1. Outline the main reasons for undertaking a competitive inhibition
radioligand binding experiment?
These type of
experiments are used to validate an assay to demonstrate that a drug binds with
the expected potency and to prove that the chosen radio-ligand has identified
the correct receptor (Motulsky, 1995-96). Competitive binding curves are also
employed to investigate the interaction between low affinity drugs and
2. What is the difference between IC50 and Ki?
(half maximal inhibitory concentration) is the concentration of unlabelled drug
that blocks half the specific binding and it is determined by three factors;
the Ki (equilibrium dissociation constant) of the receptor for the
competing drug, the radio-ligand concentration and the radio-ligands affinity
for the receptor (Kd) (Motulsky, 1995-96).
The Ki is the equilibrium dissociation constant for binding of the
unlabelled drug, it is proportional to the IC50 i.e. if the Ki
is low, the IC50 will also be low (Motulsky, 1995-96).
3. Define the Cheng Prusoff correction. Explain how it is applied in
competitive binding experiments? The
Cheng-Prusoff equation allows for the calculation of Ki from the IC50
and is as follows:
equation is based on the assumptions that the Kd value is known from
pervious experiments, that the experiment has reached equilibrium and that
binding is reversible and follows the law of mass action (Motulsky, 1995-96).
Because the IC50 value is not a direct indicator of affinity, the
Cheng-Prusoff equation is applied to derive the binding affinity of the
4. What safety measures are taken when working with 3H ligands in
Hydrogen-3 or Tritium
is a beta-emitting radioactive isotope employed in radiolabelling
experiments. Although beta radiation
cannot penetrate the outer layer of skin; ingestion, inhalation or absorption
through broken skin may result in exposure.
Minimum Personal Protective Equipment required when handling radioactive
material includes disposable gloves, lab coats, and safety glasses. For
compounds such as tritium which readily penetrate gloves, wear double gloves,
changing the outer pair at least every 20 minutes. A designated working area
should have all surfaces covered with spill paper and any aerosol-generating
procedures should be carried out in a fume hood. It is recommended that all
persons expected to handle radioactive material are trained, registered, and
documented on an approved protocol.