| The Gosling Plague (GP) is a worldwide epidemic of young goose, caused by Goose Parvovirus (GPV), which belongs to Parvoridae,Parvorinae and Parvovius. This disease has caused a huge economic losses to the young geese in poultry industry around the world ,so the diagnosis and treatment of it earlier is extremely important. This experiment attempted to take the advantage of the ELISA, s rapid diagnosed method and the monoclonal antibody, which has a high specificity to GPV ,to develop a fast and specific diagnosed method to GPV.The result of ELISA is greatly influenced by the experiment work condition , which are not cocoincided with reports. Consequently, this paper was carried on to compare each work condition of the indirect ELISA , hoping to find a stable and credible method for ELISA examination.At the first step, the best dilution concentration of the GPV and its antibody was chosen respectively by phalanx method for indirect ELISA.Secondly, the antigen at this dilution was used to coat the ELISA boards. Using different wash solution\block liquid\base materials and enzyme as work condition with constant antibody dilution concentration, the Optical Density(OD) at 492 run were measured, and then the results were analyzed.At last, the optimal work condition was ascertained, which is, the dilution degree of antigen and serum were 1:320 and 1:2000 respectively, the wash solution was the O.lmol/1 carbonate buffer, the coating temperature was 4°C ,the coating time was 12 hour, the block solution was 10% FCS PBS, the Anti-antibody was the sheep against mice IgG labeled by HRP and the base material was OPD.Using 50 percent PEG(molecular weight 3350) as fusion reagent, the hybridoma cell lines secreting monoclonal antibody which are highly specific to GPV were developed by fusing the myeloma cell lines SP2/0 and the spleen cells of BALB/C mice conventionally immunized three days early with crude GPV .The ascites was drawn out from the mouse injected hybridoma cell and analyzed to determine its titer,specificity and stability . The positive hybridoma cells were sieved by indirect ELISA, then cultured and cloned. Six hybridoma cell lines, named as 2A6A9, 4C7C5,1H3H5, 4E3D9, 2A9H1 and 3G1D5 were developed. Chromosome of the hybridoma cells were counted. Ascites was developed by injecting those positive hybridoma cells into the BALB/C mice. The monoclonal antibody was analyzed to ascertain the antibody group ,the specificity to NDV,AIV and DHV,the stability to refrigeration after 20 days .The result of the chromosome count testified the cells to be hybridoma cells. The ascites titer reached 10-5, not responded to NDV, AIV, DHV,and the hybridoma cells was stable to refrigeration.Using the developed Mabs to link the HRP, a rapid and specific method diagnosing GPV was developed. The developed HRP-labelled Mabs was used in ELISA to detect GPV,NDV,AI-H9 and DHV. The result indicated that the ELISA method had good specificity. The HRP-labelled Mabs was used in ELISA to detect GPV from geese suspected infecting GP ,healthy geese and artificial infected geese. Goose embryos were inoculated and the agar-pervasion test carried on synchronously as a comparison. Result indicated that take advantage of the beneficial effect of HRP-labelled Mabs used in ELISA, the GPV could be detected in 24 hours, shorter than that inoculated and the agar-pervasion test(P<0.05).And that method could detect viruses from more organs, and examine a LP virus. These results indicated that the method erected by us could detect GPV more rapidly and specifically and has good foreground.
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