pHSense Boost Reagent, 0.5 mL

pHSense Boost reagent: internalization assay principle

pHSense Eu reagents are europium-labeled, pH-sensitive probes that enable the real-time, no-wash detection of antibody internalization, as well as receptor- and membrane protein-mediated internalization in live-cell, plate-based assays.

pHSense Boost is a novel reagent specifically designed to optimize pHSense Eu internalization assays by addressing the limitations of narrow assay windows. It reduces background signals and enhances fold-change measurements, improving assay performance.

The efficacy of pHSense Boost depends on cell type, cell density, and incubation time. Its benefits are not universal, and must be empirically validated for each specific assay condition.

pHSense Boost reagent usage parameters

pHSense Boost reagent should be added to the medium used for preparing reagents in pHSense internalization assays.

Add the appropriate volume of pHSense Boost to 5 mL of medium, following the guidelines in the table below.

Note: For DMEM and IMDM media, the use of pHSense Boost is not required, as no improvement in assay window has been observed with these medium types.

pHSense Boost reagent effects on assay window

Raji cells were plated in a 96-well white low- volume plate (Revvity, Part#66PL96005) at a density of 12,500 cells per well, in complete culture medium.

The internalizing anti-CD20 antibody Rituximab ("Test Antibody") and the Human IgG1 Negative Control (Revvity, Part #81HUIGG1NC) were pre-incubated at a final concentration of 4 nM at room temperature for 30 minutes with pHSense Eu Fab anti-Human IgG (Revvity, Part #81HUEU1AA) at a 1:2 molar ratio. This incubation was performed in cell culture medium supplemented or not with pHSense Boost reagent.

The Fab-antibody mixtures were then added to the cells, followed by a 24-hour incubation at 37°C with 5% CO₂. The time-resolved fluorescence (TRF) signal at 620 nm was measured using a TRF-compatible plate reader.

Supplementing with pHSense Boost reagent led to a decrease in the 620 nm signal for both test antibody and Fab-only conditions. Notably, the greater reduction in Fab-only signal resulted in an increased fold-change from 21.6 to 54.3 when the culture medium was supplemented with pHSense Boost reagent.