Performance and validation of an adaptable multiplex assay for detection of serologic response to SARS-CoV-2 infection or vaccination

Abstract

Measurement of quantitative antibody responses are increasingly important in evaluating the immune response to infection and vaccination. In this study we describe the validation of a quantitative, multiplex serologic assay utilising an electrochemiluminescence platform, which measures IgG against the receptor binding domain (RBD), spike S1 and S2 subunits and nucleocapsid antigens of SARS-CoV-2. The assay displayed a sensitivity ranging from 73 to 91% and specificity from 90 to 96% in detecting previous infection with SARS-CoV-2 depending on antigenic target and time since infection, and this assay highly correlated with commercially available assays. The within-plate coefficient of variation ranged from 3.8-3.9% and the inter-plate coefficient of variation from 11 to 13% for each antigen.

Keywords: COVID-19; SARS-CoV-2; Serology.

Fig. 1

IgG levels in individuals with confirmed COVID-19 vs pre-pandemic controls. Legend: IgG levels as measured by the CEPHR assay against 4 antigens, horizontal line represents median value in each group, dotted line indicates threshold of seropositivity. RBD – receptor binding domain, S1 – S1 subunit, S2 - S2 subunit, N - nucleocapsid. Significance testing performed with Kruskall Wallis tests, *** denotes p value <0.001.

Fig. 2

Standard curves for each component antigen in the CEPHR assay. Legend – Dashed vertical lines denote upper and lower limits of detection of the assay. ECL – electrochemiluminescence, RBD – receptor binding domain, S1 – S1 subunit, S2 - S2 subunit, N - nucleocapsid.

Fig. 3

Antibody concentrations in 5 controls run on 10 plates across 3 days. Legend – Concentrations of each of the 5 controls run on 10 occasions across 3 days. Middle line represents the mean and upper and lower line represent one standard deviation from the mean.

Fig. 4

Receiver Operating Characteristic curves for each antigen. Legend: ROC curves constructed using the pROC package. Area Under the Curve (AUC) was 0.88 for RBD, 0.91 for S1, 0.96 for S2 and 0.9 for N respectively. RBD – receptor binding domain, S1 – S1 subunit, S2 - S2 subunit, N - nucleocapsid.

Fig. 5

CEPHR RBD correlation with Abbott and MSD Vplex spike assays. Legend: A: Correlation between CEPHR RBD and Abbott SARS-CoV-2 IgG II anti spike assay. Vertical dashed line represents CEPHR RBD positivity threshold, horizontal dashed line indicates Abbott positivity threshold. B: Correlation between CEPHR RBD and MSD V-PLEX Spike IgG. Vertical dashed line represents CEPHR RBD positivity threshold, no positivity threshold provided by MSD.