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Table 2 Best-fit estimations of the rate constants and the resulting K d values in kinetic real-time cell-binding experiments

From: New approaches for the reliable in vitro assessment of binding affinity based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics

Fitting approach k on [M−1 min−1] k off [min−1] K d [nM] R 2 df
2 concentrations 8.675 × 107 ± 0.414 × 107 0.077 ± 0.008 0.887 ± 0.121 0.992 216
3 concentrations 9.262 × 107 ± 0.835 × 107 0.049 ± 0.007 0.528 ± 0.118 0.991 325
4 concentrations 9.243 × 107 ± 0.919 × 107 0.051 ± 0.007 0.556 ± 0.132 0.988 434
5 concentrations 8.805 × 107 ± 0.289 × 107 0.062 ± 0.003 0.704 ± 0.054 0.994 543
6 concentrations 8.788 × 107 ± 0.292 × 107 0.061 ± 0.003 0.695 ± 0.052 0.993 652
7 concentrations 8.105 × 107 ± 0.259 × 107 0.055 ± 0.003 0.683 ± 0.056 0.993 761
8 concentrations 8.068 × 107 ± 0.260 × 107 0.057 ± 0.003 0.711 ± 0.056 0.993 870
Averaged 6.645 × 107 ± 1.423 × 107 0.043 ± 0.014 0.636 ± 0.076 nd nd
Reported affinity nd nd 0.59 ± 0.02 [29] nd nd
  1. Curve fitting parameters of the association rate constant and the dissociation rate constant of [125I]-AB-MECA on CHO-K1-hA3R cells at ambient temperature. Dedicated K d values were calculated as the ratio of k off/k on. The table summarizes best-fit estimations and corresponding goodness of fit parameters for a set of different amount of concentrations of [125I]-AB-MECA. The “averaged” value comprises data from three independent experiments. Reported K d was determined with traditional saturation binding assays using membranes from human A3R-transfected HEK-293 cells. Data in the table are expressed as mean ± SEM from individual experiments (n ≥ 4)
  2. nd not determined