鼻气管鸟杆菌间接ELISA检测方法的建立及应用  

Establishment and application of indirect ELISA method for detection of Ornithobacterium rhinotracheale

作　　者：李瑶瑶[1] 刁有祥[1] 吴海洋[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018

出　　处：《中国兽医学报》2011年第3期334-337,共4页Chinese Journal of Veterinary Science

基　　金：山东省自然科学基金资助项目(Y2006D10)

摘　　要：提取A型鼻气管鸟杆菌(ORT)外膜蛋白,经纯化后包被反应板,通过方阵滴定确定最佳抗原包被浓度、血清稀释倍数、兔抗鸡酶标二抗稀释倍数,建立了检测ORT抗体的间接ELISA方法。经方阵滴定确定最佳抗原包被质量浓度为11.85mg/L,血清样品稀释倍数为1:20,兔抗鸡IgG辣根过氧化物酶结合物最佳稀释倍数为1:1000,抗原抗体最佳结合时间为1h,血清与二抗最适反应时间为45min。试验结果确定的判定标准为:样品D_(490nm)≥0.639判定为阳性,D_(490nm)≤0.350判定为阴性,介于两者之间的为可疑。试验证明该方法具有良好的稳定性、重复性和特异性,将205份疑似ORT的鸡血清用本方法进行检测,其阳性检出率为11.7%(24/205)。Outer membrane protein（OMP） was prepared and purified from standard strain ORT of type-A,and through optimizeation for concentration of coating antigen,dilution of serum,dilution of HRP IgG by chessboard titration test to develop an indirect ELISA for detection of serum antibodies against ORT.Result of chessboard titration test showed that the optimal concentration of coating antigen was 11.85 mg/L.The optimal dilution of serum was 1：20.The optimal dilution of HRP IgG was 1：1 000,the optimal combination of antigen with antibody was 1 h,and the optimal combination of serum with HRP IgG was 45旋min.The criteria for evaluation were as follows： the serum was positive if D_（490 nm）≥0.639,the serum was negative if D_（490 nm）≤0.350.The serum was suspicious if D_（490） nm was between 0.350 and 0.639.The method showed stability,repeatability and specificity.The indirect ELISA detected a total of 205 serum samples originated from chickens to suspect ORT and the positive efficacy was 11.7%（24/205）. This research could provide a sensitive,specific and convenient method for diagnosing ORT.

关 键 词：鼻气管鸟杆菌 外膜蛋白 间接ELISA 检测 

分 类 号：S852.61[农业科学—基础兽医学]