HTRF (Homogeneous time-resolved fluorescence)
Table of contents
What is HTRF?
Homogeneous Time-Resolved Fluorescence (HTRF®) is a Time-Resolved FRET (TR-FRET) immunoassay developed by Cisbio. This technology combines Time-Resolved Fluorescence (TRF) detection with Fluorescence Resonance Energy Transfer (FRET) and is mainly used for drug screening in high-throughput.
Fluorescence resonance energy transfer is a proximity assay that takes advantage of the energy transfer happening between two fluorophores (called donor and acceptor), when they are in close contiguity. Upon excitation of the donor by a light source, the energy is transferred from the donor to the acceptor, if the distance between the two is between 20 and 90 Å. The excited acceptor emits then fluorescence at a given wavelength.
FRET is mainly used to analyse molecular binding events or interactions and can be employed to determine the binding affinity of two binding partners. These are assessed by coupling each binding partner to be investigated with a fluorophore, and by detecting the level of energy transfer taking place.
GFP/RFP and CFP/YFP steady state fluorescent FRET pairs have been widely used in fluorescence intensity assays. However, highly sensitive measurement can be hardly obtained with standard FRET. This method is negatively affected by the background noise derived from scattered excitation light and autofluorescence.
Through the adoption of time-resolved fluorescence, TR-FRET allows the elimination of the short-lived steady state background fluorescence by introducing a time delay between excitation and detection. This is made possible by the use of long-lived fluorophores called lanthanides as donors.
In fact, time-resolved FRET and HTRF assays rely on the transfer of energy between lanthanide donors and short-lived fluorophore acceptors, when in close proximity. Lanthanides are well-suited for these applications because of a large Stokes shift and because of longer emission lifetimes (from microseconds to milliseconds) compared to steady state fluorophores like fluorescein or rhodamine.
Due to these specific characteristics, time-resolved FRET and HTRF exhibit improved performance levels in stability and specificity. Buffer or medium interference is significantly reduced by the long-lived fluorescence and by the ratiometric detection of the two emission wavelengths of donor and acceptor.
The general principle of how the Homogeneous Time-Resolved Fluorescence technology is applied is very similar throughout different applications. Specific antibodies against each of the two targets (or tags), the interaction which has to be investigated, are labelled with either the donor or the acceptor. If the targets are in close proximity, the antibodies will be as well. By exciting the donor, energy will be transferred to the acceptor, which will emit because of fluorescence resonance energy transfer. The final signal is proportional to the amount of binding taking place. Alternatively, donor and acceptor can be covalently bound to the interacting partners.
Four specific fluorophores, forming different time-resolved FRET couples, are used in HTRF. The donor lanthanide fluorophore is either a europium (Eu3+) or terbium (Tb2+) cryptate (fig. 1), whereas the steady state fluorescence acceptors usually emit in the red range.
As europium and terbium ions are only poorly fluorescent on their own, they need to be embedded in a light-collecting cage to emit light. The HTRF technology makes use of a cryptate for this purpose. Similar to a chelate in other TRF and time-resolved FRET technologies, a cryptate allows both collection and transfer of energy. The acceptor fluorophore then releases this energy as light at a specific wavelength.
The lanthanide cryptate complex is not subject to photo-bleaching like several conventional fluorophores, and is said to be extremely stable under a wide range of chemical conditions.
The terbium cryptate has a 10 to 20 times increased quantum yield and a higher molar extinction coefficient compared to europium, features that improve screening performance. Both europium and terbium -based assays can be read on the same HTRF plate reader.
XL665, a pigment purified from red algae, was the first-generation developed for the HTRF technology. The second-generation d2, is similar to XL665 but much smaller. It is said to limit steric hindrance problems sometimes suspected in TR-FRET systems.
Both acceptors emit in the red range. Their emission hence is less likely to be negatively affected by intrinsic medium or compound autofluorescence. Both red acceptors can be coupled with either a europium or terbium cryptate. Terbium donors can be additionally combined with green-emitting acceptors (e.g. fluorescein), allowing for potential multiplexing (fig. 2).
Cryptate donors are typically excited at 337 nm. Acceptor and donor emit at two different wavelengths 620 nm and 665 nm, respectively. Hence, they can be optically distinguished from each other. Emission at 620 nm is the proper emission of the donor and is used as an internal reference, while emission at 665 nm is the acceptor as indicator of the energy transfer (binding event) taking place (fig. 3).
A time delay of 60 microseconds between excitation and fluorescence detection eliminates the non-specific short-lived background emission. Emission is then usually detected over a time period of 400 microseconds (fig. 4).
A ratio of the intensities of the two emission channels integrated over the 400 microseconds time span is then performed (ratiometric measurement). This normalises the signal, corrects for well to well variability or liquid handling errors, and eliminates medium interference and quenching from assay components.
Advantages of the HTRF technology
Homogeneous Time-Resolved Fluorescence is widely popular especially because of its sensitivity and versatility. The combination of time-resolved detection and ratiometric measurements significantly reduces background and increases the detection window between negative and positive signal. Moreover, the lanthanide emission is stable in time, compatible with different reagents and experimental conditions and it does not bleach.
Besides its sensitivity, another major advantage is the fact that it is a homogeneous assay. Detection of the bound FRET donor-acceptor complex does not require physical separation from the unbound components to reduce the background. Consequently, the HTRF technology does not necessitate in-between separation or washing steps and can be executed with a simple add-and-read protocol (fig.5). This minimises handling steps and makes it more convenient and less time-consuming than other methods such as ELISA or Western Blot. Hence, it is particularly suited for automation-supported screening purposes. This is one of the reasons for its large adoption in drug discovery and HTS.
However, limitations also exist. For instance, signal quenching generated by external interactions with the intramolecular excitation process or fluorescence of library compounds or biological proteins. 
HTRF plate reader
The measurement of HTRF assays is predominantly performed on microplate readers. Assay detection requires a microplate reader with TRF detection mode and the capability to measure two emission channels in one run, either sequentially or simultaneously.
As this technology is mainly used in high-throughput drug screening, HTRF plate readers should be compatible with 384 and 1536 well plates.
The HTRF plate reader basic setup consists of a light source, excitation and emission filters for wavelength selection and a photomultiplier tube (PMT) detector.
Light source: xenon lamp or laser
As the cryptate complex is typically excited at 337 nm, a xenon flash lamp or a specific excitation laser can be used as light source. A TRF laser focusses more energy at 337 nm and leads to better results with a better discrimination between low and high signals.
The TRF laser on the PHERAstar FSX with a flash frequency of 60 Hertz provides a significant speed advantage for TR-FRET- and HTRF-based detection. It allows the measurement of a full 1536 well plate in only 36 seconds while still keeping a Z´ > 0.8 for cell and biochemical assays (fig.6). This is extensively discussed in the application note “Cellular and biochemical HTRF assays measured in 1536-well microplates”.
The PHERAstar FSX HTS microplate reader is equipped with so called photon-counting PMTs for time-resolved fluorescence detection. While normally microplate readers simply give an integration value for the area under the curve during the integration time, time-resolved fluorescence -dedicated photon counting detection monitors the whole decay curve of the lanthanide.
On the PHERAstar FSX reader, photon counting detection allows the measurement and display of the emission decay curve with a time resolution of 2 microseconds. This unique feature called Decay Curve Monitoring simplifies time-resolved fluorescence assay development, helping to optimize timing parameters and thus improving detection and reducing background noise. Combined with the Integration Wizard, Decay Curve Monitoring offers a platform for the optimisation of TR-FRET assays. These solutions are very helpful in assay development and when running in-house TR-FRET assays.
Dual channel emission detection
Being a dual-wavelength emission assay, sequential or simultaneous detection of the two emission wavelengths is required. Commonly, HTRF microplate readers measure the two signals one after the other. However, the simultaneous detection of the two channels bears some advantages over sequential detection.
On the PHERAstar FSX, the Simultaneous Dual Emission (SDE) detection system uses two matched PMTs for the concurrent measurement of the 2 emission wavelengths, substantially reducing plate read times by half and increasing throughput. SDE also corrects any variation due to differences in well volumes, concentrations, or fluctuations in excitation energy. This ensures improved sensitivity and lower %CVs compared with other readers. SDE single channel measurements are then combined for the ratiometric calculation of the results from both emission channels.
The HTRF certification is given to microplate readers that meet Cisbio´s specifications in terms of S/N, DF%, CV%, sensitivity and dynamic range. Plate readers can be certified with either white or black microplates. Usually, being certified with black microplates is an indicator for a higher sensitivity of the instrument.
The following BMG LABTECH multi-mode microplate readers are HTRF-certified by Cisbio: PHERAstar FSX, CLARIOstar Plus, POLARstar Omega and FLUOstar Omega.
Signal intensity in time-resolved fluorescence detection depends on concentration and not on the overall quantity of a tracer, as it is the case for instance in radioactivity. Consequently, homogeneous time-resolved fluorescence assays can be downscaled while maintaining accuracy and reproducibility. This is further supported by the use of a highly sensitive HTRF plate reader like the PHERAstar FSX.
Assay downscaling or miniaturisation is a key step in drug screening. Its goal is the reduction of sample volumes to save on reagents and time, while maintaining reliability, robustness, and reproducibility. Although 384 and 1536 well formats are the most commonly used, assays could even be downscaled to 3456 well plates.
In our scientific talk “Successfully downscaling screening assays: Servier´s experience“, we present a case study where the use of the PHERAstar FSX high-throughput plate reader significantly contributed to the miniaturisation of a HTRF assay from the 384 to the 1536 well format. On the PHERAstar FSX, assay miniaturization was combined with “on-the-fly” detection (only one flash per well) and the further dilution of reagents. This resulted in high-quality Z´ values and a significant reduction in reagent usage and time.
HTRF measurements and applications
The Homogeneous Time-Resolved Fluorescence technology can be applied to analyse binding events in cells or in biochemical assays in 96, 384 and 1536 well plate formats for different biological settings such as protein protein (ligand-receptor) and protein DNA/RNA interactions, GPCR signaling, kinase assays, cytokines and biomarkers.
Additionally, it is suitable for most cell based procedures, allowing measurements of the cell lysate in the presence of cell culture medium.
The HTRF technology is particularly diffused in the drug screening community. Advantages include flexibility, reliability, increased assay sensitivity, higher throughput, and fewer false positive/false negative results.
The technology is also helpful in the identification of false positives. For more information see AN 359: Identification of false positives in a HTRF® screen for small molecule inhibitors of PD-1/PD-L1.
Protein protein and protein DNA/RNA interaction
Protein protein and protein nucleic acid interactions take place at almost every level in cells. This includes membrane protein communication (like receptor-ligand binding), signal transduction (like G-protein cascades), regulation of gene expression, epigenetics (like histone modifying activity), etc.
G-Protein Coupled Receptor (GPCR) assays can be divided into two categories, functional and mechanistic assays. Functional assays focus on the quantification of secondary messengers like IP1 or cAMP. As part of the signaling cascade, these may be targeted by specific inhibitors to enable their accumulation in the cell. Second messengers can be used as a readout, as their concentration correlates to receptor engagement and ligand binding. This is shown in the application notes “HTRF IP-One assay used for functional screening” and “GPCR activation is measured using Cisbio's cAMP and IP1 HTRF HTplex cell based assay”.
The mechanistic analysis focuses on receptor organization on the membrane of cells, in particular on receptor oligomerization as a means to mediate signaling.
Enzymatic activity assays, such as kinases, proteases, ubiquitination can be analysed as well. Sandwich assays can be used to measure the status change of a substrate, like phosphorylation. Competition assays can be employed to monitor other products generated in the reactions, like ADP.
Downstream signaling products can be used as biomarkers. Their analysis can identify aberrant signaling pathways that possibly play a significant role in inflammatory, metabolic and neurologic diseases. HTRF-based biomarker analysis can be hence used to test the efficacy of compounds that specifically target these diseases. Examples for biomarker analysis include “Detection of human tau protein aggregation” in neurodegenerative diseases, and “Development of a rapid HTRF insulin assay” for metabolic diseases, in particular diabetes mellitus.
Kinetic analysis of drug target binding events
In the drug discovery process, the kinetics of ligand-receptor binding have been traditionally studied only at a late time point. This is mostly because of technical difficulties and the relative low-throughput that assays provided.
However, optimizing receptor binding kinetics can be highly beneficial for drug discovery since association and dissociation rates impact on drug efficiency and on the occurrence of side-effects. Moreover, efficacy can be improved, and duration of action can be prolonged. Finally, binding kinetics seem to play a role in biased agonism.
Therefore, it is desirable to screen drug candidates for their binding kinetics at an early time point, before moving to in vivo models and clinical studies.
The study of binding kinetics can be efficiently performed on microplate readers, mainly relying on TR-FRET kinetic detection for screening purposes and the kinetic study of low-affinity compounds (fig. 7).
This is discussed in the scientific talk “A TR-FRET approach to measure the kinetics of ligand-receptor binding and its application of fragment screening”, as well as in the application note “Analyze binding kinetics with HTRF”.
The PHERAstar FSX has unique features dedicated to kinetic binding studies that make it superior to any other microplate reader currently on the market. With its high temporal resolution in HTRF detection, and dedicated hardware and software solutions, the PHERAstar FSX easily resolves binding events and calculates association and dissociation rates.
HTRF application notes
BMG LABTECH has published numerous application notes using HTRF measurements. Our HTRF plate readers include the CLARIOstar Plus and PHERAstar FSX and the Omega series.
1. Degorce et al. “HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications.” Current chemical genomics vol. 3 22-32. 28 May. 2009
HTRF® is a registered trademark of Cisbio.