| As a specific polyclonal antibody, the ducks' yolk immunoglobulin has not only the bioactivity of IgG but many advantages super to mammalian IgG. In our country, DVH is one of the severe diseases threaten to duck industry. In this assay , DHV was isolated and purified, IgG from the egg yolk of ducks immunized with DHV as well, and investigating characteristics of IgG to try to set up a Dot—ELISA for the DHV antibody. The results as follows: 1 .Preparation the anti-DHV duck yolk antibodies1.1 Proliferation DHV-1 standard strain (ATCC strain)through duck embryos, the viral antigen waspurified by extracting with chloroform, concentrating with PEG and differential centrifugation, of which the protein is 18.75mg/ml, and can be used in immunization or coating the plate.1.2 Emulsification to prepare anti-DHV vaccine, and after security test, immunizing ducks, collecting hatching eggs till the anti-serum titer is above 1:128 to provide materials for subsequent research.2. Extraction the aqueous material from egg yolk in water dilution method, and optimize the conditions of this method.2.1 pH5.1 is the optimization pH when isolate antibodies from the diluted egg yolk ,the dissolubility of its will drop when pH<5.1, but the precipitation of lipid will be affected when pH>5.12.2 The optimization of dilution for egg yolk is 1:10. when the multiple is beyond 1:10, the protein content in supernatant increased gradually, but the fat protein decreased by degrees.2.3 After dilution, the egg yolk needed at least 6h for standing, the supernatant was not significantly alter with longer time3. Isolation and purification of the anti-DHV antibodies from duck egg yolk3.1 The octoic acid-ammonium sulfate method is more sutible compared with other extraction methods3.2 Successful separation IgY and IgY (ΔFc) through ion exchange chromatography and gel filtration chromatography, the equilibration buffer is 0.02mol/L Tris-C1 and 0.02mol/L PBS separately ;the flow rate of elution is 1.0ml/min and 0.1ml/min separately4.Optimization conditions of indirect ELISA for hypso-purity yolk antibody against DHV4.1 the concentration of antigen is 16μg/ml: when the concentration is 4μg/ml, OD=0.519; and the concentration is 16μg/ml, OD is up to peak value.4.2 the concentration of primary antibodies is 28μg/ml: when the concentration ranged from 2μg/ml to 25μg/ml, the OD value increased linearly, from 16μg/ml to 32μg/ml, the OD value increased slowly, and when the concentration was above 32μg/ml, the OD value kept constant.4.3 the enzyme labelled antibody was diluted to 1:3200. The OD value decreased gradually with the augment of dilution multiple, especially when 1:3200 was diluted into 1:6400.5.The test for the stability of hypso-purity antibodies in several conditions5.1 the antibody titer was not obviously altered at 65℃, and has a slightly descent at 70℃. While at 75℃, the activity remained 35%. At 80℃, the activity only remained 19%. So the temperature beyond 75℃, the titer would descend significantly. |
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