Anti-Spike Protein Assays to Determine SARS-CoV-2 Antibody Levels: a Head-to-Head Comparison of Five Quantitative Assays

Abstract

Reliable quantification of the antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly relevant, e.g., for identifying possible vaccine failure and estimating the time of protection. Therefore, we evaluated five different anti-SARS-CoV-2 antibody assays regarding the quantification of anti-spike (S) antibodies. Sera from 69 SARS-CoV-2-naive individuals 21 ± 1 days after vaccination with a single dose of BNT162b2 (Pfizer/BioNTech) were tested using the following quantitative assays: Roche S total antibody, DiaSorin trimeric spike IgG, DiaSorin S1/S2 IgG, Abbott II IgG, and Serion/Virion IgG. Results were further compared to the percent inhibition calculated from a surrogate virus neutralization test (sVNT). Individual values were distributed over several orders of magnitude for all assays. Although the assays were in good overall agreement (ρ = 0.80 to 0.94), Passing-Bablok regression revealed systematic constant and proportional differences, which could not be eliminated by converting the results to binding antibody units (BAU) per milliliter, as suggested by the manufacturers. Seven (10%) individuals had negative sVNT results (i.e., <30% inhibition). These samples were identified by most assays and yielded significantly lower binding antibody levels. Although all assays showed good correlation, they were not interchangeable, even when converted to BAU per milliliter using the WHO international standard for SARS-CoV-2 immunoglobulin. This highlights the need for further standardization of SARS-CoV-2 serology. IMPORTANCE Reliable quantification of the antibody response to SARS-CoV-2 is highly relevant, e.g., for identifying possible vaccine failure and estimating the time of protection. We compared the performance of five CE marked tests that quantify antibodies against the viral spike protein. Our findings suggest that, although all assays showed good correlation, their results were not interchangeable, even when converted to BAU per milliliter using the WHO international standard for SARS-CoV-2 immunoglobulin. This highlights the need for further standardization of SARS-CoV-2 serology.

Keywords: SARS-CoV-2; assay standardization; comparison; immunization; quantitative antibody assays; quantitative methods; serology; vaccination.

FIG 1

Results from binding assays (A) and sVNTs (B). Solid lines mark the medians. The dotted line (B) marks the manufacturer’s threshold for positivity (30%).

FIG 2

Comparison of binding assays by linear regression (dotted lines indicate the 95% confidence interval) (A) and residual plots (B).

FIG 3

Comparison of binding assay results converted to BAU per milliliter. Given are linear regression curves and their 95% confidence intervals. Dotted diagonal lines represent lines of equality.

FIG 4

(A) Relationships between binding assay results and percent inhibition assayed using a surrogate virus neutralization test (threshold for positivity, 30% [dotted vertical lines]). Presented are quadratic regression lines and their 95% confidence intervals. (B) Comparison (Mann-Whitney tests) of standardized (BAU per milliliter) binding antibody levels (except for DiaSorin S1/2 IgG, for which a conversion factor to BAU per milliliter is not available) between samples with a positive (sVNT pos) or negative (sVNT neg) surrogate virus neutralization test result. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.