Ester-type Aconitum alkaloids (AAs), the main medicinal ingredients of Aconitum L. herbs, could cause brain and heart damage in humans and animals and have raised concerns worldwide. In the present study, we aimed to produce a high-performance and broad-spectrum antibody and establish an immunoassay method of ester-type AAs, 3-succinyl aconitine (ACO-HS) was selected as an optimal hapten from five designed haptens comparing the similarity of stereo structure, electronic distribution, and physicochemical properties using the computer-aided molecular modeling technology. The monoclonal antibody (mAb) 1A9 exhibited broad-spectrum recognition specificity of 15 ester-type AAs was obtained and had a high sensitivity with the binding affinity (half-maximum inhibition concentration, IC₅₀) of 0.73–130.36 μg L⁻¹. Through molecular docking, it was found that mAb 1A9 and ester-type AAs showed a semi-enveloped structure through hydrogen bonds and hydrophobicity interaction. The amino acid residues that responsible for recognition were ARG107, GLU55, PRO113, VAL36, and SER64, and the critical structures to be recognized of AAs were acetyl group, benzoyl group, and N-linked carbon chains. The developed indirect competitive enzyme-linked immunosorbent assay (icELISA) based on mAb 1A9 allowed a sensitive determination of 15 ester-type AAs with the limit of detection (LOD) of 0.21–43.72 μg L⁻¹, and it was suitable for the analysis of ester-type AAs in various Aconitum L. samples. These results provided an effective strategy for the preparation of targeted broad-spectrum antibodies of small molecules and proposed an icELISA method available for rapid, sensitive, and high-throughput detection of toxic ester-type AAs in Aconitum L. herbs.

酯类乌头生物碱（AAs）是乌头草本植物的主要药用成分，可能对人和动物造成脑和心脏损伤，引起了全世界的关注。在本研究中，我们旨在生产一种高性能和广谱的抗体，并建立酯型 AA 的免疫测定方法，从五种设计的半抗原中选择 3-琥珀乌头碱 (ACO-HS) 作为最佳半抗原，比较使用计算机辅助分子建模技术，立体结构、电子分布和物理化学性质的相似性。单克隆抗体 (mAb) 1A9 对 15 种酯型 AA 具有广谱识别特异性，并具有高灵敏度的结合亲和力（半最大抑制浓度，IC₅₀ ) 的 0.73–130.36 μg L ^-1。通过分子对接发现mAb 1A9与酯型AAs通过氢键和疏水相互作用呈现出半包膜结构。负责识别的氨基酸残基是 ARG107、GLU55、PRO113、VAL36 和 SER64，AA 的关键结构是乙酰基、苯甲酰基和 N 连接的碳链。开发的基于 mAb 1A9 的间接竞争性酶联免疫吸附试验 (icELISA) 可以灵敏测定 15 种酯型氨基酸，检测限 (LOD) 为 0.21–43.72 μg L ^-1，适用于分析各种乌头中的酯型AAsL. 样品。这些结果为小分子靶向广谱抗体的制备提供了有效的策略，并提出了一种可用于快速、灵敏、高通量检测乌头中毒性酯型AAs的icELISA方法。