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1. 发明申请

US20050059097A1 Quantitative binding assays using green fluorescent protein as a label 审中-公开
标题翻译：使用绿色荧光蛋白作为标签的定量结合测定
公开(公告)号：US20050059097A1
公开(公告)日：2005-03-17
申请号：US10765063
申请日：2004-01-28
申请人： Sylvia Daunert , Jennifer Lewis , Emily Hernandez
发明人： Sylvia Daunert , Jennifer Lewis , Emily Hernandez
IPC分类号： C12Q1/68 , G01N33/53 , G01N33/533 , G01N33/543 , G01N33/58 , C07K16/46
CPC分类号： G01N33/543 , G01N33/533 , G01N33/582
摘要： A heterogeneous binding assay for an analyte in a fluid sample is developed, which uses a green fluorescent protein (GFP) label. A ligand-GFP conjugate has a specific binding affinity for an anti-ligand immobilized on a support. The anti-ligand also has a specific binding affinity for the analyte. Competition between the analyte and ligand-GFP conjugate for binding sites on the anti-ligand permits an assay for an unknown amount of the analyte. Preferred specific binding pairs for use in the assay are biotin:avidin, and a selected antibody and its antigen. A preferred assay employing an antibody and its antigen is illustrated for a fusion protein containing GFP and an antigenic determinant. Picomolar amounts of analyte can be detected. The mutant of GFP that contains a six-histidine tail to facilitate purification on an immobilized metal affinity column is chemically modified to incorporate biotin moieties. The resulting conjugates retain the fluorescence characteristics of the unmodified protein and are used along with avidin-coated magnetic beads in the development of the assay.
摘要翻译：开发了流体样品中分析物的异质结合测定法，其使用绿色荧光蛋白（GFP）标记。配体-GFP缀合物对固定在载体上的抗配体具有特异性结合亲和力。抗配体还对分析物具有特异性结合亲和力。分析物与配体-GFP缀合物之间在抗配体上的结合位点之间的竞争允许测定未知量的分析物。用于测定中的优选的特异性结合对是生物素：抗生物素蛋白和选择的抗体及其抗原。对于含有GFP和抗原决定簇的融合蛋白说明了使用抗体及其抗原的优选测定法。可以检测到皮摩尔量的分析物。包含六组氨酸尾部以促进固定化金属亲和柱上的纯化的GFP突变体被化学修饰以掺入生物素部分。所得到的缀合物保留未修饰蛋白质的荧光特性，并在测定开发中与抗生物素蛋白包被的磁珠一起使用。

2. 发明申请

US20050282225A1 Quantitative binding assays using green fluorescent protein as a label 审中-公开
标题翻译：使用绿色荧光蛋白作为标签的定量结合测定
公开(公告)号：US20050282225A1
公开(公告)日：2005-12-22
申请号：US11197429
申请日：2005-08-05
申请人： Sylvia Daunert , Jennifer Lewis , Emily Hernandez
发明人： Sylvia Daunert , Jennifer Lewis , Emily Hernandez
IPC分类号： C12Q1/68 , G01N33/53 , G01N33/533 , G01N33/543 , G01N33/58
CPC分类号： G01N33/543 , G01N33/533 , G01N33/582
摘要： A heterogeneous binding assay for an analyte in a fluid sample is developed, which uses a green fluorescent protein (GFP) label. A ligand-GFP conjugate has a specific binding affinity for an anti-ligand immobilized on a support. The anti-ligand also has a specific binding affinity for the analyte. Competition between the analyte and ligand-GFP conjugate for binding sites on the anti-ligand permits an assay for an unknown amount of the analyte. Preferred specific binding pairs for use in the assay are biotin:avidin, and a selected antibody and its antigen. A preferred assay employing an antibody and its antigen is illustrated for a fusion protein containing GFP and an antigenic determinant. Picomolar amounts of analyte can be detected. The mutant of GFP that contains a six-histidine tail to facilitate purification on an immobilized metal affinity column is chemically modified to incorporate biotin moieties. The resulting conjugates retain the fluorescence characteristics of the urunodified protein and are used along with avidin-coated magnetic beads in the development of the assay.
摘要翻译：开发了流体样品中分析物的异质结合测定法，其使用绿色荧光蛋白（GFP）标记。配体-GFP缀合物对固定在载体上的抗配体具有特异性结合亲和力。抗配体还对分析物具有特异性结合亲和力。分析物与配体-GFP缀合物之间在抗配体上的结合位点之间的竞争允许测定未知量的分析物。用于测定中的优选的特异性结合对是生物素：抗生物素蛋白和选择的抗体及其抗原。对于含有GFP和抗原决定簇的融合蛋白说明了使用抗体及其抗原的优选测定法。可以检测到皮摩尔量的分析物。包含六组氨酸尾部以促进固定化金属亲和柱上的纯化的GFP突变体被化学修饰以掺入生物素部分。所得到的缀合物保留尿素化蛋白质的荧光特性，并与抗生物素蛋白包被的磁珠一起用于测定的开发。

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