Rockberg Johan, Löfblom John, Hjelm Barbara, Ståhl Stefan, Uhlén Mathias
School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden.
Curr Protoc Immunol. 2010 Aug;Chapter 9:Unit9.9. doi: 10.1002/0471142735.im0909s90.
Antibodies have proven to be invaluable tools for a vast number of applications during the last decades, including protein purification and characterization, medical diagnosis and imaging, and treatment using therapeutic antibiotics. No matter what the aims of the application are, the antibody's binding characteristics will still be the main features determining the assay's reliability. Here, we describe a protocol for determination of antibody-binding epitopes using an antigen-focused, library-based approach where library members are generated by fragmentation of antigen DNA and presented as cloned peptides on the cell surface of the Gram-positive bacterium Staphylococcus carnosus. The rigid cell structure of this organism allows for multivalent expression and permits rapid library analysis and sorting of antibody-binding cells using flow-sorting devices. Epitopes are determined by DNA sequencing of the sorted cells and alignment back to the antigen sequence. The protocol described here has been shown useful for mapping of both monoclonal and polyclonal binders with varying epitope lengths.
在过去几十年中，抗体已被证明是用于大量应用的宝贵工具，包括蛋白质纯化与表征、医学诊断与成像以及使用治疗性抗体的治疗。无论应用目的为何，抗体的结合特性仍将是决定检测可靠性的主要特征。在此，我们描述了一种使用基于文库的抗原聚焦方法来确定抗体结合表位的方案，其中文库成员通过抗原DNA片段化产生，并作为克隆肽呈现在革兰氏阳性菌肉葡萄球菌的细胞表面。这种生物体的刚性细胞结构允许多价表达，并允许使用流式分选设备对抗体结合细胞进行快速文库分析和分选。通过对分选细胞进行DNA测序并与抗原序列比对来确定表位。这里描述的方案已被证明可用于绘制具有不同表位长度的单克隆和多克隆结合物。
