HTRF® Signal Stability

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A number of homogeneous technologies (e.g. luminescence) are restricted by a reduction in signal intensity following assay measurement or prolonged incubations. Our patented HTRF® technology has many benefits including long signal stability. 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:

  • Time flexibility and assay security: In case of instrument failure, it is possible to read the microplates even after an extended period of time. The cAMP IC50 remained stable over a period of 7 days providing a large window for assay measurement (see figure 1).
  • Kinetic studies: Assays can be measured as frequently as needed (figure 2), opening 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 provide for increased assay flexibility.
  • No data loss due to reader breakdown.


cAMP 7 day signal stability

Figure 1: cAMP standard curve was run using standard assay conditions with a final assay volume of 20 µl in a low-volume 384-well plate. The signal at 620 and 665nm was detected at the times indicated following the addition of the HTRF® detection reagnets.

Kinase assay: 20 successive readouts

Figure 2: 20 successive readouts of a Kinase assay were performed without a decrease in the HTRF® ratio.



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

  • Cryptate structure, in which the lanthanide ion is tightly embedded in its macrocycle, resists harsh assay conditions or additives such as the presence of large quantities of challenging cations (Mg2+, Mn2+ ...), chelators (EDTA), solvents, or temperature. HTRF® can also be used with high serum concentrations (up to 50%), as exemplified by its application to clinical diagnostics (TRACE® technology with Kryptor® workstation)
  • Addition of fluoride ions at the time of readout or during the course of the incubation enhances assay resistance to the great majority of compound interference (quenchers), but is not mandatory for Lumi4™-Tb.
  • 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, a small organic acceptor, strengthens the signal stability for many HTRF® assays.

*Photobleaching - a phenomenon affecting many fluorophores - is the disappearance of fluorescence emission after prolonged or repeated excitation.