Ratios of the fluorescence intensities at 670 and 650 nm are calculated automatically within the DI.Control software. Dose-response curves are then fitted with the Kd model, which describes a molecular interaction with a 1:1 stoichiometry according to the law of mass action. In this model, the fraction bound f(C_ligand) at a given ligand concentration C_ligand in equilibrium is given as:

where

• K_d is the dissociation constant or binding affinity

• C_Target is the final concentration of target in the assay

 The Kd is then estimated by fitting the equation.

where

• R(C_ligand) is the 670 nm/650 nm ratio value at a given ligand concentration C_ligand

• R_unbound is the ratio value of the target alone

• R_bound is the ratio value of the complex

In the presence of ligand-induced changes in the fluorescence intensity at 650 nm, the equation is replaced by:

with

where F_bound and F_unbound are parameters obtained from fitting a sigmoidal dose-response curve to the fluorescence intensity at 650 nm. Ligand-induced changes in the fluorescence intensity at 670 nm have no effect on the ratiometric readout and, therefore, do not require a mathematical correction.