Humoral Immune Response to SARS-CoV-2

Pauline H. Herroelen, MSc; Geert A. Martens, MD, PhD; Dieter De Smet, MD; Koen Swaerts, MSc; An-Sofie Decavele, MSc


Am J Clin Pathol. 2020;154(5):610-619. 

In This Article


In this study we report on the clinical performance characteristics of 7 commercially available serology tests for detection of antibodies against SARS-CoV-2 S protein (S-RBD total antibodies, S1/S2 IgG, S1 IgA and IgG), N protein (N total antibodies, N IgG), and both proteins (N/S IgM and IgG). To our knowledge, this study is the first to report performance of Elecsys Anti-SARS-CoV-2 assay on the cobas e601 module. We specifically investigated their relative value as a complementary tool to screen for prior SARS-CoV-2 infection in individuals that were not (conclusively) tested by PCR in early stage of active viral replication up to 10 days after onset of symptoms. As a working definition for acceptable performance, we propose that such an assay should combine a minimal sensitivity for detection of SARS-CoV-2 antibodies of 95% vs a consensus estimate and a high analytical specificity above 98% in samples taken 20 days or more after onset of symptoms, also in subjects who experienced mild SARS-CoV-2 symptoms. Based on these criteria, Wantai SARS-COV-2 Ab ELISA, Elecsys Anti-SARS-CoV-2 assay, and Innovita 2019-nCoV Ab Test all showed acceptable analytical specificity. In terms of sensitivity for detection of SARS-CoV-2 antibodies vs consensus result obtained by all tests, Wantai SARS-COV-2 Ab ELISA, Elecsys Anti-SARS-CoV-2 assay, EUROIMMUN Anti-SARS-CoV-2 IgG combined with IgA, and Orient Gene COVID-19 IgG/IgM Rapid Test were acceptable. Overall, only Wantai SARS-COV-2 Ab ELISA and Elecsys Anti-SARS-CoV-2 assay fulfilled the proposed acceptance criteria, with Wantai SARS-COV-2 Ab ELISA clearly outperforming all other evaluated assays.

A strength of this study is that the parallel evaluation of several kits allowed a reliable direct comparison of clinical performance using cutoffs provided by manufacturers. Also, our patient cohorts, including not only severe COVID-19 patients but also a sizeable cohort of mild SARS-CoV-2 infections, provide a good estimate on assays' performances in the intended target population. We observed no notable differences in timing of seroconversion between severe and milder SARS-CoV-2 infections.

There are limitations to our study. Cross-reactivity analysis might require more extensive exploration. A higher number of sera from patients with PCR-confirmed HCoV infections and other common cold viruses need to be investigated. Our study did not include a sizeable cohort of fully asymptomatic SARS-CoV-2 infections, and it focused solely on qualitative analysis. Therefore, no investigation of differences in assays' performance for quantification of antibody titers was performed.

In critically ill COVID-19 patients, SARS-CoV-2 antibody levels were reported to correlate to disease severity[1] by triggering bradykinin and complement activation pathways. The assays evaluated here show large variations in their dynamic range, ranging from a good linearity for LIAISON SARS-CoV-2 S1/S2 IgG[14] to a limited dynamic range with rapid signal saturation for Wantai SARS-COV-2 Ab ELISA. With a sample volume input of 100 μL that is 10 to 20 times higher than the other evaluated assays, Wantai SARS-COV-2 Ab ELISA is clearly designed toward high sensitivity for detection of SARS-CoV-2 antibodies by maximal antibody capture. Caution is needed when comparing (semi)quantitative estimates of antibody titers across platforms before certified standards with known titers become available.

Our data are compatible with other cross-platform evaluations[15] indicating superior performance of Wantai SARS-COV-2 Ab ELISA as compared to EUROIMMUN Anti-SARS-CoV-2 IgG and IgA. The results are, however, discrepant with another study reporting a sensitivity of 100% and 99% specificity for LIAISON SARS-CoV-2 S1/S2 IgG[14] obtained on a small set of 125 samples including only 40 PCR-confirmed patients and after receiver operating characteristic optimization of assay cutoffs. Since we observed considerable lot-to-lot variations in raw signals of the 2 LIAISON SARS-CoV-2 S1/S2 IgG kits tested, we believe that caution is warranted and cutoffs should only be optimized on better data sets and proper assessment of different lots.

It was reported that antibodies against S protein appear later in infection than antibodies against N protein.[1,9] We also observed faster seroconversion of N vs S1 targeting IgG in EUROIMMUN assays. On the other hand, we observed a much faster seroconversion of total antibodies (IgA/IgM/IgG) against S-RBD (Wantai) than N protein (Elecsys). Within the same epitope/assay format (EUROIMMUN to S1), IgA antibodies clearly precede IgG. Overall, our data suggest that timing of seroconversion depends more on assay design, recombinant viral epitope, and antibody isotypes covered, and that overall sensitivity for detection of antibodies is likely enhanced when both IgA and IgG isotypes are measured.

In conclusion, this study supports clinical use of both Wantai SARS-COV-2 Ab ELISA and Elecsys Anti-SARS-CoV-2 assay for sensitive and specific screening of SARS-CoV-2 antibodies from 10 days after onset of symptoms.