Development of antibody-detection ELISA based on beta-bungarotoxin for evaluation of the neutralization potency of equine plasma against Bungarus multicinctus in Taiwan

Animal testing has been the primary approach to assess the neutralization potency of antivenom for decades. However, the necessity to sacrifice large numbers of experimental animals during this process has recently raised substantial welfare concerns. Furthermore, the laborious and expensive nature of animal testing highlights the critical need to develop alternative in vitro assays. Here, we developed an antibody-detection enzyme-linked immunosorbent assay (ELISA) technique as an alternative approach to evaluate the neutralization potency of hyperimmunized equine plasma against B. multicinctus, a medically important venomous snake in Taiwan. Firstly, five major protein components of B. multicinctus venom, specifically, α-BTX, β-BTX, γ-BTX, MTX, and NTL, were isolated. To rank their relative medical significance, a toxicity score system was utilized. Among the proteins tested, β-BTX presenting the highest score was regarded as the major toxic component. Subsequently, antibody-detection ELISA was established based on the five major proteins and used to evaluate 55 hyperimmunized equine plasma samples with known neutralization potency. ELISA based on β-BTX, the most lethal protein according to the toxicity score, exhibited the best sensitivity (75.6 %) and specificity (100 %) in discriminating between high-potency and low-potency plasma, supporting the hypothesis that highly toxic proteins offer better discriminatory power for potency evaluation. Additionally, a phospholipase A₂ (PLA₂) competition process was implemented to eliminate the antibodies targeting toxicologically irrelevant domains. This optimization greatly enhanced the performance of our assay, resulting in sensitivity of 97.6 % and specificity of 92.9 %. The newly developed antibody-detection ELISA presents a promising alternative to in vivo assays to determine the neutralization potency of antisera against B. multicinctus during the process of antivenom production.