Patent ID: 6828109
Filing Date: 2004-12-07
Classification: G01N

Abstract:
A method of detecting the presence of an intracellular analyte in one or more cells by flow cytometry, the method comprising:a) fixing and permeabilizing said cells; b) catalyzing the deposition of tyramide in said cells comprising said intracellular analyte by contacting the fixed and permeabilized cells with an antibody or fragment thereof that specifically binds said intracellular analyte, wherein said antibody is directly or indirectly bound to an enzyme, wherein said enzyme is in a medium containing an agent or agents that reduce non-specific binding to an extent at least equal to the presence of 10% fetal bovine serum and wherein said enzyme, in the presence of substrate for said enzyme and tyramide, catalyzes the deposition of tyramide in said cells comprising said intracellular analyte, and contacting the fixed and permeabilized cells with tyramide and a substrate for said enzyme; c) contacting said cells with a detectable label that directly or indirectly binds to tyramide, whereby said cells comprising said intracellular analyte are specifically labeled; and d) detecting a signal from said cells comprising said detectable label using a flow cytometric device, wherein said signal indicates the presence of said intracellular analyte, and wherein said signal is at least 10-fold greater than a signal obtainable by standard flow cytometry methods in which an immunoglobulin that does not specifically bind said intracellular analyte, and that is isotype/subtype matched to the antibody or fragment thereof of step (b) is used as a negative control.