Homogeneous proximity assays are widely implemented in high-throughput screening (HTS) of small-molecule libraries for drug and chemical probe discovery. Representative technologies include AlphaScreen, Förster/fluorescence resonance energy transfer (FRET), time-resolved FRET (TR-FRET) and homogeneous time-resolved fluorescence (HTRF), bioluminescence resonance energy transfer (BRET), and scintillation proximity assays (SPA). While highly useful, these assay technologies are susceptible to a variety of technology-related compound-mediated interferences, most notably signal attenuation (e.g., quenching, inner-filter effects, light scattering), signal emission (e.g., auto-fluorescence), and disruption of affinity capture components such as affinity tags and antibodies. These assays are also susceptible to more generalized compound-mediated interferences such as nonspecific reactivity and aggregation.
This presentation describes:
- the basic principles of homogeneous proximity assays,
- common sources of compound-mediated interference in homogeneous proximity assays, and
- counter-screens and other strategies to mitigate and identify compound-mediated assay interferences in homogeneous proximity assays.
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