Huang Aric, Jin Wei, Fahad Ahmed S, Mussman Brooklyn K, Nicchia Grazia Paola, Madan Bharat, de Souza Matheus Oliveira, Griffin J Daniel, Bennett Jeffrey L, Frigeri Antonio, Berkland Cory J, DeKosky Brandon J
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66044, USA.
Department of Chemical Engineering, The University of Kansas, Lawrence, KS 66044, USA.
Antibodies (Basel). 2022 Jun 5;11(2):39. doi: 10.3390/antib11020039.
A rapid and effective method to identify disease-specific antibodies from clinical patients is important for understanding autoimmune diseases and for the development of effective disease therapies. In neuromyelitis optica (NMO), the identification of antibodies targeting the aquaporin-4 (AQP4) membrane protein traditionally involves the labor-intensive and time-consuming process of single B-cell sorting, followed by antibody cloning, expression, purification, and analysis for anti-AQP4 activity. To accelerate patient-specific antibody discovery, we compared two unique approaches for screening anti-AQP4 antibodies from yeast antibody surface display libraries. Our first approach, cell-based biopanning, has strong advantages for its cell-based display of native membrane-bound AQP4 antigens and is inexpensive and simple to perform. Our second approach, FACS screening using solubilized AQP4 antigens, permits real-time population analysis and precision sorting for specific antibody binding parameters. We found that both cell-based biopanning and FACS screening were effective for the enrichment of AQP4-binding clones. These screening techniques will enable library-scale functional interrogation of large natively paired antibody libraries for comprehensive analysis of anti-AQP4 antibodies in clinical samples and for robust therapeutic discovery campaigns.
从临床患者中识别疾病特异性抗体的快速有效方法对于理解自身免疫性疾病以及开发有效的疾病治疗方法至关重要。在视神经脊髓炎（NMO）中，传统上鉴定靶向水通道蛋白4（AQP4）膜蛋白的抗体涉及单B细胞分选这一劳动密集且耗时的过程，随后是抗体克隆、表达、纯化以及抗AQP4活性分析。为了加速患者特异性抗体的发现，我们比较了两种从酵母抗体表面展示文库中筛选抗AQP4抗体的独特方法。我们的第一种方法，基于细胞的生物淘选，因其对天然膜结合AQP4抗原进行基于细胞的展示而具有显著优势，且成本低廉、操作简单。我们的第二种方法，使用可溶性AQP4抗原的荧光激活细胞分选（FACS）筛选，允许对特定抗体结合参数进行实时群体分析和精确分选。我们发现基于细胞的生物淘选和FACS筛选对于富集AQP4结合克隆均有效。这些筛选技术将能够对大型天然配对抗体文库进行文库规模的功能研究，以全面分析临床样本中的抗AQP4抗体，并开展强有力的治疗发现活动。
