Studies On Establishment And Application Of Dot-ELISA For Detecting The Antibody Against Riemerella Anatipesttifer

For detect the antibody against Riemerella anatipesttifer(RA),the study established Dot-ELISA(Dot-enzyme-linked immosorbent assay) method which was used Riemerella anatipesttifer dominance strain(B₈,B₁₂,B₁₅) schizolysis antigen.We optimized the reaction condition and the best working condition were:the optimization coating concentration of antigen were B₈:0.091 mg/ml,B₁₂:0.094 mg/ml,B₁₅:0.333 mg/ml;to stay overnight at 4℃was the optimal reaction condition of antigen;PBST contains 5%BSA was used to be confining liquid react 45 min;PBST contains 10%calf serum was used to be dilution for blood serum and enzyme labelled antibody;PBST was used to be cleaning solution;the reaction condition of blood serum which need to detect react 60 min at 37℃;the reaction concentration of enzyme labelled antibody was 0.25μg/mL(1:200 dilute),react 50min at 37℃;the substrate react 10 min.The comparation assay IHT was used to detect 20 serum when used Dot-ELISA,and the result showed IHT positive was 14(occupy 70%) and Dot-ELISA positive was 19(occupy 95%).Later, we used both of the assays to detect 8 serum,the result showed the antibody titer of Dot-ELISA was 16～64 times higher than IHT.The cross-reaction showed high specificity of diagnostic diaphragm:didn't react with the positive serum against Staphylococcus,Pasteurella,Escherichia,react with positive serum against Riemerella anatipesttifer.Both the test within batch and the test among batches proved the method or the diagnostic diaphragm was stable.Stored at 4℃for at least 6 months,the diagnostic diaphragm's sensitivity and specificity didn't change.We made Riemerella anatipesttifer three strains(B₈,B₁₂,B₁₅) propolis adjuvant killed vaccine.Serum antibodies were detected in 3 d,6 d,9 d,13 d,20 d,27 d,34 d,41 d,48 d,55 d and 69 d after first immune.The dynamic response about serum antibodies in immuned ducks was using supersonic wave schizolysis antigen of each strain.The result showed the young duck was produce immunity in 3 d～6 d after vaccine inoculation,the antibodies level of 3 strains were rising with the time goes by. The antibody level of once immune group reached crest value in 20 d and twice immune group reached crest value in 27 d.This high level of antibody sustained to 48 d,then slowly decreased.We can detect Riemerella anatipesttifer antibody by Dot-ELISA in 3 hours,the result is objective.We can make a decision in nake eyes and don't need other equipment. All the result showed this experiment was convenient,quickly,sensitiveness,specificly,repeatability,stabilization and reliability.The diagnostic diaphragm was easy to send and conserve;the reagent and materials can made standardization,it's very suitable for veterinary station and large duck feed company to detect antibody,diagnosis and disease extensive survey.