Journal of Pharmacopuncture (대한약침학회지)

KOREAN PHARMACOPUNCTURE INSTITUTE (대한약침학회)
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Purification of Therapeutic Serums of Snake Anti-Venom with Caprylic Acid

  • Norouznejad, Nilofar (Department of Biology, Faculty of Sciences, Science and Research Branch, Islamic Azad University) ;
  • Zolfagharian, Hossein (Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO)) ;
  • Babaie, Mahdi (Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO)) ;
  • Ghobeh, Maryam (Department of Biology, Faculty of Sciences, Science and Research Branch, Islamic Azad University)
  • Received : 2021.01.31
  • Accepted : 2022.03.31
  • Published : 2022.06.30
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Abstract

Objectives: Antivenom serums have been used extensively for over a century and are the only effective treatment option for snake bites and other dangerous animal envenomations. In therapeutic serum centers, a wide range of antivenoms is made from animal serum, mainly equine and sheep, that are immunized with single or multiple venoms. This work aimed to use caprylic acid (CA) to purify therapeutic snake antivenom. Methods: Plasma was obtained from equine immunized with a mixture of venoms. Immunized plasma was obtained by precipitation of different concentrations (2-5%) of CA. This methodology was compared to that based on ammonium sulfate (AS) precipitation. Sediment plasma proteins were purified by ion-exchange chromatography. Protein assay, SDSPAGE, and agar gel diffusion were performed. Results: The total protein precipitation with AS was higher than precipitation with CA, but the best results were obtained when CA was added to the plasma until a final CA concentration of 5% was reached. Chromatography and electrophoresis indicated a stronger band for the 5% CA, and the gel diffusion assay showed antigen-antibody interaction in the purified serum. Conclusion: The use of CA compared to the routine method for purifying hyperimmune serums is a practical and cost-effective method for preparing and producing therapeutic serums. It constitutes a potentially valuable technology for alleviating the critical shortage of antivenom in Iran.

Keywords

Acknowledgement

This work was supported by the Razi Vaccine and Serum Research Institute, Karaj, Iran.

References

  1. Guidolin FR, Caricati CP, Marcelino JR, da Silva WD. Development of equine IgG antivenoms against major snake groups in Mozambique. PLoS Negl Trop Dis. 2016;10(1):e0004325. https://doi.org/10.1371/journal.pntd.0004325
  2. Hafezi G, Rahmani AH, Soleymani M, Nazari P. An epidemiologic and clinical study of snake bites during a five-year period in Karoon, Iran. Asia Pac J Med Toxicol. 2018;7(1):13-6.
  3. Babaie M, Salmanizadeh H, Zolfagharian H. Blood coagulation induced by Iranian saw-scaled viper (echis carinatus) venom: identification, purification and characterization of a prothrombin activator. Iran J Basic Med Sci. 2013;16(11):1145-50.
  4. Babaie M, Salmanizadeh H, Zolfagharian H, Alizadeh H. Properties of biological and biochemical effects of the Iranian sawscaled viper (Echis carinatus) venom. Bratisl Lek Listy. 2014;115(7):434-8.
  5. Babaie M. Snake venom proteins and coagulopathy caused by snakebite. J Birjand Univ Med Sci. 2021;28(2):88-105.
  6. Mohamed Abd El-Aziz T, Garcia Soares A, Stockand JD. Snake venoms in drug discovery: valuable therapeutic tools for life saving. Toxins (Basel). 2019;11(10):564. https://doi.org/10.3390/toxins11100564
  7. Bochner R. Paths to the discovery of antivenom serotherapy in France. J Venom Anim Toxins Incl Trop Dis. 2016;22:20. https://doi.org/10.1186/s40409-016-0074-7
  8. de Silva HA, Ryan NM, de Silva HJ. Adverse reactions to snake antivenom, and their prevention and treatment. Br J Clin Pharmacol. 2016;81(3):446-52. https://doi.org/10.1111/bcp.12739
  9. Zolfagharian H, Dounighi NM. Progress and improvement of the manufacturing process of snake antivenom. Arch Razi Inst. 2013;68(1):1-10.
  10. Otero R, Leon G, Gutierrez JM, Rojas G, Toro MF, Barona J, et al. Efficacy and safety of two whole IgG polyvalent antivenoms, refined by caprylic acid fractionation with or without betapropiolactone, in the treatment of Bothrops asper bites in Colombia. Trans R Soc Trop Med Hyg. 2006;100(12):1173-82. https://doi.org/10.1016/j.trstmh.2006.01.006
  11. Maljaee P, Zolfagharian H, Babaie M, Mohammadpour Dounighi N. The comparison of nanoparticle adjuvant with the montanide adjuvant for hyper-immunization to produce antisnakebite serum. J Fasa Univ Med Sci. 2018;8(1):674-82.
  12. Kurtovic T, Lang Balija M, Brgles M, Sviben D, Tunjic M, Cajner H, et al. Refinement strategy for antivenom preparation of high yield and quality. PLoS Negl Trop Dis. 2019;13(6):e0007431. https://doi.org/10.1371/journal.pntd.0007431
  13. Nudel BC, Perdomenico C, Iacono R, Cascone O. Optimization by factorial analysis of caprylic acid precipitation of non-immunoglobulins from hyperimmune equine plasma for antivenom preparation. Toxicon. 2012;59(1):68-73. https://doi.org/10.1016/j.toxicon.2011.10.014
  14. Otero R, Gutierrez JM, Rojas G, Nunez V, Diaz A, Miranda E, et al. A randomized blinded clinical trial of two antivenoms, prepared by caprylic acid or ammonium sulphate fractionation of IgG, in Bothrops and Porthidium snake bites in Colombia: correlation between safety and biochemical characteristics of antivenoms. Toxicon. 1999;37(6):895-908. https://doi.org/10.1016/S0041-0101(98)00220-7
  15. Morais V, Massaldi H. A model mechanism for protein precipitation by caprylic acid: application to plasma purification. Biotechnol Appl Biochem. 2012;59(1):50-4. https://doi.org/10.1002/bab.68
  16. Wingfield P. Protein precipitation using ammonium sulfate. Curr Protoc Protein Sci. 2001;Appendix 3:Appendix 3F.
  17. Babaie M, Zolfagharian H, Zolfaghari M, Jamili S. Biochemical, hematological effects and complications of Pseudosynanceia melanostigma envenoming. J Pharmacopuncture. 2019;22(3):140-6. https://doi.org/10.3831/kpi.2019.22.018
  18. Babaie M, Ghaem panah A, Mehrabi Z, Mollaei A. Partial purification and characterization of antimicrobial effects from snake (Echis carinatus), scorpion (Mesosobuthus epues) and bee (Apis mellifera) venoms. Iran J Med Microbiol. 2020;14(5):460-77. https://doi.org/10.30699/ijmm.14.5.460
  19. Ouchterlony O. Antigen-antibody reactions in gels. Acta Pathol Microbiol Scand. 1949;26(4):507-15. https://doi.org/10.1111/j.1699-0463.1949.tb00751.x
  20. Alizadeh H, Madani R, Babaie M, Kavid N, Golchinfar F, Emami T. Preparation and purification of polyclonal antibodies against Mycobacterium avium paratuberculosis antigens in rabbit. J Fasa Univ Med Sci. 2012;2(3):168-73.
  21. Bawaskar HS. Snake bite poisoning: a neglected life-threatening occupational hazard. Indian J Crit Care Med. 2014;18(3):123-4. https://doi.org/10.4103/0972-5229.128698
  22. Gutierrez JM. Global availability of antivenoms: the relevance of public manufacturing laboratories. Toxins (Basel). 2018;11(1):5. https://doi.org/10.3390/toxins11010005
  23. Grodzki AC, Berenstein E. Antibody purification: ammonium sulfate fractionation or gel filtration. Methods Mol Biol. 2010;588:15-26. https://doi.org/10.1007/978-1-59745-324-0_3
  24. Vargas M, Segura A, Herrera M, Villalta M, Estrada R, Cerdas M, et al. Preclinical evaluation of caprylic acid-fractionated IgG antivenom for the treatment of Taipan (Oxyuranus scutellatus) envenoming in Papua New Guinea. PLoS Negl Trop Dis. 2011;5(5):e1144. https://doi.org/10.1371/journal.pntd.0001144
  25. dos Santos MC, D'Imperio Lima MR, Furtado GC, Colletto GM, Kipnis TL, Dias da Silva W. Purification of F(ab')2 anti-snake venom by caprylic acid: a fast method for obtaining IgG fragments with high neutralization activity, purity and yield. Toxicon. 1989;27(3):297-303. https://doi.org/10.1016/0041-0101(89)90177-3
  26. Gutierrez JM, Rojas E, Quesada L, Leon G, Nunez J, Laing GD, et al. Pan-African polyspecific antivenom produced by caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa. Trans R Soc Trop Med Hyg. 2005;99(6):468-75. https://doi.org/10.1016/j.trstmh.2004.09.014
  27. Cruz AM, Oscar OA, Dailin DC, Oliver PM, editor. Caprylic Acid in the purification of horse tetanus IgG immunoglobulin. Proceedings of the IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology; 2015 Oct 13-16; Medellin, Colombia. Lausanne (Switzerland): Frontiers; 2015.