| Bovine parainfluenza virus type 3(BPIV3), infectious bovine rhinotracheitis virus(IBRV), bovine viral diarrhea virus(BVDV),and bovine respiratory syncytial(BRSV) are recognized as pathogens of the most important of the known viral agents for cattle and have also been associated with bovine respiratory disease complex(BRDC) which continues to cause serious economic losses for the global cattle industry annually. BPIV3 is one of the most important pathogen associated with BRDC and a member of the genus of Respirovirus of the family Paramyxoviridae. BPIV3 is an enveloped, non-segmented negative-strand RNA virus and also referred as Shipping fever virus. So far, three genotypes A, B and C of BPIV3 have been described on the basis of genetic and phylogenetic analysis. The noted clinical signs are coughing, anorexia, pyrexia, nasal and ocular discharges, dyspnea and sometimes diarrhea. BPIV3 infection also contributes to pneumonia, bronchopneumonia and consolidation in the anterior ventral aspects of lung lobes for young and adult cattle. The BPIV3 is widespread among cattle around the world since it was firstly isolated and reported in the USA. The genotype C of BPIV3 was isolated in Shandong Province, China in 2008 for the first time(Zhu et al., 2011). So far, the research on BPIV3 has been done, but no commercial vaccine and diagnostic reagents are available for BPIV3 in China.In this study, six and eight-week-old female Balb/c mice were immunized with BPIV3 SD0835. After three immunizations, the spleen cells of the mice were fused with mouse myeloma SP2/0 cells using PEG/DMSO solution. Hybridomas were cultured in HAT selection medium and screened by indirect ELISA coated with recombinant NP of BPIV3 c strain SD0835. After three times of subcloning, seven hybridomas(2G7,6F8,6H8,7G5,7G8,7G9 and 7H5) producing monoclonal antibodies(mAbs) were got, and the ascites titer ranged from 1×105 to 1×107. The mAbs own a good reaction and specificity demonstrated by ELISA, Western blotting, and indirect immunofluorescence assay(IFA). The mAb subtype identifications indicated that all of the mAb subtypes are IgG1/κ. To identify antigenic determinants of BPIV3 NP, a panel of mAbs was tested against a series of overlapping recombinant NP fragments expressed in E.coli. Then two antigen epitopes were identified with the five mAbs(6F8, 7G5, 7G8, 7G9, and 7H5). The mAbs 7G5 7G8, 7G9, and 7H5 were reactive with the epitope 407FYKPTGG413. The second epitope 428ESRGDQDQ435 was reactive with mAb 6F8.At the same time, we also established a double antibody sandwich(DAS) ELISA with the 3% gelatin and 5% skim milk as the blocking solution, the mAb 6F8 as coating antibody, and 7G9-HRP as the detecting antibody. The cut-off value of DAS-ELISA was determined from optical density(OD) values measured at OD450 value of 30 identified negative samples for BPIV3 as 0.252. When the OD450 value was greater than 0.252, the sample was judged as positive; instead, the sample was considered as negative. The specificity test showed that the DAS-ELISA could react only with BPIV3, and did not react with IBRV, BVDV, BRSV, and bovine adenovirus type 3(BAV-3). The minimum detection of DAS-ELISA was 103.36/0.1ml of BPIV3. Furthermore, 23 positive samples and 11 negative samples including nasal swabs and lungs identified by RT-PCR were tested by DAS-ELISA, and the results indicated that the coincidence rate was 97%.In summary, we prepared seven hybridomas secreting mAbs against BPIV3 NP, and two antigen epitopes were identified by mAbs(6F8, 7G5, 7G8, 7G9, and 7H5). Meanwhile, a double antibody sandwich ELISA was established with mAbs 6F8 and 7G9. The DAS-ELISA showed a good performance in specificity test, sensitivity test, and coincidence rate test with RT-PCR. Thus, the DAS-ELISA might be used to detect the BPIV3. Also, the results in this study would be useful for studying biological structure and function of the NP protein of BPIV3 and for studying the pathogenesis of BPIV3. |
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