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The unique property to specifically bind to more or less any target of interest has made antibodies tremendously useful in numerous applications. Hence, knowledge of the precise binding site (epitope) of antibodies on the target protein is one of the most important features for understanding its performance and determining its reliability in immunoassays. Here, we describe a high-resolution method for mapping epitopes of antibodies based on bacterial surface expression of antigen fragments followed by antibody-based flow cytometric sorting. Epitopes are determined by DNA sequencing of the sorted antibody-binding cells followed by sequence alignment to the antigen sequence. The method described here has been useful for the mapping of both monoclonal and polyclonal antibodies with varying sizes of epitopes.

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Epitope Mapping of Antibodies Using Bacterial Cell Surface Display of Gene Fragment Libraries

  • 实验方案

  • 发表日期：

    2025年

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  • Anna-Luisa Volk,
  • Francis Jingxin Hu,
  • Magdalena Malm,
  • Johan Rockberg

摘要

The unique property to specifically bind to more or less any target of interest has made antibodies tremendously useful in numerous applications. Hence, knowledge of the precise binding site (epitope) of antibodies on the target protein is one of the most important features for understanding its performance and determining its reliability in immunoassays. Here, we describe a high-resolution method for mapping epitopes of antibodies based on bacterial surface expression of antigen fragments followed by antibody-based flow cytometric sorting. Epitopes are determined by DNA sequencing of the sorted antibody-binding cells followed by sequence alignment to the antigen sequence. The method described here has been useful for the mapping of both monoclonal and polyclonal antibodies with varying sizes of epitopes.