Section16:Color Quench Correction

From Assay Guidance Wiki

If colored compounds are to be tested and are present during the counting step (as in a non-separation technique such as SPA) color quenching may be present. This occurs when the photons emitted by the fluor are absorbed by the colored compound resulting in attenuation of signal. The emission spectra (~420 nm max) of SPA beads detectable in the Trilux and TopCount and the absorption spectra for common colors are shown below. (Diagrams from GE Healthcare).

For the Trilux and TopCount, compounds that are red, yellow or orange (absorption max ~400 nm) have the biggest impact on signal attenuation (with PVT or YSi SPA beads) if they are present while reading the plates.

Correction of color quenching can be performed within the software of the Trilux or TopCount using a prepared quench curve. Typically this is performed with a yellow dye such as tartrazine.