Addicted To Chlamydia Pneumoniae Antigen And Serological Testing

Chlamydophila pneumoniae (Cpn), which belongs to the chlamydiae, is an important respiratory tract pathogenic microorganism. The most common deseases caused by Cpn are pneumoniae and bronchitis, recent researches also indicated its relationship with artherosclerosis desease, bronchial asthma, sarcoidosis and asthma. Cpn is proved to be difficult to isolate and culture, which limited carrying out further study about it's pathogenic mechanism, specific diagnosis and vaccine exploitation. Imported Cpn strain was used to set up the model of Cpn infected cell, so as to obtain a great quantity of Cpn antigen to prepare spot antigen slide. These slides were used to estabilish Micro-immunofluorescence (MIF) method to detect serum antibody. According to the sequence of Major outer membrane protein (MOMP) , we also established a recombined plasmid of MOMP/pQE31 and producted recombined Cpn MOMP antigen to detect serum IgG. Through the comparison of the results of these methods, we expected to evaluate each method's specificity and sensitivity so that to help the clinical diagnosis to the Cpn infection and analyze the relationship between its infection and diseases.In the first part of this paper, 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 (DIF) Staining. Large amount of Cpn antigen was obtained after purification. It showed that the optimization culture of Cpn was in Hep-2 with 1640 added 1ug/ml cycloheximide and centrifugated on days 0, 3, 4, and 5, and kept culturing for 7 days. Thus can 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.In the second part of this paper, Cpn antigen was used to prepare Cpn spot antigen slide. We established the method of detecting serum Cpn antibody and diagnosing Cpn infection through MIF. One hundred sera of patient tested with Cpn spot antigen slide as well as Peripheral Blood Mononuclear Cell (PBMC) spot antigen slide. Cpn antigen slide got the Cpn -IgG positive rate of 61 % while the PBMC antigen slide was found to have a positive rate of 60%. Statistical analysis show that there is little difference between the positive rate of the two methods. Cpn spot antigen slide which made by Cpn infected cells is proved to be both highly specific and quite sensitive and can be use to detect serum Cpn-IgG antibody.In the third part of this paper, by establishing and expressing recombined Cpn MOMP antigen, which was purified and used as antigen in ELISA assays. Sera were obtained from 82 healthy volunteers with 207 patients. The patient samples consisted of 118 sera with cardiovascular diseases, 59 sera with diseases of respiratory system and 30 sera with other diseases. Cpn IgG-positive results were observed in 56.0% of patients and only in 3.7% of healthy subjects. There were 69 positive sera of 118 with cardiovascular diseases, 38 positive of 59 with diseases of respiratory system and 9 positive of 30 with other diseases. The positive rates respectively were 58.5%, 64.4% and 30.0%.The positive rate of recombined Cpn MOMP antigen were consistent with the ELISA kit and Cpn holoantigen.In this research, imported Cpn strain was used to infect Hep-2 cell and the result of infection was identified through Romanowsky-Giemsa Staining, AO Staining and DIF Staining. After purification, large amount of Cpn antigen was used to prepare Cpn spot antigen slide. Through comparison between the two method of Cpn spot antigen slide and PBMC antigen slide detecting serum IgG, little difference was shown between the positive rate of the two methods. Cpn spot antigen slide is proved to be both highly specific and quite sensitive and can be use to detect serum Cpn-IgG antibody. Recombined Cpn MOMP antigen was expressed, purified and used as antigen in ELISA assays, which can easily tell the difference between the health and the patient serum. Besides, the positive rate of recombined Cpn MOMP antigen were consistent with the ELISA kit, which showed to be highly specific and sensitive and could greatly help the investigation of Cpn infection. It also had considerable theoretical and pratical value.