Fluorescence polarization assay for determining histidine decarboxylase activity
AssigneeBoehringer Ingelheim Pharmaceuticals, Inc.
InventorsE. Michael August, Daniel Rajotte
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Abstract
The present invention relates to fluorescence polarization assays for determining the HDC modulating activity of a candidate compound including providing a reaction mixture comprising a HDC, histidine, a fluorescently labeled histamine probe, a candidate compound and an anti histamine antibody having selectivity for histamine at least 10 fold greater than histidine: incubating the reaction mixture; and determining whether inhibition of HDC has occurred in the presence of the test compound, wherein an increase in fluorescence signal is an indication that the test compound inhibits the activity of the HDC.
Claims17
A fluorescence polarization assay for determining the histidine decarboxylase (HDC) inhibiting activity of a candidate compound comprising the steps of:
- a) providing a reaction mixture comprising a HDC, histidine, a fluorescently labeled histamine probe, a candidate compound and an anti histamine antibody having selectivity for histamine at least 10 fold greater than histidine;
- b) incubating the reaction mixture;
- c) determining whether inhibition of HDC has occurred in the presence of the candidate compound, wherein an increase in fluorescence polarization signal relative to a reference fluorescence polarization signal is an indication that the candidate compound inhibits the activity of the HDC,
- wherein said HDC comprises the polypeptide of SEQ ID. No. 1.
A fluorescence polarization (FP) assay for determining the histidine decarboxylase (HDC) inhibiting activity of a candidate compound comprising the steps of:
- a) providing a reaction mixture comprising a HDC comprising the polypeptide of SEQ ID No. 1, histidine, a fluorescently labeled histamine probe, a candidate compound and an anti histamine antibody having selectivity for histamine at least 10 fold greater than histidine;
- b) incubating the reaction mixture for a time sufficient to produce histamine but no longer than about 180 minutes;
- c) determining the FP signal of the incubated reaction mixture;
- and
- d) comparing the FP signal of the incubated reaction mixture with a reference EP signal, wherein if the FP signal of the incubated reaction mixture is about the same as or is greater than the reference FP signal, the candidate compound is an inhibitor of HDC.
Dependencies are inferred from the claim text.