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目的制备地西泮(diazepam,DZP)单克隆抗体,并且对制备的抗体进行一系列性质鉴定。方法利用1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐[1-ethyl-3-(3-dimethyllaminopropyl)carbodi imide hydrochloride,EDC·HCl]法合成免疫原和包被原,用免疫原免疫Balb/C小鼠,当效价到1:16000以后取小鼠脾脏与SP2/0进行细胞融合。然后采用竞争结合双阳性两步筛选法,筛选出能分泌特异性抗体的杂交瘤细胞,并且利用有限稀释亚克隆方法得到单株细胞,采用体内诱生法制备腹水型单克隆抗体。利用辛酸-饱和硫酸铵法对腹水型抗体进行纯化,利用酶联免疫吸附法、表面等离子共振仪(surface plasmon resonance,SPR)等方法对纯化后的抗体进行性质鉴定。结果成功合成了地西泮免疫原和包被原,免疫Balb/C小鼠7次后效价达到1:16000,最终制备出单克隆抗体,抗体解离常数(dissociation constants,KDs)为4.0985×10-7 mol/L,且与大部分结构类似物没有明显的交叉反应。应用此抗体建立间接竞争ELISA法,抗体的IC50=10.8 ng/mL,检测范围为0.45~862.00 ng/mL。结论本研究制备出了地西泮单克隆抗体,为地西泮的免疫学检测提供了有力的支持。
Objective To prepare monoclonal antibodies against diazepam (DZP), and to characterize a series of antibodies. Methods The immunogen and package were synthesized using 1-ethyl-3- (3-dimethyllaminopropyl) carbodiimide hydrochloride, EDC · HCl Balb / C mice were immunized with the immunogen and the spleen of the mice was immunoblotted with SP2 / 0 after 1: 16000 titer. Then, the hybridoma cells secreting specific antibodies were screened by competition and double-positive two-step screening method. Single cells were obtained by sub-cloning by limiting dilution, and ascites monoclonal antibody was prepared by in vivo induction method. The ascites antibody was purified by caprylic acid - saturated ammonium sulfate method. The purified antibody was identified by enzyme - linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Results The diazepam immunogen and the coated original immunized Balb / C mice were titrated to 1: 16000 after 7 times to finally produce monoclonal antibodies with dissociation constants (KDs) of 4.0985 × 10-7 mol / L, and most of the structural analogs did not significantly cross-reaction. The indirect ELISA was used to establish this antibody. The IC50 of the antibody was 10.8 ng / mL. The detection range was 0.45 ~ 862.00 ng / mL. Conclusion This study prepared a diazepam monoclonal antibody, which provided strong support for the immunological detection of diazepam.