Detection Of Duck Swollen Head Septicemia Virus With Indirect Sandwich ELISA

A new type virosis in ducks with the characteristics of being acute, transmit rapidly and high mortality rate , has been observed in sichuan and other provinces since 1998.The disease could infect all days ducks and all kinds of breeds. Moreover its' morbidity and mortality reach over 80%, which aroused disastrous loss to local duckeries. The disease looked like Duck Plague in clinical symptoms and pathology symptoms, which handicaped us to exactly diagnose it .In order to take a good prevention and control the disease , reduce the loss, we have developed a rapid measure to diagnose and detect duck swollen head septicemia virus by Indirect Sandwich enzyme-linked inmunosorbent assay(ELISA).The paper used the duck swollen head septicemia virus XD strain which was isolated and identified by us as the antigen and purificated to inject ducks and rabbits in order to get antisera, then purified the antisera with caprylic acid and ammonium sulphate(CA-AS), and developed the indirect sandwich ELISA in the aid of purified IgG for detection the virus for the first time. Through repeatedly test and confirmed the optimization test conditions:the coating antibody against XD in ducks concentration was 8ug/ml, and the coating temperature and time was at 37℃ incubating for 1.5h.The assay buffer was 0.5% glutin and blocking for 1.5h at 37℃. Antigen incubated for 1.5h at 37℃.The concentration of rabbit anti XD was 5ug/ml, and incubated for 2h at 37℃, The optimation dilution of horseradish peroxidase(HRP)-labelled goat anti-rabbit IgG was 1: 2000, and incubated for 2h at 37℃. Substrate reacted for 30min, and wash buffer(dilution)was 0. 02mol/L PH7. 2 PBS (contain 0.15mol/L NaCl, 0.05%tween-20), wash buffer added 5% bovine serum was dilution. The cutoff of optical density(OD) at 490 nanometers(nm) wavelength was 0.412. The results of reproducibility of the Indirect Sandwich ELISA showed that coefficient of variation was between 2.0~3.5% in 3 positive samples andwas 4. 6~9. 0% in 3 negative samples, which were less than 10%.The developed Indirect Sandwich ELISA was specific, the measure could not cross react to the negative antigen of duck swollen head septicemia virus, Duck Plague Virus(DPV), E. colibacillosis, Duck Hepatictis Virus(DHV).The measure was highly sensitivity and was 1280 times than agar gel precipitation (AGP). The coated polystyrene microtiter plates can preserved at 4°C for at least 3 weeks affter washed.The measure was developed and applied to detect this virus antigen in liver, brain, kidney, lung, heart, spleen, pancreas of duck which was experimental infected with duck swollen head septicemia virus, The result showed that the positive rates in heart, liver, lung, kidney was the most high and was 100%, and the positive rates in head, spleen , pancreas was 90%, 80%, 40% respectively.