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Review
. 2023 Mar 10;24(6):5352.
doi: 10.3390/ijms24065352.

Importance, Applications and Features of Assays Measuring SARS-CoV-2 Neutralizing Antibodies

Pia Gattinger  1 , Anna Ohradanova-Repic  2 , Rudolf Valenta  1   3   4   5
Affiliations
Review

Importance, Applications and Features of Assays Measuring SARS-CoV-2 Neutralizing Antibodies

Pia Gattinger et al. Int J Mol Sci. .

Abstract

More than three years ago, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused the unforeseen COVID-19 pandemic with millions of deaths. In the meantime, SARS-CoV-2 has become endemic and is now part of the repertoire of viruses causing seasonal severe respiratory infections. Due to several factors, among them the development of SARS-CoV-2 immunity through natural infection, vaccination and the current dominance of seemingly less pathogenic strains belonging to the omicron lineage, the COVID-19 situation has stabilized. However, several challenges remain and the possible new occurrence of highly pathogenic variants remains a threat. Here we review the development, features and importance of assays measuring SARS-CoV-2 neutralizing antibodies (NAbs). In particular we focus on in vitro infection assays and molecular interaction assays studying the binding of the receptor binding domain (RBD) with its cognate cellular receptor ACE2. These assays, but not the measurement of SARS-CoV-2-specific antibodies per se, can inform us of whether antibodies produced by convalescent or vaccinated subjects may protect against the infection and thus have the potential to predict the risk of becoming newly infected. This information is extremely important given the fact that a considerable number of subjects, in particular vulnerable persons, respond poorly to the vaccination with the production of neutralizing antibodies. Furthermore, these assays allow to determine and evaluate the virus-neutralizing capacity of antibodies induced by vaccines and administration of plasma-, immunoglobulin preparations, monoclonal antibodies, ACE2 variants or synthetic compounds to be used for therapy of COVID-19 and assist in the preclinical evaluation of vaccines. Both types of assays can be relatively quickly adapted to newly emerging virus variants to inform us about the magnitude of cross-neutralization, which may even allow us to estimate the risk of becoming infected by newly appearing virus variants. Given the paramount importance of the infection and interaction assays we discuss their specific features, possible advantages and disadvantages, technical aspects and not yet fully resolved issues, such as cut-off levels predicting the degree of in vivo protection.

Keywords: SARS-CoV-2; molecular assays; neutralizing antibodies; serological assays.

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Conflict of interest statement

R.V. has received research grants from HVD Life-Sciences, Vienna, Austria, WORG Pharmaceuticals, Hangzhou, China and from Viravaxx AG, Vienna, Austria. He serves as consultant for Viravaxx AG. All other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Principle of cell-culture-based neutralization assays. (A) Conventional virus-neutralization test (VNT) and (B) pseudovirus-neutralization test (pVNT) are laboratory methods primarily used for identifying whether neutralizing antibodies (NAbs) against SARS-CoV-2 are present in the patient sample. Both tests involve incubating patient serum with the virus (A) or pseudovirus (B), and then inoculating onto cell culture. If NAbs are present and the virus is neutralized, the cells will not be infected. Accordingly, there will be a reduction in plaque formation or expression of virus proteins in the plaque-reduction neutralization assay (PRNT) and focus-reduction neutralization assay (FRNT), respectively. If NAbs are not present, the virus will infect the cells. In pVNT, a recombinant or chimeric virus with SARS-CoV-2 S protein on its surface bearing a reporter gene is used for increased safety and easier readout. A reduction in the reporter gene expression reflects the presence of NAbs.
Figure 2
Figure 2
Schematic overview of test principles used in molecular assays mimicking SARS-CoV-2 infections. There are basically two types of assays measuring the interaction of RBD and ACE2 and the ability of antibodies to prevent this interaction. As shown in (A), one possibility is that ACE2 is immobilized and differently labelled forms of RBD are incubated with serum. The other possibility as shown in (B) is that immobilized RBD is incubated with a mix of soluble ACE2 and serum. Blocking antibodies are detected as a loss or reduction in signal caused by the reduced binding of labelled ACE2 to RBD. ELISA-based competition assays as shown in (C) use immobilized RBD. After simultaneously adding ACE2 and serum, blocking antibodies competing with ACE2 for binding to RBD are detected via colorimetric reactions.
Figure 3
Figure 3
Overview of areas of application for molecular assays. Results from molecular assays mimicking SARS-CoV-2 infections can be used to determine protective antibodies of convalescent and/or vaccinated subjects or populations, as well as their blocking efficacy for emerging variants of concern. Plasma preparations and monoclonal antibodies used for preventive treatment can be tested for their neutralizing capacity.
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