A multiplex microsphere IgG assay for SARS-CoV-2 using ACE2-mediated inhibition as a surrogate for neutralization

Cameron, Andrew; Porterfield, Claire A; Byron, Larry; Wang, Jiong; Pearson, Zachary; Bohrhunter, Jessica L; Cardillo, Anthony B; Ryan-Muntz, Lindsay; Sorensen, Ryan A; Caserta, Mary; Angeloni, Steven; Hardy, Dwight J; Zand, Martin; Pecora, Nicole Danielle

The COVID-19 pandemic has highlighted the challenges inherent to the serological detection of a novel pathogen such as SARS-CoV-2. Serological tests can be used diagnostically and for surveillance, but their usefulness depends on their throughput, sensitivity and specificity. Here, we describe a multiplex fluorescent microsphere-based assay, 3Flex, that can detect antibodies to three major SARS-CoV-2 antigens--spike (S) protein, the spike ACE2 receptor-binding domain (RBD), and nucleocapsid (NP). Specificity was assessed using 213 pre-pandemic samples. Sensitivity was measured and compared to the Abbott ARCHITECT SARS-CoV-2 IgG assay using serum samples from 125 unique patients equally binned (n = 25) into 5 time intervals ([≤]5, 6 to 10, 11 to 15, 16 to 20, and [≥]21 days from symptom onset). With samples obtained at [≤]5 days from symptom onset, the 3Flex assay was more sensitive (48.0% vs. 32.0%), but the two assays performed comparably using serum obtained [≥]21 days from symptom onset. A larger collection (n = 534) of discarded sera was profiled from patients (n = 140) whose COVID-19 course was characterized through chart review. This revealed the relative rise, peak (S, 23.8; RBD, 23.6; NP, 16.7; in days from symptom onset), and decline of the antibody response. Considerable interperson variation was observed with a subset of extensively sampled ICU patients. Using soluble ACE2, inhibition of antibody binding was demonstrated for S and RBD, and not for NP. Taken together, this study described the performance of an assay built on a flexible and high-throughput serological platform that proved adaptable to the emergence of a novel infectious agent.