Volk Anna-Luisa, Hu Francis Jingxin, Rockberg Johan
School of Biotechnology, AlbaNova University Center, KTH - Royal Institute of Technology, Stockholm, Sweden.
Methods Mol Biol. 2014;1131:485-500. doi: 10.1007/978-1-62703-992-5_29.
The unique property of specific high-affinity binding 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 back 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.
抗体与几乎任何感兴趣的靶标具有特异性高亲和力结合的独特特性，这使得抗体在众多应用中极为有用。因此，了解抗体在靶蛋白上的精确结合位点（表位）是理解其性能并确定其在免疫测定中可靠性的最重要特征之一。在此，我们描述了一种基于抗原片段的细菌表面表达，随后进行基于抗体的流式细胞术分选来绘制抗体表位的高分辨率方法。通过对分选的抗体结合细胞进行DNA测序，然后将序列与抗原序列进行比对来确定表位。这里描述的方法对于绘制具有不同表位大小的单克隆抗体和多克隆抗体的表位都很有用。
