Specificity and Performance of Nucleocapsid and Spike-based SARS-CoV-2 Serologic Assays

Abstract

There is an urgent need for an accurate antibody test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this paper, we have developed 3 ELISA methods, trimer spike IgA, trimer spike IgG, and nucleocapsid IgG, for detecting anti-SARS-CoV-2 antibodies. We evaluated their performance in comparison with four commercial ELISAs, EDI™ Novel Coronavirus COVID-19 ELISA IgG and IgM, Euroimmun Anti-SARS-CoV-2 ELISA IgG and IgA, and one lateral flow assay, DPP® COVID-19 IgM/IgG System (Chembio). Both sensitivity and specificity were evaluated and the causes of false-positive reactions were determined. The assays were compared using 300 pre-epidemic samples and 100 PCR-confirmed COVID-19 samples. The sensitivities and specificities of the assays were as follows: 90%/100% (in-house trimer spike IgA), 90%/99.3% (in-house trimer spike IgG), 89%/98.3% (in-house nucleocapsid IgG), 73.7%/100% (EDI nucleocapsid IgM), 84.5%/95.1% (EDI nucleocapsid IgG), 95%/93.7% (Euroimmun S1 IgA), 82.8%/99.7% (Euroimmun S1 IgG), 82.0%/91.7% (Chembio nucleocapsid IgM), 92%/93.3% (Chembio nucleocapsid IgG). The presumed causes of positive signals from pre-epidemic samples in commercial and in-house assays were mixed. In some cases, positivity varied with assay repetition. In other cases, reactivity was abrogated by competitive inhibition (spiking the sample with analyte prior to performing the assay). In other cases, reactivity was consistently detected but not abrogated by analyte spiking. Overall, there was wide variability in assay performance using our samples, with in-house tests exhibiting the highest combined sensitivity and specificity. The causes of "false positivity" in pre-epidemic samples may be due to plasma antibodies apparently reacting with the analyte, or spurious reactivity may be directed against non-specific components in the assay system. Identification of these targets will be essential to improving assay performance.

Keywords: COVID-19; Coronavirus; Immunoassay.

Figure 1.. SARS-CoV-2 antibody assays data distribution.

The data obtained for pre-epidemic and COVID-19 samples with all evaluated assays. The yellow lines show the cut-off values or ranges recommended by commercial assays or calculated cutoff values for in-house ELISAs. Black lines indicate median values with interquartile ranges.