Establishment And Application Of Elisa For Antigen And Antibody Detection In Canine Parvovirus

The disease of canine parvovirus, caused by canine parvovirus(CPV), is acute andhighly contagious disease in canine and other carnivores, and is one of the most severeinfectious diseases in canine farming, far cultivation and wildlife conservation. In latestyears. The research about diagnosis of CPV has a lot of development. Among thesemethods, The Enzyme-linked immunosorbent assay(ELISA) has the excellence ofconvenience. Shortcut. Inexpensive and suitably spread in grass roots.At first, The canine parvovirus (CPV-GN) strain was largely cultivate in cell of F81,purified by PEG6000, mensurated the consistence of protein, in succession, CPV-GN strainwas used as antigen for developing indirect enzyme-linked immunosorbent assay (ELISA).The conditions of indirect ELISA were determined as follows: 9ug/ml of CPV-GN strainwas used to coat ELISA plate, the positive sera need 1:160 diluted. The optimal closedliquid is 1.75% glutin and its optimal woking time is 37℃1h; the woking time of thesample and antigen is 37℃2h. A series of test proved this method possessed upstandingstability and distinctness. 50 samples were detected by this method and hemagglutinationinhibitory(HI) test respectively, the concident rate was 94%. For strong positive andnegative samples, the concident rate was 100%. The indirect ELISA proved to be morespecific, rapid and easier to perform than the HI test. For evaluating the vaccinal effect,This indirect ELISA was used to detect sera collected from 160 dogs; after one year, 60dogs among these dogs was also collected sera for detection in this indirect ELISA. Theresult revealed that the immune procedure can offer enough safeguard to resisting CPVinfection. In simultaneity, The vaccinal effect was not signilicantly associated with sex andbreed, but, was signilicantly associated with age.The purified canine parvovirus (CPV-GN) strain was inoculated Balb/C mouse. Afterthe last immunity, spleen cells from Balb/C mouse was fused with SP2/0 in the fourth day,Positive clones were screened by indirect enzyme-linked immunosorbent assay (ELISA)and neutralization test (NT) and were subclonized with limited dilution. The false positiveclones were rejected by F81 cell coating ELISA plates. Three Monoclonal Antibodies (McAbs) were gained. They are named A7.C5.E9. The titer of viral neutralization isrespective 1:160.1:80 and 1:160. A series of test proved all of these three McAbs possessedupstanding stability and distinctness. The titer of ascites,which was produced from A7, wasdetermined by indiect ELISA and hemagglutination inhibitory(HI). The result is respective1:1280 and 1:640.A double antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) kitfor detection of canine parvovirus (CPV) in fecal was estabilished with the monoclonalantibodies against CPV. The result revealed that the optimal concentration of monoclonalantibody is 7ug/ml; the optimal closed liquid is 1.75% glutin and its optimal woking time is37~Clh; the woking time of the sample and McAb is 37℃2h; the reaction time ofHRP-labeled McAb is 37℃1h; the sample was direct diluted by physiological brine, aseries of test proved this method possessed upstanding stability and distinctness. 40samples were detected by the DAS-ELISA kit and hemagglutination (HA) test respectively,The results showed that the total coincident rate was 92.5%. For strong positive andnegative samples, the concident rate was 100%. The DAS ELISA kit and PCR were used todetect 20 samples, The sample which has the different result by the two method wasinoculated FS1 cell.