河豚毒素中和性单抗的制备、鉴定及TTX检测方法的建立

目的研制河豚毒素中和性单抗,建立基于河豚毒素单抗的河豚毒素检测方法。方法用TTX-KLH免疫Balb/c小鼠,用TTX-BSA间接ELISA筛选,建立杂交瘤细胞系,腹腔接种Balb/c小鼠诱生腹水,Protein A Sepharose CL4B亲和柱纯化,SDS-PAGE、间接ELISA鉴定;用常规法确定TTX对昆明小鼠的LD50;将单抗和TTX混合物注入小鼠腹腔,检测单抗对TTX的中和能力;建立检测TTX的竞争ELISA法。结果获得了2株TTX中和性单抗,腹水用Protein A Sepharose CL 4B纯化后抗体纯度大于95%;常规间接ELISA检测,显示单抗5E7的结合能力高于5E4。单抗对2 LD50 TTX攻击昆明小鼠的保护率为50%,建立了基于中和性单抗的TTX检测方法,TTX的最小检出浓度为1.56μg/mL。结论获得了TTX中和性单抗,对致死剂量TTX攻击昆明小鼠的保护率为50%,建立了基于中和性单抗的TTX检测方法,TTX的最小检出浓度为1.56μg/mL。

Establishment of a Method of Preparation and Identification of Neutralizing Monoclonal Antibody of Tetrodotoxin and Its Detection

Objective To prepare neutralizing monoclonal antibody of tetrodotoxin(TTX) and establishment of a detection method of the antibody based on competitive ELISA.Methods Blab/c mice were immunized with TTX-KLH and its monoclonal antibody was detected by TTX-BSA coated indirect ELISA.A hybrydoma cell line was established and its ascites was injected into peritoneal cavity of the mice.The monoclonal antibody was purified with protein A sepharose CL4B affinity column and identified with SDS-PAGE electrophoresis and indirect ELISA.LD50 of tetrodotoxin for Kunming mice was determined by routine method.A mixture of monoclonal antibody and TTX was injected into the peritoneal cavity of mice,and the neutralizing capacity of monoclonal antibody was detected.A detection method of TTX was also established by competitive ELISA.Results Two kinds of neutralizing monoclonal antibodies were obtained,and their purity was above 95% obtained by Protein A sepharose CL 4B affinity column.Monoclonal antibody 5E7 had a higher binding capacity with TTX than that of monoclonal antibody 5E4.The neutralizing antibody showed a protection rate of 50% for Kunming mice against 2 LD50 TTX attack.A detection method of TTX with the monoclonal antibody based on competitive ELISA was established and the detectable minimal concentration of TTX was 1.56 μg/mL.Conclusion Two kinds of neutralizing monoclonal antibody have been obtained.The protection rate was 50% for Kunming mice against 2 LD50 TTX attack.A detection method of neutralizing monoclonal antibody established based on competitive ELISA,with a minimum detection limit of 1.56 μg/mL.