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

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Title: Integrated workflow for production and in vitro characterization of equine anti-SARS-CoV-2 hyperimmune serum with epitope mapping.
Authors: 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.
Source: Journal of immunological methods [J Immunol Methods] 2026 May; Vol. 548, pp. 114059. Date of Electronic Publication: 2026 May 09.
Publication Type: Journal Article
Language: English
Journal Info: 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
MeSH Terms: 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
Abstract: 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
Database: MEDLINE
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  Data: Integrated workflow for production and in vitro characterization of equine anti-SARS-CoV-2 hyperimmune serum with epitope mapping.
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  Data: <searchLink fieldCode="AU" term="%22de+Santana+Evangelista+K%22">de Santana Evangelista K</searchLink>; Industrial Directorate, Ezequiel Dias Foundation (Funed), Belo Horizonte, MG, 30510-010, Brazil.<br /><searchLink fieldCode="AU" term="%22de+Castro+Barbosa+E%22">de Castro Barbosa E</searchLink>; Research and Development Directorate, Funed, Belo Horizonte, MG 30510-010, Brazil.<br /><searchLink fieldCode="AU" term="%22de+Souza+Andrade+A%22">de Souza Andrade A</searchLink>; Research and Development Directorate, Funed, Belo Horizonte, MG 30510-010, Brazil.<br /><searchLink fieldCode="AU" term="%22Ardisson+L%22">Ardisson L</searchLink>; Industrial Directorate, Ezequiel Dias Foundation (Funed), Belo Horizonte, MG, 30510-010, Brazil.<br /><searchLink fieldCode="AU" term="%22Martins+AA%22">Martins AA</searchLink>; Industrial Directorate, Ezequiel Dias Foundation (Funed), Belo Horizonte, MG, 30510-010, Brazil.<br /><searchLink fieldCode="AU" term="%22Boasquivis+PF%22">Boasquivis PF</searchLink>; Industrial Directorate, Ezequiel Dias Foundation (Funed), Belo Horizonte, MG, 30510-010, Brazil.<br /><searchLink fieldCode="AU" term="%22Duarte+MM%22">Duarte MM</searchLink>; Octávio Magalhães Institute Directorate, Funed, Belo Horizonte, MG, 30510-010, Brazil.<br /><searchLink fieldCode="AU" term="%22de+Oliveira+Ribeiro+B%22">de Oliveira Ribeiro B</searchLink>; 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.<br /><searchLink fieldCode="AU" term="%22de+Souza+Filho+JA%22">de Souza Filho JA</searchLink>; Research and Development Directorate, Funed, Belo Horizonte, MG 30510-010, Brazil.<br /><searchLink fieldCode="AU" term="%22Borges+MH%22">Borges MH</searchLink>; Research and Development Directorate, Funed, Belo Horizonte, MG 30510-010, Brazil.<br /><searchLink fieldCode="AU" term="%22de+Oliveira+Souza+G%22">de Oliveira Souza G</searchLink>; Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG 31270-901, Brazil.<br /><searchLink fieldCode="AU" term="%22Olórtegui+CC%22">Olórtegui CC</searchLink>; Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG 31270-901, Brazil.<br /><searchLink fieldCode="AU" term="%22Luzia+AB%22">Luzia AB</searchLink>; Industrial Directorate, Ezequiel Dias Foundation (Funed), Belo Horizonte, MG, 30510-010, Brazil.<br /><searchLink fieldCode="AU" term="%22Caldas+S%22">Caldas S</searchLink>; Research and Development Directorate, Funed, Belo Horizonte, MG 30510-010, Brazil. Electronic address: sergio.caldas@funed.mg.gov.br.
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  Data: <searchLink fieldCode="JN" term="%221305440%22">Journal of immunological methods</searchLink> [J Immunol Methods] 2026 May; Vol. 548, pp. 114059. <i>Date of Electronic Publication: </i>2026 May 09.
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  Data: <i>Publisher: </i><searchLink fieldCode="PB" term="%22Elsevier%22">Elsevier </searchLink><i>Country of Publication: </i>Netherlands <i>NLM ID: </i>1305440 <i>Publication Model: </i>Print-Electronic <i>Cited Medium: </i>Internet <i>ISSN: </i>1872-7905 (Electronic) <i>Linking ISSN: </i><searchLink fieldCode="IS" term="%2200221759%22">00221759 </searchLink><i>NLM ISO Abbreviation: </i>J Immunol Methods <i>Subsets: </i>MEDLINE
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– Name: Abstract
  Label: Abstract
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  Data: 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.<br /> (Copyright © 2026 The Authors. Published by Elsevier B.V. All rights reserved.)
– Name: Abstract
  Label: Competing Interests
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  Data: Declaration of competing interest The authors declare no conflicts of interest.
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  Data: <i>Keywords: </i>Epitope mapping; Equine hyperimmune serum; F(ab')2 antibodies; SARS-CoV-2; β-Propiolactone inactivation
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  Data: 0 (Antibodies, Viral)<br />0 (Spike Glycoprotein, Coronavirus)<br />0 (Antibodies, Neutralizing)<br />0 (spike protein, SARS-CoV-2)<br />0 (Immunoglobulin Fab Fragments)
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        Text: English
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        StartPage: 114059
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        Type: general
      – SubjectFull: Spike Glycoprotein, Coronavirus immunology
        Type: general
      – SubjectFull: Immunoglobulin Fab Fragments immunology
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