DK2912456T3 - Assay til vurdering af endosomal transport - Google Patents
Assay til vurdering af endosomal transport Download PDFInfo
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- DK2912456T3 DK2912456T3 DK13785585.4T DK13785585T DK2912456T3 DK 2912456 T3 DK2912456 T3 DK 2912456T3 DK 13785585 T DK13785585 T DK 13785585T DK 2912456 T3 DK2912456 T3 DK 2912456T3
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- molecule
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- eukaryotic cell
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- endosomes
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/952—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from bacteria
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
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- Urology & Nephrology (AREA)
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- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Toxicology (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Claims (9)
1. Assay til vurdering af et testmolekyles endosomfrigivelsesevne, hvor testmolekylet er et clostridium-neurotoxin eller en re-targeteret ikke- cytotoksisk protease (targeteret sekretionsinhibitor), hvori den ikke- cytotoksiske proteases naturlige bindingsevne er blevet modificeret ved indføring af en bindingsligand eller targeteringsenhed, og hvor testmolekylet har evnen til proteolytisk at spalte og deaktivere et SNARE-protein; hvilket assay omfatter: i) at bringe en eukaryotisk celle i kontakt med et testmolekyle, der skal vurderes for endosomfrigivelsesevne, hvor den eukaryotiske celle omfatter en cellemembran indbefattende et bindingssted på den udvendige overflade af cellens cellemembran; ii) at inkubere testmolekylet med den eukaryotiske celle og derved lade a) testmolekylet binde til og danne et bundet kompleks med bindingsstedet på den eukaryotiske celle, hvorved det bunde kompleks kan trænge ind i den eukaryotiske celle ved endocytose; b) et eller flere endosomer dannes i cellen, hvor det ene eller flere endoso-mer indeholder testmolekylet; og c) testmolekylet trænge ind i den eukaryotiske celles cytosol ved at passere den endosomale membran af det ene eller flere endosomer; iii) at fjerne overskydende testmolekyle, der ikke er bundet til bindingsstedet på de eukaryotiske celler; iv) efter et forudbestemt tidsrum at påvise mængden af testmolekylet, der er til stede i det ene eller flere endosomer, eller påvise mængden af testmolekyle, der er til stede i den eukaryotiske celles cytosol; v) at sammenligne mængden testmolekyle, der blev påvist i trin iv), med en kontrolværdi, hvor kontrolværdien repræsenterer mængden af testmolekyle i det ene eller flere endosomer eller mængden af testmolekyle i cytosolen før trin iv); vi) at beregne en endosomfrigivelseværdi for testmolekylet ved at bestemme den relative ændring i mængden af testmolekyle, der er til stede i det ene eller flere endosomer, eller bestemme den relative ændring i mængden af testmolekyle i den eukaryotiske celles cytosol; hvor trin (iv) omfatter påvisning af testmolekylet ved anvendelse af en fluorescerende markering.
2. Assay ifølge krav 1, hvor den eukaryotiske celle er valgt blandt en gærcel-le, en insektcelle, en hvirveldyrscelle, en pattedyrscelle, en plantecelle og en svampecelle.
3. Assay ifølge et hvilket som helst af de foregående krav, hvor inkube-ringstrinnet ii) fortsætter i et tidsrum på fra 5 minutter til 5 dage, såsom 1-12 timer eller 2-10 timer eller 4-8 timer eller 6-8 timer.
4. Assay ifølge et hvilket som helst af de foregående krav, hvor påvisnings-trinnet iv) udføres i mellem 5 minutter og 5 timer efter trin iii), såsom 15-240 minutter eller 30-180 minutter eller 45-150 minutter efter trin iii).
5. Assay til vurdering af de hæmmende virkninger af et blokeringsmolekyle på endosomal transport i en eukaryotisk celle, hvilket assay omfatter: i) at bringe en eukaryotisk celle i kontakt med et kontrolmolekyle, der binder til et bindingsted på overfladen af den eukaryotiske celle, hvor kontrolmoleky-let er et clostridium-neurotoxin eller en re-targeteret ikke-cytotoksisk protease (targeteret sekretionsinhibitor), hvori den ikke-cytotoksiske proteases naturlige bindingsevne er blevet modificeret ved indføring af en bindingsligand eller targeteringsenhed, og hvor kontrolmolekylet har evnen til proteolytisk at spalte og deaktivere et SNARE-protein, hvor kontrolmolekylet danner et bundet kompleks med bindingsstedet, trænger ind i den eukaryotiske celle ved endocytose, hvorved der dannes et endosom, som indeholder kontrolmolekylet, og kontrolmolekylet trænger ind i den eukaryotiske celles cytosol ved at passere endosomets endosomale membran; ii) at inkubere kontrolmolekylet med den eukaryotiske celle og derved lade a) kontrolmolekylet binde to og danne et bundet kompleks med bindingsstedet på den eukaryotiske celle, hvorved det bunde kompleks kan trænge ind i den eukaryotiske celle ved endocytose; b) et eller flere endosomer dannes i cellen, hvor det ene eller flere endoso-mer indeholder kontrolmolekylet; og c) kontrolmolekylet trænge ind i den eukaryotiske celles cytosol ved at passere den endosomale membran af det ene eller flere endosomer; iii) at bringe den eukaryotiske celle i kontakt med et testinhibitormolekyle, der skal vurderes for sin evne til at undertrykke endosomal frigivelse af kontrol-molekyle fra endosomer; iv) efter et forudbestemt tidsrum at påvise mængden af kontrolmolekylet, der er til stede i det ene eller flere endosomer, eller påvise mængden af kontrol-molekyle, der er til stede i den eukaryotiske celles cytosol; v) at sammenligne mængden kontrolmolekyle, der blev påvist i trin iv), med en kontrolværdi, hvor kontrolværdien repræsenterer mængden af kontrolmolekyle i det ene eller flere endosomer eller mængden af kontrolmolekyle i cy-tosolen før trin iii); vi) at tildele en inhiberingsværdi til testinhibitormolekylet ved at bestemme den relative ændring i mængden af kontrolmolekyle, der er til stede i det ene eller flere endosomer, eller bestemme den relative ændring i mængden af kontrolmolekyle i den eukaryotiske celles cytosol, hvor trin (iv) omfatter påvisning af kontrolmolekylet ved anvendelse en fluorescerende markering.
6. Assay ifølge krav 5, hvor den eukaryotiske celle er valgt blandt en gærcel-le, en insektcelle, en hvirveldyrscelle, en pattedyrscelle, en plantecelle og en svampecelle.
7. Assay ifølge et af kravene 5-6, hvor inkuberingstrinnet ii) fortsætter i et tidsrum på fra 5 minutter til 5 dage, såsom 1 -12 timer eller 2-10 timer eller 4-8 timer eller 6-8 timer.
8. Assay ifølge et af kravene 5-7, hvor påvisningstrinnet iv) udføres i mellem 5 minutter og 5 timer efter trin iii), såsom 15-240 minutter eller 30-180 minutter eller 45-150 minutter efter trin iii).
9. Assay ifølge et af kravene 5-8, hvor kontakttrinnet ii) udføres i mellem 5 minutter og 5 dage efter påbegyndelse af inkuberingstrinnet ii), såsom mellem 30 minutter og 12 timer eller mellem 30 minutter og 10 timer eller mellem 30 minutter og 8 timer eller mellem 1-8 timer efter påbegyndelse af inkuberingstrinnet ii).
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB1219024.5A GB201219024D0 (en) | 2012-10-23 | 2012-10-23 | Assay |
| PCT/GB2013/052765 WO2014064442A1 (en) | 2012-10-23 | 2013-10-23 | Assay for assessment of endosomal transport |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| DK2912456T3 true DK2912456T3 (da) | 2017-04-03 |
Family
ID=47359328
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK13785585.4T DK2912456T3 (da) | 2012-10-23 | 2013-10-23 | Assay til vurdering af endosomal transport |
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| Country | Link |
|---|---|
| US (1) | US9170252B2 (da) |
| EP (2) | EP3217174A1 (da) |
| JP (1) | JP6302475B2 (da) |
| CN (1) | CN104718452B (da) |
| DK (1) | DK2912456T3 (da) |
| ES (1) | ES2618521T3 (da) |
| GB (1) | GB201219024D0 (da) |
| HK (2) | HK1208527A1 (da) |
| HU (1) | HUE032518T2 (da) |
| PL (1) | PL2912456T3 (da) |
| PT (1) | PT2912456T (da) |
| RU (1) | RU2673086C2 (da) |
| WO (1) | WO2014064442A1 (da) |
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| GB9411138D0 (en) | 1994-06-03 | 1994-07-27 | Microbiological Res Authority | Toxin assay |
| 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 |
| US6277479B1 (en) | 1997-12-19 | 2001-08-21 | Kimberly-Clark Worldwide, Inc. | Microporous films having zoned breathability |
| US7183066B2 (en) * | 2002-09-27 | 2007-02-27 | Allergan, Inc. | Cell-based fluorescence resonance energy transfer (FRET) assays for clostridial toxins |
| WO2005014798A2 (en) * | 2003-03-31 | 2005-02-17 | Boston Medical Center Corporation | Novel compositions and methods for promoting, inhibiting, and detecting protein entry into cells |
| GB0321344D0 (en) * | 2003-09-11 | 2003-10-15 | Health Prot Agency | Re-targeted toxin conjugates |
| CN101063657A (zh) * | 2006-04-29 | 2007-10-31 | 中国科学院上海生命科学研究院 | 在活体细胞中筛选配体与受体结合的方法和系统 |
| AU2007278899B2 (en) * | 2006-06-23 | 2012-09-06 | Engenelc Molecular Delivery Pty. Ltd. | Targeted delivery of drugs, therapeutic nucleic acids and functional nucleic acids to mammalian cells via intact killed bacterial cells |
| CN101070547B (zh) * | 2007-05-15 | 2010-12-22 | 中国人民解放军军事医学科学院军事兽医研究所 | 主动运载外源基因的重组蛋白载体PEAⅡ-HPhA及其制备方法和应用 |
| WO2009041633A1 (ja) * | 2007-09-28 | 2009-04-02 | Kyoto University | コイルドコイルを利用した膜タンパク質標識方法 |
| WO2010085473A1 (en) * | 2009-01-20 | 2010-07-29 | Trustees Of Tufts College | Methods for the delivery of toxins or enzymatically active portions thereof |
| EP2456846B1 (en) * | 2009-07-23 | 2015-09-02 | International Technology Center | Lubricant additive comprising carbon nanoparticles |
| JP5661626B2 (ja) * | 2009-07-23 | 2015-01-28 | 国立大学法人 東京大学 | アニオン性ポリマー、該アニオン性ポリマーを用いたポリイオンコンプレックスおよび三元系ポリマー複合体、ならびに薬学組成物 |
| EP2464662A1 (en) | 2009-08-12 | 2012-06-20 | Medical Research Council | Complexing system |
| CA2770185C (en) | 2009-08-17 | 2019-04-09 | East Carolina University | Fast acting snare-cleaving enzymes |
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2012
- 2012-10-23 GB GBGB1219024.5A patent/GB201219024D0/en not_active Ceased
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2013
- 2013-10-23 JP JP2015538567A patent/JP6302475B2/ja not_active Expired - Fee Related
- 2013-10-23 PT PT137855854T patent/PT2912456T/pt unknown
- 2013-10-23 CN CN201380053659.7A patent/CN104718452B/zh not_active Expired - Fee Related
- 2013-10-23 EP EP17150909.4A patent/EP3217174A1/en not_active Withdrawn
- 2013-10-23 PL PL13785585T patent/PL2912456T3/pl unknown
- 2013-10-23 EP EP13785585.4A patent/EP2912456B1/en active Active
- 2013-10-23 US US14/429,668 patent/US9170252B2/en not_active Expired - Fee Related
- 2013-10-23 HU HUE13785585A patent/HUE032518T2/en unknown
- 2013-10-23 RU RU2015119539A patent/RU2673086C2/ru active
- 2013-10-23 WO PCT/GB2013/052765 patent/WO2014064442A1/en active Application Filing
- 2013-10-23 ES ES13785585.4T patent/ES2618521T3/es active Active
- 2013-10-23 DK DK13785585.4T patent/DK2912456T3/da active
-
2015
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2018
- 2018-03-13 HK HK18103514.1A patent/HK1244060A1/zh unknown
Also Published As
| Publication number | Publication date |
|---|---|
| RU2015119539A (ru) | 2016-12-20 |
| HUE032518T2 (en) | 2017-09-28 |
| HK1208527A1 (en) | 2016-03-04 |
| JP6302475B2 (ja) | 2018-03-28 |
| HK1244060A1 (zh) | 2018-07-27 |
| EP2912456A1 (en) | 2015-09-02 |
| EP2912456B1 (en) | 2017-01-11 |
| CN104718452A (zh) | 2015-06-17 |
| CN104718452B (zh) | 2017-07-18 |
| GB201219024D0 (en) | 2012-12-05 |
| PL2912456T3 (pl) | 2017-07-31 |
| EP3217174A1 (en) | 2017-09-13 |
| RU2673086C2 (ru) | 2018-11-22 |
| JP2015534076A (ja) | 2015-11-26 |
| US20150241408A1 (en) | 2015-08-27 |
| US9170252B2 (en) | 2015-10-27 |
| WO2014064442A1 (en) | 2014-05-01 |
| ES2618521T3 (es) | 2017-06-21 |
| PT2912456T (pt) | 2017-04-19 |
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