Rare antibody phage isolation and discrimination (RAPID) biopanning enables identification of high-affinity antibodies against challenging targets.
Rare antibody phage isolation and discrimination (RAPID) biopanning enables identification of high-affinity antibodies against challenging targets.
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DOI：
10.1038/s42003-023-05390-0
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发表时间：
2023-10-12
影响因子：
5.9
通讯作者：
Craik, Charles S.
中科院分区：
文献类型：
--
作者：
Chung, Dong Hee;Kong, Sophie;Young, Nicholas J.;Chuo, Shih-Wei;Shiah, Jamie V.;Connelly, Emily J.;Rohweder, Peter J.;Born, Alexandra;Manglik, Aashish;Grandis, Jennifer R.;Johnson, Daniel E.;Craik, Charles S.
使用合成噬菌体展示抗体库的体外生物淘选平台能够识别针对曾经被认为超出免疫范围的抗原的抗体。将这些方法应用于具有挑战性的目标仍然是一个严峻的挑战。在这里，我们提出了一种新的生物淘选流程，RAPID（稀有抗体噬菌体分离和鉴别），用于鉴定针对具有挑战性的靶标的稀有高亲和力抗体。 RAPID 生物淘选使用荧光标记的噬菌体展示片段抗原结合 (Fab) 抗体库，通过荧光激活分选来分离高亲和力结合物。随后，使用生物层干涉测量 (BLI) 方法 BIAS（生物层干涉测量抗体筛选）进行歧视性命中筛选，其中根据估计的动力学解离速率对候选结合物进行排序和优先排序。使用先前报道的抗体来开发该方法，并将 RAPID 生物淘选流程应用于三个具有挑战性的靶标（CHIP、Gαq 和 CS3D），从而能够鉴定高亲和力抗体。 FACS 辅助选择过程与所选克隆的表征相结合，有助于噬菌体展示筛选，从文库中获得针对给定靶标的抗体。
In vitro biopanning platforms using synthetic phage display antibody libraries have enabled the identification of antibodies against antigens that were once thought to be beyond the scope of immunization. Applying these methods against challenging targets remains a critical challenge. Here, we present a new biopanning pipeline, RAPID (Rare Antibody Phage Isolation and Discrimination), for the identification of rare high-affinity antibodies against challenging targets. RAPID biopanning uses fluorescent labeled phage displayed fragment antigen-binding (Fab) antibody libraries for the isolation of high-affinity binders with fluorescent activated sorting. Subsequently, discriminatory hit screening is performed with a biolayer interferometry (BLI) method, BIAS (Biolayer Interferometry Antibody Screen), where candidate binders are ranked and prioritized according to their estimated kinetic off rates. Previously reported antibodies were used to develop the methodology, and the RAPID biopanning pipeline was applied to three challenging targets (CHIP, Gαq, and CS3D), enabling the identification of high-affinity antibodies. A FACS aided selection process combined with characterization of the selected clones facilitates phage display screening to obtain antibodies against a given target from libraries.
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