The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific antibodies in the serum of an individual indicates previous infection or vaccination. However, it provides limited insight into the protective nature of this immune response. Neutralizing antibodies recognizing the viral spike protein are more revealing, yet their measurement traditionally requires virus- and cell-based systems that are costly, time-consuming, inflexible, and potentially biohazardous. Here, we present a cell-free quantitative neutralization assay based on the competitive inhibition of trimeric SARS-CoV-2 spike protein binding to the angiotensin-converting enzyme 2 (ACE2) receptor. This high-throughput method matches the performance of the gold standard live virus infection assay, as verified with a panel of 206 seropositive donors with varying degrees of infection severity and virus-specific immunoglobulin G titers, achieving 96.7% sensitivity and 100% specificity. Furthermore, it allows for the parallel assessment of neutralizing activities against multiple SARS-CoV-2 spike protein variants of concern. We used our assay to profile serum samples from 59 patients hospitalized with coronavirus disease 2019 (COVID-19). We found that although most sera had high activity against the 2019-nCoV parental spike protein and, to a lesser extent, the α (B.1.1.7) variant, only 58% of serum samples could efficiently neutralize a spike protein derivative containing mutations present in the β (B.1.351) variant. Thus, we have developed an assay that can evaluate effective neutralizing antibody responses to SARS-CoV-2 spike protein variants of concern after natural infection and that can be applied to characterize vaccine-induced antibody responses or to assess the potency of monoclonal antibodies.

在个体血清中检测到严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2) 特异性抗体表明先前感染或接种过疫苗。然而，它对这种免疫反应的保护性质提供了有限的了解。识别病毒刺突蛋白的中和抗体更具启发性，但它们的测量传统上需要基于病毒和细胞的系统，这些系统昂贵、耗时、不灵活且具有潜在的生物危害性。在这里，我们提出了一种基于三聚体 SARS-CoV-2 刺突蛋白与血管紧张素转换酶 2 (ACE2) 受体结合的竞争性抑制的无细胞定量中和试验。这种高通量方法与金标准活病毒感染检测的性能相匹配，经一组 206 名感染严重程度和病毒特异性免疫球蛋白 G 滴度不同的血清反应阳性供体验证，达到 96.7% 的敏感性和 100% 的特异性。此外，它还允许并行评估针对多种 SARS-CoV-2 刺突蛋白变体的中和活性。我们使用我们的检测分析了 59 名 2019 年冠状病毒病 (COVID-19) 住院患者的血清样本。我们发现，尽管大多数血清对 2019-nCoV 亲本刺突蛋白具有高活性，并且在较小程度上对 α (B.1.1.7) 变体具有高活性，但只有 58% 的血清样本能够有效中和含有突变的刺突蛋白衍生物存在于 β (B.1.351) 变体中。因此，