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Biologicals : journal of the International Association of Biological Standardization2012; 40(4); 240-246; doi: 10.1016/j.biologicals.2012.03.004

New equine antitoxins to botulinum neurotoxins serotypes A and B.

Abstract: Hyperimmune monovalent antitoxins to botulinum neurotoxin serotypes A and B have been produced by immunizing horses with newly developed formalin toxoids. After primary immunization, horses developed acceptable prophylactic antibody titers (1-5 IU/mL). Three horses received additional toxoid booster injections to induce hyperimmune antibody titers with antitoxin-A and antitoxin-B titers reaching peaks of approximately 2000 IU/mL and 150-625 IU/mL, respectively. Titers were quantified throughout the process by antigen-capture ELISA and by in-vivo neutralization. ELISA titers and neutralization titers correlated (R² ∼0.62-0.92), however, unique correlations between in-vitro and in-vivo titers were observed for each horse. Monovalent antitoxin pools were made by combining plasma that had been collected twice via plasmaphoresis several months after primary immunization. Neutralizing units were established for each pool relative to the current US and WHO reference standards. Titers were determined at the L(+)/10 and L(+)/40 toxin dose for Toxin types A and B, respectively, and U.S. and international units were assigned to each monovalent antitoxin. Avidity of the new Anti-A pool was equivalent to the WHO Anti-A reference at the L(+), L(+)/10 and L(+)/30 dose. Each monovalent plasma pool failed to cross-neutralize other botulinum neurotoxin serotypes indicating a high degree of specificity of each antitoxin for the toxin serotype used during immunization.
Published by Elsevier Ltd.
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Publication Date: 2012-05-05 PubMed ID: 22560800PubMed Central: PMC3393819DOI: 10.1016/j.biologicals.2012.03.004Google Scholar: Lookup
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Summary

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The research article presents a new study on the production of hyperimmune monovalent antitoxins to botulinum neurotoxin serotypes A and B, achieved by immunizing horses with newly developed formalin toxoids. Post immunization, increased antibody titers were observed, with peaks at approximately 2000 IU/mL and between 150 to 625 IU/mL, respectively. Testing established the specificity of each antitoxin for the toxin serotype used during immunization.

Immunization Procedure and Resulting Antibody Titers

  • The study started by immunizing horses with new formalin toxoids that were designed for botulinum neurotoxin serotypes A and B.
  • Following this primary immunization, it was found that the horses developed prophylactic antibody titers, measuring between 1-5 IU/mL which are within acceptable limits.
  • The process was then taken a step further, with three horses receiving more toxoid booster injections. This was done to induce hyperimmune antibody levels.
  • After these additional injections, antitoxin-A and antitoxin-B levels peaked at around 2000 IU/mL and ranged from 150-625 IU/mL respectively.

Measurement Methods and Results

  • The method of quantification of titers was two-fold: by an antigen-capture ELISA (Enzyme-Linked Immunosorbent Assay), which is a commonly used method to measure antibodies in biological fluids, and by in-vivo neutralization, a method that determines the effectiveness of the antitoxins in a living organism.
  • The researchers observed a strong correlation between the ELISA titers and neutralization titers, with an R² (a measure of how close the data are to the fitted regression line) value ranging from about 0.62 to 0.92.
  • However, the relation between the in-vitro (ELISA) and in-vivo (neutralization) titers was unique to each horse.

Preparation of Monovalent Antitoxin Pools

  • Plasma, collected twice via plasmaphoresis (a process that filters the blood and removes harmful antibodies) months after the primary immunization, was combined to make monovalent antitoxin pools.
  • The neutralizing units for each pool were established relatively to the current US and the World Health Organization (WHO) reference standards.
  • Each pool was found to have a very high degree of specificity for the toxin serotype used during the initial immunization, indicating that the pools were unable to cross-neutralize other botulinum neurotoxin serotypes.

Cite This Article

APA
Li D, Mattoo P, Keller JE. (2012). New equine antitoxins to botulinum neurotoxins serotypes A and B. Biologicals, 40(4), 240-246. https://doi.org/10.1016/j.biologicals.2012.03.004

Publication

Biologicals : journal of the International Association of Biological Standardization
ISSN: 1095-8320
NlmUniqueID: 9004494
Country: England
Language: English
Volume: 40
Issue: 4
Pages: 240-246

Researcher Affiliations

Li, D
  • US FDA, CBER, Office of Vaccines Research and Review, Bethesda, MD 20892, USA.
Mattoo, P
    Keller, J E

      MeSH Terms

      • Animals
      • Antitoxins / biosynthesis
      • Antitoxins / immunology
      • Botulinum Toxins / immunology
      • Botulinum Toxins, Type A / immunology
      • Enzyme-Linked Immunosorbent Assay
      • Horses / immunology
      • Mice
      • Neutralization Tests
      • Plasmapheresis

      Grant Funding

      • Y01 AI006153 / NIAID NIH HHS
      • Y01 AI006153-04 / NIAID NIH HHS
      • YI-AI-6153-01 / NIAID NIH HHS

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      Citations

      This article has been cited 10 times.
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