RESEARCH ARTICLE


HTRF: A Technology Tailored for Drug Discovery –A Review of Theoretical Aspects and Recent Applications



François Degorce*, 1, Amy Card2, Sharon Soh2, Eric Trinquet1, Glenn P Knapik2, Bing Xie*, 2
1 Cisbio Bioassays, Bagnols-Sur-Cèze, France
2 Cisbio US, 135 South Road, Bedford, MA 01730, USA


Article Metrics

CrossRef Citations:
0
Total Statistics:

Full-Text HTML Views: 1733
Abstract HTML Views: 1053
PDF Downloads: 233
Total Views/Downloads: 3019
Unique Statistics:

Full-Text HTML Views: 587
Abstract HTML Views: 610
PDF Downloads: 170
Total Views/Downloads: 1367



© Degorce et al.; Licensee Bentham Open.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.5/), which permits unrestrictive use, distribution, and reproduction in any medium, provided the original work is properly cited.

* Address correspondence to these authors at the François Degorce, Cisbio Bioassays, BP 84175, 30204 Bagnols-sur-Cèze Cedex, France; Tel: +33-4-6679-1931; Fax: +33-4- 6679-1920; E-mail: fdegorce@cisbio.com Bing Xie, Cisbio US, 135 South Road, Bedford, MA 01730, USA; Tel: 781-687-1410; Fax: 781-687-1510; E-mail: bxie@cisbio.us


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.

Keywords: HTRF, TR-FRET, GPCR, Kinase, Biomarker, Bioprocess.