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1. 发明申请

US20160202245A1 Means and Methods for the determination of the biological activity of Neurotoxin polypeptides in cells 审中-公开
标题翻译：用于测定细胞中神经毒素多肽的生物学活性的方法和方法
公开(公告)号：US20160202245A1
公开(公告)日：2016-07-14
申请号：US14901123
申请日：2014-06-26
申请人： MERZ PHARMA GMBH & CO. KGAA
发明人： Cornelia BRÜNN
IPC分类号： G01N33/50 , G01N21/64
CPC分类号： C07K16/18 , C07K2317/34 , G01N21/6428 , G01N33/5014 , G01N33/5058 , G01N33/5073 , G01N2021/6441 , G01N2333/33 , G01N2333/952
摘要： The present invention pertains to a method for directly determining the biological activity of a Neurotoxin polypeptide in cells, comprising the steps of: a) incubating cells susceptible to Neurotoxin intoxication with a Neurotoxin polypeptide for a time and under conditions which allow for the Neurotoxin polypeptide to exert its biological activity; b) fixing the cells and, optionally, permeabilizing the cells with a detergent; c) contacting the cells with at least a first capture antibody specifically binding to the non-cleaved and Neurotoxin-cleaved substrate and with at least a second capture antibody specifically binding to the cleavage site of the Neurotoxin-cleaved substrate, under conditions which allow for binding of said capture antibodies to said substrates; d) contacting the cells with at least a first detection antibody specifically binding to the first capture antibody, under conditions which allow for binding of said first detection antibody to said first capture antibody, thus forming first detection complexes, and at least a second detection antibody specifically binding to the second capture antibody, under conditions which allow for binding of said second detection antibody to said second capture antibody, thus forming second detection complexes; e) determining the amount of the first and second detection complexes of step d), and f) calculating the amount of substrate cleaved by said Neurotoxin polypeptide in said cells by means of the second detection complexes, thereby determining the biological activity of said Neurotoxin polypeptide in said cells. The invention further provides for a kit for carrying out the method of the invention.
摘要翻译：本发明涉及直接测定神经毒素多肽在细胞中的生物学活性的方法，包括以下步骤：a）在神经毒素多肽对神经毒素中毒敏感的情况下，使神经毒素多肽在一段时间内和在允许神经毒素多肽发挥其生物活性; b）固定细胞，并且任选地用洗涤剂使细胞透化; c）在允许细胞与至少一种特异性结合未切割和神经毒素切割的底物的第一捕获抗体和至少一种特异性结合神经毒素切割的底物的切割位点的第二捕获抗体上接触细胞，所述捕获抗体与所述底物结合; d）在允许所述第一检测抗体与所述第一捕获抗体结合从而形成第一检测复合物和至少第二检测抗体的条件下，使细胞与至少一种特异性结合第一捕获抗体的第一检测抗体接触在允许所述第二检测抗体与所述第二捕获抗体结合的条件下，特异性结合第二捕捉抗体，从而形成第二检测复合物; e）确定步骤d）的第一和第二检测复合物的量，和f）通过所述第二检测复合物计算由所述神经毒素多肽在所述细胞中切割的底物的量，由此确定所述神经毒素多肽的生物学活性在所述细胞中。本发明还提供了用于实施本发明方法的试剂盒。

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