Evaluation of commercial Anti-SARS-CoV-2 neutralizing antibody assays in seropositive subjects

Abstract

The virus neutralization test (VNT) is the reference for the assessment of the functional ability of neutralizing antibodies (NAb) to block SARS-CoV-2 entry into cells. New competitive immunoassays measuring antibodies preventing interaction between the spike protein and its cellular receptor are proposed as surrogate VNT (sVNT). We tested three commercial sVNT (a qualitative immunochromatographic test and two quantitative immunoassays named YHLO and TECO) together with a conventional anti-spike IgG assay (bioMérieux) in comparison with an in-house plaque reduction neutralization test (PRNT₅₀) using the original 19A strain and different variants of concern (VOC), on a panel of 306 sera from naturally-infected or vaccinated patients. The qualitative test was rapidly discarded because of poor sensitivity and specificity. Areas under the curve of YHLO and TECO assays were, respectively, 85.83 and 84.07 (p-value >0.05) using a positivity threshold of 20 for PRNT₅₀, and 95.63 and 90.35 (p-value =0.02) using a threshold of 80. However, the performances of YHLO and bioMérieux were very close for both thresholds, demonstrating the absence of added value of sVNT compared to a conventional assay for the evaluation of the presence of NAb in seropositive subjects. In addition, the PRNT₅₀ assay showed a reduction of NAb titers towards different VOC in comparison to the 19A strain that could not be appreciated by the commercial tests. Despite the good correlation between the anti-spike antibody titer and the titer of NAb by PRNT₅₀, our results highlight the difficulty to distinguish true NAb among the anti-RBD antibodies with commercial user-friendly immunoassays.

Keywords: Commercial tests; Competitive anti-RBD immunoassays; Neutralizing antibodies; SARS-CoV-2; Surrogate markers of protection.

Fig. 1

Comparison of performance of the two sVNT. ROC curves were built to estimate the performance of YHLO (in grey) and TECO (in black) assays for detecting the presence of neutralizing antibodies (PRNT₅₀ ≥20 (A)) and high neutralizing antibody titre (PRNT₅₀ ≥80 (B)) from samples of infected patients (n=246).

Fig. 2

Comparison of performance of the YHLO surrogate quantitative virus neutralization test and the bioMérieux anti-RBD IgG assay with reference to the plaque reduction neutralization test 50% (PRNT₅₀) from 246 serum specimens collected from convalescent patients. Panels A (YHLO assay) and B (bioMérieux assay) show the strong correlation between each test and PRNT₅₀ (the value of the Spearman correlation coefficient is shown on the upper right part of the panel for each test). ROC curves were built to estimate the performance of the YHLO (in grey) and bioMérieux (in black) assays. Two different positive thresholds were used for detecting neutralizing antibodies by PRNT₅₀: ≥20 (panel C) and ≥80 (panel D). The Delong test was used to compare the areas under the curve (AUC). No statistically significant difference was observed between the two tests for both thresholds.

Fig. 3

Correlation between the antibody titers obtained with the YHLO test (Panel A) or the BioMérieux assay (panel B) and the plaque reduction neutralization test 50% (PRNT50). Ellipses show the 95% CIs for different clades schedules, assuming multivariate normal distributions. Tests were performed with various clades (19A, Alpha, Beta, Gamma and Delta strains) on 60 samples taken from vaccinated individuals.