Great improvements in screening technologies have accelerated this process. One important assumption of the explosion in assay throughput is the development of fast and homogeneous screening assays, which can be miniaturized to save reagent costs and to minimize the quantities of limited target and compound material used in the assay. The second factor, which has made fluorescence readouts more widely used in HTS, is the development of new micro-plate readers to reach the needed sensitivity.
HTRF® - the assay principle
Time-resolved energy transfer is one of the most powerful homogeneous fluorescence readout in HTS. This dual labeling approach combines the advantages of both the time-resolved fluorescence (TRF) technique and the fluorescence resonance energy transfer (FRET) technique. FRET refers to a nonradiative energy transfer of excitation energy between a donor fluorophore and a suitable acceptor fluorophore. The efficiency of energy transfer is a function of the distance (1/d6) between donor and acceptor.1
For commonly known donor-acceptor pairs, the distance R0 (for which transfer is 50% efficient) lies within the 1-7 nm range, which allows using FRET to detect molecular proximities.
This technique has been further enhanced by using long-lived labels combined with the detection on a time-resolved fluorescence basis, allowing the minimization of prompt fluorescence interferences. The development of HTRF® by Cisbio international was made possible by extensive research into compatible donor acceptor pairs and resulted in the selection of the exceptionally well-adapted Eu3+ cryptate-XL665 combination (figure 1).2
Fig. 1: HTRF principle with Eu3+ cryptate and XL665 respectively as donor and acceptor. If the labeled macromolecules bind each other the two fluorophores will be in close proximity, allowing FRET to occur resulting in a specific long-lived fluorescence emission of XL665 at 665 nm.
HTRF® has a number of advantages. It is a true homogeneous solution assay, which can easily be miniaturized. In addition, by measuring the specific signals of both the donor and acceptor fluorophore as an internal control, the assay gives a ratio measurement that compensates for the presence of colorized compounds in the assay.
Finally, the longer distance for energy transfer (~ 9 nm), provided by Eu3+-based time-resolved energy transfer systems, means that protein-protein interactions using indirect labelling can be used successfully. Various reagents pre-labeled with HTRF® donors and acceptors, including epitope tag antibodies, streptavidin, biotin and several anti-species antibodies, are available and can be adapted to many assays.
Fluorescence readout for HTRF® relies on two essential principles. First, the instrument used should enable a measurement under time-resolved mode (i.e. by gating the detector for a short period of time and measuring only the long lasting fluorescence after the gating period). Secondly, both emissions at 620 and 665 nm have to be measured simultaneously in order to speed up the assay and maximize correction efficacy by rationing the two signals.
Fig 2: BMG LABTECH's dedicated laser-based HTRF® reader RUBYstar.
BMG LABTECH has developed two microplate readers which are designed to perform homogeneous time-resolved fluorescence measurements based on Cisbio international's proprietary HTRF® technology: the high performance laser-based plate reader RUBYstar (figure 2), which is dedicated for reading HTRF® in all plate formats up to 384-well plates, and the new multifunctional detection platform PHERAstar (figure 3), which is able to perform all four leading non-isotopic detection technologies including time-resolved fluorescence with uncompromised sensitivity even in 1536-well plates.
Fig. 3: The new multifunctional detection platform PHERAstar is designed to perform HTS assays including HTRF® - based assays in plate formats up to 1536-wells.
The two plate readers enable highly sensitive time-resolved fluorescence detection by applying europium cryptate or chelate fluorophores. These compounds are excited at 337 nm and show long fluorescence lifetimes (several hundred μs), large Stoke´s shifts and sharp and intensive fluorescence peak profiles at 620 nm.
When engaged in the FRET process, the long-lived donor induces a long-lived emission from the acceptor at 665 nm. At both wavelengths, fluorescence is simultaneously measured with a lens based dual channel PMT system after a adjustable delay of 10-100 μs to reject the medium background and the free acceptor fluorescence from the signal in order to give maximum sensitivity.
Comparison of the two HTRF® readers PHERAstar and RUBYstar in various plate formats up to 1536-wells
The time-resolved fluorescence mode was explored using the HTRF® TNFα assay (#62TNFPEB, Cisbio international, France).3 Results were obtained in black 96-well half area plates (#3694, Costar, USA), 384-well small volume plates (#784076, Greiner Bio-One, Germany) and 1536-well plates (#782076, Greiner Bio-One, Germany) according to the kit protocol.
The incubation took place overnight at room temperature and the final assay volumes were 100 μL in the 96-well plate, 20 μL in the 384-well plate and 7 μL in the 1536-well plate. The comparison between PHERAstar and BMG LABTECH's dedicated time-resolved fluorescence reader, RUBYstar, was performed in 96-well and 384-well formats. On the flashlamp-based PHERAstar the assays were run with 200 or 400 flashes per well and on the laser-based RUBYstar the assays were performed with 20 flashes. The FRET donor europium cryptate was excited at 337 nm and emission was simultaneously read at 620 nm and 665 nm on both readers.
HTRF® TNFα immunoassay
Cisbio international's HTRF® assays employ fluorescent Eu3+ cryptates (donor) and XL665 (acceptor) in homogeneous time-resolved FRET-based assays. Upon excitation, when the two entities come into close proximity, FRET can occur and XL665 re-emits a specific long-lived fluorescence at 665 nm, in addition to the donor emission at 620 nm.
Tumor Necrosis Factor alpha (TNFα), a 17 kDa cytokine, is an important mediator secreted by activated macrophages and monocytes with a large spectrum of antiviral immunoregulation, metabolic and inflammatory properties. This factor is cytotoxic for some tumor cell lines in vitro and causes the necrosis of certain tumors in vivo. TNFα acts via binding to specific cell surface receptors.
The HTRF® TNFα assay is a single step double-site immunometric assay involving two MABs conjugated either with europium cryptate or to XL665. The HTRF® TNFα assay principle is shown in figure 4.
Under its final configuration, free TNFα from calibrators or samples is sandwiched by two mouse MAbs conjugated for the first one to Eu Cryptate or to XL665 for the second one. The FRET signal generated by the simultaneous binding of the two conjugates is proportional to the amount of TNFα present in the sample. Both 665 nm and 620 nm signals were measured simultaneously on the PHERAstar. Under routine use, the 665 nm / 620 nm fluorescence ratio (US patent 5,527,684) eliminates most interference from the medium.4
Fig. 4: Assay principle of the homogeneous time-resolved fluorescence immunoassay for TNFα. The assay is run using straightforward "mix & measure" detection.
For a direct comparison of the PHERAstar with the RUBYstar, the samples were measured on both readers and the results are shown in figure 5.
Fig. 5: Direct comparison of the PHERAstar and the RUBYstar in different plate formats using the HTRF® reader comparison kit (high calibrator, n = 8) based on a TNFα immunoassay.
Delta F is a value calculated from the 665 nm / 620 nm ratios which enables the data to be normalized with respect to between-assay variations. In addition, delta F is reader independent and can be used for indicating and comparing the quality of a reader.4 A total of five PHERAstars and two RUBYstars were compared to obtain these results. When comparing the PHERAstar to the RUBYstar, the PHERAstar shows approximately 20% less signal than the RUBYstar in terms of delta F in 96- and 384-well plates. However, the %CVs and signal separation obtained with the PHERAstar still results in an excellent delta F value and good HTRF® performance.
The results from the multimode PHERAstar reader proved that HTRF® assay miniaturization (100 μL to 7 μL) in plate formats up to 1536-wells has no significant influence on the excellent reader sensitivity, dynamic range, or %CVs (2%). In addition, we have shown that the already acknowledged "HTRF® compatible" reader PHERAstar can produce high quality data for HTRF® technology assays and is comparable to the dedicated RUBYstar ("the gold standard" reader for HTRF® assays) in terms of sensitivity. Due to the fact that the RUBYstar is designed for plate formats up to 384-wells, a direct comparison in 1536-well format was not possible. However, the data shows that the PHERAstar is capable of producing excellent results for HTRF®, even in 1536-well mode, with no reduction in assay sensitivity.
The discovery of new leads though HTS is based on the ability to measure precisely biomolecular interactions and to find successful strategies in fluorescence detection that are compatible with miniaturized HTS. The criteria used in making a decision which assay and which instrumentation should be chosen are cost, sensitivity, speed, ease, and reliability.
A large number of HTS assays have now been configured using homogeneous time-resolved fluorescence. The homogeneous nature of HTRF® assays allows these assays to be much simpler, more robust, and easier to automate. Furthermore the use of HTRF® assays combined with the "HTRF® compatible" PHERAstar and RUBYstar allows straightforward assay miniaturization to plate formats up to 1536 with no influence on the excellent sensitivity of the reader. This reduces the consumption of expensive reagents and provides significant cost savings.
In addition, the specificity of HTRF® resides also in the way the signal is analyzed. When the acceptor signal at 665 nm is "ratioed" by the cryptate signal (US patent 5527684), the data obtained is then independent of the optical characteristics of the media at the excitation wavelength providing unsurpassed background reduction.
HTRF® is a registered trademark of Cisbio international.
1) Förster F: (1948) Ann. Phys. 2, 55-75
2) Mathis G: (1993) Clin. Chem. 39, 1953-9
3) Degorce, F. et al., HTRF® TNFα Kit Application Note 3, Cisbio international, France.
4) Liu, J. et al., (2004) J. Biol. Chem. 279, 15824-30.