Production, Characterization And Application Of Monoclonal Antibody Against Recombinant Protein Tp0453 From Treponema Pallidum

Objective:To express the recombinant protein encoded by the immuno-dominant epitope(28～288aa)of Tp0453 gene of Treponema pallidum,and to purify the recombinant protein and analyze its antigenicity. To fuse SP2/0 cells and spleen cells from BALB/c mouse immunized with recombinant Tp0453 of T.pallidum by hybridization technology; screen the cell lines which secrete monoclonal antibodies against recombinant Tp0453 stably and then identify and purify them. Use self-produced monoclonal antibodies to detect samples of patients suspected T. pallidum infection compared with clinic methods of silver stain of T. pallidum, and then to evaluate monoclonal antibodies as a method for syphilic diagnosis and to establish a foundation for the study on diagnosis of syphilis.Methods:Recombinant Tp0453 was purified with Ni-NTA-His affinity chromatography and protein concentration was determined by BCA assay, Then analyzed the product with SDS-PAGE and Western-blot. The BALB/c mice were immunized with purified recombinant Tp0453. The mouse's spleen cells were fused with SP2/0 cells and then screened the positive clones with ELISA. Ascites were induced to produce the mAbs and were accumulated, then identified the titer, affinity and isotype of the antibodies with indirect ELISA, and conducted the identification of hybiridoma by counting chromosomes. The clinical samples were detected with IIF and indirect ELISA, then analyze the data statistically. Results:A recombinant protein with an estimated Mr of 32kDa was obtained after expression and purification and mainly existed in the pattern of inclusion body. Western-blot proved that the recombinant protein can specifically react with Anti-His mAb. The BALB/c mice were immunized with the purified recombinant Tp0453. We got four clones that can stably secret mAbs. Indirect ELISA and Western-blot analysis indicated that the mAbs secreted by four clones respectively can react with recombinant Tp0453. Injected positive clones secret respective antibodies to the abdominal cavitise of BALB/c mice and then collected the ascites; examine the antibodies titers of ascites; purified the ascites by salt fractionation. Checked the isotypes of mAbs with Mouse Monoclonal Antibody Isotyping Kit; the isotype of mAb secreted by 2F8 strain was IgG2b, the isotype of other three mAb all belonged to IgG1. The affinities of mAbs strains 2F8,8B9,9C6 and 15B2 were 4.12×107M-1, 2.73×108M-1, 4.71×108M-1 and 3.64×107M-1, respectively. the numbers of the hybiridoma cells'chromosomes varied from 90 to 108. The results of detecting clinic samples by using self-producted mAbs to Tp0453 were accorded with the results examined by silver stain of T.pallidum.Conclusions:1. Four clones secreting monoclonal antibodies were prepared and we had proved that every monoclonal antibody can react with recombinant Tp0453.2. The mAbs against recombinant Tp0453 belonged to IgG, which all can react with native Tp0453.3. The results of detecting clinic samples by using self-producted mAbs to Tp0453 accorded with the results examined by clinic methods for diagnosis of syphilis, and indicating that the monoclonal antibodies possess potential value of diagnosis.