The Establishment And Application Of A Novel IFN-γ Assay Release In Vitro For Bovine Tubeculosis Diagnosis

Bovine tuberculosis (bTB), which is mainly caused by Mycobacterium bovis, is a chronic wasting zoonosis. In recent years, as the booming market of dairy industry, bTB severely threats the development of the dairy industry and human health. Therefore, more and more concerns are received about the diagnosis and the prevention of bTB. Investigations show that the prevalence of tuberculosis is dramatically expanded. Due to the limitations in sensitivity and specificity, traditional diagnostic methods of tuberculosis can not accurately detect Mycobacterium bovis-infected cattles.Interferon-gamma, secreted by T lymphocytes and natural killer cells which has been stimulated by antigens or mitogens (such as PHA, ConA, PWM), is a glycoprotein with multiple functions, such as anti-tumor and anti-virus functions, inhibition of proliferation of cells and immune regulation. In vitro interferon gamma assay, which has been applied into detection kits and approved to use by many countries, is the most promising method to detect Mycobacterium bovis infections. However, the application of this method in China is quite limited due to its expensive price and high false positive rate and low specificity caused by using PPD as antigen. Our research has found a new stimulation antigen and established a new effective ELISA method to detect Mycobacterium bovis infections. Our work includes:1. Preparation of monoclonal and polyclonal antibodies against bovine IFN-yThe purified recombinant protein p30-BovIFN-y is used to immunize Balb/c mice for preparation of monoclnoal antibodies (McAbs).Splenocytes from immunized mice are fused with SP2/0 myeloma cells, then the culture supernatants of hybridoma clones are screened by indirect ELISA using the purified recombinant protein as detecting antigen and using limiting dilution for recloning. Hybridoma cell lines are obtained and named as 1D9,1G9,1G11,1H3,2F6,3B3,3C7,4H9.All the McAbs secreted by these cell lines have high specificity and ELISA titers above 29×100. Polyclnoal antibodies (PcAbs) are prepared by immunization of Japanese rabbit using the purified recombinant protein p30-BovIFN-γ. ELISA titers of these PcAbs are all above 211×100.2. Establishment of BovIFN-γsandwich indirect ELISAThe 1G11 McAb is purified using caprylic acid-ammonium sulfate and the PcAbs are purified using saturated ammonium sulfate. Using 1G11 McAb capture antibody to coat ELISA plate, PcAb against BovIFN-γas primary antibody and HRP (horseradish peroxidase) labeled goat anti-rabbit antibody as second antibody, a sandwich indirect ELISA is established to detect BovIFN-γ. The working concentration of the 1G11 McAb, PcAb and secondary antibody are 1:5000,1:1600 and 1:10000 respectively. Our results show that this method has good specificity and sensitivity with a detection limit of 50pg/mL. Using BovigamTM detection kit as comparison, Our method are applied to detect 98 whole blood samples. The results of BovigamTM are 14 positive and 84 negative, and the results of our method are 12 positive and 86 negative. By comparing our method with BovigamTM, the coincidence rate of positive samples is 71.43%(10/14) and of negative samples is 97.62%(82/84), and the total coincidence rate is 93.88%(92/98), demonstrating that the coincidence rate of our method with BovigamTM is very good. Our new sandwich indirect ELISA for detecting BovIFN-γset the foundation to diagnose bTB.3. Application of new antigens to stimulate whole bloodIn the serile operating condition, Different concentrations of specific proteins ESAT-6-CFP-10 (CE) and RV3872-ESAT-6-CFP-10 (RCE) of tuberculosis are added into per ml of hepari sodium-anticoagulated whole blood. After more than 16 hours of incubation in 37℃with 5% CO2, Supernatants are harvested and the IFN-γconcentration are determined using our sandwhich ELISA. The results indicate that 40μg/mL of CE and RCE is enough to stimulate the whole blood samples which is from cattles infected by tuberculosis to release IFN-γ. We detected 37 clinical blood samples for IFN-γ, found that 17 samples are PPD positive and 20 are PPD negative. Then we compare CE with TST, the coincidence rate of positive, negative and total samples are 52.94%(9/17),95% (19/20) and 75.68%(28/37) respectively. We also compare RCE with TST, the coincidence rate of positive, negative and total samples are 64.71%(11/17),80%(16/20) and 72.97%(27/37) respectively. Additionally, we found that the special protein of tuberculosis RCE has a higher sensitivity than CE.