In India, polyvalent antivenom is the mainstay treatment for snakebite envenoming. Due to batch-to-batch variation in antivenom production, manufacturers have to estimate its efficacy at each stage of IgG purification using the median effective dose which involves 100-120 mice for each batch. There is an urgent need to replace the excessive use of animals in snake antivenom production using in vitro alternatives. We tested the efficacy of a single batch of polyvalent antivenom from VINS bioproducts limited on Echis carinatus venom collected from three different locations-Tamil Nadu (ECVTN), Goa (ECVGO) and Rajasthan (ECVRAJ)-using different in vitro assays. Firstly, size-exclusion chromatography (SEC-HPLC) was used to quantify antivenom-venom complexes to assess the binding efficiency of the antivenom. Secondly, clotting, proteolytic and PLA2 activity assays were performed to quantify the ability of the antivenom to neutralize venom effects. The use of both binding and functional assays allowed us to measure the efficacy of the antivenom, as they represent multiple impacts of snake envenomation. The response from the assays was recorded for different antivenom-venom ratios and the dose-response curves were plotted. Based on the parameters that explained the curves, the efficacy scores (ES) of antivenom were computed. The binding assay revealed that ECVTN had more antivenom-venom complexes formed compared to the other venoms. The capacity of antivenom to neutralize proteolytic and PLA2 effects was lowest against ECVRAJ. The mean efficacy score of antivenom against ECVTN was the greatest, which was expected, as ECVTN is mainly used by antivenom manufacturers. These findings pave a way for the development of in vitro alternatives in antivenom efficacy assessment.
Keywords: 3Rs; SEC-HPLC; envenomation; in vitro assays; median effective dose; polyvalent antivenom; snakebites; vipers.