.
[0105]
[Example 9]
  Preparation of endo-type reactive substrate (Part 2)
  Carboxymethyl8960 g of ultrapure water was added to 453 g of modified starch (Expprotab, manufactured by Edward Mendel Company Inc.), and the mixture was swollen by stirring for about 4 hours. To this, 1139 g of 0.5 N NaOH was added and stirred for about 1 hour for alkali treatment. Thereafter, 32 g of acetic acid was added to return to pH 7. After adjusting the pH to 5.7 with MES (2-monophorinoethanesulfonic acid) buffer, 80 g of glucoamylase enzyme solution (Toyobo, 320 k Unit / L, 5 mM MES, pH 6.0) was added. And reacted at 37 ° C. for 12 to 20 hours. After the reaction, pure water is added to make the total volume 140L, and this suspension is then converted into a ceramic membrane filter (manufactured by NGK, CEFILT-MF (registered trademark), monolith type, pore size 2 μm, membrane area 0.24 m).²). Soluble components were removed by circulating pure water through the ceramic membrane filter until the electric conductivity of the permeated water was 20 μS / cm or less. CM starch residue was collected from the filter, and pure water was added to make 100 L. About 3.6 L was centrifuged at 7500 rpm for 10 minutes, and the residue was collected. About 18 L of isopropyl alcohol was added thereto, and the precipitated white substance was collected by vacuum filtration. The operation from this centrifugation to isopropyl alcohol precipitation was performed about 26 times, and these were collected and dried at 35 ° C. for 24 hours. By the above operation, 220 g of glucoamylase-treated CM-modified starch (hereinafter also referred to as GA-treated CMS) specific to an endo-type enzyme reaction was obtained.
[0106]
CM-modified starch treated in the same manner as above except that no glucoamylase treatment was used was used as a comparative example (glucoamylase-untreated CM-modified starch: hereinafter also referred to as untreated CMS).
[0107]
[Example 10]
The degree of carboxymethylation of the GA-treated CMS and untreated CMS obtained in Example 9 and the degree of swelling when dispersed in water were examined. The degree of carboxymethylation is the ratio of glucose units modified with carboxymethyl groups to the total glucose units.¹³Investigated by C-NMR. The degree of swelling was defined as the degree of swelling after 10 mL of a 0.7% aqueous solution of each CMS was prepared, placed in a test tube and allowed to stand at 25 ° C. for 30 minutes, and then the volume (mL) of precipitated CMS was defined.
[0108]
  As shown in Table 5 below, the glucoamylase-treated CM starch has an increased degree of carboxymethylation, with 28% of all constituent glucose unitsCarboxymethylIt was found to have a group. This is due to glucoamylase treatment from the non-reducing end.CarboxymethylSince the glucoside bond was hydrolyzed until just before the conversion site, the amount of non-CM conversion glucose units in the CM conversion starch decreased, and as a result, the ratio of CM conversion glucose units increased. It is also a hydrophilic groupCarboxymethylIt is considered that since the introduction rate of the group was increased, the hydration was facilitated and the degree of swelling was increased.
[0109]
[Table 5]

[0110]
Example 11
Reactivity of GA-treated CMS with glucoamylase
The glucoamylase-treated CM starch obtained in Example 9 and the glucoamylase-untreated CM starch of Comparative Example were each prepared in 5 mM MES buffer (pH 6.0, 0.5% Block Ace (manufactured by Snow Brand Milk Products), 68 μM CaCl).₂Content) to obtain a 0.2% (W / V) dispersion. To this 0.2% CMS dispersion, 70 μL of a glucoamylase solution (manufactured by Toyobo, 320 k Unit / L, 5 mM MES, pH 6.0) was added and allowed to react at 37 ° C. for 60 minutes with shaking at 120 rpm. After the reaction, 70 μL of 10% phosphoric acid was added, and the reaction was stopped by cooling with ice. The reaction solution was centrifuged at 12,000 rpm for 4 minutes to precipitate unreacted CMS. 100 μL of the centrifugal supernatant containing the produced glucose was added to 3 mL of reaction solution A having the following composition, and the color reaction was carried out by shaking at 120 rpm for 30 minutes at 37 ° C., and the absorbance at a wavelength of 727 nm was measured. A calibration curve was prepared by adding 100 μL of glucose solution with a known concentration to 3 mL of the reaction solution and performing the same color reaction.
[0111]

[0112]
The measurement was performed 5 times for GA-treated CMS and twice for untreated CMS. As shown in Table 6, CM starch without glucoamylase treatment produced about 33 μg of glucose per gram, whereas CM starch with glucoamylase treatment in advance had an average of 0.1 μg or less of glucose. Did not produce. This indicates that most of the non-reducing terminal glucose of the GA-treated CMS of the present invention is modified or at the sugar chain branching point and does not serve as a substrate for glucoamylase, which is an exo-reactive enzyme.
[0113]
[Table 6]

[0114]
Example 12
Reactivity of GA-treated CMS with α-amylase (wet method)
The glucoamylase-treated CM starch obtained in Example 9 and the glucoamylase-untreated CM starch of Comparative Example were each prepared in 5 mM MES buffer (pH 6.0, 0.5% Block Ace (manufactured by Snow Brand Milk Products), 68 μM CaCl).₂Content) to obtain a 0.2% (W / V) dispersion. To 7 mL of this 0.2% CMS dispersion, 70 μL of α-amylase solution having various concentrations was added and reacted while shaking at 120 rpm for 60 minutes at 37 ° C. After the reaction, 70 μL of 10% phosphoric acid was added, and the reaction was stopped by cooling with ice. The reaction solution was centrifuged at 12,000 rpm for 4 minutes to precipitate unreacted CMS. 100 μL of the centrifugal supernatant containing the generated oligosaccharide was added to 3 mL of reaction solution B having the following composition, and the color reaction was performed by shaking at 120 rpm for 30 minutes at 37 ° C., and the absorbance at a wavelength of 727 nm was measured. Using a calibration curve created by adding 100 μL of an oligosaccharide (maltotetraose) solution of known concentration to 3 mL of reaction solution B and carrying out the same color reaction, a graph was created in terms of the generated oligosaccharide (see FIG. 7).
[0115]

[0116]
As shown in FIG. 7, in the glucoamylase-treated CM starch, the reactivity to α-amylase which is an endoactive enzyme (in the figure, the slope of the calibration curve) is about half that of untreated CM starch. It was decreasing. However, the background value of the untreated CMS was about 1.3 ppm, whereas the background value of the GA-treated CMS could be made almost zero.
[0117]
The reason why the oligosaccharide produced from untreated CMS is not zero even though the amount of α-amylase is zero is considered to be as follows. The vertical axis in FIG. 7 represents the oligosaccharide concentration, but in the reaction system of this example, the amount of glucose finally produced is quantified. Each CMS after reacting with α-amylase is removed by centrifugation, but a trace amount of CMS particles remains in the supernatant. If this trace amount of CMS is mixed when the supernatant is collected as the produced oligosaccharide, the CMS particles are then attacked by glucoamylase contained in the color reaction solution B. In the untreated CMS, since the terminal glucose is not removed, the terminal glucose is hydrolyzed and detected by glucoamylase. Since this amount does not depend on the amount of α-amylase used, it is almost constant, so the whole calibration curve is pushed up as a background value in FIG.
[0118]
On the other hand, CM-modified starch pretreated with glucoamylase does not have an unmodified glucose end that reacts with glucoamylase, which is an exoactive enzyme. For this reason, even if CMS particles are mixed into the color reaction system, the background value is not increased.
[0119]
  The reason why the reactivity of CM starch to α-amylase is decreased by glucoamylase treatment is considered to be as follows. α-Amylase exhibits reactivity with polysaccharides having a glucose chain length of 4 or more molecules, and the higher the degree of polymerization (long chain length), the higher the reactivity. Glucose in sugar chain is at randomCarboxymethylAs a result, the continuous portion of glucose molecules where α-amylase is highly reactive decreases. As a result, starch reacts with α-amylase by carboxymethylation. When the glucoamylase is further treated, each branch of the CM starch is decomposed and removed from the non-reducing end. If there is a CM site in the middle of the branch chain, it is hydrolyzed and removed in glucose units from the non-reducing end up to that site. For this reason, the glucoamylase-treated CM-modified starch becomes a kind of limit dextrin, and the continuous portion of glucose molecules where α-amylase shows high reactivity apparently further decreases. FIG. 7 illustrates this.
[0120]
Thus, although it is a glucoamylase-treated CM starch having reduced reactivity to α-amylase, as shown in Examples 13 and 14 below, in the environment within the dry analytical element, reactivity to α-amylase. Has increased.
[0121]
Example 13
Reactivity of GA-treated CMS with α-amylase (dry method)
Using the glucoamylase-treated CM starch obtained in Example 9, a multilayer analytical element was produced in exactly the same procedure and configuration as in Example 5.
[0122]
That is, a slide having exactly the same structure as in Example 2 was prepared except that the glucoamylase-treated CM-modified starch obtained in Example 9 was used for the substrate layer instead of the carboxymethylated starch used in Example 2, and thereafter In the same manner as in Example 5, 3 mg / m of amylase-labeled CRP-IgG was further added to the tricot knitted fabric layer serving as the substrate layer / developing layer.²A multi-layer immunoslide 5 for CRP analysis (Example 13) was prepared by applying an ethanol solution so that the coating amount would be, impregnating and drying.
[0123]
  As a comparative example, glucoamylase treatmentCarboxymethylUntreated instead of modified starchCarboxymethylA comparative slide 6 having the same configuration as that of Example 13 was prepared except that the modified starch was used as a substrate.
[0124]
Example 14
When this slide 5 and slide 6 contain CRP at concentrations of 0, 1.4, 4.2, and 10 mg / dL, 10 μL of 50 mM glycerophosphate buffer solution (pH 7) is spotted and maintained at 37 ° C. The reflection optical density from 625 nm to 625 nm was measured over time. FIG. 8 shows the change over time in the reflection optical density after spotting the CRP solution.
[0125]
When 0 mg / dL of CRP solution is spotted, α-amylase in the labeled antibody is not sterically hindered by binding to the antigen (CRP), so the hydrolytic activity on CM starch that is a non-diffusible substrate is maximum. It becomes. Thus, when the CRP concentration at which α-amylase exhibits the maximum activity is 0 mg / dL, the slide 5 using GA-treated CMS (Example 14, right side of FIG. 8) uses the comparative example (untreated CMS). Compared to the slide 6 and the left side of FIG. Using the slope of the change in reflection optical density over time as an index, the glucoamylase-treated CM starch increased the reactivity to α-amylase to about twice that of the untreated one.
[0126]
As the steric hindrance to α-amylase increases as the concentration of CRP as an antigen increases, the decrease in reactivity to α-amylase is more marked in slide 5 (Example 14, right side of FIG. 8) using GA-treated CMS. It was. This indicates that the reactivity of α-amylase to CM starch in the dry analytical element is such that the change in the reflection optical density with respect to the change in CRP concentration can be achieved by treating the α-amylase with glucoamylase in advance to make the endoactive enzyme reaction specific. Large and sensitive.
[0127]
  Finally, preferred embodiments of the present invention are summarized as follows.
  (1) Either a reaction between an antigen and an enzyme-labeled antibody, a reaction between an antigen and an antibody and an enzyme-labeled antigen, or a reaction between an antigen, a conjugate of an antigen and a polymer compound, and an enzyme-labeled antibody In an immunoassay element that analyzes the amount of antigen by measuring the change in the labeling enzyme activity caused by the reaction of
  A non-diffusible substrate comprising a substrate layer containing a non-diffusible substrate that produces a diffusible substance with the labeling enzyme, and a reagent layer containing a depolymerizing enzyme that further converts the diffusible substance into a low-molecular product. Is a substrate that reacts only with the labeling enzyme and does not react with the depolymerizing enzyme.
  (2) The non-diffusible substrate is a high molecular weight polysaccharide, the labeling enzyme is an endoactive carbohydrate degrading enzyme, and the converting enzyme is an exoactive glycolytic enzyme. (1) The immunoassay element as described.
  (3) The labeling enzyme is an endoactive carbohydrate hydrolase, the low molecular weight enzyme is an exoactive carbohydrate hydrolase, and the non-diffusible substrate is modified with non-reducing terminal glucose The immunoassay element according to (1), which is an endo-type selective reactive substrate.
  (4) The non-reducing terminal glucose is an endo-type selective reactive substrate.CarboxymethylThe immunoassay element according to (3), which is modified with a group.
  (5) The labeling enzyme is α-amylase, the depolymerizing enzyme is glucoamylase or α-glucosidase, and the non-diffusible substrate is from a non-reducing terminal glucose site.CarboxymethylLimited to the modified glucose unit site or glucose chain branch site in advance by exo-active carbohydrate hydrolaseCarboxymethylThe immunoassay element according to (3), which is a modified starch.
  (6) The immunoassay element according to (1), wherein the reagent layer is formed of a layer containing a hydrophilic polymer as a binder.
  (7) The immunoassay element according to (1), wherein the substrate layer is a porous layer made of a porous medium.
  (8) The immunoassay element according to (1), wherein the enzyme-labeled antibody is contained in the substrate layer or a layer laminated on the substrate layer.
  (9) The immunoassay element according to (1), wherein the antibody is contained in the substrate layer or a layer laminated on the substrate layer.
(10) The immunoassay element according to (1), wherein the enzyme-labeled antigen is contained in the substrate layer or a layer laminated on the substrate layer.
(11) The immunoassay element according to (1), wherein the antibody and the enzyme-labeled antigen are contained in the substrate layer or a layer laminated on the substrate layer.
(12) The conjugate of antigen and polymer compound and the enzyme-labeled antibody are contained in the substrate layer or a layer laminated on the substrate layer. ) The immunoassay element described.
(13) The reagent composition that reacts with the low-molecular-weight product to produce a dye having visible absorption is contained in the reagent layer or other water-permeable layer. Immunoassay element.
(14) The immunoassay element according to (13), wherein the reagent composition contains a leuco dye that develops color by oxidation.
(15) The immunoassay element according to (14), wherein the reagent composition comprises a dispersion of an aqueous solution of a leuco dye in an aqueous solution.
(16) The immunoassay element according to (15), wherein the reagent composition contains a peroxidase and a leuco dye.
(17) In an immunoassay element for analyzing the amount of antibody by measuring a change in labeled enzyme activity caused by a reaction between an antibody and an enzyme-labeled antigen, or a reaction between an antibody, an antigen and an enzyme-labeled antibody,
  A non-diffusible substrate comprising a substrate layer containing a non-diffusible substrate that produces a diffusible substance with the labeling enzyme, and a reagent layer containing a depolymerizing enzyme that further converts the diffusible substance into a low-molecular product. Is a substrate that reacts only with the labeling enzyme and does not react with the depolymerizing enzyme.
(18) The non-diffusible substrate is a high molecular weight polysaccharide, the labeling enzyme is an endoactive carbohydrase, and the converting enzyme is an exoactive carbohydrase. (17) The immunoassay element as described.
(19) The labeling enzyme is an endoactive carbohydrate hydrolase, the low molecular weight enzyme is an exoactive carbohydrate hydrolase, and the non-diffusible substrate is modified with non-reducing terminal glucose The immunoassay element according to (17), which is an endo-type selective reactive substrate.
(20) The endo-type selective reactive substrate is non-reducing terminal glucose.CarboxymethylThe immunoassay element according to (19), which is modified with a group.
(21) The labeling enzyme is α-amylase, the depolymerizing enzyme is glucoamylase or α-glucosidase, and the non-diffusible substrate is from a non-reducing terminal glucose site.CarboxymethylUp to the modified glucose unit site or glucose chain branch siteExoLimited degradation by active carbohydrate hydrolaseCarboxymethylThe immunoassay element according to (19), which is a modified starch.
(22) The immunoassay element according to (17), wherein the reagent layer is formed of a layer containing a hydrophilic polymer as a binder.
(23) The immunoassay element according to (17), wherein the substrate layer is a porous layer made of a porous medium.
(24) The immunoassay element according to (17), wherein the enzyme-labeled antibody is contained in the substrate layer or a layer laminated on the substrate layer.
(25) The immunoassay element according to (17), wherein the antibody is contained in the substrate layer or a layer laminated on the substrate layer.
(26) The immunoassay element according to (17), wherein the enzyme-labeled antigen is contained in the substrate layer or a layer laminated on the substrate layer.
(27) The immunoassay element according to (17), wherein the antibody and the enzyme-labeled antigen are contained in the substrate layer or a layer laminated on the substrate layer.
(28) The conjugate of antigen and polymer compound and the enzyme-labeled antibody are contained in the substrate layer or a layer laminated on the substrate layer. ) The immunoassay element described.
(29) The reagent composition that reacts with the low-molecular-weight product to produce a dye having visible absorption is contained in the reagent layer or other water-permeable layer. Immunoassay element.
(30) The immunoassay element according to (29), wherein the reagent composition contains a leuco dye that develops color by oxidation.
(31) The immunoassay element according to (30), wherein the reagent composition comprises a dispersion of an aqueous solution of a leuco dye in an aqueous solution.
(32) The immunoassay element according to (29), wherein the reagent composition contains a peroxidase and a leuco dye.
[Brief description of the drawings]
FIG. 1 is a block diagram of an embodiment of an immunoassay element of the present invention.
FIG. 2 is a block diagram of another embodiment of the immunoassay element of the present invention.
FIG. 3 is a block diagram of still another embodiment of the immunoassay element of the present invention.
FIG. 4 is a diagram showing the results of Example 4, showing changes over time in reflected optical density when a labeled enzyme-free buffer solution is spotted on the immunoassay elements of Example Slide 1 and Comparative Example Slide 2; It is.
5 is a diagram showing the results of Example 4, and is a diagram showing calibration curves of immunological analysis elements of Example Slide 1 and Comparative Example Slide 2. FIG.
6 is a diagram showing the results of Example 6, and is a diagram showing calibration curves of CRP analysis immunoassay elements of Example Slide 3 and Comparative Example Slide 4. FIG.
7 is a graph showing the results of Example 12, in which the glucoamylase-treated CM starch obtained in Example 9 and the glucoamylase-untreated CM starch of Comparative Example were compared with α-amylase in a dispersion. FIG. The reactivity was investigated.
FIG. 8 is a diagram showing the results of Example 14, showing the change in reflection optical density over time after spotting CRP solutions of various concentrations on Example Slide 5 and Comparative Example Slide 6 created in Example 13; FIG.
[Explanation of symbols]
10 Translucent support
12 Reagent layer
14 Substrate layer
16 Water-permeable layer containing antibody (or enzyme-labeled antigen)
18 Water-permeable layer containing enzyme-labeled antigen (or antibody)
20 Water-permeable layer containing antibody and enzyme-labeled antigen

Claims (2)

Either a reaction between an antigen and an enzyme-labeled antibody, a reaction between an antigen and an antibody and an enzyme-labeled antigen, or a reaction between an antigen, a conjugate of an antigen and a polymer compound, and an enzyme-labeled antibody In an immunoassay element that analyzes the amount of antigen by measuring the resulting change in labeled enzyme activity,
A substrate layer containing a non-diffusible substrate that produces a diffusible substance with the labeling enzyme, and a reagent layer containing a depolymerizing enzyme that further converts the diffusible substance into a low-molecular product,
The labeling enzyme is α- amylase, said fragmenting enzyme is glucoamylase or α- glucosidase, met substrate wherein the non-diffusible substrate is not react with unreacted said fragmenting enzyme with said labeling enzyme And carboxymethylated starch previously limited to carboxymethyl-modified glucose units by exo-active sugar hydrolase from the non-reducing terminal glucose site to the carboxymethyl-modified glucose unit site or the glucose chain branching site. Analysis element. In an immunoassay element that analyzes the amount of antibody by measuring the change in labeled enzyme activity caused by the reaction between the antibody and enzyme-labeled antigen, or the reaction between the antibody and antigen and the enzyme-labeled antibody,
A substrate layer containing a non-diffusible substrate that produces a diffusible substance with the labeling enzyme, and a reagent layer containing a depolymerizing enzyme that further converts the diffusible substance into a low-molecular product,
The labeling enzyme is α- amylase, said fragmenting enzyme is glucoamylase or α- glucosidase, met substrate wherein the non-diffusible substrate is not react with unreacted said fragmenting enzyme with said labeling enzyme And carboxymethylated starch previously limited to carboxymethyl-modified glucose units by exo-active sugar hydrolase from the non-reducing terminal glucose site to the carboxymethyl-modified glucose unit site or the glucose chain branching site. Analysis element.

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