Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this bottleneck by developing a workflow combining cell-free DNA template generation, cell-free protein synthesis, and binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to evaluate 135 previously published antibodies targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including all 8 antibodies previously granted emergency use authorization for coronavirus disease 2019 (COVID-19), and demonstrate identification of the most potent antibodies. We also evaluate 119 anti-SARS-CoV-2 antibodies from a mouse immunized with the SARS-CoV-2 spike protein and identify neutralizing antibody candidates, including the antibody SC2-3, which binds the SARS-CoV-2 spike protein of all tested variants of concern. We expect that our cell-free workflow will accelerate the discovery and characterization of antibodies for future pandemics and for research, diagnostic, and therapeutic applications more broadly.

抗体发现受到个体表达和抗原特异性命中评估的瓶颈。在这里，我们通过开发一个工作流程来解决这个瓶颈，该工作流程将无细胞 DNA 模板生成、无细胞蛋白质合成和抗体片段的结合测量相结合，整个过程需要数小时而不是数周。我们应用此工作流程来评估 135 种先前发布的针对严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 的抗体，包括先前获得 2019 年冠状病毒病 (COVID-19) 紧急使用授权的所有 8 种抗体，并证明了对最有效的抗体。我们还评估了来自用 SARS-CoV-2 刺突蛋白免疫的小鼠的 119 种抗 SARS-CoV-2 抗体，并确定了候选中和抗体，包括抗体 SC2-3、它与所有测试的相关变体的 SARS-CoV-2 刺突蛋白结合。我们期望我们的无细胞工作流程将加速抗体的发现和表征，以用于未来的流行病以及更广泛的研究、诊断和治疗应用。