Skip to content
FUll Spectrum AdobeStock_309577920

Receptor Occupancy Assays by Flow Cytometry

Understand how your biotherapeutics are engaging their targets

Receptor occupancy (RO) assays are commonly employed to measure pharmacodynamic (PD) data. These assays have a broad range of uses across preclinical and early stages of clinical trials, all the way through later stages. PD data can then be correlated with pharmacokinetic (PK) profiles to establish PK/PD relationships and guide decision-making in dosing.

Guide decision-making at every phase with RO assays

Precision provides flow cytometry services from multiple labs in the US and Europe. To help streamline projects, Precision also offers customized kitting for sample collection, a global network of sample processing labs, and logistics services to rapidly move samples to labs for analysis.

RO assays are validated for intended use and suitable for proof of concept, exploratory, secondary, or primary clinical trial endpoints.

Preclinical and discovery:

  • Optimal matrix selection: whole blood or fixed whole blood
  • Reagent specificity determined by extensive characterization during assay development
  • Guide selection of compounds

Early phase trials:

  • Measure pharmacodynamics (PD) of biomarkers of interest
  • Assess efficacy

Late phase trials:

  • Define the population PD characteristics of the biotherapeutic agent
  • Combine data with PK profiles to guide dosing and administering schedules

Common formats for receptor occupancy flow cytometry assays

Consult with Precision to decide on this critical step of RO assay development. The selection of assay format largely depends on the mechanism of action (MOA) of the drug and availability of reagents.

Free Receptor AssaysDirect Assessment of Bound ReceptorTotal Receptor AssayReceptor Modulation AssayFunctional Receptor Occupancy Assay
Function Measure unbound or unoccupied receptors on a cell using an unlabeled antibody and competing therapeutic antibody or drug-conjugated antibody Measure therapeutic antibodies bound to the receptor with an anti-therapeutic antibody detection reagent Measure binding of a therapeutic antibody to its receptor site and couple with binding of an alternative antibody that binds to a different site on the same receptor Measure effect of the therapeutic antibody binding on the function of the target receptor Measure biological effects of the therapeutic antibody binding to the target receptor
Clinical Significance Typically used for antagonistic therapeutic drugs that block ligand binding to the receptor Often applied when monoclonal antibodies to the receptor are not readily available or conjugation of the therapeutic itself may compromise binding to its receptor target Useful where receptor level or cell numbers can change over time Receptor shedding or inhibition/activation of receptor internalization over time or with increasing drug dose Cell proliferation or cytokine production, which may have implications in drug safety/efficacy

Free receptor assay

Direce assessment


Total receptor occupancy


Frequently asked questions

Which clinical phase can benefit from receptor occupancy flow cytometry?

Preclinical target verification and MOA: Determining RO through development is critical because many of these biologics have long half-lives. Understanding their binding characteristics can have an impact on lead compound selection.

Phase 1: Typically, this class of drug is very potent, and improper dosing protocols can potentially result in severe side effects or even be lethal. The identification of minimal anticipated biological effect (MABEL) model starting doses or Pharmacologically Active Doses (PAD) may require RO assessments in conjunction with PD and PK in order to appropriately guide dosing protocols.

Phase 2: Efficacy of dosing and administration protocols to help predict the levels of RO and whether the receptor is modulated up or down on cells that are engaged by the biotherapeutic.

Phase 3: Population PD for long-term safety and efficacy studies.

What is the regulatory level requested for a clinical flow cytometry panel?

Flow cytometry assays are validated and used under a fit-for-purpose approach. The parameters included in the validation of an assay should always depend on the purpose of the assay.

What are some challenges for receptor occupancy assays?

Because RO assays are typically performed on whole blood in real time and can be performed on PBMCs, sample stability must be taken into consideration.

Stability considerations:

  • Shipment time of blood from site to lab: age of blood impacts quality of results
  • Where is the site located and how long will sample shipment take?
  • Use of stabilizing fixative collection tubes (Smart Tube, Transfix, CytoChex) can be considered
  • Is the target receptor compatible with the fixative?
  • How long are the fixed samples stable for testing?

Other considerations:

  • Availability of competing or noncompeting antibodies to the target
  • Modulation of the receptor after treatment

Explore flow cytometry publications and resources

Explore best-practice methods and find useful considerations and tips for experimental design, sample prep, and data analysis.

Explore all resources

Values of Precision flow cytometry

Reproducible and valid flow cytometry data stems from experienced scientists and standardized protocols across multi-site programs. Leverage our deep network of expertise for your trials.
  • EstablishExperience

    Established experience

    Established experience running complex flow cytometry in gene therapy, cell therapy, and immuno-oncology therapies, validating up to CLIA
  • SeniorStaf

    Senior staff

    Sample testing by senior staff with >5 years of experience who understand the disease biology and gating strategy
  • QualityControl

    Quality control

    Real-time quality control during the run to ensure sample integrity

Enhance your results with complementary specialty lab services

  • Explore

    Clinical Sample Kitting & Logistics

    Kit production, clinical site training, and full sample shipment logistics
  • Explore

    PBMC & Sample Processing

    Sample processing with expertise in PBMC isolations from labs across 5 continents

  • Explore

    Genomics Services

    With NGS, NanoString, qPCR and ddPCR capabilities, we can add insights from genomic and transcriptomic technologies to your understanding of patient biology.

Explore the Precision blog

Read our blog to gain more insights and discover work that Precision has supported.