Ratiometric data reduction: a straightforward way to eliminate compound interference
HTRF technology uses either Eu3+ or Tb2+cryptate as the donor fluorophore, and either XL665 or d2 as the acceptor. For these fluorophores, we recommend measuring the fluorescence emission at 620 nm for the donor and at 665 nm for the acceptor. When a green acceptor is used in combination with Lumi4®-Tb cryptate, e.g. in some Tag-lite® assays, the acceptor emission must be measured at 520 nm.
The measurement of HTRF emissions at two different wavelengths (620 nm and 665 nm) allows the ratiometric reduction of data. This feature of HTRF is extremely advantageous, particularly for reducing well-to-well variations that may arise in homogeneous assay formats where a separation or wash step is not performed. Compounds and/or media additives left in the plate may change the photophysical properties in a given sample, and the degree to which this occurs can vary from sample to sample. By using the ratio of the donor and acceptor emission signals, it is possible to eliminate compounds that are simply interfering with detection.
Cisbio Bioassays has developed and patented a ratiometric measurement that uses both the emission wavelength of the donor and of the acceptor (patent US 5,527,684 and foreign equivalents) to correct for well-to-well variability and signal quenching from assay components and media. Emissions at 620 nm (donor) are used as an internal reference, while emissions at 665 nm (acceptor) are used as an indicator of the biological reaction being assessed. Measurement of the total (positive control) and background (negative control) signal was carried out as well as the signal generated in the presence of a colored compound possessing quenching capabilities. As expected, the positive and negative controls show a clear difference in the absolute signal intensity at 665 nm and not at 620nm (see diagrams on the right).
This corresponds to an appropriate change in the HTRF ratio for the two sample types. However, in the sample containing the colored compound, a similar decrease in both 620 nm and 665 nm signals is observed. Since the degree of sample quenching in both emissions is similar, the HTRF ratio remains relatively unchanged. This shows that the compound is nonspecifically interfering with the total emission in the sample and demonstrates the benefit of using the ratiometric data reduction to eliminate interfering compounds in an assay.
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. This clearly demonstrates the usefulness of calculating the acceptor/donor ratio for each well.