10.1177/1087057103252304ARTICLETurek-Etienne et al.Fluorescent Compound Interference in 4 FP Assays Evaluation of Fluorescent Compound Interference

With the increasing use of fluorescence-based assays in high-throughput screening (HTS), the possibility of interference by fluorescent compounds needs to be considered. To investigate compound interference, a well-defined sample set of biologi-cally active compounds, LOPAC™, was evaluated using 4 fluorescein-based fluorescence polarization (FP) assays. Two kinase assays, a protease assay, and a phosphatase assay were studied. Fluorescent compound interference and light scattering were observed in both mixture- and single-compound testing under certain circumstances. In the kinase assays, which used low levels (1-3 nM) of fluorophore, an increase in total fluorescence, an abnormal decrease in mP readings, and negative inhi-bition values were attributed to compound fluorescence. Light scattering was observed by an increase in total fluorescence and minimal reduction in mP, leading to false positives. The protease and phosphatase assays, which used a higher concentra-tion of fluorophore (20-1200 nM) than the kinase assays, showed minimal interference from fluorescent compounds, demon-strating that an increase in the concentration of the fluorophore minimized potential fluorescent compound interference. The data also suggests that mixtures containing fluorescent compounds can result in either false negatives that can mask a potential “hit ” or false positives, depending on the assay format. Cy ™ dyes (e.g., Cy3B ™ and Cy5™) excite and emit further into the red region than fluorescein and, when used in place of fluorescein in kinase 1, eliminate fluorescence interference and light scattering by LOPAC ™ compounds. This work demonstrates that fluorescent compound and light scattering interference