Quantification of Antibody Persistence for Cell Surface Protein Labeling.

Abstract

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. 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 10days. Relative contributions by the antibody removal mechanisms were quantified based on differences between the four culture conditions. Greater than 90% of each antibody tested was no longer present on the surface of the two cell types after 5days, with removal observed in as little as 1day 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. 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. The online version contains supplementary material available at 10.1007/s12195-021-00670-3.