Monolith and Dianthus detect biomolecular interactions by following changes in target fluorescence properties upon binding of ligands. Ligand binding can directly lead to a change of initial fluorescence. It can manifest both, as a ligand concentration dependent fluorescence decrease, or as a ligand dependent fluorescence increase. In both cases, the impact of this initial fluorescence change on the measured ratio and F_norm data is accounted for in the K_d- and Hill-Fit Model. In case of an initial fluorescence change smaller than ± 20 % of the target’s initial fluorescence, the initial fluorescence change can be ignored. In case of an initial fluorescence change higher than ± 20 % of the target’s initial fluorescence, it is necessary to perform some control experiments to rule out possible artifacts. The possible causes for an artifact and the necessary control measurements are described below:

• Ligand autofluorescence: A ligand concentration dependent increase in initial fluorescence can be caused by ligand autofluorescence. If the ligand itself is fluorescent, the complex fluorescence will be higher than target fluorescence alone. In this case, the assay setup must be adjusted. 

  Control: Ligand autofluorescence can be tested by comparing the fluorescence of a buffer only capillary and a ligand only capillary in absence of target.
   

• Quenching/inner filter effect: A ligand concentration dependent decrease in initial fluorescence (i.e. quenching) can also be caused if the ligand absorbs light in the relevant range (inner filter effect). In this case, the complex fluorescence will be lower than target fluorescence. This effect is not binding-specific, so it is independent of the target molecule. The phenomena will also occur in a sample that includes fluorophore and ligand, but not the target. 

  Control:  To test for this, compare the fluorescence signal of the fluorophore (dye, fusion protein) alone, without being attached to a target molecule, to a combination of fluorophore and ligand.
   

• Aggregation/adsorption: If neither of the above cases could be confirmed as a cause for the initial fluorescence change, fluorescence variations in both directions can also arise if the addition of the ligand to the target is influencing target aggregation or target adsorption to the reaction tubes or other labware. This means that material is lost, and the resulting measurement will be unreliable. 

  Controls: To rule out this possibility, please perform a specificity test (see SD-Test for covalent labeling and ECP-test for tris-NTA labeling).

LIFC Factor: When control measurements demonstrate that changes in the target’s initial fluorescence are binding-specific, the resulting effects on Fnorm and Spectral Shift data must be mathematically corrected. This correction can be achieved by applying a ligand-induced fluorescence change (LIFC) factor in MO.Control 2. The Software automatically applies this LIFC factor, once a ligand dependent fluorescence intensity change by more than ±20% is detected. The LIFC factor can be displayed in the Details tab via "Show Details" (see MO.Control 2 screenshot below). The correction factor will not be applied if the reason for Ligand Induced Fluoresces Change is manually set to "Non-specific change".



The principles and mathematical derivation of this correction factor are further outlined in Evaluation of ligand-induced fluorescence changes (LIFC) in MO.Control 2 and Dianthus Software (see Calculation-of-ligand-induced-fluorescence-changes-LIFC-in-MO-Control-2-and-Dianthus-Software).