Multiplexing fluorescence polarization assays to increase information content per screen: applications for screening steroid hormone receptors.

As the push to reduce cost per well in high-throughput screening reaches the practical limitations of liquid handling, future cost savings will likely arise from an increase in information content per well. One strategy to increase information content is to perform discreet assays against multiple targets in a single well. In such assays, reagent usage and liquid handling steps do not scale-up in direct proportion to the increase in information content, providing for a simple method to increase data points per screen without further reductions in assay volume. The authors have used tracers incorporating the spectrally distinct fluorophores fluorescein and TAMRA to develop a high-throughput assay to identify selective estrogen receptor alpha or progesterone receptor ligands. Selectivity is assessed immediately in this assay, with no requirement for separate follow-up screening to determine selectivity. This methodology is easily adaptable to other target classes.