Fast and Accurate Surrogate Virus Neutralization Test Based on Antibody-Mediated Blocking of the Interaction of ACE2 and SARS-CoV-2 Spike Protein RBD

Abstract

The humoral response to the SARS-CoV-2 S protein determines the development of protective immunity against this infection. The standard neutralizing antibodies detection method is a live virus neutralization test. It can be replaced with an ELISA-based surrogate virus neutralization test (sVNT), measuring the ability of serum antibodies to inhibit complex formation between the receptor-binding domain (RBD) of the S protein and the cellular ACE2 receptor. There are conflicting research data on the sVNT methodology and the reliability of its results. We show that the performance of sVNT dramatically improves when the intact RBD from the Wuhan-Hu-1 virus variant is used as the plate coating reagent, and the HRP-conjugated soluble ACE2 is used as the detection reagent. This design omits the pre-incubation step in separate tubes or separate microplate and allows the simple quantification of the results using the linear regression, utilizing only 3–4 test sample dilutions. When this sVNT was performed for 73 convalescent plasma samples, its results showed a very strong correlation with VNT (Spearman’s Rho 0.83). For the RBD, bearing three amino acid substitutions and corresponding to the SARS-CoV-2 beta variant, the inhibitory strength was diminished for 18 out of 20 randomly chosen serum samples, and the magnitude of this decrease was not similar to the change in overall anti-RBD IgG level. The sVNT assay design with the ACE2-HRP is preferable over the assay with the RBD-HRP reagent and is suitable for mass screening of neutralizing antibodies titers.

High Heterogeneity of Virus-Neutralizing and RBD-Binding Activities of COVID-19 Convalescent Sera

“…Note that recently our compatriots, D. Kolesov et al [ 14 ], published work similar to that presented here. Their study was also performed on the Moscow population and with the participation of patients who had been infected with Wuhan or similar variants of SARS-CoV-2 [ 14 ]. However, in our study, we obtained lower correlation coefficients between the results of cVNT, sVNT, and the anti-RBD test.…”

“…Notably, the largest number of such patients ( n = 23) was detected using sVNT, a test that detects only RBD-neutralizing antibodies. It can be assumed that some plasma samples from this group of recovered patients contained neutralizing antibodies that bind to the S protein outside the RBD region [ 14 ]. Indeed, it has been previously shown that antibodies to the N-terminal domain or the S2 region of the S-protein can also be involved in the neutralization of the virus [ 15 ].…”

Humoral Immune Responses in Patients with Severe COVID-19: A Comparative Pilot Study between Individuals Infected by SARS-CoV-2 during the Wild-Type and the Delta Periods

“…Extracellular domain of the human angiotensin-converting enzyme 2 (residues 319–541) was fused to an immunoglobulin G crystallizable fragment (ACE2-Fc) as described earlier [ 16 ]. The ACE2-Fc was stably expressed in a DHFR-negative Chinese hamster ovary (CHO) DG-44 cell line (Thermo Fischer Scientific, Waltham, MA, USA) and was purified from the conditioned-culture medium using chromatography on MabSelect SuRe column (Cytiva, Marlborough, MA, USA).…”

Characteristics of the Reactions in Tests for Antibodies to Viruses and Their Significance for Standard Assays and Adequate Routine Tests

“…In 2022, Kolesov et al [15] published a simpler version allowing reaction in the ELISA plates, where the coating agent was the RBD viral protein while ACE2 was the reactant in the enyme conjugate. A third version may appear in the future, where the reactant in the detecting enzyme conjugate is replaced with a polyclonal, or alternatively a monoclonal, speci c viral antibody.…”