A Novel Cell-\nand Virus-Free SARS-CoV‑2 Neutralizing\nAntibody ELISA Based on Site-Specific Labeling Technology

The\nemergence of severe acute respiratory syndrome coronavirus\n2 (SARS-CoV-2) led to the global spread of coronavirus disease 2019\n(COVID-19), creating an urgent need for updated methods to evaluate\nimmune responses to vaccines and therapeutic strategies. In this study,\nwe introduce a novel cell-free, virus-free SARS-CoV-2 neutralizing\nantibody ELISA (NAb-ELISA), which is based on competitive inhibition\nof the receptor binding domain (RBD) of spike protein binding to the\nangiotensin-converting enzyme 2 (ACE2) receptor. In this method, site-specific\nbiotinylated hACE2-Fc-Avi recombinant protein is immobilized onto\na 96-well plate for capture, and the RBD-Fc-vHRP recombinant proteins\nserve as detection probes. Evaluation of sera from wild type (WT)\nor Delta RBD-immunized mice using the NAb-ELISA and pseudovirus neutralization\ntests (pVNTs) demonstrated strong correlations between assays (<i>R</i><sup>2</sup> = 0.91 and 0.90 for the WT and Delta groups,\nrespectively). Additionally, the NAb-ELISA successfully detected cross-neutralizing\nactivity in sera, though with slightly lower correlation to pVNT (<i>R</i><sup>2</sup> = 0.70–0.83). By employing NAb-ELISA\ninstead of an indirect ELISA for hybridoma screening, five monoclonal\nantibodies (mAbs) with neutralizing activities against WT, Delta,\nand BA.2 pseudoviruses were obtained. This assay offers a straightforward,\nrapid, and safe approach to characterizing vaccine-induced antibody\nresponses and mAb neutralization activity. Notably, the NAb-ELISA\nplatform can be quickly adapted to assess neutralizing antibody responses\nagainst emerging mutant strains, addressing the rapid mutation of\nthe virus.