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

The COVID-19 pandemic has highlighted challenges inherent to serological detection of a novel pathogen like SARS-CoV-2. Serological tests can be used diagnostically and for surveillance, but their usefulness depends on throughput, sensitivity and specificity. Here, we describe a multiplex fluorescent microsphere-based assay, 3Flex, that can detect antibodies to three 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 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.