Studies On Soluble Antigens Of Trichomonas Vaginalis

Infection of Trichomonas vaginalis is the most common sexually transmitted disease (STD) worldwide, reported to have an annual incidence of 170 million cases . T. vaginalis is a flagellated parasitic protozoan that elicits a broad range of clinical symptoms . It is estimated that up to 50% of infected women are asymptomatic (with normal vaginal pH and flora), with about one third of these women developing symptoms within 6 months. In acute symptomatic infections, clinical manifestations can include punctate hemorrhagic spots on the vaginal and cervical mucosa and yellow-green discharge . In chronic infections, symptoms are milder and may include itching and pain during sexualintercourse . Trichomoniasis has also been associated with cervical cancer ,atypical pelvic inflammatory disease , and infertility . Pregnant women with T. vaginalis infections are predisposed to premature rupture of the placental membranes, premature labor, and low-birth-weight babies . Men generally remain asymptomatic and are classified as carriers, although some develop urethritis and prostatitis .Given the worldwide distribution of T. vaginalis, data suggesting an epidemiologic link with HIV-1.The prevalence of T. vaginalis is likely to be underestimated because there are no guidelines for T. vaginalis screening of women, and clinicians often rely upon insensitive diagnostic methods.The most common method of T. vaginalis detection is wet-mount microscopy. Although this technique is inexpensive and provides immediate results, it is a subjective test that require clinical experience and access to a microscope. Even in the hands of trained observers, the wet mount is only 35 to 80% sensitive compared to culture .The present "gold standard" for the diagnosis of T. vaginalis is culture. Selective media for culture include Diamond's, Trichosel, and InPouch TV (BioMed Diagnostics, San Jose, Calif). No single culture appears to have 100% sensitivity for T. vaginalis infection. And it is time-consuming and expensive. a lot of studies on immunodiagnosis of trichomonas vaginalis have been carried out at home and abroad.Because the antigenic components are very complicated.when the antigen was used for the immunodiagnosis of trichomonas vaginalis,they often occurred cross-reaction with sera from pations with other parasitic diseases and result in false-positive reactions .so it is very difficult to make specific diagnosis for T. vaginalis.in order to find specific antigens for the diagnosis of T. vaginalis,the components of T. vaginalis soluble antigens were analyzed by SDS-PAGE technology,then these antigens were used to react with sera by using immuno-blotting and the sera was extracted from the body of BALB/c mice which was immunized with T. vaginalis solates.Materials and methods1.Culture of T.vaginalis T.vaginalis were obtained from vaginal secretions from women and then inoculated into culture of Hepar-peptone-glucose medium.After 24h growth, culture tubes were centrifuged at 500×g for 10min at room temperature. The pellets were combined into one tube and washed in phosphate-buffered saline(PBS;pH7.2)three times by centrifugation at 500×g.2.Preparation for soluble proteins of T.vaginalis Then the cells of T.vaginalis were frozen and melted, ultrasonicated, centrifuged, respectively,the supernate was collected and used as the soluble antigens. Protein concentrations of antigens were assayed. 3. Preparation for polyclonal antibody of T.vaginalis Five BALB/c mice were divided into one group randomly.The cell of T. vaginalis and immune adjuvent (freund's complete adjuvant and freund's incomplete adjuvant)were injected into the body of the mice through derm at different sports on back.The mice were immunized one time per two weeks.For the first time,the freund's complete adjuvant was used,and the second time ,the freund's incomplete adjuvant.Adjuvant and antigens were mixed 1:1 in volume. After six weeks,we could get blood by extirpating eyeball of the mice. Through disposal,then the sera were made.4. Test for Antibody titer The test for antibody titer with ELISA.5. Preparation for sample of SDS-PAGE Supernatants of the samples were diluted 1:4 with 4% SDS sample buffer, heated to 100C for 5 min and then processed as antigens.6.SDS-PAGE For the SDS-PAGE, stacking and separating gels of 5% and 10% acrylamide were prepared in the Electrophoresis. The gel was stained and bleached sufficiently, different electrophoresis bands were revealed. According to the migrating rate of standard molecule weight protein bands and the logarithm of molecule, standard curve was draw, and the corresponding molecule weight was checked out.7. Western blot These antigens can be transfered to nitrocellulose and analyzed usingimmunoblotting.results1. The test for Antibody titer with ELISA After BALB/c mice had been immunized by T.vaginalis forthree times, two weks later ,the results of ELISA show that there were high antibody titer of 1:100000 inimmune mice serum.2.Choice of protein contents of electrophoresis sample:There Were differencesamong the electrophoresis bands, when the protein concentration was different insample hole. The higher the sample concentration is, the less the quality is needed, thebetter the band is revealed. The protein content was about 18μg/hole in theelectrophoresis system of Bio-Rad company.3.Analysis of T.vaginalis soluble antigen by SDS-PAGE .soluble antigen revealed thecomplicated band pattern consisted of 32 protein bands. The bands with molecularweight(MW) ranged from 15kDa -97 kDa, and 12 protein bands with MW more than 100 kDa. There are 8 major protein bands with MW of 110,97,75,60,57,34,26 kDa and15kDa.4. The identification of components in soluble antigen of T.vaginalis by Western blot :The immuno-blotting results show that the protein bands of soluble antigens have a wide reaction with immune sera from mice with cell of T.vaginalis .Among 32 protein bands , protein bands of 86,75kDa和22kDa show no immunogenicity. Especially,The protein bands of 110,97,60,26kDa show more clear after being stained by DAB, were major reactioning bands with stronger immunogenicity.Conclusion1.Analysis of T.vaginalis soluble antigen by SDS-PAGE: soluble antigen revealed the complicated band pattern consisted of 32 .Protein bands. protein bands with MW of 110,97,75,60,57,34,26 kDa and 15kDa.are major components.2.The identification of components in soluble antigen of T.vaginalis by Western blot: The immunogenic protein components with 110,97,60,26kDa in cell of Trichomonas vaginalis soluble antigens are good targets for the development of sensitive and specific diagnostic assays of trichomoniasis.