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。
Abstract:
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(E1protein).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 at1∶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 1virus 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.
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