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ADC Internalization Detection Antibody – Labeled with pH-Sensitive Fluorescent Dye

Antibody-drug conjugates (ADCs) are targeted cancer treatments consisting of a monoclonal antibody linked to a cytotoxic drug. The antibody targets specific cancer cell antigens, allowing the drug to be delivered directly to tumors while reducing off-target effects. A key step in the mechanism of action (MOA) of ADCs is receptor-mediated endocytosis.

Internalization testing is not only a critical step in drug screening but also a core tool for mechanism optimization. By quantifying internalization efficiency, researchers can accurately predict ADC behavior in vivo, providing key data support for clinical trials.

To support ADC internalization research, we have developed a specialized Antibody Internalization Detection Reagent (Cat. No. IGG-PZF2001), featuring a pH-sensitive red fluorescent dye that labels the human IgG Fc region. The reagent forms a stable fluorescent complex within 10 minutes, enabling rapid assessment of antibody internalization processes. After labeling, antibodies can be detected using various methods including flow cytometry or cell imaging techniques.

Classic mechanism of action of ADC drugs

Product List

Cat. No.	Product Description	Tests
IGG-PZF2001	Antibody Internalization Detection reagent	100tests/500tests

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Product Features

High Signal-to-Noise Ratio: Strong fluorescence with minimal background

Quick Labeling: Complete in just 10 minutes

pH-Sensitive: Bright signal in acidic intracellular compartments

Fab Region Preserved: Not affect antibody binding

Principles & Applications

Principles

Schematic of antibody internalization detection reagent principle

Applications

Discovery stage:
To identify antibodies that not only bind specifically to tumor-associated antigens but also internalize efficiently.
Lead optimization:
To evaluate how variations in antibody structure or conjugation chemistry affect internalization and intracellular trafficking.
Preclinical development:
To confirm that the selected ADC candidate exhibits consistent internalization behavior across relevant cell models.
Mechanism-of-action studies:
To validate the intracellular delivery of the payload and its correlation with cytotoxic effects.

Validation Data

FACS Analysis of Antibody Internalization

Anti-CD20 Abs and Human IgG1 isotype control were labeled with Antibody Internalization Detection Reagent (Cat.No.IGG-PZF2001). Raji cells were treated with Anti-CD20 Abs-Internalization Detection Reagent conjugate and Isotype control-Internalization Detection Reagent conjugate separately for 2 hours, then analysis by Flow cytometric. APC signal was used to evaluate the activity (Routine tested).

Protocol

Anti-Her2 Abs and Human IgG1 isotype control were labeled with Antibody Internalization Detection Reagent (Cat.No.IGG-PZF2001). SK-BR-3 cells were treated with Anti-Her2 Abs-Internalization Detection Reagent conjugate and Isotype control-Internalization Detection Reagent conjugate separately for 2 hours, then analysis by Flow cytometric. APC signal was used to evaluate the activity (Routine tested).

Protocol

Fluorescence Imaging of Antibody Internalization

SK-BR-3 cells were treated with CellLights Lysosome GFP (green) for 16 hours followed by treatment with Anti-Her2 Abs-Internalization Detection Reagent conjugate and IgG1 Isotype-Internalization Detection Reagent conjugate separately for 16 hours (red), then stained with NucBlue Live ReadyProbes(blue) for 20 minutes and imaged on the EVOS M7000. A. Antibody Internalization Detection Reagent (Cat.No.IGG-PZF2001). B. IgG1 Isotype-Internalization Detection Reagent conjugate. C. Anti-Her2 Abs-Internalization Detection Reagent conjugate. D. Anti-Her2 Abs-Internalization Detection Reagent conjugate(Z-stacking).

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Resources

Webinar playback and appointment

Addressing Challenges in Antibody-Drug Conjugate Development

Here is the brief recap of the webinar:ADCs have the potential to enable 'precision medicine' with a wide market reach.Challenges in ADCs include managing Target Affinity, enhancing payload conjugation, and assessing Payload Delivery in vivo Pharmacokinetics. We can provide high-quality solutions to address these challenges.

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Building the Perfect Antibody-based Therapeutic from Selection to Engineering and Manufacturing

In this webinar, discover how advanced techniques such as Al-driven candidate selection optimization of antibody sequences, and precise conjugation methods come together to address some of the current challenges in antibody-based therapeutics.