Preparation Of Monoclonal Antibody Against Nucleocapsid Protein Of Bovine Parainlfuenza Virus3, Establishment And Application Of A Solid-phase Blocking ELISA

Bovine parainfluenza virus3(BPIV3) is a member of Respirovirus genus within the familyParamyxoviridae,and also named as shipping fever virus. The infection of BPIV3causes clinical dieasesuch as enzootic pneumonia and atypical interstitial pneumonia, and also causes huge economic lossesto cattle industry every year. The BPIV3was firstly isolated by Reisinger et al.(1959) in the USA. TheChinese BPIV3strains of the new genotype C were firstly isolated by Zhu et al.(2011) in2008inShandong Province, China. The research on BPIV3is still at the primary stage in China. There are notany commercial vaccine and assay kit in China.Nucleocapsid protein（NP）has the most quantity in BPIV3virion and it is highly conserved. In thisstudy,6to8weeks-old BALB/c mice were immunized with purified recombinant NP(rNP) expressed byE.coli. After four immunization, the spleen cells of the immunized BALB/c mice were fused with themyeloma cells Sp2/0. One positive hybridomas (5E5) was obtained after screening by indirect ELISAbased on BPIV3. The MAb had ascites titer of2×106and1×105as detected by rNP protein and BPIV3itself, respectively. The MAb secreted by hybridomas5E5had highly reactivity and specificity inindirect ELISA, western blotting, IFA and identified as IgG1with a light chain of κ by indirect ELISA.Five days after the guinea pigs were intranasally inoculated with BPIV3,5E5MAb could react with thelung tissues collected from the inoculated guinea pigs with BPIV3by immunohistochemistry test butthere was no reaction with the control group. This indicates that BPIV3can be detected in animalsinfected with BPIV3in immunohistochemistry with the5E5MAb. The5E5MAb could be used toestablish diagnosis method for BPIV3and study the structure or function of NPof BPIV3.ELISA plates were coated by BPIV3which were diluted into1:200with carbonate buffer saline(CBS). The bloking epitope is which5E5MAb recognizes. The optimum reaction conditions ofSPB-ELISA were confirmed by square titration test.A total of120bovine sera which had beenconfirmed by neutralization test were used as the standard reference sera. The cut-off point ofSPB-ELISA was ultimately identified as30%of serum inhibition, the weak positive was from30%to40%of serum inhibition. We can regard it is positive when the serum inhibition is more than40%andnegative when the serum inhibition is lower than30%. SPB-ELISA was used to detect positive BPIV3,IBRV, BVDV bovine sera. Consequently, only positive BPIV3bovine serum could be detected by thismethod. It indicated that this method had a good specificity. The result of sensitivity test indicated thismethod could detect the sera completely when they had a neutralizing antibody titer more than1:32.The CV of intra-repeatability assay and inter-repeatability assay were both lower than10%indicatedthat this method had a good repeatability.100positive sera which had been tested by virusneutralization test were used for SPB-ELISA. There were93positive sera of all. It indicated that thismethod had a good coincidencerate. The SPB-ELISA was used to detect231sera from the immunizedcattles and54sera from surrounding areas in Harbin. Consequently, there were220positive sera of231 sera and34positive sera of54, respectively. SPB-ELISA has been widely used to detect sera of thepathogenic virus for its high efficacy and convenience. The establishment of SPB-ELISA in this studycan be used for the surveillance of BPIV3epidemic in cattle.