When assessing a ligand Dose Response Curve in DI.Screening Analysis, the system evaluates the response amplitude, resulting in a response quality check. The response values are obtained from the selected Response Evaluation Mode.

In single-dose screening, all data points for a ligand have to show a consistent significant deviation from the references for the ligand to be considered a Hit. The minimum requirements to be considered a hit are defined on the Data page of DI.Screening Analysis, either as a manual minimum or as a multiple of the global standard deviation over all references of the assay. In addition, no data point must deviate from the ligand average by more than 20 %. Otherwise, the Inhomogeneity Quality is set to negative.

In affinity screening, a dose-response curve is fitted to the response values by either the Kd Fit Model or the Hill Model.

The fit succeeds when the following three criteria are met.

• The fitted response amplitude is larger than the response amplitude threshold of 0.018 for Spectral Shift or variable for TRIC, depending on the On-time analysis (see table below).
• The signal-to-noise ratio is larger than 5, and the baseline is clearly visible (the gap between baseline and model value at the lowest concentration must be less than 25 % of the response amplitude).
• The saturation is reached (the gap between saturation and model value at the highest concentration must be less than 50 % of the response amplitude)

If all other qualities are positive, this ligand is considered a Binder. If a level of saturation is reached between 25% and 50% and all other criteria are fulfilled, then the ligand is considered a Weak Binder.

The following table shows the response amplitude threshold required for binders in TRIC measurements, depending on the measured/selected On-time.

The automatic setting of the quality can be overridden by the user as described in Quality Checks.