EA039761B1 - In vitro и клеточные анализы измерения активности ботулинических нейротоксинов - Google Patents
In vitro и клеточные анализы измерения активности ботулинических нейротоксинов Download PDFInfo
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- EA039761B1 EA039761B1 EA201990929A EA201990929A EA039761B1 EA 039761 B1 EA039761 B1 EA 039761B1 EA 201990929 A EA201990929 A EA 201990929A EA 201990929 A EA201990929 A EA 201990929A EA 039761 B1 EA039761 B1 EA 039761B1
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| CN114540419A (zh) * | 2022-03-04 | 2022-05-27 | 中国人民解放军军事科学院军事医学研究院 | 一种分析包膜病毒膜融合效率的三功能报告系统 |
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| WO2024217577A1 (zh) * | 2023-04-21 | 2024-10-24 | 深圳琅技生命科技有限公司 | 一种肽以及含有该肽的发光复合物 |
| GB202404021D0 (en) | 2024-03-20 | 2024-05-01 | Ipsen Biopharm Ltd | Cell-based neurotoxin assay |
| CN119039432B (zh) * | 2024-10-31 | 2025-01-28 | 兰州生物技术开发有限公司 | 一种检测肉毒毒素活性或突触体相关蛋白的方法和试剂盒 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005038029A2 (en) * | 2003-10-10 | 2005-04-28 | Promega Corporation | Luciferase biosensor |
| US6890745B1 (en) * | 2000-07-19 | 2005-05-10 | Chemicon International, Inc. | Protease specific cleavable luciferases and methods of use thereof |
Family Cites Families (23)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4737462A (en) | 1982-10-19 | 1988-04-12 | Cetus Corporation | Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of interferon-β |
| US4518584A (en) | 1983-04-15 | 1985-05-21 | Cetus Corporation | Human recombinant interleukin-2 muteins |
| US4950588A (en) | 1985-07-10 | 1990-08-21 | Molecular Diagnostics, Inc. | Prolonged enhanced chemiluminescence |
| DE3537877A1 (de) | 1985-10-24 | 1987-04-30 | Geiger Reinhard | Luciferin-derivate und immunoassays unter einsatz derartiger luciferin-derivate |
| US5004565A (en) | 1986-07-17 | 1991-04-02 | The Board Of Governors Of Wayne State University | Method and compositions providing enhanced chemiluminescence from 1,2-dioxetanes |
| US5374534A (en) | 1990-07-19 | 1994-12-20 | Charm Sciences, Inc. | Method of preparing D-luciferin derivatives |
| DE4210759A1 (de) | 1992-04-01 | 1993-10-07 | Boehringer Mannheim Gmbh | Substituierte Thiazolin-Dioxetan-Substrate, Verfahren zur Herstellung und Verwendung |
| JP3523879B2 (ja) | 1994-05-31 | 2004-04-26 | アレルガン インコーポレイテッド | 輸送タンパク質用クロストリジウム属細菌毒素の修飾 |
| GB9508204D0 (en) | 1995-04-21 | 1995-06-07 | Speywood Lab Ltd | A novel agent able to modify peripheral afferent function |
| GB9617671D0 (en) | 1996-08-23 | 1996-10-02 | Microbiological Res Authority | Recombinant toxin fragments |
| GB9721189D0 (en) | 1997-10-08 | 1997-12-03 | Speywood Lab The Limited | Analgesic conjugates |
| EP1064360B1 (en) | 1998-03-27 | 2008-03-05 | Prolume, Ltd. | Luciferases, gfp fluorescent proteins, their nucleic acids and the use thereof in diagnostics |
| US8753831B2 (en) | 2007-06-05 | 2014-06-17 | City Of Hope | Methods for detection of botulinum neurotoxin |
| EP3181687B1 (en) | 2009-05-01 | 2018-08-08 | Promega Corporation | Synthetic oplophorus luciferases with enhanced light output |
| US20140287433A1 (en) * | 2011-07-19 | 2014-09-25 | ETH Zürich | Means and methods for determining clostridial neurotoxins |
| EP2823053B1 (en) | 2012-03-07 | 2017-08-23 | Merz Pharma GmbH & Co. KGaA | Means and methods for determining neurotoxin activity based on a modified luciferase |
| CA2886939C (en) * | 2012-10-16 | 2020-11-03 | Merz Pharma Gmbh & Co. Kgaa | Cellular test systems for the determination of the biological activities of neurotoxin polypeptides |
| HK1210190A1 (en) * | 2012-11-21 | 2016-04-15 | Merz Pharma Gmbh & Co. Kgaa. | Means and methods for determination of botulinum neurotoxin biological activity |
| EP4177261A1 (en) * | 2013-03-15 | 2023-05-10 | Promega Corporation | Activation of bioluminescence by structural complementation |
| PL3014267T3 (pl) * | 2013-06-28 | 2019-05-31 | Merz Pharma Gmbh & Co Kgaa | Środki i sposoby określania aktywności biologicznej polipeptydów neurotoksyny w komórkach |
| EP3981783A1 (en) * | 2015-03-26 | 2022-04-13 | President And Fellows Of Harvard College | Engineered botulinum neurotoxin |
| CA3040507A1 (en) | 2016-10-20 | 2018-04-26 | President And Fellows Of Harvard College | In vitro and cell based assays for measuring the activity of botulinum neurotoxins |
| PH12021551398A1 (en) * | 2018-12-11 | 2022-11-07 | Q32 Bio Inc | Fusion protein constructs for complement associated disease |
-
2017
- 2017-10-19 CA CA3040507A patent/CA3040507A1/en active Pending
- 2017-10-19 BR BR112019007831-2A patent/BR112019007831A2/pt active Search and Examination
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- 2017-10-19 HU HUE17801528A patent/HUE064332T2/hu unknown
-
2019
- 2019-04-15 MX MX2023004733A patent/MX2023004733A/es unknown
- 2019-04-18 SA SA519401607A patent/SA519401607B1/ar unknown
- 2019-04-26 ZA ZA2019/02654A patent/ZA201902654B/en unknown
-
2022
- 2022-03-10 US US17/691,848 patent/US11788069B2/en active Active
- 2022-10-21 JP JP2022169039A patent/JP2023011700A/ja not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6890745B1 (en) * | 2000-07-19 | 2005-05-10 | Chemicon International, Inc. | Protease specific cleavable luciferases and methods of use thereof |
| WO2005038029A2 (en) * | 2003-10-10 | 2005-04-28 | Promega Corporation | Luciferase biosensor |
Non-Patent Citations (3)
| Title |
|---|
| Society for Neuroscience ; 1 January 2001 (2001-01-01), LENG J, ET AL.: "CLEAVALITETM: A NOVEL BIOLUMINESCENT CASPASE ACTIVITY ASSAY", XP008005347 * |
| TURTON, K. CHADDOCK, J.A. ACHARYA, K.R.: "Botulinum and tetanus neurotoxins: structure, function and therapeutic utility", TRENDS IN BIOCHEMICAL SCIENCES, ELSEVIER, AMSTERDAM, NL, vol. 27, no. 11, 1 November 2002 (2002-11-01), AMSTERDAM, NL , pages 552 - 558, XP004390740, ISSN: 0968-0004, DOI: 10.1016/S0968-0004(02)02177-1 * |
| WAUD J P, ET AL: "Engineering the C-terminus of firefly luciferase as an indicator of covalent modification of proteins", BIOCHIMICA ET BIOPHYSICA ACTA., ELSEVIER, NL, vol. 1292, no. 1, 1 January 1996 (1996-01-01), NL , pages 89 - 98, XP002325291, ISSN: 0006-3002 * |
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