Traditional antibody generation, using either phage display or animal immunization, relies on purified antigens. Many membrane proteins, such as G protein-coupled receptors, solute carriers, or ion channels, are important drug targets but very challenging for the formation of antibodies due to the difficulty of protein purification. Whole-cell panning is an alternative approach for generating antibodies without the need for antigen purification. However, it often suffers from background interference and therefore requires extensive screening with low success rates. Here, we develop a new phage selection method, dubbed affinity-tag-guided proximity selection (A-GPS), to efficiently isolate specific antibodies directly from the antigen-presenting cells. By engineering a genetically fused affinity tag for the target antigen, A-GPS confines the proximity labeling reaction near the target antigen and preferentially enriches the phage bound to the target antigen. Using surface-presented GFP on human cells as a model antigen, we demonstrated that A-GPS successfully enriched the antigen-specific clones in two rounds of selection. Among the 46 randomly picked clones, >95% of clones showed great affinity and specificity for GFP over the background of HEK293T surface proteins. One of the best clones expressed as a Fab fragment showed subnanomolar binding affinity for GFP. This clone was successfully applied to common biological applications, such as immunofluorescence and flow cytometry, reflecting the usefulness of A-GPS for generating commercial-grade antibodies.

中文翻译：

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使用亲和标记引导的邻近选择对细胞进行直接抗体分离

使用噬菌体展示或动物免疫的传统抗体产生依赖于纯化的抗原。许多膜蛋白，例如G蛋白偶联受体，溶质载体或离子通道，都是重要的药物靶标，但由于蛋白纯化困难，对于抗体的形成非常具有挑战性。全细胞淘选是无需抗原纯化即可产生抗体的另一种方法。然而，它经常遭受背景干扰，因此需要以低成功率进行广泛的筛选。在这里，我们开发了一种新的噬菌体选择方法，称为亲和标记引导的邻近选择（A-GPS），可以直接从抗原呈递细胞中有效分离出特异性抗体。通过对目标抗原进行基因融合的亲和标签，A-GPS将邻近标记反应限制在目标抗原附近，并优先富集与目标抗原结合的噬菌体。使用人细胞表面呈递的GFP作为模型抗原，我们证明了A-GPS在两轮选择中成功地富集了抗原特异性克隆。在46个随机选择的克隆中，> 95％的克隆在HEK293T表面蛋白的背景下显示出对GFP的巨大亲和力和特异性。表达为Fab片段的最好的克隆之一显示出对GFP的亚纳摩尔结合亲和力。该克隆已成功应用于常见的生物学应用，例如免疫荧光和流式细胞术，反映了A-GPS产生商业级抗体的有用性。