Venom detectives: a sandwich ELISA platform for precision diagnosis of snakebite envenoming

Background Clinical management of snakebite in India relies predominantly on polyspecific antivenom and is complicated by difficulties in identifying the causative snake species and monitoring venom levels in circulation. Reliable detection and quantification of venom in blood could support species identification, guide antivenom selection and dosing, monitor venom levels following treatment, and facilitate the future use of monospecific or recombinant antivenoms. Methods To address this critical diagnostic gap, we developed a highly sensitive sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for five medically important Indian vipers: D. russelii, E. carinatus, H. hypnale, C. malabaricus , and C. gramineus . Rabbits were immunised with individual venoms, and the resulting monospecific IgGs were purified and used as capture and biotinylated detection antibodies, with signal detection using a streptavidin-HRP conjugate. Results The assay was highly reproducible and detected viper venoms at concentrations as low as 2.2 -5.23 ng/mL. Binding to non-target venoms was at least threefold lower than to the corresponding target venom, except for cross-reactivity between C. malabaricus and C. gramineus . The assay also detected circulating venom in clinical samples from envenomed patients, providing proof-of-concept evidence for its potential clinical application. Conclusion Our findings establish a sensitive platform for species-specific venom detection and quantification, with potential applications in the diagnosis and clinical management of snakebite in India.