| The laboratory dog is one of the animals used widely in teaching and researchingcurrently. There are a large number of dogs every year for drug safety evaluation andcardiopulmonary bypass experiments. Quality control is a major problem inlaboratory dogs standardization process currently.According to China’s national standard, GB14922.2-2011"Laboratory animal-Microbiological standards and monitoring", Canine Distemper virus must be detectedin laboratory dogs. What is more，specified-pathogens free dogs can not immueCanine Distemper virus vaccine, must be Canine Distemper virus antibody negative.Currently, the technologies for the detection of canine serum antibody includeimmunofluorescence, ELISA and other methods, which have a common disadvantage:one test can only detect one type pathogenic antibody so that they can not be achievedon real-time monitoring of various pathogenic antibodies. With the features of easyoperation, strong sensitivity, flexibility, wide detection range and so on，SuspensionArray is a new biochip technology platform, whose greatest advantage is that it cansimultaneously detect various pathogenic antibodies in one serum sample. We canmonitor various pathogenic antibodies in real-time by Suspension Array.In this study, mouse IgG was conjugated with fluorescent microspheres D6.Inorder to optimize the coupling process, we evaluated the efficiency of couplingreaction by coupling verification test.We finaly confirmed that the amount ofDTT(1M) was2μL, the activation time1h, the amount of sulfo-SMCC(2mg/mL)2μL, sulfo-SMCC reaction time1h, the reaction time of the protein-sulfo-SMCCcomplex and activated microspheres1h, the amount of NEM(2mg/mL)2μL, NEMreaction time20min.In this study，we predicted Canine Distemper virus N protein B cell epitopes andsynthesised. The result contained eight sections amino acid sequences. We purified Canine Distemper virus solution by differential centrifugation. The recombinant Hprotein of Canine Distemper virus,which was expressed in293cell, was acquiredfrom United States Immune Tech Company. We used the synthetic peptides, full-virussolution and recombinant H protein to couple with fluorescent microspheres D6. Thenwe used capture microspheres coupled with different antigen to detect CanineDistemper virus antibody positive and negative serum. The results showed that thecapture microspheres coupled with the recombinant H protein can be used to establishsuspension array. The result of optimization of influence factors was that the amountof capture microspheres was1μL, diluted to50μL by PBS(0.01M,pH7.2), thereaction time of capture microspheres and serum1h, the dilution of PE-labeled goatanti-canine antibody1:8; PE-labeled goat anti-canine antibody reaction time1h. Theresult of evaluation of suspension array was that it had no cross reaction with Rabiesvirus antibody positive serum, Canine Parvovirus antibody positive serum or CanineAdenovirus type2antibody positive serum.The detection limit of antibody titer was1:6800. CV in group were all less than5%. And CV between groups was2.98%. Itcan be stable at4℃for three months, stable at room temperature for7days. WithELISA kit detection result, coincidence rate was90%.In X2paired test, P was lessthan0.01. Suspension array built by us for the detection of Canine Distemper virusantibody was confirmed to have strong specificity, strong sensitivity, a goodreproducibility and a good stability. Suspension array can be used as an alternativetechnology to ELISA. This study lay the foundation of how the suspension array forthe detection of canine various pathogeny antibodies should be established. |
