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BackgroundAntivenom remains the standard and most effective treatment for snakebite envenomation, yet batch-to-batch variability in neutralization potency, even under standardized immunization protocols, continues to present a significant challenge in antivenom production worldwide. This issue is particularly evident in the manufacturing of Deinagkistrodon acutus antivenom in Taiwan, where some hyperimmunized equines fail to achieve the required potency despite identical conditions. Previous studies identified a lower antibody titer against acutolysin A—a key metalloproteinase toxin in D. acutus venom—as a potential contributor to reduced efficacy.Methodology/principal findingsIn this study, acutolysin A was purified from D. acutus venom using a two-step HPLC workflow, and specific antibodies were isolated from high-potency equine plasma via affinity chromatography. When supplemented into low-potency plasma, these antibodies markedly improved survival in a murine lethality model, providing direct in vivo evidence that anti-acutolysin A antibodies are a critical determinant of antivenom efficacy. To explore strategies for enhancing these antibody levels, we evaluated a supplemental boosting approach in which purified acutolysin A replaced crude venom during later immunization rounds. This regimen significantly increased titers against acutolysin A but reduced immunoreactivity toward other venom proteins, raising concerns about narrowing antigenic breadth.Conclusions/significanceCollectively, our results highlight anti-acutolysin A antibodies as key determinants of D. acutus antivenom potency and demonstrate that their targeted supplementation can enhance neutralization efficacy, supporting antibody-based enhancement as a potential strategy to improve the potency of D. acutus antivenoms.