Development Of The Elisa Methods For The Detection Of Canine Distemper Virus

Canine distemper(CD), which is caused by canine distemper virus(CDV), is an acute, highly contagious and fatal disease. CD generally occurs worldwide, and frequently in China. In recent years, CDV has infected a growing number of animals, and there were even cases of atypical symptoms in some areas. Although we already have vaccines, we can not stop its outbreaks in some areas. So it is important to develop rapid and specific detection methods for the prevention and treatment of CD. As a specificity, high sensitivity, easy to operate detection technology, ELISA has a very good application prospects for detecting CDV. In this study, two ELISA for detecting CDV were established:a sandwich ELISA based on the monoclonal antibodies and an indirect ELISA using recombinant N protein as antigenthe monoclonal antibodies.Part one:Establishment and application of the monoclonal antibodies-based sandwich ELISA for detecting canine distemper virusIn order to detect CDV, we established the sandwich ELISA based on the monoclonal antibodies against canine distemper virus (CDV). The purified monoclonal antibody (McAb)1D9was used to capture CDV antigen and McAb1E7conjugated with Horseradish peroxidase (HRP) as trace antibody in the sandwich ELISA. Reaction conditions were as follows. The amount of the coated antibody was at2.4μg/mL, and the amount of the trace antibody conjugated with Horseradish peroxidase (HRP) was at a dilution of1:4000, and the blocking reagent was10%calf serum. The sandwich ELISA had no cross-reaction with canine parvovirus(CPV), canine parainfluenza virus (CPIV), Canine adenovirus type-1(CAV-1) and Canine coronvirs (CCV). The Sensitivity of the sandwich ELISA was102TCID50/mL. The variation coefficients of the intra-assay and inter-assay were less10%. There were80serum samples detected by this method and RT-PCR, and the agreement ratio between the them was90%. The sandwich ELISA established by our laboratory was specific, sensitive and stable, and it provided an effective method for the detection of CD.Part two:Establishment and application of an indirect ELISA for detection of antibody against Canine Distemper VirusAn indirect ELISA was established for detection of antibodies against Canine Distemper Virus (CDV), using the N protein expressed in recombinant baculovirus system as coating antigen. The results of optimizing reaction conditions were as follows. The amount of the recombinant N protein coated antigen at4.75μg/mL and incubated for overnight at4℃. The blocking reagent was10%calf serum. The serum dilution was100times.The rabbit anti-canine IgG HRP conjugate was added at1:2000and incubated for1h at37℃.The cut-off value was0.148(OD450nm)-The OD450nm values were still higher than the cut-off value, when the standard positive serum dilution of1:3200. The variation coefficients of the intra-assay and inter-assay were less10%. There were128serum samples detected by this method and commercial ELISA kit, and the agreement ratio between the them was93.8%. The indirect ELISA was sensitive and stable for the diagnosis of CD.