General Principle 7211 Saturation Assays

Saturation assays are used to characterize the affinity of the radioligand to the target as well as to determine the target density. In saturation assays, in a series of experiments a constant target concentration (Ttot) is incubated with an increasing concentration of marker (Mtot). The amount of bound radioligand determined after the equilibrium is reached represents total binding. Nonspecific binding is defined in a control experiment where the target is incubated with the marker in the presence of a great excess of a competitor, i.e. another ligand with high

Straight Isotherms

Fig. 7.1 Typical saturation binding experiment: (a) Hypothetical binding isotherm for total binding (---) and straight line for nonspecific binding (---). (b) Saturation binding isotherm for specific binding after subtraction of nonspecific binding from total binding (Bmax = 10 pM, Kd = 100 pM).

Fig. 7.1 Typical saturation binding experiment: (a) Hypothetical binding isotherm for total binding (---) and straight line for nonspecific binding (---). (b) Saturation binding isotherm for specific binding after subtraction of nonspecific binding from total binding (Bmax = 10 pM, Kd = 100 pM).

affinity to the target. Plotting the specific binding of the marker (target-marker complex, TM, calculated from total and nonspecific binding) against the concentration of the free marker (M) gives a saturation isotherm (Fig. 7.1). This provides the equilibrium dissociation constant (Kd) of the marker represented by the concentration of the free marker yielding a target occupancy of 50% [see Eqs (1), (2)].

From the plateau of the saturation isotherm, the maximal occupancy of the target Bmax can be determined that corresponds to the target concentration in the binding assay. Preferably, the target is used in concentrations of Ttot « Kd, as under these conditions the concentration of TM remains low compared to the concentration of the free marker M and therefore M can be replaced by Mtot for the analysis of the binding experiment [7, 16, 17, 21].

7.2.1.2 Competition Assays

Competition assays are used to determine indirectly, that is by quantifying the binding of a radioligand, the affinity of a test compound to a target. In such an experiment the target is incubated in the presence of a constant concentration of Mtot (Mtot @ Kd) and of varying concentrations of the test compound under conditions that allow an equilibrium to be attained. As a result of the competition between marker and test compound the binding of the marker to the target is reduced with increasing concentrations of the test compound. Plotting the specific binding of the marker against the free concentration of the test compound gives a

250 I 7 MS Binding Assays - An Alternative to Radioligand Binding I / cps

250 I 7 MS Binding Assays - An Alternative to Radioligand Binding I / cps

Ic50 Calculator
Fig. 7.2 Typical competitive binding experiment. The hypothetical test compound is an inhibitor of marker binding with an IC50 of 0.66 nM (log c = -9.2). Specific binding covers approx. 980 units of signal intensity (/).

competition curve (Fig. 7.2) yielding the IC50 value, i.e. the concentration of test compound required to reduce the specific binding of the radioligand by 50%.

The affinity constant of the test compound Ki can in turn be calculated from the IC50 value, the Kd of the marker and the concentration of M according to Cheng-Prusoff [see Eq. (3)] [22]. Since the concentration of free marker (M) is usually not determined in radioligand binding assays, it should be noted that the calculation of Ki according to Eq. (3) based on Mtot yields only reliable results if the depletion of the marker is negligible (i.e. <5% to 10% [7, 17, 21]). In competition experiments performed under typical conditions (Ttot « Kd, Mtot @ Kd) this criterion is fulfilled.

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  • vittoria
    What are saturation assays?
    8 years ago
  • ella
    What is saturation isotherm?
    8 years ago

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