A型肉毒毒素单克隆抗体的制备和鉴定

摘要

Objective:To prepare and identify mouse monoclonal antibody against heavy chain carboxy terminal of botulinum neurotoxin type A(BoNT/AHc).Methods:BALB/c mice were immunized with BoNT/AHc and monoclonal antibodies(McAbs) were harvested by hybridoma technique.Isotyping assay,SDS-PAGE,ELISA and Western blot were used to identify the McAbs.Results:Three McAbs were obtained which designated as 1B1,1B2,1C6 respectively.Our result shows that heavy chains belong to IgG1,and the light chains belong to κ type,and the purity of them is over 90％.The McAbs combine with BoNT/AHc specifically and the value of Kaff is 1.43× 108,2.31×108 and 1.44×109 L/mol respectively.The result of additive ELISA indicates that 3 McAbs bind to the same or similar site of BoNT/AHc.Conclusion:Three mouse McAbs were obtained specifically binding to BoNT/AHc,which may be applied to develop therauptic antibodies against BoNT/A and establish a rapid and effective method for detection of BoNT/A.%目的:制备并鉴定抗A型肉毒毒素重链C端(BoNT/AHc)鼠源单抗.方法:以BoNT/AHc蛋白为抗原免疫小鼠,利用杂交瘤技术获得鼠源单抗,通过抗体分型、SDS-PAGE、Western印迹及ELISA法分析鉴定.结果:筛选得到3株鼠单抗1B1、1B2、1C6,重链类型均为IgG1,轻链类型均为κ型;3株纯化抗体的纯度达90％以上,均能与抗原BoNT/AHc特异性结合,亲和力常数分别为1.43×1108、2.31×108、1.44×109 L/mol;叠加ELISA结果显示3株抗体与抗原BoNT/AHc结合位点相同或相近.结论:筛选得到3株能与BoNT/AHc特异性结合的鼠源单抗,为A型肉毒毒素中和抗体的研发,以及快速有效的肉毒毒素检测方法的建立奠定了基础.