抗肺炎衣原体蛋白酶样活性因子单克隆抗体在冠状动脉硬化患者肺炎衣原体感染诊断中的初步应用  

Preliminary Application of the Monoclonal Antibodies Against Chlamydial Protease-Like Activity Factor From Chlamydia Pneumoniae to Diagnose Coronary Arteriosclerosis

作　　者：唐熟能[1] 郑江花[2,3] 莫小辉 王继平 

机构地区：[1]厦门大学附属第一医院检验科,福建省厦门市361003 [2]上海市(复旦大学附属)公共卫生临床中心,上海市201508 [3]南华大学附属第二医院医学检验科,湖南省衡阳市421001 [4]上海市皮肤性病医院中心实验室,上海市200050 [5]上海市第六人民医院中心实验室,上海市200233

出　　处：《中国动脉硬化杂志》2013年第4期359-363,共5页Chinese Journal of Arteriosclerosis

基　　金：湖南省自然科学基金(10JJ3014);复旦大学医院优势学科建设项目"感染性疾病实验诊断"

摘　　要：目的制备抗肺炎衣原体蛋白酶样活性因子重组蛋白的单克隆抗体,检测冠状动脉硬化患者的外周血单核细胞和冠状动脉瘤斑块中肺炎衣原体特异性抗原。方法以肺炎衣原体蛋白酶样活性因子重组蛋白免疫小鼠,与SP2/0细胞融合后,采用间接ELISA和Western blot筛选阳性杂交瘤细胞,经亚克隆建株;小鼠体内诱生腹水法制备单克隆抗体,用硫酸铵沉淀法对腹水中的单克隆抗体进行纯化,测定其亚类和效价;检测肺炎衣原体标准株以鉴定其特异性。建立间接ELISA方法检测冠状动脉硬化患者外周血单核细胞和冠状动脉瘤斑块标本中的肺炎衣原体抗原。结果获得4株稳定分泌抗蛋白酶样活性因子重组蛋白的杂交瘤细胞株,分别命名为3F8、7B9、8C4和11B5,其效价分别为1∶28000、1∶16000、1∶38000和1∶12000;经亚类鉴定,3F8和7B9杂交瘤细胞分泌的单克隆抗体为IgG2b,其它2株均为IgG1。以制备的单克隆抗体建立间接ELISA检测肺炎衣原体抗原,与经典PCR方法的检测结果比较,检出率具有较高的一致性。结论成功获得了抗肺炎衣原体蛋白酶样活性因子重组蛋白的特异性单克隆抗体,能特异地识别天然肺炎衣原体蛋白酶样活性因子抗原,有望应用于冠状动脉硬化患者肺炎衣原体感染的抗原诊断。Aim To prepare and identify the monoclonal antibodies against recombinant protein of chlamydial protease-like activity factor from chlamydia pneumoniae for clinical diagnosing C. pneumoniae infection. Methods The chlamydial protease-like activity factor recombinant protein was used as antigen for the immunization of female BALB/ c mice, and the spleen cells of mice were fused with SP2/0 myelomas. Indirect ELISA and Western blot analysis were used to screen the hybridomas secreting antibodies and then subcloning positive clones were carried out to establish stable cell lines by limiting dilution. Ascites were induced to produce MAbs and their specificity were identified by indirected immunofluorescence （IIF） test and Western blot analysis based C. pneumoniae type strain. The new indirect ELISA was estabilished to examine coronary artery plaques from patients and the results were analyzed by statistical method. Re- sets Four hybridoma cell lines secreted the monoclonal antibodies stably were finally obtained and named as 3F8,7139, 8C4 and llBS, respectively; the subtype of the monoclonal antibodies secreted by 3F$ and 7B9 strain was IgG2b, the other two monoclonal antibodies were IgG1 and their titers in ascites were 1 ： 28000, 1 ： 16000, 1 ： 38000 and 1 ： 12000, respec- tively. The Western blot and IIF analysis showed that these monoclonal antibodies have excellent specificity to C. pneu- moniae. The results of detecting clinical samples by the new indirect ELISA based on self-produced the monoclonal anti- bodies had better consistenc~ with the results of PCR kits for C. pneumoniae infection. Conclusion The specific mon- oclonal antibodies against chlamydial protease-like activity factor recombinant protein of C. pneumoniae were obtained. All the 4 monoclonal antibodies belonged to IgG, which can react with native chlamydial pretease-like activity factor from C. pneumoniae. The self-producted monoclonal antibodies are suitable for C. pneumoniae antigen diagnosis to coronary ar- teriosclerosis patients.

关 键 词：肺炎衣原体 冠状动脉硬化 衣原体蛋白酶样活性因子重组蛋白 单克隆抗体 抗原诊断 

分 类 号：R44[医药卫生—诊断学]