Detection and Quantification of SARS-CoV-2 Receptor Binding Domain Neutralization by a Sensitive Competitive ELISA Assay

Abstract

This protocol describes an ELISA-based procedure for accurate measurement of SARS-CoV-2 spike protein-receptor binding domain (RBD) neutralization efficacy by murine immune serum. The procedure requires a small amount of S-protein/RBD and angiotensin converting enzyme-2 (ACE2). A high-throughput, simple ELISA technique is employed. Plate-coated-RBDs are allowed to interact with the serum, then soluble ACE2 is added, followed by secondary antibodies and substrate. The key steps in this procedure include (1) serum heat treatment to prevent non-specific interactions, (2) proper use of blank controls to detect side reactions and eliminate secondary antibody cross-reactivity, (3) the addition of an optimal amount of saturating ACE2 to maximize sensitivity and prevent non-competitive co-occurrence of RBD-ACE2 binding and neutralization, and (4) mechanistically derived neutralization calculation using a calibration curve. Even manually, the protocol can be completed in 16 h for >30 serum samples; this includes the 7.5 h of incubation time. This automatable, high-throughput, competitive ELISA assay can screen a large number of sera, and does not require sterile conditions or special containment measures, as live viruses are not employed. In comparison to the ‘gold standard’ assays (virus neutralization titers (VNT) or plaque reduction neutralization titers (PRNT)), which are laborious and time consuming and require special containment measures due to their use of live viruses. This simple, alternative neutralization efficacy assay can be a great asset for initial vaccine development stages. The assay successfully passed conventional validation parameters (sensitivity, specificity, precision, and accuracy) and results with moderately neutralizing murine sera correlated with VNT assay results (R2 = 0.975, n = 25), demonstrating high sensitivity.

Humoral immunity in dually vaccinated SARS-CoV-2-naïve individuals and in booster-vaccinated COVID-19-convalescent subjects

“…ELISA formats to determine SARS-CoV-2 neutralizing antibodies that compete with ACE2-receptor binding have been described to correlate well with virus neutralization [ 13 – 15 ]. In principle, the S-protein or its RBD is immobilized on a solid phase, incubated with serum, ACE2 is added and its binding in comparison to a non-serum-bound control is quantified.…”

The potential of developing a protective peptide‐based vaccines against SARS‐CoV‐2

“…The RBM‐derived epitopes were also found to be neutralizing (e.g., SARS‐2‐S 451–470 and S 491–510 ) in mice when conjugated to diphtheria toxoid and adjuvanted with alum or emulsion‐based adjuvants (Pandey et al, 2021 ; Shalash et al, 2022 ). When sera of mice immunized with two different epitopes were combined, synergistically inhibited RBD/ACE2 binding when examined using competitive ELISA (Pandey et al, 2021 ; Shalash et al, 2021a ). Furthermore, 14–24‐mer peptides were used as vaccine antigens to identify neutralizing epitopes in BALB/c mice.…”

“…Vaccine antigen selection should not only focus on original SARS‐2 lineage sequences, but also consider new mutant variant sequences; especially, as several mutations have increased the virulence and also have compromised the efficacy of approved vaccines. Several in vitro methods have been employed to evaluate SARS‐2‐S neutralization efficacy (Shalash et al, 2021a , 2021b ; Shalash et al, 2022 ), and these methods can be adapted to evaluate vaccine efficacy against emerging variants. Further, since the immune correlates of protection of several approved vaccines were evaluated in animal infection challenge models (e.g., ferrets and non‐human primates) and in humans, validation of a translation model is possible to infer higher confidence in the relevance of animal infection challenge outcomes to efficacy in humans (Shalash et al, 2021b ).…”

L’exploration de l’immunogénicité vaccinale

“…L’avidité prend donc en compte la valence des anticorps et des antigènes, elle pourrait se résumer par la somme de toutes les affinités du complexe immun. De nombreux Elisa de compétition de différentes configurations ont d’ailleurs été développés dans le cadre de la pandémie de Sars-CoV-2 [ 8 ]. Dans celui proposé par Byrnes et al , la protéine RBD ( receptor binding domain ) du Sars-CoV-2 est adsorbée au fond du puits puis l’échantillon du patient et le récepteur ACE-2 (angiotensin I converting enzyme 2) du Sars-CoV-2 sont incubés en même temps sur l’antigène.…”