氯胺酮单克隆抗体的制备及生物学特性的研究

Preparation and characterization of monoclonal antibody against ketamine

目的制备抗氯胺酮(ketamine)的单克隆抗体(mAb),并鉴定其特性,为氯胺酮的胶体金免疫试纸条研制奠定基础。方法氯胺酮-BSA免疫BALB/c小鼠,采用杂交瘤技术制备抗氯胺酮的mAb。采用间接ELISA、竞争抑制ELISA和Western blot对mAb的特异性进行鉴定。采用间接ELISA法鉴定mAb的Ig亚类、检测其腹水效价及相对亲和力,并进行表位分析。结果获得2株可分泌特异性mAb的杂交瘤细胞(FB12b和CH10),其Ig亚类分别为IgG2a和IgG3;腹水mAb的效价均为1∶106;mAb FB12b和CH10的相对亲和力均在105以上。ELISA相加实验的结果显示,2株mAb可识别不同的抗原表位。结论成功制备抗氯胺酮的2株mAb,为建立快速特异检测氯胺酮滥用的现场检测方法提供了有力的工具。

Objective To prepare monoclonal antibody （mAb） against ketamine, to be used as the gold colloidal reagent strip for rapid detection of ketamine,and to determine its biological characterization. Methods BALB/c mice were immunized by ketamine conjugated BSA. Anti-ketamine mAbs were prepared by using hybridoma technique. The specificity of mAb was determined by indirect ELISA, competitive inhabitation ELISA and Western blot. The indirect ELISA was used to identity Ig subgroup, detect its titer in ascetic fluid and relative affinity, and analyze antigen-binding epitope of mAbs. Results Two hybridoma cells （ FB12b and CH10）, secreting anti- ketamine mAbs were obtained. The Ig subgroups of FB12b and CH10 were IgG2a and IgG3 , respectively. The mAb＇s titer in ascetic fluid was 1 ： 10^6. The relative affinity of mAb FB12b and CH10 was more than 10^5. The result of additive ELISA showed that two mAbs could recognize different antigen epitopes. Conslution The successful preparation of two anti-ketamine mAbs provides a powerful tool for the development of a rapid method of detecting ketamine abuse.