Development And Application Of Monoclonal Antibodies Against Canine Parvovirus

Canine parvovirus(CPV) is a small nonenvelop virus. It possesses single- stranded DNA that codes for the two capsid protein(VP1,VP2) and two nonstructural protein(NS1,NS2). The appearance of CPV is roundity or hexagon under transmission electron microscope, and it's diameter is about 20~22nm. It can withstand acid, heat and aether. Canine parvovirus(CPV) is a significant pathogen for domestic dog. It causes acute haemorrhagic diarrhoea and sometimes fatal myocarditis in young dogs. CPV affects puppies much more frequently than it affects adult dogs. In very young puppies, it can infect the heart muscle and lead to"sudden"death. Because this virus was(and is) shed in gigantic numbers by infected animals and is especially hardy in the environment, worldwide distribution of the virus rapidly occurred. It is imminent to prevent CPV for its high mortality to dogs. And seting up fast and effective detection methods is the focus of the CPV study.Conventional diagnosis of CPV is mainly based on epidemiological data and clinical symptoms to give a preliminary diagnosis. But confirming the diagnosis shoud be through the hemagglutination assay(HA) and hemagglutination inhibition (HI)test, virus isolation(VI), electron microscopy(EM) and immune electron microscopy(ME), enzyme-linked immunosorbent assay(ELISA), polymerase chain reaction(PCR), immunofluorescence assay(FA), nucleic acid probe detection methods and so on. Fluorescent antibody diagnostic technique is widely used in these laboratory diagnosise metheds of CPV. Given the specificity and high affinity of monoclonal antibodies, the test is carried out to preparare fluorescent antibody against canine parvovirus. The fluorescent antibody against canine parvovirus will provide the necessary precondition for seting up a fast, easy, highly sensitive and specifical detective method.Monoclonal antibody against VP2 protein of CPV was developed by means of the conventional protocol. By using the CPV as antigen, Monoclonal antibody(McAb) was prepared, and two positive clones, designated as 3G4 and 5H8 respectively, were obtained from hybridoma cell lines. The BALB/c mice were immunized subcutaneously with CPV, which was purified with polyethylene glycol. After being immunized three times, the mice were immunized intraveinally with equal the dose for the final immunization. Three days later, spleen cells from immunized mice were fused with SP2/0 myeloma cells. Indirect enzyme linked immunosorbent assay (iELISA) was used to screen hybridoma cells, and limiting dilution method was applied to subclone positive hybridoma cells three times, and two positive clones, designated as 3G4 and 5H8 respectively, were obtained from hybridoma cell lines. The indirect ELISA results showed that the McAbs ascites titer were 1:106,1:105; No cross reaction was found when McAbs reacted with RV,CDV,CAV and CCV. 3G4 was subtyped to be IgG1, and 5H8 was subtyped to be IgG2a; The results of Dot-ELISA and indirect ELISA indicated this two monoclonal antibodies are both against VP2 protein of CPV .One McAb(3G4) was purified by protein G affinity chromatography method and labelled with FITC. The results of absorption test and blocking test showed that the fluorescent antibody(FA) has highly specificity and sensitivity, and has no interaction with CDV, RV and CAV. The reaction activity of fluorescent antibody to F81 cells infected with CPV was test by using direct immunofluorescent assay. These results suggested that the fluorescent antibody is useful for the rapid and high specific diagnosis for CPV.