Botulinum neurotoxin type E (BoNT/E), the fastest acting toxin of all BoNTs, cleaves the 25 kDa synaptosomal associated protein (SNAP-25) in motor neurons, leading to flaccid paralysis. Specific detection and quantification of BoNT/E-cleaved SNAP-25 neoepitope is essential for diagnosis of BoNT/E intoxication as well as for characterization of anti-BoNT/E antibody preparations. In order to isolate highly specific monoclonal antibodies suitable for in vitro immuno-detection of the exposed neoepitope, mice and rabbits were immunized with an eight amino acid peptide composed of the C-terminus of the cleaved SNAP-25. Immunized rabbits developed a specific and robust polyclonal antibody response, whereas immunized mice mostly demonstrated a weak antibody response that could not discriminate between the two forms of SNAP-25. An immune scFv phage-display library was constructed from the immunized rabbits and a panel of antibodies was isolated. Sequence alignment of the isolated clones revealed high similarity between both heavy and light chains, with exceptionally short HCDR3 sequences. A chimeric scFv-Fc antibody was further expressed and characterized, exhibiting a selective, ultra-high affinity (pM) towards the SNAP-25 neoepitope. Moreover, this antibody enabled sensitive detection of the cleaved SNAP-25 in BoNT/E treated SiMa cells with no cross reactivity with the intact SNAP-25. This novel antibody can be further used to develop an in vitro cell-based assay to diagnose BoNT/E intoxication and to characterize antitoxin preparations, thus eliminating the use of animals in the standard mouse bioassay.

中文翻译：

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靶向暴露于 SNAP-25 新表位的 E 型肉毒杆菌神经毒素的高度特异性单克隆抗体的分离和表征

E 型肉毒杆菌神经毒素 (BoNT/E) 是所有 BoNT 中作用最快的毒素，可切割运动神经元中的 25 kDa 突触体相关蛋白 (SNAP-25)，导致弛缓性麻痹。BoNT/E 切割的 SNAP-25 新表位的特异性检测和定量对于 BoNT/E 中毒的诊断以及抗 BoNT/E 抗体制剂的表征至关重要。为了分离适用于暴露的新表位的体外免疫检测的高度特异性单克隆抗体，小鼠和兔用由裂解的 SNAP-25 的 C 端组成的八氨基酸肽进行免疫。免疫兔产生了特异性强的多克隆抗体反应，而免疫小鼠大多表现出微弱的抗体反应，无法区分两种形式的 SNAP-25。从免疫的兔子构建了一个免疫 scFv 噬菌体展示文库，并分离出一组抗体。分离克隆的序列比对揭示了重链和轻链之间的高度相似性，具有特别短的 HCDR3 序列。嵌合 scFv-Fc 抗体被进一步表达和表征，表现出对 SNAP-25 新表位的选择性、超高亲和力 (pM)。此外，该抗体能够灵敏检测 BoNT/E 处理的 SiMa 细胞中裂解的 SNAP-25，与完整 SNAP-25 无交叉反应。这种新型抗体可进一步用于开发基于体外细胞的检测，以诊断 BoNT/E 中毒并表征抗毒素制剂，从而消除在标准小鼠生物检测中使用动物的情况。