JPH05505239A - 改良された免疫組織化学的染色法およびそのための試薬 - Google Patents
改良された免疫組織化学的染色法およびそのための試薬Info
- Publication number
- JPH05505239A JPH05505239A JP3505979A JP50597991A JPH05505239A JP H05505239 A JPH05505239 A JP H05505239A JP 3505979 A JP3505979 A JP 3505979A JP 50597991 A JP50597991 A JP 50597991A JP H05505239 A JPH05505239 A JP H05505239A
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- antibody
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- stabilized
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Abstract
Description
Claims (1)
- 【特許請求の範囲】 1、実質的に塩不含の水溶液および表面張力を減少して水性リンス溶液の平らな シートを形成するために十分な量の非イオン性の生物学的洗浄剤から本質的に成 る、スライドのアッセイ領域のリンシングに使用する水性リンス溶液。 2、前記非イオン性洗浄剤は0.01〜5%(v/v)の濃度で存在する、請求 の範囲第1項記載の水性リンス溶液。 3、前記非イオン性洗浄剤はポリオキシエチレンソルビタンおよびポリオキシエ チレンエーテルから選択される非イオン性洗浄剤である、請求の範囲第1項記載 の水性リンス溶液。 4、前記非イオン性洗浄剤はポリオキシエチレンソルビタンモノラウレートおよ びポリオキシエチレン23ラウリルエーテルから選択される、請求の範囲第3項 記載の水性リンス溶液。 5、さらに有効量の保存剤を含む、請求の範囲第1項記載の水性リンス溶液。 6、実質的に塩不含の水溶液はトリス(Tris)溶液である、請求の範囲第1 項記載の水性リンス溶液。 7、前記実質的に塩不含の水溶液は水である、請求の範囲第1項記載の水性リン ス溶液。 8、約0.05〜約0.1モルのトリス(Tris)、0.01〜5%(v/v )の濃度のポリオキシエチレンソルビタンおよびポリオキシエチレンエーテルか ら選択される非イオン性洗浄剤、および、必要に応じて、有効量の保存剤から本 質的に成る、スライドのアッセイ領域のリンシングに使用する水性リンス溶液。 9、実質的に塩不含の水溶液および表面張力を減少して水性リンス溶液の平らな シートを形成するために表面張力を減少するために十分な量の非イオン性の生物 学的洗浄剤から本質的に成る溶液で、スライドのアッセイ領域をリンスすること からなる、アッセイ試薬または試料をスライドのアッセイ領域に結合させる改良 されたアッセイ方法。 10、前記アッセイ領域は組織の切片を結合するスライドの区域からなる、請求 の範囲第9項記載の方法。 11、スライドの試薬を適用したアッセイ領域に蒸発抑制液体でカバーすること からなる、前記アッセイ領域に適用した試薬の蒸発を防止する方法。 12、前記蒸発抑制液体は中程度の連鎖のアルカン族の油から本質的に成る、請 求の範囲第11項記載の方法。 13、前記アルカンは10〜16個の炭素を有する、請求の範囲第12項記載の 方法。 14、前記蒸発抑制液体はペンタデカンから本質的に成る、請求の範囲第13項 記載の方法。 15、中程度の連鎖のアルカン族の油から本質的に成る蒸発抑制液体でアッセイ 領域をカバーすることからなる、アッセイ試薬または試料をスライドのアッセイ 領域に結合する、改良されたアッセイ法。 16、前記方法は免疫組織化学的染色法である、請求の範囲第15項記載の方法 。 17、前記方法は正常部位ハイブリダイゼーションである、請求の範囲第15項 記載の方法。 18、a、40〜60%のグリコール、b、生理学的緩衝液、 c、有効量の還元剤、 d、酵素の安定性を増大するために十分な濃度のカルシウムイオン源、 e、有効量のタンパク質分解酵素、およびf、必要に応じて、有効量の保存剤、 からなる、安定化されたタンパク質分解酵素の溶液。 19、前記酵素はVIII型アルカリ性プロテアーゼである、請求の範囲第18 項記載の安定化されたタンパク質分解酵素の溶液。 20、前記緩衝液はトリス(Tris)緩衝液pH7.0〜7.5である、請求 の範囲第18項記載の安定化されたタンパク質分解酵素の溶液。 21、前記緩衝液は0.01〜0.1モルのトリス(Tris)/HCI、pH 7.4である、請求の範囲第20項記載の安定化されたタンパク質分解酵素の溶 液。 22、前記還元剤はジチチオスオスレイトール、アスコルビン酸およびメタ重亜 硫酸ナトリウムから成る群より選択される、請求の範囲第18項記載の安定化さ れたタンパク質分解酵素の溶液。 23、前記還元剤は0.0005〜0.05%の濃度のメタ重亜硫酸ナトリウム である請求の範囲第22項記載の安定化されたタンパク質分解酵素の溶液。 24、前記グリコールはポリエチレングリコールである、請求の範囲第18項記 載の安定化されたタンパク質分解酵素の溶液。 25、前記カルシウムイオン源は1〜10ミリモルの塩化カルシウムである、請 求の範囲第18項記載の安定化されたタンパク質分解酵素の溶液。 26、前記保存剤が存在し、そして0.001%〜0.1%の範囲の濃度のチメ ロサルである、請求の範囲第18項記載の安定化されたタンパク質分解酵素の溶 液。 27、a、有効量のVlII型アルカリ性プロテアーゼ、b、生理学的トリス( Tris)緩衝液pH7.0〜7.5、c、0.0005〜0.05%のメタ重 亜硫酸ナトリウム、d、40〜60%のプロピレングリコール、e、1〜10ミ リモルの塩化カルシウム、およびf、0.001%〜0.1%のチメロサル、か らなる、安定化されたタンパク質分解酵素の溶液。 28、抗原をアンマスキングする組織の切片を、生理学的緩衝液、40〜60% のグリコール、有効量の還元剤、酵素の安定性を増大するために十分な濃度のカ ルシウムイオン源、有効量のタンパク質分解酵素、および必要に応じて、有効量 の保存剤からなる安定化されたタンパク質分解酵素の溶液で、前記抗原をアンマ スキングするために十分な時間の間カバーすることからなる、前記組織の切片を 免疫組織化学的に染色する改良された方法。 29、a、生理学的緩衝液、および b、非特異的抗体の結合を阻害するために十分なタンパク質濃度の第2抗体の種 からの非免疫血清のグロブリン分画、から本質的に成る、第1および第2の抗体 を有する免疫化学的方法において使用するための改良された抗体の希釈剤。 30、前記グロブリンは0.1〜5%の濃度で存在する、請求の範囲第29項記 載の改良された抗体の希釈剤。 31、前記第2抗体はヤギ抗体であり、そして前記グロブリンはヤギグロブリン である、請求の範囲第29項記載の改良された抗体の希釈剤。 32、前記ヤギグロブリンはコーン分画II/IIIである、請求の範囲第31 項記載の改良された抗体の希釈剤。 33、前記生理学的緩衝液はリン酸塩緩衝化生理的塩類溶液pH7.0〜7.5 である、請求の範囲第29項記載の改良された抗体の希釈剤。 34、前記リン酸塩緩衝化生理的塩類溶液は0.01〜0.5モルのである、請 求の範囲第33項記載の改良された抗体の希釈剤。 35、希釈剤の平らなシートを形成するために表面張力を樹脂するために十分な 量の洗浄剤をさらに含む、請求の範囲第29項記載の改良された抗体の希釈剤。 36、前記洗浄剤はポリオキシエチレンソルビタンおよびポリオキシエチレンエ ーテルから選択される非イオン性洗浄剤である、請求の範囲第35項記載の改良 された抗体の希釈剤。 37、前記非イオン性洗浄剤はポリオキシエチレンソルビタンモノラウレートお よびポリオキシエチレン23ラウリルエーテルから選択される、請求の範囲第3 6項記載の改良された抗体の希釈剤。 38、有効量の保存剤をさらに含む、請求の範囲第29項記載の改良された抗体 の希釈剤。 39、a、0.01〜0.5モルのリン酸塩緩衝化生理的塩類溶液pH7.0〜 7.5、 b、0.1〜5%の前記第2抗体の種のグロブリン、c、0.1〜5%(v/v )の塩化カルシウムのポリオキシェチレンソルビタンおよびポリオキシエチレン エーテルから選択される非イオン性洗浄剤、および d、において、有効量の保存剤、 から本質的に成る、第1および第2の抗体を有する免疫化学的方法において使用 するための改良された抗体の希釈剤。 40、第1および第2の抗体を、生理学的緩衝液および非特異的抗体の結合を阻 害するために十分なタンパク質濃度の第2抗体の種からの非免疫血清のグロブリ ン分画からに成る希釈剤中で、前記抗体を抗原と組み合わせる前に、希釈するこ とからなる、第1および第2の抗体を使用する改良された免疫化学的方法。 41、a、酵素の働く範囲の濃度のペルオキシダーゼ発色団、b、6.0より低 いpHを維持することができる酸性緩衝液、c、有効量の還元剤、および d、1〜10%のグリコール、 から本質的に成る、安定化された水性ペルオキシダーゼ発色団溶液。 42、前記ペルオキシダーゼ発色団は3,3′−ジアミノベンジジンテトラヒド ロクロライド(DAB)であり、そして前記酸性緩衝液は5〜10ミリモルのク エン酸塩−リン酸塩緩衝液である、請求の範囲第41項記載の安定化された水性 ペルオキシダーゼ発色団溶液。 43、前記ペルオキシダーゼ発色団は3−アミノ−9−エチルカルバゾール(A EC)であり、そして酸性緩衛液は0.01〜0.5モルの酢酸塩緩衝液である 、請求の範囲第41項記載の安定化された水性ペルオキシダーゼ発色団溶液。 44、前記還元剤はジチチオスオスレイトール、アスコルビン酸およびメタ重亜 硫酸ナトリウムから成る群より選択される、請求の範囲第41項記載の安定化さ れた水性ペルオキシダーゼ発色団溶液。 45、前記還元剤はメタ重亜硫酸ナトリウムである、請求の範囲第44項記載の 安定化された水性ペルオキシダーゼ発色団溶液。 46、前記グリコールはポリエチレングリコールであり、請求の範囲第41項記 載の安定化された水性ペルオキシダーゼ発色団溶液。 47、ポリエチレングリコールは約1〜約10%の濃度で存在する、請求の範囲 第41項記載の安定化された水性ペルオキシダーゼ発色団溶液。 48、前記溶液の平らなシートを形成するために表面張力を減少するために十分 な量の洗浄剤をさらに含む、請求の範囲第41項記載の安定化された水性ペルオ キシダーゼ発色団溶液。 49、a、2ミリモルのDAB、 b、5〜10ミリモルのクエン酸塩−リン酸塩緩衝液pH5.0〜5.5、 c、0.0005〜0.05%のメタ重亜硫酸ナトリウム、d、1〜10%のポ リエチレングリコール、およびe、必要に応じて、0.01〜5%(v/v)の ポリオキシエチレンソルビタンおよびポリオキシエチレンエーテルから選択され る非イオン性洗浄剤、 からなる安定化された水性3,3′−ジアミノベンジジンテトラヒドロクロライ ド(DAB)溶液。 50、a、2ミリモルのAEC、 b、0.01〜0.5モルの酢酸塩緩衝液pH4.5〜5.5、c、0.008 〜0.008%のメタ重亜硫酸ナトリウム、d、1〜10%のポリエチレングリ コール、およびe、必要に応じて、0.01〜5%(v/v)のポリオキシエチ レンソルビタンおよびポリオキシエチレンエーテルから選択される非イオン性洗 浄剤、 からなる安定化された水性3−アミノ−9−エチルカルバゾール(AEC)溶液 。 51、酵素の働く範囲の濃度のペルオキシダーゼ発色団、6.0より低いpHを 維持することができる酸性緩衝液、有効量の還元剤、および前記ペルオキシダー ゼ発色団を安定化するために有効な濃度のグリコールから本質的に成る安定化さ れた水性ペルオキシダーゼ発色団溶液をペルオキシダーゼと組み合わせることか らなる、改良されたペルオキシダーゼに基づく免疫化学的方法。 52、次の順序: a、組織の切片を、実質的に塩不含の水溶液および表面張力を減少して水性リン ス溶液の平らなシートを形成するために十分な量の非イオン性の生物学的洗浄剤 から本質的に成る水性リンス溶液でリンスし、 b、前記組織の切片をカバーするために十分な量の蒸発抑制液体を適用し、 c、一次抗体を、生理学的緩衝液および非特異的抗体の結合を阻害するために十 分なタンパク質濃度の第2抗体と同一の種からの非免疫血清のグロブリン分画か ら本質的に成る希釈剤中で、前記組織の切片と、抗体の結合を実質的に完結する ために十分な時間の間、組み合わせ、 d、前記組織の切片を、実質的に塩不含の水溶液および表面張力を減少して水性 リンス溶液の平らなシートを形成するために十分な量の非イオン性の生物学的洗 浄剤から本質的に成る水性リンス溶液でリンスし、 e、前記組織の切片をカバーするために十分な量の蒸発抑制液体を適用し、 f、標識した第2抗体を、生理学的緩衝液および非特異的抗体の結合を阻害する ために十分なタンパク質濃度の前記第2抗体と同一の種からの非免疫血清のグロ ブリン分画から本質的に成る希釈剤中で、前記組織の切片と、抗体の結合を実質 的に完結するために十分な時間の間、組み合わせ、 g、前記組織の切片を、実質的に塩不含の水溶液および表面張力を減少して水性 リンス溶液の平らなシートを形成するために十分な量の非イオン性の生物学的洗 浄剤から本質的に成る水性リンス溶液でリンスし、 h、前記組織の切片をカバーするために十分な量の蒸発抑制液体を適用し、 i、発色試薬を前記組織の切片と、発色のために十分な時間の間、組み合わせ、 そして j、前記組織の切片を、実質的に塩不含の水溶液および表面張力を減少して水性 リンス溶液の平らなシートを形成するために十分な量の非イオン性の生物学的洗 浄剤から本質的に成る水性リンス溶液でリンスする、 ことからなる、改良された免疫組織学的方法。 53、工程(b)の後にかつ工程(c)の前に、i、過酸化水素溶液を前記組織 の切片と、内因性ペルオギシダーゼ活性を実質的に排除するために十分な時間の 間、組み合わせ、ii、前記組織の切片を、実質的に塩不含の水溶液および表面 張力を減少して水性リンス溶液の平らなシートを形成するために十分な量の非イ オン性の生物学的洗浄剤から本質的に成る水性リンス溶液でリンスし、そして iii、前記組織の切片をカバーするために十分な量の蒸発抑制液体を適用する 、 ことをさらに含む、請求の範囲第52項記載の方法。 54、工程(d)の後にかつ工程(e)の前に、i、前記組織を、40〜60% のグリコール、生理学的緩衝液、有効量の還元剤、酵素の安定性を増大するため に十分な濃度のカルシウムイオン源、有効量のタンパク質分解酵素、および必要 に応じて、有効量の保存剤からなる安定化されたタンパク質分解酵素の溶液で、 組織の抗原をアンマスキングするために十分な時間の間、カバーし、そして ii、前記組織の切片をカバーするために十分な量の蒸発抑制液体を適用する、 ことをさらに含む、請求の範囲第53項記載の方法。 55、前記標識した第2抗体はビオチニル化抗体であり、そして方法はさらに、 工程(h)の後にかつ工程(i)の前に、i、ペルオキシダーゼ標識したアビジ ンを、適当な希釈剤中で、前記組織の切片と、ビオチン−アビジンの結合を実質 的に完結するために十分な時間の間、組み合わせ、ii、前記組織の切片を、実 質的に塩不含の水溶液および表面張力を減少して水性リンス溶液の平らなシート を形成するために十分な量の非イオン性の生物学的洗浄剤から本質的に成る水性 リンス溶液でリンスし、そして iii、前記組織の切片をカバーするために十分な量の蒸発抑制液体を適用する 、 ことをさらに含む、請求の範囲第54項記載の方法。
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Cited By (6)
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JPH08136542A (ja) * | 1994-11-04 | 1996-05-31 | Sanyo Chem Ind Ltd | 改良された免疫分析用洗浄液 |
JPH09257795A (ja) * | 1996-03-21 | 1997-10-03 | Nippon Dpc Corp | スライドプレート洗浄液及びそれを含むキット |
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- 1991-02-27 US US07/924,053 patent/US5322771A/en not_active Expired - Lifetime
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
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JPH08136542A (ja) * | 1994-11-04 | 1996-05-31 | Sanyo Chem Ind Ltd | 改良された免疫分析用洗浄液 |
JPH09257795A (ja) * | 1996-03-21 | 1997-10-03 | Nippon Dpc Corp | スライドプレート洗浄液及びそれを含むキット |
JP2002517725A (ja) * | 1998-06-02 | 2002-06-18 | ヴェンタナ メディカル システムズ インコーポレイテッド | 顕微鏡スライド上で不安定試薬を合わせることによる生物学的検体を染色する方法 |
JP2013156262A (ja) * | 2006-01-13 | 2013-08-15 | Ventana Medical Syst Inc | 生体サンプル処理用組成物及び方法 |
JP2017519191A (ja) * | 2014-04-30 | 2017-07-13 | ヴェンタナ メディカル システムズ, インク. | ヘマトキシリン沈殿物の洗浄方法及びシステム |
JP2018520647A (ja) * | 2015-05-10 | 2018-08-02 | ヴェンタナ メディカル システムズ, インク. | 自動多重組織染色アッセイの間のアルカリホスファターゼ及びペルオキシダーゼ酵素の同時不活化のための組成物と方法 |
US11162877B2 (en) | 2015-05-10 | 2021-11-02 | Ventana Medical Systems, Inc. | Compositions and methods for simultaneous inactivation of alkaline phosphatase and peroxidase enzymes during automated multiplex tissue staining assays |
US11971337B2 (en) | 2015-05-10 | 2024-04-30 | Ventana Medical Systems, Inc. | Compositions and methods for simultaneous inactivation of alkaline phosphatase and peroxidase enzymes during automated multiplex tissue staining assays |
Also Published As
Publication number | Publication date |
---|---|
EP1030178B1 (en) | 2006-02-15 |
EP0517818B1 (en) | 2002-05-08 |
JP2001231552A (ja) | 2001-08-28 |
EP0517818A4 (en) | 1995-03-22 |
US5225325A (en) | 1993-07-06 |
ATE317978T1 (de) | 2006-03-15 |
EP0517818B9 (en) | 2003-01-08 |
WO1991013336A1 (en) | 1991-09-05 |
ES2177521T3 (es) | 2002-12-16 |
CA2077451C (en) | 1996-06-18 |
US5322771A (en) | 1994-06-21 |
DE69133508D1 (de) | 2006-04-20 |
DE69133006T2 (de) | 2002-12-12 |
EP1030178A1 (en) | 2000-08-23 |
ATE217418T1 (de) | 2002-05-15 |
DK1030178T3 (da) | 2006-06-12 |
JP2966522B2 (ja) | 1999-10-25 |
JP3168195B2 (ja) | 2001-05-21 |
JP2000046827A (ja) | 2000-02-18 |
EP0517818A1 (en) | 1992-12-16 |
JP3503890B2 (ja) | 2004-03-08 |
CA2077451A1 (en) | 1991-09-03 |
DE69133006D1 (de) | 2002-06-13 |
US5418138A (en) | 1995-05-23 |
DE69133508T2 (de) | 2006-09-28 |
ES2258422T3 (es) | 2006-09-01 |
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