Establishment Of A Double Antibody Sandwich ELISA And Colloidal Gold Immunochromatographic Strip For The Detection Of Infectious Bursal Disease Virus

Infectious bursal disease virus(IBDV), the etiologieal agent of Infectious bursal disease（IBD）,cause severe immunodepression in chickens,mainly at ages of3-6weeks,bydestruction of lymphocytes in the bursa of fabricius. IBD had caused great economic loss topoultry industry.In recent years,emergence of variant strains and vvIBDV brought difficultiesto clinical diagnosis and treatment.So it was important to develop the a quick and accuratemethod for preventing and curing IBD.In this study,we prepared three strains of monoclonalantibodies against infecious bursal disease virus and established double antibodies sandwichELISA and colloidal gold immunochromatographic assay for detecting infectious bursaldisease virus. There are three patrs in this study.Part one: Establishment of the hybridoma secreting monoclonal antibodies againstInfecious Bursal Disease VirusThree hybridomas secreting antibodies against IBDV were established by fusing SP2/0with spleen cells from BALB/c mice immunized with purified IBDV, designated as1D11,2G8and2E5. The subtypes of immunoglobulin were IgG1κ, IgG2bκ and IgG1κ respectively. TheELISA antibody titers of the ascites were107,106and107respectively. The three mAbs did notreact with IBV、EDS-76V、AIV-H9N2、NDV in specific analysis assay by indirect ELISA.IFA proved that the three mAbs could react with IBDV specifically. In western blot analysis,monoclonal antibody1D11and2E5displayed specific protein bands with purified IBDV inthe40kDa and32kDa, while2G8did not show specific protein bands. Neutralization testindicated that the mAb2G8had neutralizing ability to IBDV, with the neutralizing titres of104. Addition ELISA revealed that1D11and2G8recognized similar antigen epitopes, while2G8and2E5recognized quite different antigen epitopes. Part two: Establishment of a monoclonal antibodies sandwich ELISA for the detectionof Infectious Bursal Disease VirusA double antibodies sandwich ELISA (DAS-ELISA) was established using2G8ascapture antibody and2E5as enzyme-labeled antibody after purified by HiTrapTM Protein Gaffinity chromatography. The sandwich ELISA had no cross-reaction with InfectiousBronchitis Virus（IBV）, Egg Drop Syndrome Virus （EDS-76V）, H9N2subtype of AvianInfluenza Virus(AIV-H9N2) and Newcastle Disease Virus（NDV）.The minimum detectableconcentration of IBDV was102TCID50/mL. The DAS-ELISA detected the virus in the clinicalsamples, and the results were confirmed by RT-PCR with a rate of95.3%, indicating that theDAS-ELISA was a suitable method for the detection of IBDV. This method was rapid andsensitive and could be convenient for the diagnosis and detecting of IBDV.Part three: Development of a colloidal gold immunochromatographic assay for thedetection of Infectious Bursal Disease VirusThe purified monoclonal antibody2E5was labeled with colloidal gold as captureantibody and the purified monoclonal antibody2G8was immobilized on the test line asdetection antibody, while a sheep anti-mouse IgG antibody was coated on the control line ofthe nitrocellulose membrane.Then the colloidal gold immunochromatographic strip for thedetection of IBDV was assembled in regular sequence. The detection results indicated that thetest strip did not react with IBV, EDS-76V, AIV-H9N2and NDV. The lowest detection limit ofthe strip was103TCID50units of IBDV. The results are consistent with different batches ofthe strip. The test strip was specific, sensitive, reproducible and stable for diagnosis anddetecting of IBDV.