米酵菌酸单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibodies of bongkrekic acid

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1×10^(5)。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。

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 1×10^(5).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.