制备肺炎衣原体抗原片检测血清抗体  

Preparation of Chlamydia pneumoniae Antigen Slide for Detecting Serum Antibody

作　　者：陈学勤[1] 陈园园[1] 李雪萍[1] 王卫群[2] 余竹元[2] 钱利生[1] 

机构地区：[1]复旦大学上海医学院病原生物系,上海200032 [2]复旦大学附属金山医院,上海200032

出　　处：《生物技术通讯》2006年第6期904-907,共4页Letters in Biotechnology

基　　金：上海市卫生局科技发展基金项目(024006)

摘　　要：目的:探索肺炎衣原体抗原片检测血清抗体法在诊断Cpn感染中的实际应用前景。方法:应用进口肺炎衣原体(Cpn)毒株感染Hep-2细胞,分别以瑞氏-姬母萨染色、吖啶橙染色和直接免疫荧光染色等3种方法鉴定Cpn感染细胞。纯化获取大量Cpn抗原,用于制备斑点抗原片。建立微量免疫荧光染色法(MIF)检测血清抗体,诊断Cpn感染。结果:Cpn感染Hep-2细胞的最适条件是用含1μg/mL放线菌酮的维持液,在35℃、5%CO2孵箱中培养7d,并在培养的第0、3、4、5天以2600r/min离心1h,感染成功率极高。染色反应显示,瑞氏-姬母萨染色可将Cpn包涵体染成蓝紫色或红紫色;吖啶橙染色则使Cpn感染的Hep-2细胞呈现鲜明的橘红色;免疫荧光抗体染色后,在Cpn感染细胞内可见亮苹果绿色包涵体。通过斑点抗原荧光抗体染色的方法抽样检测了100份病人血清中的Cpn抗体,其中抗Cpn-IgG抗体的阳性血清共61份,阳性率为61%。与Cpn-外周血单核细胞(Cpn-PBMC)抗原片比较,阳性检出率无明显差别。结论:用Cpn感染细胞制作的Cpn斑点抗原片可用于临床检测血清Cpn-IgG抗体,且具有特异性、敏感性高的特点,但要求检测人员有一定的经验。Objective： To explore the application perspective of Chlarnydia pneumoniae（Cpn） spot antigen slide diagnosing Cpn infection. Methods： Imported Cpn strain was used to infect Hep-2 cell and the result of infection was identified trough Romanowsky-Giemsa staining, acridine orange （AO） staining and direct immunofluorescence （DI） staining. Large amount of Cpn antigen was obtained after purification and was used to prepare Cpn spot antigen slide. The method of micro-immunofluorescence （MIF） detecting serum Cpn antibody and diagnosing Cpn infection was established. Results： It showed that the optimization culture of Cpn was in Hep-2 with 1640 added 1 μg/mL cycloheximide and centrifugated on days 0, 3, 4, and 5, and kept culturing for 7 days. Thus could make a really high infection ratio. It also showed Cpn infected Hep-2 cell would turn amethyst or magenta using Romanowsky-Giemsa staining, bright orange using AO staining and apple green cytorrhyctes using DI staining. One hundred sera of patient tested with Cpn spot antigen slide were found to have Cpn-IgG in 61 sera. The positive rate of 61% appeared no significant difference contrast to the detection rate of the antibody to Cpn-PBMC spot antigen slide. Conclusion： Cpn spot antigen slide which made by Cpn infected cells can be use to detect serum Cpn-IgG antibody. This method is beth highly specific and quite sensitive. However it requires experimenter with abundant experience.

关 键 词：肺炎衣原体 肺炎衣原体斑点抗原片 肺炎衣原体-PBMC斑点抗原片 微量免疫荧光法 

分 类 号：R392.1[医药卫生—免疫学]