抗炭疽pa15单克隆抗体的制备及免疫学检测方法 - 南京医科大学学报.doc

抗炭疽PA15单克隆抗体的制备及免疫学检测方法的建立 方国平1,2，唐甜2,4，仇镇宁1，冯振卿1,2＊，朱进1,3＊，管晓虹1 （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南京医科大学病理学系，江苏 南京210029；3南京军区军事医学研究所，江苏 南京 210002；4广州医科大学附属第二医院广州 510260? [摘要] 目的：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ELISA、Western blot及IP和蛋白质谱ELISA检测方法。2株抗PA15单克隆抗体，命名为3D7和8E9。SDS电泳可见抗体的重链和轻链，间接ELISA、Western blot发现单抗3PA15、PA63特异性结合，IP和蛋白质谱抗3PA83特异性结合，双抗体夹心ELISA方法检测炭疽感染血清保护性抗原的最低检出浓度为16 ng/ml。结论：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保护性抗原。 [关键词] 炭疽病；炭疽芽胞杆菌；保护性抗原；单克隆抗体 Generation and characterization of monoclonal antibodies against Bacillus anthracis PA15 and establishment of AC-ELISA detection system Fang Guoping1,2, Tang Tian2,4,Qiu Zhenning1,Feng Zhenqing1,2＊,Zhu Jin1,3＊,Guan Xiaohong1 （1Key Laboratory of Antibody Technique of Ministry of Health, 2Department of Pathology,NJMU,Nanjing,China,210029;3Huadong Medical Institute of Biotechniques, Nanjing, China, 210002;4The Second Affiliated Hospital of Guangzhou Medical University,Guangzhou, China, 510260） [Abstract] Objectives：To produce antibodies against PA with hybridoma technology, and quantitative sandwich-ELISA for?protective?antigen with antibody obtained was developed. Methods: To produce hybridoma cell lines excreting monoclonalantibodies against PA by using B cell hybridoma technique. A cell line named 3D7, which could secrete monoclonal antibody against PA15 was selected. Anti-PA15 monoclonal antibody was purified, which the titration is about 1:12800. Results：Data obtained using clinical samples showed that protective antigen could be detected by AC-ELISA detection system using the 3D7 Mab. The limited concentration of detection was 16 ng/ml. Conclusion：We have developed murine monoclonal antibody against PA. It was potentially useful for the further development of highly sensitive, easily handled, and relatively rapid detection kits/tools for anthrax surveillance in those areas where Bacillus anthracis is endemic. [Key words] Anthrax; Bacillus anthracis; Protective antigen; Monoclonal antibody 通讯作者，＊E-ma