As the neutralization capacity of an antivenom is often tested through in vivo animal neutralization assays, these studies are mostly preceded by tests of the ability of the antivenom to cross-react with different snake species, meaning its ability to cross-bind to toxins from several snake species. It is therefore highly desirable for antivenoms to be able to neutralize different toxins in venoms from multiple snake species, a trait called cross-neutralization. Additionally, the immunogenicity of different groups of snake toxins are highly variable, which can result in antivenoms with variable neutralizing abilities against certain toxins. However, snake venoms differ greatly, both between different species, as well as between specimens from the same species inhabiting different geographical locations. In producing such antivenoms, the snake venoms included in the immunization mixtures (homologous venoms) are chosen based on which snakebites the antivenom is intended to treat. The current treatment of snakebite envenoming is based on polyclonal antibodies obtained from the plasma of animals hyper-immunized with snake venom. This has renewed the interest in antivenom research and will hopefully lead to new advances within the field. Snakebite envenoming was added to the list of the world’s most neglected tropical diseases by the World Health Organization in 2017. A special focus is given to antivenomics and high-density peptide microarray technology as these high-throughput methods have recently been introduced in this field and may enable more detailed assessments of antivenom cross-reactivity. This review provides an overview of both the classical and new methods used to investigate antivenom cross-reactivity, the advantages and disadvantages of each method, and examples of studies using the methods. Antivenomics involves a top-down assessment of the toxin-binding capacities of antivenoms, whereas high-density peptide microarray technology may be harnessed to provide in-depth knowledge on which toxin epitopes are recognized by antivenoms. In recent years, new methods for determination of cross-reactivity have emerged, including surface plasmon resonance, antivenomics, and high-density peptide microarray technology. Traditionally, the methods used for analyzing cross-reactivity have been immunodiffusion, immunoblotting, enzyme-linked immunosorbent assay (ELISA), enzymatic assays, and in vivo neutralization studies. Antivenom cross-reactivity has been investigated for decades to determine which antivenoms can be used to treat snakebite envenomings from different snake species.
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