鸭坦布苏病毒E蛋白包被抗原间接ELISA方法的建立  

Indirect ELISA based on Protein E from Duck Tembusu Virus

作　　者：傅秋玲[1] 陈珍[1] 施少华[1] 傅光华[1] 程龙飞[1] 陈红梅[1] 万春和[1] 林建生[1] 黄瑜[1] 

机构地区：[1]福建省农业科学院畜牧兽医研究所,福建福州350013

出　　处：《福建农业学报》2015年第1期1-5,共5页Fujian Journal of Agricultural Sciences

基　　金：国家现代农业产业技术体系建设专项(CARS-43);国家自然科学基金项目(31201936);福建省种业创新与产业化工程项目(2011FJZY-9);福建省农业科学院导师制青年基金项目(2013DQB-3)

摘　　要：以鸭坦布苏病毒E蛋白的主要优势抗原表位区E1蛋白作为包被抗原,建立检测鸭坦布苏病毒中和抗体效价的间接ELISA方法。优化后确定最佳的抗原包被浓度为1.548mg·L-1,血清的最佳稀释度为1∶100,酶标二抗的稀释度为1∶6 000。特异性表明,用建立的ELISA方法对禽流感、新城疫病毒、1型鸭肝炎病毒、胰腺型鸭甲肝病毒、禽霍乱和鸭疫里默氏杆菌的阳性血清进行检测,均未发生交叉反应;批内和批间重复试验的平均变异系数都小于10%;敏感性达1∶3 200。该方法与以全病毒为包被抗原的间接ELISA检测结果相关性达到95.1%。利用该方法对237份来自福建省开产麻鸭的血清样品进行检测表明,其血清阳性率达77.9%,表明福建省开产麻鸭感染鸭坦布苏病毒阳性率很高。本试验建立的间接ELISA检测方法具有特异性强,重复性好,敏感性高,为鸭坦布苏病毒抗体检测提供了简单快捷的检测方法。An indirect enzyme-linked immunosorbent assay (ELISA ) method was developed to detect serum antibodies for duck Tembusu virus (DTV).ELISA plates were coated with the main antigenic determinant of the envelop proteins (E1 protein).The optimized test conditions included:concentration of E1 coating on the ELISA plate of 1. 548 mg · L-1 ,serum sample dilution at 1∶100,and HRP-labeled goat anti-duck IgG dilution at 1∶6 000.The specific tests showed no cross-reactions on anti-sera against the avian influenza virus,Newcastle disease virus,duck hepatitis virus-I,duck hepatitis type 1 virus with pancreatitis,pasteurellosis or riemerella anatipestifer,indicating a high specificity of the ELISA against DTV.The coefficients of variation of the intro- and inter-batch duplicability tests were below 10%.The sensitivity was 1∶3 200.Compared with the indirect ELISA coated with DTV,the coincidence rate of indirect ELISA was 95. 1%.Out of the 237 laying duck serum samples collected from Fujian,194 (i.e.,77. 9% of the total)were found positive on the indirect ELISA.The newly developed indirect ELISA methodology appeared to have desirable specificity,repeatability,as well as sensitivity, and could potentially be applied for routine DTV detection.

关 键 词：鸭坦布苏病毒 E蛋白 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]