Homogeneous time-resolved fluorescence

HTRF is a homogeneous technology which combines an FRET process with time-resolved fluorescence detection to probe biomolecular interactions. This combination is made possible through the use of a long lifetime fluorescent FRET donor, a lanthanide cryptate. Cryptates are formed by the inclusion of a rare earth ion e.g. 

Europium cryptates are excited in the UV wavelength range either by a xenon flash lamp or by a nitrogen laser. Their fluorescence occurs in a wavelength range between 550 and 710 nm with typical narrow emission lines. Since the electronic transitions of the europium ion are forbidden by quantum mechanical rules, the cryptate fluorescence lifetime is exceptionally long, in the range of 100-1000 J,s. 

Europium cryptates can be associated with various acceptors, originally with a cross-linked allophycocyanin called XL665 or with small fluorescent near infrared dyes that have been selected so that their photophysical properties fit those of the europium cryptate for an efficient FRET process  

The high FRET efficiency obtained in HTRF assays enables a large variety of biological events to be probed. Protein/protein interactions, enzymatic activities (e.g. kinases or proteases), or a large number of biomarkers have been detected with a very low detection limit. Like the other homogeneous fluorescent technologies like FP or FCS, HTRF can be easily automated and miniaturized down to a 1536 weUs plate format for the HTS of large libraries of chemical compounds.  

Moreover, the HTRF signal readout minimizes the instances of compound interference in HTS the ratiometric signal measurement overcomes the influence of highly coloured compounds while the time-resolved fluorescence detection discards 

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Homogeneous Time Resolved Fluorescence (HTRF) (Cisbio International) is an assay based on the proximity of a lanthanide cryptate donor and a fluorescent acceptor molecule whose excitation wavelength overlaps that of the cryptate s emission. The utility of this technique is based on the time resolved fluorescence properties of lanthanides. Lanthanides are unique in the increased lifetime of their fluorescence decay relative to other atoms, so a delay in collection of the emission intensity removes the background from other fluorescent molecules. An example of the HTRF assay is a generic protein-protein interaction assay shown in Fig. 2. 

Cencic, R., Yan, Y., and Pelletier, J. (2007). Homogenous time resolved fluorescence assay to identify modulators of cap-dependent translation initiation. Comb. Chem. High Throughput Screen 10, 181-188. 

Maurel, D., Kniazeff, J., Mathis, G., Trinquet, E., Pin, J. P. and Ansanay, FI. (2004). Cell surface detection of membrane protein interaction with homogeneous time-resolved fluorescence resonance energy transfer technology. Anal. Biochem. 329, 253-62. 

Liang, A.M., Claret, E., Ouled-Diaf, J., Jean, A., Vogel, D., Light, D.R., Jones, S.W., Guilford, W.J., Parkinson, J.F., and Snider, R.M. (2007) Development of a homogeneous time-resolved fluorescence leukotriene B4 assay for determining the activity of Leukotriene A4 hydrolase. J. Biomol. Screen. 12, 536-545. 

HTRF (homogeneous time-resolved fluorescence) LANCE (lanthanide chelate excitation) Cisbio International PerkinElmer Life Sciences Lanthanide TR-FRET using Eu3+ cryptate/chelate-donor fluorophore and cross-linked aUophycocyanin-acceptor fluorophore... 

Kolb, A.J., Burke, J.W., and Mathis, G., Homogeneous, time-resolved fluorescence method for drug discovery, in High Throughput Screening, The Discovery of Bioactive Substances, Devlin, J.P., Ed., Marcel Dekker, New York, 1997, chap. 19. 

Ferrer, M. et al. 2003 Miniaturizable homogeneous time-resolved fluorescence assay for carboxypeptidase B activity. Anal. Biochem. 317. 94-98. 

Karvinen, J. et al. 2002. Homogeneous time-resolved fluorescence quenching assay (LANCE) for caspase 3. J. Biomol. Screen. 1, 223-231. 

Primary Homogeneous Time-Resolved Fluorescence (HTRF) HTS Assay.84... 

PRIMARY HOMOGENEOUS TIME-RESOLVED FLUORESCENCE (HTRF) HTS ASSAY... 

Inherent fluorescence in natural product samples rules out the use of frank fluorescence as an assay readout. In the early 1990s, the development of homogeneous time-resolved fluorescence (HTRF) allowed the use of fluorescence with such samples (Kolb, Yamanaka, and Manly, 1996 Kolb and Manly, 1997 Bazin, Trinquet, and Mathis, 2002 Mathis, 1999). This technique... 

Because of the very stringent reliability requirements for diagnostic applications, the only homogeneous techniques that have survived in this field are the fluorescence polarization (FP) methods originally developed by AbbotE and the homogeneous time resolved fluorescence (HTRF) and TRACE approaches developed later by CIS bio international. 

The homogeneous TRF assay formats HTRF ( homogeneous time-resolved fluorescence ) by GisBio International [115, 120, 136, 137] and LANGE ( lantha-... 

HTRF/LANCE - homogeneous time-resolved fluorescence/lanthanide cryptate excitation SPA - scintillation proximity assay - homogeneous assay which detects radioisotopes in close proximity to a solid scintillant ... 

A study at Novartis compared hits obtained by screening 30,000 compounds for tyrosine kinase activity in three different assays a scintillation proximity assay (SPA), a homogeneous time-resolved fluorescence energy transfer assay (HT-FRET), and a fluorescence polarization (FP) assay.Library compounds were screened as mixtures of five in a well, which meant that after active wells were identified, the five constituents would be deconvoluted by screening each compound individually or using other methods to establish which of the compounds was active. " When this was done SPA, HT-FRET, and FP turned up 30, 59, and 64 hits respectively. A low degree of overlap was observed between the sets. Only four or seven compounds, depending on where the activity bars were set, would have been identified... 

An example of a recent development in assay technology that combines speed, sensitivity and safety is homogeneous time resolved fluorescence (HTRF , Packard). HTRF has the inherent sensitivity associated with fluorescence, but is designed to eliminate or compensate for problems such as high background, color interference and fluorescence quenching often found in biological assays. 

The name Homogeneous Time-Resolved Fluorescence can be used as a starting point to explain how this method works. 

Kolb, A., Burke, J., Mathis, G. (1997). Homogeneous, Time-Resolved Fluorescent Method for Drug Discovery. In High Throughput Screening The Search for Bioactive Molecules. J. Devlin (ed) pp 345-360. Marcel Dekker, New York. 

Bazin, H. Preaudat. E. Trinquet, E. Mathis. G. Homogeneous time resolved fluorescence resonance energy transfer using rare earth cryptates as a tool for probing molecular interactions in biology. Spectrochim. Acta. Part A 2001. 57. 2197-2211. 

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