Fluorescent capture assay for kinase activity
AssigneePharmacopiea, Inc.
InventorsJ. Fraser Glickman, James Inglese, Bassam Damaj, Maria L. Webb, Jonathan J. Burbaum
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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 fluorescent label, for instance, Cy5, Cy5.5 or Cy7 or 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.
Claims32
A method for measuring protein 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) determining the level of kinase activity in said biological sample by measuring the amount of a second anti-phosphotyrosine antibody which binds to said phosphorylated tyrosine-containing molecules.
The method of claim of 3, wherein label on said labeled anti-phosphotyrosine antibody is selected from Cy5, Cy5.5, Cy7 and a lanthanide ion.
A method for screening a substance for the ability to modulate tyrosine kinase activity comprising the steps:
- (a) exposing a portion of a biological sample to said substance;
- (b) determining the level of tyrosine kinase activity in said portion by:
- (1) contacting said portion with a solid phase coated with a first anti-phosphotyrosine antibody so that phosphorylated tyrosine-containing molecules in the biological sample are bound by the antibody to the solid phase;
- and
- (2) measuring the amount of a second anti-phosphotyrosine antibody which binds to said phosphorylated tyrosine-containing molecules.
A method for screening a substance for the ability to modulate tyrosine kinase activity comprising the steps:
- (a) exposing a portion of a cell population to said substance;
- (b) preparing cell fractions from cells of said portion of said cell population;
- and
- (c) determining the level of tyrosine kinase activity in said cell fractions by:
- (1) contacting said cell fractions with a solid phase coated with a first anti-phosphotyrosine antibody so that phosphorylated tyrosine-containing molecules in the biological sample are bound by the antibody to the solid phase;
- and
- (2) measuring the amount of a second anti-phosphotyrosine antibody which binds to said phosphorylated tyrosine-containing molecules.
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 label selected from Cy5, Cy5.5, Cy7 and a lanthanide ion;
- and
- (c) instructions for carrying out a method for using said kit.
Dependencies are inferred from the claim text.