Validation of a combined ELISA to detect IgG, IgA and IgM antibody responses to SARS-CoV-2 in mild or moderate non-hospitalised patients

Abstract

Background: Frequently SARS−CoV−2 results in mild or moderate disease with potentially lower concentrations of antibodies compared to those that are hospitalised. Here, we validated an ELISA using SARS−CoV−2 trimeric spike glycoprotein, with targeted detection of IgG, IgA and IgM (IgGAM) using serum and dried blood spots (DBS) from adults with mild or moderate disease. Methods: Targeting the SARS−CoV−2 trimeric spike, a combined anti−IgG, IgA and IgM serology ELISA assay was developed using 62 PCR−confirmed non-hospitalised, mild or moderate COVID−19 samples, ≥14 days post symptom onset and 624 COVID−19 negative samples. The assay was validated using 73 PCR−confirmed non−hospitalised COVID−19 and 359 COVID−19 negative serum samples with an additional 81 DBSs, and further validated in 226 PCR−confirmed non−hospitalised COVID−19 and 426 COVID−19 negative clinical samples. Results: A sensitivity and specificity of 98.6% (95% CI, 92.6−100.0), 98.3% (95% CI, 96.4−99.4), respectively, was observed following validation of the SARS−CoV−2 ELISA. No cross−reactivities with endemic coronaviruses or other human viruses were observed, and no change in results were recorded for interfering substances. The assay was stable at temperature extremes and components were stable for 15 days once opened. A matrix comparison showed DBS to correlate with serum results. Clinical validation of the assay reported a sensitivity of 94.7% (95% CI, 90.9−97.2%) and a specificity of 98.4% (95% CI, 96.6−99.3%). Conclusions: The human anti−IgGAM SARS−CoV−2 ELISA provides accurate and sensitive detection of SARS−CoV−2 antibodies in non-hospitalised adults with mild or moderate disease. The use of dried blood spots makes the assay accessible to the wider community.

SARS -CoV-2 T-cell immunity to variants of concern following vaccination

“…The clinical specificity of this assay is reported to be 98.4% [95% CI, 96.6–99.3%], and sensitivity 94.7% [95% CI, 90.9–97.2%]. 2…”

Site-specific steric control of SARS-CoV-2 spike glycosylation

“…is important for therapeutic and vaccine design to understand whether similar glycoforms arise across different protein expression platforms from different sources to ensure the antigenic surface of the S protein remains consistent when used as an immunogen or in serological assays [44][45][46][47] .This is particularly important as glycan processing can be impacted by adverse protein conformations 48 . Here, we describe an integrated approach including liquid chromatography-mass spectrometry (LC-MS) and MD simulations to understand the glycosylation features across recombinant S proteins from different sources.…”

Longitudinal protection following natural SARS-CoV-2 infection and early vaccine responses: insights from a cohort of community based dental health care professionals

“…The percentage of individuals who lose detectable serological responses after 3 months (30%) is higher than that reported in other studies. This is most likely due to the sensitivity of the screening assay that measures the total antibody against the spike glycoprotein [10]. This approach identifies individuals who are exposed to the virus with high sensitivity but some individual appear to mount modest serological responses that are not associated with durable immunity.…”

“…Serological analysis was performed using a commercially available, CE marked, IgGAM ELISA that measures the total antibody response (IgG, IgA and IgM simultaneously) against the spike glycoprotein (Product code: MK654, The Binding Site (TBS), Birmingham). Detailed descriptions of this assay including its construction, validation and verification have been published previously [10, 11]. The assay is optimized for seroepidemiological studies and demonstrates 98.3% (95% CI: 96.4-99.4%) specificity and 98.6% sensitivity (95% CI: 92.6-100%) in detecting a serological response to the SARS-CoV-2 spike glycoprotein following PCR-positive, non-hospitalised, mild-to-moderate COVID-19.…”