Development And Application Of Monoclonal Antibodies Against Canine Parvovirus

Canine parvovirus infection is a potent cause of infectious diseases caused by canine parvovirus,mainly hazards puppies,especially the puppies with age of 2 to 6 months,both Morbidity and mortality are high,is prevalent all over the world,is one of the most important infectious diseases that are harmful to dogs.Because China's army, police and experiment with dogs and pet dog breeding,and the increase of foreign exchange,viruses mutate constantly,make the disease prevention is more difficult to present,diagnosis and treatment of preparation of immunology has not caused in clinically misdiagnosed,and cure serious decline.Therefore,the development of the epidemic strain in CPV monoclonal antibody to improve the diagnostic accuracy and specific treatment is important.The purpose of this study is preparation anti-monoclonal antibody by separate strain of canine parvovirus that is prevalent around the city of Changchun,For clinical treatment of CPV infection in the mid-prophase and lay the foundation for to prepare CPV kit and single-chain antibody.In this study,first of all,collet the excrement of the dogs that initial clinical diagnosis of canine parvovirus infected,succeed in isolation of a strain of canine parvovirus(CPV-ch5) and proceed its hemagglutinin and hemagglutination inhibition test,Electron microscope observation,Physical and chemical properties of the sensitivity tests PCR identification,confirm this viral isolation strain is CPV epidemic strain,can use it as antigen used for immunization to prepare CPV monoclonal antibody.Secondly,CPV using monoclonal antibody preparation.CPV-ch5 cell cultures, depurated by sucrose density gradient centrifugation,used as antigen to immunize 4 to 6 week-old BALB /C female mice,and then the application of hybridoma technology,twice cell fusion,Indirect ELISA screening,Limited dilution cloning 3 times,to obtain 3 strain hybridoma cell that secret of canine parvovirus monoclonal antibody stably,named 1F3,2A9,2G5.Prepared for the experiments then carried out CPV McAb biological characterization:Indirect ELISA using three hybridoma cell culture supernatant titer followed by 1:3018,1:2138 and 1:1857,hybridoma cell injection to BALB/C mice induced ascites,Indirect ELISA to mensure ascites titer discern to achieve 3.2×10~6,3.0×10~5,1.6×10~5;Rodent monoclonal antibody used to identify subclasses of the monoclonal antibody kit for identification of sub-categories, The results showed that 1F3 is IgG-type,2A9 and 2G5 are IgM-type;3 hybridoma cell line Serial Passage for 30 generations,liquid nitrogen to survive for 6 months,detect cells activity and the ability of secretory antibody,to prove 3 hybridoma cell line can secrete CPV McAb stably;CPV McAb that 3 hybridoma cell line secret correspondence affinity are 1F3＞2A9＞2G5;Specificity mensuration showed that CPV McAb only response to CPV,but never response to CDV or other Viruses; microculture neutralization experiment showed that1:320 diluent ascites(1F3 and 2A9) protect 50%FK81 cells don't produce CPE,1F3 and 2A9 can protect FK81 cells effectively,can used to make Initial treatment of clinical canine parvovirus infection.Finally,CPV McAb used in the clinical treatment of dogs suffering from canine parvovirus infection,tearted dogs that suffering form canine paevovirus for 79 altogether,In addition to 10 cases of death,the rest of the dogs are essential to cure, recovery rate is 87.3%,played a good treatment Effectiveness.This research on CPV epidemic strains were separated and identified,as foundation,we carried out CPV monoclonal antibody preparation,in the clinical trials, the preliminary validation monoclonal antibodies have good curative effect,but application must also further clinical batch test.This experiment preparation for establishment of McAb CPV ELISA and colloidal gold specificity of diagnosis method lays the foundation.