Preparation Of Monoclonal Antibodies Against Newcastle Disease Virus And Establishment Of Double Antibodies Sandwich ELISA

Newcastle disease is an acute and highly contagious disease chickens and avian caused by Newcastle disease virus. At present new castle disease is listed in an infectious disease by world Animal Health Organization (OIE). It is one of the most important diseases of world aviculture, because it is highly contagious and high mortalities and can cause tremendous economic losses to the poultry industry, especially to chicken industry. Now and future our country face to prevent and control new castle disease is a tough mission, therefore, it is very important to investigate Newcastle disease diagnosis kit.Methods, to establishment of new castle disease detection method, we must first prepare anti NDV monoclonal antibodies. In this study, we using the purified NDV antigen to immune BALB/c female mice with age of 6 to 8 weeks, after the basic immunization and booster immunization, blood samples were obtained in mice to test antibody titers. Spleen cells from immunized mouse with SP2/Ocells induced fusion in 50% PEG. McCabe cell strains were determined by both hem agglutinin inhibition (HI), Immunoflurescence assay (IFA), indirect ELISA coated with purified virus. Cloning of positive hybridoma cell lines by limiting dilution, after detecting and screening, at last gained 5 positive specificial McAbs of NDV, they are named respectively 1H10、2H3、4E4、5B11 and 5E9. Then BALB/c female mice were inoculated intraperitoneally with hybridoma cells, gathered ascites after 1 week, centrifuged and collected the supernatant. The 5 specificial McAbs of NDV ELISA titers all to fill the establishment of Double Antibodies Sandwich ELISA.The 5 positive hybridoma cell lines producing monoclonal antibodies had satisfactory thermal stability.The serum antibody purified through caprylic cid precipitation method, protein levels between 2.46mg/mL and 4.9mg/mL. The results of ELISA test proved that the 2 specificial McAbs of NDV did not cross react with AIV、MDV、REV、IBV、IBDV and EDS-76. These 2 monoclonal antibodies laid the foundation of the newcastle disease virus investigate.In this study, the double antibody sandwich ELISA method was constructed on the basic of specificia monoclonal antibody.We utilized the 2H3 as the coating antibody, screened the testingantibody pair horseradish peroxidase-4E4(abbreviation:4E4-HRP) using one HRP-labeled strain as capture antibody and another strain of monoclonal antibody as detecting antibody for detecting specific newcastle disease virus. the optimum conditions were achieved,McAb was diluted for 1:1600, HRP-labled antibody was diluted for 1:320.Coating antibody Incubate overnightand and HRP-labled antibody optimum reaction conditions is 37℃,45min.AIV、MDV、REV、IBV、IBDV and EDS-76 were detected by the sandwich ELISA and the result showed that there was no cross reaction.The detecting method of NDV had excellent specialty, high sensibility, has a wonderful application to future in detection of NDV.