Precision Medicine
Flow Cytometric Assays

Flow Cytometry


Receptor Occupancy Assays

Flow cytometry-based Receptor Occupancy (RO) assays are rapid, powerful, and customisable tools in precision medicine. RO assays are designed to quantify and characterize the binding capabilities of therapeutic drugs to their target receptors on the cell surface [1, 2]. RO assays can also be used to assess other bioactive substances such as neurotransmitters and hormones but spotlight in clinical platforms tend to fall on therapeutic drugs due to their vast applications in medicine. These drugs are often antibody-based therapeutics that are used to target specific receptors to manipulate downstream signalling in diseases. Together with pharmacokinetic and pharmacodynamic (PKPD) data, the assessment of specificity of these antibody-based drugs is essential to identify their mechanism of action, adverse events, and effectiveness in patients [2].  RO assays are used to determine binding affinity, efficacy, potency, therapeutic dose, and duration of action of these drugs. There are several RO assay formats that can be used to assess these parameters, and the choice of assay depends on the specific research or clinical objectives and mechanisms of action [1]. PeploBio offers several RO assay formats, and we provide expert advice to help you choose a suitable assay design for your requirements.

Total receptor binding assay

This assay measures the total number of receptors which includes both free and drug‐occupied receptors. A non-competing fluorescent labelled ligand is used to bind, detect, and quantify the target receptors of the drug. Determining the expression levels of specific receptors on different cell types [1, 2]. This assay format is useful for:
Assessing changes in receptor expression over time or under different conditions.
Evaluating the binding capacity of cells or particles for specific ligands.
Screening for potential ligands or drugs that target specific receptors.

Free receptor binding assay

A free receptor assay measures the proportion of specific receptors on the surface of cells or particles that are unoccupied or free. A fluorescently labelled ligand is used to bind the epitope of interest on the target receptor. The assay then quantifies the remaining number of receptors unoccupied by the labelled ligand [1, 2]. Application of this method includes:
Evaluating the availability of binding sites for a ligand on target cells or particles.
Assessing competitive binding between different ligands to the same receptor.
Investigating the effects of treatments or interventions on ligand-receptor interactions.
Studying ligand-receptor kinetics and affinity.

Drug occupied receptor binding assay

The drug occupied receptor binding RO format is used to measure the extent to which a specific receptor on the surface of cells or particles is bound or occupied by a drug molecule. A fluorescently labelled drug molecule that specifically binds to the target receptor of interest measured in this assay.  This type of assay is valuable in drug development and pharmacology to assess the binding affinity, potency, and effectiveness of a drug candidate in binding to its target receptor [1, 2]. This RO assay format is crucial for:
Evaluating the binding characteristics of drug candidates to target receptors.
Assessing the competitive binding of drugs with other ligands.
Determining the drug binding affinity and ability to activate the target receptor.
Investigating the effects of drug modifications or formulations on receptor binding.

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[1] Stewart JJ, Green CL, Jones N, Liang M, Xu Y, Wilkins DE, Moulard M, Czechowska K, Lanham D, McCloskey TW, Ferbas J. Role of receptor occupancy assays by flow cytometry in drug development. Cytometry Part B: Clinical Cytometry. 2016 Mar;90(2):110-6.
[2] Liang M, Schwickart M, Schneider AK, Vainshtein I, Del Nagro C, Standifer N, Roskos LK. Receptor occupancy assessment by flow cytometry as a pharmacodynamic biomarker in biopharmaceutical development. Cytometry Part B: Clinical Cytometry. 2016 Mar;90(2):117-27.

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