HTRF assay

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. 

Fig. 2 A schematic representation of an HTRF assay for a protein-protein interaction. One protein is tagged with a fluorescent molecule whose emission spectra overlaps with the excitation of another fluorescent molecule. When they are in close proximity (above) the energy is transferred. When they diffuse apart (below) or are inhibited from coming together by a small molecule no FRET occurs...

A homogeneous and sensitive HTRF binding assay was developed to allow prosecution of an HTS campaign for novel small molecule Hsp90 inhibitors. The HTRF assay was based on a non-radio-active resonance energy transfer between a donor label (europium chelate) and an acceptor label (allophycocyanin [APC]) brought into close proximity by a specific binding interaction. 

Testing compounds of interest from the HTRF assay in the TRF binding assay helped eliminate compounds that exhibit false positive signals in the HTRF assay. All compounds that were active in the TRF assay also worked in the functional Hsp90 ATPase and cell-based target modulation assays. 

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. ... 

Figure 2.4. The schematic shows the basis of a tyrosine kinase hTRF assay. The reaction (a) uses a biotinylated peptide substrate that becomes phosphorylated on tyrosine in the presence of kinase, ATP, and magnesium ions. The reaction product is then detected (b).

The strategies for labeling biomolecules with EuK and XL665 and HTRF assay conditions have been reported [4,5,6]. Samples were counted on the Discovery HTRF Analyzer (Packard) in black, low fluorescent HTRF-96 or HTRF-384 microplates. 

The prolactin immunoassay results shown in Table 2 demonstrate the ability to miniaturize HTRF assays. The recommended volume of 300 pL was used in the 96-well plate. This was reduced to 75 pL for the 384-well assay. The result of most importance is the ratio. For every prolactin standard, the ratio in the 384-well is the same as in the 96-well. It should also be noted that although the volume in the 384-well was only 25% of the 96-well, the fluorescent counts were 50%. This results from the difference in the ratio of the liquid column height to volume of the two plates. This presents the possibility that an optimized microplate geometry will allow a degree of miniaturization with HTRF that is not possible with other labels. 

Compound 16 had excellent selectivity against the nicotinic acid receptor with no activity observed at a concentration up to 30 pM. Most surprisingly, compound 16 did not show any agonistic activity in our in-house hu and rat GPR81 cAMP HTRF assay. Furthermore, the compound in our hands was not active in primary human or rat adipocytes either. 

There are cAMP assays that can be measured using a variety of techniques [35], including HTRF (CisBio) [36], bioluminescence (cAMP-Glo, Promega) [37], TR-FRET (CisBio) [38], LANCE [39], Alphascreen (Perkin Elmer) [40] and Enzyme Fragment Complementation (EFC) [41] (Hithunter, DiscoverX). This review will focus on the LANCE, the TR-FRET and ECF assays as they are the most used in our laboratories. 

Parameters Radiometric proximity assays (SPA, Flashplate) Fluorescence polarization (FP) Time- resolved fluorescence (HTRF) Amplified luminescence (ALPHAScreen) Enzyme (p-galactosidase) complementation Electrochemilumines cence... 

Fig. 3. Comparison between IP-One kit versus calcium mobilization assay (384-well format). Human embryonic kidney (HEK) 293 cells expressing a chemokine receptor were evaluated on HTRF IP-One kit (CisBio, Bedford, MA) and fluroescent imaging plate reader (FLIPR) with Calcium 3 kit (Molecular Devices, Mountain View, CA).

Preaudat, M. et al. 2002 A homogeneous caspase-3 activity assay using HTRF technology. J. Biomol. Screen. 7, 267-274. 

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

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

For any HTRF binding assay, it is desirable to achieve saturating concentrations with detection reagents. Under those conditions, each binding event would produce a transfer of energy and... 

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... 

For example, in an FRET assay for the research of a receptor ligand inhibitor, the presence of a library componnd which flnoresces at the acceptor wavelength will lead to a false negative resnlt (compensation of the decrease of the FRET signal by the fluorescent componnd) on the contrary, the same compound would give a false positive result in a cytokine assay nsing a sandwich format with two monoclonals (compound fluorescence will be interpreted as an FRET increase). In both cases, the use of HTRF would have corrected for the fluorescence of the compound in the acceptor channel. 

---