Antibody Internalization

Antibodies specific to cell surface antigens induce endocytosis which leads to the cellular internalization of those antibodies along with any molecules conjugated to them. This process, termed Antibody Internalization (ABI), and has many powerful applications for research and clinical discovery, such as delivery of highly toxic drugs to specific cancer cells via antibody drug conjugates (ADCs), removal or degradation of surface receptors from cancer cells (i.e. EGFR), and antibody immunotherapies used to identify tumor cells for immune cell killing (i.e. ADCC or ADCP).

The therapeutic significance of antibodies as effective treatments for disease takes in a wide variety of areas including cancer, autoimmune disorders, cardiovascular disease, inflammation and infectious diseases. In order to develop antibodies for clinical use, it is imperative to assess their potential therapeutic significance through effective in vitro functional assays. Traditional techniques for measuring ABI often:

  • Have low-throughput acquisition and long sample times due to the requirement of large sample volumes (e.g. traditional flow cytometry)
  • Use assays that are complicated to configure due to the optimization of signal resolution and compensations
  • Are laborious and time-consuming, requiring steps such as antibody labeling, protocol optimization, fixation and repetitive washes
  • Require complicated manual data analysis
  • Necessitate the use of large volumes of precious sample and antibody

The iQue® Antibody Internalization assay utilizes the iQue® Antibody Internalization Kit in combination with the iQue® Advanced High-throughput Flow Cytometer, for high-throughput measurement of antibody internalization through a simplified, streamlined workflow. Save precious samples, time and get real time pharmacological analysis using integrated iQue® Forecyt software. Quantifying Antibody Internalization for a panel of antibodies across adherent or suspension cells has never been easier!


Concept

Figure 1. The pH-sensitive fluorescent probe principle. 

A novel pH-sensitive fluorescent probe enables a rapid one-step, no-wash labeling of isotype-matched antibodies in complete, serum-containing medium. A fluorescent signal is generated as internalized antibody is processed into the acidic endosome and lysosome pathway. Antibody internalization is quantified as the percentage of live cells positive for the fluorescent probe. Cellular viability is measured using the included iQue® Cell Membrane Integrity dye.  

Key Advantages

Key Advantages

Maximize your productivity

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.

Flexible assay format 

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.  

Streamline data acquisition and 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.     

Gain more biological insights from your precious samples

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.

Frequently Asked Questions

The iQue® Antibody Internalization Kits have been validated using an acquisition range of every hour over the course of a total of 4 hours.  This range was found to be suitable for a number of receptors however optimization is advised for every receptor being analyzed. 

The antibody internalization reagent is conjugated to antibodies by binding the Fc region.  Whatever antibody type being used it must have this Fc region in order to be labelled.

The iQue® is an advanced flow cytometer giving it significant advantage over traditional flow cytometry and confocal microscopy which are other methods used for antibody characterization.  Traditional techniques have lengthy acquisition times due to either large sample volumes (traditional flow) or needing to be imaged and these assays are generally more complex to optimize.  These alternative methods are also much more laborious and time-consuming requiring steps such as antibody labelling, protocol optimization, fixation and repetitive washes.  And unlike the iQue® Antibody Internalization Kit, these methods require complicated manual data analysis. 

Ordering Information

Platform: Compatible with iQue® 3/iQue® Screener Plus - VBR Configuration
Product

Size

Catalog Number

iQue® Human Antibody Internalization Reagent

1 x 96 wells
1 x 384 wells

90564
90565

Platform: Compatible with iQue® 3/iQue® Screener Plus - VBR Configuration
Product

Size

Catalog Number

iQue® Mouse Antibody Internalization Reagent

1 x 96 wells
1 x 384 wells

90566
90567

Platform: Compatible with iQue® 3/iQue® Screener Plus - VBR Configuration

Product

Size

Catalog Number

iQue® Human Multiplex Antibody Internalization Kit

1 x 96 wells
5 x 96 wells
1 x 384 wells
5 x 384 wells

97046
97047
97048
97049

Platform: Compatible with iQue® 3/iQue® Screener Plus - VBR Configuration

Product

Size

Catalog Number

iQue® Mouse Multiplex Antibody Internalization Kit

1 x 96 wells
5 x 96 wells
1 x 384 wells
5 x 384 wells

97050
97051
97052
97053

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Resources

Literature and Documentation

iQue® Brochure

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Brochure: iQue® Reagent Kits

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Product Guide

Antibody Internalization

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Protocol

Adherent Cell Lifting

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White Paper

Antibody internalization: Advanced flow cytometry and Live-cell analysis give rich insights during antibody profiling

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Poster

High throughput, multiple antibody internalization assay

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