Assessing the neutralization capability of nonlethal but medically relevant toxins in venom has been a challenging task. Nowadays, neutralization efficacy is evaluated based simply on the survival rates of animals injected with antivenom together with a predefined dose of venom, which can determine potency against neurotoxicity but not validate the capability to neutralize cytotoxin-induced complications. In this study, a high correlation with in-vivo and in-vitro neutralization assays was established using the immunoreactive peptides identified from short-chain neurotoxin and cytotoxin A3. These peptides contain conserved residues associated with toxin activities and a competition assay indicated that these peptides could specifically block the antibody binding to toxin and affect the neutralization potency of antivenom. Moreover, the titers of peptide-specific antibody in antivenoms or mouse antisera were determined by enzyme-linked immunosorbent assay (ELISA) simultaneously, and the results indicated that Taiwanese bivalent antivenom (BAV) and Vietnamese snake antivenom-Naja (SAV-Naja) exhibited superior neutralization potency against the lethal effect of short-chain neurotoxin (sNTX) and cytotoxicity of cardiotoxin/cytotoxin (CTX), respectively. Thus, the reported peptide ELISA shows not only its potential for antivenom prequalification use, but also its capability of justifying the cross-neutralization potency of antivenoms against Naja atra venom toxicity.
