A scalable serology solution for profiling humoral immune responses to SARS-CoV-2 infection and vaccination

Abstract

BACKGROUND: Testing for antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in detecting previous exposures and analyzing vaccine-elicited immune responses. Here, we describe a scalable "Made-in-Canada" solution that can detect and quantify SARS-CoV-2 antibodies, discriminate between natural infection- and vaccination-induced responses, and assess antibody-mediated inhibition of the spike-angiotensin converting enzyme 2 (ACE2) interaction. METHODS: We developed a set of methods and reagents to detect SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA). The main assays focus on the parallel detection of immunoglobulin (Ig)Gs against the spike trimer, its receptor binding domain (RBD), and the nucleocapsid (N) protein. These antigens are complemented by a detection antibody (human anti-IgG fused to horseradish peroxidase (HRP)) and a positive control reference antibody (recombinant IgG against the RBD), permitting intra- and inter-laboratory comparisons. Using this toolkit and commercial reagents, we optimized automated ELISAs on two different high throughput platforms to measure antibody responses to SARS-CoV-2 antigens. The assays were calibrated to a reference standard from the World Health Organization. We also automated a surrogate neutralization (sn)ELISA that measures inhibition of ACE2-Spike or -RBD interactions by antibodies using biotinylated ACE2. RESULTS: Our individual IgG-based ELISAs measure antibody levels in single-point measurements in reference to a standard antibody curve to accurately distinguish non-infected and infected individuals (area under the curve > 0.96 for each assay). Positivity thresholds can be established in individual assays using precision-recall analysis (e.g., by fixing the false positive rate), or more stringently, by scoring against the distribution of the means of negative samples across multiple assays performed over several months. For seroprevalence assessment (in a non-vaccinated cohort), classifying a sample as positive if antibodies were detected for at least 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti-spike and -RBD (but not -N) antibodies are observed. Here, we present detailed protocols to perform these assays using either serum/plasma or dried blood spots both manually and on two automated platforms, and to express the results in international units to facilitate data harmonization and inter-study comparisons. We also demonstrate that the snELISA can be performed automatically at single points, increasing the scalability of this functional assay for large seroprevalence studies. INTERPRETATION: The ability to measure antibodies to three viral antigens and identify neutralizing antibodies capable of disrupting spike-ACE2 interactions in high-throughput assays enables large-scale analyses of humoral immune responses to SARS-CoV-2 infection and vaccination. The "Made-in-Canada" set of protein reagents, produced at the National Research Council of Canada are publicly available to enable the up-scaling of standardized serological assays, permitting nationwide data comparison and aggregation.

Differences in mRNA-1273 (Moderna) and BNT162b2 (Pfizer-BioNTech) SARS-CoV-2 vaccine immunogenicity among patients undergoing dialysis

“…We measured SARS-CoV-2 anti-spike, anti-RBD and anti-NP immunoglobulin G (IgG) antibodies on an automated enzyme-linked immunosorbent assay (ELISA) platform as reported previously 7 – 9 at 6–7 and 12 weeks after 2-dose vaccination. Antibody levels were reported as relative ratios to a synthetic standard included as a calibration curve on each assay plate.…”

What factors converged to create a COVID-19 hot-spot? Lessons from the South Asian community in Ontario

“…Blood was collected on the WhatmanTM 903® Protein Saver Cards. Batched specimens were sent for analysis to the CITF-funded lab of our collaborator (Dr. Langlois) for analysis and were processed and analyzed using a high throughput ELISA assay (10,11). The main assays focused on the parallel detection of immunoglobulin IgG against the spike trimer (S), and the nucleocapsid (N) protein with standardized antigens to S, and N produced by the National Research Council of Canada laboratory.…”

“…The false discovery rate (FDR) was set at 2% for the IgG S, and 3% for IgG to N; These cut-offs have > 98% accuracy when validated by the National Microbiology Laboratory reference panel (11). The background seasonal coronavirus antibody mean titers in the Ottawa region was used as reference (10,13).…”

SARS-CoV-2 Virus-Like Particle Neutralizing Capacity in Blood Donors Depends on Serological Profile and Donor-Declared SARS-CoV-2 Vaccination History

“…For this study, 1,500 specimens each from January, February, and March of 2021 (total: n = 4,500) were analyzed with previously published assays ( 5 , 6 , 19 ) for SARS-CoV-2 antigens: anti-Spike (S), anti-receptor-binding domain (RBD), and anti-nucleocapsid (N) (Sinai Health anti-N and Abbott anti-N) ( Fig. 1 ).…”

“…IgG analysis was undertaken at the Lunenfeld-Tanenbaum Research Institute, Sinai Health, on all retained specimens. Assays were anti-spike (anti-S), anti-receptor-binding domain (anti-RBD), and anti-nucleocapsid protein (anti-N), as previously described ( 19 ). Ratio-converted enzyme-linked immunosorbent assay (ELISA) reads were undertaken as previously described ( 5 , 20 ), and cutoffs (positive) of each of the targets were ≥0.396 for N, ≥0.186 for RBD, and ≥0.190 for S ( 2 ).…”