| Encephalomyocarditis virus (EMCV) is a new kind of zoonoses virus. It has a very wild of host species including a variety of mammals, birds, insects and so on. Infection with EMCV can cause severe economic losses on pig production due to acute myocarditis and sudden death in preweaned piglets and reproductive failure in sows. EMCV is classified in the cardiovirus genus of the picornavirus family. It is a single-stranded positive-sense RNA virus. The virus particle, without envelope, is composed of four structural proteins including VP1, VP2, VP3and VP4, among which VP1is most antigenic. The VP1protein can induce the production of neutralization antibody and protective immunity against EMCV. In this study, ten monoclonal antibodies against EMCV VP1protein and one monoclonal antibody against VP2protein were obtained, five linear epitopes were identified in VP1protein. What’s more, a blocking ELISA method for EMCV was developed based on the recombinant VP1protein and its specific monoclonal antibody. The details as follow:1. Preparation and identification of monoclonal antibodies against porcine encephalomyocarditis virusThe purified porcine encephalomyocarditis virus (EMCV) was used to immunize BALB/c mice. The stimulated splenocytes were fused with myeloma cells of SP2/0to produce hybridomas. Eleven stable murine monoclonal cell lines producing antibodies (McAbs) against EMCV were generated and named as1D1,2A2,2A5,2B6,4B2,4B9,4E2,4F1,5A1,5A11and5G1, respectively. The titers of the eleven McAbs in the supernatant of cell culture were1:1600,1:6400,1:3200,1:6400,1:1600,1:6400,1:3200, 1:6400,1:6400,1:6400and1:3200respectively, and the titers of ascites were1:1.63×106,1:3.28×106,1:3.28×106,1:1.13×106,1:1.63×106,1:1.63×106,1:5.63×105,1:3.28×106,1:1.63×106,1:3.28×106and1:1.631106respectively. Subclass identification results showed that1D1,2A2,2A5,4B2,4B9,4F1,5A1,5A11and5G1belonged to IgGl subclass, while2B6and4E2were found to be of IgG2b subclass. All of the McAbs had k light chain. Western blot analysis indicated that1D1,2A2,2A5,4B2,4B9,4E2,4F1,5A1,5A11and5G1could react with VP1protein of EMCV,2B6was able to recognize the VP2protein of EMCV. The results of indirect immunofluorescence assay showed that all of them could react with BHK-21cells infected by EMCV, which indicating that the eleven McAbs had good specificity.2. Identification of B cell epitopes of VP1of porcine encephalomyocarditis virus41truncated VP1protein were expressed successfully as GST-fusion protein in E. coli. system to determine the epitopes of monoclonal antibodies (McAbs) against the EMCV VP1. Through the identification of ELISA and Western blot, Epitope mapping results indicated that three McAbs (6E11,7A7,7C9) specifically recognized the epitope V (2) ENAEK (7), the McAb2C8recognized A (17) DFVA (21), ten McAbs (1D1,1F3,2A2,2A5,4B2,4B9,4F1,5A1,5A11,5G1) recognized the epitope F (19) VAQPVY(25), two McAbs (1G8,3A9) recognized P (42) IGAFTVK (49) and the McAb4E2recognized F (36) YDRSSPIGAFTVKS (50). Protein sequence alignment of VP1with16derivate EMCV isolates indicated that sequences of A (17) DFVA (21) and F (19) VAQPVY (25) were conserved in all the reference strains, the epitopes of P (42) IGAFTVK (49) and F (36) YDRSSPIGAFTVKS (50) were conserved among the strains isolated from China, Whereas there were variable amino acids in different site of this region among the reference strains isolated from other countries. In addition, the epitope V (2) ENAEK (7) was relatively conserved among all EMCV strains, except for a K7â†'R7change in GXLC, D variant, EMC-B and EMC-D strains. All the results herein might promote the future investigations of the antigen structure and function of VP1of EMCV and facilitate the development of diagnostic methods for EMCV infection.3. Development of blocking ELISA for the detection of antibody to EMCV by a monoclonal antibodyA blocking ELISA method was developed based on EMCV VP1protein and its specific monoclonal antibody (7C9). The reaction conditions for each step were optimized as:The coating antigen was1μg/mL, and the coating time was12h at4℃; The sealing buffer was1%BSA, and the sealing time was3h at37℃; Sera samples diluted by1fold and incubated2h at37℃; MAb-HRP dilution was15000fold and incubated0.5h at37℃; The TMB substrate was added and incubated at37℃for5min before terminated with stop solution. The results of30EMCV negative serum samples were statistically analyzed, and the cutoff value of blocking ELISA was determined:samples with a calculated percentage inhibition (PI) of≤29.46%were treated as negative, samples presenting a percentage inhibition (PI) of≥39.10%were considered as positive and those presenting a blocking effect between29.46%and39.10%were considered doubtful. For30serum samples, the results of Serum Neutralization test (SNT) and blocking ELISA indicated that, the sensitivity of the blocking ELISA was better than SNT, and the coincidence of the two methods is90%. In the repeatability test, both the intra-batch and inter-batches coefficient of variation were less than10%. Recombinant antigen had no cross reaction with the antibodies to FMDV, PRV, PRRSV, CSFV and PCV2. This method was used to detect580clinical serum samples from different areas of China, resuming a positive rate of41.03%. These results suggested that the established blocking ELISA is specific, sensitive and reproducible. And it could be used to detect the antibody to EMCV in the future. |
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