Establishment And Preliminary Application Of Hybridoma Cell Lines Secreting Monoclonal Antibodies Against Newcastle Disease Virus

The Newcastle Disease viruses (NDV) of strains F48Eg and N-79 prepared from embryonated eggs were purified by one differential centrifugation. Balb/c mice with an age of eight weeks were immuned with the purified NDV antigens. Splenocytes of vaccinated Balb/c mice were fused with mouse myelomas cells strain NSO by 50% PEG 4000, and selected with HAT medium. Hybridoma clones secreting monoclonal antibodies against NDV were screened by indirect ELISA ,and positive clones were subcloned three times by limited dilution. One clone which can stably secrete McAb was obtained and designated cell strain FG1. The average number of chromosome of the hybridoma cell strain FG1 was 93.7. The ELISA titers of monoclonal antibodies in cell supernatant and ascite were 1:512 and 1:64000 respectively. Western-blotting proved that FG1 McAb specifically recognizes hemagglutinin neuramidinase (HN) proteins of NDV . FG1 McAb has the hemagglutination inhibition(Ffl) activity and the HI titers of supernatant and ascite is 31og2 and 100 × 31og2 respectively. TheMcAb shows no cross reactivity to other avian viruses of chicken including Infectious Bursal Disease virus(IBDV), Infectious Laryngotracheitis virus(ILTV), Infectious Bronchitis virus(IBV), Avian Influenza virus(AIV) and Egg Drop Syndrome virus(EDS). Strain FG1 hybridoma cell can also stably secrete McAb after tweenty serial passages and freezing and revivfmg three times. Sandwich ELISA for detecting NDV were developed by using monoclonal antibody strain FG1. The optimal coating concentration of rabbit-anti-NDV hyperimmune serum was 1:1600, and the working, concentration of HRP-McAb was 1:200. The sensitivity to NDV of the sandwich ELISA was 300ng/ml. The sandwich ELISA is specific and higher sensitive to NDV and has no cross reaction to other avian disease viruses including IBDV, IBV, ILTV, AIV and EDS-76. The McAb-mediated sandwich ELISA established in the present study should be further consummated to suit producing rapid diagnosis strip by combining gold-labeled chromatography to be applied in the clinical practice.