抗炭疽PA15单克隆抗体的制备及免疫学检测方法的建立  

Generation of monoclonal antibodies against Bacillus anthracis PA15 and establishment of AC-ELISA detection system

作　　者：方国平[1,2] 唐甜[2,3] 仇镇宁[1] 冯振卿[1,2] 朱进[1,4] 管晓虹[1] 

机构地区：[1]南京医科大学卫生部抗体技术重点实验室,江苏南京210029 [2]南京医科大学卫生部病理学系,江苏南京210029 [3]广州医科大学附属第二医院病理科,广东广州510260 [4]南京军区军事医学研究所,江苏南京210002

出　　处：《南京医科大学学报（自然科学版）》2014年第6期734-740,共7页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金资助(31170884)

摘　　要：目的:制备抗炭疽保护性抗原15(protective antigen,PA15)单克隆抗体,初步建立双抗体夹心ELISA检测炭疽感染者血清中的保护性抗原。方法:以纯化的PA63蛋白为免疫原免疫小鼠,利用杂交瘤技术制备单克隆抗体,SDS-PAGE电泳检测抗体纯度,间接ELISA、Western blot、免疫沉淀(IP)和蛋白质谱分析单抗特异性,并建立双抗体夹心ELISA检测方法。结果:制备了2株抗PA15单克隆抗体,命名为3D7和8E9。SDS-PAGE电泳可见抗体的重链和轻链,间接ELISA、Western blot发现单抗3D7和8E9可与PA15、PA63特异性结合,IP和蛋白质谱分析发现单抗3D7可与PA83特异性结合,双抗体夹心ELISA方法检测炭疽感染血清中保护性抗原的最低检出浓度为16 ng/ml。结论:成功制备了抗PA15单克隆抗体,并建立了双抗体夹心ELISA方法检测炭疽感染血清中的保护性抗原。Objective：To produce monoclonal antibodies against Bacillus anthracis PA15,and preliminarily establish double antibody sandwich-ELISA to detect the protective antigen obtained from serum of patient infected with Bacillus anthracis.Methods：Purified PA63 proteins were performed as immunogen to immunize mice.Hybridoma technology was performed to produce monoclonal antibodies.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） was performed to detect the purity of antibodies.Indirect ELISA,Western blot,immunoprecipitation（IP） and protein profiling were performed to analyze the specificity of monoclonal antibodies.Furthermore,double antibody sandwich-ELISA method was performed.Results：Two monoclonal antibodies against PA15were obtained,named 3D7 and 8E9.SDS-PAGE showed the heavy and light chains of antibodies.Indirect ELISA and Western blot detected that monoclonal antibodies 3D7 and 8E9 can specifically bind PA15 and PA63.IP and protein profiling analyses showed that monoclonal antibody 3D7 can specifically bind PA83.Double antibody sandwich-ELISA detected that the limited detectable concentration of protective antigen from serum infected with Bacillus anthracis was 16 ng/ml.Conclusion：We have successfully developed monoclonal antibody against PA15,and established double antibody sandwich-ELISA to detect anthrax infected serum protective antigen.

关 键 词：炭疽病 炭疽芽胞杆菌 保护性抗原 单克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]