Academic Journal
Integrated workflow for production and in vitro characterization of equine anti-SARS-CoV-2 hyperimmune serum with epitope mapping.
| Τίτλος: | Integrated workflow for production and in vitro characterization of equine anti-SARS-CoV-2 hyperimmune serum with epitope mapping. |
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| Συγγραφείς: | de Santana Evangelista K; Industrial Directorate, Ezequiel Dias Foundation (Funed), Belo Horizonte, MG, 30510-010, Brazil., de Castro Barbosa E; Research and Development Directorate, Funed, Belo Horizonte, MG 30510-010, Brazil., de Souza Andrade A; Research and Development Directorate, Funed, Belo Horizonte, MG 30510-010, Brazil., Ardisson L; Industrial Directorate, Ezequiel Dias Foundation (Funed), Belo Horizonte, MG, 30510-010, Brazil., Martins AA; Industrial Directorate, Ezequiel Dias Foundation (Funed), Belo Horizonte, MG, 30510-010, Brazil., Boasquivis PF; Industrial Directorate, Ezequiel Dias Foundation (Funed), Belo Horizonte, MG, 30510-010, Brazil., Duarte MM; Octávio Magalhães Institute Directorate, Funed, Belo Horizonte, MG, 30510-010, Brazil., de Oliveira Ribeiro B; Research and Development Directorate, Funed, Belo Horizonte, MG 30510-010, Brazil; Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG 31270-901, Brazil., de Souza Filho JA; Research and Development Directorate, Funed, Belo Horizonte, MG 30510-010, Brazil., Borges MH; Research and Development Directorate, Funed, Belo Horizonte, MG 30510-010, Brazil., de Oliveira Souza G; Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG 31270-901, Brazil., Olórtegui CC; Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG 31270-901, Brazil., Luzia AB; Industrial Directorate, Ezequiel Dias Foundation (Funed), Belo Horizonte, MG, 30510-010, Brazil., Caldas S; Research and Development Directorate, Funed, Belo Horizonte, MG 30510-010, Brazil. Electronic address: sergio.caldas@funed.mg.gov.br. |
| Πηγή: | Journal of immunological methods [J Immunol Methods] 2026 May; Vol. 548, pp. 114059. Date of Electronic Publication: 2026 May 09. |
| Τύπος έκδοσης: | Journal Article |
| Γλώσσα: | English |
| Στοιχεία περιοδικού: | Publisher: Elsevier Country of Publication: Netherlands NLM ID: 1305440 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1872-7905 (Electronic) Linking ISSN: 00221759 NLM ISO Abbreviation: J Immunol Methods Subsets: MEDLINE |
| Imprint Name(s): | Publication: Amsterdam : Elsevier Original Publication: Amsterdam, North-Holand |
| Ιατρικοί όροι (MeSH): | Epitope Mapping*/methods , Antibodies, Viral*/immunology , Antibodies, Viral*/blood , SARS-CoV-2*/immunology , Antibodies, Neutralizing*/immunology, COVID-19/immunology ; Spike Glycoprotein, Coronavirus/immunology ; Immunoglobulin Fab Fragments/immunology ; Animals ; Horses ; Workflow ; Neutralization Tests ; Enzyme-Linked Immunosorbent Assay ; Humans |
| Περίληψη: | 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. (Copyright © 2026 The Authors. Published by Elsevier B.V. All rights reserved.) |
| Competing Interests: | Declaration of competing interest The authors declare no conflicts of interest. |
| Contributed Indexing: | Keywords: Epitope mapping; Equine hyperimmune serum; F(ab')2 antibodies; SARS-CoV-2; β-Propiolactone inactivation |
| Substance Nomenclature: | 0 (Antibodies, Viral) 0 (Spike Glycoprotein, Coronavirus) 0 (Antibodies, Neutralizing) 0 (spike protein, SARS-CoV-2) 0 (Immunoglobulin Fab Fragments) |
| Entry Date(s): | Date Created: 20260511 Date Completed: 20260613 Latest Revision: 20260730 |
| Update Code: | 20260730 |
| DOI: | 10.1016/j.jim.2026.114059 |
| PMID: | 42114615 |
| Βάση Δεδομένων: | MEDLINE |
| ISSN: | 1872-7905 |
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| DOI: | 10.1016/j.jim.2026.114059 |