Primary screenings are usually conducted in a single-dose setup and are best and most comfortably analyzed using DI.Screening Analysis. Each Ligand is tested at a single, high concentration. This experimental setup provides a simple yes/no answer for ligand binding and is suited for screening a larger number of ligands compared to the quantitative Affinity Screening. If the ligand binds the Target, it will change the target TRIC signal and is therefore a Potential Hit. The used high ligand concentration can be adjusted to pre-select for ligands with desired affinities, e.g. limiting this concentration to 50 μM omits the detection of weak binders with significantly lower affinities.

A single-dose screening requires the following controls and setup details.

• A reference sample. The reference sample includes a target in the final assay buffer but no ligand. The reference is required to set thresholds for hits and Non-Binders and control for signal stability over the screening duration. For best results, a reference sample must be measured regularly (several times per plate). Oftentimes, the reference sample is equivalent to a DMSO control.
• A positive control. The positive control interaction includes target and a ligand known to bind to it. This is to control for target functionality (its capability to bind ligands) over the screening duration.
• If possible, ligands should be run in duplicate.

When planning a single-dose screening, please familiarize yourself with “Normalization ID”. It is an important determinant for how data is analyzed in DI.Screening Analysis.