engleski

Towards the novel in vitro alternative to the in vivo assay for estimation of antivenom potency

Long-nosed viper (Vipera a. ammodytes) is the most poisonous European snake. Its venom has been used for immunobiological production of antivenoms, the only specific therapy of snake envenomation, for over 50 years. The method for assessment of neutralization potency of antivenoms requires the use of large number of experimental animals. The method, although painful for the animals, is legally approved, since there has been no in vitro alternative yet. The goal of our work was to investigate the immunology of the most toxic long-nosed viper venom components and to gain knowledge that would enable the development of the novel in vitro method correlating to neutralization potency of antivenoms against long-nosed viper. Here we present the data on the correlation between the protective efficacies of antisera raised in rabbits against the venom of V. a. ammodytes (expressed as number of lethal toxic doses 50 that could be neutralised by 1 ml of undiluted serum) and the quantities of IgG-s specific for the venom and its most toxic venom components, ammodytoxins (Atx) and haemorrhagins, (determined by respective ELISAs). The highest correlation (rS= 0.884, p<0.0001) was observed between anti-AtxA content and protective efficacy of antisera. Accordingly, high correlation (rS=0.842, p<0.0001) was obtained between the ability of serum to neutralise phospholipase activity of the venom and protective efficacy of antisera. ELISA assays and Western blot analyses of antisera raised against pure toxic and non-toxic phospholipases, respectively, demonstrated complete lack of cross reactivity between the two. The obtained results have lead so far to the hypothesis that anti-ammodytoxin antibodies might be key molecules for the design of the novel in vitro alternative to the assay for estimation of antivenom potency.

long-nosed viper venom; antivenom; ED50; AtxA; IgG

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