Evaluation of neutralization efficacy is a critical step for qualification of antivenom products. According to the regulation guidelines regarding the preclinical testing of cobra antivenom neutralizing potency, the only criteria for acceptance is based on the neutralization of venom lethality, which not only raises ethical concerns about using large numbers of animals, but faces a challenge in assessing the neutralizing capability on the non-lethal but medically relevant toxins. In this study, several immunoreactive epitopes of the short-chain neurotoxin and cardiotoxin A3, the two most relevant toxins identified in venom of N.atra, were identified by peptide mapping and some of the antibody recognition sites were found to be preserved among toxin variants. In addition, two epitopes detected near the proposed functional sites responsible for toxicity were found to interact with antibodies associated with neutralization against either neurotoxicity or cytotoxicity. Enzyme-linked immunosorbent assay (ELISA) was employed to determine antibody titers in commercial antivenoms against these epitopes which demonstrated high correlation with in vivo and in vitro neutralization assays. As such, a peptide-based ELISA could be developed as an in vitro complemented assay for potency evaluation at the middle process of manufacturing before moving to the preclinical assessment.
