• Comprehensive platform

    Comprehensive platform

    • 8 diverse assay formats available for ADC evaluation
    • 80+ free assay positive controls provided with dose-dependent curves
    • 200+ tumor cells and 100+ overexpression cell lines
  • Upgraded strategy

    Upgraded strategy

    • Toxin-based cytotoxicity assay eligible for hybridoma supernatant screening
    • Real-time live cell imaging-based internalization assay
  • Extensive experience

    Extensive experience

    • 50+ executed internalization projects
    • Dozens of targets verified including CD20, HER2, ROR1 and TROP2

Service Highlight

GenScript ProBio provides comprehensive solutions for antibody internalization, cell cytotoxicity assays and bystander effect, including 6 different assay formats tailored to meet the specific needs of our clients. Our services cover naked antibodies as well as ADCs.

Scope Internalization assay Cytotoxicity assay Bystander effect
Assay format Live-cell imaging based assay Toxin-conjugated mAb based cytotoxicity assay pH-indicator based internalization Temperature shift-based internalization Cell growth inhibition assay Co-culture assay Medium transfer assay
Molecule Naked Antibody ADC or Toxin ADC ADC
Price Low Low High Medium Low High Low
Analyzer Live-Cell Analyzer Microplate Reader Flow cytometer Flow cytometer Microplate Reader / Live-Cell Analyzer Flow cytometer Live-Cell Analyzer Microplate Reader
Feature Real-time and kinetics analysis High sensitivity
High throughput Close to the cytotoxic MOA
Classic pH-sensitive method Cost-effective Susceptible to multiple factors Cytotoxic MOA Cost-effective Specific Real-time Specific Cost-effective

・Live cell imaging-based assay

The Incucyte® live-cell analysis system enables direct detection of antibody internalization in a 96-well plate. By one step of dye addition, a rapid, kinetic and high-throughput analysis of antibody internalization can be achieved.

  • Advantages:

    Single experiment evaluates time-dependent changes and concentration-dependent curves of antibody internalization, suitable for pharmacological dynamic quantitative analysis.

    Real-time observation of live cells, with continuous monitoring up to 72 hours with a minimum detection interval of 15 minutes.

    Applicable for high-throughput screening.

Figure 2a depicts the internalization of anti-ROR1 antibody (UC-961) or hIgG1 isotype control labeled with Incucyte® FabFluor, where the internalization level of anti-ROR1 antibody gradually increases with time. Figure 2b shows CHO-K1/DDR1 cells treated with anti-DDR1 antibody (AB3E3) rapidly increasing in the red target area and displaying a dose-response curve.

Figure 2: real-time live cell imaging-based internalization screening assay.

・Toxin-based cytotoxicity assay

  • GenScript ProBio employs a recombinant immunotoxin (IT) conjugated to the test antibody. Upon internalization of the mAb-IT complex by target cells, the IT is released into the cytosol, causing ADP-ribosylation of elongation factor (EF)-2, which subsequently inhibits protein translation and ultimately leads to cell cytotoxicity. The internalization efficiency of the antibody is assessed by measuring the cell killing effect.

  • Figure 3: Mechanism of immunotoxin induced cytotoxicity


Strong specificity: only when the mAb-IT conjugate is internalized by target cells, a significant decrease in cell viability can be observed, and free immunotoxins do not cause cell toxicity.

High efficiency: mAb-IT conjugates have a more stable molecular weight and higher internalization efficiency compared to the traditional mAb-ZAP method.

Wide applicability: suitable for early screening of antibodies in hybridoma supernatant ; applicable to different species (such as human, mouse, rabbit, and goat), and different antibody subtypes.

Low cost: easy to prepare while simulating the MOA of ADC drugs.

  • Figure 4: Toxin-based cytotoxicity assay.

  • Dose-dependent internalization rate of trastuzumab was determined by the immunotoxin-induced cell toxicity upon binding to SK-BR-3 cells after 24-48 hours of incubation.

・Classical internalization assay

  • pH probe-based assay: antibodies labeled with pH probes sensitive to pH are only able to release fluorescence signals when they are engulfed by target cells and enter acidic intracellular compartments. The fluorescence signals can be captured and analyzed using flow cytometry.

    Cell surface fluorescence quenching: the internalization level of the test antibody can be traced by incubating at 37°C and 4°C respectively, and using the secondary antibody labeling method to track the antibodies left on the surface.

  • pH probe-based assay

These two classical internalization screening methods have been widely applied in ADC naked antibody screening due to their simplicity and flow cytometry-based detection. However, the endpoint detection nature of these methods can limit their ability to capture dynamic changes sequentially and may result in changes in cell status due to a lack of environmental control, thereby affecting the accuracy of the results.

  • Case study 1: cell surface fluorescence quenching

    Analysis of internalization internalization can be performed by quantifying the remaining antibody on the cell surface using secondary antibodies. This indirect method can reflect the internalization efficiency of the cells.

  • Case study 2: pH probe-based assay

    Antibodies labelled with pH probes undergo internalization and sequentially translocate to the lysosomes, where the acidic pH induces fluorescence enhancement of the pH probes.

・Cytotoxicity assay

Cell viability assays can be used to measure the ability of cytotoxic compounds to cause cellular damage or death, and are a critical factor in evaluating the efficacy and intended use of such compounds.

Quantification of cell viability using the CellTiter-Glo® assay kit revealed that the dose-response curve data exhibited suppression of SK-BR-3 cell viability after addition of the tested ADC samples.

Service package

Assay format Assay description Sample Deliverable Timeline
Live cell imaging-based assay Dose-dependent testing and time course testing Antibodies and/or ADCs (toxin molecules) (GenScript ProBio offers 80+ free assay positive controls for free) Raw data, processed data, figures, and summary report. 2-3 weeks
Toxin-based cytotoxicity assay Dose-dependent testing 2-3 weeks
pH probe-based assay Dose-dependent testing 2-3 weeks
Time course testing 2-3 weeks
Cell surface fluorescence quenching Dose-dependent testing 2-3 weeks
Time course testing 2-3 weeks
Cytotoxicity assay Dose-dependent testing 2-3 weeks
Bystander effect High, medium, low concentration testing 3-4 weeks