Antibody Internalization

Measure cellular internalization of a panel of antibodies in a single well, enabling high throughput antibody discovery 

Figure 2. Efficient assessment of antibody activity using iQue Forecyt^®.

T-cells were used in an antibody internalization assay to generate high-content data in each well of a 384 well plate. Multiple antibodies were labeled with the antibody internalization reagent before incubation with T-cells in the same well. (A) A plate based heatmap of the internalization of the different antibodies in T-cells for easy visualization. (B) Serial dilution curves show internalization profiles of multiple labeled antibodies in T-cells.

Facilitate your discoveries using validated reagents for rapid quantification of ADCP in 96 or 384-well plates.

Figure 3. Functional screening and viability analysis in both non-adherent and adherent cell types. 

With a slight alteration to the protocol the assay can be adapted to be used with adherent cell types. Data shows concentration related activity for two therapeutic antibodies, Rituxan (anti-CD20) in suspension Ramos cells and Herceptin (anti-Her2) in adherent AU565 cells with concurrent assessment of cellular viability. Cells were seeded in 96-well plates, antibodies complexed with ABI reagent were added in combination with the iQue^® Cell Membrane Integrity dye and incubated for 3h. Suspension cells were immediately analyzed on the iQue^® platform, while adherent cells were carefully detached and resuspended prior to analysis.  

Collect, analyze and present complex data quickly and easily, driving rapid decision making 

Figure 4. Internalization of Herceptin antibody isotypes in Her2+ AU565 and Her2- MDA-MB 468 cells.

Cells were seeded in a 96 well plate and treated with varying isotypes of Herceptin complexed with the ABI reagent (A. AU565, B. MDA-MB-468). No internalization was measured in the Her2- cells.  Serial dilution curves were also performed for the Herceptin isotypes in AU565 (C) displaying varying degrees of activity.     

Simultaneously assess specific antibody internalization in phenotypically distinct cell types co-cultured in a single well

Figure 5. Internalization analysis of co-cultured cells in a single well provides robust data from small volumes of sample. 

Prior to seeding cells in a 96 well plate, cells were stained with different concentrations of the non-perturbing iQue^® Cell Encoding Dye (V/Blue). Both mono and co-cultures were left to adhere overnight. Herceptin or hIgG1, complexed with the antibody internalization reagent were added for 3h prior to analysis using the iQue^® platform. The scatter plot (A) enables easy identification of all three cell types when mixed in the same well, based on the encoding dye. Internalization can be assessed in each population (B), maximizing the information obtained from the test antibody while minimizing the quantity of precious sample and test antibody used. 

Figure 6. Schematic of the iQue^® Antibody Internalization Kit highlighting its simplicity and ease of use. 

The assay consists of 3 components, each 10 µL: labelled test antibody, cells (Violet encoding optional), and iQue^® Cell Membrane Integrity Dye (B/Green). Each component is prepared at 3X before addition for a final concentration of 1X and an assay volume of 30 µL. 

Easy labelling step can be performed in full media suitable for early stage hybridoma screening, low quantity of antibody required, can be tested on multiple cell types for multiplex screening – internalization and cell health readout.