Study Of The Monoclonal Antibodies-based Sandwich ELISA And Colloidal Gold Immunochromatographic Strip For The Detection Of Canine Distemper Virus

Canine Distemper (CD) which caused by the canine distemper virus (CDV)，is highlycontagious,infectious disease, the incidence of mortality up to90%. Although the past20years has been inoculated susceptible animals with CDV vaccine widely, but with the CDVstrain mutating, a large number of the outbreak of CD or epidemic of CD in CDV attenuatedvaccine-induced immune dogs have been reported, CD is still one of the major epidemicsharm dogs, fur economy of animal and wildlife.CDV belongs to the paramyxovirus Measlesvirus, for unsegmented piece envelopednegative-sense linear RNA viruses, only one serotype. Virus particles by the nucleocapsidprotein (N), hemagglutinin protein (H), the fusion protein (F), large protein (L), the matrixmembrane protein (M) and phosphoprotein (P). N protein is the most abundant and the mostconservative immunogenic protein. H protein and F protein play an important role in CDimmunization and prevention. CDV has extensive tissue cell tropism and can infect a varietyof cells and tissues, which ecotropic epithelial cells and lymphocytes strongest, damage thebody’s humoral and cellular immunity seriously, leading to immune suppression, mixedinfections caused by other bacterial or viral. Therefore, early and rapid detection and accuratediagnosis is the key of prevention and control.Simple, rapid, sensitive, practical CDV rapiddetection method is still an urgent problem.So far, there have been reported the methods of detection CDV are isolation,neutralization test, immunofluorescence antibody technology, immunohistochemistry,RT-PCR, but these methods are tedious or repetitive, they are difficult tograssroots application.This study was designed to establish a monoclonal antibody (MAb) sandwich ELISAdetection method and colloidal gold immunochromatographic test strip, to provide technicalsupport for the rapid detection of CDV. Part one: Establishment of the hybridomas secreting monoclonal antibodiesagainst canine distemper virusCDV NJ01strain of this experiment was pre-isolated from the tissues of atypical CDdogs, The splenocyte of BALB/c mice immunized with purified canine distemper virus (CDV)were fused with SP2/0cells,using indirect ELISA and indirect immunofluorescence assay(IFA) screened four stable secretion of anti-CDV antibody hybridoma cell lines were namedA2, H4, F7and G12. Indirect immunofluorescence assay (IFA) test results showed that thefour MAbs reaction with Vero cells infected with CDV, green fluorescence, while nofluorescence with normal Vero cells, which have better specificity. Indirect ELISA fordetection of the four MAb cell supernatants and ascites titer determination, the results of thesupernatant antibody titers are103induced ascites antibody titer of105to106. Hybridoma celllines was determined by non-antigen-dependent subgroups, measurement results of fourhybridoma cell lines subclasses were IgG1, the kappa light chain type were κ. Neutralizationtest indicated that these four strains of MAbs having the ability to neutralize the virus, and theA2and the G12monoclonal antibodies ascites titer of104.Part two: Development of a sandwich ELISA based on monoclonalantibodies against canine distemper virusTo develop a method for the detection of canine distemper virus (CDV), four hybridomasA2, F7, H4and G12secreting monoclonal antibodies(MAb) to CDV were established by thefusion of SP2/0cells and splenocyte from BALB/C mice immunized with the purified CDV.Additivity ELISA revealed that the four mAbs recognized spatially independent epitopes ofCDV. Purified mAb G12and mAb A2conjugated with HRP were used to establish asandwich ELISA for the rapid diagnosis of CDV. There was no cross reactivity with othercanine virus and the minimum detection limit was5μg/mL. The coefficient of variation ofreproducibility was less than6%. A total of57clinical sanples were detected by the ELISAand RT-PCR with agreement rate of100%. The result indicated that the sandwich ELISA wasapplicable to the clinical detection of CDV.Part three: Development of the monoclonal antibodies-based colloidal goldimmunochromatography strip for the detection of canine distemper virusMAbA2fixed as gold-labeled antibody with purified glass cellulose membrane, purifiedG12fixed as a capture antibody in the NC membrane, Goat anti-mouse IgG antibody as acontrol antibody, assembling pairsantibody sandwich method CDV colloidal goldimmunochromatographic test strip. Optimize the test conditions of colloidal gold immunochromatographic strip synchronous detection CDV, CPV CPIV, CAV-1, CCV, Verocell cultures and negative canine serum tests showed that the method has good specificity,andcan be used for the rapid diagnosis of CD clinical.