Real-Time SPR Biosensing to Detect and Characterize Fast Dissociation Rate Binding Interactions Missed by Endpoint...

Area of Science:

• Biomolecular interactions
• Biophysics
• Assay development

Background:

• Accurate detection of biomolecular interactions is vital for clinical diagnostics, drug development, and basic research.
• Traditional endpoint assays can yield false negatives for interactions with fast kinetics.
• Real-time detection methods, such as surface plasmon resonance (SPR), mitigate this risk by monitoring interactions dynamically.

Purpose of the Study:

• To compare the efficacy of a fluorescence endpoint assay versus real-time SPR detection for analyzing biomolecular interactions.
• To illustrate the limitations of endpoint assays when dealing with transient interactions and fast dissociation rates.
• To emphasize the importance of assay selection and reagent choice in binding studies.

Main Methods:

• Utilized cell-free expressed proteins captured on glass or SPR biosensors.
• Employed two commercial antibodies targeting HaloTag antigens with varying off-rates.
• Compared results from a fluorescence endpoint assay with real-time sensor-integrated proteome on chip (SPOC®) SPR detection.

Main Results:

• The fluorescence endpoint assay demonstrated limitations in detecting transient interactions with fast dissociation rates.
• Real-time SPOC® SPR provided a more comprehensive analysis of biomolecular interactions, especially those with rapid kinetics.
• The study underscored the impact of antibody off-rates on assay outcomes.

Conclusions:

• Real-time SPR detection is superior to endpoint assays for characterizing biomolecular interactions with fast kinetics.
• Careful consideration of binding kinetics, protein ligand states, and reagent suitability is essential for reliable binding assay results.
• Accurate assessment of therapeutic off-target interactions necessitates methods that can resolve transient binding events.