抗A型肉毒毒素鼠源性单克隆抗体的制备和鉴定

目的：制备与鉴定抗A型肉毒神经毒素重链C端（ heavy chain of botulinum neurotoxin serotype A， BoNT／AHc）特异性鼠单克隆抗体。方法通过纯化BoNT／AHc抗原、免疫BALB／c小鼠并建立杂交瘤细胞以制备鼠单抗，用ELISA、Western印迹实验和抗体分型试剂盒进行分析鉴定，并通过ELISA检测鼠单抗与BoNT／AHc突变体结合，初步鉴定其在BoNT／AHc的结合表位。结果得到纯度较高的BoNT／AHc抗原，制备了4株特异性鼠单抗1A4、3H3、3H7、5H8。间接ELISA结果显示，单抗细胞上清效价均＞3．0×103；Western印迹检测结果显示，单抗均能与BoNT／AHc特异性结合；抗体亚型鉴定结果为1A4、3H7属于IgG1（Κ），3H3属于IgM（Κ），而5H8属于IgG2b （Κ）；叠加ELISA实验表明，4株单抗抗原识别表位相近；用ELISA检测单抗与BoNT／AHc突变体结合实验结果初步确定了4株单抗与BoNT／AHc的结合表位。结论对抗A型肉毒毒素鼠源性抗体完成了制备和鉴定，为肉毒毒素中和抗体的开发与阐明中和抗体的表位奠定了基础。

Preparation and characterization of monoclonal antibodies against botulinum neurotoxins serotype A

Objective To prepare and characterize specific monoclonal antibodies( McAbs) against the heavy chain of botulinum neurotoxin serotype A ( BoNT/AHc ) .Methods BALB/c mice were immunized with purified BoNT/AHc protein.After the fusion of mouse splenic cells with SP2/0 cells, hybridoma cell lines secreted McAbs against BoNT/AHc. The McAbs obtained were characterized by indirect ELISA, Western blotting and rapid isotypingassay before being used in ELISA to detect interaction sites in McAbs and BoNT/AHc preliminarily.Results Antigen protein BoNT/AHc of high purification was obtained.Four hybridoma cell lines secreting McAbs against BoNT/AHc were screened,named 1A4,3H3, 3H7 and 5H8,respectively.Their titers of McAbs were all above 3.0 ×103 .They were specifically combined with BoNT/AHc protein by Western blotting.The isotype of 1A4 and 3H7 was IgG1(Κ),that of 3H3 was IgM(Κ),and that of 5H8 was IgG2b(Κ).Additive ELISA showed that epitopes recognized by the four McAbs were close.ELISA analysis confirmed the interaction epitopes in McAbs and BoNT/AHc.Conclusion Monoclonal antibodies against BoNT/AHc are prepared and characterized,providing effective tools for studying the neutralizing antibody and antibody epitopes of BoNT/AHc.