米酵菌酸单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibodies of bongkrekic acid

中文关键词:  米酵菌酸  单克隆抗体  酶联免疫吸附法

英文关键词:fermentative acid  monoclonal antibody  ELISA

中文摘要:

      目的 建立鲜湿米粉中米酵菌酸(bongkrekic acid, BA)的免疫学快速检测方法, 制备特异性识别BA的单克隆抗体并进行评价。方法 利用碳二亚胺[1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EDC]法将BA半抗原与牛血清白蛋白(bovine serum albumin, BSA)和卵清蛋白(ovalbumin, OVA)载体偶联, 分别合成BA免疫原(BA-BSA)和包被原(BA-OVA), 用BA-BSA免疫Balb/C小鼠, 取免疫效果好的小鼠脾脏与小鼠NS-1骨髓瘤细胞进行融合。采用间接竞争酶联免疫吸附法(indirect competitive enzyme-linked immunosorbent assay, icELISA)进行筛选, 筛选出能分泌所需特异性抗体的杂交瘤细胞, 利用有限稀释法进行亚克隆得到单株能稳定分泌所需抗体的细胞; 采用体内诱生法制备腹水型单克隆抗体。利用正辛酸-饱和硫酸铵法纯化腹水型抗体, 通过酶联免疫吸附法测定纯化后的抗体效价。结果 成功合成了BA免疫原BA-BSA和BA包被原BA-OVA, 筛选获得BA杂交瘤细胞株BA-3F1E9, 单克隆抗体的效价为1×105。结论 本研究建立了制备高特异性BA单克隆抗体的方法, 为开发鲜湿米粉中BA快速检测试剂盒提供了理论依据。

英文摘要:

      Objective To establish an immunological rapid detection method for bongkrekic acid (BA) in fresh wet rice noodles, and prepare and evaluate monoclonal antibodies that specifically recognize BA. Methods BA semi-antigen was coupled with bovine serum albumin (BSA) and ovalbumin (OVA) carriers using the carbodiimide 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) method to synthesize BA immunogen (BA-BSA) and encapsulant (BA-OVA), respectively. Balb/C mice were immunized with BA-BSA, and the spleens of mice with good immunization results were taken and fused with NS-1 myeloma cells of mice. Indirect competitive enzyme-linked immunosorbent assay (icELISA) was used to screen the hybridoma cells that could secrete the desired specific antibody, and subcloning was performed using limited dilution to obtain a single cell that could stably secrete the desired antibody; in vivo ascites-type monoclonal antibody was prepared by in vivo induction method. The ascites-type antibody was purified by n-octanoic acid-ammonium saturated sulfate method, and the potency of the purified antibody was determined by enzyme-linked immunosorbent assay. Results Immunogen BA-BSA and coating antigen BA-OVA were successfully synthesized and the hybridoma cell line BA-3F1E9 was obtained by screen. The titer of this monoclonal antibody was 1×105. Conclusion A method for preparing highly specific monoclonal antibody against BA has been established, which will provide a theoretical basis for developing a rapid detection kit for BA in fresh wet rice noodles.