Snake Venom Protease Detection and Inhibition in Serum

Snake bites remain major threats with only limited diagnostic and therapeutic options. Current antibody-based antivenoms show only limited effectiveness against local tissue damage and require complex manufacturing and cold chain logistics. To overcome these limitations, a fluorescence-based assay was established to enable the sensitive detection of snake venom metalloprotease (SVMP) and serine protease (SVSP) activities in crude viper venoms from Crotalus atrox, Bothrops jararaca, and Echis carinatus. The assays showed species-specific activity profiles with detection limits in the sub-microgram per 45 and 100 µL range for SVSP and SVMP, respectively. Notably, protease activity in bovine and human blood sera, with only a slight loss of sensitivity compared to isolated venom in buffer, can be measured. Inhibitory effects of small molecule protease inhibitors, batimastat, marimastat, ilomastat, nafamostat, and leupeptin, were determined, showing strong SVMP or SVSP inhibition, respectively. Based on this proof-of-concept, the combination of such activity-based assays with selective small-molecule inhibitors could open new opportunities for rapid venom detection in diagnosis and complementary therapy, particularly in resource-poor settings.