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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