Sensitive intracellular calcium assay
View Patent ↗A sensitive intracellular calcium assay is disclosed comprising conveniently a reagent comprised of a dye precursor capable of entering cells and being hydrolyzed to a dye, whereby the dye complexes with calcium in the cells and provides a luminescent signal, an antibody specific for the dye and conjugated with a quencher, and a cellular anion exchange enzyme inhibitor. In performing the assay, the reagent is combined with cells expressing a receptor responsive to a ligand resulting in a change in cytosolic calcium. After incubation for the dye precursor to permeate the cells, the calcium may be determined by exciting the dye precursor and determining the peak fluorescence over a time course. The method can be used for measuring the effect of an agent on cytosolic calcium by binding to a cell surface membrane receptor.
1. A method for determining intracellular calcium comprising:
adding to cells a dye precursor capable of entering the cells and being hydrolyzed to a dye, whereby the dye complexes with calcium in said cells and provides a luminescent signal, an antibody specific for the dye and not the dye precursor, said antibody conjugated with a quencher for quenching extracellular hydrolyzed dye precursor, said quencher being a p-aminophenylazo compound or a 4,5-dimethoxyfluorescein, and a cellular anion exchange enzyme inhibitor for inhibiting cell entry of extracellular calcium;
incubating for sufficient time for said dye precursor to enter said cells; and determining in the presence of said dye precursor over a time span the change in the luminescent signal from said dye with decreased extracellular dye signal as a measure of intracellular calcium.
2. A method according to claim 1 , wherein said luminescent signal is fluorescence and said determining comprises illuminating said cells with excitation light.
3. A method according to claim 1 , wherein said dye precursor is a xanthene ester and said antibody is a monoclonal antibody specific for the hydrolyzed xanthene ester.
4. A method according to claim 1 , wherein said dye is selected from the group consisting of Fluo¾, Fura⅔, and calcein green.
5. A method according to claim 1 , wherein the signal is at least about twice the signal at 1000 nM carbachol as the signal at 10 nM carbachol.
6. A method according to claim 1 , wherein said quencher conjugated antibody has on the average from about 2 to 15 quencher molecules per antibody.
7. A method according to claim 1 , further comprising an agent wherein said agent binds to a neuronal receptor.
8. A method according to claim 1 , further comprising an agent wherein said agent binds to an ion channel.
9. A method according to claim 1 , wherein said cells are genetically modified to express a cell surface membrane receptor.