| In this test, All Infectious bursal disease virus (IBDV) BC 6/85 F4 and IBDVHE BEI isolated strain were propagated on specific-pathogen-free (SPF) 10-day-old embryonated eggs and on chicken-embryo fibroblast (CEF) monolayer that were prepared from SPF embryonating chicken eggs. All IBDV BC 6/85 F4 and IBDV HE BEI isolated strain were obtained from allantochonon and allantoic fluid of died embryonated eggs in 2-5 days after inoculating and virus-infected cell fluid that were frozen and thawed three times after approximately 80% of the monolayer exhibited cytopathic effects. Highly purified IBDV growing on SPF embryonating chicken eggs was got respectively and essentially through freezing and thawing three times, precipitated virus with chloroform, concentrated by polyethylene glycol 6000 and ultracentrifugated in sucrose density gradient and IBDV propagated on CEF cell was concentrated by using polyethylene glycol 6000 and percolated with method of gel filtration with Sephadex G-200. The hyperimmune serum of rabbit and mouse anti-IBDV was prepared by immunization in rabbit and mouse with purified IBDV. Mainly immunoglobulin G (IgG) of sheep anti-rabbit, rabbit and mouse anti-IBDV was obtained by ammonium sulphate precipitation subsequently unsalted with Sephadex G-25 and chromatography over diethylamino-ethyl-cellulose-32 (DEAE-32). The sheep anti-rabbit IgG conjugated to horseradish peroxidase(HRP) and labeled with colloidal gold was individually made by the method of sodium periodate and sodium citrate-tannic acid. Three immunology methods (indirect double antibody sandwich enzyme-linked immunosorbent assay (ELISAX Dot-ELISA and Dot immunogold-silver staining (Dot-IGSS) ) were established by the optimal concentration fixation of rabbit, mouse anti-IBDV IgG and sheep anti-rabbit IgG according to the phalanx determination to detect IBDV antigen. The optimal incubating antibody concentration in sandwich ELISA method was 1 .' 100, the optimal rabbit anti-IBDV IgG concentration was 1 : 200 and in HRP labeled sheep anti-rabbit antibody, optimal concentration was 1 : 400. In Dot-ELISA test reactive concentration of rabbit anti-IBDV was 1 I 100, the optimal working concentration of HRP labeled sheep anti-rabbit antibody was 1 : 200.The incubating concentration of rabbit anti-IBDV in Dot-IGSS test is 1 : 200 and sheep anti-rabbit labeled withcolloidal gold was 1 : 10.Three methods proved to be superior in terms of specificity and duplication through the specificity blocking test, cross test and duplication test and they had no antigen-crossing react with new castle virus (NDV). IBV and EDV. Comparing the result of sensitivity test with Agar Gel Precipitin Test (AGP) , sandwich ELISA had the highest sensitivity in detecting IBDV antigen and its lowest detective concentration was 0.3192@g/ml,then was Dot-IGSS test which detective concentration was 0.798@g/ml,and the last was Dot-ELISA which had the lowest concentration ,only 1.596@g/ml.All the established three methods were better then AGP in sensitivity. Sandwich ELISA was one hundred times than AGP, Dot-IGSS was forty times than AGP, Dot-ELISA was twenty times than AGP. Through the compare of sequence in genome segment A of IBDV different strains, a pair of oligonucleotide primers were synthesized in the overlapping area of VP5 and VP2 where the sequence is highly homologous according to the sequence of genome segment A of classic strains "STC" from Genebank and 267bp intent nucleotide fragment were amplified .The same size of intent gene fragment were got from the test of specificity, sensitivity and replicate detecting IBDV antigen and its sensitivity got to 1.995ng/ml.This method was most sensible one in whole method of detecting IBDV antigen. Our test also verified PCR method have the excellence of celerity, nicety, easy to performing, high sensitivity, easy to reading, high specificity and good repeating and this was a most nicety and most available method for detecting IBDV, which can be used for detecting a majority of IBDV specimens.
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