JP3618168B2 - Measuring method of reverse transcriptase activity blocking antibody - Google Patents

Description

【００５５】
表１に示されるように、どのマグネシウムイオン濃度でも逆転写酵素と検体との不可逆的結合による鋳型ＲＮＡからのＤＮＡ伸長反応阻害抗体群を活性阻害量として定量できた。
また、オリゴｄＴ−ポリＡ固相化プレートでの逆転写酵素の結合反応の時に、０．１％トリトンＸ−１００、逆転写酵素５ｍＵ／１００μｌ溶液中にマグネシウムが濃度０で試験した結果は、ＨＩＶ感染者Ｈ．Ｋ．で５６％、Ｋ．Ｋ．で６３％、Ｋ．Ｓ．で７１％、ＨＩＶ非感染者Ｍ．Ｔ．で１０３．４％、Ｔ．Ｅ．で１００．３％、Ｔ．Ｔ．で１１０．４％の残存活性であった。
【００５６】
【実施例２】
実施例１の（１）オリゴｄＴ−ポリＡ固相化プレートの調製に従って、オリゴｄＴ−ポリＡ固相化プレートを調製した。調製したプレートに実施例１の（２）オリゴｄＴ−ポリＡ固相化プレートでの逆転写酵素の結合反応の中の塩化マグネシウムのかわりに塩化マンガンまたは塩化カルシウムを用いて逆転写酵素結合プレートを調製した。調製した酵素結合プレートを実施例１の（３）逆転写酵素結合プレートと被検液との反応、（４）逆転写酵素活性検出、（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。その結果を表２に示した。
【００５７】
【表２】

【００５８】
表２に示されるように、用いたどの金属イオンにおいても、逆転写酵素と検体との不可逆的結合による鋳型ＲＮＡからのＤＮＡ伸長反応阻害抗体群を活性阻害量として定量でき、しかもどのイオン濃度でも定量できた。
【００５９】
【実施例３】
実施例１の（１）オリゴｄＴ−ポリＡ固相化プレートの調製における、オリゴｄＴ_{１９−２４} のかわりにオリゴｄＴ_５ 、オリゴｄＴ_１０（シグマ社製）、オリゴｄＴ_{２５−３０} 、ポリｄＴ（平均鎖長７９４塩基）（ファルマシア社製）を用いて、オリゴｄＴ−ポリＡ固相化プレートを調製した。調製したプレートに実施例１の（２）オリゴｄＴ−ポリＡ固相化プレートでの逆転写酵素の結合反応の中の塩化マグネシウム濃度を５０ｍＭとして用い、逆転写酵素結合プレートを調製した。調製した酵素結合プレートを実施例１の（３）逆転写酵素結合プレートと被検液との反応、（４）逆転写酵素活性検出、（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。その結果を表３に示した。
【００６０】
【表３】

【００６１】
表３に示されるように、用いたどの長さのオリゴｄＴでも、逆転写酵素と検体との不可逆的結合による鋳型ＲＮＡからのＤＮＡ伸長反応阻害抗体群を活性阻害量として定量できた。
【００６２】
【実施例４】
実施例１の（１）オリゴｄＴ−ポリＡ固相化プレートの調製におけるポリＡのかわりにオリゴＡ_{１２ー１８} 、ポリＡ（平均鎖長５３４塩基）（ファルマシア社製）を用いて、オリゴｄＴ−ポリＡ固相化プレートを調製した。調製したプレートに実施例１の（２）オリゴｄＴ−ポリＡ固相化プレートでの逆転写酵素の結合反応の中の塩化マグネシウム濃度を５０ｍＭとして用い、逆転写酵素結合プレートを調製した。調製した酵素結合プレートを実施例１の（３）逆転写酵素結合プレートと被検液との反応、（４）逆転写酵素活性検出、（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。その結果を表４に示した。
【００６３】
【表４】

【００６４】
表４に示されるように、用いたどの長さのポリＡでも、逆転写酵素と検体との不可逆的結合による鋳型ＲＮＡからのＤＮＡ伸長反応阻害抗体群を活性阻害量として定量できた。
【００６５】
【実施例５】
実施例１の（１）オリゴｄＴ−ポリＡ固相化プレートの調製に従いオリゴｄＴ−ポリＡ固相化プレートを調製した。調製したプレートに実施例１の（２）オリゴｄＴ−ポリＡ固相化プレートでの逆転写酵素の結合反応の中の塩化マグネシウム濃度を５０ｍＭとして用い、逆転写酵素量を０．１、１、１０、１００、１０００、１００００、１０００００ｍＵ／１００μｌの濃度の逆転写酵素を用いて、逆転写酵素結合プレートを調製した。調製した酵素結合プレートを用い、実施例１の（３）逆転写酵素結合プレートと被検液との反応、における、血清をＨＩＶ感染血友病患者５名のを用いて反応を行った。
【００６６】
その後実施例１の（４）逆転写酵素活性検出、（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。 また、これとは別に、上記手法の中で、逆転写酵素量を０．１、１、１０μＵ／１００μｌの濃度の逆転写酵素を用いた時、実施例１の（４）逆転写酵素活性の検出において、２時間の逆転写酵素反応のかわりに２４時間の逆転写酵素反応を行い、その他の操作条件は、同一で実施した。その結果を表５に示した。
【００６７】
【表５】

【００６８】
表５に示されるように、用いたどの濃度の逆転写酵素でも、逆転写酵素と検体との不可逆的結合による鋳型ＲＮＡからのＤＮＡ伸長反応阻害抗体群を活性阻害量として定量できた。
【００６９】
【実施例６】
実施例１の（１）オリゴｄＴ−ポリＡ固相化プレートの調製に従いオリゴｄＴ−ポリＡ固相化プレートを調製した。調製したプレートに実施例１の（２）オリゴｄＴ−ポリＡ固相化プレートでの逆転写酵素の結合反応の中の塩化マグネシウム濃度を５０ｍＭとして用い、逆転写酵素結合プレートを調製した。調製した酵素結合プレートを実施例１の（３）逆転写酵素結合プレートと被検液との反応における血清の希釈を１、１０、１００、１０００、１００００、１０００００倍希釈液をそれぞれ用いて行いて抗原抗体反応を行わせ、その後実施例１の（４）逆転写酵素活性検出、（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。その結果を表６に示した。
【００７０】
【表６】

【００７１】
表６に示されるように、用いたどの希釈濃度でも、逆転写酵素と検体との不可逆的結合による鋳型ＲＮＡからのＤＮＡ伸長反応阻害抗体群を活性阻害量として定量できた。
【００７２】
【実施例７】
（６）実施例１の（１）オリゴｄＴ−ポリＡ固相化プレートの調製に従いオリゴｄＴ−ポリＡ固相化プレートを調製した。
（７）逆転写酵素と被検液との抗原抗体反応
試験管に０．１％トリトンＸ−１００、ＨＩＶ−１型逆転写酵素（組換大腸菌による発現体；生化学工業社製）０．２ｍＵ／１００μｌ溶液中に０．０２、０．２、２、２０、２００、２０００ｍＭの塩化マグネシウム濃度となるように調製し酵素液とした。この酵素液に別の試験管に用意したＨＩＶ感染血友病患者５名およびＨＩＶ非感染者５名の血清をそれぞれ１０ｍＭリン酸緩衝液（ｐＨ７．４）、１５０ｍＭ塩化ナトリウム、０．１％トリトンＸ−１００溶液で２００倍に希釈した被検液を別の試験管中で２００μｌずつ等量混合し、３７℃で１時間反応させた。また、対照群として血清希釈液のかわりに１０ｍＭリン酸緩衝液（ｐＨ７．４）、１５０ｍＭ塩化ナトリウム、０．１％トリトンＸ−１００溶液を用いて同様に操作した。
（８）オリゴｄＴ−ポリＡ固相化プレートへの逆転写酵素の結合反応
（７）の反応後、反応液の１００μｌを（６）で調製したオリゴｄＴ−ポリＡ固相化プレートへ添加し、３７℃で１時間インキュベートした。インキュベート後、液を捨て、プレートを０．０２％のポリオキシエチレン（２０）ソルビタンモノラウレートを含むＴＢＳ溶液で５回洗浄した。洗浄したプレートを実施例１の（４）逆転写酵素活性検出、（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。その結果を表７に示した。
【００７３】
【表７】

【００７４】
表７に示されるように、どのマグネシウムイオン濃度でも、酵素と鋳型−プライマーとの結合阻害抗体群を活性阻害量として定量できた。
また、逆転写酵素と被検液との抗原抗体反応時に酵素液濃度５ｍＵ／１００μｌ、塩化マグネシウム濃度を０濃度としたときに結果は、ＨＩＶ感染者Ｈ．Ｋ．で２３．２％、Ｋ．Ｋ．で５４．４％、Ｋ．Ｓ．で４８．０％、ＨＩＶ非感染者Ｍ．Ｔ．で１１１．．％、Ｔ．Ｅ．で１０４．８％で、Ｔ．Ｔ．で１０２．７％の残存活性であった。
【００７５】
【実施例８】
実施例７の（６）オリゴｄＴ−ポリＡ固相化プレートの調製に従いオリゴｄＴ−ポリＡ固相化プレートを調製した。試験管に実施例７の（７）逆転写酵素と被検液との抗原抗体反応における塩化マグネシウムのかわりに塩化マンガンまたは塩化カルシウムを用いて反応させた。反応液を実施例７の（８）オリゴｄＴ−ポリＡ固相化プレートへの逆転写酵素の結合反応、実施例１の（４）逆転写酵素活性検出、（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。その結果を表８に示した。
【００７６】
【表８】

【００７７】
表８に示されるように、用いたどの金属イオンにおいても、酵素と鋳型−プライマーとの結合阻害抗体群を活性阻害量として定量でき、しかもどのイオン濃度でも定量できた。
【００７８】
【実施例９】
実施例７の（６）オリゴｄＴ−ポリＡ固相化プレートの調製における、オリゴｄＴ_{１９−２４} のかわりにオリゴｄＴ_５ 、オリゴｄＴ_１０（シグマ社製）、オリゴｄＴ_{２５−３０} 、ポリｄＴ（平均鎖長７９４塩基）（ファルマシア社製）を用いて、オリゴｄＴ−ポリＡ固相化プレートを調製した。試験管中で、実施例７の（７）逆転写酵素と被検液との抗原抗体反応における塩化マグネシウム濃度を５０ｍＭとして同様に反応させ、調製しておいたオリゴｄＴ−ポリＡ固相化プレートへ反応液１００μｌを添加し、３７℃１時間インキュベート後、液を捨て、０．０２％のポリオキシエチレン（２０）ソルビタンモノラウレートを含むＴＢＳ溶液で５回洗浄した。洗浄したプレートを実施例１の（４）逆転写酵素活性検出、（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。その結果を表９に示した。
【００７９】
【表９】

【００８０】
表９に示されるように、用いたどの長さのオリゴｄＴでも、酵素と鋳型−プライマーとの結合阻害抗体群を活性阻害量として定量できた。
【００８１】
【実施例１０】
実施例７の（６）オリゴｄＴ−ポリＡ固相化プレートの調製におけるポリＡのかわりにオリゴＡ_{１２ー１８} 、ポリＡ（平均鎖長５３４塩基）（ファルマシア社製）を用いて、オリゴｄＴ−ポリＡ固相化プレートを調製した。試験管中で、実施例７の（７）逆転写酵素と被検液との抗原抗体反応における塩化マグネシウム濃度を５０ｍＭとして同様に反応させ、調製しておいたオリゴｄＴ−ポリＡ固相化プレートへ反応液１００μｌを添加し、３７℃１時間インキュベート後、液を捨て、０．０２％のポリオキシエチレン（２０）ソルビタンモノラウレートを含むＴＢＳ溶液で５回洗浄した。洗浄したプレートを実施例１の（４）逆転写酵素活性検出、（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。その結果を表１０に示した。
【００８２】
【表１０】

【００８３】
表１０に示されるように、用いたどの長さのポリＡでも、酵素と鋳型−プライマーとの結合阻害抗体群を活性阻害量として定量できた。
【００８４】
【実施例１１】
実施例７の（６）オリゴｄＴ−ポリＡ固相化プレートの調製に従いオリゴｄＴ−ポリＡ固相化プレートを調製した。試験管中で実施例７の（７）逆転写酵素と被検液との抗原抗体反応における塩化マグネシウム濃度を５０ｍＭとして用い、逆転写酵素量を０．２、２、２０、２００、２０００、２００００、２０００００ｍＵ／１００μｌの濃度の逆転写酵素を用い、血清をＨＩＶ感染血友病患者５名のものを用いて同様に反応させた。この反応液を調製しておいたオリゴｄＴ−ポリＡ固相化プレートへ１００μｌ添加し、３７℃１時間インキュベート後、液を捨て、０．０２％のポリオキシエチレン（２０）ソルビタンモノラウレートを含むＴＢＳ溶液で５回洗浄した。
【００８５】
洗浄したプレートを実施例１の（４）逆転写酵素活性検出、（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。 また、これとは別に、上記手法の中で、逆転写酵素量を０．２、２、２０μＵ／１００μｌの濃度の逆転写酵素を用いた時、実施例１の（４）逆転写酵素活性の検出において、２時間の逆転写酵素反応のかわりに２４時間の逆転写酵素反応を行い、その他の操作条件は、同一で実施した。その結果を表１１に示した。
【００８６】
【表１１】

【００８７】
表１１に示されるように、用いたどの濃度の逆転写酵素でも、酵素と鋳型−プライマーとの結合阻害抗体群を活性阻害量として定量できた。
【００８８】
【実施例１２】
実施例７の（６）オリゴｄＴ−ポリＡ固相化プレートの調製に従いオリゴｄＴ−ポリＡ固相化プレートを調製した。試験管中で実施例７の（７）逆転写酵素と被検液との抗原抗体反応における塩化マグネシウム濃度を５０ｍＭとして用い、血清の希釈を２、２０、２００、２０００、２００００、２０００００倍希釈液をそれぞれ用いて同様に抗原抗体反応を行わせた。この反応液を調製しておいたオリゴｄＴ−ポリＡ固相化プレートへ１００μｌ添加し、３７℃１時間インキュベート後、液を捨て、０．０２％のポリオキシエチレン（２０）ソルビタンモノラウレートを含むＴＢＳ溶液で５回洗浄した。洗浄したプレートを実施例１の（４）逆転写酵素活性検出、（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。その結果を表１２に示した。
【００８９】
【表１２】

【００９０】
表１２に示されるように、用いたどの希釈濃度でも、酵素と鋳型−プライマーとの結合阻害抗体群を活性阻害量として定量できた。
【００９１】
【実施例１３】
（９）オリゴｄＴ−ポリＡ固相化プレートの調製
実施例１の（１）オリゴｄＴ−ポリＡ固相化プレートの調製に従い、オリゴｄＴ−ポリＡ固相化プレートを調製した。
（１０）オリゴｄＴ−ポリＡ固相化プレート上での逆転写酵素の結合反応
調製したオリゴｄＴ−ポリＡ固相化プレートから、実施例１の（２）オリゴｄＴ−ポリＡ固相化プレートでの逆転写酵素の結合反応に従い、逆転写酵素結合プレートを調製した。
（１１）抗原抗体反応と逆転写酵素反応の同時反応
調製したプレートに、ＨＩＶ感染血友病患者５名およびＨＩＶ非感染者５名の血清をそれぞれ１０ｍＭリン酸緩衝液（ｐＨ７．４）、１５０ｍＭ塩化ナトリウム、０．１％トリトンＸ−１００溶液で１００倍に希釈した溶液５０μｌを添加し、さらに、１８０ｍＭＨＥＰＥＳ（ｐＨ７．８）、２５２ｍＭ塩化カリウム、１８ｍＭ塩化マグネシウム、０．９ｍＭジチオスレイトール、２．１６ｍＭ還元型グルタチオン、２％トリトンＸ−１００、３．６％エチレングリコール、２３４μＭデオキシチミジントリフォスフェート（ｄＴＴＰ）、６μＭビオチン標識デオキシウリジントリフォスフェート（Ｂｉｏ−ｄＵＴＰ；ベーリンガーマンハイム社製）溶液を５０μｌ添加し３７℃で２時間インキュベートした。インキュベート後５Ｍ塩化ナトリウム溶液を１０μｌ添加しプレートを室温で５分静置したのち液を捨て、０．０２％のポリオキシエチレン（２０）ソルビタンモノラウレートを含むＴＢＳ溶液で５回洗浄した。
【００９２】
プレートに、５０ｍＭトリス−塩酸緩衝液（ｐＨ７．５）、０．５Ｍ塩化ナトリウム、５ｍＭ塩化マグネシウム、２％牛血清アルブミン溶液中にアルカリフォスファターゼ標識ストレプトアビジン（ジャクソンイムノリサーチラボラトリー社製）を２００００倍希釈した溶液を各ウエルに１００μｌずつ添加し、３７℃で１時間インキュベート後、０．０２％のポリオキシエチレン（２０）ソルビタンモノラウレートを含むＴＢＳ溶液で５回洗浄した。
【００９３】
洗浄後、ジエタノールアミン−塩酸緩衝液（ｐＨ９．５）、１ｍＭ塩化マグネシウム溶液に１ｍｇ／ｍｌの濃度となるように溶解したパラニトロフェニルフォスフェートジソディウム（シグマ社製）溶液を１５０μｌ添加し３７℃で３０分インキュベート後、１Ｎの水酸化ナトリウム溶液を５０μｌ添加し、４０５ｎｍの吸光度をプレートリーダーにて測定した。この後、実施例１の（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。その結果を表１３に示した。
【００９４】
【表１３】

【００９５】
表１３に示されるように、どのマグネシウムイオン濃度でも、逆転写酵素と抗体との競合反応による鋳型ＲＮＡからのＤＮＡ伸長反応阻害抗体群を活性阻害量として定量できた。
また、オリゴｄＴ−ポリＡ固相化プレートでの逆転写酵素の結合反応の時に、０．１％トリトンＸ−１００、逆転写酵素５ｍＵ／１００μｌ溶液中にマグネシウムが濃度０で試験した結果は、ＨＩＶ感染者Ｈ．Ｋ．で３３．２％、Ｋ．Ｋ．で２８．４％、Ｋ．Ｓ．で４９．２％、ＨＩＶ非感染者Ｍ．Ｔ．で１０２．６％、Ｔ．Ｅ．で１０６．５％、Ｔ．Ｔ．で１０７．１％の残存活性であった。
【００９６】
【実施例１４】
実施例１３の（９）オリゴｄＴ−ポリＡ固相化プレートの調製に従って、オリゴｄＴ−ポリＡ固相化プレートを調製した。調製したプレートに実施例１３の（１０）オリゴｄＴ−ポリＡ固相化プレート上での逆転写酵素の結合反応における塩化マグネシウムのかわりに塩化マンガンまたは塩化カルシウムを用いて逆転写酵素結合プレートを調製した。調製した酵素結合プレートを実施例１３の（１１）抗原抗体反応と逆転写酵素反応の同時反応に従い同様に操作した。その後、実施例１の（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。その結果を表１４に示した。
【００９７】
【表１４】

【００９８】
表１４に示されるように、用いたどの金属イオンにおいても、逆転写酵素と抗体との競合反応による鋳型ＲＮＡからのＤＮＡ伸長反応阻害抗体群を活性阻害量として定量でき、しかもどのイオン濃度でも定量できた。
【００９９】
【実施例１５】
実施例１３の（９）オリゴｄＴ−ポリＡ固相化プレートの調製における、オリゴｄＴ_{１９−２４} のかわりにオリゴｄＴ_５ 、オリゴｄＴ_１０（シグマ社製）、オリゴｄＴ_{２５−３０} 、ポリｄＴ（平均鎖長７９４塩基）（ファルマシア社製）を用いて、オリゴｄＴ−ポリＡ固相化プレートを調製した。調製したプレートに実施例１３（１０）オリゴｄＴ−ポリＡ固相化プレート上での逆転写酵素の結合反応における塩化マグネシウム濃度を５０ｍＭとして用い、逆転写酵素結合プレートを調製した。調製した酵素結合プレートを実施例１３の（１１）抗原抗体反応と逆転写酵素反応の同時反応に従い同様に操作した。その後、実施例１の（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。その結果を表１５に示した。
【０１００】
【表１５】

【０１０１】
表１５に示されるように、用いたどの長さのオリゴｄＴでも、逆転写酵素と抗体との競合反応による鋳型ＲＮＡからのＤＮＡ伸長反応阻害抗体群を活性阻害量として定量できた。
【０１０２】
【実施例１６】
実施例１３の（９）オリゴｄＴ−ポリＡ固相化プレートの調製におけるポリＡのかわりにオリゴＡ_{１２ー１８} 、ポリＡ（平均鎖長５３４塩基）（ファルマシア社製）を用いて、オリゴｄＴ−ポリＡ固相化プレートを調製した。調製したプレートに実施例１３の（１０）オリゴｄＴ−ポリＡ固相化プレート上での逆転写酵素の結合反応における塩化マグネシウム濃度を５０ｍＭとして用い、逆転写酵素結合プレートを調製した。調製した酵素結合プレートを実施例１３の（１１）抗原抗体反応と逆転写酵素反応の同時反応に従い同様に操作した。その後、実施例１の（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。その結果を表１６に示した。
【０１０３】
【表１６】

【０１０４】
表１６に示されるように、用いたどの長さのポリＡでも、逆転写酵素と抗体との競合反応による鋳型ＲＮＡからのＤＮＡ伸長反応阻害抗体群を活性阻害量として定量できた。
【０１０５】
【実施例１７】
実施例１３の（９）オリゴｄＴ−ポリＡ固相化プレートの調製に従いオリゴｄＴ−ポリＡ固相化プレートを調製した。調製したプレートに実施例１３の（１０）オリゴｄＴ−ポリＡ固相化プレート上での逆転写酵素の結合反応における塩化マグネシウム濃度を５０ｍＭとして用い、逆転写酵素量を０．１、１、１０、１００、１０００、１００００、１０００００ｍＵ／１００μｌの濃度の逆転写酵素を用いて、逆転写酵素結合プレートを調製した。調製した酵素結合プレートを実施例１３の（１１）抗原抗体反応と逆転写酵素反応の同時反応、における血清をＨＩＶ感染血友病患者５名のものを用いその他は同様に操作した。その後、実施例１の（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。
【０１０６】
また、これとは別に、上記手法の中で、逆転写酵素量を０．１、１、１０μＵ／１００μｌの濃度の逆転写酵素を用いた時、実施例１３の（１１）抗原抗体反応と逆転写酵素反応の同時反応において、２時間の逆転写酵素反応のかわりに２４時間の逆転写酵素反応を行い、その他の操作条件は、同一で実施した。その結果を表１７に示した。
【０１０７】
【表１７】

【０１０８】
表１７に示されるように、用いたどの濃度の逆転写酵素でも、逆転写酵素と抗体との競合反応による鋳型ＲＮＡからのＤＮＡ伸長反応阻害抗体群を活性阻害量として定量できた。
【０１０９】
【実施例１８】
実施例１３の（９）オリゴｄＴ−ポリＡ固相化プレートの調製に従いオリゴｄＴ−ポリＡ固相化プレートを調製した。調製したプレートに実施例１３の（１０）オリゴｄＴ−ポリＡ固相化プレート上での逆転写酵素の結合反応における塩化マグネシウム濃度を５０ｍＭとして用い、逆転写酵素結合プレートを調製した。調製した酵素結合プレートを実施例１３の（１１）抗原抗体反応と逆転写酵素反応の同時反応における、血清の希釈を１、１０、１００、１０００、１００００、１０００００倍希釈液をそれぞれ用いて行いて抗原抗体反応を行わせ、同様に操作した。その後、実施例１の（５）逆転写酵素活性阻止抗体による活性阻止の定量を行った。その結果を表１８に示した。
【０１１０】
【表１８】

【０１１１】
表１８に示されるように、用いたどの希釈濃度でも、逆転写酵素と抗体との競合反応による鋳型ＲＮＡからのＤＮＡ伸長反応阻害抗体群を活性阻害量として定量できた。
【０１１２】
【発明の効果】
逆転写酵素活性阻止抗体の研究用試薬において、簡便かつ再現良く活性阻止のエピトープ群を３つに分類することでウイルスの変異を抗体によって追跡検討でき、この分野の研究コストの低減化がはかれる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring a reverse transcriptase activity blocking antibody derived from a human immunodeficiency virus in a sample. Specifically, in a method for measuring a reverse transcriptase activity blocking antibody derived from a human immunodeficiency virus in a specimen, at least a hybrid of at least a substantial oligodeoxythymine nucleotide on a solid phase and a substantial adenine ribopolynucleotide and reverse transcription In the binding reaction with an enzyme, at least the following four steps are performed, and preferably in the following step A, the binding reaction is performed in the presence of a water-soluble metal salt that generates a divalent metal ion. A method for measuring a reverse transcriptase activity blocking antibody, characterized in that step A: a substantial oligodeoxythymine nucleotide immobilized on a transparent or translucent or colored solid phase and a substantial hybridized thereto Binding reaction between adenine ribopolynucleotide and reverse transcriptase, step B: enzyme reacted in step A and unreacted enzyme After washing and separation, the sample containing a reverse transcriptase activity blocking antibody is reacted, and after washing and separating the antibody reacted in Step C: Step B and the unreacted antibody, deoxygenation is performed by reverse transcriptase on the solid phase. A step of synthesizing a nucleic acid complementary to the template RNA from mononucleotide triphosphate, and a step D: quantifying the inhibition of the enzyme activity of the antibody based on the amount of the synthesized nucleic acid.
[0002]
Alternatively, in a method for measuring a reverse transcriptase activity blocking antibody derived from human immunodeficiency virus in a specimen, at least a hybrid of at least a substantial oligodeoxythymine nucleotide and a substantial adenine ribopolynucleotide on a solid phase and a reverse transcriptase In the binding reaction, at least the following four steps are performed, and preferably in the following step F, the binding reaction is performed in the presence of a water-soluble metal salt that generates a divalent metal ion. In the method for measuring a reverse transcriptase activity blocking antibody, Step E: reacting a reverse transcriptase with a specimen containing a reverse transcriptase activity blocking antibody, and Step F: on a transparent, translucent or colored solid phase Immobilized solid oligodeoxythymine nucleotide and its hybridized adenine ribopolynucleotide After binding and reacting with the reaction solution of E, Step G: The reaction product in Step F and the unreacted product are washed and separated, and then complementary to the template RNA from deoxymononucleotide triphosphate by reverse transcriptase on the solid phase. Step H: A step of quantifying the inhibition of the enzyme activity of the antibody based on the amount of the synthesized nucleic acid.
[0003]
Alternatively, in a method for measuring a reverse transcriptase activity blocking antibody derived from human immunodeficiency virus in a specimen, at least a hybrid of at least a substantial oligodeoxythymine nucleotide and a substantial adenine ribopolynucleotide on a solid phase and a reverse transcriptase In the binding reaction, at least the following three steps are performed, and preferably in the following step I, the binding reaction is performed in the presence of a water-soluble metal salt that generates a divalent metal ion. In the method for measuring a reverse transcriptase activity blocking antibody, step I: a substantial oligodeoxythymine nucleotide immobilized on a transparent, translucent or colored solid phase and a substantial adenine hybridized thereto Ribopolynucleotide and reverse transcriptase are allowed to bind and react, Step J: Unreacted with the enzyme reacted in Step I After washing and separating the element, a sample containing a reverse transcriptase activity blocking antibody and deoxymononucleotide triphosphate are added simultaneously to synthesize a nucleic acid complementary to the template RNA by reverse transcriptase on the solid phase. Step K: relates to a step of quantifying inhibition of enzyme activity of an antibody based on the amount of synthesized nucleic acid.
[0004]
[Prior art]
Human immunodeficiency virus (HIV) infection has not yet been completely treated, and the process leading to the onset after a latent period of 5 to 10 years after infection has not been clarified. Therefore, the prediction of the onset time has not yet been established, and most of the conventional diagnosis of this infectious disease has been a method for determining the presence or absence of viral infection.
[0005]
Until now, as a method for diagnosing HIV infection, a method of detecting an antigen or antibody present in body fluid such as serum, plasma, saliva, urine, etc. of an infected person has been common.
As a method for detecting the antigen, for example, an antibody against the coat protein of HIV is immobilized on a solid phase such as a polystyrene ball, and serum as a specimen is added thereto to cause a binding reaction with the viral protein. This antibody-virus protein complex is further reacted with an enzyme-labeled antibody that recognizes another site of the virus protein to form an antibody-virus protein-antibody-enzyme complex, which finally exists on the solid support. The presence of the virus is confirmed by measuring the enzyme activity. This method has already been marketed as a diagnostic kit (Abbott: HIV antigen EIA Abbott) and has been applied in the medical field.
[0006]
However, the detection method of antigen by this enzyme immunoassay is only detected in the acute infection period immediately after viral infection or when viremia is caused by the onset of AIDS, and it can hardly be detected in the incubation period. When used as a marker for the pathological condition or the characteristics of the virus, it could only be used at a limited time when virus detection was possible.
[0007]
There is also a method for detecting a viral antigen in the latent period by another method. For example, the peripheral lymphocytes of a patient suspected of being infected are isolated and infected with non-infected Molt-4 cells. After culturing the cells for about 2 weeks, the presence of the virus in the cell culture supernatant is determined by the enzyme immunization. Judgment is made by measuring or measuring reverse transcriptase activity (Journal of Infectious Diseases, 1990, Vol. 64, No. 10, pp. 1287 to 1294). However, with this method, not all infected individuals are isolated, and the properties of the isolated virus must be examined individually, and it takes 2 weeks to make a decision, and the incubation period is 5 to 10 years. In view of the great labor and continuous testing cost for cell culture, it has not been common for the diagnosis of HIV infection that requires the treatment of multiple specimens.
[0008]
On the other hand, instead of antigen detection, a technique for measuring antibodies against HIV virus present in serum can be considered. In fact, enzyme immunoassay is used and is also commercially available (DuPont: DuPont HIV ELISA). For example, the specific method is to add a serum-diluted sample to a 96-well microplate on which an HIV antigen is immobilized, incubate at room temperature for 2 hours, wash and remove the reaction solution, and add an enzyme-labeled anti-human antibody. After incubating at room temperature for 1 hour, the reaction solution is washed and removed, an enzyme substrate is added, color reaction is performed at 37 ° C. for 30 minutes, and the absorbance is measured by a plate reader or the like. Since this antibody measurement method can be detected even in the latent period of HIV infection, it is frequently used for screening tests for infected persons.
[0009]
For purposes other than screening tests, this antibody assay can also be used as a disease state marker. It has been reported that HIV-infected individuals tend to decrease antibodies against p24 antigen, which is the core protein of HIV, when the pathology progresses from AISY-related complex (AC) to AIDS-related complex (ARC). DA, et al, J. Med. Viol., 22: 357, 1987). However, these methods detect all antibodies against viral proteins such as gp120, gp41, p24, p15, etc., and do not measure a specific epitope in a limited manner, and correspond to viral mutations after infection. Due to the drawback of not being able to see antibody changes, it has not been used as a marker to reflect the latent pathology of an infected person and the properties of the virus at some point.
[0010]
In the antibody measurement method, it is considered to classify and measure the epitope of an antibody against HIV antigen protein and examine the relationship between the amount and the disease state. For example, a method is considered in which several types of synthetic peptides are prepared using a synthetic method and the reactivity of the antibody in the infected patient sample is examined. However, considering that the antibody recognizes the three-dimensional structure of the substance, it is not always possible to detect an antibody against a virus-derived antigen molecule that is close to nature.
[0011]
In addition, a technique for measuring an antibody that inhibits reverse transcriptase activity has been devised as a technique for limiting the epitope of an antibody in an infected host. The amount of antibody is determined by adding an antibody that inhibits the activity of reverse transcriptase present in the serum of the infected person during the reverse transcriptase reaction and quantifying how much the enzyme reaction has been inhibited for a certain amount of enzyme. Is known (Sano K., et al, J. Clin. Microbiol. 25: 2415-2417, 1987, JP-A-3-47098). This method differs from the antibody measurement method described above in that only the antibody that inhibits the enzyme activity against the reverse transcriptase, which is an antigen, is detected to limit and measure the epitope.
[0012]
However, this commonly used method requires a special facility to be measured in accordance with the enzyme activity measurement method using a radioisotope, and handles many specimens such as HIV infected persons due to the complexity of operation. It is unsuitable for having to. Even if this method is used, epitopes may be used to quantitate antibodies that recognize sites mainly consisting of the active center of reverse transcriptase activity, etc. It was difficult to track changes such as those with antibodies.
[0013]
In addition, as a method for measuring a reverse transcriptase activity blocking antibody on a solid phase, a hybrid product of oligo dT and polyadenine ribonucleotide, which serves as a template for reverse transcriptase, is previously placed on a solid phase on a 96-well plate for EIA. In this case, reverse transcriptase, deoxythymine mononucleotide, biotin-labeled deoxyuridine mononucleotide, and an activity-blocking antibody-containing analyte are added and reacted simultaneously. After the unreacted product was washed away, the extended biotinylated nucleic acid was reacted with streptavidin-alkaline phosphatase, the unreacted product was washed away, the alkaline phosphatase activity on the solid phase was measured, and the reverse transcriptase activity blocking antibody was A measuring method is known (EP Publication No. 480408).
[0014]
However, it is known that conventional reverse transcriptase derived from HIV has RNase H activity, which is an enzymatic activity that degrades only RNA in a hybrid of DNA and RNA (LA Kohlstedt, et al. , Science, 1992, Vol. 256, 1783-1790), therefore, the DNA-RNA hybrid on the solid phase and the reverse transcriptase are simply brought into contact to form a hybrid of the hybrid and the reverse transcriptase. The subsequent reverse transcriptase reaction was assumed to be difficult to measure because the hybridized product is expected to be degraded by the RNase H activity.
[0015]
Furthermore, recently, a technique has been conceived in which an epitope is further identified and measured as an antibody group competing between a template-primer and an antibody or an antibody group inhibiting binding (Anthony, L.D. et al, AIDS Research and Human Retroviruses, 1994, Vol. 10, Number 8, 953-960). Specifically, when measuring HIV-derived reverse transcriptase activity using a radioisotope substrate, a method of measuring by adding diluted serum containing an activity-inhibiting antibody, the amount of template-primer used for activity measurement was varied. The inhibition of activity for each amount is quantified, and the competitive reaction or binding inhibition reaction between the template primer and the activity blocking antibody can be detected over the template primer recognition site of the enzyme. However, since the reverse transcriptase used in this method is an enzyme prepared from HIV particles, it is measured in a system in which a virus-derived template-primer is mixed in the enzyme activity measurement system, and the activity inhibition is accurate and reproducible. There was a fault that could not be measured. Moreover, it was lacking in convenience from the aspect of biohazard.
[0016]
More recently, a technique for measuring reverse transcriptase activity on a carrier in which a template-primer is immobilized on a solid phase has been developed (Nakano et al., Journal of Infectious Diseases, 1994, Vol. 68, No. 7). 923-931). In this method, a template-primer is immobilized on a 96-well microplate, and reverse transcriptase and biotin-labeled deoxyuridine triphosphate, which is a DNA synthesis substrate for reverse transcriptase, are added to cause a reverse transcriptase reaction. In this method, the amount of biotinylated DNA finally formed on the solid phase is quantified by a non-radioisotopic method. Using this method, in order to further measure the epitope of the activity-inhibiting antibody by limiting to the binding-inhibiting antibody group with the template-primer enzyme, the reverse transcriptase and the activity-blocking antibody are reacted in advance, and then fixed. It is possible to imagine a method in which a template-primer on a phase is combined with a reverse transcriptase, and then a DNA synthesis substrate is added to each other to cause a reverse transcriptase reaction.
[0017]
However, since HIV-derived reverse transcriptase actually has ribonuclease H activity, even if the reverse transcriptase is preliminarily bound to the template-primer on the solid phase, the template-primer Since it was not possible to cause a stable and efficient and reproducible binding reaction by degradation, it has not been practical to measure antibodies by separating the epitope groups of reverse transcriptase activity blocking antibodies using the method of Nakano et al. It was.
[0018]
As described above, it has been difficult to study the mutation of a virus using an antibody possessed by an infected person because the antibody produced in the body is polyclonal. Moreover, the method of classifying the epitope of an antibody that recognizes a viral antigen, which is considered as a method for solving this, is also difficult as described above, and therefore reflects the nature of the virus or the pathology of the infected person in HIV infection. As a possible diagnostic marker, an excellent technique using an antibody has not been established, and has been made only at the research stage. And since the method is extremely complicated, research in this field has been under pressure in terms of both research cost and labor. Therefore, a measurement system that can easily and reproducibly limit and classify epitopes among antibodies to proteins present in virus antigens as a reagent for research in this field and can quantify the amount of those antibodies has been desired. .
[0019]
[Problems to be solved by the invention]
Of the antibodies to proteins present in the viral antigens, it will be difficult to detect during the incubation period, such as AC, if a measurement system that can easily and reproducibly limit the epitope and quantify the amount of antibody can be established. It is possible to study the relationship with virus properties by using antibodies instead of viruses, and the properties of viruses that have been mutated over a long period of 5 to 10 years, and what resistance can be given by drug administration It is possible to study from the antibody measurement whether it was acquired.
[0020]
In addition, it is expected that changes on the host side including the ability to produce antibodies in the infected host will be captured more accurately and rapidly as the disease progresses. Furthermore, since an antibody is used, it is easy to obtain a specimen, and the labor for culturing viruses is reduced, which can be a very effective means for items with extremely large test amounts such as diagnosis of HIV. In addition, the measurement using an antibody is an inevitable requirement in the field of research on HIV infection from the viewpoint of biosafety, such as the advantage that the antibody is more stable than the virus antigen itself and the ability to treat the sample with a surfactant. Thus, the present invention provides a method for measuring reverse transcriptase activity blocking antibodies by dividing them into epitope groups.
[0021]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventor has found that the first step of the reverse transcriptase reaction; the binding reaction between the template-primer and the reverse transcriptase, and the second step; We have found a measurement system that measures enzyme activity in two stages of the DNA synthesis reaction. In particular, we have discovered a stable and reproducible reaction method in the first stage. In addition, when a reverse transcriptase is preliminarily bound to a template-primer immobilized on a solid support, it is preferably reproducibly stable by reacting in the presence of a water-soluble metal salt capable of generating a divalent metal ion. And completed the method of measuring the activity-inhibiting antibody by dividing it into three epitope groups by utilizing the inhibition mode of the reverse transcriptase activity-blocking antibody. That is, 1. 1. a group of antibodies that inhibit DNA elongation reaction from template RNA by irreversible binding of reverse transcriptase and specimen (antibody); There are three groups: an enzyme template-primer binding inhibition antibody group, 3, and a DNA elongation reaction inhibition antibody group from the template RNA due to the reaction between the reverse transcriptase and the specimen (antibody).
[0022]
Specifically, the principle is as follows. One inhibitory antibody group includes a hybrid of a substantial oligodeoxythymine nucleotide (oligo dT) and a substantial adenine ribopolynucleotide (poly A) on a solid phase. A certain amount of HIV-derived reverse transcriptase is preferably subjected to a binding reaction in the presence of a water-soluble metal salt that generates at least a divalent metal ion, and then unreacted enzyme is washed away, and then reverse transcription is performed. After reacting with a sample containing an enzyme activity blocking antibody and washing and separating the unreacted sample, an enzyme reaction solution containing deoxymononucleotide triphosphate, which is a DNA synthesis substrate for reverse transcriptase, is added to cause a reverse transcriptase reaction. Finally, the amount of nucleic acid synthesized is quantified, and based on the amount, the enzyme activity inhibited by reacting with the sample is quantified to measure the reverse transcriptase activity blocking antibody. Have found a way to.
[0023]
The present invention has been completed based on the above findings, and in the method for measuring an HIV-derived reverse transcriptase activity-inhibiting antibody, at least the following four steps are performed. Step A: Binding reaction between a substantial oligo dT immobilized on a transparent, translucent or colored solid phase and a substantial poly A hybridized thereto and reverse transcriptase, step B : Wash and separate the enzyme reacted in step A and the unreacted enzyme, then react with a specimen containing a reverse transcriptase activity blocking antibody, and step C: wash the antibody reacted in step B and the unreacted antibody After separation, a nucleic acid complementary to the template RNA is synthesized from deoxymononucleotide triphosphate by reverse transcriptase on the solid phase. Step D: Based on the amount of the synthesized nucleic acid, the enzyme activity of the antibody is blocked. Quantitative And said that process.
[0024]
In particular, in step A in the method for measuring a reverse transcriptase activity blocking antibody, the binding reaction is performed in the presence of a water-soluble metal salt that generates at least a divalent metal ion.
The reverse transcriptase activity blocking antibody in the sample in the present invention is an immunoglobulin A, an immunoglobulin G, an immunoglobulin M, an immunoglobulin D, an immunoglobulin E, or the like that inhibits the reverse transcriptase activity. Examples of such biological fluids include serum, plasma, urine, saliva, tears and the like, but are not particularly limited thereto, and preferably serum or plasma.
[0025]
The specimen to be subjected to the reaction can be measured in a dilution range of 1 to 1000000 times, but preferably used in a range of 1 to 100000 times dilution.
The solid phase carrier may be transparent, translucent, or colored, and any solid phase carrier can be used as long as oligo dT can be solid-phased. Commonly used are EIA plates, magnetic beads, and EIA plastics. For example, polystyrene, polypropylene, Teflon, polyethylene, methylbenten resin (TPX), fluororesin, acrylic resin, polycarbonate, polyurethane, vinyl chloride resin, stainless steel, aluminum, titanium, etc. And metals such as glass, rubber and the like.
[0026]
The solid phase immobilization method of the nucleic acid on the base material is a method of adsorbing on the solid phase carrier or a method of chemically binding directly from the 5 ′ end of the nucleic acid to the base material (V. Lundet. Al., Nucleic Acids Research, Vol. 10861-10880 1988), biotin, hapten, receptor, etc., modified on the 5 ′ end, 3 ′ end of nucleic acid or purine or pyrimidine ring portion of nucleic acid, and solidified on the base material in advance. It can be easily immobilized by binding to a ligand.
[0027]
The binding reaction in the present invention is not a reverse transcriptase reaction that transcribes RNA into DNA, but means a reaction in which reverse transcriptase recognizes and binds to a template-primer. The purpose of the reaction is to allow the enzyme template-primer binding reaction and the DNA extension reaction to be performed separately. Therefore, the binding reaction is different from the enzyme reaction, and in the present invention, it has been found that the binding reaction is stably and efficiently performed in the presence of a water-soluble metal salt that generates a divalent metal ion. It is.
[0028]
The conditions in Step A, which is a binding reaction between a reverse transcriptase and a template-primer, are preferably reacted in the presence of a water-soluble metal salt that generates a divalent metal ion. As a kind of metal ion which can produce a valent cation, for example, magnesium (Mg), manganese (Mn), calcium (Ca), zinc (Zn), cadmium (Cd), copper (Cu), etc. Water-soluble salts such as chlorides, fluorides, iodides, nitrates, sulfates, hydrogen carbonates that can release divalent cations such as Mg, Mn, Ca, Zn, Cd, Cu, etc. Suitable metal salts thereof include, for example, MgCl ₂ , MnCl ₂ , CaCl ₂ ZnCl ₂ , CdCl ₂ , CuCl ₂ , MgSO ₄ , MnSO ₄ , CdSO ₄ , CuSO ₄ Etc., preferably MgCl ₂ , MnCl ₂ , CaCl ₂ . Its concentration is MgCl ₂ , MnCl ₂ , CaCl ₂ Whichever of these is used, the reaction can be carried out at 0.01 mM to 1 M, preferably 0.1 mM to 500 mM.
[0029]
The pH in the binding reaction solution may be an optimum value taking into consideration the optimum pH for binding, and may be pH 3 to 10, preferably pH 5 to 9, but is particularly limited thereto. is not. The buffer used for the reaction solution may be selected according to the set pH. For example, one or more types selected from Tris buffer, phosphate buffer, acetate buffer, Good buffer, etc. may be used. . Then, if necessary, a salt such as NaCl or KCl is added, and an SH protecting agent such as dithiothreitol, dithioerythritol, or glutathione is added, or a protein such as albumin or IgG is added depending on the type of storage container. Any liquid can be used as long as it is a liquid in which a mono- or polyamino acid such as lysine or poly-L-lysine and a surfactant are added as an adsorption inhibitor.
[0030]
The reaction temperature may be at or above the temperature at which the solution freezes and maintained at such a level that enzyme activity can be measured, and the range may be 0 ° C to 50 ° C. The reaction time may be any time as long as the reaction is such that the enzyme bound to the template-primer can be detected, and the range may be 1 second to 24 hours.
[0031]
In addition, the reaction conditions for the enzyme and antibody in Step B may be those used for normal antigen-antibody reaction, and the pH in the reaction solution may be set to an optimum value in consideration of the optimum pH. The pH is 3 to 10, preferably 5 to 9, but not particularly limited thereto. For example, the optimum pH for the antigen-antibody reaction may be adjusted with an appropriate buffer, and the type may be selected according to the pH. As the buffer, one or more of Tris buffer, phosphate buffer, acetate buffer, Good buffer, etc. may be selected, and a salt such as NaCl or KCl may be added if necessary. SH protective agents such as dithiothreitol, dithioerythritol and glutathione can be added, and depending on the type of storage container, proteins such as albumin and IgG, mono or polyamino acids such as lysine and poly-L-lysine Any liquid can be used as long as it is a liquid to which a surfactant is added as an adsorption inhibitor. It can also be used by adding ethylene glycol or a ribonuclease inhibitor to stabilize the nucleic acid on the solid phase.
[0032]
In addition, the reaction time of the step B is sufficient if the antigen-antibody reaction can be performed to such an extent that the reverse transcriptase activity blocking antibody can be detected, and the time may be 1 second to 24 hours.
In addition, the reaction conditions of the reverse transcriptase in the step C can be anything as long as the reverse transcriptase reaction proceeds. For example, the pH may be an optimum value in consideration of the optimum pH, and may be any pH from 3 to 10, preferably from pH 5 to 9, but is not particularly limited thereto. For example, the pH optimum for the antigen-antibody reaction may be adjusted with an appropriate buffer, and the type may be selected according to the pH. The buffer may be selected from one or more of Tris buffer, phosphate buffer, acetate buffer, Good buffer, etc., and monovalent salt such as NaCl or KCl as required. Alternatively, a divalent metal salt such as magnesium chloride or manganese chloride is added, and an SH protective agent such as dithiothreitol, dithioerythritol, or glutathione is added. Depending on the type of storage container, albumin, IgG, Use any solution that contains a protein, lysine, poly-L-lysine, or other mono- or polyamino acid, and a surfactant added as a desorption inhibitor and deoxymononucleotide triphosphate as a substrate. Can do. It can also be used by adding ethylene glycol or a ribonuclease inhibitor to stabilize the nucleic acid on the solid phase.
[0033]
The oligo dT on the solid phase should have a substantial structure that is recognized as a transcription initiation site of reverse transcriptase, for example, a structure having a base number of at least 3 or preferably 5 or more as the length of the oligo dT. It is also commercially available and can be easily obtained (Pharmacia, Sigma, etc.). In addition, it can be easily prepared by a synthetic method, and can be commissioned (Takara Shuzo, Nippon Gene, etc.). Furthermore, when an oligo dT having a length of 700 bases or more is required, it can be easily prepared in-house enzymatically. For example, an enzyme (T4 RNA ligase; Takara Shuzo Co., Ltd.) having the above-mentioned commercially available oligo dT as a substrate and linking its 5 ′ phosphate group and 3 ′ hydroxyl group to a single-stranded DNA by a phosphodiester bond. (Commercially available from the company). Another enzyme can also be obtained by polymerizing deoxythymine nucleotides from the 3 ′ hydroxyl group of oligo dT with terminal deoxynucleotidyl transferase (commercially available from Takara Shuzo). In the present invention, the oligo dT may have a length of 3 to 100,000 bases, preferably 5 to 100,000 bases.
[0034]
Poly A is only required to be capable of hybrid reaction with oligo dT on a solid phase and to be recognized and synthesized as a template RNA for reverse transcriptase, and is also commercially available and readily available (Pharmacia). Moreover, it is easy to prepare by a synthetic method, and consignment preparation is possible (Takara Shuzo). Furthermore, when a poly A having a length of 500 bases or more is required, it can be easily prepared in-house enzymatically. For example, an enzyme (T4 RNA ligase; Takara Shuzo Co., Ltd.) having the above-mentioned commercially available poly A as a substrate and linking its 5 ′ phosphate group and 3 ′ hydroxyl group to a single-stranded RNA by a phosphodiester bond. (Commercially available from the company). In the present invention, the length of the poly A is 3 to 100,000 bases, preferably 5 to 100,000 bases. The length of the base is 3 to 100,000 bases, preferably 5 to 100,000 bases.
[0035]
The reverse transcriptase used is an HIV-derived enzyme having an activity of synthesizing DNA having a complementary base sequence with RNA as a template, and types 1 and 2 are known. These enzymes can be easily obtained by separating the enzyme directly from the virus, for example, LAV-1 strain, LAV-2 strain, GH-1 strain, GH-2 strain, GH-3 strain, GH-4 strain, GH- It can be easily prepared by treating virus particles such as 5 strains and GH-6 strains with a surfactant such as Triton X-100 (Triton X-100). Can be disassembled and separated. In addition, the enzyme obtained by expressing a large amount of the recombinant in E. coli is commercially available and can be easily obtained (Seikagaku Corporation), but it was preferably expressed in E. coli that does not contain the template nucleic acid. Recombinant reverse transcriptase should be used. The amount of reverse transcriptase obtained when a test solution using a sample such as serum is added is 0% when the enzyme activity obtained when no test solution is added is 100%. However, the concentration may be in the range of 0.1 μU / ml to 10000 U / ml, and preferably in the range of 2 μU / ml to 1000 U / ml. Good.
[0036]
Deoxymononucleotide triphosphate is deoxythymidine mononucleotide triphosphate as an unlabeled substance, biotin-labeled deoxyuridine mononucleotide triphosphate, digoxigenin-labeled deoxyuridine mononucleotide triphosphate, tritium or ³² There are P-labeled deoxythymidine mononucleotide triphosphates, etc., and one or more of these may be used. Preferably, deoxythymidine mononucleotide triphosphate as a non-labeled substance and biotin-labeled deoxyuridine mononucleotide triphosphate as a labeled substance are combined It is good to use. The amount may be any as long as the reverse transcriptase reaction can be performed and the enzyme activity can be detected from the labeled body. The ratio of the labeled body to the unlabeled body is, for example, that the unlabeled body is 1 label. Measurement can be performed from a ratio of 0 molecule to a ratio of 1 molecule of labeled body to 500 molecules of unlabeled body.
[0037]
Next, 2. Specific principles for detecting a group of antibodies that inhibit the binding of the enzyme to the template-primer shown in FIG. First, a certain amount of HIV-derived reverse transcriptase and a specimen containing a reverse transcriptase activity-blocking antibody are contact-reacted, and the reaction solution is mixed with a substantial oligo dT and a substantial poly A on the solid phase. A sample containing a reverse transcriptase activity blocking antibody after the binding reaction is preferably carried out in the presence of a water-soluble metal salt that generates at least a divalent metal ion, and then the unreacted product is washed away. After reacting and washing and separating the unreacted test solution, an enzyme reaction solution containing deoxymononucleotide triphosphate, which is a DNA synthesis substrate for reverse transcriptase, is added to cause reverse transcriptase reaction and finally synthesized. The amount of nucleic acid was quantified, and the enzyme activity inhibited by reacting with the specimen based on the amount was quantified to find a method for measuring a reverse transcriptase activity blocking antibody.
[0038]
That is, in the method for measuring a reverse transcriptase activity blocking antibody derived from HIV, the method for measuring a reverse transcriptase activity blocking antibody comprising performing at least the following four steps: Step E: reverse transcriptase and reverse transcriptase Reacting with an analyte containing an activity blocking antibody, step F: substantial oligo dT immobilized on a transparent or translucent or colored solid phase and substantial poly A hybridized thereto and step E Step G: The reaction product in Step F and the unreacted product are washed and separated, and then complementary to the template RNA from deoxymononucleotide triphosphate by reverse transcriptase on the solid phase. Step H: a step of quantifying the inhibition of the enzyme activity of the antibody based on the amount of the synthesized nucleic acid.
[0039]
In Step F of the above-described method for measuring a reverse transcriptase activity blocking antibody, it is preferable to carry out a binding reaction in the presence of a water-soluble metal salt that generates at least a divalent metal ion.
2. In the step of detecting the antibody group, the binding reaction between the reverse transcriptase and the template-primer is carried out in step F. This corresponds to the step A of antibody group detection. Accordingly, the reaction conditions in Step F are the same as those in Step A above, and the binding reaction between the reverse transcriptase and the template-primer is carried out in the presence of a water-soluble metal salt that generates a divalent metal ion. If it is made to react, it is sufficient. And the kind and density | concentration, reaction temperature, and the various components used suitably for other reaction also have a preferable composition and conditions of the said process A.
[0040]
The components used in Step F may be added in advance at the time of antigen-antibody reaction between the antibody and enzyme in Step E, and the reaction solution may be used directly in Step F. And you may use the liquid for the process F.
In addition, 2. Step E for detecting the antibody group of Antigen-antibody reaction between the enzyme corresponding to step B and the reverse transcriptase activity-blocking antibody for detecting the antibody group, and therefore the reaction conditions in step E are the same as those in step B above. Is preferred.
[0041]
And 2. In the measurement of the inhibitory antibody group shown in the above, the solid phase carrier, the method of immobilizing nucleic acid on the substrate, the oligo dT on the solid phase, poly A, the reverse transcriptase used, deoxymononucleotide 3-phosphorus The acid may be It may be the same as the antibody group shown in.
Next, 3. The specific principle for detecting the DNA elongation reaction inhibiting antibody group from the template RNA by the competitive reaction between the reverse transcriptase and the specimen (antibody) shown in FIG. In the presence of a water-soluble metal salt that produces a hybrid of a substantial oligo dT and a substantial poly A on a solid phase and a certain amount of HIV-derived reverse transcriptase, at least a divalent metal ion. After the unreacted enzyme is washed and removed, an enzyme reaction solution containing a sample containing a reverse transcriptase activity blocking antibody and deoxymononucleotide triphosphate which is a reverse transcriptase DNA synthesis substrate is added simultaneously. The method of measuring the reverse transcriptase activity blocking antibody by quantifying the amount of nucleic acid finally synthesized by reacting with the reverse transcriptase and quantifying the enzyme activity suppressed by reacting with the sample based on the amount. I found it.
[0042]
That is, in the method for measuring a reverse transcriptase activity blocking antibody derived from HIV, the method for measuring a reverse transcriptase activity blocking antibody comprising performing at least the following three steps: Step I: transparent or translucent or A substantial oligo dT immobilized on a colored solid phase, a substantial poly A hybridized with the oligo dT, and a reverse transcriptase are subjected to a binding reaction, Step J: an enzyme reacted in Step I and an unreacted enzyme And a sample containing a reverse transcriptase activity blocking antibody and deoxymononucleotide triphosphate are added simultaneously to synthesize a nucleic acid complementary to the template RNA by reverse transcriptase on the solid phase, Process K: It is characterized by quantifying the inhibition of enzyme activity of an antibody based on the amount of synthesized nucleic acid.
[0043]
In Step I of the above-described method for measuring a reverse transcriptase activity blocking antibody, it is preferable that the binding reaction be performed in the presence of a water-soluble metal salt that generates at least a divalent metal ion.
3. In the step of detecting the antibody group, the binding reaction between the reverse transcriptase and the template-primer is carried out in Step I. Step A of antibody group detection in the above, or 2. This corresponds to the antibody group detection step F. Therefore, the reaction conditions in Step I are the same as those in Step A above, and the binding reaction between the reverse transcriptase and the template-primer is carried out in the presence of a water-soluble metal salt that generates a divalent metal ion. If it is made to react, it is sufficient. And the kind and density | concentration, reaction temperature, and the various components used suitably for other reaction also have a preferable composition and conditions of the said process A.
[0044]
In step J, the reverse transcriptase reaction and the antigen-antibody reaction between the reverse transcriptase and the activity-blocking antibody are simultaneously performed. 1. Step B and C for detection of antibody group This corresponds to steps E and G of antibody group detection. The reaction conditions in step J may be any conditions as long as the reaction can be performed to such an extent that a reverse transcriptase reaction can be detected. DNA synthesis substrate of reverse transcriptase, for example, in the reaction condition of step B in the detection of antibody group of ³ HdTTP, ³² PdTTP and biotin-labeled dUTP may be added to such an extent that enzyme activity can be detected. In addition, 1. The reaction composition used in the normal antigen-antibody reaction only needs to be included in the solution under the reaction conditions in step C in the antibody group detection. As the composition included, for example, the optimum pH for the antigen-antibody reaction is adjusted with an appropriate buffer, and one type is selected from Tris buffer, phosphate buffer, acetate buffer, Good buffer, etc. according to the pH. It is sufficient if it is selected or more, and if necessary, a salt such as NaCl or KCl is added, and an SH protective agent such as dithiothreitol, dithioerythritol or glutathione is added. Accordingly, any liquid can be used as long as it is a solution in which a protein such as albumin or IgG, a mono- or polyamino acid such as lysine or poly-L-lysine, and a surfactant are added as an adsorption inhibitor. It can also be used by adding ethylene glycol or a ribonuclease inhibitor to stabilize the nucleic acid on the solid phase.
[0045]
3. In the measurement of the inhibitory antibody group shown in the above, the solid phase carrier, the method of immobilizing nucleic acid on the substrate, the oligo dT on the solid phase, poly A, the reverse transcriptase used, deoxymononucleotide 3-phosphorus The acid may be It may be the same as the antibody group shown in.
And in these three antibody group measurement methods, a new method for detecting nucleic acid on the solid phase formed by reverse transcriptase may use a radioisotope substrate as a reverse transcriptase substrate. ³ HdTTP and ³² When PdTTP is used, the radioactivity remaining on the solid phase may be measured after washing away the unreacted substrate after the reverse transcriptase reaction. Preferably, biotin-labeled dUTP, which is a non-radioisotope substrate, is used as the reverse transcriptase substrate. After the reverse transcriptase reaction, the unreacted substrate is washed away, reacted with avidin or streptavidin labeled with peroxidase or alkaline phosphatase, and the unreacted product is washed away and the enzyme activity remaining on the solid phase is measured. That is fine, and this method is a commonly used method (Nakano et al., Journal of Infectious Diseases, 1994, Vol. 68, No. 7, pages 923-931).
[0046]
More specifically, the method is described as follows: When measuring a group of antibodies that inhibit the DNA elongation reaction from the template RNA due to irreversible binding between the reverse transcriptase and the specimen (antibody) shown in Fig. 1, for example, reverse transcriptase is dissolved in a 50 mM magnesium chloride solution, This solution is added as a template-primer to a 96-well EIA plate on which oligo dT-poly A is immobilized, and incubated at 37 ° C. for 1 hour to bind the enzyme and the template primate.
[0047]
Thereafter, unreacted enzyme is washed away, and then a test solution containing a reverse transcriptase activity-inhibiting antibody diluted with 1% Triton X-100 or the like is added, and incubated at 37 ° C. for 1 hour. After washing and removing the reaction specimen, a reverse transcriptase reaction substrate solution containing biotin-labeled dUTP is added, and a reverse transcriptase reaction is performed at 37 ° C. for 2 hours. After the reaction, the unreacted substrate was washed away and then alkaline phosphatase-labeled streptavidin was added. After incubation at 37 ° C. for 1 hour, the unreacted product was washed away, and then the alkaline phosphatase substrate paranitrophenyl phosphate was added. The color development at 37 ° C. for 30 minutes is measured with a plate reader at an absorbance of 405 nm. At that time, it is possible to easily determine the residual activity% from the absorbance when the sample is used, with the absorbance when measured using only the 1% Triton X-100 solution diluted with the sample instead of the sample as 100%. .
[0048]
In addition, 2. When measuring a group of antibodies that inhibit the binding of the enzyme to the template-primer, for example, a serum sample containing an HIV-derived reverse transcriptase and a reverse transcriptase activity blocking antibody is mixed in advance in a separate test tube. Is prepared to be 50 mM Hepes buffer (pH 7.5) and 150 mM sodium chloride, and incubated at 37 ° C. for 1 hour to cause an antigen-antibody reaction between the enzyme and the antibody. Magnesium chloride is added to the reaction solution so that the magnesium chloride concentration in the reaction solution is 50 mM. This solution is added as a template-primer to a 96-well EIA plate on which oligo dT-poly A is immobilized, and incubated at 37 ° C. for 1 hour to bind the enzyme and the template primer. Thereafter, unreacted substances are washed away, a reverse transcriptase reaction substrate solution containing biotin-labeled dUTP is added, and a reverse transcriptase reaction is performed at 37 ° C. for 2 hours. In order to detect newly formed nucleic acid on the solid phase, 1. It can be easily quantified if it is carried out according to the technique for detecting the antibody group.
[0049]
In addition, when quantifying the DNA elongation reaction-inhibiting antibody group from the template RNA by the competitive reaction between the reverse transcriptase and the specimen (antibody) shown in 3, for example, HIV-derived reverse transcriptase is placed in a 50 mM magnesium chloride solution. The solution is dissolved, and this solution is added as a template-primer to a 96-well EIA plate on which oligo dT-polyA is immobilized, and incubated at 37 ° C. for 1 hour to bind the enzyme and the template primer. Thereafter, unreacted enzyme is washed away, and then a test solution containing a reverse transcriptase activity-inhibiting antibody diluted with 1% Triton X-100 or the like and a reverse transcriptase reaction substrate solution containing biotin-labeled dUTP Are simultaneously added and allowed to undergo reverse transcriptase reaction at 37 ° C. for 2 hours. In order to detect newly formed nucleic acid on the solid phase after washing away unreacted substrate after the reaction, 1. It can be easily quantified if it is carried out according to the technique for detecting the antibody group.
[0050]
DETAILED DESCRIPTION OF THE INVENTION
Next, the present invention is further illustrated by examples, but the present invention is not limited thereby.
[0051]
[Example 1]
(1) Preparation of oligo dT-poly A solid phase plate
100 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (abbreviated as CDI; manufactured by Peptide Institute) and 100 mM 1-methyl-imidazole-HCl buffer (pH 7.0; abbreviated as IMD); In aqueous solution containing Sigma) _19-24 (Average chain length: 22 bases; manufactured by Sigma) was dissolved to 200 ng / 100 μl, and 100 μl of each was dispensed into each well of a 96-well amino plate (manufactured by Sumitomo Bakelite) and reacted at room temperature for 24 hours. I let you. After the reaction, this solution was discarded, and the wells were washed three times with 200 μl of 0.15 M sodium chloride and 0.1 M Tris-HCl buffer (pH 7.5; hereinafter referred to as TBS). In this well, poly A (manufactured by Pharmacia) becomes 10 μg / 100 μl in 80 mM Tris-HCl (pH 7.5), 0.6 M sodium chloride, 4 mM disodium ethylenediaminetetraacetate, 0.1% sodium dodecyl sulfate solution. 100 μl of the prepared solution was added and incubated at 37 ° C. for 16 hours. After discarding the poly A solution on the plate, the plate was washed 3 times with TBS to obtain an oligo dT-poly A solid phased plate.
(2) Binding reaction of reverse transcriptase on oligo dT-poly A solid phase plate
0.1% Triton X-100, HIV-1 type reverse transcriptase (expressed by recombinant Escherichia coli; manufactured by Seikagaku Corporation) in 0.1 mU / 100 μl solution, 0.01, 0.1, 1, 10, It prepared so that it might become a magnesium chloride density | concentration of 100 and 1000 mM, and dispensed 100 microliters at a time to oligo dT-poly A solid-phase plate, and incubated at 37 degreeC for 1 hour. After incubation, the plate was washed 5 times with a TBS solution containing 0.02% polyoxyethylene (20) sorbitan monolaurate to obtain a reverse transcriptase-binding plate.
(3) Reaction between reverse transcriptase-binding plate and test solution
Serum of 5 HIV-infected hemophilia patients and 5 non-HIV infected individuals was diluted 200 times with 10 mM phosphate buffer (pH 7.4), 150 mM sodium chloride, and 0.1% Triton X-100 solution, respectively. 100 μl was added to the reverse transcriptase binding plate and incubated at 37 ° C. for 1 hour. After the incubation, the reaction solution was discarded and washed 5 times with a TBS solution containing 0.02% polyoxyethylene (20) sorbitan monolaurate. In addition, as a control group, instead of serum dilution, 100 μl of 10 mM phosphate buffer (pH 7.4), 150 mM sodium chloride, 0.1% Triton X-100 solution was added to the reverse transcriptase-binding plate and the same operation was performed. did.
(4) Reverse transcriptase activity detection
To the plate washed in (3), 90 mM HEPES (pH 7.8), 126 mM potassium chloride, 9 mM magnesium chloride, 0.45 mM dithiothreitol, 1.08 mM reduced glutathione, 1% Triton X-100, 1.8% 100 μl of ethylene glycol, 117 μM deoxythymidine triphosphate (dTTP), 3 μM biotin-labeled deoxyuridine triphosphate (Bio-dUTP; manufactured by Boehringer Mannheim) was added, and a reverse transcriptase reaction was performed at 37 ° C. for 2 hours.
[0052]
After the reaction, 10 μl of 5M sodium chloride solution was added and the plate was allowed to stand at room temperature for 5 minutes. The plate is diluted 20000 times with 50 mM Tris-HCl buffer (pH 7.5), 0.5 M sodium chloride, 5 mM magnesium chloride, 2% bovine serum albumin solution and alkaline phosphatase-labeled streptavidin (Jackson ImmunoResearch Laboratories). 100 μl of each solution was added to each well, incubated at 37 ° C. for 1 hour, and then washed 5 times with a TBS solution containing 0.02% polyoxyethylene (20) sorbitan monolaurate.
[0053]
After washing, 150 μl of diethanolamine-hydrochloric acid buffer solution (pH 9.5), paranitrophenyl phosphate disodium (manufactured by Sigma) dissolved in 1 mM magnesium chloride solution to a concentration of 1 mg / ml was added at 37 ° C. After incubation for 30 minutes, 50 μl of 1N sodium hydroxide solution was added, and the absorbance at 405 nm was measured with a plate reader.
(5) Quantification of activity inhibition by reverse transcriptase activity inhibition antibody
In the absorbance obtained in (4), the absorbance when using a serum sample is divided by the absorbance obtained when no serum sample is used, and the enzyme activity when not using the serum sample is 100%. The residual activity% was calculated. The results are shown in Table 1.
[0054]
[Table 1]

[0055]
As shown in Table 1, the DNA elongation reaction inhibitory antibody group from the template RNA due to irreversible binding of the reverse transcriptase and the sample could be quantified as the activity inhibition amount at any magnesium ion concentration.
In addition, at the time of reverse transcriptase binding reaction on oligo dT-poly A solid phase plate, 0.1% Triton X-100, reverse transcriptase 5 mU / 100 μl solution was tested at a magnesium concentration of 0, HIV infected person K. 56%; K. 63%, K. S. 71%, non-HIV infected T.A. 103.4%, T. E. At 100.3%, T.I. T.A. The residual activity was 110.4%.
[0056]
[Example 2]
An oligo dT-poly A immobilized plate was prepared according to the preparation of (1) oligo dT-poly A immobilized plate in Example 1. A reverse transcriptase-binding plate was prepared by using manganese chloride or calcium chloride instead of magnesium chloride in the reverse transcriptase binding reaction in (2) oligo dT-poly A solid phase immobilized plate of Example 1 on the prepared plate. Prepared. The prepared enzyme-bound plate was subjected to (1) reaction between the reverse transcriptase-bound plate and the test solution in Example 1, (4) detection of reverse transcriptase activity, and (5) quantification of activity inhibition by the reverse transcriptase activity-blocking antibody. went. The results are shown in Table 2.
[0057]
[Table 2]

[0058]
As shown in Table 2, for any metal ion used, the DNA elongation reaction inhibiting antibody group from the template RNA due to irreversible binding between the reverse transcriptase and the sample can be quantified as an activity inhibition amount, and at any ion concentration. Quantification was possible.
[0059]
[Example 3]
Example 1 (1) Oligo dT in Preparation of Oligo dT-Poly A Immobilized Plate _19-24 Instead of oligo dT ₅ , Oligo dT ₁₀ (Sigma), oligo dT _25-30 Poly dT (average chain length 794 base) (manufactured by Pharmacia) was used to prepare an oligo dT-poly A solid phase plate. A reverse transcriptase-binding plate was prepared using the prepared plate with the magnesium chloride concentration in the binding reaction of reverse transcriptase in (2) oligo dT-poly A solid phase-immobilized plate of Example 1 as 50 mM. The prepared enzyme-bound plate was subjected to (1) reaction between the reverse transcriptase-bound plate and the test solution in Example 1, (4) detection of reverse transcriptase activity, and (5) quantification of activity inhibition by the reverse transcriptase activity-blocking antibody. went. The results are shown in Table 3.
[0060]
[Table 3]

[0061]
As shown in Table 3, with any length of oligo dT used, it was possible to quantify the group of antibodies that inhibit the DNA elongation reaction from the template RNA due to irreversible binding between the reverse transcriptase and the sample as the activity inhibition amount.
[0062]
[Example 4]
Example 1 (1) Oligo A instead of Poly A in the preparation of oligo dT-poly A immobilized plate _12-18 Poly (A) (average chain length of 534 bases) (Pharmacia) was used to prepare oligo dT-poly A solid phased plates. A reverse transcriptase-binding plate was prepared using the magnesium chloride concentration of 50 mM in the reverse transcriptase binding reaction on the prepared plate (2) oligo dT-poly A solid phased plate in Example 1. The prepared enzyme-linked plate was subjected to (3) reaction between the reverse transcriptase-conjugated plate and the test solution in Example 1, (4) detection of reverse transcriptase activity, and (5) quantification of activity inhibition by the reverse transcriptase activity blocking antibody. went. The results are shown in Table 4.
[0063]
[Table 4]

[0064]
As shown in Table 4, with any length of poly A used, the group of antibodies that inhibit the DNA elongation reaction from the template RNA due to irreversible binding of the reverse transcriptase and the sample could be quantified as the activity inhibition amount.
[0065]
[Example 5]
An oligo dT-poly A solid-phase plate was prepared according to the preparation of (1) oligo dT-poly A solid-phase plate in Example 1. In the prepared plate, the magnesium chloride concentration in the reverse transcriptase binding reaction in Example 2 (2) oligo dT-poly A solid phase immobilized plate was used as 50 mM, and the reverse transcriptase amount was 0.1, 1, Reverse transcriptase binding plates were prepared using reverse transcriptase at concentrations of 10, 100, 1000, 10000, 100000 mU / 100 μl. Using the prepared enzyme-binding plate, the serum was reacted using 5 HIV-infected haemophilia patients in Example 1, (3) Reaction of reverse transcriptase-binding plate and test solution.
[0066]
Thereafter, (4) detection of reverse transcriptase activity in Example 1 and (5) inhibition of activity by a reverse transcriptase activity blocking antibody were quantified. Separately, in the above method, when reverse transcriptase was used at a concentration of 0.1, 1, 10 μU / 100 μl, the amount of reverse transcriptase activity in Example 1 (4) In the detection, a 24-hour reverse transcriptase reaction was performed instead of the 2-hour reverse transcriptase reaction, and the other operating conditions were the same. The results are shown in Table 5.
[0067]
[Table 5]

[0068]
As shown in Table 5, at any concentration of reverse transcriptase used, the DNA elongation reaction inhibitory antibody group from the template RNA due to irreversible binding between the reverse transcriptase and the sample could be quantified as an activity inhibition amount.
[0069]
[Example 6]
An oligo dT-poly A immobilized plate was prepared according to the preparation of Example 1 (1) oligo dT-poly A immobilized plate. A reverse transcriptase-binding plate was prepared using the magnesium chloride concentration of 50 mM in the reverse transcriptase binding reaction on the prepared plate (2) oligo dT-poly A solid phased plate in Example 1. The prepared enzyme-linked plate was diluted with the 1, 10, 100, 1000, 10000, and 100000-fold diluted solutions of the serum in the reaction between the reverse transcriptase-linked plate and the test solution in Example 1 (3). The antigen-antibody reaction was performed, and then (4) reverse transcriptase activity detection in Example 1 and (5) activity inhibition by the reverse transcriptase activity blocking antibody were quantified. The results are shown in Table 6.
[0070]
[Table 6]

[0071]
As shown in Table 6, at any dilution concentration used, the DNA elongation reaction inhibiting antibody group from the template RNA due to irreversible binding between the reverse transcriptase and the sample could be quantified as an activity inhibition amount.
[0072]
[Example 7]
(6) An oligo dT-poly A solid-phase plate was prepared according to the preparation of Example 1 (1) oligo dT-poly A solid-phase plate.
(7) Antigen-antibody reaction between reverse transcriptase and test solution
In a test tube, 0.1% Triton X-100, HIV-1 reverse transcriptase (expressed by recombinant Escherichia coli; manufactured by Seikagaku Corporation) in a 0.2 mU / 100 μl solution, 0.02, 0.2, 2 , 20, 200, and 2000 mM magnesium chloride concentrations were prepared as enzyme solutions. Serum of 5 HIV-infected hemophilia patients and 5 non-HIV-infected persons prepared in separate test tubes was added to this enzyme solution in 10 mM phosphate buffer (pH 7.4), 150 mM sodium chloride, 0.1% Triton, respectively. The test solution diluted 200 times with the X-100 solution was mixed in an equal volume of 200 μl in a separate test tube and reacted at 37 ° C. for 1 hour. As a control group, the same operation was performed using a 10 mM phosphate buffer (pH 7.4), 150 mM sodium chloride, and 0.1% Triton X-100 solution instead of the serum dilution.
(8) Binding reaction of reverse transcriptase to oligo dT-poly A solid phase plate
After the reaction of (7), 100 μl of the reaction solution was added to the oligo dT-poly A solid-phase plate prepared in (6) and incubated at 37 ° C. for 1 hour. After incubation, the solution was discarded, and the plate was washed 5 times with a TBS solution containing 0.02% polyoxyethylene (20) sorbitan monolaurate. The washed plate was subjected to (4) detection of reverse transcriptase activity in Example 1 and (5) quantification of activity inhibition by a reverse transcriptase activity inhibition antibody. The results are shown in Table 7.
[0073]
[Table 7]

[0074]
As shown in Table 7, at any magnesium ion concentration, the binding inhibition antibody group between the enzyme and the template-primer could be quantified as the activity inhibition amount.
In addition, when the antigen-antibody reaction between the reverse transcriptase and the test solution was performed, the results were as follows: the enzyme solution concentration was 5 mU / 100 μl, and the magnesium chloride concentration was 0. K. 23.2%; K. 54.4%, K. S. 48.0%, HIV non-infected persons T.A. 111. . %, T.M. E. At 104.8% and T.W. T.A. The residual activity was 102.7%.
[0075]
[Example 8]
An oligo dT-poly A solid-phase plate was prepared according to the preparation of Example 6 (6) oligo dT-poly A solid-phase plate. The test tube was reacted with manganese chloride or calcium chloride instead of magnesium chloride in the antigen-antibody reaction of Example 7 (7) reverse transcriptase and test solution. The reaction solution was subjected to the reaction of Example 7 (8) reverse transcriptase binding reaction to oligo dT-poly A solid phase plate, Example 1 (4) reverse transcriptase activity detection, and (5) reverse transcriptase activity blocking antibody. Quantification of activity inhibition by was carried out. The results are shown in Table 8.
[0076]
[Table 8]

[0077]
As shown in Table 8, for any metal ion used, the binding inhibitory antibody group between the enzyme and the template-primer could be quantified as an activity inhibition amount, and at any ion concentration.
[0078]
[Example 9]
Example 6 (6) Oligo dT in the preparation of oligo dT-poly A immobilized plate _19-24 Instead of oligo dT ₅ , Oligo dT ₁₀ (Sigma), oligo dT _25-30 Poly dT (average chain length 794 base) (manufactured by Pharmacia) was used to prepare an oligo dT-poly A solid phase plate. An oligo dT-poly A solid-phased plate prepared in the same manner by reacting in a test tube with a magnesium chloride concentration of 50 mM in the antigen-antibody reaction of (7) reverse transcriptase and test solution in Example 7 After adding 100 μl of the reaction solution and incubating at 37 ° C. for 1 hour, the solution was discarded and washed 5 times with a TBS solution containing 0.02% polyoxyethylene (20) sorbitan monolaurate. The washed plate was subjected to (4) detection of reverse transcriptase activity in Example 1 and (5) quantification of activity inhibition by a reverse transcriptase activity inhibition antibody. The results are shown in Table 9.
[0079]
[Table 9]

[0080]
As shown in Table 9, with any length of oligo dT used, the binding inhibition antibody group between the enzyme and the template-primer could be quantified as the activity inhibition amount.
[0081]
[Example 10]
Example 6 (6) Oligo A instead of Poly A in preparation of oligo dT-poly A solid phase plate _12-18 Poly (A) (average chain length of 534 bases) (Pharmacia) was used to prepare oligo dT-poly A solid phased plates. An oligo dT-polyA solid phase plate prepared in the same manner by reacting in a test tube in the same manner as in Example 7 (7) Magnesium chloride concentration in the antigen-antibody reaction between the reverse transcriptase and the test solution at 50 mM. After adding 100 μl of the reaction solution and incubating at 37 ° C. for 1 hour, the solution was discarded and washed 5 times with a TBS solution containing 0.02% polyoxyethylene (20) sorbitan monolaurate. The washed plate was subjected to (4) detection of reverse transcriptase activity in Example 1, and (5) quantification of activity inhibition by a reverse transcriptase activity inhibition antibody. The results are shown in Table 10.
[0082]
[Table 10]

[0083]
As shown in Table 10, with any length of poly A used, the binding inhibition antibody group between the enzyme and the template-primer could be quantified as an activity inhibition amount.
[0084]
Example 11
An oligo dT-poly A solid-phase plate was prepared according to the preparation of Example 6 (6) oligo dT-poly A solid-phase plate. In a test tube, the magnesium chloride concentration in the antigen-antibody reaction of Example 7 (7) reverse transcriptase and test solution was used as 50 mM, and the amount of reverse transcriptase was 0.2, 2, 20, 200, 2000, 20000. Using reverse transcriptase at a concentration of 200000 mU / 100 μl, the serum was reacted in the same manner using 5 HIV-infected hemophilia patients. Add 100 μl of this reaction solution to the prepared oligo dT-poly A solid phase plate, incubate at 37 ° C. for 1 hour, discard the solution, and add 0.02% polyoxyethylene (20) sorbitan monolaurate. Washed 5 times with TBS solution.
[0085]
The washed plate was subjected to (4) detection of reverse transcriptase activity in Example 1, and (5) quantification of activity inhibition by a reverse transcriptase activity inhibition antibody. In addition, when reverse transcriptase with a concentration of 0.2, 2, 20 μU / 100 μl was used in the above method, the amount of reverse transcriptase activity in Example 1 (4) In the detection, a 24-hour reverse transcriptase reaction was performed instead of the 2-hour reverse transcriptase reaction, and the other operating conditions were the same. The results are shown in Table 11.
[0086]
[Table 11]

[0087]
As shown in Table 11, at any concentration of reverse transcriptase used, the binding inhibitory antibody group between the enzyme and the template-primer could be quantified as an activity inhibition amount.
[0088]
Example 12
An oligo dT-poly A solid-phase plate was prepared according to the preparation of Example 6 (6) oligo dT-poly A solid-phase plate. In a test tube, the magnesium chloride concentration in the antigen-antibody reaction of Example 7 (7) reverse transcriptase and test solution was used as 50 mM, and the dilution of serum was 2, 20, 200, 2000, 20000, 200,000 times diluted solution. The antigen-antibody reaction was performed in the same manner using each of. Add 100 μl of this reaction solution to the prepared oligo dT-poly A solid phase plate, incubate at 37 ° C. for 1 hour, discard the solution, and add 0.02% polyoxyethylene (20) sorbitan monolaurate. Washed 5 times with TBS solution. The washed plate was subjected to (4) detection of reverse transcriptase activity in Example 1 and (5) quantification of activity inhibition by a reverse transcriptase activity inhibition antibody. The results are shown in Table 12.
[0089]
[Table 12]

[0090]
As shown in Table 12, the antibody-template-primer binding inhibition antibody group could be quantified as the activity inhibition amount at any dilution concentration used.
[0091]
Example 13
(9) Preparation of oligo dT-poly A solid phase plate
According to the preparation of (1) oligo dT-poly A immobilized plate in Example 1, an oligo dT-poly A immobilized plate was prepared.
(10) Reverse transcriptase binding reaction on oligo dT-poly A solid phase plate
A reverse transcriptase-binding plate was prepared from the prepared oligo dT-poly A solid-phased plate in accordance with the reverse transcriptase binding reaction on the oligo dT-poly A solid-phased plate in Example 1 (2).
(11) Simultaneous reaction of antigen-antibody reaction and reverse transcriptase reaction
On the prepared plate, sera from 5 HIV-infected hemophilia patients and 5 non-HIV infected individuals were each added with 10 mM phosphate buffer (pH 7.4), 150 mM sodium chloride, and 0.1% Triton X-100 solution. 50 μl of the diluted solution was added, and 180 mM HEPES (pH 7.8), 252 mM potassium chloride, 18 mM magnesium chloride, 0.9 mM dithiothreitol, 2.16 mM reduced glutathione, 2% Triton X-100, 3. 50 μl of 6% ethylene glycol, 234 μM deoxythymidine triphosphate (dTTP), 6 μM biotin-labeled deoxyuridine triphosphate (Bio-dUTP; manufactured by Boehringer Mannheim) was added and incubated at 37 ° C. for 2 hours. After incubation, 10 μl of 5M sodium chloride solution was added and the plate was allowed to stand at room temperature for 5 minutes. Then, the solution was discarded, and the plate was washed 5 times with a TBS solution containing 0.02% polyoxyethylene (20) sorbitan monolaurate.
[0092]
The plate is diluted 20000 times with 50 mM Tris-HCl buffer (pH 7.5), 0.5 M sodium chloride, 5 mM magnesium chloride, 2% bovine serum albumin solution and alkaline phosphatase-labeled streptavidin (Jackson ImmunoResearch Laboratories). 100 μl of each solution was added to each well, incubated at 37 ° C. for 1 hour, and then washed 5 times with a TBS solution containing 0.02% polyoxyethylene (20) sorbitan monolaurate.
[0093]
After washing, 150 μl of diethanolamine-hydrochloric acid buffer solution (pH 9.5), paranitrophenyl phosphate disodium (manufactured by Sigma) dissolved in 1 mM magnesium chloride solution to a concentration of 1 mg / ml was added at 37 ° C. After incubation for 30 minutes, 50 μl of 1N sodium hydroxide solution was added, and the absorbance at 405 nm was measured with a plate reader. Thereafter, the activity inhibition by the antibody (5) reverse transcriptase activity inhibition in Example 1 was quantified. The results are shown in Table 13.
[0094]
[Table 13]

[0095]
As shown in Table 13, at any magnesium ion concentration, the group of antibodies that inhibit the DNA elongation reaction from the template RNA by the competitive reaction between the reverse transcriptase and the antibody could be quantified as the activity inhibition amount.
In addition, during the reverse transcriptase binding reaction on the oligo dT-poly A solid phase plate, the test result of magnesium at a concentration of 0 in a 0.1% Triton X-100, reverse transcriptase 5 mU / 100 μl solution is as follows: HIV infected person K. At 33.2%; K. 28.4%, K. S. 49.2%, non-HIV infected M. T. T. et al. At 102.6%, T.W. E. At 106.5%, T.I. T. T. et al. The residual activity was 107.1%.
[0096]
Example 14
An oligo dT-poly A immobilized plate was prepared in accordance with (9) preparation of oligo dT-poly A immobilized plate in Example 13. A reverse transcriptase-binding plate was prepared using manganese chloride or calcium chloride instead of magnesium chloride in the binding reaction of reverse transcriptase on the prepared plate (10) oligo dT-poly A solid phased plate in Example 13 did. The prepared enzyme-binding plate was similarly operated according to the simultaneous reaction of (11) antigen-antibody reaction and reverse transcriptase reaction in Example 13. Thereafter, the activity inhibition by the antibody (5) reverse transcriptase activity inhibition in Example 1 was quantified. The results are shown in Table 14.
[0097]
[Table 14]

[0098]
As shown in Table 14, in any of the metal ions used, the group of antibodies that inhibit DNA elongation reaction from the template RNA due to the competitive reaction between the reverse transcriptase and the antibody can be quantified as an activity inhibition amount, and quantification is possible at any ion concentration. did it.
[0099]
Example 15
Oligo dT in the preparation of (9) oligo dT-poly A immobilized plate of Example 13 _19-24 Instead of oligo dT ₅ , Oligo dT ₁₀ (Sigma), oligo dT _25-30 Poly dT (average chain length 794 base) (manufactured by Pharmacia) was used to prepare an oligo dT-poly A solid phase plate. A reverse transcriptase-binding plate was prepared using the prepared plate with a magnesium chloride concentration of 50 mM in the binding reaction of reverse transcriptase on Example 13 (10) oligo dT-poly A-immobilized plate. The prepared enzyme-binding plate was similarly operated according to the simultaneous reaction of (11) antigen-antibody reaction and reverse transcriptase reaction in Example 13. Thereafter, the activity inhibition by the antibody (5) reverse transcriptase activity inhibition in Example 1 was quantified. The results are shown in Table 15.
[0100]
[Table 15]

[0101]
As shown in Table 15, with any length of oligo dT used, the group of antibodies that inhibit the DNA elongation reaction from the template RNA due to the competitive reaction between the reverse transcriptase and the antibody could be quantified as the activity inhibition amount.
[0102]
Example 16
Instead of poly A in preparation of (9) oligo dT-poly A solid phase plate in Example 13, oligo A _12-18 Poly d (average chain length 534 bases) (Pharmacia) was used to prepare oligo dT-poly A solid phased plates. A reverse transcriptase-binding plate was prepared using the magnesium chloride concentration in the reverse transcriptase binding reaction on the prepared plate (10) oligo dT-poly A-immobilized plate of Example 13 as 50 mM. The prepared enzyme-binding plate was similarly operated according to the simultaneous reaction of (11) antigen-antibody reaction and reverse transcriptase reaction in Example 13. Thereafter, the activity inhibition by the antibody (5) reverse transcriptase activity inhibition in Example 1 was quantified. The results are shown in Table 16.
[0103]
[Table 16]

[0104]
As shown in Table 16, with any length of poly A used, the group of antibodies that inhibit the DNA elongation reaction from the template RNA due to the competitive reaction between the reverse transcriptase and the antibody could be quantified as the activity inhibition amount.
[0105]
[Example 17]
An oligo dT-poly A immobilized plate was prepared according to the preparation of Example 13 (9) oligo dT-poly A immobilized plate. In the prepared plate, the magnesium chloride concentration in the reverse transcriptase binding reaction on the (10) oligo dT-poly A solid phase immobilized plate of Example 13 was used as 50 mM, and the reverse transcriptase amount was 0.1, 1, 10 , 100, 1000, 10000, 100000 mU / 100 μl concentrations of reverse transcriptase were used to prepare reverse transcriptase binding plates. The prepared enzyme-binding plate was treated in the same manner as in Example 13, except that the serum in 5 (11) antigen-antibody reaction and reverse transcriptase reaction was the same as that of 5 HIV-infected hemophilia patients. Thereafter, the activity inhibition by the antibody (5) reverse transcriptase activity inhibition in Example 1 was quantified.
[0106]
Separately from this, when reverse transcriptase of 0.1, 1, 10 μU / 100 μl was used in the above method, the reverse of (11) antigen-antibody reaction of Example 13 In the simultaneous reaction of the photoenzyme reaction, a 24-hour reverse transcriptase reaction was performed instead of the 2-hour reverse transcriptase reaction, and the other operating conditions were the same. The results are shown in Table 17.
[0107]
[Table 17]

[0108]
As shown in Table 17, at any concentration of reverse transcriptase used, the group of antibodies that inhibit the DNA elongation reaction from the template RNA due to the competitive reaction between the reverse transcriptase and the antibody could be quantified as the activity inhibition amount.
[0109]
Example 18
An oligo dT-poly A immobilized plate was prepared according to the preparation of Example 13 (9) oligo dT-poly A immobilized plate. A reverse transcriptase-binding plate was prepared using the magnesium chloride concentration in the reverse transcriptase binding reaction on the prepared plate (10) oligo dT-poly A-immobilized plate of Example 13 as 50 mM. The prepared enzyme-binding plate was diluted with 1, 10, 100, 1000, 10000, and 100,000-fold diluted solutions in the simultaneous reaction of Example 13 (11) antigen-antibody reaction and reverse transcriptase reaction, respectively. An antigen-antibody reaction was performed and the same operation was performed. Thereafter, the activity inhibition by the antibody (5) reverse transcriptase activity inhibition in Example 1 was quantified. The results are shown in Table 18.
[0110]
[Table 18]

[0111]
As shown in Table 18, at any dilution concentration used, the DNA elongation reaction inhibiting antibody group from the template RNA due to the competitive reaction between the reverse transcriptase and the antibody could be quantified as the activity inhibition amount.
[0112]
【The invention's effect】
By classifying the epitope group of activity blocking into three in the reagent for research of reverse transcriptase activity blocking antibody, the virus mutation can be traced by the antibody, and the research cost in this field can be reduced.

Claims (27)

A method for measuring a reverse transcriptase activity-blocking antibody comprising performing at least the following four steps in a method for measuring a reverse transcriptase activity-blocking antibody derived from a human immunodeficiency virus in a sample. Step A: Binding reaction between a substantial oligodeoxythymine nucleotide immobilized on a transparent or translucent or colored solid phase and a substantial adenine ribopolynucleotide hybridized thereto and reverse transcriptase, step B: After washing and separating the enzyme reacted in step A and the unreacted enzyme, reacting with a specimen containing a reverse transcriptase activity blocking antibody, and step C: reacting the antibody reacted in step B with the unreacted antibody. After washing and separation, a nucleic acid complementary to the template RNA is synthesized from deoxymononucleotide triphosphate by reverse transcriptase on the solid phase. Step D: Enzyme activity inhibition of antibody based on the amount of synthesized nucleic acid Quantifying the process. 2. The method for measuring a reverse transcriptase activity blocking antibody according to claim 1, wherein in step A according to claim 1, the binding reaction is carried out in the presence of a water-soluble metal salt that generates a divalent metal ion. The method for measuring a reverse transcriptase activity blocking antibody according to claim 2, wherein the divalent metal ion is 0.1 mM to 500 mM. The method for measuring a reverse transcriptase activity blocking antibody according to claim 2, wherein the divalent metal ion is magnesium, calcium or manganese ion. The method for measuring a reverse transcriptase activity blocking antibody according to claim 1, wherein the oligodeoxythymine nucleotide has 5 to 100,000 bases. The method for measuring a reverse transcriptase activity blocking antibody according to claim 1, wherein the adenine ribopolynucleotide has 5 to 100,000 bases. The method for measuring a reverse transcriptase activity-blocking antibody according to claim 1, wherein the reverse transcriptase is 2 µU / ml to 1000 U / ml. The method for measuring a reverse transcriptase activity blocking antibody according to claim 1, wherein the deoxymononucleotide triphosphate is at least biotin-labeled deoxyuridine triphosphate. The method for measuring a reverse transcriptase activity blocking antibody according to claim 1, wherein the specimen is obtained from serum or plasma and diluted 1-fold to 100,000-fold. A method for measuring a reverse transcriptase activity-blocking antibody, comprising performing at least the following four steps in a method for measuring a reverse transcriptase activity-blocking antibody derived from a human immunodeficiency virus in a sample. Step E: reacting a reverse transcriptase with a specimen containing a reverse transcriptase activity blocking antibody, Step F: a substantial oligodeoxythymine nucleotide immobilized on a transparent or translucent or colored solid phase Substantially adenine ribopolynucleotide hybridized thereto and the reaction solution of Step E are reacted with binding, Step G: The reaction product and the unreacted product in Step F are washed and separated, and then reverse transcriptase on the solid phase. A step of synthesizing a nucleic acid complementary to the template RNA from deoxymononucleotide triphosphate, and step H: a step of quantifying the inhibition of the enzyme activity of the antibody based on the amount of the synthesized nucleic acid. The method for measuring a reverse transcriptase activity blocking antibody according to claim 10, wherein in step F of claim 10, the binding reaction is carried out in the presence of a water-soluble metal salt that generates a divalent metal ion. The method for measuring a reverse transcriptase activity blocking antibody according to claim 11, wherein the divalent metal ion is 0.1 mM to 500 mM. The method for measuring a reverse transcriptase activity blocking antibody according to claim 11, wherein the divalent metal ion is magnesium, calcium or manganese ion. The method for measuring a reverse transcriptase activity blocking antibody according to claim 10, wherein the oligo dT has 5 to 100,000 bases. The method for measuring a reverse transcriptase activity blocking antibody according to claim 10, wherein the adenine ribopolynucleotide has 5 to 100,000 bases. The method for measuring a reverse transcriptase activity-blocking antibody according to claim 10, wherein the reverse transcriptase is 2 μU / ml to 1000 U / ml. The method for measuring a reverse transcriptase activity blocking antibody according to claim 10, wherein the deoxymononucleotide triphosphate is biotin-labeled deoxyuridine triphosphate. The method for measuring a reverse transcriptase activity-blocking antibody according to claim 10, wherein the specimen is obtained from serum or plasma and diluted 1-fold to 100000-fold. A method for measuring a reverse transcriptase activity blocking antibody, comprising performing at least the following three steps in a method for measuring a reverse transcriptase activity blocking antibody derived from a human immunodeficiency virus in a sample. Step I: Binding reaction between a substantial oligodeoxythymine nucleotide immobilized on a transparent or translucent or colored solid phase and a substantial adenine ribopolynucleotide hybridized thereto and a reverse transcriptase, Step J: After washing and separating the enzyme reacted in Step I and the unreacted enzyme, a reverse transcriptase on the solid phase is added by simultaneously adding a specimen containing a reverse transcriptase activity blocking antibody and deoxymononucleotide triphosphate. Step K: a step of quantifying the inhibition of the enzyme activity of the antibody based on the amount of the synthesized nucleic acid. 20. The method for measuring a reverse transcriptase activity blocking antibody according to claim 19, wherein in step I of step 19, a binding reaction is carried out in the presence of a water-soluble metal salt that generates a divalent metal ion. The method for measuring a reverse transcriptase activity blocking antibody according to claim 20, wherein the divalent metal ion is 0.1 mM to 500 mM. The method for measuring a reverse transcriptase activity blocking antibody according to claim 20, wherein the divalent metal ion is magnesium, calcium, or manganese ion. 20. The method for measuring a reverse transcriptase activity blocking antibody according to claim 19, wherein oligo dT has 5 to 100,000 bases. The method for measuring a reverse transcriptase activity blocking antibody according to claim 19, wherein the adenine ribopolynucleotide has 5 to 100,000 bases. 20. The method for measuring a reverse transcriptase activity blocking antibody according to claim 19, wherein the reverse transcriptase is 2 [mu] U / ml to 1000 U / ml. The method for measuring a reverse transcriptase activity blocking antibody according to claim 19, wherein the deoxymononucleotide triphosphate is biotin-labeled deoxyuridine triphosphate. The method for measuring a reverse transcriptase activity-blocking antibody according to claim 19, wherein the specimen is obtained from serum or plasma and diluted 1-fold to 100000-fold.