Calculation of affinity constants directly from homologous displacement curves

Homologous displacement experiments are described in which the binding of labelled ligand (tracer) was inhibited with the same ligand unlabelled (inhibitor). Data were evaluated with a direct curve-fitting method. The equation used employs the concentration of unlabelled ligand as the independent variable and the fraction of tracer bound (p) as the dependent variable. The other entities of the equation are the dissociation constant (Kd), the maximal fraction of tracer bound (Po) and the tracer concentration (T). Different options exist regarding the insertion of Po and T as parameters or constants in the curve-fitting. When applied to experimental data obtained from the binding of dinitrophenyl-hapten by a monoclonal antibody, the least interexperimental variation in affinity was obtained when both Kd and Po were estimated as parameters, and T was inserted as a constant. This calculation procedure achieves more reproducible results than those obtained with the Scatchard plot and the Langmuir curve, even after exclusion of the most scattered data points.