Development And Preliminary Application Of Bovine IFN-γ Capturing ELISA

Bovine tuberculosis is one of important zoonoses caused by Mycobacterium bovis(MB). More than 20 homoiothermal animals such as cow, deer and badger could be infected by MB. It could also infect humanbeing through respiratory tract and alimentary tract. About 10% human tuberculosis is caused by MB infection. Bovine tuberculosis not only causes great economic losses but is also a kind of serious threat to public health.A lot of countries take'detect-slaughter'policy as a kind of useful measure for the control of Bovine tuberculosis as there's no effectual vaccine to prevent this infection. Tuberculin skin test (TST) is a kind of diagnostic method for Bovine tuberculosis since it had been used . But it is bewildered by the low sensitivity, low specificity and it's failure to diagnose the early infection. IFN-γis a kind of cytokine as an indication of the Th1 cell mediated immunity (CMI) when infected by MB. This could become a target for the detection of Bovine tuberculosis. A MB antigen specific BoIFN-γantibody sandwich ELISA as a substitution for TST was developed by Wood in 1990 and achieved great success. However it couldn't be widely used in China for the high price. It needs to have our own ELISA Kit for the detection of BoIFN-γ.Three monoclonal antibodies (McAbs) against natural BoIFN-γwere screened from thirteen mouse anti-rHis-BoIFN-γMcAbs. The attemptation of the development of a kind of sandwich ELISA with two McAbs was not successful. However, BoIFN-γcapturing ELISA were developed by one McAb paired with rabbit serum anti-rHis-BoIFN-γpolyclonal antibodies. The coincidence of BoIFN-γcapturing ELISA compared with Mycobacterium bovis Gamma Interferon Test Kit for cattle was 83.9%. It laid the base for the development of BoIFN-γantibody sandwich ELISA and ELISPOT. It also provides us a useful diagnostic reagent for Bovine tuberculosis.1. Identification of anti-BoIFN-γMcAbsThe reactivity of McAbs with rHis-BoIL-4,rGST-BoIL-4,rHis-BoIFN-γ,rGST-BoIFN-γ,rHis-ChIFN-γ,rHis-ChIL-4,rGST-ChIL-4 and the supernate of the lysate of BL21(DE3)(pET) and BL21(pGEX-6P-1) induced by IPTG were identified by Dot-ELISA. Western-blot was used as a kind of method for the identification of the reactivity of McAbs with BL21(DE3)(pET) and BL21(DE3)(pET-BoIFN-γ). The reactivity of McAbs with standard BoIFN-γwas also identified by indirect ELISA. At last all 13 McAbs could only react with rBoIFN-γ. The titers of the ascites of 13 McAbs are all above 1∶80000 except McAb 9G11.2. Development of BoIFN-γcapturing ELISAHighly purified antibodies which had good characteristics were accepted by purification of caprylic acid and ammonium sulfate. Two McAbs against different epitopes of BoIFN-γidentified by additive ELISA, 6F8 and 1G5 were biotinylated. And the titer of the Biotinylated 6F8 was 1∶10000. Meanwhile the titer of the HRP labelled McAb 8D3 was 1∶3200. Sandwich ELISA for the detection of rHis-BoIFN-γwas developed directly with McAbs 3E6/1C12 and Biotinylated 6F8 with a sensitivity of 0.5ng/mL. There was no good result in the detection of natural BoIFN-γthough standard BoIFN-γcould also be detected by the sandwich ELISA. So the BoIFN-γcapturing ELISA was further explored.Rabbit serum polyclonal antibodies against rHis-BoIFN-γwere prepared with a titer of 1∶200000. McAbs 5E11,6F8 and 8D3 reacted with natural BoIFN-γwere screened from 13 McAbs by natural BoIFN-γin the whole blood of cattle induced by pokeweed motigen (PWM). And the BoIFN-γcapturing ELISA was developed by McAb 5E11 paired with rabbit serum polyclonal antibodies.3. Preliminary application of BoIFN-γcapturing ELISAUsing Mycobacterium bovis Gamma Interferon Test Kit for cattle as a tool for the surveillance of Bovine tuberculosis in two herds of cattle in Jiangsu province, 15 cattle infected by Mycobacterium avium and 3 suspects were detected in 117 samples in farm A. Nine Bovine tuberculosis positive cases, 2 cases infected by Mycobacterium avium as well as ten suspects were detected in 59 cattles in farm B. The coincidence of IFN-γdetection in the supernates of the induced whole blood in the clinical samples between BoIFN-γcapturing ELISA compared with Mycobacterium bovis Gamma Interferon Test Kit for cattle is 83.9%.The success of the ELISA assay laid the base for the development of BoIFN-γantibody sandwich ELISA and ELISPOT. It also provides us a useful diagnostic reagent for Bovine tuberculosis.