A Multiplex Microsphere IgG Assay for SARS-CoV-2 Using ACE2-Mediated Inhibition as a Surrogate for Neutralization

Abstract

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 Abbott⃝ ARCHITECT⃝ 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.

Antibody Mediated Immunity to SARS-CoV-2 and Human Coronaviruses: Multiplex Beads Assay and Volumetric Absorptive Microsampling to Generate Immune Repertoire Cartography

“…This was true even when serum samples with high-level antibodies against other HCoVs were assayed. These results are consistent with our previously published SARS2 antibody clinical study (13).…”

“…For a positive control standard, we used a mixture of equal volumes from four serum specimens selected from a COVID-19 positive cohort collected in April 2020 from 140 unique patients (13). These sere were selected for high IgG titers against SARS-CoV-1, SARS-CoV2, HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E.…”

“…In-depth serologic analyses are essential for understanding the prevalence of, and immunity to, SARS-CoV-2. Recently, a number of antibody binding assays based on the S or N antigens have become available, primarily enzyme-linked immunosorbent assays (ELISAs) (11) and lateral flow assays (LFAs) (12), as well some Luminex assay based multiplex assays (13)(14)(15)(16). However, these assays are primarily focused on the SARS-CoV-2 S and/or N proteins, and not the broad range of CoV Cross-reactivity.…”

A flexible, pan-species, multi-antigen platform for the detection and monitoring of SARS-CoV-2-specific antibody responses

“…During the course of this work, many reports of serological diagnostic assays, including those based upon the Luminex format (15)(16)(17)(18)(19)(20)(21)(22)(23), have been presented. Rather than developing an additional diagnostic platform, we emphasize here the utility of the Luminex system as a research tool -for monitoring changes in antibody responses over time, detecting antibody responses in different sample matrices and for rapid adaptation and evaluation of new antigen formulations.…”

“…Using this tool, we reconfirm the wide variation in the generation and maintenance, as well as antigen-specificity of SARS-CoV-2-specific antibodies in different individuals/animals and using multiple sample types, as well as the various patterns of change in these antibody levels over time post-infection. Including multiple SARS-CoV-2 antigens in a single assay not only potentially increases the sensitivity of detection of infection history (17), it also enhances the relative depth of the data on immune response patterns, without the time and sample volume requirements of individual assays. The inclusion of reference antigens such as influenza nucleoproteins and tetanus toxoid provide confidence that the observed changes in SARS-CoV-2-specific responses are not the result of sample quality/storage.…”