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P2046
A Sensitive & Simple Homogeneous QRET Technology for Biomolecular Screening
Presenter Harri Härmä, University of Turku, Finland
Additional Authors: Anita Rozwandowicz, Eija Martikkala, Mirva Koskelin, Pekka Hänninen, Heini Frang
Single labeling strategy has been developed for biomolecular screening studies allowing simplified and sensitive assays. TR-FRET has been widely applied in drug development studies. The technique requires labeling of two binding partners and close proximity in order to meet the limitation of Forster distances. We have developed a new approach to overcome the need for dual labeling and difficulties related to labeling of binder receptors. The detection of the QRET technology relies on a single labeled binding partner in combination with a soluble quencher. The QRET technology is a versatile detection tool for drug development studies. We have applied the technique for receptor-ligand screening study of ß2-adrenoreceptor and signaling pathway studies of cyclic adenosine monophosphate (cAMP).The ß2-adrenoreceptor was stably expressed in transfected human embryonic kidney 293. The receptor-ligand assay was carried out with the use of intact cells and single labeling strategy of europium labeled ligand in a homogeneous assay format. Unbound Eu-pindolol was quenched with a strong solution-based quencher. The IC50 values were for antagonist and agonist in nanomolar and micromolar ranges, respectively, and the CV was below 10% in 30-minutes assays.
Signaling pathway studies of cAMP was performed in a competitive homogenous assay format using non-labeled polyclonal anti-cAMP antibody and Eu-labeled cAMP. The signal of unbound Eu-labeled cAMP was strongly reduced with a soluble quencher. Present assay development has reached a low nanomolar detection limit for spiked cAMP with less than 6% CV in a 10-minutes assay. The future work will be focused on cell-based studies and cell stimulation assays.The QRET technology is fast, simple, efficient and low reagent consuming method being highly potential technique for biomolecular screening studies. Moreover, the QRET technology reduces risks related dual labeling strategies in assay development.