Development Of Monoclonal Antibodies Against Bovine IFN-γ And A Antibody Sandwich ELISA For The Detection Of Bovine Tuberculosis

The recombinant protein rHIS-BovIFN-γwas used to immunize BALB/c mice, splenocytes from immunized mice were fused with Sp2/0-Ag-14 myeloma cells, the supernatants of hybridoma clones were screened by indirect ELISA, using rGST-BovIFN-γprotein as detecting antigen. Six hybridoma cell lines were obtained and named as 1C12, 4A10, 5F5, 6E12, 2D10, 6F6. The results showed that 6 McAb secret high specific antibodies against rBovIFN-γ, and they didn't react with rCHIFN-γand rCHIL-2. The isotypes of 6 McAbs were IgG1, IgM, IgM, IgM, IgG2a, IgM respectively, and the ELISA titers of their culture supernatant were 1:1600, 1:400, 1:3200, 1:800, 1:800, 1:1600, and those of the ascites were 1:12800, 1:3200, 1:25600, 1:12800, 1:6400, 1:12800 respectively. Two of them 1C12, 2D10 were purified and labeled with HRP. The quanlity of HRP-1C12 and HRP-2D10 was identified. Their E/P were 1.01 and 1.06 respectively, while the rate of labeling were 0.378 and 0.368.After screened the six antibodies, the mixture antibodies of 5F5 and 6F6 (1:1, 13μg/ml) was selected and used as coating antibody, and HRP-1C12 (5μg/ml) used as the second antibody in a antibody sandwich ELISA. Based on the optimization assay, the phosphate buffered saline(PBS) containing 10% FCS was determined as the blocking solution, and the PBS containing 0.5% BSA and 0.05% Tween-20 was used as diluting solution. The optimal reaction time courses were determined as 75min as reaction time of antigen, 60min as reaction time of labeled antibody, meanwhile 10min as substrate reaction time. As to the critical value, it was concluded that the positive critical OD492 values was equal or higher than 0.185, and the negtive critical OD492 values was lower than 0.155, between two values were suspicious. The detecting limit of this antibody sandwich ELISA was 0.5μg/ml of BovIFN-γ, and it didn't react with rCHIFN-γand rCHIL-2.182 bovines were investigated for TB by TST, bovine IFN-γEIA and antibody sandwich ELISA respectively. Compared with the TST, it was concluded that the sensitivity of bovine IFN- γEIA was 30.30%, the specificity was 82.05%, the consistency was 39.56%; meanwhile, the sensitivity of antibody sandwich ELISA was 39.69%, the specificity was 70.27%, the consistency was 48.35%. Compared with the bovine IFN-γEIA, the sensitivity of antibody sandwich ELISA was 78.18%, the specificity was 82.3%, the consistency was 74.72%. These results showed that compared with TST, both bovine IFN-γEIA and antibody sandwich ELISA's consistency were lower, while the consistency of bovine IFN-γEIA and antibody sandwich ELISA was higher. The result also provide some useful data for three assay's further study.This study developed six monoantibodies and an antibody sandwich ELISA successfully, it may be a valuable tool for bovine tuberculosis detection and be helpful for futher application.