Fig. 7

Verification and performance analysis of kinetic-real time cell-binding studies. Best-fit estimations for k _on and k _off were obtained using Eqs. (5) and (6), as detailed in the methods, and resulting K _d were calculated as the ratio of k _off/k _on. Fitted rate constants were determined using eight different concentrations (0.05–5 nM) of [¹²⁵I]-AB-MECA to fully cover the concentration span needed for proper affinity estimation. The corresponding K _d (all) was compared to K _d values, calculated from best-fit estimations for the rate constants using two (2 conc.), three (3 conc.), four (4 conc.), five (5 conc.), six (6 conc.) or seven (7 conc.) concentrations of the radioligand, respectively (a). Differences between the K _d values are statistically not significant (ns = P > 0.05), using ordinary one-way ANOVA with Tukey’s multiple comparisons correction (see Table 2 for detailed summary of k _on, k _off and K _d values). Differences between group means of the dedicated K _d , calculated using the full set of concentrations and the K _d , determined using less concentrations of the radioligand with the corresponding 95% confidence intervals (b). Data are expressed as mean ± SEM from individual experiments (n ≥ 4)