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Quantification of Antibody Persistence for Cell Surface Protein Labeling

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Abstract

Introduction

Antibodies are an essential research tool for labeling surface proteins but can potentially influence the behavior of proteins and cells to which they bind. Because of this, researchers and clinicians are interested in the persistence of these antibodies, particularly for live-cell applications. We developed an easily adoptable method for researchers to characterize antibody removal timelines for any cell–antibody combination, with the benefit of studying broad, hypothesized mechanisms of antibody removal.

Methods

We developed a method using four experimental conditions to elucidate the contributions of possible factors influencing antibody removal: cell proliferation, internalization, permanent dissociation, and environmental perturbation. This method was tested on adipose-derived stem cells and a human lung fibroblast cell line with anti-CD44, CD90, and CD105 antibodies. The persistence of the primary antibody was probed using a fluorescent secondary antibody daily over 10 days. Relative contributions by the antibody removal mechanisms were quantified based on differences between the four culture conditions.

Results

Greater than 90% of each antibody tested was no longer present on the surface of the two cell types after 5 days, with removal observed in as little as 1 day post-labeling. Anti-CD90 antibody was primarily removed by environmental perturbation, anti-CD105 antibody by internalization, and anti-CD44 antibody by a combination of all four factors.

Conclusions

Antibody removal mechanism depended on the specific antibody tested, while removal timelines for the same antibody depended more on cell type. This method should be broadly relevant to researchers interested in quantifying an initial timeframe for uninhibited use of antibody-labeled cells.

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Acknowledgments

The authors would like to acknowledge Ryan Dubay for creating the MATLAB image analysis script used in the study. Funding support was provided by NIH/NIAMS (R01 AR063642, EMD) and Brown University’s Undergraduate Teaching and Research Award (OWB).

Author Contributions

MED, OWB, and EMD designed all experiments. OWB conducted preliminary optimization experiments, initial antibody removal iterations, and antibody dilution/concentration experiments. MED carried out all remaining experiments and iterations as well as conducted final analysis and interpretation of data. MED and EMD wrote the manuscript with figure contributions from OWB.

Conflict of interest

Megan E. Dempsey, Olivia Woodford-Berry, and Eric M. Darling declare that they have no conflicts of interest.

Ethical Standards

No human or animal studies were carried out by the authors for this article.

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Authors and Affiliations

  1. Center for Biomedical Engineering, Brown University, Providence, RI, 02912, USA

    Megan E. Dempsey & Eric M. Darling

  2. Departmant of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI, 02912, USA

    Olivia Woodford-Berry & Eric M. Darling

  3. School of Engineering, Brown University, Providence, RI, 02912, USA

    Eric M. Darling

  4. Departmant of Orthopaedics, Brown University, Providence, RI, 02912, USA

    Eric M. Darling

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  1. Megan E. Dempsey
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Correspondence to Eric M. Darling.

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Dempsey, M.E., Woodford-Berry, O. & Darling, E.M. Quantification of Antibody Persistence for Cell Surface Protein Labeling. Cel. Mol. Bioeng. 14, 267–277 (2021). https://doi.org/10.1007/s12195-021-00670-3

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