key: cord-292578-co5essuw
authors: Johnson, Marina; Wagstaffe, Helen R.; Gilmour, Kimberly C.; Mai, Annabelle Lea; Lewis, Joanna; Hunt, Adam; Sirr, Jake; Bengt, Christopher; Grandjean, Louis; Goldblatt, David
title: Evaluation of a novel multiplexed assay for determining IgG levels and functional activity to SARS-CoV-2
date: 2020-08-02
journal: J Clin Virol
DOI: 10.1016/j.jcv.2020.104572
sha: 
doc_id: 292578
cord_uid: co5essuw

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.

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) was first recognised in January 2020 and rapidly spread world-wide (1) . Tests designed to measure antibodies to SARS-CoV-2 antigens were rapidly developed and are important for diagnostics and seroprevalence studies. The latter could help inform disease burden estimates, studies of transmission dynamics and modelling of the epidemic. Antibody tests are particularly important in the context of mild or asymptomatic disease where a swab reverse transcriptase polymerase chain reaction (RT-PCR) test may be negative. For this reason, an understanding of the sensitivity and specificity of the tests being used is critical.

The trimeric spike (S) protein of SARS-CoV-2 is present on the viral surface and in most cases is cleaved by host proteases into the S1 and S2 subunits, responsible for receptor recognition and membrane fusion respectively. S1 uses a region of the molecule, known as the receptor binding domain (RBD) to bind to host ACE-2 receptor and thereby gain entry to the cell (2) . Specific immunoglobulin-G (IgG) and IgM antibody responses to SARS-CoV-2 S, N and RBD of the spike protein develop between 6-15 days following disease-onset (4) .

Despite a rapid increase in the number and availability of SARS-CoV-2 serologic assays, most have undergone minimal external evaluation and validation (5) . A recent large scale Spanish seroprevalence study used a point of care IgG test with a stated sensitivity of 97.2% but on verification found it to have a sensitivity of either 82.1%, 89.7%, 99.6% or 100% depending on the sample sets used for evaluation (6) . All assays currently suffer from the absence of a defined standard serum so results are reported as positive or negative or as optical density readouts complicating the comparison between assays and studies and for many binding assays the relationship between antibody concentration and function is unclear.

We have evaluated a novel assay designed to simultaneously measure IgG to four SARS-CoV-2 antigens; full-length trimeric S, RBD and NTD of spike as well as N protein. The assay, based on Meso Scale Discovery (MSD) technology, utilises a 96-well based solid-phase antigen printed plate and an electrochemiluminescent detection system. In addition this assay can measure the ability of serum to inhibit the interaction between spike protein components and soluble ACE-2, also called a pseudo-neutralisation assay (7) . To evaluate the sensitivity and specificity of the MSD assay, we were able to utilise a relatively large number of samples obtained from SARS-CoV-2 RT-PCR positive health care workers or patients as well as antibody positive health care staff enrolling in a large SARS-CoV-2 cohort study. 

Samples were screened for IgG to SARS-CoV-2 N protein using a commercially available kit (Epitope Diagnostics Inc, San Diego, USA) as previously described (8) . To measure IgG antibodies, plates were blocked with MSD Blocker A following which reference standard, controls and samples diluted 1:500 in diluent buffer were added. After incubation, detection antibody was added (MSD SULFO-TAG™ Anti-Human IgG Antibody) and then MSD GOLD™ Read Buffer B was added and plates read using a MESO® SECTOR S 600 Reader.

Plates were blocked and washed as above, assay calibrator (COVID-19 neutralising antibody; monoclonal antibody against S protein; 200µg/ml), control sera and test sera samples diluted 1 in 10 in assay diluent were added to the plates. Following incubation Plates an 0.25µg/ml solution of MSD SULFO-TAG™ conjugated ACE-2 was added after which plates were read as above. Percentage inhibition was calculated relative to the assay calibrator (maximum 100% inhibition).

Statistical analysis was performed using MSD Discovery Workbench and GraphPad Prism version 8.0 (GraphPad, San Diego, CA). Antibody concentration in arbitrary units (AU) was interpolated from the ECL signal of the internal standard sample using a 4-parameter logistic curve fit. ROC curves showing the sensitivity and specificity (plotted as 100%-specificity %) calculated using each value in the data as a cut-off were plotted for each antigen. A cut-off antibody concentration was chosen based on the lowest value leading to a positive likelihood ratio (LR) of >10, in order to maximise sensitivity while providing strong evidence to rule-in J o u r n a l P r e -p r o o f infection (9) . For S antigen binding, all LR's were above 10, therefore the LLOD was used as the cut-off for this antigen. Comparisons between groups were performed by Kruskal-Wallis one-way ANOVA with Dunn's correction for multiple comparisons. Correlation analysis was performed using Spearman correlation. P values of <0.05 were considered as significant.

Latent class models with two classes were fitted with the binary antibody responses as outcome variables, using the poLCA package in the R statistical environment. The code used for the latent class analysis is available on request. The lower limit of detection (LLOD) was assigned as 1% of the standard value in AU, and upper limit of detection (ULOD) was assigned for NTD and RBD only as the S and N antigen did not reach an upper limit (Table 1) . For statistical purposes, ULOD was assigned the highest calculated concentration plus 20% and LLOD as 0.5%.

The mean coefficient of variation (CV) between duplicates was <15% for all except NTD (17.4%, data not shown). The mean intra-assay CV was 6.2% and inter-assay variation <15% across all SARS-CoV-2 antigens except NTD (19.0%) on one of four samples (Supplementary The specificity for S, RBD and N assays are shown in Table 2 Table 2 : Assay specificity calculated for each SARS-CoV-2 antigen from the control cohort. Table 3 .

Sensitivity and specificity was calculated for groups 0-7d, >7d, >14d and >21d since the onset of symptom The S antigen was the most sensitive of the three, with a sensitivity of 96.2% and 97.9% >14 days and >21 days respectively (Table 3) . ROCs were plotted to visualise the trade-off between sensitivity and specificity for S and RBD neutralisation. Cut-offs (LR>10) were 0.162% for S and 0.524% for RBD (shown by the dotted line on Figure 5A -B). Sensitivity and specificity for S were 97.8% and 97.9% respectively but lower for RBD (77.2% and 92.8% respectively). In the COVID-19 cohort there were some IgG positive sera that did not demonstrate neutralisation (below cut-off, n= 4 for S and 36 for RBD).

These sera were predominantly those taken soon after the onset of symptoms; 22 between 0-7 days, 9 over 14 days and 5 over 21 days. Using a carefully defined cohort of known SARS-CoV-2 exposed individuals and relevant controls we were able to show the sensitivity and specificity of the assay for the four antigens of interest. Comparing the performance of S and RBD assays in a recently published systematic review and metanalysis of the diagnostic accuracy of serological tests for COVID-19 (10) the S assay we evaluated had superior sensitivity to all of the assays included in the review while RBD performance was superior to most. The reason for this could be related to the technical aspects of the assay itself including the integrity of the antigen used and the sensitivity of the detection platform but also the use of a well-defined cohort of individuals with known exposure to SARS-CoV-2. Only the N terminal domain of the spike protein did not perform well in this assay with poor sensitivity due to the overlap in antibody titres between the COVID-19 cohort and controls.

The assay format permitted the measurement of antibody against spike protein derived from SARS-1, MERS and two seasonal coronaviruses, but the results of antibody binding to these antigens could not be assessed in the same way as for the SARS-CoV-2 antigens due to the absence of defined negative and positive serum sets.

An advantage of this assay is its ability to measure antibody induced inhibition of ACE-2 receptor-spike interaction thought to be the major mechanism by which SARS viruses, including SARS-CoV-2 attach to host cell surfaces (11, 12) . In the COVID-19 cohort, there was a good correlation between anti-S and anti-RBD IgG and function although a few sera bound antigen but did not neutralize. These were dominated by sera taken soon after infection and as recently described, could be non-neutralising and targeting epitopes outside the RBD (13).

Few of the control cohort sera had any pseudo-neutralisation activity despite pre-existing IgG to seasonal Coronavirus spike proteins suggesting season Coronavirus exposure is unlikely to modify interaction with SARS-CoV-2. Other cross reactive immunological mechanisms (eg T cells) cannot be ruled out and may explain the varied clinical response following exposure to SARS-CoV-2 (14) . This pseudo-neutralisation assay has been shown to correlate well with neutralisation assays using live SARS-CoV-2 (MSD, personal communication).

In summary, the MSD multiplexed coronavirus panel assay evaluated in this study is highly reproducible, specific and sensitive for the detection of anti-SARS-CoV-2 antibody over 14 days since the onset of COVID-19 symptoms. The assay can be adapted to measure antibody function which corelated well with spike protein antibody concentration.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

None

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The study team would like to thank Meso Scale Discovery for the donation of the plates and reagents that allowed us to complete the work, the COSTARS study team at Great Ormond Street Children's Hospital, staff in the Great Ormond Street Children's Hospital Clinical Immunology Laboratory for additional support and the NIHR UCL Great Ormond Street J o u r n a l P r e -p r o o f
