Development of an innovative magnetic-beads based methodology to enhance antivenomics capabilities.

Snake envenomation remains a significant public health crisis in Africa, the Middle East, Asia and subtropical regions, mainly affecting rural populations. Each year, between 80,000 and 138,000 people die from snakebite envenomation, while three times as many survivors are left with long-term disabilities despite treatment. The main available treatments are antivenoms, which are sera-containing immunoglobulins G (IgGs) or IgG fragments targeting venom toxins. Such IgGs are purified from the blood of hyperimmunized horses or sheep. While antivenoms save many lives, they also come with significant limitations. Purified sera contain not only toxin-specific IgGs but also IgGs unrelated to venom exposure. Unfortunately, these non-specific IgGs may trigger severe adverse effects for people already in a critical situation. Moreover, antivenoms are thermally unstable and are difficult to store in regions where they are most needed. To address these challenges, the European ADDovenom project (2020–2025), funded by the European Commission (FET-Open), aims to develop a novel generation of antivenom based on ADDomers construct. ADDomers are thermally stable megadalton virus-like particles, produced at low cost and featuring 60 high-affinity binding sites, offering a promising alternative to conventional antivenoms. Here, we present a novel method for antivenomics, providing a fully automatable, faster process. Magnetic beads were coupled with EchiTab G, a monospecific antivenom targeting Echis ocellatus venom. The beads were incubated with E. ocellatus venom, and both the eluate and supernatant were analyzed using LC-MS and tryptic digestion to identify and quantify the specific toxins immuno-recognized by EchiTab G. Cross-reactivity of the antivenom was also assessed with E. leucogaster and E. coloratus venoms. This innovative method reduces the need for large amounts of solvent and valuable biological materials (venoms and antivenoms). Unlike the conventional method using NHS-activated sepharose matrices and UV-visible detection, which is impractical for small-scale analysis, our method using magnetic beads and LC-MS significantly improves sensitivity and scalability. Additionally, a novel relative quantification method for toxins in venoms, based on the three most intense, unique ions from tryptic digestion, enables quantitative measurement of the different toxin families captured by the beads. Once the methodology is optimized for traditional antivenoms, it will be used to assess the efficacy of ADDobody—firstly against synthetic phospholipases A2 targeted by ADDobodies and secondly against E. coloratus raw venom. Finally, the efficacy of the ADDomers structures will be evaluated. This method could accelerate antivenom development, making it more efficient, adaptable and industry ready.