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. 2020 Sep:130:104572.
doi: 10.1016/j.jcv.2020.104572. Epub 2020 Aug 2.

Evaluation of a novel multiplexed assay for determining IgG levels and functional activity to SARS-CoV-2

Marina Johnson  1 , Helen R Wagstaffe  1 , Kimberly C Gilmour  2 , Annabelle Lea Mai  2 , Joanna Lewis  1 , Adam Hunt  1 , Jake Sirr  1 , Christopher Bengt  1 , Louis Grandjean  3 , David Goldblatt  4
Affiliations

Evaluation of a novel multiplexed assay for determining IgG levels and functional activity to SARS-CoV-2

Marina Johnson et al. J Clin Virol. 2020 Sep.

Abstract

Background: The emergence of SARS-CoV-2 has led to the development of serological assays that could aid in an understanding of the burden of COVID-19 disease. Many available tests lack rigorous evaluation and therefore results may be misleading.

Objectives: The aim of this study was to assess the performance of a novel multiplexed immunoassay for the simultaneous detection of antibodies against SARS-CoV-2 trimeric spike (S), spike receptor binding domain (RBD), spike N terminal domain and nucleocapsid antigen and a novel pseudo-neutralisation assay.

Methods: A multiplexed solid-phase chemiluminescence assay (Meso Scale Discovery) was evaluated for the simultaneous detection of IgG binding to four SARS-CoV-2 antigens and the quantification of antibody-induced ACE-2 binding inhibition (pseudo-neutralisation assay). Sensitivity was evaluated with a total of 196 COVID-19 serum samples (169 confirmed PCR positive and 27 anti-nucleocapsid IgG positive) from individuals with mild symptomatic or asymptomatic disease. Specificity was evaluated with 194 control serum samples collected from adults prior to December 2019.

Results: The specificity and sensitivity of the binding IgG assay was highest for S protein with a specificity of 97.4 % and sensitivity of 96.2 % for samples taken 14 days and 97.9 % for samples taken 21 days following the onset of symptoms. IgG concentration to S and RBD correlated strongly with percentage inhibition measured by the pseudo-neutralisation assay.

Conclusion: Excellent sensitivity for IgG detection was obtained over 14 days since onset of symptoms for three SARS-CoV-2 antigens (S, RBD and N) in this multiplexed assay which can also measure antibody functionality.

Keywords: Covid19; Immunoassays; Immunology; SARS-CoV-2.

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Figures

Fig. 1
Fig. 1
Anti-SARS-CoV-2 IgG concentration. The concentration of SARS-CoV-2 antibody against (a) spike (S), (b) receptor binding domain (RBD), (c) nucleocapsid (N) and (d) N terminal domain (NTD) was measured using the MSD coronavirus panel. Graphs show data in arbitrary units (AU) (based on the calibrated internal standard serum) in the COVID-19 cohort (n = 196) and controls (n = 194, pre-December 2019). Line shows positive/negative discrimination cut-off.
Fig. 2
Fig. 2
Receiver Operating Characteristic (ROC) curves for each SARS-CoV-2 antigen. Sensitivity and specificity were calculated using each value in the data table as a cut-off value (n = 390). Graphs show the sensitivity vs 100 %-specificity of SARS-CoV-2 antigen (a) spike (S), (b) receptor binding domain (RBD), (c) nucleocapsid (N) and (d) N terminal domain (NTD). The area under curve (AUC) and 95 % CI is also shown for each antigen.
Fig. 3
Fig. 3
Anti-SARS-CoV-2 IgG concentration according to time since onset of symptoms. Graphs show the concentration of SARS-CoV-2 antibody against (a) spike (S), (b) receptor binding domain (RBD) and (c) nucleocapsid (N) in arbitrary units (AU) (based on the calibrated internal standard serum) of the COVID-19 cohort split in to intervals of 0−7 days, 8–14 days, 15–21 days and over 21 (>21) days since symptom onset (to sample collection). Error bars show geometric mean with 95 % CI, line shows positive/negative discrimination cut-off, *p < 0.05, ** p < 0.01 determined by Dunn’s multiple comparisons test. Comparisons across interval groups had p < 0.0001 by one-way ANOVA Kruskal-Wallis test. The assay sensitivity at each time point is shown in Table 3.
Fig. 4
Fig. 4
IgG concentration relationship between antigens. Correlation between anti-SARS-CoV-2 antibody concentration of all COVID-19 group samples (n = 196) (a) S vs RBD, (b) S vs N and (c) N vs RBD. r and p value were determined by Spearman correlation. p values of <0.05 were considered as significant.
Fig. 5
Fig. 5
Percentage inhibition by anti-SARS-CoV-2 S and RBD antibody measured by MSD pseudo-neutralisation assay. Inhibition of ACE-2 binding by SARS-CoV-2 antibody against (a) spike (S) and (b) receptor binding domain (RBD) was measured using the MSD coronavirus pseudo-neutralisation assay. 183 COVID-19 cohort samples and 194 control samples were analysed. Graphs show median and 95 % CI with a line showing neutralisation assay positive/negative discrimination cut-off determined by ROC. The correlation between antibody concentration and percentage inhibition of (c) S and (d) RBD antigens in all positive group samples was assessed and r and p was determined by Spearman correlation, line shows binding assay positive/negative discrimination cut-off.
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