Monitor antibody and ADC internalization with pHSense Eu probes

Summary

This technical note presents a no-wash, time-resolved fluorescence (TRF) assay using pHSense™ Eu Fab probes to monitor antibody and antibody-drug conjugate (ADC) internalization in live cells. The study evaluates probe performance across different IgG isotypes and pH conditions, and demonstrates antibody internalization in HER2- and EGFR-expressing cell models. Experimental results include dose-response analysis, EC₅₀ determination, and detection of HER2-targeted ADCs Kadcyla® and Enhertu®. Findings demonstrate the assay's ability to quantify antibody and ADC internalization with high sensitivity and minimal background fluorescence.

How does the technology work?

pHSense Eu probes detect antibody and ADC internalization through pH-dependent fluorescence activation:

Binding → Internalization → Acidification → Signal Generation → Quantification

As antibody–probe complexes enter cells, they encounter increasingly acidic compartments, from early endosomes to lysosomes. This activates the europium-based fluorescence signal, which can be measured using a TRF-compatible plate reader to quantify internalization.

What can you measure?

• Internalization kinetics
• Dose-response curves and EC₅₀ values
• Uptake differences across cell models
• Target-dependent internalization

Applications in ADC research

• ADC candidate screening and characterization
• HER2-targeted ADC research, including Kadcyla® and Enhertu®
• EGFR-targeted antibody internalization studies
• Mechanism-of-action and preclinical characterization studies

Download the technical note to explore experimental validation, assay performance, and dose-response data demonstrating antibody and ADC internalization.