Comparison of the Anti-SARS-CoV-2 Surrogate Neutralization Assays by TECOmedical and DiaPROPH-Med with Samples from Vaccinated and Infected Individuals

Abstract

Anti-SARS-CoV-2-specific serological responses are a topic of ongoing evaluation studies. In the study presented here, the anti-SARS-CoV-2 surrogate neutralization assays by TECOmedical and DiaPROPH -Med were assessed in a head-to-head comparison with serum samples of individuals after vaccination as well as after previous infection with SARS-CoV-2. In case of discordant results, a cell culture-based neutralization assay was applied as a reference standard. The TECOmedical assay showed sensitivity and specificity of 100% and 61.3%, respectively, the DiaPROPH-Med assay 95.0% and 48.4%, respectively. As a side finding of the study, differences in the likelihood of expressing neutralizing antibodies could be shown for different exposition types. So, 60 of 81 (74.07%) of the samples with only one vaccination showed an expression of neutralizing antibodies in contrast to 85.71% (60 of 70 samples) of the samples with two vaccinations and 100% (40 of 40) of the samples from previously infected individuals. In conclusion, the both assays showed results similar to previous assessments. While the measured diagnostic accuracy of both assays requires further technical improvement of this diagnostic approach, as the calculated specificity values of 61.3% and 48.4%, respectively, appear acceptable for diagnostic use only in populations with a high percentage of positive subjects, but not at expectedly low positivity rates.

Keywords: COVID-19; SARS-CoV-2; neutralization; serology; test comparison; vaccination.

Figure 1

Principle of the TECOmedical SARS-CoV-2 Neutralization Antibody ELISA. (A) Serum samples and conjugate (HRP-labelled SARS-CoV-2-SP-RBD) are premixed. (B) During incubation, neutralizing antibodies (anti-SARS-CoV-2-RBD-IgG) and the conjugate form antibody–antigen complexes. (C) The mixture is added to the solid phase; preformed complexes are unable to interact with ACE2-receptors; unbound conjugate can interact freely. (D) Tetramethylbenzide substrate is added and reacts with HRP; the measured optical density is inversely correlated with the concentration of neutralizing antibodies. Yellow square: color change after enzymatic reaction.

Figure 2

Principle of the DIA-SARS-CoV-2-nAb assay. (A) Serum samples and conjugate (HRP-labelled recombinant SARS-CoV-2-spike-protein) are simultaneously added to the solid phase. (B) Present neutralizing antibodies (anti-SARS-CoV-2-RBD-IgG) are bound to the conjugate and consequently block the interaction between the receptor-binding domain of the spike protein and recombinant human ACE-2-receptors on the solid phase. (C) Tetramethylbenzide substrate is added and reacts with HRP; the measured optical density is inversely correlated with the concentration of neutralizing antibodies in the sample. Yellow square: color change after enzymatic reaction.

Figure 3

Principle of the DIA-SARS-CoV-2-S-IgG-av avidity assay. (A) Serum samples are added to the solid phase in duplicate (reference wells and test wells); present anti-SARS-CoV-2-RBD-IgG are bound to the antigen (recombinant SARS-CoV-2-spike protein) on the solid phase. (B) The test wells are treated with dissociation solution to destroy antibody-antigen complexes formed by low avidity antibodies. (C) Remaining antibody-antigen complexes are detected by the conjugate (HRP-labelled monoclonal anti-human-IgG). (D) Tetramethylbenzide substrate is added and reacts with HRP; the reference well indicates whether the sample contains a sufficient amount of IgG or not, if the first applies, the test well is used to calculate the RAI (Relative Avidity Index). Yellow square: color change after enzymatic reaction.