Fluorescent capture assay for kinase activity employing anti-phosphotyrosine antibodies as capture and detection agents
AssigneePharmacopeia, Inc.
InventorsJ. Fraser Glickman, James Inglese, Bassam Damaj
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Abstract
A method for determining the level of tyrosine kinase activity in a biological sample is disclosed. The method employs an anti-phosphotyrosine antibody as both the capture agent and the detecting agent. The detecting antibody is labeled with a lanthanide ion, such as europium, as the signal generating entity. The method is particularly well suited to high throughput screening, for example, for compounds which modulate tyrosine kinase activity.
Claims12
A method for measuring protein tyrosine kinase activity in a biological sample, said method comprising the steps:
- (a) coating a solid phase with a first anti-phosphotyrosine antibody;
- (b) contacting a biological sample with said solid phase coated with said first anti-phosphotyrosine antibody so that phosphorylated tyrosine-containing molecules in the biological sample are bound by the antibody to the solid phase;
- (c) contacting bound phosphorylated tyrosine-containing molecules with a second anti-phosphotyrosine antibody;
- (d) removing unbound second anti-phosphotyrosine antibody;
- and
- (e) determining the level of tyrosine kinase activity in said biological sample by measuring the amount of said second anti-phosphotyrosine.
A kit for use in measuring the level of tyrosine kinase activity in a biological sample comprising:
- (a) a solid phase coated with a first anti-phosphotyrosine antibody;
- (b) a second anti-phosphotyrosine antibody, wherein said second anti-phosphotyrosine antibody is labeled with a lanthanide ion;
- and
- (c) instructions for carrying out a method for using said kit.
Dependencies are inferred from the claim text.