DT3C Protein: A Convenient Tool for Antibody Internalization Analysis

DT3C Protein: A Convenient Tool for Antibody Internalization Analysis

Simple preparation

Multi species applicability

Rapid evaluation

High internalization efficiency

DT3C is a recombinant protein mainly used for in vitro evaluation of internalization efficiency of cancer cell towards monoclonal antibodies (mAb), thereby enabling the screening of more effective monoclonal antibodies. This protein consists of a diphtheria toxin (DT) protein without a receptor binding domain and Streptococcus protein G.

DT3C schematic diagram

Application Areas of DT3C:

  • In vitro evaluation of antibody internalization efficiency
  • ADC drug research
  • Antibody screening and evaluation

Technical Advantages of DT3C:

Compared to currently common techniques for evaluating antibody internalization efficiency, such as acid buffer elution, mAb-ZAP, pHrodo technology, the DT3C recombinant protein assay offers the advantages of simplicity in operation and precise quantification. It exhibits broad application prospects in the field of antibody internalization.

  • Simple preparation: mAb-DT3C conjugates can be formed by incubating at room temperature for 30 minutes.
  • Multi species applicability: It can can combine with any IgG from different species to form mAb-DT3C conjugates.
  • Rapid evaluation: The internalization efficiency of mAb on cell surfaces can be quickly and accurately detected using techniques such as flow cytometry or fluorescence microscopy.
  • High internalization efficiency: The internalization efficiency of antibodies is significantly higher than that of Mab ZAP.
In vitro detection of ADC antibody internalization efficiency
In vitro detection of ADC antibody internalization efficiency

In vitro detection of ADC antibody internalization efficiency

Reference: M. Yamaguchi et al. Biochemical and Biophysical Research Communications 454 (2014) 600-603.

DT3C Recombinant Protein from CUSABIO:

The DT3C recombinant protein from CUSABIO (CSB-EP360556CQR1) is expressed from Escherichia coli expression system. It's verified by SDS-PAGE and detected by SEC-HPLC, with a purity of over 90%.

DT3C protein SDS-PAGE
DT3C protein with CCR8 antibody

After incubating different concentrations of DT3C protein with CCR8 antibody at 37°C for 30 minutes, the mixture was added to a culture plate and incubated for 48 hours. The range of EC50 values are 0.5795-1.400 μg/mL and 0.5608-1.318 μg/mL respectively.

CT26/Human CCR8 Stable Cell Line

CT26/Human CCR8 Stable Cell Line: 5000 cells/well
BMK-1/2: 10μg/mL

How to use DT3C to detect antibody internalization efficiency?

Experimental steps:

  • Cell culture conditions: Cultivate the cells to be tested under appropriate culture conditions.
  • Antibody and DT3C conjugation: Incubate mAb and DT3C at room temperature for 30 minutes to form mAb-DT3C conjugates.
  • Cell treatment: Add mAb-DT3C conjugates to the cell culture medium to interact with cells.
  • Evaluation of internalization efficiency: Detect the internalization efficiency of mAb on the cell surface using flow cytometry, fluorescence microscopy, or other methods.
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