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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

Kinase assay principle. The substrates used in HTRF kinase assays are tagged by fusion proteins, peptides, or biotin. An antibody to these proteins or peptides, or streptavidin (for biotin in this case) is labeled with acceptor. An antibody against phosphorylated substrate is labeled by donor cryptate. In the enzymatic reaction, once substrate is phosphorylated, donor and acceptor can be brought into proximity by affinity interactions of antibodies/substrate or streptavidin/biotin.
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Figure 7: Kinase assay principle. The substrates used in HTRF kinase assays are tagged by fusion proteins, peptides, or biotin. An antibody to these proteins or peptides, or streptavidin (for biotin in this case) is labeled with acceptor. An antibody against phosphorylated substrate is labeled by donor cryptate. In the enzymatic reaction, once substrate is phosphorylated, donor and acceptor can be brought into proximity by affinity interactions of antibodies/substrate or streptavidin/biotin.

Mentions: Protein kinases are key elements in intracellular signaling pathways that control many physiological processes and the activities of protein kinases are altered in a number of human diseases such as cancer and autoimmune disorders [31-33]. The rapidly growing interest in kinases as potential targets for therapeutic intervention has prompted the development of many kinase assay technologies. HTRF-based tools for HTS could apply to all kinases, with specific applications for Tyrosine (Tyr) and Serine/Threonine (Ser/Thr) kinases. This kinase screening platform offers the ability to use a single technology for both universal and specific kinase assays, saving valuable development time and cost (Fig. 7). A comprehensive review summarized the recent literatures and meeting reports on HTRF kinase assay [34].


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)

Kinase assay principle. The substrates used in HTRF kinase assays are tagged by fusion proteins, peptides, or biotin. An antibody to these proteins or peptides, or streptavidin (for biotin in this case) is labeled with acceptor. An antibody against phosphorylated substrate is labeled by donor cryptate. In the enzymatic reaction, once substrate is phosphorylated, donor and acceptor can be brought into proximity by affinity interactions of antibodies/substrate or streptavidin/biotin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Kinase assay principle. The substrates used in HTRF kinase assays are tagged by fusion proteins, peptides, or biotin. An antibody to these proteins or peptides, or streptavidin (for biotin in this case) is labeled with acceptor. An antibody against phosphorylated substrate is labeled by donor cryptate. In the enzymatic reaction, once substrate is phosphorylated, donor and acceptor can be brought into proximity by affinity interactions of antibodies/substrate or streptavidin/biotin.
Mentions: Protein kinases are key elements in intracellular signaling pathways that control many physiological processes and the activities of protein kinases are altered in a number of human diseases such as cancer and autoimmune disorders [31-33]. The rapidly growing interest in kinases as potential targets for therapeutic intervention has prompted the development of many kinase assay technologies. HTRF-based tools for HTS could apply to all kinases, with specific applications for Tyrosine (Tyr) and Serine/Threonine (Ser/Thr) kinases. This kinase screening platform offers the ability to use a single technology for both universal and specific kinase assays, saving valuable development time and cost (Fig. 7). A comprehensive review summarized the recent literatures and meeting reports on HTRF kinase assay [34].

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