Detection of cytotoxins by sandwich-ELISA for discrimination of cobra envenomation and indication of necrotic severity.

Snake envenoming is both a healthcare and socioeconomic problem for developing countries and underserved communities. In Taiwan, clinical management of Naja atra envenomation is a major challenge, since cobra venom-induced symptoms are usually confused with hemorrhagic snakebites and current antivenom treatments do not effectively prevent venom-induced necrosis for which early surgical debridement should be administered. Identification and validation of biomarkers of cobra envenomation is critical for progress in setting a realistic goal for snakebite management in Taiwan. Previously, cytotoxin (CTX) was determined as one of potential biomarker candidates; however, its ability to discriminate cobra envenoming remains to be verified, especially in clinical practice. In this study, we selected a monoclonal single-chain variable fragment (scFv) and a polyclonal antibody to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for CTX detection, which successfully recognized CTX from N. atra venom over that from other snake species. Using this specific assay, the CTX concentration in envenoming mice was shown to remain consistent in about 150 ng/mL during the 2-hour post-injection period. The measured concentration was highly correlated with the size of local necrosis in mouse dorsal skin, which the correlation coefficient is about 0.988. Furthermore, our ELISA method displayed 100 % of specificity and sensitivity in discriminating cobra envenoming among snakebite victims through CTX detection and the level of CTX in victim plasma was ranged from 5.8 to 253.9 ng/mL. Additionally, patients developed tissue necrosis at plasma CTX concentrations higher than 150 ng/mL. Thus, CTX not only serves as a verified biomarker for discrimination of cobra envenoming but also a potential indicator of severity of local necrosis. In this context, detection of CTX may facilitate reliable identification of envenoming species and improve snakebite management in Taiwan.