Antivenomics by mass spectrometry: use of magnetic beads.

Snakebite is classified as a Category A neglected tropical disease by the WHO, as it causes the death of about 150,000 people every year, and up to 4 times more people suffer long-term morbidity, mostly in rural and poor areas of the world. Snake envenoming is classically treated by injecting antivenoms containing antitoxin antibodies (Igs), produced by hyperimmunized animals. As antitoxin Igs represent only 10-15% of the total antivenom Ig content, such treatments have poor efficacy and the administration of animal Igs may induce immunological adverse reactions, possibly severe for the patient. Moreover, venom compositions strongly differ from species, gender, and habitat, explaining why providing broadly effective antivenoms is a real challenge. An effort has been made to reduce snakebite deaths and disability by 50% by 2030 [1].
In this context, quantitatively evaluating the toxin-binding (/neutralizing) capability of any antivenom is crucial to improve the production of effective snakebite therapeutics. In this study, we propose an alternative methodology for the so-called ‘antivenomics’ methodology. Indeed, affinity columns coupled to mass spectrometry have been demonstrated performant [2], but their preparation and their lifetime represent strong constraints to the increase of antivenom evaluation throughput. In this work, we exploit the potential of magnetic beads, LC-MS and shotgun proteomics mass spectrometry to speed up antivenom efficacy characterization. The antivenom Igs are bound to magnetic beads, before being incubated in the presence of various venoms. Comparative MS analysis of the toxins remaining in suspension (not recognized by Ig) and those remaining on the beads (recognized by Ig) allows the binding selectivity of the antivenom to be determined. The strategy is demonstrated here with venom from the medically important African viper Echis ocellatus.