High-throughput epitope identification for snakebite antivenom

Abstract

In this study, a high-throughput peptide microarray approach was used to identify linear epitopes in 966 toxin sequences from pit vipers (family Crotalidae), focusing mainly on snake venom metalloproteinases and phospholipase A₂s—two major toxin families present in the venoms used to produce the ICP Crotalidae antivenom. Peptide libraries were generated in silico, synthesized on microarrays, and screened for antibody binding. The resulting peptide signal intensities were mapped back onto toxin amino acid sequences and structural models to localize epitopes. The identified epitopes were found to be surface-exposed and highly conserved among pit viper species, indicating potential cross-recognition by the antivenom. The study demonstrates that custom high-density peptide microarrays can efficiently map epitopes across hundreds of toxins and predict antivenom cross-reactivity based on sequence conservation, providing a valuable tool for the rational design of next-generation snakebite antivenoms.