检测鸭肿头败血症病毒的间接夹心ELISA的建立及应用

为了快速诊断和检测鸭肿头败血症病毒抗原,用分离自典型鸭肿头败血症的XD株病毒,制备鸭抗XD IgG、兔抗XD IgG,建立了检测鸭肿头败血症病毒(DSHSV)抗原的间接夹心ELISA法。试验的最佳条件为:抗体包被量为8μg/mL,封闭液为0.5%明胶,抗原孵育条件为37℃1.5 h,兔抗XD病毒IgG浓度5μg/mL,酶标羊抗兔抗抗体浓度为1∶2000,洗涤液为0.02 mol/LpH7.2 PBS。阴阳性结果判定的临界OD值为0.412。重复性试验显示变异系数小于10%。建立的间接夹心ELISA法不与鸭肿头败血症阴性抗原、鸭大肠杆菌抗原、鸭瘟抗原、鸭肝炎病毒抗原呈现交叉反应。敏感性比琼脂扩散实验高1280倍。包被的酶标板在4℃至少可以保存3周。用此方法对人工感染鸭组织脏器中鸭肿头败血症病毒检测结果显示,心、肝、肺、肾检出率最高,脑、脾、胰次之。

Detection of Duck Swollen Head Septicemia Virus with Indirect Sandwich ELISA

In order to rapidly diagnose and detect Duck Swollen Head Septicemia Virus antigen, the Indirect Sandwich ELISA for detection of the Duck Swollen Head Septicemia Virus was established with XD virus separated from Duck Swollen Head Septicemia. The optimization test conditions were 8μg/mL of the coating antibody, 0.5 % glutin of the assay buffer, antigen incubated for 1.5 h at 37 ℃, 5μg/mL of the concentration of rabbit anti XD,1:2000 of the dilution of HRP-labelled goat anti-rabbit IgG, 0.02 mol/L pH7.2 PBS of the wash buffer(dilution). The cutoff of OD at 490 nm wavelength was 0.412. The coefficient of variation was less than 10 %. The established Indirect Sandwich ELISA had no cross reaction to the negative antigen of duck swollen head septicemia virus, DPV,E.colibacillosis, DHV. The sensitivity was 1280 times than AGP. The coated polystyrene microtiter plates could be preserved for at least 3 weeks at 4 ℃. The Duck Swollen Head Septicemia virus antigen in various tissues of the infected ducks was detected with the method. The results demonstrated that positive rates were higher in heart, liver, lung, kidney than that in brain, spleen, pancreas.