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目的 :制备抗肉毒毒素A(BoNT/A)的单克隆抗体 (mAb)。方法 :用纯化的重组BoNT/A Hc片段免疫BALB/c小鼠 ,取其脾细胞与骨髓瘤Sp2 /0融合 ,经间接ELISA筛选和克隆化制备杂交瘤细胞系 ,及Western免疫印迹分析等方法对mAb进行特异性鉴定。结果 :获得 3株杂交瘤细胞株 :命名为4A8、2F7和 4F2 ,IgG亚类鉴定均为IgG1,腹水mAb的效价在 1× 10 -4～ 1× 10 -6之间。其中 ,4A8和 4F2能稳定分泌抗BoNT AmAb,并可特异性地识别重组BoNT/A和天然BoNT/A ,特别是 4A8可保护小鼠抵抗 10LD50 BoNT/A的攻击。结论 :成功地制备 3株特异性抗BoNT/AmAb ,并有 1株属于中和性mAb ,为BoNT/A的检测和肉毒中毒的临床治疗奠定了基础
OBJECTIVE: To prepare a monoclonal antibody (mAb) against botulinum toxin A (BoNT / A). METHODS: BALB / c mice were immunized with the purified recombinant BoNT / A Hc fragment. The splenocytes were fused with Sp2 / 0 of myeloma. The hybridoma cell lines were selected by ELISA and cloned by indirect ELISA. Western Blot analysis The mAbs were specifically identified. Results: Three hybridoma cell lines were obtained, named as 4A8, 2F7 and 4F2. The IgG subclasses were all identified as IgG1, and the titer of ascites mAb was between 1 × 10 -4 and 1 × 10 -6. Among them, 4A8 and 4F2 can stably secrete anti-BoNT AmAb, and can specifically recognize recombinant BoNT / A and native BoNT / A, in particular, 4A8 can protect mice against 10LD50 BoNT / A challenge. Conclusion: Three specific anti-BoNT / AmAb and one neutralizing mAb were successfully prepared, which laid the foundation for the detection of BoNT / A and the clinical treatment of botulism