Standardization of enzyme-linked immunosorbent assays for serosurveys of the SARS-CoV-2 pandemic using clinical and at-home blood sampling

Abstract

The extent of SARS-CoV-2 infection throughout the United States population is currently unknown. High quality serology is a key tool to understanding the spread of infection, immunity against the virus, and correlates of protection. Limited validation and testing of serology assays used for serosurveys can lead to unreliable or misleading data, and clinical testing using such unvalidated assays can lead to medically costly diagnostic errors and improperly informed public health decisions. Estimating prevalence and clinical decision making is highly dependent on specificity. Here, we present an optimized ELISA-based serology protocol from antigen production to data analysis. This protocol defines thresholds for IgG and IgM for determination of seropositivity with estimated specificity well above 99%. Validation was performed using both traditionally collected serum and dried blood on mail-in blood sampling kits, using archival (pre-2019) negative controls and known PCR-diagnosed positive patient controls. Minimal cross-reactivity was observed for the spike proteins of MERS, SARS1, OC43 and HKU1 viruses and no cross reactivity was observed with anti-influenza A H1N1 HAI titer during validation. This strategy is highly specific and is designed to provide good estimates of seroprevalence of SARS-CoV-2 seropositivity in a population, providing specific and reliable data from serosurveys and clinical testing which can be used to better evaluate and understand SARS-CoV-2 immunity and correlates of protection.

SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness

“…In early January 2020, a novel CoV (nCoV) was identified as the cause of a respiratory virus outbreak occurring in Wuhan, China. Within 24 hours of the release of the SARS-CoV-2 isolate sequences (then known as “2019-nCoV”) on January 10 th , the 2P mutations were substituted into S positions aa986 and 987 to produce prefusion-stabilized SARS-CoV-2 S (S-2P) protein for structural analysis 22 and serological assay development 23,24 in silico without additional experimental validation. Within 5 days of sequence release, current Good Manufacturing Practice (cGMP) production of mRNA/LNP expressing the SARS-CoV-2 S-2P as a transmembrane-anchored protein with the native furin cleavage site (mRNA-1273) was initiated in parallel with preclinical evaluation.…”

Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays

“…To support an NIH-led serosurvey on the extent of the coronavirus immune response in~10,000 human samples, our lab was tasked to produce RBD and spike proteins for ELISA assay optimization and deployment [4]. We found our RBD production yield to be similar to published reports, however, spike production was problematic with our initial attempts producing inconsistent results and lower yields than https://doi.org/10.1016/j.pep.2020.105686 Received 29 May 2020; Received in revised form 1 June 2020; Accepted 1 June 2020 Abbreviations: AnSEC, analytical size exclusion chromatography; CV, column volume; ELISA, enzyme-linked immunosorbent assay; IMAC, immobilized metal ion affinity chromatography; RBD, receptor binding domain; SEC, size exclusion chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TEM, transmission electron microscopy; TFF, tangential flow filtration; VRC, Vaccine Research Center * Corresponding author.…”

Optimizing high-yield production of SARS-CoV-2 soluble spike trimers for serology assays

“…To support an NIH-led serosurvey on the extent of the coronavirus immune response in ~ 10,000 human samples, our lab was tasked to produce RBD and spike proteins for ELISA assay optimization and deployment [4]. We found our RBD production yield to be similar to published reports, however, spike production was problematic with our initial attempts producing inconsistent results and lower yields than those reported in the literature.…”

“…In order to assess batch-to-batch reproducibility of spike antigens, purified VRC spike proteins from five different purifications were used as antigens in an ELISA with positive control CoV-2 serum at multiple dilutions. The ELISA protocol was carried out as reported [4] using 100 ng per well of the various spike proteins and dilutions of 1:500, 1:10,000, and 1:100,000.…”

“…Taken together, these improvements allowed the production of sufficient spike antigen for more than 500 ELISA plates per liter of culture and generated enough protein from a single 4-liter expression to support a robust serosurvey being conducted by the NIH [4]. Florian Krammer (Icahn School of Medicine, Mt.…”