Development And Preliminary Application Of Rapid Detecting Tuberculosis By Bovine IFN-γ Level

Bovine tuberculosis (TB) is one of an important zoonotic, chronic and marantic infectious disease which is caused by mycobacterium bovis (MB). Human beings can be infected with mycobacterium bovine tuberculosis to develop tuberculosis. As a result, bovine tuberculosis does not only relate to the development of husbandry, but also directly affect human health. Hitherto, all countries still generally take the measures of "quarantine-slaughter" to control the disease. Altough there are many TB test methods, the rapid, sensitive and specific method is not established in our country. In this study, a bovine IFN-γcompetitive inhibition ELISA method and a colloidal gold immunochro-matographic assay for TB test were developed with monoclonal antibodies against BoIFN-γ. The results of clinical preliminary application showed that the coincidence rate was 83.4% between the results of ELISA and tuberculin skin test (TST),78.6% between the results of the colloidal gold immunochromatographic assay and TST,85.7% between the results of the colloidal gold immunochromatographic assay and Austrailia commercial kits,78.6% between the results of Austrailia commercial kits and TST. These methods will provide good tools for control and prevention of bovine TB in China.1. Development and identification of monoclonal antibody to BoIFN-γThree monoclonal antibodies against BoIFN-γ, named BoIFN-γ-5G7,BoIFN-γ-6G7 and BoIFN-γ-6D11 respectively were developed by fusions between SP2/0 cells and spleen cells from BALB/C mice immuned with GST-BoIFN-γfusion protein expressed in E.coli. Western blot results showed that all of the monoclonal antibodies can react with the recombinant GST-BoIFN-γfusion protein expressed by E. coli but didn't react with the GST. The results of indirect immunofluorescence assay (IFA) indicated that the recombinant BoIFN-y expressed in Sf9 cells infected with recombinant baculovirus could be specifically recognized by the monoclonal antibodies. The immunoglobulin subtypes of monoclonal antibodies were identified with a commercial capture-ELISA kit. The results were IgG2a for monoclonal antibody 6G7 and 5G7, IgM for monoclonal antibodies 6D11. With ELISA, the ascite titer of 5G7 antibody was 1:409600 and cell supernatant titer was 1:20000. The ascites and cell supernatant titers of 6G7 monoclonal antibody were 1:204800 and 1:4000 respectively. The ascites and cell supernatant titers of 6D11 monoclonal antibody were 1:12800 and 1:2560 respectively. Only monoclonal antibody 6G7 can rightly neutralize rBoIFN-γexpressed in E. coli and Sf9 cells in neutralization analysis.2. The establishment and application of competitive inhibition ELISA (ciELISA) for detecting BoIFN-γELISA Plates were coated with different concentration of GST-BoIFN-y purified by GST Sepharose FF column. Then the plates were reacted with different dilutions of monoclonal antibody BoIFN-y-6G7, which was purified with the Hi-Trip Protein G column. The best concentration of coat antigen and the saturation antibody were determined. Samples for test were first mixed with the saturation antibody and then the mixture was added to the ELISA plates coated with the best concentration of antigen.The rabbit anti-mouse antibodies conjugated with HRP at work concentration and substrate were as results indicator. The ciELISA for detecting BoIFN-γwas finally established. With the ciELISA,23 clinical samples (positive in commercial kit) from Beijing and 30 samples from Jiangsu cow farm were detected. The results demonstrated that all the samples from Beijing were positive in ciELISA,8 positive and 3 suspicious were found in 30 samples from Jiangsu cow farm. Compared with the TST, the coincidence rate was 83.4%.3. The establishment and application of colloidal gold immunochromatography test for BoIFN-γAbout 30nm colloidal gold solution at pH 8.3 was labeled with monoclonal antibody 6G7 at 3.3μg/ml and stabled with 10% BSA and 10% PEG-20000. The gold-labeled compound diluted with PB after centrifuge was printed in the glass-fiber membrance. T line and C line with 0.6μl/cm and 0.8μl were printed at NC membrane respectively. The test strips were assembled.14 blood samples from Jiangsu cow farm were detected with the test strips. Seven samples were positive, three were suspicious and four were negative. The coincidence rates between test strips and TST were 78.6%, between test strips and Australia commercial kit were 85.7%, between Australia commercial kit and TST was 78.6%.These results showed that ciELISA and colloidal gold immunochromatography assay for BoIFN-γwere effective. They provided simple, rapid and specific detection methods for the control and prevention of bovine TB in China.