| Rabbit hemorrhagic disease (RHD) is a highly contagious and fatal disease by rabbit hemorrhagic disease virus (RHDV). It is associated with necrosis in hepatic cells and congestion in parenchymatous organs, which caused serious economical losses in rabbitry.In order to diagnose intuitively RHD and provide a good tool for the rapid detection of RHDV, five monoclonal antibody (McAb) against RHDV were developed in this research, two methods of detection of RHDV, the immunochromatograpic colloidal gold dipsticks for Rabbit hemorrhagic disease virus and the indirect ELISA for detection of antibodies against Rabbit hemorrhagic disease virus, have been developed. The details are divided into three parts as following.1. Development of monoclonal antibody against RHDVSix-week old BALB/c mice were immunized subcutaneously with engineering VP60 protein and purified complete virus particle of RHDV, respectively. Mouse spleen cells were fused with SP2/0 myeloma cells, and hybridoma culture supernants were screened by indirect ELISA. Five hybridoma cell strains against RHDV, named A3C,A,B,C and D, respectively, were developed after four times cloning via limiting dilution assay. The indirect ELISA titers of the ascites of five McAbs were 327600,3200,6400,3200 and 6400, respectively. The five McAbs specificly reacted with complete virus particle and engineering VP60 protein of RHDV in Western-blot assay analysis.2. Development of the immunochromatographic colloidal gold dipsticks for RHDVGold immunochromatography assay (GICA) has been used widely in epidemiology investigations and clinical diagnosis especially in infection diseases because of its specificity, sensitivity and rapidness. The goal of this study is to establish a sandwich GICA to detect RHDV. The colloidal gold particle with diameter of 20-30nm was selected to be labeled with polyclone antibody protect against RHDV and the A3C McAb was used as coating antibody. Based on the laminating ability and application effect, we screened the GICA materials and determined the optimal working concentration of buffers and antibodies. The detection sensitivity of the prepaered immunochromatograpic colloidal gold dipsticks against RHDV was higher than erythrocyte agglutination test, and it has not cross-reacted with other bacterias in rabbit. Different batch of GICA dipsticks were showed similarity results in repeating assay. In destructing testing, the prepared dipsticks showed well reproducibility when preserved 11.5-day-post stroe at 37℃condition which is equivalently about 17 months store at 2-8℃condition. The erythrocyte agglutination test and the dipstick test were completely coincidence in detection of 127 samples of rabbit liver tissue, which indicated that appropriate for applicaton in clinical diagnose.3. Development of the indirect ELISA for detection antibodies against RHDVThe indirect enzyme linked immunosorbent assay (iELISA) method was developed to detect rabbit antibody against RHDV, with the capsid protein (VP60 protein) as coating antigen in the concentration of 4.617μg/mL and the serum sample as 1:200 dilution. The coincidence of the iELISA and HI test was 91.3%. For total of 1127 serum samples to be detected,955 out of these samples were positive by indirect ELISA test and 857 out of these samples were positive by HI test. The other tests were confirmed that the iELISA was specific, sensitive, reproducible and easily operate in detection, which indicated that this method was appropriate for applicaton in clinical diagnose. |
PDF Full Text Request