eISSN: 1644-4124
ISSN: 1426-3912
Central European Journal of Immunology
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2/2024
vol. 49
 
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Comparison of cytotoxicity methods for studying Vipera ammodytes venom and the anticytotoxic potency of antivenom

Ivana Lukic
1
,
Veljko Blagojevic
1
,
Rajna Minic
1
,
Sasa Ivanovic
2
,
Suncica Borozan
3
,
Vitomir Cupic
2
,
Irena Zivkovic
1

  1. Institute of Virology, Vaccines and Sera “Torlak”, Belgrade, Serbia
  2. Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, University of Belgrade, Belgrade, Serbia
  3. Faculty of Veterinary Medicine, Department of Chemistry, University of Belgrade, Belgrade, Serbia
Cent Eur J Immunol 2024; 49 (2): 94-104
DOI: https://doi.org/10.5114/ceji.2024.142417
Online publish date: 2024/08/26
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Introduction:
Alternative in vitro tests that can be used instead of animal experiments are those that can most closely evaluate the biological activity of the drug of interest. For testing the potency of antivenom, these are the methods used to assess cytotoxicity. The aim of this study was to evaluate the most commonly used cytotoxicity methods for determining the protective potency of the antivenom Viekvin, which neutralizes Vipera ammodytes venom.

Material and methods:
The selected methods are based on different biological mechanisms: MTT assay, based on the activity of cell oxidoreductase enzymes; crystal violet staining, based on the degree of cell adhesion; trypan blue staining, based on cell membrane permeability, and propidium iodide staining, based on measurement of nucleic acids of dead cells. The pro-apoptotic effect of the venom was also determined with annexin V staining.

Results:
The IC50 value of V. ammodytes venom obtained by these methods was very similar, while the EC50 values differed significantly.

Conclusions:
We concluded that the choice of the method used to measure the anticytotoxic anti­venom potency depends on the immunogenicity of the venom components that cause cell death; for each venom/antivenom pair, it is necessary to select the appropriate assay separately, and at present, none of the standard cytotoxic methods can be universally applied to determine antivenom potency.

keywords:

cytotoxicity, venom, antivenom, Vipera ammodytes, apoptosis, necrosis
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