Preparation, Identification And Clinical Application Of The Monoclonal Antibodies Against Reticuloendotheliosis Virus

In order to produce the Commercial test kit For detecting REV> depurating the parents Flocks and quarantine of import and export- The polyclonal antibody and monoclonal antibody were prepared and also the specific testing methods were established. In Clinic, McAb were Conducted for detecting RIIV which showed Its values.96 micro titer wells with C'EFs were inoculated with REV, diluted from 10"' tolO"10 in culture medium. 7 days Later, This was followed by O.lml of cold alcohol-acetone mixture for 2min to fix monolayers ; O.lml polyclonal antibody at a dilution of 1:20 was added .then following with (J.I ml of fluorescein-isothiocyanate-conjugated anti-chicken IgG at a dilution of 1:100 . Fluorescent foci were read with an FA microscope .the 7 weeks to 10 weeks old chicken were infected with 10" TClD50/ml REV by belly and vein . Chicken were bled 3 weeks after infection, ELISA titer reach 1:3200. At the Same time ELISA was conducted.The standard strain REV-DIA was purified and used to immunize BALB/c mice.The immunized splenocytes of mice were fused with the sp2/0 myeloma cells which were tamed by 8-AG. bydetection with IFA ,three hybridoma cell strains which secret REV-McAb were obtained .these three cell strains were named C18 D31 G5 which were able to secret McAbs stably by series passage in vitro culturing ,freezing and recovering .The titer of cell culture media and ascitic fluid were 10-2-10-3 and 10-4-10-5 respectively by IFA .there was no cross -reaction with MDV and ALV. Both of the additivity ELISA and Western-blotting showed that McAb of C,8 and D3, were specific for the same epitope or very closed related epitope, The McAb of G5 has no much relations of epitopes with C18 and D31For REV separation and propagation , Chicken embryo kidney cell (CEKS) prepared from 19-day-old embryos were cultured for three generations. The morphology of REV preparations was verified by electron microscopy at the Same time, monoclonal-antibody mediated enzyme-linked immunosorbent Assay and immunofluorescent antibody test were conducted to detect the viremin of field infected and artificially infected birds, the Percentage of the REV viremin in the tested birds is 100% So the monoconal antibody has been proved very sensitive and specific to REV.