pHSense Eu Fab Anti-Mouse IgG2, 2 x 96 wells

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 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.

EGFR antibody internalization assay

Breast cancer adenocarcinoma cells were seeded in a 96-well white culture-treated plate at a density of 50,000 cells per well in complete culture medium, and then incubated overnight at 37°C with 5% CO₂. An anti-EGFR internalizing antibody was pre-incubated with pHSense Eu Fab at a 1:2 molar ratio in cell culture medium for 30 minutes at room temperature. The Fab–antibody mix was then serially diluted in medium, and 50 µL of each dilution was added to the cells, followed by a 4H incubation at 37°C with 5% CO₂. The fluorescence signal was recorded using an HTRF-compatible plate reader.

Results show the internalizing anti-EGFR antibody induced a dose-dependent increase in signal. No signal increase was observed when an irrelevant mouse IgG2 was used as a negative control.

pHSense-based detection of GLP1R internalization following Exendin-4 stimulation

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 then incubated overnight at 37°C with 5% CO₂. An anti-GLP1R antibody (15 nM) was pre-incubated at room temperature for 30 minutes with pHSense Eu Fab at a 1:2 ratio in cell culture medium. Subsequently, 40 µL of the Fab-antibody mixture was added to the cells, followed by a 1 hour incubation at room temperature. Exendin-4 was serially diluted in cell culture medium, and 10 µL of each dilution were added to the wells. Following incubation at 37°C, the signal was recorded using an HTRF-compatible plate reader. Results show Exendin-4 induced a dose-dependent increase in signal, with an EC₅₀ value consistent with values described in the scientific literature.