摘要
目的 研制青霉素结合蛋白2a（PBP2a）单克隆抗体（McAb）,为建立耐甲氧西林金黄色葡萄球菌（MRSA）免疫层析检测方法提供检测用抗体.方法 以基因工程抗原rPBP2a免疫BALb/c小鼠,通过常规小鼠B淋巴细胞杂交瘤技术制备单克隆抗体,采用免疫印迹技术（Western blotting）分析单克隆抗体特异性.选取敏感、特异的单克隆抗体进行金标记和硝酸纤维膜包被,建立胶体金免疫层析检测方法.结果 共获得11株分泌抗rPBP2a的杂交瘤细胞,其中6株分泌的单克隆抗体能够与天然PBP2a呈阳性反应.用其中2株单克隆抗体建立的PBP2a胶体金免疫层析方法,可在5～ 20 min内完成检测.结论 获得了特异性针对PBP2a蛋白的单克隆抗体,并初步建立了检测PBP2a蛋白的胶体金免疫层析检测方法,为临床快速、简便检测产生PBP2a的细菌提供了检测方法.
Objective To prepare monoclonal antibodies against PBP2a protein in order to develop a gold immunochroma- tographic assay for the detection of MRSA. Methods BALB/c mice were immunized by injection of recombinant PBP2a （rPBP2a）. Anti-PBP2a monoclonal antibodies were produced by fusion of mouse myeloma cells （Sp2/0） with spleen cells isolated from immunized BALB/c mouse. The specificity of monoclonal antibodies were identified by Western blotting. The monoclonal antibodies with good sensitivity and specificity were selected to develop gold immunochromatographie assay. One of the monoclonal antibodies was conjugated with gold colloid particles, and another antibody was immobilized on a nitrocellulose membrane. Results 11 strains of mouse hybridoma cells secreting anti-rPBP2a were obtained, 6 of them were specific to native PBP2a. One pairs of McAb was selected to develop gold immunochromatographic assay. This assay could be finished within 5-20 minutes. Conclusion McAbs against PBP2a were obtained and the preliminarily established gold immunoehromatographic assay could be used to detect PBP2a. The results of our work provided a method for simple and rapid detection of PBP2a-producing cells in clinical laboratories.
出处
《微生物学免疫学进展》
2015年第3期5-8,共4页
Progress In Microbiology and Immunology
基金
上海市普陀区科技自主创新资助项目(项目编号:普KW11103)
