Development Of Monoclonal Antibodies Against Porcine Circovirus Type 2 (PCV2) And Immunohistochemistry Based On The McAbs And Its Potential Application In The PCV2 Antigen Detection From Lymph Nodes Of Commercial Slaughter Pigs

According to the pathogenicity and genomic characteristics, porcine circovirus (PCV) can be divided into non-pathogenic type 1 (PCV1) and pathogenic type 2 (PCV2). Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) in pigs, it is involved also in many diseases related, such as porcine dermatitis nephropathy syndrome (PDNS), proliferative necrotizing pneumonia (PNP), porcine respiratory disease complex (PRDC), reproductive, congenital trembled, enteritis and other diseases, which have taken place and spreaded in the world. The wide spreading of the diseases posed serious threat to pig industry and huge loss in many nations and regions in the world. The more serious thought was that the infection of PCV2 could impair the pig immune system and lead to the second infection of other pathogens. Then, the more huge loss was faced. Now the infection with PCV2 has contributed one of main economic burdens to the pig industry. So far, there is no sensitive, specific antibodies ready to be used in the median tissue culture infective dose determination of PCV2 and in the serological diagnosis of PCV2 infection. The purpose of this study was to develop PCV2 specific monoclonal antibodies (McAbs) and to provide sensitive, specific reagents for determination of the median tissue culture infective doses of PCV2, and to develop the immunohistochemistry (IHC) based on these monoclonal antibodies aimed at detection of PCV2 antigens in clinical specimens.To generate McAbs against PCV2, inactivated chimeric PCV1-2 viruses were used as the antigens for the immunization of Balb/C mice. Four hybridoma cell lines named 1E8,3F9,5G4 and 7G6, respectively, were screened and obtained by immunofluorescence assay (IFA) and indirect enzyme-linked immunosorbent assay (ELISA) after fusion between SP2/0 myeloma cells and spleen cells of Balb/C mice immunized with inactivated chimeric PCV1-2 viruses. The results of IFA indicated that McAb 1E8 was specific to the PCV2 antigenic epitope, McAb 5G4 reacted with both PCV1 and PCV2 epitopes, while McAb 3F9 and McAb 7G6 not reacted with PCV1 or PCV2 in the IFA. The results of ELISA indicated that McAb 1E8 and McAb 7G6 were specific to the purified PCV2 protein, while McAb 3F9 and McAb 5G4 reacted with either PCV1 or PCV2 purified protein. The ascites from McAbs were evaluated by IFA, ELISA, western-blot, IHC, virus neutralization (VN). The McAb 1E8 reacted with PCV2 in IFA at a titer of 1:3200, the McAb 1E8 and McAb 7G6 reacted with purified PCV2 protein in ELISA at a titer of 1:12800 and 6400, respectively. Western-blot analyses showed that all of four McAbs reacted with a 27.8-kDa peptide derived from purified PCV1 or PCV2 proteins. The McAbs 1E8 and 5G4 were found to behave VN activity against PCV2, with VN titers of 1:267 and 1:93 respectively. No neutralizing activity against PCV2 was discovered in respect of McAbs 3F9 and 7G6. The results of IHC indicated that both McAb 1E8 and McAb 5G4 gave a positive reaction with PCV2 antigens in tested lymph node tissues while McAb 3F9 and McAb 7G6 did not. Classes and subclasses of the McAbs were determined using a sandwich ELISA, and all of their immunoglobulin isotypes were IgGl.A total of 104 inguinal lymph nodes were collected from pigs for slaughter in Yangzhou district. Lymph nodes were detected for PCV2 by PCR and IHC, the results showed that 22.1% and 27.9% of the pigs were positive for PCV2 in PCR and IHC, respectively, while 14.4% of tested pigs experienced positive results for PCV2 both in PCR and IHC. The coincidence rate between the PCR and the virus isolation was 64.7% while the coincidence rate between the IHC and the virus isolation was 79.4%. The main microscopical findings in the lymph nodes were documented by histopathology. In total, 48.0% of the examined lymph nodes was observed lymphoid dedpletion; histiocytic infiltration presented in 36.5% of the tested lymph nodes.12.5%,4.8%,1.9%,21.2% of indistinct follicles, neutrophils in sinus system, presence of multinucleated giant cells and increasing of eosinophile granulocytes were detected in lymphoid follicles of tested lymph nodes, respectively.