Preparation And Application Of The Monoclonal Antibodies Against Canine Parvovirus

canine parvovirus disease (CPVD) is a kind of highly contagiously disease caused by canine parvovirus (CPV). CPV can spread across the dogs of all ages and gender, the sick dogs are characteristics of hemorrhagic enteritis or purulent myocarditis. Puppies’ mortality rates is as high as 70%, the adults is relatively low but can detoxificate to the environments continually. Furthermore, CPVD is one of the most severe infectious diseases in canine farming. So it is important to develop rapid and specific detection methods for the prevention of CPVD. In this study, we preparated the monoclonal antibodies (mAbs) against canine parvovirus. Based on the mAbs, sandwich ELISA and colloidal gold immunochromatographic assay for detecting canine parvovirus were established. The paper contains four parts:Part Ⅰ:Isolation of canine parvovirus and analysis of the VP2 geneJS12 was isolated from a dog with immunoprophylaxis defeat. It could agglutinate porcine’s erythrocytes and was stable to the treatments of 56℃ for 1 h, pH 3.0 and lipid solvents. Sequence analysis showed that the VP2 gene is composed of 1755 nucleotides, encoding 584 amino acids. Clustering analysis indicated that the homology of the VP2 gene were 98.9%-99.6% in nucleotide and 98.3%～99.7% in amino acid based on the sequences of CPV isolates available in GenBank. The phylogenetic tree based on VP2 gene showed that 16 Chinese isolates were in different groups and JS12 was in the same subgroup with a Korea strain. Comparing the structure of JS12 with the vaccine strain, there were nine amino acid residues variation, six located on the capsid protein surface, five located in the antigenic area. SDS-PAGE analysis indicated that the recombinant VP2 protein was 67.0 ku. Western blot showed that the recombinant VP2 protein could be recognized by CPV positive serum.Part II:Establishment of the hybridomas secreting monoclonal antibodies against canine parvovirusEight hybridomas secreting antibodies against canine parvovirus (CPV) were established by fusing SP2/0 with spleen cells from BALB/c mice immunized with purified CPV, naming 1A4.1G3.1G8、1E4.2H2、3A5.3F3 and 3H1. The Ig isotypes of mAbs were IgG2bκ、IgG2bκ、IgGlκ、IgGlK、IgG1κ、IgGlκ、IgG2aκ and IgG1k. Indirect immunofluorescence assay (IFA) showed that the eight mAbs could combine with natural CPV specifically. In western blotting, mAb-1A4 and mAb-1G8 could react with the VP2 protein of CPV. Addition ELISA revealed that the eight mAbs could recognize different antigen epitopes of CPV. Neutralization test indicated that mAb-3A5 had neutralizing ability to CPV, with the neutralizing titers of 210 and 106 in cultural supernatant and ascites respectively.Part III:Establishment of the monoclonal antibodies-based sandwich ELISA for detecting canine parvovirusThe purified mAb-2H2 was labeled with horseradish peroxidase as HRP antibody and the purified mAb-3A5 was used to capture CPV antigen, then the double antibody sandwich ELISA was established for detecting CPV. The results showed that the sandwich ELISA had no cross-reaction with F81 cells, canine distemper virus (CDV), canine adenovirus type-1 (CAV-1), canine parainfluenza virus (CPIV) and canine coronvirs (CCV). The Sensitivity of the sandwich ELISA was 102TCID50/mL. The variation coefficients of the intra-assay and inter-assay were less 6%. The sandwich ELISA was specific, sensitive and stable for the diagnosis of CPVD.Part IV:Development of the monoclonal antibodies-based colloidal gold immunochromatography strip for the detection of canine parvovirusThe monoclonal antibodies-based colloidal gold immunochromatography strip was developed for the detection of canine parvovirus (CPV). The mAb-2H2 conjugated with colloidal gold was used to capture CPV antigen and mAb-3A5 to bind colloidal-gold mAb-CPV complexes at a test (T) line on the nitrocellulose strip. A downstream control (C) line of goat anti-mouse immunoglobulin G (IgG) antibody was used to capture excess free colloidal-gold conjugated 2H2 to validate test performance. The detection results indicated that the strip was specific to CPV and the detection limits of CPV was 102.5 TCID50/mL, which was equivalent to the sandwich ELISA established by using the same monoclonal antibodies. The monoclonal antibodies-based colloidal gold immunochromatography strip is more convenient, rapid and simple for the clinical diagnosis of CPVD.