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.

Determining the presence of antibodies to the SARS-CoV-2 antigens is the best way to identify infected people, regardless of the development of symptoms of COVID-19. The nucleoprotein (NP) of the SARS-CoV-2 is an immunodominant antigen of the virus; anti-NP antibodies are detected in persons previously infected with the virus with the highest titers. Many test systems for detecting antibodies to SARS-CoV-2 contain NP or its fragments as antigen. The sensitivity and specificity of such test systems differ significantly, which can be explained by variations in the antigenic properties of NP caused by differences in the methods of its cultivation, isolation and purification. We investigated this effect for the Escherichia coli-derived SARS-CoV-2 NP, obtained from the cytoplasm in the soluble form. We hypothesized that co-purified nucleic acids that form a strong complex with NP might negatively affect NP’s antigenic properties. Therefore, we have established the NP purification method, which completely eliminates the RNA in the NP preparation. Two stages of RNA removal were used: treatment of the crude lysate of E. coli with RNase A and subsequent selective RNA elution with 2 M NaCl solution. The resulting NP without RNA has a significantly better signal-to-noise ratio when used as an ELISA antigen and tested with a control panel of serum samples with antibodies to SARS-CoV-2; therefore, it is preferable for in vitro diagnostic use. The same increase of the signal-to-noise ratio was detected for the free N-terminal domain of the NP. Complete removal of RNA complexed with NP during purification will significantly improve its antigenic properties, and the absence of RNA in NP preparations should be controlled during the production of this antigen.

DNA aptamers are oligonucleotides specifically bound to target molecules that can serve as antibodies of nucleic acid nature. For diagnosing the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), methods using antibodies specific to antigens on the virus are broadly used. We generated by classical SELEX a number of aptamers, interacting with the receptor-binding domain of SARS-CoV2 spike protein (SARS-CoV2 Spike RBD) from Wuhan-Hu-1 strain. The sequence identification was performed using a novel methodology based on the nanopore sequencing. For sequence identification of selected aptamers, we created the novel protocol for aptamer identification based on nanopore sequencing. We identified the best aptamer candidate named MEZ. It was chemically synthesized and tested for binding with SARS CoV2 Spike RBD domain of the S-protein from different strains. Kd of the complex is 6.5 nM being comparable with known aptamers. Virus neutralization tests demonstrate similar results for already known and MEZ aptamers. We identified differences for aptamers binding to SARS-CoV-2 Spike RBD from Wuhan-Hu-1 and Omicron strains. MD simulations reveal that the number of hydrogen bonds between the protein and aptamer is higher for the more stable complex. Moreover, dynamic network analysis show that the motions of the aptamer and protein are correlated to a higher extent in a more stable complex. Based on the experimental data and computational results we can conclude that the authentic RBD-aptamer complex has two specific points for interaction and the 3'-end of aptamer is responsible for strain identification. Therefore, the selected aptamer based on experimental data can be an alternative biological element for the development of SARS-CoV-2 diagnostic testing with strain specificity and cost efficiency due to the short length of aptamer being 31 nucleotides.