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1. 发明授权

US07927871B2 Visualization and quantitation of cellular cytotoxicity using cell-permeable fluorogenic protease substrates and caspase activity indicator markers 有权
标题翻译：使用细胞渗透性荧光蛋白酶底物和胱天蛋白酶活性指示标记物可视化和定量细胞细胞毒性
公开(公告)号：US07927871B2
公开(公告)日：2011-04-19
申请号：US11669080
申请日：2007-01-30
申请人： Beverly Packard , Akira Komoriya
发明人： Beverly Packard , Akira Komoriya
IPC分类号： C12N5/00 , C12Q1/37 , G01N33/53
CPC分类号： G01N33/505 , A61K38/07 , A61K38/08 , G01N33/5047 , G01N33/5055 , G01N33/533 , G01N33/582
摘要： This invention provides a non-radioactive assay to monitor and quantify the target-cell killing activities mediated by cytotoxic T lymphocytes (CTLs). This assay is predicated on the discovery that apoptosis pathway activation and, in particular, granzyme B activity, provides a measure of cytotoxic effector cell activity. In one embodiment, measurement of CTL-induced granzyme B activation in target cells is achieved through detection of the specific cleavage of fluorogenic granzyme B substrates. This assay reliably detects antigen-specific CTL killing of target cells, and provides a more sensitive, more informative and safer alternative to the standard 51Cr-release assay most often used to quantify CTL responses. The assay can be used to study CTL-mediated killing of primary host target cells of different cell lineages, and enables the study of antigen-specific cellular immune responses in real time at the single-cell level. As such, the assay can provide a valuable tool for studies of infectious disease pathogenesis and development of new vaccines and immunotherapies.
摘要翻译：本发明提供了用于监测和定量由细胞毒性T淋巴细胞（CTL）介导的靶细胞杀伤活性的非放射性测定。该测定基于发现凋亡途径活化，特别是颗粒酶B活性提供细胞毒性效应细胞活性的量度的发现。在一个实施方案中，通过检测荧光颗粒酶B底物的特异性切割来实现靶细胞中CTL诱导的颗粒酶B活化的测量。该测定可靠地检测靶细胞的抗原特异性CTL杀伤，并且提供了更常用于定量CTL反应的标准51Cr释放测定法更灵敏，更有信息和更安全的替代方法。该测定可用于研究CTL介导的不同细胞系主要宿主靶细胞的杀伤，并能够在单细胞水平实时研究抗原特异性细胞免疫应答。因此，该测定可以为感染性疾病发病机理的研究和新疫苗和免疫疗法的开发提供有价值的工具。

2. 发明申请

US20090325168A1 HOMO-DOUBLY LABELED COMPOSITIONS FOR THE DETECTION OF ENZYME ACTIVITY IN BIOLOGICAL SAMPLES 审中-公开
标题翻译：用于检测生物样品中酶活性的双酚酞标记组合物
公开(公告)号：US20090325168A1
公开(公告)日：2009-12-31
申请号：US12422127
申请日：2009-04-10
申请人： Beverly S. Packard , Akira Komoriya
发明人： Beverly S. Packard , Akira Komoriya
IPC分类号： C12Q1/68 , C12Q1/02 , C12N5/06
CPC分类号： C07K7/06 , C07K7/08 , C12Q1/37 , C12Q1/44 , G01N33/533 , G01N33/542 , G01N2333/918 , G01N2333/96425 , G01N2333/96469
摘要： The present invention provides for novel reagents whose fluorescence changes upon cleavage or a change in conformation of a backbone. The reagents comprise a backbone (e.g. nucleic acid, polypeptide, etc.) joining two fluorophores of the same species whereby the fluorophores form an H-dimer resulting in quenching of the fluorescence of the fluorophores. When the backbone is cleaved or changes conformation, the fluorophores are separated, no longer forming an H-type dimer, and are de-quenched thereby providing a detectable signal. The use of a single fluorophore rather than an “acceptor-donor” fluoresecence resonance energy transfer system offers synthesis and performance advantages.
摘要翻译：本发明提供了新的试剂，其荧光在裂解时发生变化或骨架的构象变化。试剂包含连接相同物质的两个荧光团的骨架（例如核酸，多肽等），由此荧光团形成H-二聚体，导致荧光团的荧光猝灭。当主链被切割或改变构象时，荧光团被分离，不再形成H型二聚体，并且被淬灭，从而提供可检测的信号。使用单个荧光团而不是“受体 - 供体”荧光共振能量转移系统提供综合和性能优势。

3. 发明授权

US07541143B2 Homo-doubly labeled compositions for the detection of enzyme activity in biological samples 失效
标题翻译：用于检测生物样品中酶活性的双重标记的组合物
公开(公告)号：US07541143B2
公开(公告)日：2009-06-02
申请号：US11015864
申请日：2004-12-15
申请人： Beverly Packard , Akira Komoriya
发明人： Beverly Packard , Akira Komoriya
IPC分类号： C12Q1/68 , C12N5/00
CPC分类号： C07K7/06 , C07K7/08 , C12Q1/37 , C12Q1/44 , G01N33/533 , G01N33/542 , G01N2333/918 , G01N2333/96425 , G01N2333/96469
摘要： The present invention provides for novel reagents whose fluorescence changes upon cleavage or a change in conformation of a backbone. The reagents comprise a backbone (e.g. nucleic acid, polypeptide, etc.) joining two fluorophores of the same species whereby the fluorophores form an H-dimer resulting in quenching of the fluorescence of the fluorophores. When the backbone is cleaved or changes conformation, the fluorophores are separated, no longer forming an H-type dimer, and are de-quenched thereby providing a detectable signal. The use of a single fluorophore rather than an “acceptor-donor” fluoresecence resonance energy transfer system offers synthesis and performance advantages.
摘要翻译：本发明提供了新的试剂，其荧光在裂解时发生变化或骨架的构象变化。试剂包含连接相同物质的两个荧光团的骨架（例如核酸，多肽等），由此荧光团形成H-二聚体，导致荧光团的荧光猝灭。当主链被切割或改变构象时，荧光团被分离，不再形成H型二聚体，并且被淬灭，从而提供可检测的信号。使用单个荧光团而不是“受体 - 供体”荧光共振能量转移系统提供综合和性能优势。

4. 发明授权

US6037137A Fluorogenic peptides for the detection of protease activity 失效
标题翻译：荧光肽用于检测蛋白酶活性
公开(公告)号：US6037137A
公开(公告)日：2000-03-14
申请号：US802981
申请日：1997-02-20
申请人： Akira Komoriya , Beverly S. Packard
发明人： Akira Komoriya , Beverly S. Packard
IPC分类号： C12N15/09 , C07K7/06 , C07K7/08 , C12Q1/37 , C12Q1/68 , G01N33/542 , A61K38/00 , G01N21/00 , G01N21/76
CPC分类号： C07K7/06 , C07K7/08 , C12Q1/37 , G01N33/542 , G01N2333/96469
摘要： The present invention provides for novel reagents whose fluorescence increases in the presence of particular proteases. The reagents comprise a characteristically folded peptide backbone each end of which is conjugated to a fluorophore. When the folded peptide is cleaved, as by digestion with a protease, the fluorophores provide a high intensity fluorescent signal at a visible wavelength. Because of their high fluorescence signal in the visible wavelengths, these protease indicators are particularly well suited for detection of protease activity in biological samples, in particular in frozen tissue sections. Thus this invention also provides for methods of detecting protease activity in situ in frozen sections.
摘要翻译：本发明提供了在特定蛋白酶存在下荧光增加的新型试剂。试剂包括特征性折叠的肽主链，其每个末端与荧光团缀合。当折叠的肽被切割时，通过用蛋白酶消化，荧光团提供可见波长的高强度荧光信号。由于其可见光波长的高荧光信号，这些蛋白酶指示剂特别适用于检测生物样品中蛋白酶活性，特别是在冷冻组织切片中。因此，本发明还提供了在冷冻切片中原位检测蛋白酶活性的方法。

5. 发明授权

US07312302B2 Compositions for the detection of enzyme activity in biological samples and methods of use thereof 失效
标题翻译：用于检测生物样品中酶活性的组合物及其使用方法
公开(公告)号：US07312302B2
公开(公告)日：2007-12-25
申请号：US09874350
申请日：2001-06-04
申请人： Beverly S. Packard , Akira Komoriya
发明人： Beverly S. Packard , Akira Komoriya
IPC分类号： C12Q1/37 , C07K7/00
CPC分类号： C07K7/06 , C07K7/08 , C12Q1/37 , G01N33/533 , G01N33/542 , G01N2333/96425 , G01N2333/96469
摘要： The present invention provides for novel reagents whose fluorescence increases in the presence of particular proteases. The reagents comprise a characteristically folded peptide backbone conjugated to two fluorophores such that the fluorophores are located opposite sides of a cleavage site. When the folded peptide is cleaved, as by digestion with a protease, the fluorophores provide a high intensity fluorescent signal at a visible wavelength. Because of their high specificity and their high fluorescence signal in the visible wavelengths, these protease indicators are particularly well suited for detection of protease activity in biological samples, in particular in frozen tissue sections. Thus this invention also provides for methods of detecting protease activity in situ in frozen sections.
摘要翻译：本发明提供了在特定蛋白酶存在下荧光增加的新型试剂。试剂包含与两个荧光团缀合的特征性折叠的肽主链，使得荧光团位于切割位点的相对侧。当折叠的肽被切割时，通过用蛋白酶消化，荧光团提供可见波长的高强度荧光信号。由于它们的高特异性和在可见波长中的高荧光信号，这些蛋白酶指示剂特别适用于检测生物样品中的蛋白酶活性，特别是在冷冻组织切片中。因此，本发明还提供了在冷冻切片中原位检测蛋白酶活性的方法。

6. 发明授权

US5364619A Oncoimmunins 失效
标题翻译：免疫原性
公开(公告)号：US5364619A
公开(公告)日：1994-11-15
申请号：US764695
申请日：1991-09-23
申请人： Beverly Packard , Akira Komoriya
发明人： Beverly Packard , Akira Komoriya
IPC分类号： A61K38/00 , C07K14/47 , C07K14/515 , C07K14/71 , A61K37/02
CPC分类号： C07K14/515 , C07K14/4702 , C07K14/71 , A61K38/00
摘要： The present invention relates, in general, to oncoimmunins. In particular, the present invention relates to oncoimmunin-lymphoid factor and oncoimmunin-myeloid factor, pharmaceutical compositions of said factors, and methods of use of said factors.
摘要翻译：本发明一般涉及免疫原。特别地，本发明涉及免疫原性蛋白 - 淋巴因子和免疫蛋白 - 髓细胞因子，所述因子的药物组合物和所述因子的使用方法。

7. 发明授权

US4889916A Protein label and drug delivery system 失效
标题翻译：蛋白质标签和药物输送系统
公开(公告)号：US4889916A
公开(公告)日：1989-12-26
申请号：US151186
申请日：1988-02-01
申请人： Beverly Packard , Michael Edidin , Akira Komoriya
发明人： Beverly Packard , Michael Edidin , Akira Komoriya
IPC分类号： A61K47/48 , C07D311/82 , C07K1/107 , G01N33/533
CPC分类号： C07K1/1077 , A61K47/48715 , C07D311/82 , G01N33/533
摘要： A bridging molecule carrying a drug, or a label such as a fluorophore, which adds across disulfide bonds of molecules, particularly proteins, and methods of manfacturing and using the bridging molecules, are disclosed. The bridging molecule is reactive with sulfhydryl groups formed by the reduction of disulfide bonds of the protein. The functional groups of the bridging molecules are typically --SH groups.
摘要翻译：公开了携带药物的桥接分子或诸如荧光团的标记，其分子加二分子，特别是蛋白质，以及制造和使用桥接分子的方法。桥连分子与通过还原蛋白质二硫键形成的巯基反应。桥连分子的官能团通常是-SH基团。

8. 发明申请

US20080199898A1 COMPOSITIONS FOR THE DETECTION OF ENZYME ACTIVITY IN BIOLOGICAL SAMPLES AND METHODS OF USE THEREOF 有权
标题翻译：用于检测生物样品中酶活性的组合物及其使用方法
公开(公告)号：US20080199898A1
公开(公告)日：2008-08-21
申请号：US11941766
申请日：2007-11-16
申请人： Beverly S. Packard , Akira Komoriya
发明人： Beverly S. Packard , Akira Komoriya
IPC分类号： C12Q1/37 , C12N5/00
CPC分类号： C07K7/06 , C07K7/08 , C12Q1/37 , G01N33/542 , G01N2333/96469
摘要： The present invention provides for novel reagents whose fluorescence increases in the presence of particular proteases. The reagents comprise a characteristically folded peptide backbone conjugated to two fluorophores such that the fluorophores are located opposite sides of a cleavage site. When the folded peptide is cleaved, as by digestion with a protease, the fluorophores provide a high intensity fluorescent signal at a visible wavelength. Because of their high specificity and their high fluorescence signal in the visible wavelengths, these protease indicators are particularly well suited for detection of protease activity in biological samples, in particular in frozen tissue sections. Thus this invention also provides for methods of detecting protease activity in situ in frozen sections.
摘要翻译：本发明提供了在特定蛋白酶存在下荧光增加的新型试剂。试剂包含与两个荧光团缀合的特征性折叠的肽主链，使得荧光团位于切割位点的相对侧。当折叠的肽被切割时，通过用蛋白酶消化，荧光团提供可见波长的高强度荧光信号。由于它们的高特异性和在可见波长中的高荧光信号，这些蛋白酶指示剂特别适用于检测生物样品中的蛋白酶活性，特别是在冷冻组织切片中。因此，本发明还提供了在冷冻切片中原位检测蛋白酶活性的方法。

9. 发明申请

US20070184493A1 VISUALIZATION AND QUANTITATION OF CELLULAR CYTOTOXICITY USING CELL-PERMEABLE FLUOROGENIC PROTEASE SUBSTRATES AND CASPASE ACTIVITY INDICATOR MARKERS 有权
标题翻译：使用细胞渗透性荧光素酶基质和CASPASE活性指示标记的细胞生物学活性的可视化和定量
公开(公告)号：US20070184493A1
公开(公告)日：2007-08-09
申请号：US11669080
申请日：2007-01-30
申请人： Beverly Packard , Akira Komoriya
发明人： Beverly Packard , Akira Komoriya
IPC分类号： G01N33/567 , A61K38/10
CPC分类号： G01N33/505 , A61K38/07 , A61K38/08 , G01N33/5047 , G01N33/5055 , G01N33/533 , G01N33/582
摘要： This invention provides a non-radioactive assay to monitor and quantify the target-cell killing activities mediated by cytotoxic T lymphocytes (CTLs). This assay is predicated on the discovery that apoptosis pathway activation and, in particular, granzyme B activity, provides a measure of cytotoxic effector cell activity. In one embodiment, measurement of CTL-induced granzyme B activation in target cells is achieved through detection of the specific cleavage of fluorogenic granzyme B substrates. This assay reliably detects antigen-specific CTL killing of target cells, and provides a more sensitive, more informative and safer alternative to the standard 51Cr-release assay most often used to quantify CTL responses. The assay can be used to study CTL-mediated killing of primary host target cells of different cell lineages, and enables the study of antigen-specific cellular immune responses in real time at the single-cell level. As such, the assay can provide a valuable tool for studies of infectious disease pathogenesis and development of new vaccines and immunotherapies.
摘要翻译：本发明提供了用于监测和定量由细胞毒性T淋巴细胞（CTL）介导的靶细胞杀伤活性的非放射性测定。该测定基于发现凋亡途径活化，特别是颗粒酶B活性提供细胞毒性效应细胞活性的量度的发现。在一个实施方案中，通过检测荧光颗粒酶B底物的特异性切割来实现靶细胞中CTL诱导的颗粒酶B活化的测量。该测定可靠地检测靶细胞的抗原特异性CTL杀伤，并且为最常用于定量CTL应答的标准 Cr释放测定提供更灵敏，更有信息和更安全的替代方案。该测定可用于研究CTL介导的不同细胞系主要宿主靶细胞的杀伤，并能够在单细胞水平实时研究抗原特异性细胞免疫应答。因此，该测定可以为感染性疾病发病机理的研究和新疫苗和免疫疗法的开发提供有价值的工具。

10. 发明申请

US20050158766A1 Homo-doubly labeled compositions for the detection of enzyme activity in biological samples 失效
公开(公告)号：US20050158766A1
公开(公告)日：2005-07-21
申请号：US11015864
申请日：2004-12-15
申请人： Beverly Packard , Akira Komoriya
发明人： Beverly Packard , Akira Komoriya
IPC分类号： C07K7/06 , C07K7/08 , C12N1/21 , C12N15/12 , C12Q1/37 , C12Q1/44 , G01N33/533 , G01N33/542 , C12Q1/68 , C07H21/04
CPC分类号： C07K7/06 , C07K7/08 , C12Q1/37 , C12Q1/44 , G01N33/533 , G01N33/542 , G01N2333/918 , G01N2333/96425 , G01N2333/96469
摘要： The present invention provides for novel reagents whose fluorescence changes upon cleavage or a change in conformation of a backbone. The reagents comprise a backbone (e.g. nucleic acid, polypeptide, etc.) joining two fluorophores of the same species whereby the fluorophores form an H-dimer resulting in quenching of the fluorescence of the fluorophores. When the backbone is cleaved or changes conformation, the fluorophores are separated, no longer forming an H-type dimer, and are de-quenched thereby providing a detectable signal. The use of a single fluorophore rather than an “acceptor-donor” fluoresecence resonance energy transfer system offers synthesis and performance advantages.

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