Spectrum Bias and Individual Strengths of SARS-CoV-2 Serological Tests—A Population-Based Evaluation

Abstract

Antibody testing for determining the SARS-CoV-2 serostatus was rapidly introduced in early 2020 and since then has been gaining special emphasis regarding correlates of protection. With limited access to representative samples with known SARS-CoV-2 infection status during the initial period of test development and validation, spectrum bias has to be considered when moving from a “test establishment setting” to population-based settings, in which antibody testing is currently implemented. To provide insights into the presence and magnitude of spectrum bias and to estimate performance measures of antibody testing in a population-based environment, we compared SARS-CoV-2 neutralization to a battery of serological tests and latent class analyses (LCA) in a subgroup (n = 856) of the larger population based TiKoCo-19 cohort (n = 4185). Regarding spectrum bias, we could proof notable differences in test sensitivities and specificities when moving to a population-based setting, with larger effects visible in earlier registered tests. While in the population-based setting the two Roche ELECSYS anti-SARS-CoV-2 tests outperformed every other test and even LCA regarding sensitivity and specificity in dichotomous testing, they didn’t provide satisfying quantitative correlation with neutralization capacity. In contrast, our in-house anti SARS-CoV-2-Spike receptor binding domain (RBD) IgG-ELISA (enzyme-linked-immunosorbant assay) though inferior in dichotomous testing, provided satisfactory quantitative correlation and may thus represent a better correlate of protection. In summary, all tests, led by the two Roche tests, provided sufficient accuracy for dichotomous identification of neutralizing sera, with increasing spectrum bias visible in earlier registered tests, while the majority of tests, except the RBD-ELISA, didn’t provide satisfactory quantitative correlations.

Humoral immunity in dually vaccinated SARS-CoV-2-naïve individuals and in booster-vaccinated COVID-19-convalescent subjects

“…To exclude the possibility of asymptomatic breakthrough-infection, the absence of antibodies specific for SARS-CoV-2’s nucleoprotein (N) in the serum sample taken 6 month after complete vaccination was furthermore analyzed using Roches Elecsys N-Test (data not shown). As a prerequisite of such analysis, the Elecsys N-Test has been reported to be highly specific and sensitive [ 9 ].…”

“…Anti-SARS-CoV-2-specific antibody levels in serum were detected by an ELISA utilizing the SARS-CoV-2-spike protein’s receptor-binding domain (RBD) as antigen, as previously described [ 10 ]. The assay is able to detect IgM-, IgA- and IgG-SARS-CoV-2 antibody responses separately with high specificity and sensitivity and the detected antibody levels were shown to correlate well with the virus neutralization capacity of the respective serum sample [ 9 ]. The IgG antibody levels in serum samples after vaccination and booster vaccination, respectively, were titrated in eight steps of twofold dilutions, starting at a dilution of 1:200.…”

Association between Adverse Reactions and Humoral Immune Response No Longer Detectable after BNT162b2 Booster Vaccination

“…To identify possible breakthrough infections that may have occurred since the initial vaccination, SARS-CoV-2 nucleocapsid-specific antibodies were detected using the Elecsys Anti-SARS-CoV-2 N assay (Roche Diagnostics GmbH, Penzberg, Germany). The assay has been shown to be highly sensitive and specific [ 19 ]. The measurements were performed on a Roche COBAS pro e 801 clinical chemistry analyzer according to the manufacturer’s recommendations.…”

“…SARS-CoV-2 neutralization tests were performed as described previously [ 19 ]. In brief, we used Vesicular Stomatitis Virus (VSV–Δ G*FLuc) [ 20 ] pseudotyped with SARS-CoV-2-Spike-ΔER (wildtype) and determined pseudoviral titers by limited dilution and fluorescence microscopy.…”

Comparative Immunogenicity of COVID-19 Vaccines in a Population-Based Cohort Study with SARS-CoV-2-Infected and Uninfected Participants

“…(ii) Our validated in-house ELISA detecting IgA antibody responses to the SARS-CoV-2 spike protein's receptor-binding domain (RBD) was performed as described earlier [15]. (iii) The neutralization assay using the Vesicular Stomatitis Virus (VSV-∆ G*FLuc) pseudotyped with wt-SARS-CoV-2-Spike-∆ER was performed as described earlier [14]. In brief, an inoculum of 25,000 ffu was neutralized with a 2-fold serum dilution series for 1 h, and luciferase activity was determined 20 h post infection of HEK293T-ACE2 + -cells using BrightGlo (Promega Corp, Madison, WI, USA).…”