| Canine distemper (CD) is a highly contagious and lethal diseases, which was infect the predators such as dogs. It is one of the most serious epidemics hazards dog breeding industry, fur animal breeding industry, and wildlife conservation industry. This study was designed to establish methods of ELISA and colloidal gold immunochromatography test for the rapid detection. The experiment consist of two parts.Experiment one:Use the hybridoma cell line1D7and3H11inject into BALB/c mice’s abdominal respectively, with the scretion of canine distemper virus monoclonal antibody. A double antibody sandwich ELISA for CDV antigen was developed using1D7as capture antibody and HRP marked3H11as detection antibody. The reaction conditions were optimized. The results showed that the best concentration of the monoclonal antibody at20μg/mL and incubated for overnight at4℃. The blocking reagent as5%skim milk powder, followed by adding IgG HRP conjugate at1:4000. Coated distemper antibody has no cross-reaction with canine parvovirus (CPV), canine coronavirus(CCV), canine parainfluenza virus(CPIV), canine adenovirus Ⅰ (CAV-Ⅰ). The lowest levels of virus detected was102TCID50/mL through the double antibody sandwich ELISA, the coefficient of variation between0.5%and6.3%. To test50clinical samples, the assay had92%agreement with the commercial kit. The above test results show that the establishment sandwich ELISA method has high sensitivity and stability.Experiment two:The gold particle in colloidal solution was coupled with purified monoclonal antibody3H11, and then sprayed on the glass fibers. The purified monoclonal antibody1D7served as the capture-antibody was coated on the nitrocellulose membrane. The optimization of antibody concentration is17μg/mL under pH8.0. The concentration of antibody, which is coating on NC, is2.0mg/mL, Colloidal gold immunochromatographic test strip has no cross-reaction with canine parvovirus (CPV), canine coronavirus(CCV), canine parainfluenza virus(CPIV), canine adenovirus I (CAV-I). The lowest levels of virus detected was103TCIDso/mL, the sensitivity is slightly lower than the double antibody sandwich ELISA. The testing strip can be stored under the condition of4℃or25℃for six months. The detection results indicated that the IC strip has a good sensitivity and stabillity. And we can see the results in1-5min. So this IC strip is suit for rapid diagnosis and clinical samples as the scene detection. |
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