论文部分内容阅读
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 prepare anti-anthrax protective antigen (PA15) monoclonal antibody and to establish a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of protective antigen in anthrax infected patients. Methods: The purified PA63 protein was used as immunogen to immunize mice. The hybridoma technique was used to prepare the monoclonal antibody. The purity of the antibody was detected by SDS-PAGE electrophoresis. The specificity of McAb was analyzed by indirect ELISA, Western blot, immunoprecipitation (IP) and protein profile , And established double antibody sandwich ELISA detection method. Results: Two anti-PA15 monoclonal antibodies were prepared and named 3D7 and 8E9. SDS-PAGE electrophoresis showed heavy and light chains of antibody. Indirect ELISA and Western blot showed that mAbs 3D7 and 8E9 could specifically bind to PA15 and PA63. IP and protein spectrum analysis showed that mAb 3D7 specifically bound to PA83. The minimum detectable concentration of anti-anthrax serum in the antibody sandwich ELISA was 16 ng / ml. Conclusion: The anti-PA15 monoclonal antibody was successfully prepared and a sandwich ELISA method was established for the detection of protective antigen in anthrax-infected sera.