酚酞单克隆抗体的制备

Preparation of monoclonal antibody of phenolphthalein illegally added in health products

中文关键词:  酚酞  单克隆抗体  酶联免疫吸附法

英文关键词:phenolphthalein  monoclonal antibody  enzyme-linked immunosorbent assay

中文摘要:

      目的 建立免疫学快速检测减肥类保健食品中非法添加的酚酞, 制备酚酞单克隆抗体并进行评价。方法 利用碳二亚胺(carbodiimide, EDC)法合成免疫原和包被原, 用免疫原免疫Balb/C小鼠, 取小鼠脾脏与SP2/0鼠骨髓瘤细胞融合。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, 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鉴定纯化后的抗体。结果 成功合成了酚酞-BSA免疫原和酚酞-OVA包被原, 筛选获得酚酞杂交瘤细胞株FT/BSA/2019, 单克隆抗体的效价1×105。结论 本研究初步建立了特异性高的酚酞单克隆抗体的制备方法。

英文摘要:

      Objective To establish immunological rapid detection of illegally added phenolphthalein in dietetic health foods, and to prepare and evaluate phenolphthalein monoclonal antibodies. Methods The immunogen and coating antigen were synthesized using a carbodiimide (EDC) method. Balb/C mice were immunized with the synthetic immunogen, and the mouse spleen was fused with SP2/0 mouse myeloma cells. The hybridoma cells capable of secreting specific antibodies were screened by adopting a competition and double-positive two-step screening method, individual cell line was obtained by using a limited dilution subcloning method, and the ascites-type monoclonal antibody was prepared by adopting an in-vivo induction assay. The ascites-type antibody was purified by caprylic acid-saturated sulfuric acid method, and the purified antibody was identified by enzyme-linked immunosorbent assay (ELISA). Results Phenolphthalein -BSA immunogen and phenolphthalein-OVA encapsulant were successfully synthesized, FT/BSA/2019 of phenolphthalein hybrid tumor cell line was screened, and the titer of monoclonal antibody was 1×105. Conclusion This paper establish the preparation method of the specific high-specificity phenolphthalein monoclonal antibody.