Quantification of Antibody Persistence for Cell Surface Protein Labeling

Area of Science:

• Cell biology
• Immunology
• Biotechnology

Background:

• Antibodies are crucial research tools for cell surface protein labeling.
• Antibody binding can alter cell and protein behavior, impacting live-cell applications.
• Understanding antibody persistence is vital for experimental design and interpretation.

Purpose of the Study:

• To develop and validate a method for quantifying antibody removal timelines from cell surfaces.
• To investigate the mechanisms driving antibody removal, including cell proliferation, internalization, dissociation, and environmental perturbation.
• To assess the impact of cell type and antibody specificity on removal rates and mechanisms.

Main Methods:

• A novel method was employed using four distinct experimental conditions to isolate factors influencing antibody removal.
• The method was tested on adipose-derived stem cells and human lung fibroblasts using antibodies against CD44, CD90, and CD105.
• Antibody persistence was monitored daily for 10 days using fluorescent secondary antibodies.

Main Results:

• Over 90% of tested antibodies were removed from cell surfaces within 5 days, with significant removal observed as early as 1 day post-labeling.
• The primary removal mechanism varied: anti-CD90 by environmental perturbation, anti-CD105 by internalization, and anti-CD44 by a combination of factors.
• Antibody removal timelines were more dependent on cell type than the specific antibody used.

Conclusions:

• Antibody removal mechanisms are antibody-specific, while removal rates are influenced by cell type.
• The developed method provides a quantitative framework for determining antibody removal timeframes.
• This research is broadly applicable to scientists using antibody-labeled cells in live-cell applications.