Preparation Of Anti-bovine IFN-γ Monoclonal Antibody And Establishment Of Double Antibody Sandwich ELISA Method

Bovine tuberculosis is a zoonotic chronic infectious disease,which is caused by Mycobacterium bovis.There is no effective vaccine for the prevention of bovine tuberculosis.Currently,tuberculin skin test（TST）and interferon-gamma（IFN-γ）in vitro release test integrated global radiosonde archive are often used for detection.Among them,the national animal tuberculosis quarantine industry standard is TST,but many factors affect its accuracy.IGRA is an auxiliary diagnosis method for bovine tuberculin intradermal allergy designated by the World Organization for Animal Health（Office international desépizooties,OIE）.IFN-γenzyme linked immunosorbent assay（ELISA）detection reagents are often used the cassette detects IFN-γprotein released in vitro.The test kit needs to be imported from abroad,but the price is high and it is difficult to popularize in the country.In this study,the target gene Bo IFN-γwas ligated with the vector p ET28a,and the ligation product was transformed into E.coli BL21（DE3）,induced by IPTG.The expression of Bo IFN-γprotein was analyzed by SDS-PAGE electrophoresis.Purify by Ni-NTA affinity chromatography and CM ion exchange chromatography with His Trap FF,and explore the best purification conditions.The target protein was verified by antiviral activity experiment to verify the biological activity of the protein,and mice were immunized,and anti-Bo IFN-γmonoclonal antibody was prepared by optimizing electrofusion conditions.The polyclonal antibody of chicken egg yolk was prepared from laying hens,and the antibody was purified by supersaturated ammonium sulfate precipitation method.The antibodies were identified by ELISA and Western blot.The monoclonal antibody was labeled with horseradish peroxidase（HRP）and paired with the polyclonal antibody to establish a preliminary double-antibody sandwich ELISA detection method.Determine the best concentration of capture antibody and detection antibody by checkerboard titration,and the best time of action of antigen and enzyme-labeled secondary antibody.The established double antibody sandwich ELISA method was used to detect different concentrations of standard Bo IFN-γprotein,and the sensitivity of the method was analyzed.The main experimental results are as follows:（1）The recombinant plasmid p ET28a-Bo IFN-γwas successfully constructed and expressed in E.coli BL21（DE3）.The target protein was soluble and expressed by SDS-PAGE electrophoresis analysis.After being purified twice by Ni-NTA column and CM ion exchange chromatography column,Bo IFN-γprotein with a purity of over 95%and a biological activity of1×10⁶IU/m L can be obtained.（2）The ratio of spleen lymphocytes to SP2/0 cells is 3:1,the number of single fusion cells is 3×10⁷,the alternating electric field voltage is 40 V,and the DC pulse voltage is 500 V,the cell fusion rate can reach 0.41%,PEG The cell fusion rate of the method is 0.07%.Obtained three hybridoma cell lines that can stably secrete a single anti-Bo IFN-γmonoclonal antibody:2E9,3F7,and 5B7.The highest antibody titer can reach 1:25600.All three monoclonal antibodies can recognize the standard Bo IFN-γprotein.The affinities are 2.3 L·mol^-1,3.9L·mol^-1,3.6 L·mol^-1.Three chicken polyclonal antibodies were obtained,all of which can non-specifically recognize standard Bo IFN-γprotein.（3）HRP successfully labeled 3 monoclonal antibodies,and the highest titer was 1:4000.2E9-HRP and 3F7-HRP were successfully paired with chicken polyclonal antibody No.2,and HRP-3F7 and chicken polyclonal antibody No.2 had the best pairing effect.The optimal coating concentration of No.2 polyclonal antibody is 5μg/m L,the optimal dilution factor of3F7-HRP monoclonal antibody is 1:2000,the optimal reaction time of antigen is 1 h,and the reaction time of enzyme-labeled secondary antibody is 30 min.The minimum antigen detection concentration of this method is 20 ng/m L.When it is between 40 ng/m L and 10μg/m L,a standard curve is drawn:y=0.7886x-1.0374,R²is 0.9773.The x is lgα（α:antigen concentration in ng/m L）.