Bovine Interferon-γ Fluorescence Immunoassay Based On The Spore@Zr⁴⁺ Microspheres

Bovine tuberculosis（TB） is a chronic, infectious, and zoonotic disease in cattle caused by Mycobacterium bovis, and seriously affects the health of bovine and human. Bovine interferon gamma（Bo IFN-γ） released by sensitized T cells can be specifically used for the early diagnosis of Bovine tuberculosis. Based on the catalytic of HRP for H2O2 oxidized Amplex Red to produce resorufin with strong fluorescent, we established a novel Bo IFN-γ fluorescence immunoassay sensing technology using spore@Zr⁴⁺ microspheres.The results are listed as follows:1. The treated Bacillus megaterium spores show good stability and dispersity. Zr⁴⁺ ions can be successfully adsorbed on the surface of treated spores via –NH2, –COOH, and–OH groups. The adsorption behavior consisted with the Langmuir adsorption model.2. A good linear correlation was found between the fluorescence intensity and the concentrations of Bo IFN-γ in the range of 0.10 ng/m L to 20.00 ng/m L（R2 = 0.998）.The limit of detection（LOD） was 40.00 pg/m L（S/N = 3）. This method showed excellent repeatability and specificity. The developed method show good accuracy for the quantitative analysis of Bo IFN-γ in serum or plasma sample. The recoveries in newborn calf serum were between 97.6% and 110.0%. The contrast of this method and commercial ELISA kit was conducted and the relative error was between-3.3%and 4.2%. Fifty four clinical cow plasma respectively stimulated by bovine PPD,avian PPD and sterile PBS were analyzed using this developed method. The results were compared with that obtained by commercial Bovigam TM kit, and yielded a sensitivity of 82.6%, specificity of 90.3%, and efficiency of 87.0%.