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1. 发明授权

US06818748B2 Cloning, expression, sequencing, and functional enhancement of monoclonal ScFv antibody against Venezuelan equine encephalitis virus (VEE) 有权
标题翻译：针对委内瑞兰马脑炎病毒（VEE）的单克隆ScFv抗体的克隆，表达，测序和功能增强
公开(公告)号：US06818748B2
公开(公告)日：2004-11-16
申请号：US10096246
申请日：2002-03-13
申请人： R. Elaine Fulton , Leslie Nagata , Azhar Alvi
发明人： R. Elaine Fulton , Leslie Nagata , Azhar Alvi
IPC分类号： C12P2108
CPC分类号： C07K16/10 , C07K2317/567 , C07K2317/622 , Y10S530/866
摘要： A single chain variable fragment (ScFv) antibody from a monoclonal antibody (Mab) against Venezuelan equine encephalitis (VEE) virus, is generated by cloning linked variable regions of the heavy (VH) and the light (VL) chain antibody genes. Mab clone 1A4A1 in E. coli strain TG-1 was successfully cloned as ScFv. Results were reproduced in E. coli strain HB2151, expressing the same clone, A116, though displaying weak binding specificity to VEE due to a frame shift in the N-terminal region of the VL domain, upstream to the complementarity-determining region 1. A PCR-based site-directed mutagenesis approach was adopted to re-introduce the three single-base deletions in the 5 prime region of the VL gene of A116, corresponding to framework-1 region, producing mutant MA116, correcting a localized frame-shift in framework-1 region to consensus framework-1 amino acid sequence. A MA116 clone, MA116-15, shows comparable reactivity to the parental Mab in recognizing VEE antigen.
摘要翻译：通过克隆重（VH）和轻链（VL）链抗体基因的连锁可变区，产生来自针对委内瑞兰马脑炎（VEE）病毒的单克隆抗体（Mab）的单链可变片段（ScFv）抗体。大肠杆菌菌株TG-1中的Mab克隆1A4A1成功克隆为ScFv。结果在大肠杆菌菌株HB2151中复制，表达相同的克隆A116，尽管显示出对VEE的弱结合特异性，这是由于VL域的N末端区域在互补决定区1上游的帧移位。采用基于PCR的定点诱变方法，重新引入A116 VL基因的5个主要区域，与框架1区域相对应，产生突变体MA116，校正局部帧位移框架-1区域到共有框架-1氨基酸序列。 MA116克隆MA116-15在识别VEE抗原中显示与亲本Mab的可比反应性。

2. 发明授权

US07767419B2 Calibrator for NT-proBNP immunoassay 有权
标题翻译：用于NT-proBNP免疫测定的校准物
公开(公告)号：US07767419B2
公开(公告)日：2010-08-03
申请号：US10359028
申请日：2003-02-04
申请人： Azhar Alvi , William Yajima , Roshana Sikora , George Jackowski , Mee-Ra Hong
发明人： Azhar Alvi , William Yajima , Roshana Sikora , George Jackowski , Mee-Ra Hong
IPC分类号： C12N15/12 , C12N15/63 , C12N15/70 , C12P19/34 , G01N33/531 , C07H21/04
CPC分类号： G01N33/74 , G01N2333/58 , Y10S436/811 , Y10S530/806 , Y10T436/10
摘要： The invention provides a method to efficiently express high levels of a recombinant untagged NT-proBNP for use as a calibrator in NT-proBNP immunoassays.
摘要翻译：本发明提供了一种在NT-proBNP免疫测定中有效表达高水平重组未标记的NT-proBNP用作校准物的方法。

3. 发明申请

US20050118569A1 Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to venezuelan equine encephalitis virus 失效
标题翻译：链霉亲和素结合肽标记的单链可变片段抗体对委内瑞拉马脑炎病毒的遗传工程
公开(公告)号：US20050118569A1
公开(公告)日：2005-06-02
申请号：US10784305
申请日：2004-02-24
申请人： R. Fulton , Leslie Nagata , Azhar Alvi , Weigang Hu
发明人： R. Fulton , Leslie Nagata , Azhar Alvi , Weigang Hu
IPC分类号： C07K14/47 , C07K16/08 , C07K16/10 , C07K19/00 , C12N15/10 , C12N15/66 , G01N33/53 , G01N33/569 , C12Q1/70 , C07H21/04 , C12N5/06 , C12P19/34 , C12Q1/68
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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