engleski

THE ROAD TOWARDS IN VITRO ALTERNATIVE TO IN VIVO SNAKE VENOM TOXICITY AND ANTIVENOM POTENCY ASSAYS

Continuous quality control of venoms and antivenoms is necessary for a successful production of the only specific treatment of victims of venomous snake bites in Europe. Control is based on two in vivo tests: (a) the venom lethal toxicity assay (determination of median lethal dose of venom or LD50) and (b) test for determining the neutralization potency (effectiveness) of produced antivenoms (determination of mean effective antivenom dose or ED50). Both these tests, which cause suffering, pain and death of the experimental animals and also require a large number of animals, were identified by ECVAM (European Centre for Validation of Alternative Methods) as assays that are necessary to be replaced with alternative methods. Snake venoms are complex mixtures of more than a hundred mostly proteins and peptides, and non-protein compounds with different biochemical and pharmacological effects. Such complex mixtures are used as an antigen for animal immunization in antivenom production. For the development of in vitro tests, it is necessary to identify the components that contribute to the venom’s overall toxicity or specificity of antibodies that effectively neutralize them. So far several molecules, mainly from the family of metalloproteinases (haemorrhagins, the dominant cause of the human envenomation pathology) and phospholipase A2 (ammodytoxins, the most toxic molecules in the venom known to date) have been isolated and characterized from the European vipers’ venom. However, there were no literature data for their immunogenicity. Our study showed that ammodytoxin content, determined by two developed biochemical methods, HPLC and the "sandwich" ELISA, highly correlated with the lethal toxicity of the whole viper venom. Further we examined the role of ammodytoxin (Atx)- and haemorrhagin (H)-specific antibodies in the venom lethal toxicity neutralization. The results showed that functional anti-Atx antibodies were only partially involved in the neutralization of the venom toxicity. On the other hand, functional anti-H antibodies did not provide protection at all. Developed methods for anti-Atx or anti-H determination, due to only limited involvement of the mentioned antibodies in venom toxicity neutralization, cannot be a substitute for the in vivo assays. Deeper insight into snake venom composition at the molecular level and the participation of the particular components in the venom toxicity has yet to be done before solving this task. On the other hand, Atx content determination in different venom batches could be a good screening method in the selection of the best antigen for immunisation.

quality control; snake venom; LD50; snake antivenom; ED50; in vitro alternative

nije evidentirano