Production of a Human Recombinant Polyclonal Fab Antivenom against Iranian Viper Echis carinatus

Document Type : Original Articles

Authors

1 Department of Immunization and Plasma Production, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

2 Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

3 Department of Medical Biotechnology, School of Medicine, Tarbiat Modares University, Tehran, Iran

4 Department of Venomous Animals, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran

5 Department of Foot and Mouth Disease, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran

6 Department of Virology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran

7 Department of Biotechnology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran

Abstract

Venomous snakebite is a life-threatening injury in many tropical and subtropical areas including Iran. The gold standard treatment option for human envenomation is the use of antivenoms. Despite the unique effects of horse-derived antivenoms on the treatment of snakebite, they are not fully perfect and need improvements. In this study, human recombinant Fab fragment antivenom was produced in Rosetta-g bacterium using a gene library constructed in the previous study. The prepared Fab was purified in several steps, desalted, and lipopolysaccharide-depleted using ammonium sulfate solution and dialysis against phosphate buffer and Triton X-114 solution, respectively. Subsequently, the product was initially confirmed by the sodium dodecyl sulfate polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. Finally, the neutralization potency of the product was investigated in laboratory Syrian Mice. The obtained results showed corresponding reduced bands to Fab fragment with the molecular weight of about 28 kDa at a concentration of 3.1 mg/ml. There was a significant difference between the groups in terms of ELISA test (P<0.05). The neutralization potency of the product against the venom of Echis carinatus (E. carinatus) was about 7 LD50/ml (54.6 µg/ml) when tested on mice. Based on the results, the Fab fragment antivenom had the ability to neutralize the in vivo biological activity of the venom of Iranian E. carinatus. However, further studies are recommended to reach a suitable concentration of antivenom fragment.

Keywords

Main Subjects

Article Title [French]

Production d'un de Sérum Recombinant Humain de Fab Polyclonal contre Echis carinatus Iranien

Abstract [French]

La morsure de serpent venimeux est une blessure mortelle dans de nombreuses régions tropicales et subtropicales, y compris Iran. L'etraitement de référence en cas d'envenimation humaine est l'utilisation d'anti-venirs. Malgré l'efficacité uniques des sérums antivenimeux dérivés du cheval sur le traitement des morsures de serpents, ils présent néanmoins des limitations et doivent être améliorés. Dans cette étude, un fragment de sérum recombinant humain de Fab Polyclonal a été produit dans la bacterie Rosetta-g en utilisant une bibliothèque de gènes construite dans l’étude précédente. Le Fab préparé a été purifié en plusieurs étapes, dessalé et appauvri en lipopolysaccharide en utilisant une solution de sulfate d'ammonium et une dialyse contre un tampon phosphate et une solution de Triton X-114, respectivement. Par la suite, le produit a été analysé par électrophorèse sur gel de polyacrylamide de dodécyl sulfate de sodium (SDS-PAGE) et par dosage immuno-enzymatique (ELISA), respectivement. Enfin, à capacité de neutralisation du produit a été étudiée chez des souris syriennes de laboratoire. Les résultats obtenus ont montré des bandes réduites correspondantes au fragment Fab avec un poids moléculaire d'environ 28 kDa à une concentration de 3,1 mg / ml. Il y avait une différence significative entre les groupes en termes de test ELISA (p <0,05). La capacité de neutralisation de ce produit contre le venin Echis carinatus était d'environ 7 LD50 / ml (54,6 µg / ml) lorsqu'il a été testé sur des souris. Sur la base des résultats, l'antivenin du fragment Fab avait la capacité de neutraliser l'activité biologique in vivo du venin E. carinatus d'Iran. Cependant, des études complémentaires sont recommandées pour atteindre une concentration appropriée de fragment antivenin.

Keywords [French]

  • Echis carinatus
  • Fragment Fab
  • Bibliothèque de Gènes
  • Antivenin
  • Polyclonal

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Archives of Razi Institute
Volume 73, Issue 4
December 2018
Pages 287-294

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