鸡传染性贫血重组抗原间接ELISA诊断方法的建立

Development of an indirect ELISA assay for detecting chicken infectious anemia virus antibody with recombinant antigen

本研究应用重组抗原,成功建立了检测鸡传染性贫血病毒血清抗体的间接ELISA诊断方法。确定了抗原最适包被浓度为5μg/mL,血清最适稀释度为1∶100,其作用时间为60 min,酶标抗体最适作用时间为60 min,S/P≥0.24为阳性,≤0.19为阴性,介于二者之间为可疑的判定标准。该抗原不与鸡其他疾病的10种阳性血清反应,具有良好的特异性;批内重复试验,变异系数小于10%,批间重复试验,变异系数小于15%,具有良很好的重复性;该方法可检出人工接毒后20 d的血清抗体,直到试验结束110 d时,仍能检出鸡血清阳性率为81%与全病毒包被抗原ELISA方法的检测结果符合率达92%以上,与进口诊断试剂盒的符合率为97%。本研究为现地免疫鸡群抗体检测和进行CIA流行病学调查提供了一种简便的血清学诊断方法。

We utilized recombinant antigen and developed an indirect ELISA assay for detecting CIAV antibodies. The optimal conditions were determined for the ELISA. The concentration of antigen was 5μg/mL. The dilution of sera was 1 ： 100. The reaction time of sera was 60 rain. The reaction time of conjugate was 60 rain. The standard of judgment was that S/P≥0.24 was positive and S/P≤0.16 was negative.The recombinant antigen had no cross reaction with other 10 sera of avian diseases,and it showed this assay was highly specific.The assay could detect antibodies in the chicken inoculated CIAV from 20th to 110th day.At the end of test,81% positive rate was detected in the 110th-day-sera.The result suggested the assay was sensitive. In the intro-batch duplicativity test, the variation coefficient is less than 10 % and in the inter-batch duplicativity test, the vartiation coefficient is less than 15 %. The result showed the assay had good replicativity. When compared with antigen from cultures in MDCC-MSB1, above 92 % agreement was obtained; in compare with the commercial Kit, 97 % agreement was obtained. The application of indirect ELISA assay would provide a simple and rapid means of detecting anti-CIAV in monitoring CIAV infection or assessing vaccination in the field.