| Chlamydia trachomatis( Chlamydia trachomatis C.t) is a kind of major pathogens which can cause sexually transmitted disease and genitourinary tract infections diseases.According to WHO statistics,the new chlamydial infection cases amount keep a 92 millions pace of speed every year worldwide.In recent years,urogenital infection caused by Chlamydia trachomatis showed growth year after year in the world,and ranked the first in sexually transmitted diseases?According to the epidemiological investigation, the symptoms of chlamydia infection are rarely to be found, so the patients usually not to be found and not being treated,eventually would cause a variety of serious disease such as pelvic inflammatory disease, ectopic pregnancy, infertility and male prostatitis.C.t also co-infected with the human papillomavirus(HPV) and can increase the risk of HIV transmission,and also positively correlated with the incidence of cervical squamous cell carcinoma.Therefore, the early screening of Chlamydia trachomatis infection,early detection of infected population and asymptomatic carriers,and adequate treatment provided after detection all make a great significance for the prevention and control of the Chlamydia trachomatis infection spread.However, there are many defects existing in the variety of detection methods such as painful of the samples taken,low sensitivity and specificity, and determines also subjectivity,thus not suitable as a screening tool for C.t infection.Currently in our country the main target proteins used for C.t detection kits are Chlamydial LPS(Lipopolysaccharides),Chlamydial MOMP(major outer membrane protein, MOMP)or heat shock protein 60(Chlamydial heat shock protein60, HSP60).MOMP and HSP-60 has good antigenicity and specificity,but their antigenicity all rank after the top 20 immunodominant proteins.Lps lack of specificity,and can easy cross-react with other pathogens' Lps ntibodies and then affect the detection accuracy.Therefore, in order to optimize the laboratory screening of Chlamydia trachomatis infection and improve the detection sensitivity and specificity,on the basis of our previous studies we selected a combination of this three immunodominant proteins:Pgp3, CT694 and CT875.We collected 300 cases ofserums of sexually active population aging among 15-40 years old in non-STD clinic of Tianjin Medical University General Hospital.Using enzyme linked immunosorbent assay(ELISA) method and micro immunofluorescence(MIF) method to detect the antibody of the three proteins mentioned above respectively,through the comparison between this two kinds of methods,to learn the sensitivity and specificity of the newly established ELISA method.Object:To express three kinds of C.t immunodominant proteins: Chlamydia plasmids encoding protein 3(Pgp3), Ct875 encoded protein(CT875), Ct694 encoded protein(CT694).Using the ELISA method to detect the three proteins' antibodies in the serum,and then detect by the MIF method,through the comparison between this two kinds of methods,to learn the sensitivity and specificity of the newly established ELISA method.Method Transformed the recombinant plasmids which encoded the 3 proteins and blank plasmid into E. coli, respectively.Use the Agarose gel electrophoresis to confirm the transformation is successful,expanding culture the E. Coli,The proteins' expression were induced by IPTG. Broke the bacterial by ultrasonic.Purify the protein with the GST Magbeads, and then SDS electrophoresis to confirm the expression is successfull.Collect 300 cases of serums of sexually active population aging among15-40 years old in non-STD clinic of Tianjin Medical University General Hospital.The lysates were used to coat the glutathione precoated 96-well ELISA plates.Added the prepared serum ? murine monoclonal antibody as antibody I,HRP-conjugated goat anti-human?goat anti-mouse Ig G as antibody II,ELISA method was used to detect the presence of all three proteins in serum samples.Make the chlamydia EB antigen tablets. which were used to link with the rabbit anti-EB antibodies and human serum antibodies separately.Using MIF method to detect the presence of all three proteins in serum samples.Finally, comparing the detected results of both the two methods.Results: 1 three proteins recombinant plasmid was transformed successfully, The proteins were successful expressed.2.ELISA results:300 cases of serums screening results of the sexually active population in non-STD clinic are as follows:Totally 63 patients positive forchlamydia infection.The positive rate of Chlamydia trachomatis infection is 21%.15 to 20 age group, 7 cases were positive, the positive rate is 21.88%;21 to 25 age group,9 cases were positive, the positive rate is 19.15%;26 to 30 age group,22 cases were positive, the positive rate is 25.88%;31 to 35 age group,17 cases were positive, the positive rate is 21.52%;36 to 40 age group, 8 cases were positive, the positive rate is14.04%.3.MIF Results: Totally 65 cases positive and 235 cases negative for Chlamydia trachomatis infection.4.Two methods results contrast:In the 63 cases by ELISA positive serum result, 62 cases were positive for MIF, one case negative.In the 237 cases of negative ELISA result of serum, 234 cases of micro immunofluorescence were negative, 3 were positive.Make MIF as a standard,The sensitivity of this newly established screening method is 95.38%, and the specificity is 99.57%.Conclusion:1.The detection rate of these 3 kinds of immunodominant protein antibody of Tianjin sexually active population is 21%.2.The newly established ELISA method has higher sensitivity and specificity,and it's fit for screening for the infection of chlamydia trachomatis?... |
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