HTRF® signal stability

HTRF® signal stability benefits

Time flexibility and assay security! In case of instrument failure, it is possible to read the microplates even after an extended period of time Kinetic studies. Assays can be measured as frequently as desired, opening up the opportunity for kinetic measurement of interactions for many assay configurations - Flexible assay formats. Many chemical and biological additives, e.g. culture media with FCS, BSA, DMSO, detergents, enzyme quenchers (EDTA), do not interfere with assay measurement and therefore allow increased assay flexibility.

Figure 1: Seven-day signal stability of cAMP assay

A number of homogeneous technologies (e.g. luminescence) are restricted by a reduction in signal intensity following assay measurement or prolonged incubations. Long signal stability is among the many benefits of our patented HTRF technology. HTRF fluorescence is not dampened by assay measurement, assay additives such as DMSO, or extended incubations prior to reading. The advantages of the tremendous stability of HTRF fluorescence include read time flexibility during HTS measurements, the ability to perform kinetic studies, and increased sample compatibility. The cAMP IC50s, as shown on the left, remained stable over a period of 7 days.

Figure 2: Twenty successive readouts of kinase assay

HTRF technology and signal robustness are due to both the photophysical and the chemical stability of the fluorophores involved. These properties are detailed below:

Cryptate structures, in which either the europium or the terbium ion is tightly embedded in its macrocycle, resist harsh assay conditions or additives: presence of large quantities of challenging cations (Mg2+ , Mn2+ …), chelators (EDTA), solvents, pH or temperature variations. HTRF can also be used with high serum concentrations (up to 50%), as exemplified by its application for clinical diagnostics (TRACE® technology with Kryptor® workstation).
For Europium cryptate based assays, addition of fluoride ions at the time of readout or during the incubation enhances assay resistance to the great majority of compound interferences (e.g. quenchers). Fluoride ion supplementation is not mandatory for Lumi4®-Tb based assays.
Cryptates do not photobleach*. There is no loss of signal after multiple readings.
HTRF acceptors are compatible with a broad range of assay conditions. The introduction of d2 and green dye, small organic acceptors, has strengthened the long signal stability of HTRF assays even further.

* Photobleaching – a phenomenon affecting many fluorophores - is the disappearance of fluorescence emission after prolonged or repeated excitation. The fluorophore reaches permanent excitation state without any deactivation (i.e. fluorescence emission).