Development of Camelid Nanobody-Based Nanobioconjugates for the Detection of Bothrops and Crotalus Venoms

Snakebite envenoming remains an important global public health problem, and Brazil ranks among the countries with the highest notification rates, especially in the Northern region. Therapeutic intervention is decisive in snakebite cases, as early administration of antivenom reduces the severity of systemic and local manifestations. However, in the absence of a diagnostic test, identification of the snake responsible for envenoming remains restricted to clinical and epidemiological assessment. In this context, this study demonstrates the potential for developing a diagnostic assay for snakebite envenoming using nanobioconjugates composed of antivenom VHHs against rattlesnakes and jararacas, medically important snakes in the country. In dot blot assays, the anti-Bothrops nanobioconjugates showed visible reactivity against Bothrops jararacussu venom down to 10 ng/µL and more consistent reactivity at concentrations ≥ 50 ng/µL. The anti-Crotalus nanobioconjugates detected Crotalus durissus terrificus venom at concentrations as low as 400 ng/µL and also showed reactivity toward three additional Crotalus subspecies venoms. In control assays, no cross-reactivity was observed with the heterologous venoms evaluated. These results provide initial evidence for the potential use of VHH-based nanobioconjugates for venom detection, while indicating that further optimization of analytical sensitivity is required before clinical application.