Development and Evaluation of a Set of Spike and Receptor Binding Domain-Based Enzyme-Linked Immunosorbent Assays for SARS-CoV-2 Serological Testing

Abstract

The implementation and validation of anti-SARS-CoV-2 IgG serological assays are reported in this paper. S1 and RBD proteins were used to coat ELISA plates, and several secondary antibodies served as reporters. The assays were initially validated with 50 RT-PCR positive COVID-19 sera, which showed high IgG titers of mainly IgG1 isotype, followed by IgG3. Low or no IgG2 and IgG4 titers were detected. Then, the RBD/IgG assay was further validated with 887 serum samples from RT-PCR positive COVID-19 individuals collected at different times, including 7, 14, 21, and 40 days after the onset of symptoms. Most of the sera were IgG positive at day 40, with seroconversion happening after 14–21 days. A third party conducted an additional performance test of the RBD/IgG assay with 406 sera, including 149 RT-PCR positive COVID-19 samples, 229 RT-PCR negative COVID-19 individuals, and 28 sera from individuals with other viral infections not related to SARS-CoV-2. The sensitivity of the assay was 99.33%, with a specificity of 97.82%. All the sera collected from individuals with infectious diseases other than COVID-19 were negative. Given the robustness of this RBD/IgG assay, it received approval from the sanitary authority in Mexico (COFEPRIS) for production and commercialization under the name UDISTEST-V2G®.

In Vitro and In Vivo Characterization of a Broadly Neutralizing Anti-SARS-CoV-2 Antibody Isolated from a Semi-Immune Phage Display Library

“…IgG-A7 was subjected to an analytical platform of validated methodologies to assess its physicochemical profile for pharmaceutical developability and biological activity [ 17 , 18 , 19 ]. This platform considers determining protein content, purity, identity/integrity, and thermal stability.…”

Evolution of Anti-SARS-CoV-2 Therapeutic Antibodies

“…Five out of the nine therapeutic antibodies except Etesevimab, Sotrovimab, Cilgavimab, and Tixagevimab are hIgG1 without Fc modifications. We [ 96 ] and other authors [ 97 ] have shown that IgG1 predominates the anti-SARS-CoV-2 immune response, followed by the IgG3 isotype. IgG2 and IgG4 are almost inexistent.…”

Anti-SARS-CoV-2 Omicron Antibodies Isolated from a SARS-CoV-2 Delta Semi-Immune Phage Display Library

“…The expression, purification, and characterization of the RBD-WT and RBD-DT have been described elsewhere [ 27 ]. In brief, the plasmid containing the RBD-WT (or mutated in our laboratory to obtain the RBD-DT) donated by Dr Florian Kammer at Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.…”

“…This platform considers the determination of purity, identity/integrity, and thermal stability by analytical size exclusion chromatography (SEC), denaturing polyacrylamide electrophoresis (SDS-PAGE), and Protein Thermal Shift™ assay. All of these analytical techniques were performed using standard and well-known physicochemical methods for proteins [ 27 , 28 , 29 ].…”