Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, CB2 1GA Cambridge, U.K.
Antibody Discovery and Protein Engineering, R&D, AstraZeneca, Milstein Building, Granta Park, Cambridge CB21 6GH, U.K.
Anal Chem. 2021 Feb 2;93(4):2166-2173. doi: 10.1021/acs.analchem.0c03992. Epub 2021 Jan 4.
The precise determination of affinity and specificity is a crucial step in the development of new protein reagents for therapy and diagnostics. Paradoxically, the selection of protein binders, e.g., antibody fragments, from large combinatorial repertoires is a rapid process compared to the subsequent characterization of selected clones. Here we demonstrate the use of suspension bead arrays (SBA) in combination with flow cytometry to facilitate the post-selection analysis of binder affinities. The array is designed to capture the proteins of interest (POIs) covalently on the surface of superparamagnetic color-coded microbeads directly from expression cell lysate, based on SpyTag-SpyCatcher coupling by isopeptide bond formation. This concept was validated by analyzing the affinities of a typical phage display output, i.e., clones consisting of single-chain variable fragment antibodies (scFvs), as SpyCatcher fusions in 12- and 24-plex SBA formats using a standard three-laser flow cytometer. We demonstrate that the equilibrium dissociation constants () obtained from multiplexed SBA assays correlate well with experiments performed on a larger scale, while the antigen consumption was reduced >100-fold compared to the conventional 96-well plate format. Protein screening and characterization by SBAs is a rapid and reagent-saving analytical format for combinatorial protein engineering to address specificity maturation and cross-reactivity profiling of antibodies.
准确确定亲和力和特异性是开发用于治疗和诊断的新型蛋白质试剂的关键步骤。矛盾的是，与随后对选定克隆的表征相比，从大型组合库中选择蛋白质结合剂（例如抗体片段）是一个快速的过程。在这里，我们展示了悬浮珠阵列（SBA）与流式细胞术结合使用，以促进结合物亲和力的选择后分析。该阵列旨在根据异肽键形成的 SpyTag-SpyCatcher 偶联，直接从表达细胞裂解物中将感兴趣的蛋白质（POI）共价捕获到超顺磁性彩色编码微珠的表面上。通过分析典型噬菌体展示产物的亲和力来验证该概念，即由单链可变片段抗体（scFv）组成的克隆作为 SpyCatcher 融合体，在 12 plex 和 24-plex SBA 格式中使用标准三激光流式细胞仪进行分析。我们证明，从多路复用 SBA 测定获得的平衡解离常数（Kd）与在更大规模上进行的实验很好地相关，而与传统的 96 孔板格式相比，抗原消耗减少了 100 多倍。通过 SBAs 进行蛋白质筛选和表征是一种快速且节省试剂的组合蛋白质工程分析方法，可用于解决抗体的特异性成熟和交叉反应性分析。
