Research On Monoclonal Antibodies Against Toxoplasma Gondii And Development Of Sandwich Elisa

The aim of my research is to lay foundations for diagnosis and preparation for subunit vaccine of toxoplasmosis,and Separation and purification of antigens and Explore of Protective immunity.The tachyzoites of Toxoplasma gondii RH strain were purified by discontinuous Fluid lymphocytes gradients,then which was treated by filtration and centrifugation,freeze-thawed and sonicated.Balb/c mice were immunized for six times with purified Tachyzoites of Toxoplasma gondii and the ELISA titers were all above 1:6400 in the end.Indirect Enzyme-linked Immunosorbent Assayz(ELISA) method was developed to detect antibodies against Toxoplasma gondii.The optimum conditions of the ELISA are set as follows:Toxoplasma gondii crude antigen diluted by carbonate buffer solution(CBS,pH 9.6,0.05mol/L)in 1:20 as a coated antigen dissolved,1% BSA as a blocking agent post-coating,horse radish heroxidase-labeled sheep anti-mouse IgG(HRP-anti IgG)diluted by PBST in 1:5000 as a secondary antibody,and TMB(3,3'5,5'-tetramethyl benzidine dihydrochloride)as a shustrate.The specificity and repetitiveness of the ELISA were primely proved by the blocking test.The ELISA was conducted to detect the antisera of mouse and presented good results.Spleen cells of the mice were selected to fuse with NSO cells. The positive wells were selected by indirect ELISA,and after 3 to 4 generations of clone by limited dilution,4 hybridized cell clones(3A3,3G3,4F4,4G3)against Toxoplasma gondii were obtained that the solution titers ranged from 1:800 to 1:12,800,and ascites titers were 1:51200 showing good stability.Identification results of monoclonal antibody subclass was IgG1.The monoclonal ascites were examined with indirect immunofluorescence assay,was against Tachyzoites.Three ascite clones were purified and passed through SDS-PAGE electrophoresis,the result showed that the chain identified by T.gondii Ag strains was approximately 45 kDa.Molecular weights of bands identified by various McAb strains were all over 55kDa.Immunogen was prepared using purified Toxoplasma gondii with the same methods as above,pig had been injected six times with oil-adjuvant-vaccines and liquid-phase-vaccines to produce antibody against Toxoplasma gondii.HRP was used to label pure anti-T. gondii IgG. The mass concentration of IgG was5.73mg·mL-1.McAb 3A3 was selected to develop sandwich ELISA with the HRP-pig-antibody.The best dilution of McAb was 1:100-1:200.The specificity test showed that the method has higher discernment to T. spiraLis ES,C.suis.A. suum,Ⅰ. suis,NPM which belonged to the same family,the OD value of them was remarkable lower than that of Toxoplasma gondii.The establishment of a double decker sandwhich-ELISA provided a simple,fast,sensitive and universally used method for the diagnosis of Toxoplasmosis and Public health,and laid foundations for a rapid diagnostic system for toxoplasmosis.