Antibody library screens using detergent-solubilized mammalian cell lysates as antigen sources
Antibody library screens using detergent-solubilized mammalian cell lysates as antigen sources
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DOI：
10.1093/protein/gzq029
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发表时间：
2010-07-01
影响因子：
2.4
通讯作者：
Shusta, Eric V.
中科院分区：
文献类型：
--
作者：
Cho, Yong Ku;Shusta, Eric V.
针对细胞组分的抗体的高通量产生目前是蛋白质组学、治疗开发和其他生物应用中的挑战。由于膜蛋白在水溶液中的不溶性，产生靶向膜蛋白的抗体特别具有挑战性。为了解决这些问题，直接针对靶细胞系的去污剂溶解的和生物素化的裂解物有效地筛选人单链抗体片段（scFv）的酵母展示文库，从而避免膜蛋白不溶性的问题并消除对抗原的异源表达或纯化的需要。根据所应用的生物素化方法，鉴定特异性结合质膜蛋白或细胞内蛋白的抗体克隆。也可以使用细胞裂解物作为抗原来源来鉴定针对预定靶标的抗体，如通过选择针对转铁蛋白受体（TfR）的scFv所证明的。当从酵母分泌并纯化时，所选择的scFv在不存在去污剂的生理条件下具有活性。此外，该方法允许靶抗原的简易表征，因为它与酵母展示免疫沉淀相容。我们期望该方法将证明可用于多重亲和试剂生成和靶向抗体筛选。
High-throughput generation of antibodies against cellular components is currently a challenge in proteomics, therapeutic development and other biological applications. It is particularly challenging to raise antibodies that target membrane proteins due to their insolubility in aqueous solutions. To address these issues, a yeast display library of human single-chain antibody fragments (scFvs) was efficiently screened directly against detergent-solubilized and biotinylated lysates of a target cell line, thereby avoiding issues with membrane protein insolubility and eliminating the need for heterologous expression or purification of antigens. Antibody clones that specifically bind plasma membrane proteins or intracellular proteins were identified, depending on the biotinylation method applied. Antibodies against a predetermined target could also be identified using cell lysate as an antigen source as demonstrated by selecting an scFv against the transferrin receptor (TfR). When secreted from yeast and purified, the selected scFvs are active under physiological conditions in the absence of detergents. In addition, this method allows facile characterization of target antigens because it is compatible with yeast display immunoprecipitation. We expect that this method will prove useful for multiplex affinity reagent generation and in targeted antibody screens.