抗肠出血性大肠杆菌O157:H7 EspA单克隆抗体的制备与鉴定
[Preparation and characterization of monoclonal antibody against Enterohemorrhagic Escherichia coli O157:H7 EspA].
作者信息
Yu Shu, Luo Ping, Chen Hong-zhang, Li Hai-xia, Mao Xu-hu
机构信息
Department of Clinical Microbiology, Third Military Medical University, Chongqing 400038, China.
出版信息
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Jul;23(7):657-9.
AIM
To prepare hybridoma cell lines were obtained by fusing Sp2/0 with spleen cells from BALB/c mice immunized with killed enterohemorrhagic E.coli O157:H7 EspA (EHEC O157:H7 EspA).
METHODS
The subclass isotype and the specificity of monoclonal antibodies (mAb) were determined and identified by ELISA, Western blot and immune fluorescence staining.
RESULTS
Isotype of 3 mAb was IgG1kappa, IgG1lambda, and IgG2kappa, respectively, and the affinity constant were 3.0x10(9), 2.8x10(9), 1.9x10(9). As demonstrated by Western blot, these 3 mAb specifically reacted with EspA protein and EHEC O157:H7. Useing immune fluorescence staining, EHEC O157:H7 could adhere to the membrace of Hela cell.
CONCLUSION
Three hybridoma cell lines can stably secrete anti-EspA mAb with high-titer and high-specific have been established. It can be used to deeply study EHEC O157:H7 pathopoiesis mechanism.
目的
通过将Sp2/0细胞与用灭活的肠出血性大肠杆菌O157:H7 EspA（EHEC O157:H7 EspA）免疫的BALB/c小鼠脾细胞融合，获得杂交瘤细胞系。
方法
采用酶联免疫吸附测定（ELISA）、蛋白质免疫印迹法（Western blot）及免疫荧光染色法对单克隆抗体（mAb）的亚类、亚型及特异性进行测定和鉴定。
结果
3株单克隆抗体的亚型分别为IgG1κ、IgG1λ和IgG2κ，亲和常数分别为3.0×10⁹、2.8×10⁹、1.9×10⁹。蛋白质免疫印迹法结果显示，这3株单克隆抗体均能与EspA蛋白及EHEC O157:H7发生特异性反应。免疫荧光染色结果显示，EHEC O157:H7可黏附于Hela细胞膜。
结论
已成功建立3株能稳定分泌高效价、高特异性抗EspA单克隆抗体的杂交瘤细胞系，可用于深入研究EHEC O157:H7的致病机制。
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