IP3 protein binding assay
OwnerTabassum Naqvi, Riaz Rouhani +3inventors
InventorsTabassum Naqvi, Riaz Rouhani, Peter A. Fung, Richard M. Eglen, Rajendra Singh
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Abstract
Protein binding assays are provided for determining IP3 in a sample employing as reagents a conjugate of IP3 joined at the 2-oxy through a bond or linking group to a detectable label and a truncated portion of the extracellular fragment of an IP3R. The reagents are combined with the sample and the amount of IP3 determined by means of the detectable label. The conjugate with the enzyme donor fragment of β-galactosidase or a fluorescer is specifically described.
Claims21
A protein binding assay for measuring IP3 in a sample employing as reagents a conjugate of IP3 and a detectable label joined through a bond or linker at the 2-hydroxyl position, and a truncated extracellular portion of an IP3R having at least about 200 times the affinity for IP3 than the intact IP3R, said method comprising:
- combining in an assay medium said sample, said conjugate and said binding protein and incubating said mixture for sufficient time for any IP3 and said conjugate to bind to said binding protein;
- and
- detecting the bound or unbound label as a measure of the IP3 present in the sample.
A protein binding assay for measuring IP3 in a sample using a homogeneous format, employing as reagents a conjugate of IP3 and an ED of from 37 to 60 amino acids derived from β-galactosidase joined through a linker at the 2-hydroxyl position, and a truncated extracellular portion of an IP3R having at least about 200 times the affinity for IP3 than the intact IP3R, said method comprising:
- combining in an assay medium assay components in the following order:
- said sample, said binding protein, said conjugate and EA, and incubating after each combining for sufficient time for complex formation between said assay components;
- adding substrate for said β-galactosidase;
- and
- detecting the turnover of said β-galactosidase of said substrate as a measure of the IP3 present in the sample.
A protein binding assay for measuring IP3 in a sample using a homogeneous format, employing as reagents a conjugate of IP3 and a fluorescer joined through a linker at the 2-hydroxyl position, and a truncated extracellular portion of an IP3R having at least about 200 times the affinity for IP3 than the intact IP3R, said method comprising:
- combining in an assay medium assay components:
- said sample, said binding protein, and said conjugate, and incubating for sufficient time for complex formation between said assay components;
- and
- detecting the change in fluorescence polarization as a measure of the IP3 present in the sample.
A protein binding assay for measuring IP3 in a sample employing as reagents a conjugate of IP3 and a detectable label joined through a bond or linker at the 2-hydroxyl position, and a truncated extracellular portion of an IP3R having at least about 200 times the affinity for IP3 than the intact IP3R, said method comprising:
- combining in an assay medium said sample, said conjugate, said binding protein and a chemical reductant and incubating said mixture for sufficient time for any IP3 and said conjugate to bind to said binding protein;
- and
- detecting the bound or unbound label as a measure of the IP3 present in the sample.
A compound of the formula:
- 4
- wherein:
- R is a neutral linking group of from 4 to 20 carbon atoms bonded to the oxygen through a saturated carbon atom or carbonyl;
- Z is a functionality for linking X to the oxygen at the 2-position;
- X is an enzyme donor fragment of β-galactosidase of from 27 to 60 amino acids;
- and
- n is 1 or 2.
A compound of the formula:
- 5
- wherein:
- R is a neutral linking group of from 2 to 20 carbon atoms bonded to the oxygen through a saturated carbon atom;
- Z is a functionality for linking X to the oxygen at the 2-position;
- and
- X is a fluorescer.
A kit for performing an IP3 assay comprising a conjugate of IP3 and a detectable label joined through a bond or linker at the 2-hydroxyl position, a truncated extracellular portion of an IP3R having at least about 200 times the affinity for IP3 than the intact IP3R and instructions for performing said assay.
Dependencies are inferred from the claim text.