A simple protein-based surrogate neutralization assay for SARS-CoV-2

Abstract

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector-based assay.

Keywords: Adaptive immunity; Antigen; Immunoglobulins; Infectious disease.

Figure 1. Establishment of a surrogate neutralization ELISA (snELISA) to monitor the spike-ACE2 interaction.

(A) SARS-CoV-2 attachment to the host cell requires a direct interaction between the host cell receptor, ACE2 (blue), and the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (peach). (B) Ninety-six–well plates are set up in a similar manner for both the detection of anti–SARS-CoV-2 antibodies and neutralizing antibodies. Antigens are adsorbed overnight and incubated with diluted patient serum/plasma samples, monoclonal antibodies, and other affinity reagents. Antigen-specific antibodies are colored black. (C) Principle of direct binding ELISA detection using HRP-conjugated anti–human IgG/A/M. (D) Principle of the snELISA, which uses biotinylated ACE2 for the detection of RBD or spike epitopes that have not been blocked by neutralizing antibodies (using polyHRP-streptavidin). (E) Results of the snELISA using either the RBD or the spike trimer immobilized on the plate (see Supplemental Figures 1 and 2 for the antigen cloning, expression, and purification). The dashed lines are from a sample (CBS39) that was negative for direct RBD/spike binding, while the solid lines are from a positive sample (CBS50; see Supplemental Figure 4A for the direct binding results and Supplemental Table 1 for all OD₄₅₀ values). (F) snELISA (immobilized RBD) for an expanded set of 4 positive controls with high anti-RBD signals in a single-point direct-binding ELISA (green), 8 negative samples acquired before COVID (red), and 4 samples with low anti-RBD levels (blue; Supplemental Figure 4A). The 5-point curves in E–F were generated from 1 experiment.

Figure 2. Application of the snELISA to a larger cohort.

(A) Representative direct binding ELISAs with titrations of different samples from a patient cohort sampled by Canadian Blood Services (all 58 ELISA curves are shown in Supplemental Figure 6; see Supplemental Figure 7 for an extended titration of the most abundant samples). (B) snELISA results for the samples shown in A; see Supplemental Figure 8 for all curves. The 5-point curves in A and B were generated from 1 experiment. (C) Correlation between the AUCs for the direct and snELISAs for all samples profiled (see an expanded view in Supplemental Figure 9). (D) Outliers in the correlation plot C were calculated using the total least squares (TLS) method; points with a TLS > 0.4 (labeled) were marked as outliers (see Supplemental Figure 10 for selected examples with side-by-side direct and snELISAs).

Figure 3. Validation of the snELISA results using orthogonal methods.

(A) Results of the plaque reduction neutralization tests on the same samples overlaid on the AUC curves from Figure 2C (n = 57 samples). Color coding indicates the PRNT50 titers, while negative/positive hits on the PRNT90 assay are displayed with a different-sized dot (see Supplemental Figure 11 for additional PRNT results and Supplemental Figure 12 for spike pseudotyped virus results). (B) Correlation between the lentiviral spike pseudotyped virus assay calculated IC₅₀ values and the AUC results from the snELISA. (C and D) Assessment of the ability of monoclonal or affinity reagents to block the interaction between ACE2 and the RBD in the snELISA (C) or the lentiviral spike pseudotyped assay (D) (see Supplemental Figure 13–16 for the direct binding and viral neutralization assays and for additional reagents tested). Values for C were generated from 3 independent experiments, and underlayed points have been placed along the x axis. Values for D were obtained in 1 experiment in technical triplicate. Data are presented as mean ± SEM.