| Canine distemper virus(CDV), as measles virus and peste-des-petits-ruminants virus,belongs to genus Morbilli virus, family Paramyxoviridae. CD is one of the most important virusdisease in dogs,and spreads throughout the world. The host of CDV ranges wildiy including allcanine animal (dog, dingoes, foxes, wolves, coyotes, jackals), the raccoon family(raccoons, longkiss raccoons, pandas), mustelidae (weasels，ferrets, mink, badgers, skunks, otters) and most ofthe members of the cat family(lion, leopard, cheetah, tiger). CDV is highly contagious andseveral kinds of forms,the most common acute type which has two stages elevated bodytemperature (dual phase heat)，with severe in the second phase of the heat of white blood cellsreduce disease, and respiratory symptoms or gastrointestinal symptoms.In subacute neurologicalsymptoms, it will causes a central system of permanent sequelae. The dangers always exist todomestic pets, fur animals, and many wild animals also have some adverse effects when itoutbreaks. Therefore,it needs a quick, simple and efficient method for the diagnosis of thedisease. The research of CDV monoclonal antibody and the colloidal gold test strip has greatsignificance to the diagnosis and treatment of canine distemper.This experiment is composed of the four parts: First, the purification of canine distemper.After CDV virus to cell debris, the virus was concentrated and dialysis with using PEG6000precipitation, then purified by Sephrose4FF gel column chromatography purification.Electronmicroscopy (sem) observation, the purification methods can obtain canine distemper. Theprotein content up to2.17mg/mL by the determination; Second, production and characterizationof monoclonal antibodies against canine distemper. Using the canine distemper virus immunizedBalb/C mice. PEG fusion method to SP2/0myeloma cells and immune mice spleen cellfusion.The stable secrete antibody hybridomas were obtained after several times of cloning andfiltered by indirect ELISA method, which were named062-1,062-2,respectively. Used toestablish ELISA method to detect the titer, clear on those strains hybridoma supernatant titerswere1:105，ascites titers were1:106, respectively. Subtype identification results accorded themonoclonal antibodies were belong to subtype IgG1. Indirect immunofluorescence test resultswere shown that these monoclonal antibodies have great specificity.Third, preparation andbiological characteristics identification of canine distemper polyclonal antibody. Rabbits wereimmunized with virus, then blood serum was obtained from jugular vein. Using the establishELISA method to determined the titer as follows:1:107. Indirect immunofluorescence test resultis the polyclonal antibody has false positive.Fourth, the preparation and preliminary applicationof colloidal gold test strip for canine distemper. Application of sodium citrate reductionpreparation of colloidal gold particles and tag monoclonal antibody, for detection of caninedistemper colloidal gold immune chromatography test paper, and the preparation of technicalappraisement to detect CDV colloidal gold strip with good specificity.Colloidal gold test strip obtained in this experiment richer distemper detection method,monoclonal and polyclonal antibody obtained distemper for future research to provide goodconditions. |
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