Preparation Of Monoclonal Antibody Against Feline Parvovirus And Establishment Of The Virus Detection Method

Feline parvovirus is a highly contagious viral disease of kittens with a high mortality caused by feline panleukopenia virus (FPV), a member of parvoviridae. The FPV is characteristed by sudden bipolar high fever, vomiting , diarrhea, generous decreases of white blood cell and hemorrhagic enteritis. So FPV is the most important infectious diseases of cat. Not only can FPV infect all members of the cat family, but also other wild animals are susceptible to infection, such as tiger , raccoons, coatimundis, ringtails and so on, which has posed a serious threat to the economic animals and wildlife. Felid of all ages can be infected but mainly in the kitten of one years old, particularly 2 to 5 months old. FPV is one of the pathogens that give a great threat to China's special economic animal breeding and wildlife conservation The serological survey on antibodies against FPV showed that infection of FPV occurs worldwide, and there were high positive rates in the aera of wild animals in China. The clinical diagnosis of FPV is difficult because of the broad spectrum of signs. Therefore, FPV on the clinical and laboratory diagnosis need to establish a simple, rapid, specific and sensitive detection methods.Monoclonal antibodies (McAbs), however, assure a reliable source of antibodies and very often achieve greater sensitivity and specificity than polyclonal antibodies. In recent years, Monoclonal antibody technology has been widely used in the diagnosis of FPV. In order to provide more specific and standardized reference antibodies for the diagnosis, pathogenesis and epidemiological studies of FPV, we prepared anti-FPV monoclonal antibody. The FPV was purified through polyethyleneglycol 6000 precipitation and differential centrifugation from the infected cell cultures, then inject the BALB/c mice with the purified FPV. An indirect enzyme-linked immunosorbent assay(ELISA) was developed to detect specific antibody to infectious Feline panleukopenia virus(FPV) in serum with prepared diagnostic antigen. Select the potency of the highest mice was facilitatory immunized after they were to strengthen the immune about four times. The spleen B lymphocytes with myeloma cells (SP2/0) to carry out the integration after 3 days immunization. The mediums of hybridoma cells were screened by indirect enzyme-link immunosorbent assay. Hybridomas whose supernatants show stronger positive were subcloned by limited dilution methods 3~4 times, until the positive rate come up to 100%. And then subcloned and expanded training, through repeated cryopreservation, the recovery, we obtained two stable Feline parvovirus antibody secretion of hybridoma cell line, named for the 5G9, 1A2.Throngh the identification of monoclonals obtained from the two hybridoma cell :①Both 5G9's and 1A2's subtypes are IgGl;②We will get the integration of cells into mouse peritoneal and used to prepare the ascites.The titers of cell supernatant and hybridoma cells were 10×29,10×27 and100×28,100×26;③Chromosome analysis showed an average of 96 pairs;④The specificity assay and cross-reaction test of monoclonal antibody proved that these McAbs were specificity and broad-spectrum. The highly effective and specific Mabs plays an important role in diagnosis, treatment and prevention of the infection of Feline panleukopenia virus.HRP marked by a post-1A2 McAb, with another strain 5G9 McAb secreted by the establishment of double-antibody sandwich ELISA detection methodsdetection of FPV. The test note for the 5G9 and 1A2 against FPV need different epitope of McAb cell line. The use of 5G9, 1A2 for FPV anti-ELISA detection method, research by ELISA conditions were optimized respectively.According to exploring and optimizing the condition such as the best dilution of Concentration of monoclonal antibody-coated is 1:5000, and the best coating is 4℃overnight; the best solution is 1% BSA and the best closing time is 37℃1h; the best dilution of antigen is between 1:40 and 1:50; the best dilution of HRP-McAb is 1:5000 and the best reaction time is 37℃1h; substrate best time is between 10min and 15min and so on.Finally, the double antibody filled ELISA detection of FPV was preliminary established. he detection Method Established by double-antibody sandwich ELISA can test for FPV, which lays a significant foundation for the next step of the development of diagnostic test kits.