Published September 20, 2016
| Version v1
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Long fluorescence lifetime or label-free? A comparative study of high throughput screening methodologies to minimise assay interferences
Description
Fluorescence based assay methodologies are one of the most commonly used detection technologies for modern high throughput screening (HTS). However, the majority of fluorescence-based techniques are affected by a number of compound interferences, most notably; quenching, auto-fluorescence and light scattering. Fluorescence lifetime (FLT) is an under-used fluorescence screening methodology that measures the time a fluorophore spends in the excited state before returning to the ground state. Detecting fluorescence lifetime is an attractive assay readout due to its ability to mitigate some of the aforementioned compound-related assay interferences, in addition to being insensitive to well-to-well volume variations and less prone to turbidity by precipitating particles.
The utilisation of Mass spectrometry (MS) as a label-free, direct-detection methodology for screening of enzyme targets has been demonstrated to reduce assay interferences, when compared to indirect, fluorescent technologies. The Aglient RapidFire platform, which utilises solid phase extraction based techniques, has become widely used in drug discovery and is considered the optimal approach for low interference, medium throughput, enzyme compound screening and profiling. There are however disadvantages to using MS-based technologies, including the cost and complexity of the instrumentation and limitations in compound screening throughput.
To assess the suitability of FLT for use as as both a high throughput screening and hit qualification assay technology we have generated comparative data sets, using both the ?gold standard? RapidFire MS and the widely used, HTS-suitable, Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) methodology. These data demonstrate that FLT has a low incidence of interference, correlates well with RapidFire MS and is ammeable for use as a HTS assay format. Furthermore, we demonstrate that using lifetime, combined with features of the ameon FLT microplate reader and FLEXYTE assay reagents, we enable fluorescence recovery of true inhibitors and qualification of compound interference mechanisms.
The utilisation of Mass spectrometry (MS) as a label-free, direct-detection methodology for screening of enzyme targets has been demonstrated to reduce assay interferences, when compared to indirect, fluorescent technologies. The Aglient RapidFire platform, which utilises solid phase extraction based techniques, has become widely used in drug discovery and is considered the optimal approach for low interference, medium throughput, enzyme compound screening and profiling. There are however disadvantages to using MS-based technologies, including the cost and complexity of the instrumentation and limitations in compound screening throughput.
To assess the suitability of FLT for use as as both a high throughput screening and hit qualification assay technology we have generated comparative data sets, using both the ?gold standard? RapidFire MS and the widely used, HTS-suitable, Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) methodology. These data demonstrate that FLT has a low incidence of interference, correlates well with RapidFire MS and is ammeable for use as a HTS assay format. Furthermore, we demonstrate that using lifetime, combined with features of the ameon FLT microplate reader and FLEXYTE assay reagents, we enable fluorescence recovery of true inhibitors and qualification of compound interference mechanisms.