DT3C, a New Tool for Antibody Internalization Assay

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

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 is expressed from Escherichia coli expression system. It's verified by SDS-PAGE and detected by SEC-HPLC, with a purity of over 90%.

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: 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.