An ELISA Platform for the Quantitative Analysis of SARS-CoV-2 RBD-neutralizing Antibodies As an Alternative to Monitoring of the Virus-Neutralizing Activity

Abstract

Monitoring of the level of the virus-neutralizing activity of serum immunoglobulins ensures that one can reliably assess the effectiveness of any protection against the SARS-CoV-2 infection. For SARS-CoV-2, the RBD-ACE2 neutralizing activity of sera is almost equivalent to the virus-neutralizing activity of their antibodies and can be used to assess the level of SARS-CoV-2 neutralizing antibodies. We are proposing an ELISA platform for performing a quantitative analysis of SARS-CoV-2 RBD-neutralizing antibodies, as an alternative to the monitoring of the virus-neutralizing activity using pseudovirus or "live" virus assays. The advantage of the developed platform is that it can be adapted to newly emerging virus variants in a very short time (1-2 weeks) and, thereby, provide quantitative data on the activity of SARS-CoV-2 RBD-neutralizing antibodies. The developed platform can be used to (1) study herd immunity to SARS-CoV-2, (2) monitor the effectiveness of the vaccination drive (revaccination) in a population, and (3) select potential donors of immune plasma. The protective properties of the humoral immune response in hospitalized patients and outpatients, as well as after prophylaxis with the two most popular SARS-CoV-2 vaccines in Russia, were studied in detail using this platform. The highest RBD-neutralizing activity was observed in the group of hospitalized patients. The protective effect in the group of individuals vaccinated with Gam-COVID-Vac vaccine was 25% higher than that in outpatients and almost four times higher than that in individuals vaccinated with the CoviVac vaccine.

Keywords: COVID-19; CoviVac; Gam-COVID-Vac; SARS-CoV-2; Sputnik V; antibodies; virus-neutralizing activity.

Fig. 1

Scheme of the quantitative determination of the activity of SARS-CoV-2 RBD-specific neutralizing antibodies in serum or plasma (sVNT). Antibodies in the serum sample interact with the recombinant RBD adsorbed in the wells. If the sample contains RBD-neutralizing antibodies (A), they block the binding of RBD to ACE2. If the sample does not contain neutralizing antibodies (B), the RBD adsorbed on the plate binds to recombinant ACE2. This binding is detected by peroxidase-labeled antibodies against the 3×FLAG sequence (3×FLAG) contained in recombinant ACE2. Therefore, the colorimetric signal recorded in the assay is inversely proportional to the concentration of the neutralizing antibodies in the sample. Designations: Ab – antibodies without neutralizing activity; ACE2 – recombinant human ACE2 receptor; HRP – antibodies to the FLAG epitope labeled with horseradish peroxidase; nAB – antibodies with RBD-neutralizing activity; RBD – the recombinant receptor-binding domain of the coronavirus SARS-CoV-2 S protein

Fig. 2

Validation of the RBD neutralization test by comparison with the conventional and pseudovirus neutralization assays. (A) Plot showing the activity of SARS-CoV-2 RBD-specific neutralizing antibodies in sera obtained by sVNT against serum titers that yield 50% virus neutralization (IC₅₀) using the cVNT test (26 samples). (B) Plot showing the activity of SARS-CoV-2 RBD-specific neutralizing antibodies in sera obtained by sVNT against sera titers at which pseudovirus neutralization of 50% (IC₅₀) was achieved (pVNT test) (29 samples). r2 is the coefficient of determination. In all the serum samples where no anti-RBD neutralizing antibodies were detected (25 samples), neutralization of the SARS-CoV-2 infection was not detected in all the tests

Fig. 3

Frequency of seropositive serum samples in the analyzed groups. (A) Frequency of anti-RBD IgG positive serum samples per group. (B) Frequency of RBD-nAb seropositive serum samples among RBD-IgG-positive samples. (C) Frequencies of occurrence of RBD-nAb-positive samples in the groups. Statistical significance of the intergroup differences was determined using the Fisher’s exact test (* p < 0.05; **** p < 0.0001)

Fig. 4

Concentration of SARS-CoV-2 RBD-specific IgG and the activity of RBD-ACE2 neutralizing antibodies in seropositive serum samples measured by sVNT. (A) Concentration of SARS-CoV-2 RBD-specific IgG in seropositive serum samples. (B) Neutralizing activity of RBD-specific antibodies (RBD-nAb) in seropositive serum samples. The statistical significance of intergroup differences was determined using the Kruskal–Wallis test (* p < 0.05; ** p < 0.01; **** p < 0.0001)

Fig. 5

The frequency of occurrence of SARS-CoV-2 RBD-specific immunoglobulin class G subclasses among RBD-nAb+ samples. Statistical significance of intergroup differences was determined using the Fisher’s exact test (* p < 0.05; **** p < 0.0001)

Fig. 6

Linear regression analysis of the serum antibody RBD-ACE2 neutralizing activity and RBD-specific IgG concentration. Double seropositive (RBD-IgG+ and RBD-nAb+) serum samples are shown as white circles; negative samples are shown as gray circles. The 95% confidence intervals and activity and concentration thresholds are shown with dotted lines. R is the Pearson correlation coefficient; K is the slope of the regression line; CI is the 95% confidence interval

Fig. 7

The virus-neutralizing activity of the humoral immunity in the study groups. The protectivity index of the group Outpatients was taken as 100%