地西泮单克隆抗体的制备及其酶联 免疫吸附检测方法的建立

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EDC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。接着利用辛酸-饱和硫酸铵法对腹水型抗体进行纯化,利用酶联免疫吸附,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(KDs)为4.0985×10_-7M,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=10.8ng/mL,检测范围为0.45ng/mL-862ng/mL。结论 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。

Preparation of anti-diazepam monoclonal antibody and establishment of its enzyme-linked immunosorbent assay method

Objectives To establish a rapid and effective method for the detection of diazepam (DZP), we prepared anti-DZP monoclonal antibody and identified the characteristics of the antibody. Methods The immunogen and coating antigen were synthesized by using the method of EDC and the mice were immunized with the immunogen until the titer reach 1:16000. To obtain the hybridoma, splenocytes from immunized mice were fused with SP2/0 under the condition of PEG4000. Then, hybridoma clones were screened by indirect complete enzyme-linked immunosorbent assay (ELISA) and the positive cell lines were subclone three times using the method of limit dilution assay. In order to prepare ascites monoclonal antibody, the positive cell lines were injected into the Balb/C mice which have been injected into paraffin oil seven days ago. The ascites monoclonal antibody was purified by bitterness-ammonium sulfate precipitation and the characteristics were determined by a method based on ELISA. Results The immunogen and coating antigen were synthesized successfully and the titer reach 1:16000 after seven times immunization. The results showed that the limit of detection (LOD) was 0.45 ng/mL and IC50=10.8 ng/mL. The dissociation constants (KDs) of the monoclonal antibody by surface plasmon resonance (SPR) was 4.0985×10-7 mol/L and the cross-reaction was not obvious except temazepam (35.4%). Conclusion The anti-DZP monoclonal antibody was prepared successfully which was very meaningful for the rapid detection of DZP.