Antibody Internalization and Degradation Assays for ADC Discovery

pHrodo Deep Red has a lower pKA than pHrodo red and green, which means it will not fluoresce until later in the endocytic pathway. This delayed fluorescence allows for the monitoring of different stages within the endocytic pathway, providing valuable insights into the internalization process.

Figure 1.Low pKa pHrodo Deep Red is optimized for late endosomes and lysosomes. Herceptin (monoclonal antibody used to treat breast and stomach cancers) was labeled with pHrodo Deep Red. SKBR3 cells were treated with pHrodo Deep Red–labeled Herceptin and CellLight Early Endosome-RFP (left), CellLight Late Endosome-RFP (center), and CellLight Lysosomes-RFP right). After overnight incubation, samples were stained with NucBlue Live nuclear dye for 30 minutes at 37°C. Using the EVOS M7000 cell imaging system, fluorescent images were acquired and overlaid. Overlapping fluorescent signals (seen as white) demonstrate that internalized Herceptin antibody labeled with pHrodo Deep Red (magenta) fluoresces in late endosomes and lysosomes, but not early endosomes. These data suggest that the more acidified pH of 5.5–6.0 found in late endosomes and lysosomes is required to activate the pHrodo Deep Red from non-fluorescent to a deep red fluorescence.

Anti-dye antibodies are valuable quench-based assays for studying the internalization of fluorescently labeled antibodies. These antibodies specifically quench the fluorescent antibodies that remain on the cell surface, allowing researchers to distinguish fluorescence from the internalized antibodies. 

Anti-Alexa Fluor antibodies will specifically bind to the Alexa Fluor dyes that remain outside the cell. Once bound, the anti-Alexa Fluor antibody will effectively reduce the signal of the Alexa Fluor dye. Since quenching only occurs for the fluorescent dyes on the cell surface, the remaining fluorescence indicates the internalized antibodies. This method helps provide a clear and quantitative measure of antibody uptake by the cells. 

Find anti-dye antibodies 

Figure 4. Internalization of 15cl12 antibody. (A) Quantitative analysis of internalization: Percent quenched fluorescence of the 15cl12 antibody was measured using an Alexa Fluor 488 quenching antibody. Edox cells were treated with 15cl12 or a non-binding control antibody, cetuximab (CX), for 0.5, 2, and 4 hours at 37°C or on ice. The percent quenched fluorescence calculated as (1–mean fluorescence intensity (MFI) (quenched sample)/MFI (non-quenched sample) × 100. This analysis highlights the internalization efficiency over time and under different conditions. (B) Microscopic visualization: Fluorescence microscopy images show the internalization and localization of the 15cl12 antibody (green) in Edox and Cdox cell lines, with Hoechst 33342 staining (blue) marking the nuclei. The images were taken after incubation on ice and at 37°C, providing visual confirmation of antibody internalization. The scale bar represents 20 μm. Figure reproduced from International Journal of Molecular Sciences, Rodak et al., 2023, CC BY license.

Rodak, A.; Stadlbauer, K.; Bobbili, M.R.; Smrzka, O.; Rüker, F.; Wozniak Knopp, G. Development of a Cytotoxic Antibody–Drug Conjugate Targeting Membrane Immunoglobulin E-Positive Cells. Int. J. Mol. Sci. 2023, 24, 14997. https://doi.org/10.3390/ijms241914997