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1. 发明申请

WO2009139919A2 METHODS FOR DIAGNOSIS OF CLOSTRIDIUM DIFFCILE AND METHODS AND VECTORS FOR RECOMBINANT TOXIN EXPRESSION 审中-公开
标题翻译：用于诊断骨质疏松症的方法和用于重组蛋白表达的方法和载体
公开(公告)号：WO2009139919A2
公开(公告)日：2009-11-19
申请号：PCT/US2009003055
申请日：2009-05-15
申请人： UNIV TUFTS , FENG HANPING , TZIPORI SAUL , YANG GUILIN
发明人： FENG HANPING , TZIPORI SAUL , YANG GUILIN
IPC分类号： G01N33/569
CPC分类号： G01N33/56911 , G01N33/5014 , G01N33/5055 , G01N2333/33
摘要： Cell-based methods for rapid real time assay of a presence of Clostridium difficile toxin and/or cells are provided, using an assay having a toxin-enhancing antibody and a sensitive cell line carrying Fc?R receptors, and kits for this assay. An ultrasensitive cell- based immunocytotoxicity assay for detecting less then 1 pg/ml of C. difficile toxins in clinical samples. The assay is simple, has a turnaround time of approximately 3 hours, and detects less than about 1 pg/ml of toxin A. The presence of C. difficile toxins in the fecal and serum specimens of experimentally infected piglets was detected with this assay. The cellular effects of TcdA were substantially enhanced via an opsonizing antibody through Fc gamma receptor I (Fc?RI)-mediated endocytosis. A TcdA-specific monoclonal antibody, A1H3, was found to significantly enhance the cytotoxicity of TcdA to macrophages and monocytes. The A 1H3 -dependent enhancement of glucosyltransferase activity, cytoskeleton disruption, and TNF-a production induced by TcdA was demonstrated also in RAW 264.7 cells. The interaction of Fc?RI with A1H3 underlay the antibody-dependent enhancement of cellular effects of TcdA. Expression of Fc?RI in CHO cells strikingly enhanced their sensitivity to TcdA complexed with A1H3. Presence of A1H3 facilitated the cell surface recruitment of TcdA, conxributing to the antibody-dependent, Fc?RI -mediated enhancement of TcdA activity. The effect of chlorpromazine and endosomal acidification inhibitors indicated an important role of the endocytic pathway in AlH3-dependent enhancement of TcdA activity. Methods for high level recombinant expression of C. difficile toxins in Bacillus cells, and vectors for expression, strains of Bacillus carrying the vectors are provided.
摘要翻译：使用具有毒素增强抗体和携带FcγR受体的敏感细胞系的测定法和用于该测定的试剂盒提供了用于快速实时测定艰难梭菌毒素和/或细胞存在的基于细胞的方法。用于在临床样品中检测少于1pg / ml艰难梭菌毒素的超敏感的基于细胞的免疫细胞毒性测定。该测定很简单，具有约3小时的周转时间，并且检测到小于约1pg / ml的毒素A.使用该测定法检测艰难梭菌毒素在实验感染的仔猪的粪便和血清标本中的存在。通过Fcγ受体I（FcγRI）介导的内吞作用的调理性抗体，TcdA的细胞效应显着增强。发现TcdA特异性单克隆抗体A1H3显着增强TcdA对巨噬细胞和单核细胞的细胞毒性。在RAW 264.7细胞中也证明了由TcdA诱导的葡萄糖基转移酶活性，细胞骨架破坏和TNF-α产生的A 1H3依赖性增强。 FcγRI与A1H3的相互作用支持TcdA的细胞效应的抗体依赖性增强。 CHO细胞中FcγRI的表达显着提高了它们对与A1H3复合的TcdA的敏感性。 A1H3的存在促进了TcdA的细胞表面募集，促进了抗体依赖性，FcγRI介导的TcdA活性的增强。氯丙嗪和内体酸化抑制剂的作用表明，内吞途径在AlH3依赖性增强TcdA活性中起重要作用。提供了在芽孢杆菌细胞中高水平重组表达艰难梭菌毒素的方法和用于表达载体的载体载体的芽孢杆菌菌株。

2. 发明申请

WO2009139919A4 METHODS FOR DIAGNOSIS OF CLOSTRIDIUM DIFFCILE AND METHODS AND VECTORS FOR RECOMBINANT TOXIN EXPRESSION 审中-公开
标题翻译：用于诊断骨质疏松症的方法和用于重组蛋白表达的方法和载体
公开(公告)号：WO2009139919A4
公开(公告)日：2010-06-24
申请号：PCT/US2009003055
申请日：2009-05-15
申请人： UNIV TUFTS , FENG HANPING , TZIPORI SAUL , YANG GUILIN
发明人： FENG HANPING , TZIPORI SAUL , YANG GUILIN
IPC分类号： C12Q1/04
CPC分类号： G01N33/56911 , G01N33/5014 , G01N33/5055 , G01N2333/33
摘要： Cell-based methods for rapid real time assay of a presence of Clostridium difficile toxin and/or cells are provided, using an assay having a toxin-enhancing antibody and a sensitive cell line carrying FcyR receptors, and kits for this assay. An ultrasensitive cell based immunocytotoxicity assay for detecting less then 1 pg/ml of C. difficile toxins in clinical samples. A TcdA-specific monoclonal antibody, AIH3, was found to significantly enhance the cytotoxicity of TcdA to macrophages and monocytes. The AIH3-dependent enhancement of glucosyltransferase activity, cytoskeleton disruption, and TNF-a production induced by TcdA was demonstrated also in RAW 264.7 cells. Methods for high level recombinant expression of C. difficile toxins in Bacillus cells, and vectors for expression, strains of Bacillus carrying the vectors are provided.
摘要翻译：使用具有毒素增强抗体和携带FcyR受体的敏感细胞系的测定法和用于该测定的试剂盒，提供了用于快速实时测定艰难梭菌毒素和/或细胞存在的基于细胞的方法。用于在临床样品中检测少于1pg / ml的艰难梭菌毒素的基于超敏感的基于细胞的免疫细胞毒性测定。发现TcdA特异性单克隆抗体AIH3显着增强TcdA对巨噬细胞和单核细胞的细胞毒性。在RAW 264.7细胞中也证明了由TcdA诱导的葡萄糖基转移酶活性，细胞骨架破坏和TNF-α产生的AIH3依赖性增强。提供了在芽孢杆菌细胞中高水平重组表达艰难梭菌毒素的方法和用于表达载体的载体载体的芽孢杆菌菌株。

3. 发明申请

WO2010006326A3 METHODS AND COMPOSITIONS FOR SPORE-BASED VACCINES 审中-公开
标题翻译：基于孢子的疫苗的方法和组合物
公开(公告)号：WO2010006326A3
公开(公告)日：2010-05-06
申请号：PCT/US2009050356
申请日：2009-07-13
申请人： UNIV TUFTS , HERRMANN JOHN E , BELITSKY BORIS R , SONENSHEIN ABRAHAM L , TZIPORI SAUL
发明人： HERRMANN JOHN E , BELITSKY BORIS R , SONENSHEIN ABRAHAM L , TZIPORI SAUL
IPC分类号： A61K39/12 , A61K39/02 , A61K39/39 , A61P37/00
CPC分类号： A61K39/15 , A61K39/0016 , A61K39/12 , A61K2039/522 , A61K2039/523 , A61K2039/542 , A61K2039/543 , A61K2039/545 , A61K2039/55544 , C12N2720/12334 , C12N2830/007 , C12N2830/55
摘要： Methods for immunizing a subject to an infectious agent are provided, using vegetative cytoplasmic expression of an antigen encoding an infectious agent or spore surface display of the antigen, and contacting the subject with a composition including a spore or a vegetative cell or both with or without an adjuvant. Also included are a thermally- stable vaccine composition using the method described above and a kit for its use.
摘要翻译：提供了使用营养细胞质表达编码感染因子或孢子表面展示抗原的细胞质表达，并使受试者与包含孢子或营养细胞或含有或不含有营养细胞的组合物接触的方法，佐剂。还包括使用上述方法的热稳定疫苗组合物及其使用的试剂盒。

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