The patent, already granted in Europe and Japan, relates to a screening method for modulators for calcium-coupled proteins by measuring the intracellular calcium. The order of addition of reagents is reversed compared to what is normally done. In this new method, cells expressing apoaequorin and calcium-coupled protein are incubated with a cofactor (coelenterazine) needed for the detection of intracellular calcium.
Subsequently, aliquots of cells to be assayed are added to the compounds expected to be modulators for the target proteins. Finally, the activity of modulators is measured via an aequorin signal, a unique tool for the search of natural or surrogate ligands for any calcium coupled-protein.
The present invention would allow the detection of biological active substances at a high-throughput scale, which could be adapted to specific recipients such as microtiter plates without requiring the modification of the high-throughput screening device.
A lot of G-protein-coupled receptors (GPCR) trigger, upon binding of an agonist, a transient increase in intracellular calcium concentration. This variation acts as an internal secondary messenger and is an important modulator of many physiological mechanisms.
Measurement of intracellular calcium concentration in cells expressing a GPCR can thus be used to monitor the efficacy of activation of a GPCR by various compounds known, or suspected, to be a ligand for this GPCR.
Euroscreen already has the worldwide exclusive license on additional patents, which cover the aequorin gene for use in cell-based assays from the University of Georgia Research Foundation.
"This protocol permits higher throughputs, saves in the cost of reagents, plastic ware, and greatly decreases the handling time," said Dr Vincent Dupriez, head of molecular biology and co-inventor of this method.
"It also eliminates the need to grow adherent cells on assay plates, consequently eliminating the need for damaging wash procedures prior the assay, and therefore offers clear advantages in terms of the requirement for incubators for cell culture," he added.