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.

中文翻译：

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细胞表面蛋白标记抗体持久性的量化

介绍

抗体是标记表面蛋白的重要研究工具，但可能会影响与它们结合的蛋白质和细胞的行为。正因为如此，研究人员和临床医生对这些抗体的持久性感兴趣，特别是对于活细胞应用。我们为研究人员开发了一种易于采用的方法来表征任何细胞-抗体组合的抗体去除时间线，这有助于研究广泛的、假设的抗体去除机制。

方法

我们开发了一种方法，使用四种实验条件来阐明影响抗体去除的可能因素的贡献：细胞增殖、内化、永久解离和环境扰动。该方法在脂肪来源的干细胞和人肺成纤维细胞系上用抗 CD44、CD90 和 CD105 抗体进行了测试。在 10 天内每天使用荧光二抗探测一抗的持久性。基于四种培养条件之间的差异，量化了抗体去除机制的相对贡献。

结果

5 天后，超过 90% 的每种测试抗体不再存在于两种细胞类型的表面上，在标记后仅 1 天就观察到去除。抗CD90抗体主要通过环境扰动去除，抗CD105抗体通过内化去除，抗CD44抗体通过所有四种因素的组合去除。

结论

抗体去除机制取决于测试的特定抗体，而同一抗体的去除时间线更多地取决于细胞类型。这种方法应该与有兴趣量化不受限制地使用抗体标记细胞的初始时间框架的研究人员广泛相关。