抗T-2毒素单克隆抗体的制备与初步鉴定

Preparation and characterization of a monoclonal antibody against T-2 toxin

目的制备和鉴定抗T-2毒素单克隆抗体。方法将T-2毒素与牛血清白蛋白(BSA)交联,制备T-2毒素与BSA交联物(T-2-BSA),以T-2-BSA免疫BALB/c小鼠,取其脾脏细胞与小鼠骨髓瘤细胞Sp2/0融合,以HAT培养液筛选杂交瘤细胞,用有限稀释法进行克隆化。用筛选的阳性单克隆细胞株,按常规方法制备腹水,腹水用饱和硫酸铵沉淀后,用阴离子交换法纯化,获得纯化的单克隆抗体。采用ELISA鉴定抗体的亚类,并测定血清和腹水中抗体的效价。结果制备出T-2毒素与BSA交联物T-2-BSA,免疫小鼠血清的效价在1×10-6以上。获得1株能稳定分泌抗T-2毒素抗体的杂交瘤细胞系,该抗体(T2mAb)属于IgG1亚类,轻链为κ链。腹水效价为1×10-6。ELISA法证明T2mAb可与T-2毒素发生特异性反应。结论成功制备出抗T-2毒素单克隆抗体,为建立测定食品、饲料和组织液中T-2毒素的免疫学方法打下了坚实的基础。

Objective To prepare and characterize a monoclonal antibody against T - 2 toxin. Methods T - 2 toxin was conjugated with bovine serum album （BSA）, and the conjugated immunogen （ T - 2 - BSA） was employed to immunize BALB/c mice. After spleen cells from immunized mice were fused with Sp2/0 cells, hybridoma cell lines secreted antibody against T - 2 were selected by HAT and cloned by limiting dilution. The mAb was extracted and purified through saturated ammonium sulphate precipitation and anion exchange chromatography column from ascitic fluid, which was prepared according to the routine protocol. The subclass and titer of the mAb were detected by double antibodies sandwich ELISA and indirect ELISA, respectively. Results Conjugated immunogen of T - 2 - BSA was produced. The titers of sera from all BALB/c mice immunized with T - 2 - BSA were over 1 × 10 ^-6. One hybridoma cell line secreting mAb against T - 2 toxin was obtained. The mAb. designed as T2mAb belonged to IgG1 （κ） subclass, and the titer of ascitic fluid was 1 × 10^-6. In ELISA assay, T2mAb could react with T -2 toxin specifically. Conclusion A monoclonal antibody against T -2 toxin was prepared, which makes a solid foundation for establishment of immunological methods for detecting T - 2 toxin levels in food, feed and biological fluid.