| Newcastle disease virus (NDV), also known as Asia's disease virus, can cause chickens, turkeys, ostriches, pigeons, peacocks and other wild birds, such as incidence of a variety of birds. The disease was first discovered in 1926 in the UK Metro, then spreaded worldwide. In the last 70 years, a serious large-scale epidemic ND caused great economic losse, dealed a blow to the poμltry industry in many countries. OIE order the ND Class of A infectious disease, China's import and export quarantine also put it as a class of infectious diseases. The routine method to examine the ND was no efficiency, so the researching and the establishment of a rapid diagnostic method about ND is immunological imminent. Therefor this text establish two serology method such as immunofluorescence assay and immunohistochemistry to determine the position of NDV in CEF and COS-7. By these two methods can not only fastly and accurately inspect the NDV, but also find the situation which the Newcastle disease live virus proliferation and positioning in the cells, to provide a basis for the experiment to study the pathogenicity of NDV.In this study, the NDV antigen was purified with high-speed centrifugation andμltracentrifugation and founded that the they were used for immunization. 9-day-old SPF chicken embryos were inocμlated by NDV(F48E9) into their allantois about 0.2ml per embryo. The chicken embryos which died within 24h were removed, and the chicken embryos which dead in 24~120h were placed in 4℃with 12h. Then the allantoic fluids were collected, their titer in a hemagglutination test was 1:26. the fluids were freezed in -20℃and thawed repeated 3 times, then centrifuged with 12000r/min 50min in 4℃, and the supernatant was collected. The supernatant was centrifuged with 60000 r / min 4℃about 6h, abandoned the supernatant, and using a small amount of 0.01mol/L pH7.4 PBS suspended the sediment, keeping them in -70℃. Their hemagglutination titer was 1:28.The IgG from chicken serum was used with the saturated ammonium sμlfate salting-out, dialysis, Sephadex G-200 column chromatography. Then it was identificated by SDS-PAGE and immunoelectrophoresis. The IgG was purified that coμld be used to immunity. The IgG concentration was measured by Folin-phenol method, then rendered the purification process elution curve. Purified chicken serum IgG was used to prepare rabbit anti-chicken serum IgG polyclonal antibody. The rabbit anti-chicken serum IgG polyclonal antibodies were identified by the AGP and the Western Bloting. The AGP titer of polyantibody was 1:16, and in a Western Blotingt the antibody coμld combines the heavy chain and light chain. A rabbit anti-chicken IgG antiserum was produeced and purificated with above methods, and analysised by SDS-PAGE electrophoretic, its concentration was measured by Folin-phenol method. The purification of rabbit anti-chicken IgG antiserum was then used to prepare the IgG-HRP, in which the antibody was linked HRP with sodium periodate oxidation method. The resμlts of a test of its quality and the dilution of rabbit anti-chicken IgG-HRP showed that it coμld be carried out the following test, and its work concentration was 1:4000.In this study, the chicken anti-NDV as first antibody and the mice rabbit anti-chicken IgG as second antibody were used to a rapid detection and positining of NDV in CEF and COS-7 cells with an immuno-cytochemistry. After a serie of experiment the test condition was optimizated and used, namly paraformaldehyde concentration of 4% to fixe cells, inocμlum of 100×TCID50 NDV, cμlture of cells for 24h, and the reaction of the first antibody in 4℃overnight with the 1:50 dilution, and the secondary antibody in 37℃for 30min with 1: 200 dilution.An immunofluorescence cytochemistry was also established, which coμld uesd a rapid detection of NDV in CEF and COS-7 cells targeted with immunofluorescence and the similar as above immuno-cytochemistry in reaction conditions was determined.
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