HTRF involves several carefully selected fluorophores. Obviously, FRET partners must fulfill multiple compatibility criteria. First, their emission spectra must show non-overlapping regions in order to be able to measure each partner's fluorescence individually. Second, the FRET quantum yield (i.e. its efficacy) must be as high as possible. Third, fluorescence emission must occur within a region of the spectrum remote from that naturally produced by proteins; in other words, a red-shifted emission is theoretically better in order to avoid medium-intrinsic fluorescence. HTRF uses a combination of several fluorophores forming different TR-FRET systems. HTRF's central element, the energy donor, consists of a rare earth complex in which the lanthanide ion (Europium or Terbium) is tightly embedded in a macrocycle. This very unique type of structure gives HTRF donors their long-lived fluorescence properties, as well a robustness that enables these molecules to be used in most assay conditions.
Two donors are currently included in HTRF products:
Figure 1: Europium cryptate structure typically consists of a tris bipyridine macrocycle in which the lanthanide ion is tightly embedded.
These macrocyclic structures allow both the collection and transfer of energy to the Eu3+ or the Tb2+ ions, which ultimately release this energy in a specific fluorescent pattern (Figure 2). Both cryptates show long-lived emission in the range of 1 to 2 msec, a characteristic that is fundamental for a time-resolved detection.
HTRF acceptors have been optimized for Eu3+ and Tb2+ cryptate donors, and in particular to match their emission properties. Eu3+ cryptates are mainly compatible with near infrared acceptors showing a peak emission at 665 nm, whereas Lumi4-Tb can be paired to the same red acceptors as well as green ones like fluorescein or GFP, emitting around 520 nm (Figure 2).
The first acceptor developed for HTRF was XL665, a phycobilliprotein pigment purified from red algae. XL665 is a large hetero hexameric edifice of 105 kDa, cross-linked after isolation for better stability and preservation of its photophysical properties in HTRF assays. The second generation of acceptors is characterized by organic structures 100 times smaller, displaying a series of photophysical properties very close to those of XL665. These acceptors fulfill the previously-mentioned compatibility criteria. Their excitation spectra overlap those of both Eu3+ and Lumi4-Tb cryptate emissions, thereby allowing the donor to excite the acceptor, whose maximum emission at 665 nm spans a region where HTRF cryptates do not emit or do so only weakly. In the end, energy transfer with the donor occurs with a high quantum yield.
The comparison of d2 to XL665 was achieved by screening 14,700 compounds on an assay for quantifying a phosphorylated peptide. As shown in Figure 3, the correlation between the two systems was extremely close, and validated the integration of d2 in a number of different HTRF assays, notably cAMP and IP-One. As it is a much smaller entity, d2 limits the steric hindrance problems sometimes suspected in XL665-based systems. Evaluation also showed that the new acceptor contributed to significantly greater stability of immuno-competitive assays, and in some cases to better assay sensitivity.
In addition to XL665 and d2, green acceptors are fully compatible with the spectral characteristics of Lumi4-Tb cryptate. These properties have led to the development of Tag-lite® ligand binding assays in which the second partner, either a ligand or a second receptor respectively, is labeled with a green synthetic acceptor. The use of encoded fluorophores such as GFP was also demonstrated, opening the possibility for cell-based assays with GFP fusion proteins as one of the assay partners.
Figure 2: HTRF donor and acceptor emission spectra. Red acceptor emissions occur in a region where the donor does not emit significantly. Long-lived fluorescence detected at this specific wavelength is therefore characteristic of the emission of the acceptor engaged in the FRET process. The same is true for green acceptors emitting around 520 nm when combined with a Lumi4-Tb donor.
Figure 3: XL665 and d2 were compared in an HTRF assay involving an anti-phosphotyrosine antibody conjugated to Eu3+ cryptate (PT66), a phosphorylated biotinylated substrate, and streptavidin alternatively labeled with XL665 and d2. Each system was tested with a library of 14,700 compounds (Schering AG). Comparison shows that the two acceptors behave very similarly and that d2 represents a possible alternative to XL665.