The response amplitude is the difference between the signal of the Target alone (unbound) and the Complex (bound).

For the analysis, the response amplitude can be negative or positive, depending on the exact influence of ligand binding on the target. The response amplitude will be negative if the signal of the complex is lower than the target alone and positive if the complex signal is higher than that of the target alone. Both directions are common, and for evaluation, the direction of the response amplitude is irrelevant.

When a flat dose-response is obtained, one can differentiate between a non-binder ligand from a saturated at the lowest concentrations by comparing the values of the reference points (i.e DMSO) to the assay points. If the lower concentrations of the ligand are significantly different from the reference, the interaction is already saturated and the interaction needs to be tested at lower ligand doses. But, if the values between the reference and the highest concentrations of the assay points are similar, the compound is not a binder under those conditions.

In Spectral Shift assays, the 670nm/650nm ratio is calculated by the software, and the response amplitude is the difference between the ratio of the Bound and Unbound complexes. Since Spectral Shift assays are measured at room temperature and last only one second, there is only one possible amplitude.

For TRIC experiments, on the contrary, the response amplitude depends on the measured on-time and the selected on-time for analysis. The area response for a single dose, on the other hand, only depends on the measured on-time. Generally, one would expect higher amplitudes for 5-sec measurements than for 3-sec measurements.