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    • 6. 发明申请
    • MEANS AND METHODS FOR THE DETERMINATION OF THE BIOLOGICAL ACTIVITY OF BONT/E IN CELLS
    • 用于确定细胞中BONT / E生物活性的方法和方法
    • WO2016097243A1
    • 2016-06-23
    • PCT/EP2015/080395
    • 2015-12-18
    • MERZ PHARMA GMBH & CO. KGAA
    • BRÜNN, CorneliaMANDER, Gerd
    • C07K16/18G01N33/50G01N33/94
    • G01N33/5014C07K16/18C07K2317/34G01N2333/33G01N2800/709
    • The present invention pertains to a polyclonal or monoclonal antibody specifically binding to BoNT/E-cleaved SNAP-25. Further, the invention provides a method for directly determining the biological activity of BoNT/E in cells, comprising the steps of: a) incubating cells susceptible to BoNT/E intoxication with BoNT/E for a time and under conditions which allow for the BoNT/E 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 non-cleaved and BoNT/E-cleaved SNAP-25, and with at least a second capture antibody specifically binding to BoNT/E-cleaved SNAP-25, wherein the second capture antibody is an antibody of the invention, under conditions which allow for binding of said capture antibodies to the indicated 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 with 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, and wherein the first detection antibody is different from the second detection antibody; e) determining the amount of the first and second detection complexes of step d); and f) calculating the amount of SNAP-25 cleaved by BoNT/E in said cells by means of the second detection complexes, thereby determining the biological activity of BoNT/E in said cells. Furthermore, the invention relates to a kit for carrying out the method of the invention.
    • 本发明涉及与BoNT / E切割的SNAP-25特异性结合的多克隆或单克隆抗体。 此外,本发明提供了一种用于直接测定细胞中BoNT / E的生物学活性的方法,包括以下步骤:a)在BoNT / E中与BoNT / E中毒易感的细胞一段时间并在允许BoNT / E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)使细胞与至少一种特异性结合未切割的和BoNT / E切割的SNAP-25的第一捕获抗体以及与至少一种特异性结合于BoNT / E切割的SNAP-25的第二捕获抗体接触,其中 第二捕获抗体是本发明的抗体,在允许所述捕获抗体与指定底物结合的条件下; d)在允许所述第一检测抗体与所述第一捕获抗体结合的条件下,使所述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,从而形成第一检测复合物,并至少具有第二检测 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,特异性结合第二捕获抗体的抗体,从而形成第二检测复合物,并且其中第一检测抗体不同于第二检测抗体; e)确定步骤d)的第一和第二检测复合物的量; 和f)通过第二检测复合物计算BoNT / E在所述细胞中切割的SNAP-25的量,由此确定所述细胞中BoNT / E的生物学活性。 此外,本发明涉及一种用于实施本发明方法的试剂盒。
    • 7. 发明申请
    • METHODS FOR THE DETERMINATION OF THE BIOLOGICAL ACTIVITIES OF NEUROTOXIN POLYPEPTIDES
    • 测定神经毒素多糖生物活性的方法
    • WO2016079310A1
    • 2016-05-26
    • PCT/EP2015/077245
    • 2015-11-20
    • MERZ PHARMA GMBH & CO. KGAA
    • EISELE, Karl-Heinz
    • G01N33/569C07K14/00
    • G01N33/6809C07K14/70571C07K2319/61G01N33/56911
    • The present invention pertains to a method for determining the biological activity of a neurotoxin, the method comprising the steps of: (a) expressing a fusion protein comprising (i) an anchor protein, (ii) a reporter protein and (iii) a neurotoxin cleavage site intervening the anchor protein and the reporter protein, in neurotoxin-sensitive cells; (b) incubating the neurotoxin-sensitive cells of (a) with a neurotoxin and cultivating the cells under conditions which allow the neurotoxin to exert its biological activity; (c) permeabilizing the neurotoxin-sensitive cells of (b) under conditions which allow the release of the reporter protein but not the release of the anchor protein from the permeabilized neurotoxin-sensitive cells; and (d) quantifying the activity of the reporter protein released from the cells, thereby determining the biological activity of the neurotoxin. In addition, the invention relates to a fusion protein comprising (i) an anchor protein, (ii) a reporter protein, and (iii) a neurotoxin cleavage site intervening the anchor protein and the reporter protein, for determining the biological activity of a neurotoxin, in neurotoxin-sensitive cells. Further encompassed by the present invention is a kit comprising the fusion protein of the invention. Finally, the invention pertains to the use of a fusion protein of the invention for determining the biological activity of a neurotoxin, in neurotoxin-sensitive cells.
    • 本发明涉及一种用于测定神经毒素生物活性的方法,所述方法包括以下步骤:(a)表达融合蛋白,其包含(i)锚蛋白,(ii)报道蛋白和(iii)神经毒素 在神经毒素敏感细胞中插入锚蛋白和报告蛋白的切割位点; (b)将(a)的神经毒素敏感细胞与神经毒素孵育并在允许神经毒素发挥其生物活性的条件下培养细胞; (c)在允许释放报告蛋白但不从透化的神经毒素敏感细胞释放锚蛋白的条件下使(b)的神经毒素敏感细胞透化; 和(d)定量从细胞释放的报道蛋白的活性,从而确定神经毒素的生物活性。 此外,本发明涉及融合蛋白,其包含(i)锚蛋白,(ii)报道蛋白,和(iii)插入锚蛋白和报道蛋白的神经毒素切割位点,用于确定神经毒素的生物学活性 ,在神经毒素敏感细胞。 本发明进一步包括的是包含本发明的融合蛋白的试剂盒。 最后,本发明涉及本发明的融合蛋白在神经毒素敏感细胞中用于测定神经毒素的生物学活性的用途。