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1. 发明授权

US07622111B2 Fusion protein of human IgG1 heavy chain constant region and scFv antibody against equine encephalitis virus 失效
标题翻译：人IgG1重链恒定区的融合蛋白和针对马脑炎病毒的scFv抗体
公开(公告)号：US07622111B2
公开(公告)日：2009-11-24
申请号：US10378832
申请日：2003-03-05
申请人： Leslie P. Nagata , R. Elaine Fulton , Weigang Hu , Azhar Z. Alvi
发明人： Leslie P. Nagata , R. Elaine Fulton , Weigang Hu , Azhar Z. Alvi
IPC分类号： A61K39/42
CPC分类号： C07H21/04 , C07K16/1081 , C07K2317/21 , C07K2317/52 , C07K2317/622 , C07K2319/00
摘要： Construction of a recombinant gene fusion encoding a human IgG1 heavy chain constant region and a single-chain variable fragment antibody of 1A4A1 monoclonal antibody is disclosed. The recombinant antibody of the present invention confers human immune effector functions on murine antibodies. After expression in bacteria as inclusion bodies, the recombinant antibody was purified and refolded in vitro. The recombinant soluble antibody retains high antigen-binding affinity to VEE and possesses some human IgG crystallizable fragment domain functions. On non-reducing gel electrophoresis analysis, disulfide bond formation was found in the hinge region of the recombinant antibody. The present invention shows that the recombinant antibody is in a native, functionally active form and it provides the basis to characterize the recombinant antibody for efficacy in vivo.
摘要翻译：公开了编码人IgG1重链恒定区的重组基因融合物和1A4A1单克隆抗体的单链可变片段抗体的构建。本发明的重组抗体赋予人抗体上的人免疫效应子功能。在细菌中作为包涵体表达后，重组抗体在体外纯化和重折叠。重组可溶性抗体对VEE具有高抗原结合亲和力，并且具有一些人IgG可结晶的片段结构域功能。在非还原凝胶电泳分析中，在重组抗体的铰链区发现二硫键形成。本发明显示重组抗体是天然的功能活性形式，并且为体内重组抗体的功能表征提供了基础。

2. 发明授权

US07309566B2 Genetically biotinylated recombinant antibody in immunofiltration assay by light addressable potentiometric sensor for identification of Venezuelan equine encephalitis virus 失效
标题翻译：遗传生物素化重组抗体在免疫过滤测定中通过光寻址电位传感器鉴定委内瑞兰马脑炎病毒
公开(公告)号：US07309566B2
公开(公告)日：2007-12-18
申请号：US10807194
申请日：2004-03-24
申请人： R. Elaine Fulton , Leslie P. Nagata , Azhar Z. Alvi , Weigang Hu
发明人： R. Elaine Fulton , Leslie P. Nagata , Azhar Z. Alvi , Weigang Hu
IPC分类号： C07K16/10 , C07K16/08 , G01N33/543 , G01N33/569 , C12Q1/70
CPC分类号： C07K16/1081 , C07K2317/622 , G01N33/543 , G01N33/56983
摘要： A genetically biotinylated single chain fragment variable (scFv) antibody against Venezuelan equine encephalitis virus (VEE) being applied in a system consisting of an immunofiltration-enzyme assay (IFA) with a light addressable potentiometric sensor (LAPS) for the rapid identification of VEE is disclosed. The IFA entails formation of an immunocomplex sandwich consisting of VEE, biotinylated antibody, fluoresceinated antibody and streptavidin, capturing the sandwich by filtration on biotinylated membrane, and detecting the sandwich by anti-fluorescein urease conjugate. The concentration ratio of biotinylated to fluoresceinated antibodies is investigated and optimized. The IFA/LAPS assay sensitivity was approximately equal to that of a conventional enzyme-linked immunosorbant assay utilizing polystyrene plates and a chromogenic substrate, however, less time and effort were required for performance of the IFA/LAPS assay.
摘要翻译：在由用于快速鉴定VEE的可光寻址电位传感器（LAPS）组成的系统组成的系统中应用针对委内瑞兰马脑炎病毒（VEE）的遗传生物素化单链片段变量（scFv）抗体是披露 IFA需要形成由VEE，生物素化抗体，荧光抗体和链霉抗生物素蛋白组成的免疫复合物夹心，通过在生物素化膜上过滤捕获夹心物，并通过抗荧光素脲酶缀合物检测夹心。研究并优化生物素化至荧光抗体的浓度比。 IFA / LAPS测定灵敏度大约等于使用聚苯乙烯板和显色底物的常规酶联免疫吸附测定法，然而，对于IFA / LAPS测定的性能需要较少的时间和精力。

3. 发明授权

US07527796B2 Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to Venezuelan equine encephalitis virus 失效
标题翻译：针对委内瑞兰马脑炎病毒的链霉抗生物素蛋白结合肽标记的单链可变片段抗体的遗传工程
公开(公告)号：US07527796B2
公开(公告)日：2009-05-05
申请号：US10784305
申请日：2004-02-24
申请人： R. Elaine Fulton , Leslie P. Nagata , Azhar Z. Alvi , Weigang Hu
发明人： R. Elaine Fulton , Leslie P. Nagata , Azhar Z. Alvi , Weigang Hu
IPC分类号： A61K39/00 , A61K39/42
CPC分类号： C07K16/1081 , C07K2317/622 , C07K2319/22
摘要： A recombinant gene encoding a single-chain variable fragment (scFv) antibody against Venezuelan equine encephalitis virus (VEE) being cloned into a prokaryotic T7 RNA polymerase-regulated expression vector was disclosed. A streptavidin-binding peptide (SBP) sequence fused to a 6His tag is then attached downstream to the scFv gene. The recombinant fusion protein is expressed in bacteria and then purified by immobilized metal affinity chromatography. ELISA and Western blotting results revealed that the fusion protein not only retained VEE antigen binding and specificity properties similar to those of its parent native monoclonal antibody, but also possessed streptavidin-binding activity. This discovery obviates the need for chemical biotinylation of antibodies and the risk associated with antibody denaturation and provides a stable and reproducible reagent for rapid and efficient immunoassay of VEE.
摘要翻译：公开了克隆到原核T7RNA聚合酶调节表达载体中的编码针对委内瑞兰马脑炎病毒（VEE）的单链可变片段（scFv）抗体的重组基因。然后将与6His标签融合的链霉抗生物素蛋白结合肽（SBP）序列连接到scFv基因的下游。重组融合蛋白在细菌中表达，然后通过固定化金属亲和层析纯化。 ELISA和Western印迹结果表明，融合蛋白不仅保留了VEE抗原结合和特异性，与其母体天然单克隆抗体相似，而且具有链霉抗生物素蛋白结合活性。这一发现消除了对抗体的化学生物素化和与抗体变性相关的风险的需要，并且提供用于快速和有效的VEE免疫测定的稳定且可重现的试剂。

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