| Porcine rotavirus, a member of genus Rotavirus, the family Reoviridae, was a causative agent of piglets diarrhea caused by virus. Organisms infected by PoRV showed diarrhea, vomit, dehydration, acid-base imbalance and dies at last. Porcine rotavirus had many serotypes and there was no or little cross-protection among different serotypes. It showed that porcine rotavirus infection was mainly group A in our country, and viral protein 6 was group antigen with high conservation. Routin detection methods for rotavirus had many shorcomings, such as needing special equipments, having noxious chemical materials, multiplicity and so on. A new process with high sensitivity and specificity to detect rotavirus and control porcine rotavirus infection was needed badly.In this experment OSU strain (G5 serotype) , represent of group A rotavirus was used as immunogen, and purified it by differential centrifugation and sucrose density gradient centrifugation, and immunized the BALB/c mouse. By the hybridoma technique and three times cloning, 8 hybridoma cells secreting monoclonal antibody were established and named 1D5, 1B5, 1G9, 1C7, 3C10, 1F10, 1D11, 1F3 respectively. By indirect ELISA, the antibody titers of them were 1:6400,1:12800,1:25600,1:12800,1:6400,1:6400,1:25600 and 1:6400. 1D5, 1B5, 1C7 were belonging to IgM, and 1G9, 3C10, 1F10, 1F3 were belonging to IgG2b, and 1D11 belonged to IgG2a, and the light chains of the antibodies were allκchains, which were analyzed by ELISA using a commercial kit. Western-blot indicated that 1D5, 1G9, 1C7, 1F10, 1D11 had special reaction with the VP6 protein of PoRV, and 1B5, 3C10, 1F3 had special reaction with the VP7 protein of PoRV. The result of additive ELISA showed that 1D5 and 1F310 monoclonal antibody recognized the same antigen epitope, 1B5 and 1F3 monoclonal antibody recognized the same antigen epitope, 1C7 and 1D11 monoclonal antibody recognized the same antigen epitope, 1G9 and 3C10 recognized two antigen epitopes respectively, and the relative affinity is 1B5>1D11>1D5(1C7,1F10,1F3)>1G9>3C10.Colloidal gold chromatography techniques was convenient, accurate, and had good specificity and sensitivity combinated with monoclonal antibody. This experiment developed colloidal gold chromatography strip with monoclonal antibody 1G9 and 1D11, which had good bioactivity and specificity to VP6. Colloidal gold with about 20 nm and 40 nm particle diameter were prepared by trisodium citratec reduction. It was proved that colloidal gold with 20 nm diameter was uniform, without fragments, without agglutination, and it could be conserved at 4℃or room temperature for long time. The monoclonal antibody 1G9 was labelled with colloidal gold of 20 nm particle diameter at a rate of 8.57μg monoclonal antibody per 1 mL colloidal gold. The compound was purified by differential centrifugation and combined with the fiberglass to form conjugated pad. Spraying another monoclonal antibody 1D11 and goat-anti-mouse IgG to nitrocellulose membrane (Milipore 135) in turns at a dose of 2.4μg/cm and 3.2μg/cm especilly as the test line and the control line. Assembled them and incised into lateral test strips. Sensitivity test showed that the colloidal gold immunochromatography strip could detect 5.4μg/mL purified porcine rotavirus antigen and 1.0×103.4 TCID50/mL porcine rotavirus. The test strip showed high specificity with porcine rotavirus OSU strain (G5 serotype) , JS strain (G9 serotype), bovine rotavirus NCDV (G6 serotype), caprine rotavirus LLR-85 (G10 serotype), and had no cross reactions with other viruses such as porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus, porcine circovirus type 2, pseudorabies virus. 37 specimens form some pig farms were detected by the colloidal gold immuno -chromatography strip and the result compared with the PoRV Ag rapid test kit. The result showed that the strip could detect 11 PoRV positive specimens, and had a high coincidence (91.7 %) with the PoRV Ag rapid test kit.The result of this experiment showed that the colloidal gold immunochromatography strip developed with monoclonal antibody against VP6 of porcine rotavirus OSU strain (G5 serotype) has good sensitivity and specificity, and had high coincidence with the Rota Ag rapid test kit. The development of this method provides a rapid, time-saving and convenient means to detect the porcine rotavirus clinically, and it could also be used in porcine rotavirus epidemiology investigation. So the colloidal gold immunochromatography strip had great contribution on the prevention and control of porcine rotavirus diarrhea.
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