Abstract In light of the COVID‐19 pandemic, there is an ongoing need for diagnostic tools to monitor the immune status of large patient cohorts and the effectiveness of vaccination campaigns. Here, we present 11 unique nanobodies (Nbs) specific for the SARS‐CoV‐2 spike receptor‐binding domain (RBD), of which 8 Nbs potently inhibit the interaction of RBD with angiotensin‐converting enzyme 2 (ACE2) as the major viral docking site. Following detailed epitope mapping and structural analysis, we select two inhibitory Nbs, one of which binds an epitope inside and one of which binds an epitope outside the RBD:ACE2 interface. Based on these, we generate a biparatopic nanobody (bipNb) with viral neutralization efficacy in the picomolar range. Using bipNb as a surrogate, we establish a competitive multiplex binding assay (“NeutrobodyPlex”) for detailed analysis of the presence and performance of neutralizing RBD‐binding antibodies in serum of convalescent or vaccinated patients. We demonstrate that NeutrobodyPlex enables high‐throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, to monitor immune status or to guide vaccine design. This study describes the generation of biparatopic nanobodies as surrogates for monitoring neutralizing immune responses in SARS‐CoV‐2 infected individuals. NeutrobodyPlex enables high‐throughput screening and detailed analyses of infected or vaccinated individuals.
【저자키워드】 immunology, immune response, Neutralizing antibodies, structural biology, SARS‐CoV‐2, Microbiology, Virology & Host Pathogen Interaction, Serological assay, nanobodies, 【초록키워드】 Efficacy, ACE2, vaccination, pandemic, antibody, Vaccine design, diagnostic, docking, COVID‐19, SARS‐CoV‐2, serum, nanobody, RBD, Effectiveness, Neutralizing, viral neutralization, epitope mapping, multiplex, convalescent, epitope, patients, binding, Immune status, Interaction, Analysis, Structural analysis, enzyme, infected individuals, vaccinated individuals, receptor‐binding domain, MONITOR, inhibitory, bind, inhibit, generate, unique, analysis, large patient cohort, 【제목키워드】 SARS‐CoV‐2, nanobody, Neutralizing,