Description
pHSense™ Eu Fab reagent is a covalently europium-labeled, pH-sensitive Fab fragment designed to monitor antibody or antibody-drug conjugate (ADC), as well as receptor- and membrane protein-mediated internalization in real time. It binds with high affinity to the Fc region of test antibodies, particularly mouse IgG2 subtypes, and remains minimally fluorescent at neutral extracellular pH (≥7). Upon internalization, the Fab-antibody complex encounters increasingly acidic intracellular compartments such as early and late endosomes and lysosomes, where the europium signal becomes progressively stronger. This novel pH sensitive europium complex is compatible with a time-resolved fluorescence (TRF) detection, effectively eliminating most fluorescence background and significantly enhancing the signal-to-background ratio. Its unique photophysical properties enable simple and robust no-wash detection of receptor-mediated endocytosis of antibodies in plate-based assays with live cells.
pHSense Eu Fab Anti-human IgG: Internalization assay principle
pHSense Eu Fab reagent is a covalently europium-labeled, pH-sensitive Fab fragment designed to monitor antibody or antibody-drug conjugate (ADC) as well as receptor- and membrane protein-mediated internalization in real time. It binds with high affinity to the Fc region of test antibodies, and remains minimally fluorescent at neutral extracellular pH (≥7). Upon internalization, the Fab-antibody complex encounters increasingly acidic intracellular compartments such as early and late endosomes and lysosomes, where the europium signal becomes progressively stronger.
Assay protocol for monitoring antibody or ADC internalization
The assay begins by culturing cells in a 96-well plate. The test antibody or ADC is pre-incubated with pHSense Eu Fab at a 1:2 molar ratio in cell culture medium for 30 minutes at room temperature. The resulting complex is then added to the cells and then incubated at 37°C, followed by kinetic or endpoint fluorescence measurement using an HTRF-compatible plate reader.
Assay protocol for monitoring receptor and membrane protein-mediated internalization
The assay begins by culturing cells in a 96-well plate. The antibody directed against the target of interest is pre-incubated with pHSense Eu Fab at a 1:2 molar ratio in cell culture medium for 30 minutes at room temperature. This mixture is then added to the cells and incubated for 1 hour at room temperature. Following this incubation, cells are stimulated with a pharmacological compound. Fluorescence is then measured either kinetically or at endpoint using an HTRF-compatible plate reader.
pHSense-based monitoring of internalizing antibody and ADCs
BT-474 cells were seeded in a 96-well white culture-treated plate at a density of 20,000 cells per well in complete culture medium, and incubated overnight at 37°C with 5% CO₂. The anti-HER2 internalizing antibody IgG1 Trastuzumab and two derived ADCs (Trastuzumab-Emtansine and Trastuzumab-Deruxtecan) were pre-incubated at room temperature for 30 minutes with pHSense Eu Fab at a 1:2 molar ratio in cell-culture medium. The Fab–antibody mixtures were then serially diluted in cell-culture medium and 50 µL of each dilution was added to the cells, followed by 1H incubation at 37°C with 5% CO2.
The fluorescence signal was recorded using an HTRF-compatible plate reader.
A dose-dependent increase of the signal demonstrated that pHSense Eu Fab can specifically detect the internalization of both anti-HER2 antibody and its ADC derivatives. Results obtained with the 2 tested ADCs suggest that the assay is not affected by the nature of the payload or the linker used in ADC design. No signal increase was observed when an irrelevant human IgG1 was used as negative control.
pHSense-based monitoring of Dulaglutide internalization: a GLP-1 analog covalently linked to Fc Fragment of IgG4
Tag-Lite® GLP1R cells (stable cell line, Part #: C1SU1GLP1, Revvity) were seeded in a 96-well white, culture-treated plate at a density of 80,000 cells per well in complete culture medium and incubated overnight at 37°C with 5% CO₂. Dulaglutide, a GLP-1R agonist consisting of a GLP-1 analog covalently linked to the Fc fragment of human IgG4, was pre-incubated at room temperature for 30 minutes with pHSense Eu Fab at a 1:2 molar ratio in cell culture medium. The Fab–Dulaglutide mixture was then serially diluted in cell-culture medium and 50 µL of each dilution were added to the cells, followed by 20 min incubation at 37°C with 5% CO₂. The fluorescence signal was recorded using an HTRF-compatible plate reader. A dose-dependent increase in signal with Dulaglutide, and the absence of signal with an irrelevant human IgG4, demonstrated that pHSense Eu Fab can specifically monitor GLP1R internalization.
