Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA.
Biointerfaces Institute, University of Michigan, Ann Arbor, MI, USA.
Methods Mol Biol. 2022;2491:471-490. doi: 10.1007/978-1-0716-2285-8_22.
Conformational antibodies specific for amyloid-forming peptides and proteins are important for a range of biomedical applications, including detecting, inhibiting, and potentially treating protein aggregation disorders ranging from Alzheimer's to Parkinson's diseases. Generation of anti-amyloid antibodies is greatly complicated by the complex, heterogeneous and insoluble nature of amyloid antigens. Here we describe systematic methods for isolating and affinity maturing anti-amyloid antibodies using yeast surface display. Magnetic-activated cell sorting is used to sort single-chain antibody libraries positively for binding to amyloid antigens and negatively against the corresponding disaggregated antigens to remove antibodies that bind in a conformation-independent manner. Isolated lead antibody clones with conformational specificity are affinity matured via targeted CDR mutagenesis and magnetic-activated cell sorting.
针对淀粉样形成肽和蛋白质的构象抗体在一系列生物医学应用中非常重要，包括检测、抑制和潜在治疗从阿尔茨海默病到帕金森病等蛋白质聚集疾病。由于淀粉样抗原的复杂、异质和不溶性，产生抗淀粉样抗体的过程非常复杂。在这里，我们描述了使用酵母表面展示来分离和亲和成熟抗淀粉样抗体的系统方法。磁激活细胞分选用于正向分选与淀粉样抗原结合、负向分选与相应解聚抗原结合的单链抗体文库，以去除以构象非依赖性方式结合的抗体。具有构象特异性的分离先导抗体克隆通过靶向 CDR 突变和磁激活细胞分选进行亲和力成熟。
