An update on the application of alternative methods for evaluating antivenom potency

The use of alternative methods for in vivo lethality neutralization assays in mice (potency tests) during antivenom production has been encouraged by the World Health Organization, based on the 3Rs principle (Replacement, Reduction, and Refinement). In addition to animal welfare-associated benefits, alternative methods can reduce the time, cost, and intrinsic variability linked with animal-based assays. Several studies have been conducted using different methodologies, including immunological assays and tests for inhibiting enzymatic and toxic activities of venom, to assess their level of agreement with the in vivo method, which is considered the gold standard. However, the results varied depending on the methodology applied, as well as the specificity and avidity of antibodies toward the toxic and non-toxic venom fractions, and the composition of each venom. This literature review examined these studies and their outcomes, revealing that the Enzyme-Linked Immunosorbent Assay (ELISA) has historically been the most evaluated alternative to the in vivo antivenom potency test. Despite promising results, the development and implementation of substitute methodologies face significant challenges, primarily in their validation, which requires substantial time and resources from the institutions that produce them. Efforts should focus on developing, validating, and applying alternative methods for potency assays in antivenom production, particularly in the early and intermediate stages, where regulatory requirements are less stringent.