Modelling ligand depletion for simultaneous affinity and binding site quantification on cells and tissue.

Area of Science:

• Biochemistry
• Pharmacology
• Cell Biology

Background:

• Quantifying biological targets and their binding kinetics is crucial for drug development and medical imaging.
• Current methods for quantifying membrane antigens on live cells are often manual, labor-intensive, and lack kinetic information.

Purpose of the Study:

• To present a novel real-time method for simultaneously quantifying kinetic binding parameters and the number of available binding sites on live cells and tissue.
• To validate the method's feasibility using simulated and experimental data for various molecular tracers.

Main Methods:

• Measuring molecular interactions in real-time under ligand depletion conditions.
• Utilizing simulated data to optimize assay design.
• Validating the method with experimental data using radiotracers and fluorescent antibodies.

Main Results:

• The method accurately quantifies both kinetic binding parameters (affinity, rates) and the number of accessible target sites.
• It does not require knowledge of the absolute signal generated per ligand molecule.
• Feasibility was demonstrated with peptide and antibody-based tracers.

Conclusions:

• This real-time approach offers a simplified and more accurate workflow for quantifying molecular interactions and target sites.
• It is applicable to both radioligands and fluorescent binders, enhancing drug discovery and imaging applications.