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    Kinase receptor activation assay
    5.
    发明授权
    Kinase receptor activation assay 失效
    激酶受体活化测定

    公开(公告)号：US5766863A

    公开(公告)日：1998-06-16

    申请号：US286305

    申请日：1995-08-05

    申请人： Paul J. Godowski ,  Melanie R. Mark ,  Michael D. Sadick ,  David L. Shelton ,  Wai Lee Tan Wong

    发明人： Paul J. Godowski ,  Melanie R. Mark ,  Michael D. Sadick ,  David L. Shelton ,  Wai Lee Tan Wong

    IPC分类号： A61K38/00 ,  A61K39/00 ,  A61K39/395 ,  A61P43/00 ,  C07H21/04 ,  C07K14/435 ,  C07K14/705 ,  C07K14/71 ,  C07K14/715 ,  C07K16/28 ,  C12N1/21 ,  C12N5/02 ,  C12N9/12 ,  C12N15/02 ,  C12N15/09 ,  C12N15/12 ,  C12N15/54 ,  C12P21/08 ,  C12Q1/48 ,  C12R1/19 ,  C12R1/91 ,  G01N33/543 ,  G01N33/566 ,  G01N33/573 ,  G01N33/567 ,  C07K16/00 ,  G01N33/53

    CPC分类号： C07K16/28 ,  C07K14/705 ,  C07K14/71 ,  C07K14/715 ,  C12N9/12 ,  C12Q1/485 ,  G01N33/54306 ,  G01N33/566 ,  G01N33/573 ,  A61K38/00 ,  G01N2333/705 ,  G01N2333/912 ,  G01N2333/9121 ,  G01N2500/00 ,  Y10S435/975

    摘要： An assay for measuring activation (i.e., autophosphorylation) of a tyrosine kinase receptor of interest is disclosed. (a) A first solid phase is coated with a substantially homogeneous population of cells so that the cells adhere to the first solid phase. The cells have either an endogenous tyrosine kinase receptor or have been transformed with DNA encoding a receptor or "receptor construct" and the DNA has been expressed so that the receptor or receptor construct is presented in the cell membranes of the cells. (b) A ligand is then added to the solid phase having the adhering cells, such that the tyrosine kinase receptor is exposed to the ligand. (c) Following exposure to the ligand, the adherent cells are solubilized, thereby releasing cell lysate. (d) A second solid phase is coated with a capture agent which binds specifically to the tyrosine kinase receptor, or, in the case of a receptor construct, to the flag polypeptide. (e) The cell lysate obtained in step (c) is added to the wells containing the adhering capture agent so as to capture the receptor or receptor construct to the wells. (f) A washing step is then carried out, so as to remove unbound cell lysate, leaving the captured receptor or receptor construct. (g) The captured receptor or receptor construct is exposed to a labelled antiphosphotyrosine antibody which identifies phosphorylated residues in the tyrosine kinase receptor. (h) Binding of the anti-phosphotyrosine antibody to the captured receptor or receptor construct is measured.

    摘要翻译： 公开了用于测量感兴趣的酪氨酸激酶受体的活化（即自身磷酸化）的测定法。 （a）第一固相涂覆有基本上均匀的细胞群，使得细胞粘附到第一固相。 细胞具有内源性酪氨酸激酶受体或已经用编码受体或“受体构建体”的DNA转化，并且已经表达了DNA，使得受体或受体构建体存在于细胞的细胞膜中。 （b）然后将配体加入到具有粘附细胞的固相中，使得酪氨酸激酶受体暴露于配体。 （c）暴露于配体后，粘附细胞溶解，从而释放细胞裂解物。 （d）第二固相包被与酪氨酸激酶受体特异性结合的捕获剂，或在受体构建体的情况下，与标记多肽结合。 （e）将步骤（c）中获得的细胞裂解物加入含有粘附的捕获剂的孔中，以将孔或受体构建体捕获到孔中。 （f）然后进行洗涤步骤，以除去未结合的细胞裂解物，留下捕获的受体或受体构建体。 （g）将捕获的受体或受体构建体暴露于标记的抗磷酸酪氨酸抗体，其鉴定酪氨酸激酶受体中的磷酸化残基。 （h）测量抗磷酸酪氨酸抗体与捕获的受体或受体构建体的结合。