Homogeneous Time-Resolved Fluorescence (HTRF) assay is a laboratory technique used to study molecular interactions. It enables researchers to detect and measure the binding of biological molecules, such as proteins or DNA, in a sample. This robust method analyzes various biological processes without complex sample preparation steps.
Understanding HTRF Assay
HTRF assay is a type of immunoassay that combines Time-Resolved Fluorescence (TRF) and Förster Resonance Energy Transfer (FRET). This combination allows for sensitive and specific detection of molecular interactions in a homogeneous format, meaning no wash steps are required. The assay relies on energy transfer between two fluorescent molecules, or fluorophores, when they are in close proximity.
The “time-resolved” aspect of HTRF uses long-lived donor fluorophores, such as lanthanide chelates like Europium cryptate or Terbium cryptate. This minimizes background interference from short-lived fluorescence signals in the sample.
The Mechanism Behind HTRF Assay
The HTRF assay operates on the principle of Förster Resonance Energy Transfer (FRET), a phenomenon where energy is transferred non-radiatively from a donor fluorophore to an acceptor fluorophore when they are very close, typically within 20 to 90 Å. In HTRF, a specific donor fluorophore, often a lanthanide cryptate such as Europium cryptate (Eu3+ cryptate) or Lumi4™-Tb (Tb2+ cryptate), is excited by a light source, usually a flash lamp or laser at around 337 nm. This excitation causes the donor to emit light with a long decay time, lasting for hundreds of microseconds.
If the donor and an appropriate acceptor fluorophore, like XL665 or d2, are brought into close proximity due to a molecular interaction, the donor’s energy is transferred to the acceptor. This energy transfer then causes the acceptor to emit its own distinct fluorescence at a different wavelength, typically around 665 nm for XL665 or d2, while the donor also emits at 620 nm. The time-resolved aspect of HTRF is implemented by introducing a delay, often between 50 to 150 microseconds, between the excitation pulse and the measurement of the emitted light. This delay allows any short-lived background fluorescence from the sample components to decay, leaving only the long-lived signal from the lanthanide donor and the FRET-induced acceptor emission. The ratio of the acceptor emission to the donor emission is then measured, providing a ratiometric signal that corrects for variations in well-to-well conditions and signal quenching.
Where HTRF Assay is Used
HTRF assay finds extensive use across various scientific disciplines, particularly in drug discovery and biomedical research. A primary application is identifying potential drug candidates by determining if a compound binds to a specific target receptor. This indicates a successful binding event.
HTRF is also broadly applied to study protein-protein interactions, which are fundamental to many biological processes. The technology facilitates research into protein-DNA/RNA interactions, protein-peptide interactions, and the activity of enzymes like protein kinases. It is also employed for the detection and quantification of various biomarkers, including cytokines, in cellular or biochemical assays. HTRF supports research in areas such as G protein-coupled receptors (GPCRs), epigenetics, and the characterization of biotherapeutics.
Key Benefits of HTRF Assay
The HTRF assay offers several advantages over traditional methods. One benefit is its homogeneous, “add-and-read” format, which eliminates the need for wash or separation steps common in assays like ELISA. This simplification reduces assay time and makes the process highly amenable to automation, improving efficiency in high-throughput screening campaigns.
The time-resolved detection mechanism, coupled with long-lived lanthanide fluorophores, substantially reduces background noise from sample components and media. This leads to increased assay sensitivity and a wider detection window between positive and negative signals. HTRF assays also consume small sample volumes, which conserves precious reagents. The robustness of the HTRF signal, which is stable over time, provides flexibility in read times and allows for kinetic studies.