| Porcine circovirus type2(PCV2) is one of the most important economically viral pathogens severally effected the worldwide swine producing countries. The virus infection is associated with a number of collection of disease syndromes in pigs known as porcine circovirus diseases (PCVD) or porcine circovirus-associated diseases (PCVAD). PCV2is a small, non-enveloped virus with a single-strand circular DNA genome, which contain four open reading frames, ORF1～4. Among them, ORF1～3encode the non-stucture proteins of the virus, and ORF2encodes the major structural protein, Cap protein, which is the main antigenic determinant protein and plays an important role in the process of anti-virus infection. In this study, the purified PCV2were used as immunogen for Balb/c mice, and three McAbs specific to PCV2Cap protein were obtained successfully. By using one of the three McAbs and anti-PCV2polyclonal rabbit antibody, we developed a sandwich ELISA method for detecting PCV2antigen, which would be useful for PCVAD dignosis and functional study of viral protein. The main contents of the research were as following:1. Preparation and identification of McAbs against PCV2To prepare monoclonal antibodies (McAbs) against porcine circovirus2, BALB/c mice were immunized with purified PCV2, and3stable hybridomas were produced, named as3E5,3E6and5A6, respectively. The titers of the McAbs to PCV2were confirmed steadily during20passages of the cell clones in vitro. The titers of the McAbs in supernatant of cell culture were1:12800,1:1600and1:6400for3E5,3E6and5A6, respectively. Correspondingly, the titers in ascites were1:6400000,1:640000and1:800000. All McAbs belonged to IgG2a subclass and had κ chain. Results of indirect immunofluorescende assay (IFA) showed the McAbs could react strongly with PCV2-infected PK-15cells. The McAbs of3E5and3E6but not5A6were confirmed to have the neutralizing ability to PCV2in the neutralizing assay. Western blot analysis showed that all McAbs were able to react with PCV2Cap protein. And some of residues42to50at the N terminus of the capsid protein are required for binding of all the three McAbs. These McAbs would be useful tools for the molecular diagnostic and antigen epitope analysis of the virus.2. Development and preliminary application of a sandwich ELISA for detection of PCV2In this study, a sandwich ELISA method was developed by using an anti-PCV2McAb as capture antibody and polyclonal rabbit antibody against PCV2as detective antibody. The reaction condition for each step were optimized as follows:The coating McAb was diluted to2μg/ml, and incubated for2h at37℃;5%free-fat milk was optimized as the blocking solution and was blocked for1h at37℃; antigen samples was diluted by2fold at least and incubated for2h at37℃; PcAb against PCV2at a dilution of1:8000was incubated for1h at37℃; goat anti-rabbit IgG-HRP was at a dilution of1:10000in blocking solution and incubated for0.5h at37℃; The TMB substrate was added and incubated at37℃for10min before terminated with stop solution. The cut-off value of sandwich ELISA was determined as follows:the OD450nm value of samples more than or equal to0.209were considered as positive and the OD450nm value less than0.191was considered as negative and the others were suspicious. The ELISA method was proved of having no cross-reaction with antigens of PCV1, PEDV, PCMV, PRRSV, EMCV and Hps. The limit of detection of PCV2was102.59TCID50/mL. The variation coefficients of intra-batch and inter-batches were less than10%. By dilution of PCV2standard, a standard curve was made in the working range from390.6to25000TCID50/mL which had an correlation coefficient of R>0.99, which could be used for quantization of PCV2. And the results of quantifying PCV2doses of6cultured samples by sandwich ELISA method were in accordance with IFA. Primarily application of this method showed that it could be used for the detection of inactive PCV2by β-propiolactoneas, the recombinant Cap protein of PCV2expressed in baculovirus expression system, but not inactive PCV2by formaldehyde.And the clinical detecting results of ELISA showed that it were positive correlated between the PCV2doses in lymphoid tissues from the field piglets and its PMWS clinical signs. So, it indicated that this sandwich ELISA provided a convenient tool for quantization of PCV2and PCVAD diagnosis in the future. In summary, these McAbs had a good reaction with PCV2in Western blot, IFA and virus neutralization assay, which could be as a diagonisis agent for PCV2antigen. The sandwich ELISA developped in this research had a well quaility of specificity, sensitivity and repeatability and could be used as a convenient tool for PCVAD diagnosis and epidemiological investigation in the future. |
