The saga of the development of laboratory tests to evaluate the neutralizing efficacy of antivenoms for the treatment of snakebite envenoming

The parenteral administration of animal-derived antivenoms has been, since 1894, the mainstay of the therapy of snakebite envenoming, a neglected tropical disease that exerts a high toll of morbidity and mortality on a global basis. A key test in the development and quality control of antivenoms has been the neutralization of the lethal effect of venoms. Since the early times of antivenoms, the neutralization of venom's lethality has been predominantly based on the incubation of venom and antivenom prior to the injection in laboratory animals. The present narrative review summarizes the main milestones in the development of venom neutralization assays by antivenoms, starting with the pioneer work of Albert Calmette and Vital Brazil. Despite various initiatives along the 20th century to standardize these tests, the antivenom efficacy assays have been highly heterogeneous between laboratories and countries. Efforts carried out to understand the main variables that determine the lethality neutralization tests are discussed, together with the introduction of assays to assess the neutralization of other relevant toxic and enzymatic activities of venoms, which complement the lethality assay. In the last decades, international efforts coordinated by the World Health Organization (WHO) led to the publication of the WHO guidelines for the production and control of antivenoms, which comprise detailed descriptions of preclinical neutralization assays, including the essential test, i.e., lethality, and supplementary assays that assess other relevant toxic effects. The pain and suffering of animals in the traditional antivenom neutralization tests urge the implementation of the 3Rs principles (replacement, reduction, refinement) in antivenom efficacy assessment, and innovation is urgently needed in this field.