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

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).