Studies On Establishment And Application Of Dot-ELISA For Detecting The Antibody Against Newcastle Disease Virus(NDV)

A dot-enzyme-linked immnosorbent assay (Dot-ELISA) for the detection of antidodies against Newcastle Diesease Virus (NDV) was established by using purified NDV and self-made enzyme-labeled anti-chicken IgG, The mehod was also evaluated through it's application. In the studies, the virulent F48E9 strain and avirulent LaSota strain were used for purifying antigen and comparision was done between them.Different methods had been used to purify antigen and the discontinuous sucrose density gradieng was determined to be the optimal one. Purified chicken IgG was used to immune rabbits ,then the rabbit antichicken IgG was purified and the rabbit antichicken IgG conjugated with HRP was made. The optimum conditions of Dot-ELISA was determined:the optimal coating concentration was 8.9 u g/ml ,1:400 was the best working concentration of HRP-laleled rabbit antichicken IgG,the other steps and reagentes were also optimized. The high specificity of Dot-ELISA was proved by the specific blocking test and the cross-reaction test in which the diagnostic diaphragm didn't react with the positive serum against IBDV, IBV,EDS-76,Pox ,IBV,ILTV, Salmonellosis JC.Both the test within batch and the test among batches proved the method or the diagnostic diaphragm was stable .Stored at 4C for at least 6 months ,the diagnostic diaphragm's sensitivity and specificity didn't change. Parallel test was done between Dot-ELISA and heamagglutination test (HI) by using them to detect 150 serum ,the result show 92 serum were positive (the positive rate is 61.3%)by Dot-ELISA while only 77 serum was positive (the positive rate is 51.3%); Among the positive serum ,the serum titers by Dot-ELISA were 2 to 4 tilers higher than that by HI .Through parallel test ,1:128 was preliminarily detennned to be the crical defensive point of Dot-ELISA.Using Dot-ELISA to detect antibody can be finished within 2.5 hours and the result can be determined by nakedeye without needing special instruments.All the results above have shown that Dot-ELISA has many advantages.Firstly,it is a convenient ,rapid .sensitive .specific method for detecting antibody.Besides,the diagnostic diaphragm is convenient to post or store because of it's stability. Fourhtermore ,all the materials and reagents can be easily standardized . So Dot-ELISA is believed to be very useful for the surveillane of antibody against NDV, epidemiologic investigation etc.