Establishment And Application Of Two Rapid Detection Methods For Canine Parvovirus Enteritis

Canine parvovirus enteritis which caused by canine parvovirus is an acute,strong, height of contact infectious diseases of canid clinically characterized bysevere vomiting, diarrhea, hemorrhagic enteritis and non-suppurative myocarditis,and is prejudicial to our country’s kennel industry and wild animals. At present, thedisease has no specific treatment, and relies mainly on the immune prevention.Therefore, it is significance to develop a rapid, simple, sensitive and specificdetection of canine parvovirus antigen and antibody for the disease’s prevention,diagnosis, treatment and epidemiological analysis. To establish two rapid detectionmethods for the Canine parvovirus enteritis which one was an indirect ELISA fordetecting canine parvovirus antibody, and another was colloidal gold test strip forcanine parvovirus, this study include three aspects as follows:1. Development of an indirect ELISA for detecting canine parvovirus antibodyThe virus-like particles of canine parvovirus (CPV-VLPs) which werecomposed of VP2protein were expressed and assembled successfully in insectbaculovirus expression system. The CPV-VLPs were purified through (NH4)2SO4sedimentation and sucrose density gradient ultracentrifugation, and the purificationresults were analyzed by electron microscope, SDS-PAGE and Western-blotting.After purification, the purity of CPV-VLPs was higher than90%. An indirectenzyme-linked immunosorbent assay (ELISA) was developed and optimized withthe purified CPV-VLPs as coating antigen. The optimal conditions of the indirectELISA were determined as follows: the coating concentration of purifiedCPV-VLPs was5.0ug/ml at4℃overnight;1%BSA as blocking agent at37℃for2h; the serum sample was diluted into1:40and incubated at37℃for1.5h; theenzyme labeled antibody was diluted into1:20,000at incubated at37℃for1h; thesubstrate TMB for ELISA being incubated for30min at room temperature. Themethod could detect canine parvovirus antibodies specific and show no cross-reaction with the positive sera of the four dog diseases (CDV, ICHV, CCVand RABV). The result showed that the sensitivity was1:640. Coefficient ofvariability percent (C.V%) of inter-batch and extro-batch duplicative texts was lessthan10%. The coincidence rate was90.48%with HI in detecting42clinical serumsamples.2. Development and characteristics identification of monoclonal antibodiesagainst canine parvovirusIn this study, CPV was grown in F81cell. The purified CPV which was dealedwith polyethylene glycol sedimentation and sucrose density gradientultracentrifugation was used as antigen to immunize6to8week-old Balb/c femalemice. After being immunized three times, the antibody titer in serum attained1:105detected by indirect ELISA. PEG induced fusion methods was adopted to improvethe immune mice spleen cells and SP2/0myeloma cell fusion. Indirect ELISA wasused to screen positive hybridoma cells, and limiting dilution method was applied toclone positive hybridoma cells. After three times cloning, three strain hybridomacell cecreting monoclonal antibodies stably was obtained and named1A3,6B10,7F12. The indirect ELISA results showed that monoclonal antibodies hybridomacell culture supernatant titer were1:103,1:103,1:103, and the ascites titer were1:106,1:105,1:107. The HI results showed that monoclonal antibodies hybridoma cellculture supernatant titer were28,28,28, and the ascites titer were215,214,215.Subtype identification results showed that1A3,6B10,7F12belonged to IgG2a,IgG3, IgG2b respectively. Indirect ELISA method were used to detect the threestrains hybridoma cells culture supernatant which comed from consecutivegenerations and freezed. The results showed that the three strains monoclonalantibodies have a great stability. Specificity results detected by indirect ELISA,indirect immunofluorescence, western blotting Data above showed that threemonoclonal antibodies not only reacted with CPV specificitily, but also had thespecificity of the inhibitiory of hemagglutination3. Preparation of colloidal gold test strip for canine parvovirusThe1A3,7F12monoclonal antibodies were purified through (NH4)2SO4sedimentation and protein G affinity chromatography method, the purified proteinconcentration is3.21mg/mL and5.421mg/mL respectively. Sodium citrate was used for the preparation of colloidal gold which was used to lable1A3monoclonalantibodies. The purified7F12monoclonal antibodies and the goat anti mouseantibodies was coated on the test and control lines of the nitrocellulose membrane(NC), respectively. Canine parvovirus immune colloidal gold test strip was preparedaccording to the principle of double antibodies sandwich. The detection resultsindicated that the test strip has good specificity to CPV, MEV, FPV and don’tcross-react with CCV, CDV, RABV, ICHV. The sensitivity of the test strip was80inhibition of hemagglutination unit per millilitre. Using different methods to detectethe90clinical faeces samples, compared with PCR, the sensitivity and specificity ofthe CPV immune colloidal gold test strip prepared by this study were94.4%and100%, the coincidence rate was95.6%. Compared with PCR, the sensitivity andspecificity of the CPV immune colloidal gold test strip prepared by South KoreanBioindist were97.3%and88.9%, the coincidence rate was95.6%. The above resultsshowed that the preparation of parvovirus immune colloidal gold test strip whichhad a good specificity and sensitivity, was suitable for rapid detection of largeclinical samples.