Abstract：
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.
摘要：
几十年来，动物试验一直是评估抗蛇毒血清中和效力的主要方法。然而,在此过程中牺牲大量实验动物的必要性最近引起了大量的福利问题。此外，动物试验的费力和昂贵的性质突出了开发替代体外试验的关键需求。这里,我们开发了一种抗体检测酶联免疫吸附测定（ELISA）技术，作为一种替代方法来评估超免疫马血浆对B。台湾一种医学上重要的毒蛇。首先,多重蛇毒的五种主要蛋白质成分，具体来说,α-BTX,β-BTX,γ-BTX,MTX,和NTL，被隔离。对它们的相对医学意义进行排名，使用毒性评分系统。在测试的蛋白质中，得分最高的β-BTX被认为是主要的毒性成分。随后，抗体检测ELISA是基于5种主要蛋白建立的,并用于评估55份具有已知中和效力的超免疫马血浆样品.基于β-BTX的ELISA,根据毒性评分，最致命的蛋白质，在区分高效力和低效力血浆方面表现出最佳的敏感性（75.6%）和特异性（100%），支持高毒性蛋白质为效力评估提供更好的辨别能力的假设。此外,实施磷脂酶A2(PLA2)竞争过程以消除靶向毒理学无关结构域的抗体.这种优化大大提高了我们的检测性能，敏感性为97.6%，特异性为92.9%。新开发的抗体检测ELISA为体内测定提供了一种有前途的替代方法，以确定抗血清在抗蛇毒血清生产过程中对多蛇毒杆菌的中和效力。
