Isolation Of A Canine Parainfluenza Virus(CPIV) And Preparation Of Monoclonal Antibodies Against CPIV

Canine Parainfluenza Virus(CPIV) is one of the main pathogens of canine respiratory disease. Binn et al. isolated a strain of CPIV for the first time from diseased dogs with respiratory syndrome by using Madin-Darby Canine Kidney(MDCK) cells in 1967.A CPIV strain named CPIVyz0506 was isolated from a one-year-old Pomeranian dog died with high fever, cough, sneezes, pursiness in June, 2005. The biological properties of the strain were determined, and the results indicated that the virus were sensitive to chloroform, and could agglutinate red blood cells coming from dog, chicken and the'O'type of human origin. The polymorphism of the virus was observed by electro-microscope with a diameter of 80nm²00nm. The virus possessed a set of 6 structural proteins, and produced CPE in Vero cells. CPIV could be inspected from snivel five days later after the dog infected the isolate by RT-PCR.The 8-week-old female BALB/c mice were immunized with CPIVyz0506 emulsified in Freund's complete adjuvant for 5 times. After immunization, spleen cells from immunized mice were fused with SP2/0-Ag-14 myeloma cells. Four hybridomas secreting monoclonal antibodies(McAbs) against CPIV were prepared after screening by indirect enzyme linked assay(ELISA) and cloning by limiting dilution. These four hybridomas, named 4F3B6, 5B4C9, 4G7F4, 4C9D8, were able to screte McAbs stably. The indirect ELISA titers of mice ascites of 4F3B6, 5B4C9 and 4G7F4 were all above 1:10⁶. The indirect ELISA titer of mice ascites of 4C9D8 was above 1:10⁴. The immunoglobulin subclass of 4C9D8, 5B4C9 and 4G7F4 were IgM. The immunoglobulin subclass of 4F3B6 was IgG1. The result of Western-blot indicated that 4G7F4 was specific for the protein band about 60kD(hemagglutinin-neuraminidase), and 4F3B6 was specific for the protein band about 51kD(fusion protein), 5B4C9 and 4G7F4 didn't react with CPIVyz0506 protein after change. The result of indirect immunofluorescence experiment indicated their sensitivity of the four McAbs, they all showed strong specific perinuclear fluorescence in CPIVyz0506 infected cells. The result of indirect ELISA indicated that 4C9D8 could react with NDV, CDV, while 4F3B6, 5B4C9 and 4G7F4 were specific for CPIVyz0506. LgPD₅₀ of 4F3B6, 5B4C9, 4G7F4 and 4C9D8 were -3,-3.3,-2.7,-2.3 respectively.