Patent ID: 6025145
Filing Date: 2000-02-15
Classification: A61K,C07K,C12N,C12Q,G01N

Abstract:
A method for measuring autophosphorylation of a tyrosine kinase receptor comprising the steps of:(a) coating a first solid phase with a homogeneous population of eukaryotic cells so that the cells adhere to the first solid phase, wherein, positioned in their membranes, the cells have a receptor construct comprising a flag polypeptide and the tyrosine kinase receptor;(b) exposing the adhering cells to an analyte which is known to contain or is suspected of containing a ligand for the tyrosine kinase receptor;(c) solubilizing the adhering cells, thereby releasing cell lysate therefrom;(d) coating a second solid phase with a capture agent which binds specifically to the flag polypeptide so that the capture agent adheres to the second solid phase;(e) exposing the adhering capture agent to the cell lysate obtained in step (c) so that the receptor construct adheres to the second solid phase, wherein the cell lysate is not concentrated nor clarified prior to exposure to said adhering capture agent;(f) washing the second solid phase so as to remove unbound cell lysate;(g) exposing the adhering receptor construct to an anti-phosphotyrosine antibody which identifies phosphorylated tyrosine residues in the tyrosine kinase receptor; and(h) measuring binding of the anti-phosphotyrosine antibody to the adhering receptor construct, wherein the amount of anti-phosphotyrosine antibody binding to the adhering receptor construct is proportional to the amount of autophosphorylation of said tyrosine kinase receptor.