The area response represents a robust approach to analyze the Response Amplitude and the Signal Quality of a TRIC assay. In contrast to the Fnorm approach the area response is less dependent on a one second snapshot of the data but takes all measured data points of a TRIC-trace into account. Therefore, in Single-Dose Screening the analysis method cannot artificially induce false-negatives during Hit identification. To quantify the area response the software calculates all possible areas between all possible pairs of TRIC traces. There are three types of areas.
- Reference areas are areas between two Reference traces
- Ligand areas are areas between two ligand/positive control traces
- Signal areas are areas between a ligand trace and a reference trace.
From the areas the Signal Quality is then calculated in Binding Check and Buffer Screen experiments to yield a good measure for assay quality. In general one should aim for small reference and ligand areas (areas smaller 0.15) and large signal areas (areas larger 1). Also, the different areas calculated should not show high variation within their own group.