Preparation And Application Of The Monoclonal Antibodies Against Infectious Bursal Disease Virus

Infectious bursal disease virus(IBDV), the etiologieal agent of Infectious disease,cause severe immunodepression in chickens,mainly at ages of3-6weeks,by destruction of lymphocytes in the bursa of fabricius. IBD had caused great economic loss to poultry industry.In recent years,emergence of variant strains and vvIBDV brought difficulties to clinical diagnosis and treatment. So it was important to develop the a quick and accurate method for preventing and curing IBD.In this study,we preparated monoclonal antibodies against infecious bursal disease virus VP2protein,established. double antibody sandwich ELISA and colloidal gold immunochromatographic assay for detecting infectious bursal disease virus. The paper contains three patrs.Part one:Establishment of the hybridoma secreting monoclonal antibodies against infecious bursal disease virus VP2proteinThree hybridomas secreting antibodies against IBDV VP2ptotein were established by fusing SP2/0with spleen cells from BALB/c mice immunized with purified IBDV, naming1D7,2A3and2C8. The Ig isotypes of mAbs were IgG2bK,IgG2bK and IgG1κ,respectively. The ELISA antibody titers of the ascites were108,106and108respectively. The three mAbs did not react with four other avian virus in the specific analysis by indirect ELISA. IFA proved that the three mAbs could react with IBDV and vp2portein specifically.In the western blot, only2C8displayed the stained protein band. The result of neutralization test showed that2A3did not have neutralizing activity, neutralization titer of1D7.2C8were106,107.Part two:Establishment of the monoclonal antibody sandwich ELISA for detecting infectious bursal disease virus The purified monoclonal antibody2C8was labeled with horseradish peroxidase as HRP antibody and the purified monoclonal antibody1D7was coated,then double antibody sandwich ELISA was establishment for detecting IBDV. The results showed that double antibody sandwich ELISA had no cross-reaction with other avian viruses. Its the lowest detection limit was1025TCID50. Repetition test showed that variant coefficients was less than6.3%.The double antibody sandwich ELISA was specific,sensitive and repetitive for diagnosis and detecting IBDV.Part three:Development of the colloidal gold immunochromatographic stip for the detection of infectious bursal disease virusThe purified monoclonal antibody2C8was labeled with colloidal gold as capture antibody and the purified monoclonal antibody1D7was immobilized on the test line as detection antibody,while a sheep anti-mouse IgG antibody was coated on the control line of the nitrocellulose membrane.Then the colloidal gold immunochromatographic strip for the detection of IBDV was assembled in regular sequence. The detection results indicated that the test strip did not react with other avian viruses.The lowest detection limitation of the strip was103TCID50IBDV. The results are consistent with different batches of the strip. The test strip was specific, reproducible and stable. The sensitivity of the test strip was as same as sandwich ELISA,100times higher than that of agar diffusion test. The result of the detection could be obtained in1-5min. The strip was more convenient and rapid for the detection of IBDV.