Development of an Europium(III) DOTA-based Luminescence Assay for Detection of Ligand-Receptor Interactions

摘要

Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improved sensitivity and affordability in high throughput screening while eliminating the use of radioactivity [1]. Accordingly, dissociation enhanced lanthanide fluoroimmunoassay (DELFIA) technology is rapidly emerging as a bioanalytical tool using Eu(III)-coordinated chelators such as DTPA (diethylenetriaminepentaacetic acid) and DTTA (diethylenetriaminetetraacetic acid) linked to peptides targeted towards a specific cell surface receptor. In DELFIA assays, a ligand is labeled with a lanthanide(III) ion [such as Eu(HI) or Tb(III)] that is competed off with screened ligand or directly used in saturation binding assays [2,3]. The amount of specifically bound lanthanide-labeled ligand is then analyzed by adding an "enhancement solution" which is capable of completely releasing Eu(III) ions from their nonphotoactive chelator and rearranging them into highly luminescent coordination complexes. This process enhances the lanthanide-based luminescence by up to 10~7-fold [4]. Despite the widespread usage, DOT A (l,4,7,10-tetraaza-l,4,7,10-tetrakis(carboxymethyl)-cyclododecane) and its derivatives offer the highest thermodynamic and kinetic stability with a selection of metal ions, and therefore have potential for implementation with a wide range of in vivo imaging applications. However, their application for these purposes has been limited due to the incomplete release of lanthanide(III) ions under standard DELFIA. Here we report our preliminary results towards developing an improved DELFIA assay for whole cell ligandreceptor binding studies, using lanthanide(III)-DOTA labeled ligands coupled with an acid treatment protocol.