Integrated workflow for production and in vitro characterization of equine anti-SARS-CoV-2 hyperimmune serum with epitope mapping.

This study establishes an integrated workflow linking antibody production, neutralization assessment, and epitope mapping, supporting the identification of antigenic regions recognized by polyclonal antibodies generated in vivo. The viral suspension was inactivated with β-propiolactone under biosafety level 3 (BSL-3) conditions, with no cytopathic effect or plaque formation observed, confirming loss of viral replicative capacity. The produced immunogen retained antigenic activity, as demonstrated by reactivity with IgG antibodies from vaccinated and previously infected individuals. Protein analysis by Western blot revealed the presence of major structural proteins, spike and nucleocapsid, in different molecular forms, consistent with previous reports. Sequential immunizations in horses induced robust humoral responses, with antibody titers increasing up to 16-fold after the booster cycle. The purified F(ab')₂ fraction showed 80.4% purity, as estimated by densitometry, and retained antigen binding activity in ELISA assays, supporting the effectiveness of the purification process. The preparation achieved PRNT₅₀ values of 1:97,000 against SARS-CoV-2 under the conditions tested. The use of chemically inactivated whole virus as the immunogen provides a robust approach for generating antibodies recognizing a broad repertoire of viral epitopes while preserving structural complexity. This workflow, which can be safely implemented in BSL-3 laboratories, represents an alternative to recombinant protein-based approaches and may be adaptable to emerging infectious threats. Moreover, the identification of multiple linear immunogenic peptides recognized by the generated antibodies provides experimentally derived information on antibody-binding profiles, supporting potential applications in diagnostics and immunobiotechnology.