Preparation Of Antibodies And Establishment The Sandwich ELISA Method For Toxoplasma Gondii MIC10 And GRA6 Protein

Toxoplasmosis is a kind of zoonosis caused by Toxoplasma gondii.It can result suddely death of fatting pig,abortion and stillbirth of sows.What’s more,the toxoplasmosis of human most caused by eating pork of infected pig.So toxoplasmosis not only harms the production of pig industry,but also has huge effects on human health.Circulating antigen(CAG)is a kind of excretory and secretory protein released by Toxoplasma gondii,when it invasion into host cells.It mainly includes rhoptry proteins(ROPS),dense granule antigen(GRAS)and micronema protein(MICs);CAg usually as the marker for acute infection of Toxoplasma gondii,because it appeared early in the blood and disappeared soon.In this study,we expressed MIC10 and GRA6 protein,prepared polyclonal antibodies from different animal sources,respectively!And established two kind of sandwich ELISA methods,and used two methods to detect clinical pig serum samples at different stages,in order to provide technology for early diagnosis of swine toxoplasmosis.1.Prokaryotic expression of MIC10 and GRA6 proteinThe results of SDS-PAGE showed that MIC10-His was expressed as soluble protein,the molecular weight is 37 k Da.Recording to the results of ELISA,we know MIC10-His protein have a good response with positive serum of Toxoplasmosis.What’s more,the p ET-32a-GRA6 expression results showed no significant expression was observed;therefore,we established pet-32a-t GRA6 vector ues the truncated GRA6 genes(t GRA6),and it express successfully.The SDS-PAGE showed that the t GRA6-His protein was mainly expressed by inclusion body protein with 43 k Da.It also have a good response with positive serum of Toxoplasmosis.2.Preparation of polyclonal antibodies against MIC10 and GRA6 proteinUse MIC10-His and t GRA6-His protein as immunizingantigen to immunize BALB/c mice and New Zealand white rabbits,respectively!Subsequently,blood was collected to prepare antiserum,and anti-MIC10 and t GRA6 serum were purified by the octanoic acid ammonium sulfate method,respectively.The results of ELISA and Western blot demonstrate polyclonal antibodies made in this study can react specifically with the corresponding antigens.3.Development and application of sandwich ELISA methodUse rabbit polyclonal antibody as capture antibody,and the mouse polyclonal antibody was used as the detection antibody.The MIC10 and GRA6 sandwich ELISA were developed,respectively.The repeatability and sensitivity test of MIC10 ELISA showed that it’s detection limit is 7.8 ng/ml MIC10-His protein and the coefficient of variation was less than 12%;The repeatability and sensitivity test of GRA6 ELISA showed that it’s detection limit is 1.95 ng/ml t GRA6-His protein and the coefficient of variation was less than 10%.Use the MIC10 and GRA6 sandwich ELISA methods to detect 368 swine serum,the seropositive rate was 42.12%and 50%,respectively!In addition,the two self-built sandwich ELISA methods and commercial kits were used to detect the 89 same serum samples.The results showed that the positive rate of MIC10 sandwich ELISA,GRA6sandwich ELISA method and commercial kits was 49.4%,33.7%and 29.2%,respectively!The self-built two kinds of double antibody sandwich test results almost same,and partly consistent with the detection results of the commercial kit.4.Preparation of anti-MIC10 monoclonal antibodyBy constructed the p GEX-4T-1-MIC10 expression vector,expressed and purified the MIC10-GST protein,using the MIC10-GST protein as the detection antigen to screening of hybridoma cells which acquired from SP2/0 fused with the spleen cells of mice immunitied by MIC10-His,Result,1 hybridoma cell that could stably secreting monoclonal antibodies was successfully screened,named as 13-2B;The titers of ascites prepared from 4 mice were all in more than 1×10~6,and the results of Western-blot showed that,13-2B can react specifically with MIC10 protein.In summary,In this study,we has established two sandwich ELISA methods for detection Toxoplasma gondii circulating antigen,and the two kind of methods were specific,sensitive,and easy to perform.