Time-resolved fluorescence resonance energy transfer (TR-FRET) is a technology that generates a measurable fluorescent signal when two biomolecules are brought close together. This makes it ideal to study a vast range of biological events, including many that characterize cellular signaling pathways. Until recently, TR-FRET has been considered the exclusive domain of specialized high-throughput screening laboratories. However, the growing commercial availability of ready-to-use TR-FRET immunoassay kits has opened up the technique to far greater mainstream use. This article explains how researchers choosing to integrate TR-FRET into workflows can benefit.
Unlike a conventional sandwich ELISA, which involves multiple incubations interspersed by time-consuming wash steps, TR-FRET follows a simple, no-wash, add-and-read protocol. Not only does this provide faster time to results, but it also improves assay robustness by minimizing the number of steps where variability might be introduced. According to experts, by using a pre-optimized TR-FRET immunoassay kit, hands-on time can be as little as 5 minutes, and off-the-shelf kits enhance experimental reproducibility, thereby reducing the number of repeat tests that need to be carried out.
A typical TR-FRET immunoassay uses two antibodies for sandwich detection. Each recognizes a distinct epitope on the target antigen, yet while one antibody (the donor) is conjugated to a lanthanide, the other (the acceptor) is conjugated to a small molecule fluorescent dye. When both antibodies bind the target, excitation of the donor results in energy transfer to the nearby acceptor molecule, producing a signal that is directly proportional to the target concentration. In comparison to technologies such as western blot that use only one antibody to deliver a qualitative readout, TR-FRET delivers a highly specific and quantitative measurement as a result of the sandwich assay configuration.
By using lanthanides like Europium chelate as donors, TR-FRET benefits from very low background signal. This is attributable to the exceptionally long fluorescent lifetime (up to 1 ms) of lanthanides, which allows them to provide both temporal and spectral discrimination against background fluorescence. Because the long-lasting fluorescence results in high signal-to-noise ratios, TR-FRET is often more sensitive than ELISA, making it an ideal technology for phospho-protein detection.
A further advantage of lanthanides’ long fluorescent lifetime is that it reduces fluorescence interference, an issue that can be especially problematic when working with autofluorescent cells or lysates, or the fluorescent chemicals that are often found within compound libraries. Since the delay between excitation and emission for lanthanides can be much longer than that of traditional fluorescent dyes, replacing a traditional, fluorescence-based immunoassay with TR-FRET is an effective approach to address this issue.
As well as a long fluorescent lifetime, lanthanides also have a large Stokes shift. An advantage of this is that it allows for the use of acceptor molecules that produce visible, near-infrared or infrared emission spectra. While such acceptors demonstrate excellent overlap with the emission spectra of Europium chelate or similar molecules, they suffer far fewer interferences than many other fluorescent dyes yet can still be read using existing TR-FRET instrumentation/filters for easy integration into existing workflows.
In contrast to the fluorescent dyes used for detection in many ELISAs, the donor and acceptor molecules used for TR-FRET do not suffer from photobleaching. The stable signal therefore provides researchers with much greater flexibility, for instance eliminating the requirement to keep reagents and assay plates in the dark and avoiding the need to read plates as soon as an experiment is complete. TR-FRET also provides the opportunity to read each plate multiple times, for example during a kinetic study or allowing a re-read should a plate reader issue occur.
While assays based on absorbance can be difficult to miniaturize because the signal is concentration-dependent, scaling down TR-FRET is much more straightforward. Fluorescent readouts allow TR-FRET assays to be run with as little as 15 µL cell lysate per well without compromising accuracy, a feature that translates to more tests per sample than can be achieved with many ELISAs.
Using commercially available TR-FRET immunoassay kits, there is no need to perform assay development and optimization in-house. Instead, off-the-shelf kits contain carefully selected, highly compatible donor/acceptor TR-FRET antibody pairs and are provided with validated positive control material to ensure robust assay performance. Pre-optimized kits also follow a standardized assay protocol, contributing to more consistent operator-to-operator performance and enhanced intra- and inter-site reproducibility.
Biorbyt offers TR-FRET based immunoassays for detection of phosphorylated or total proteins in cell lysates. More information can be found here.