The median effective dose (ED50)

The preclinical efficacy of an antivenom, in effect, its capability to neutralise the lethal systemic effects of snake venom, can be evaluated by calculating the ED₅₀ value, which is defined as the minimum amount of antivenom required to protect 50% of mice injected with lethal doses of venom [40]. For these experiments, we used the Premium Serums antivenom, as this product was found to recognise the Naja venoms to a greater extent than all of the other Indian marketed antivenoms, as determined by our in vitro assays. We chose to test only the most promising of the marketed Indian antivenoms in these experiments to reduce the burden of suffering on experimental animals. Venom doses equivalent to five LD₅₀ determined in the experiments above were used as the ‘challenge dose’. Different volumes of antivenom were mixed with the challenge dose of venom, followed by an incubation period of 30 min at 37° C. Immediately after incubation, each venom-antivenom mixture (n = 4 per venom) was intravenously injected into a group of five male CD-1 mice (18–22 g). A group of five male mice injected with 1× LD₅₀ of venom, served as the positive control. The ED₅₀ values of the antivenom against each venom were calculated using Probit statistics [41]. Antivenom neutralisation potency was calculated as described before [14,42].

Here, n is equal to the number of LD₅₀ used as the challenge dose.