HTRF Products

• GPCR Solutions
• Kinase & Oncology
• Inflamation Metabolic Diseases, CNS
• Bioprocess
• Labeling
• Anti-tag
• Streptavidins
• Toolbox Reagents

Services

• Custom Labeling
• Assay Development
• Training

HTRF® technology uses either Eu3+ or Tb2+ cryptate as the donor fluorophore, and either XL665 or d2 as the acceptor fluorophore. For these fluorophores, we recommend measurement of the fluorescence emission at 620 nm for the donor and at 665 nm for the acceptor.
For more information on HTRF chemistry »

• Ratio theory
• Calculating HTRF ratio
• Delta F

HTRF® emissions are measured at two different wavelengths, 620nm and 665nm. This feature of HTRF® is extremely advantageous, particularly for reducing well-to-well variations that may arise in homogeneous assay formats. Homogeneous formats do not require washing or separation steps so all the assay components are present at the time of assay readout. Because wells contain a range compounds and/or medium additives, each well will have different photophysical properties and cause varying degrees of signal interference and therefore altered signal intensities. This well-to-well signal variation is not due to true differences in light transition. This can lead to misleading results if only a single emission wavelength is measured.

Cisbio Bioassays has developed a unique ratiometric measurement of two emission wavelengths (patent US 5,527,684 and foreign equivalents) that corrects for well-to-well variability and signal quenching from assay components and medium variability. Emissions at 620nm (donor fluor) are used as an internal reference while emissions at 665nm (acceptor fluor) are used as an indicator of the biological reaction being assessed. Because both the 620nm and 665nm emissions are decreased by sample interferences, the ratio remains unchanged (Figure 1). Figure 3 summarizes the results of an HTRF® assay run in the presence of increasing concentrations of Commassie blue and depicts the use of the HTRF® ratio to correct for quenching effects. These data clearly show the ability of the ratio to correct for signal interference.

Figure 1: While the absolute signal intensity decreases due to lower transmission, the ratio of the signal intensities at 620nm and 665nm remains unaffected.

Figure 2: An HTRF assay was carried out in the presence of increasing concentrations of commassie blue and emissions at 620 and 665nm detected. The absolute signal decreases as the concentration of commassie blue increases, however, the ratio remains unchanged.

The HTRF® ratio is calculated using the equation:



The 10⁴ multiplying factor is used solely for the purpose of easier data handling. The ratio for each well within an assay is calculated and the mean and standard deviation of replicates determined using these values.

In the example below, negative and positive controls were run in addition to a positive control in which a colored compound was introduced to produce signal interference. The positive and negative controls show a clear difference in the absolute signal intensity at 665nm, as expected, as well as a corresponding change to the HTRF® ratio. In samples that had color compounds added, a decrease in both 620nm and 665nm signals is observed, while the HTRF® ratio remains relatively unchanged. If the ratio had not been calculated, the sample would be falsely identified as an inhibitor of the biological reaction being tested in the assay. The calculation of the ratio for each well is critical for decreasing false positives as well as improving assay and well-to-well reproducibility.

Delta F Calculation: