Establishment And Preliminary Application Of Immunochromatographic Strips For Detection Of Antibodies Against M.bovis

Mycobacterium bovis is the causative agent of Bovine tuberculosis in a range of animal species and human beings. The prevalence and circulation of this disease not only influence the development of stock raising severely, but also threaten food safety and human health . at present,The unique method of exterminate Bovine tuberculosis is use quarantine and slaughter measure weed out positive bovis.Thus,the high accuracy and timely diagnostic method is very important for control the Bovine tuberculosis. For the detection of bTB, tuberculin skin test (TST) is the only method recommended by world organisation for animal health (Office International DesEpizooties, OIE), but the method has serious non-specific reactions and is mostly false negative for seriously diseased cows.people began to question the veracity of TST.So it has a significance for control and wipe out Bovine tuberculosis to exploit a handy,sensibility and specificity diagnostic approach.Three major antigens of Mycobacterium tuberculosis called MPB70,MPB83 and ESAT-6 were analyzed by DNAstar. Synthesis the epitope which is in a high antigenic index And cloned into the pET-28a(+) expression vector. To construct a recombinant plasmid Called pET28a-mpb-70-83-esat-6.the recombinant plasmid was transformed into E.coli BL21 competent cells and then induced by1mmol/L IPTG.SDS-PAGE. The result showed that a band of approximately 21 KU in molecular weight was expressed from the induced E.coli BL21 component cells.use the NI-NTA purification the recombination protein.,western-blot analysis indicated that the protein can be discriminated by Mycobacterium tuberculosis polyclonal antibody, indicaed that the protein has a good antigenicityStrips were prepared with the principle of colloidal gold immunochromato- graphyassay(GICA).Bovis IgG and MPB70-83-ESAT-6 was used as the Quality Control reagent and test capture reagent respectively to detect the corresponding antibodies, and determine it's optimization operating conditions. The best conditioning fluid of the sample and combine pad were 3% BSA,0.1% Tween-20 Tris buffer and 3% BSA ,5%sucrose,0.1%Tween-20 Tris buffer The optimal concentration of capture reagent MPB70-83 -ESAT-6 was 2.5mg/ mL,The strips did not react with positive sera of other unrelated bovine diseases and displayed high specificity. The minimun detection dilution of the strips was 1:640 . The clinical serum samples were detected by the strips, then Comparied with tuberculin skin test (TST) and M.bovis isolation . The coincidence were 80% , Seal the strips in a dry place in a room temperature for five month,The detection results do not had any change compare within five mouth previously. This novel colloidal gold test strip for bTB antibody detection is sensitive, specific, convenient to be used, rapid to get results. It will be a good method in bTB survey used alone or as a supplementary diagnostic method for TST in bTB eradication program.