In a recent study published on the medRxiv* preprint server, researchers validate a quick, highly efficient, and automated assay to detect neutralizing antibodies (nAb) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This assay is based on the blockade of human angiotensin-converting enzyme 2 (hACE2) binding (BoAb).
Study: Rapid, high throughput, automated detection of SARS-CoV-2 neutralizing antibodies against native-like vaccine and delta variant spike trimers. Image Credit: anyaivanova / Shutterstock.com
The levels of nAb induced by SARS-CoV-2 vaccination or natural infection strongly correlate with sterilizing immunity, which can prevent severe forms of coronavirus disease 2019 (COVID-19). Thus, the reliable detection and quantification of SARS-CoV-2 nAb levels have significant implications in public health scenarios to determine the waning effect of vaccines and the need for boosters.
The traditional gold standards such as live virus neutralization and cellular pseudovirus neutralization assays for measuring SARS-CoV-2 nAbs are tedious and laborious. Hence, these approaches are not suitable for use in laboratories on a routine basis to determine the level of immune protection achieved following natural SARS-CoV-2 infection or vaccination.
About the study
In the present study, the researchers developed and validated a rapid and high throughput BoAb assay technique for SARS-CoV-2 nAb level measurement against trimeric spike (S) proteins similar to SARS-CoV-2 variants. The assay was based on BoAb in an automated digital immunoassay on the Quanterix HD-X platform. The SARS-CoV-2 strains used in the assay include the Delta variant and the wild-type (WT) strain.
Samples from a previous study that evaluated the neutralization determinants of SARS-CoV-2 Delta and WT strains in myeloma patients using live virus and pseudovirus neutralization assays were used for the current study. The SARS-CoV-2 neutralization activity of samples from individuals with or without SARS-CoV-2 exposure or vaccination status was also examined using a single-dilution screening version of the current BoAb assay.
Blockade of ACE-2 Binding (BoAb) assay design.
Study findings
Purified SARS-CoV-2 WT and Delta strains used in the present BoAb assay maintained one receptor-binding domain (RBD) in the open conformation and two in the closed conformation, which is the required conformational state for the viral RBD-host ACE2 interaction. Additionally, a strong interaction between the SARS-CoV-2 S and the hACE2 receptor was observed through the biolayer interferometry (BLI) assay.
A significant correlation was observed between the neutralization of SARS-CoV-2 variants in the pseudovirus and live virus assays and the BoAb assays using the native-like trimeric S proteins of the Delta variant and WT viral strains.
Moreover, a strong correlation was found between the RBD endpoint titers of the SARS-CoV-2 WT and Delta strains in the live virus and pseudovirus neutralization results and the BoAb measurements. The BoAb assay demonstrated a sensitivity of more than 94% against SARS-CoV-2 WT strain and more than 90% against Delta, with 100% specificity against both of these SARS-CoV-2 strains. The pattern of resistance of the Delta variant to neutralization was comparable among samples containing paired Delta variant and WT strain BoAb measurements.
Only the samples from patients exposed to SARS-CoV-2 or vaccinees demonstrated a significant nAb activity against SARS-CoV-2 S in the BoAb assay as compared to those who did not have SARS-CoV-2 exposure.
Conclusions
The study findings show that, theoretically, the BoAb assay provides a timely, robust, and expandable substitute for the existing standard methods that are costly, time-consuming, and require expertise for quantifying SARS-CoV-2 nAb levels. Taken together, BoAb assays will be beneficial in evaluating the neutralization of all SARS-CoV-2 variants, as well as other potential pathogenic viruses in the future.
The study indicated a strong correlation between the gold standards of nAb detection like the live virus neutralization assay and the BoAb assay. However, in contrast to existing methods, the present assay technique does not require extensive manual liquid handling, biosafety level 3 (BSL3) facilities, or cell culture.
The BoAb assay employs the S proteins of SARS-CoV-2 variants with the native trimeric structure to capture the extent of infection and vaccine-induced Ab responses bound epitopes. Therefore, this approach is of prime significance in the rollout of SARS-CoV-2 booster vaccines resulting in broadened Ab response.
Since the current study findings are limited to available antibody data from the gold-standard live virus neutralization assay, further direct correlations of nAb activity between BoAb measurements and current cell-based assays are required.
The present findings with the BoAb assay are in line with other studies employing gold standard methods stating that SARS-CoV-2 neutralization capacity and Ab responses are directly proportional. However, further investigations are required to determine the impact of neutralization and Ab response on natural infection and vaccine-induced protection.
*Important notice
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
