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High throughput assay systems and methods for identifying agents that alter surface expression of integral membrane proteins

机译:高通量检测系统和方法,用于鉴定可改变完整膜蛋白表面表达的试剂

摘要

Disclosed is a method of identifying an agent that alters the level of surface expression of an integral membrane protein in a mammalian cell, said integral membrane protein being a human membrane ion channel, said method comprising; a) preparing a first medium containing mammalian cells that express a first mutant form of said membrane protein, wherein said first mutant form is expressed on the surface of said cells at a level less than a wild-type form of said protein. b) adding to said first medium containing mammalian cells a test amount of a candidate agent; c) incubating said cells in the presence of said candidate agent for a predetermined period of time; d) adding to said first medium containing mammalian cells at least one antibody which binds to at least one extracellular epitope of said mutant form of said membrane protein; and e) determining the level of binding of said at least one antibody to said extracellular epitope, wherein a change in said level of binding relative to a first control indicates that said candidate agent alters the level of surface expression of said mutant form of said membrane protein, wherein said first control comprises said first medium containing mammalian cells that express said first mutant form of said membrane protein in an amount equal to that in step (a) and said at least one antibody in an amount equal to that added in step (d).
机译:公开了一种鉴定改变哺乳动物细胞中整合膜蛋白表面表达水平的试剂的方法,所述整合膜蛋白是人膜离子通道,所述方法包括: a)制备含有表达所述膜蛋白的第一突变体形式的哺乳动物细胞的第一培养基,其中所述第一突变体形式在所述细胞表面上的表达水平低于所述蛋白的野生型形式。 b)向所述含有哺乳动物细胞的第一培养基中加入测试量的候选试剂; c)在所述候选试剂存在下将所述细胞孵育预定时间; d)向所述含有哺乳动物细胞的第一培养基中加入至少一种与所述膜蛋白的所述突变形式的至少一个细胞外表位结合的抗体; e)确定所述至少一种抗体与所述细胞外表位的结合水平,其中相对于第一对照,所述结合水平的改变表明所述候选试剂改变了所述膜的所述突变形式的表面表达水平蛋白,其中所述第一对照包含所述第一培养基,所述第一培养基包含以等于步骤(a)中的量表达所述膜蛋白的所述第一突变形式的哺乳动物细胞和以等于步骤(a)中添加的量的所述至少一种抗体。 d)。

著录项

  • 公开/公告号NZ545081A

    专利类型

  • 公开/公告日2009-04-30

    原文格式PDF

  • 申请/专利权人 CHANXPRESS INC.;

    申请/专利号NZ20040545081

  • 申请日2004-07-15

  • 分类号A61K31/495;C12Q1/02;G01N33/50;G01N33/53;G01N33/68;

  • 国家 NZ

  • 入库时间 2022-08-21 19:26:08

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