Comparative in vitro immunoreactivity and protein analysis of Trimeresurus albolabris and Tropidolaemus wagleri venoms

Snakebite envenomation remains a significant global health concern, with antivenoms being the primary treatment. However, variations in venom composition can affect antivenom efficacy, leading to differences in immunoreactivity. This study aimed to evaluate and compare the immunological reactivity of venom components in Trimeresurus albolabris and Tropidolaemus wagleri venoms and further investigate the differences in antigenic properties of a key protein between two species that may influence antivenom recognition. The levels of immunological reactivity of monovalent (homospecific) antivenom and hemato polyvalent antivenom to Trimeresurus albolabris and Tropidolaemus wagleri venoms were evaluated using indirect ELISA. The immunoreactive levels of both antivenoms to antigenic proteins in Trimeresurus albolabris venom were comparable. In addition, both antivenoms reacted immunologically with antigens in Tropidolaemus wagleri venom. However, the hemato polyvalent antivenom showed greater reactivity to Tropidolaemus wagleri venom than the monovalent antivenom. The overall reactivity of the antivenoms to Trimeresurus albolabris venom was higher than that to Tropidolaemus wagleri venom. Using two-dimensional (2DE) immunoblotting and liquid chromatography mass-spectrometry-based proteomic technology (LC-MS/MS), immunoreactive and non-reactive proteins in both pit viper venoms were characterized and identified. Trimeresurus albolabris venom comprised a total of 235 spots, while Tropidolaemus wagleri venom contained 72 spots. Immunorecognition between the polyvalent antivenom and specific proteins in both venoms was mostly detected in proteins with a size over 30 kDa. Among the nine protein families identified in both venoms, the most frequently reactive proteins found in Trimeresurus albolabris venom were snake venom metalloproteinases (SVMP) and snake venom serine proteases (SVSP), while in Tropidolaemus wagleri venom, the most frequent were members of the L-amino acid oxidase (LAAO) family. For the non-immunoreactive proteins, we detected the highest identity numbers of phospholipase A₂ (PLA₂) in Trimeresurus albolabris venom and SVSP in Tropidolaemus wagleri venom. The distinctive characteristics between the non-reactive SVSP in Tropidolaemus wagleri venom and the reactive SVSP in Trimeresurus albolabris venom were investigated. The antigenic properties and predicted B cell epitopes were further analyzed using a computational approach. Structural and physicochemical analyses indicated that Loop 2 (residues 100-110) in the immunoreactive SVSP from Trimeresurus albolabris venom exhibited higher hydrophilicity and surface accessibility compared to the non-immunoreactive SVSP from Tropidolaemus wagleri venom. These findings provide important insights into the differences in antivenom reactivity to specific proteins across different snake venoms and may contribute to future research aimed at optimizing antivenom formulations.