Fig. 6

Graphical representation of the kinetic real-time cell-binding approach. Observed association time-courses of [¹²⁵I]-AB-MECA to CHO-K1-hA₃R at several concentrations (0.05–5 nM) spanning the K _d (a). Real-time binding data were globally fitted in GraphPad Prism 6.0 using “association kinetics—two or more conc. of hot” (solid lines). Best-fit estimations for k _on and k _off were obtained from Eqs. (5) and (6). Resulting rate constants were used to determine the K _d (see Table 2 for summary of dedicated k _on, k _off and K _d values). To verify these results, k _obs values were determined using Eq. (2) and plotted against the corresponding concentration of [¹²⁵I]-AB-MECA (b). Values for k _on and k _off calculated from linear regression analysis are in good agreement with values obtained from the global fit estimation. An estimation of the associated target concentration of each single petri dish was used to confirm homogenous receptor expression within the whole binding experiment (c). Values corresponding to the maximum amount of target receptors were obtained using Eq. (7). The K _d of [¹²⁵I]-AB-MECA obtained from the novel kinetic real-time cell-binding studies and from traditional binding assays, as previously reported in the literature (see Table 2 for detailed summary of K _d values), was compared using two-tailed, unpaired Student’s t test with Welch’s correction (d). Differences between the K_d values are statistically not significant (ns = P > 0.05). Data in the graph (a) are shown from one representative experiment. Data in the graphs (b–d) are shown as mean ± SEM from individual experiments (n ≥ 4)