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HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications.

Degorce F, Card A, Soh S, Trinquet E, Knapik GP, Xie B - Curr Chem Genomics (2009)

Bottom Line: Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation.Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient.This review addresses the general principles of HTRF and its current applications in drug discovery.

View Article: PubMed Central - PubMed

Affiliation: Cisbio Bioassays, 30204 Bagnols-Sur-Cèze, France. fdegorce@cisbio.com

ABSTRACT
HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS). This technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation. The HTRF assay is usually sensitive and robust that can be miniaturized into the 384 and 1536-well plate formats. This assay technology has been applied to many antibody-based assays including GPCR signaling (cAMP and IP-One), kinases, cytokines and biomarkers, bioprocess (antibody and protein production), as well as the assays for protein-protein, proteinpeptide, and protein-DNA/RNA interactions.Since its introduction to the drug-screening world over ten years ago, researchers have used HTRF to expedite the study of GPCRs, kinases, new biomarkers, protein-protein interactions, and other targets of interest. HTRF has also been utilized as an alternative method for bioprocess monitoring. The first-generation HTRF technology, which uses Europium cryptate as a fluorescence donor to monitor reactions between biomolecules, was extended in 2008 through the introduction of a second-generation donor, Terbium cryptate (Tb), enhancing screening performance. Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient. In addition to being compatible with the same acceptor fluorophors used with Europium, it can serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm. Moreover, all Terbium HTRF assays can be read on the same HTRF-compatible instruments as Europium HTRF assays.Overall, HTRF is a highly sensitive, robust technology for the detection of molecular interactions in vitro and is widely used for primary and secondary screening phases of drug development. This review addresses the general principles of HTRF and its current applications in drug discovery.

No MeSH data available.


Related in: MedlinePlus

The energy pulse from the excitation source (flash lamp, laser) is followed by a time delay, allowing interfering short-lived fluorescence (compounds, proteins, medium etc.) to decay. The red line: FRET signal intensity generated at 665 nm; black line, emission of donor cryptate at 620 nm; orange line, fluorescent signal generated from acceptor fluorophores.
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Figure 2: The energy pulse from the excitation source (flash lamp, laser) is followed by a time delay, allowing interfering short-lived fluorescence (compounds, proteins, medium etc.) to decay. The red line: FRET signal intensity generated at 665 nm; black line, emission of donor cryptate at 620 nm; orange line, fluorescent signal generated from acceptor fluorophores.

Mentions: The principle behind FRET is based on the transfer of energy between two fluorophores, a donor (long-lived fluorescence) and an acceptor (short-lived fluorescence), when in close proximity [3, 4]. Molecular interactions between biomolecules can be assessed by coupling each partner with a fluorescent label and detecting the level of energy transfer (Fig. 1). In the past, organic fluorescent compounds such as fluorescein and rhodamine have been widely used in the regular fluorescence assay. However, these bioassays have great disadvantages in that fluorescent detection because it is dramatically inhibited by noise in the back ground derived from scattered excitation light and significantly interfered by fluorescence from coexisting material in the sample (fluorescent compounds and dust/line), making it difficult to obtain a highly sensitive measurement. Through time-resolved measurement of fluorescence, HTRF allows the elimination of short-lived background fluorescence. Introducing a time delay (50-150 microseconds) between the initial light excitation and fluorescence measurement minimizes the contribution of all non-specific short-lived fluorescence emissions. In contrast, HTRF acceptor fluorophores emit long-lived fluorescence when engaged in the TR-FRET process with a long-lived donor fluorophore, signifying energy transfer through proximity of the labeled biomolecules (Fig. 2).


HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications.

Degorce F, Card A, Soh S, Trinquet E, Knapik GP, Xie B - Curr Chem Genomics (2009)

The energy pulse from the excitation source (flash lamp, laser) is followed by a time delay, allowing interfering short-lived fluorescence (compounds, proteins, medium etc.) to decay. The red line: FRET signal intensity generated at 665 nm; black line, emission of donor cryptate at 620 nm; orange line, fluorescent signal generated from acceptor fluorophores.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2802762&req=5

Figure 2: The energy pulse from the excitation source (flash lamp, laser) is followed by a time delay, allowing interfering short-lived fluorescence (compounds, proteins, medium etc.) to decay. The red line: FRET signal intensity generated at 665 nm; black line, emission of donor cryptate at 620 nm; orange line, fluorescent signal generated from acceptor fluorophores.
Mentions: The principle behind FRET is based on the transfer of energy between two fluorophores, a donor (long-lived fluorescence) and an acceptor (short-lived fluorescence), when in close proximity [3, 4]. Molecular interactions between biomolecules can be assessed by coupling each partner with a fluorescent label and detecting the level of energy transfer (Fig. 1). In the past, organic fluorescent compounds such as fluorescein and rhodamine have been widely used in the regular fluorescence assay. However, these bioassays have great disadvantages in that fluorescent detection because it is dramatically inhibited by noise in the back ground derived from scattered excitation light and significantly interfered by fluorescence from coexisting material in the sample (fluorescent compounds and dust/line), making it difficult to obtain a highly sensitive measurement. Through time-resolved measurement of fluorescence, HTRF allows the elimination of short-lived background fluorescence. Introducing a time delay (50-150 microseconds) between the initial light excitation and fluorescence measurement minimizes the contribution of all non-specific short-lived fluorescence emissions. In contrast, HTRF acceptor fluorophores emit long-lived fluorescence when engaged in the TR-FRET process with a long-lived donor fluorophore, signifying energy transfer through proximity of the labeled biomolecules (Fig. 2).

Bottom Line: Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation.Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient.This review addresses the general principles of HTRF and its current applications in drug discovery.

View Article: PubMed Central - PubMed

Affiliation: Cisbio Bioassays, 30204 Bagnols-Sur-Cèze, France. fdegorce@cisbio.com

ABSTRACT
HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS). This technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation. The HTRF assay is usually sensitive and robust that can be miniaturized into the 384 and 1536-well plate formats. This assay technology has been applied to many antibody-based assays including GPCR signaling (cAMP and IP-One), kinases, cytokines and biomarkers, bioprocess (antibody and protein production), as well as the assays for protein-protein, proteinpeptide, and protein-DNA/RNA interactions.Since its introduction to the drug-screening world over ten years ago, researchers have used HTRF to expedite the study of GPCRs, kinases, new biomarkers, protein-protein interactions, and other targets of interest. HTRF has also been utilized as an alternative method for bioprocess monitoring. The first-generation HTRF technology, which uses Europium cryptate as a fluorescence donor to monitor reactions between biomolecules, was extended in 2008 through the introduction of a second-generation donor, Terbium cryptate (Tb), enhancing screening performance. Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient. In addition to being compatible with the same acceptor fluorophors used with Europium, it can serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm. Moreover, all Terbium HTRF assays can be read on the same HTRF-compatible instruments as Europium HTRF assays.Overall, HTRF is a highly sensitive, robust technology for the detection of molecular interactions in vitro and is widely used for primary and secondary screening phases of drug development. This review addresses the general principles of HTRF and its current applications in drug discovery.

No MeSH data available.


Related in: MedlinePlus