Validation of a commercially available SARS-CoV-2 serological immunoassay

Abstract

Objectives: To validate the diagnostic accuracy of a Euroimmun SARS-CoV-2 IgG and IgA immunoassay for COVID-19.

Methods: In this unmatched (1:2) case-control validation study, we used sera of 181 laboratory-confirmed SARS-CoV-2 cases and 326 controls collected before SARS-CoV-2 emergence. Diagnostic accuracy of the immunoassay was assessed against a whole spike protein-based recombinant immunofluorescence assay (rIFA) by receiver operating characteristic (ROC) analyses. Discrepant cases between ELISA and rIFA were further tested by pseudo-neutralization assay.

Results: COVID-19 patients were more likely to be male and older than controls, and 50.3% were hospitalized. ROC curve analyses indicated that IgG and IgA had high diagnostic accuracies with AUCs of 0.990 (95% Confidence Interval [95%CI]: 0.983-0.996) and 0.978 (95%CI: 0.967-0.989), respectively. IgG assays outperformed IgA assays (p=0.01). Taking an assessed 15% inter-assay imprecision into account, an optimized IgG ratio cut-off > 2.5 displayed a 100% specificity (95%CI: 99-100) and a 100% positive predictive value (95%CI: 96-100). A 0.8 cut-off displayed a 94% sensitivity (95%CI: 88-97) and a 97% negative predictive value (95%CI: 95-99). Substituting the upper threshold for the manufacturer's, improved assay performance, leaving 8.9% of IgG ratios indeterminate between 0.8-2.5.

Conclusions: The Euroimmun assay displays a nearly optimal diagnostic accuracy using IgG against SARS-CoV-2 in patient samples, with no obvious gains from IgA serology. The optimized cut-offs are fit for rule-in and rule-out purposes, allowing determination of whether individuals in our study population have been exposed to SARS-CoV-2 or not. IgG serology should however not be considered as a surrogate of protection at this stage.

Keywords: COVID-19; ELISA; Pseudovirus neutralisation assay; Recombinant immunofluorescence assay; SARS-CoV-2; Serological assays; Serological testing strategy.

Fig. 1

A and B OD ratio of negative control samples (n=326) and PCR-confirmed CoVID-19 patients (n=181) were determined using an IgG and IgA ELISA. C and D ROC curve analysis for IgG and IgA OD ratio results including the area under the curve (AUC) and 95% confidence intervals. E IgG OD ratios of COVID-19 patients at different days post onset of symptoms (dpos, dots) or days post diagnosis (dpd, triangles). Red dots or triangles show samples that are confirmed by whole spike recombinant immunofluorescence analysis. Dotted and dashed lines indicate the Euroimmun- (EI) or Geneva- (GE) cut-offs for negative, indeterminate and positive samples, respectively.

Fig. 2

Graphical overview or serological testing strategies in low (<10%) and high (>35%) pre-test probability settings. PPV: positive predictive value; NPV: negative predictive value; rIFA: recombinant immunofluorescence assay; 95%CI: 95% confidence intervals.