Production, Characterization And Application Of Monoclonal Antibody Against Recombinant Protein MOMP_VD2-VD3 From Chlamydia Pneumoniae

To express the recombinant vector containting the gene encodingimmuno- dominant epitope of major outer membrane protein (Omp) of Chlamydiapneumoniae (Cpn). Then purify the recombinant protein and analyse its antigenicity.To fuse SP2/0 cells and spleen cells from BALB/c mouse immunized withrecombinant Major Outer Membrane Protein (MOMP) with hybridizationtechnology; screen the cell lines which secrete monoclonal antibodies againstrecombinant MOMP_VD2-VD3V stably and then identify and purify them. Usemonoclonal antibodies we produced to detect samples of patients confirmedChlamydia pneumoniae infections at the same time. Compare the results and then toidentify the specificity of monoclonal antibodies as a method of Chlamydiapneumoniae infections diagnosis so that to establish a foundation for study ondiagnosis of Chlamydia pneumoniae infections and related diseases.Methods: (1) Recombinant MOMP_VD2-VD3 was purified with Ni-NTA-His affinitychromatography and protein concentration was determined by BCA assay. Thenanalyze the product with SDS-PAGE and Western blot. (2) The BALB/c mice wereimmunized with purified recombinant MOMP_VD2-VD3. The mouse's spleen cells werefused with hybridoma cells and then screened the positive clones with ELISA. (3) Theascites caused by immunization of positive clones were accumulated and thenidentified the titer and the specificity of the antibodies with ELISA, Monoclonal Antibodies Isotyping Kits and MIE (4) Human peripheral blood mononuclear cells ofpatients and health people were detect with MIF and then analyze the datastatistically.Results: A recombinant protein with molecular weight of 24KDa was obtained afterexpression and purification and mainly existed in the pattern of inclusion body.Western blot proved that the recombinant protein can specifically react with Anti-HisMAb. The BALB/c mice were immunized with the purified recombinantMOMP_VD2-VD3. We got three clones that can secret monoclonal antibodies stably andWestern-Blot analysis indicated that the monoclonal antibodies secreted by threeclones respectively can act with recombinant MOMP_VD2-VD3. We named the threeclones with 5D6, 7G3 and 8C9 respectively. Western blot proved that three kinds ofMAb can react with MOMP_VD2-VD3 and recombinant bacteria. Immunize the BALB/cmouse with positive clones secret respective antibodies and then collect the ascites;Examine the antibodies titers of ascites with ELISA and the titer is 1:51200,1:25600and 1:25600 respectively; Purify the ascites through vitriol deposition; Calculate theaffinity of monoclonal antibodies through MAbs affinity fomula and the affinity is52μg/mL,48μg/mL and 47μg/mL respectively; Check the isotypes of MAbs withMouse Monoclonal Antibody Isotyping Kit and found the three MAb are all IgG1 inheavy chain andκin light chain; Examine the PBMCs of 96 patients that wereconfirmed the Chlamydia pneumoniae infection with MIF, and the results prove thatour monoclonal antibodies almost have the same sensitivity with clinical IgGdiagnostic kits and the coincidence was 96.9%.Conclusion:(1) Three clones secreting monoclonal antibodies are prepared and we haveproved that the three monoclonal antibodies all can react with recombinantMOMP_VD2-VD3 indicating that the specificity of monoclonal antibodies(2) IgGs against to recombinant MOMP_VD2-VD3 are prepared. (3) The monoclonal antibodies we produced own the same specificity andsensibility proved by the detection of clinical samples.