Concentration And Purification Of Anti-canine Parvovirus Antibody

Canine parvovirus（CPV）is the causative agentof acute hemorrhagic enteritis and myocarditis in dogs with high pathogenicity and mortality and resulted in heavy lossin breeding dog industry in C hina.CPV-2 was first recognized in 1977 and since then it has been well established as an enteric pathogen of dogs throughout the world and has been gradually replaced by CPV-2a and CPV-2b,which continuously emerged antigenic mutants.CPV-2c,which was discovered in 2000,is characterized with increased pathogenicity in dogs and expanded host range and even poses a threat to the survival of wild animals.Vaccination is an ideal approach to control CPV,but many factors including maternal antibodies may lead to immune failure.Thus once the dogs were infected,support therapy and symptomatic treatment combined with hyper-immune serum against CPV are applied.In the present study,expanded culture of strains CPV-S5（New CPV-2a）and CPV-1401（New CPV-2b）,which were isolated in Guangdong area and preserved in our lab,in F81cells were performed under the optimal growth conditions,respectively,and viral solution with HA titer 2¹⁴ were obtained through the compact tangential flow ultrafiltration.The virus concentrates were then mixed withβ-propiolactone（β-propiolactone:virus solution=1:2000）,respectively,and the mixtures were inactivated at 4℃for 24 hours,followed by incubation in a 37℃water bath for 2 hours.After validation,the virus concentrates were mixed with the water-soluble adjuvant MONTANIDE GEL at a ratio of MONTANIDE GEL:inactivated virus concentrate=1:9,respectively,and sterility physical and chemical characterization of these two vaccines met standards.Subcutaneous injection of 1mL of each vaccine was administered simultaneously to CPV HI negative dogs and booster immunization was given 28 days after first immunization.O n day 10 postsecond immunization,blood was collected via the carotid artery and mixed and the serum was sepreated.The HI titer of the hyper-immune serum was detected and the antibody level was 1:3072.This hyper-immune serum was preliminarily treated using20%-50%saturated ammonium sulfate to remove most of the protein impurities.The optimum test conditions for the third step were as follows:33%saturated ammonium sulfate,with p H=7.0.Desalination was followed with a 5mL HiTrap?Desalting prepacked column and then the compact tangential flow ultrafiltration.Sterility and physical and chemical characterization of this preparation complied with safety standards,with a HI titer of 1:10240.No adverse reaction was found after this preparation was injected into dogs.