Receptor occupancy (RO) Assays by flow cytometry are methods of assessment of binding of a therapeutic to its target receptor.  RO assays can be as simple as measuring the number of cell surface receptors bound by a biotherapeutic agent or highly complex such as measuring receptor dynamics, depending on the scientific questions being studied.  Assessment of receptor occupancy by flow cytometry can take several approaches:

  • Free Receptor Assay – Shown in the Left Panel is an illustration of free receptors that are not occupied by biotherapeutic agent detected using either a fluorescently labeled competitive antibody or biotherapeutic agent.
  • Free and Total Receptor Assay – Shown in the Middle Panel is an illustration of free and total receptor assay design which depicts the total receptors available, recognized by a noncompetitive antibody bound to a different epitope on the receptor from the one recognized by the biotherapeutic agent. This design can be used determine the ratio of free (unoccupied) sites to total sites available.
  • Direct Assessment of Bound Receptor – Shown in the Right Panel is an illustration of direct assessment of bound receptor with biotherapeutic agent, typically detected with a fluorescently labeled anti-drug antibody.