新型鸭呼肠孤病毒σB和σC蛋白间接ELISA方法的建立

摘要

以纯化的新型鸭呼肠孤病毒（NDRV)NP03株重组σB 和σC 蛋白作为包被抗原，对反应条件进行优化，建立检测 NDRV抗体的间接 ELISA方法。优化后的最佳条件为：σB 蛋白包被浓度为12．5μg · mL－1；σC蛋白包被浓度为6．25μg·mL－1；封闭液为15％ FBS；血清稀释度为1∶80；酶标二抗稀释度为1∶400；底物37℃显色5 min。该方法对 MDRV、DHV、MPV 和 MD-GPV 阳性血清均无交叉反应，具有良好的特异性。该方法与 NDRV全病毒间接 ELISA相比较，符合率高达92．5％。本研究进一步丰富了 NDRV抗体的检测方法。%An indirect ELISA was developed using the purified recombinant novel duck reovirus (NDRV)σB andσC proteins as coating antigens. The optimized conditions for the methodology were determined to include:concentrations ofσB at 12.5μg·mL-1 andσC at 6.25μg·mL-1 ,a blocking buffer of 15% FBS,serum samples being diluted 80 times,goat anti-duck HRP-IgG being diluted 400 times,and substrate incubation at 37℃ for 5 min.The assay was found to be specific without any cross reaction with antibodies of MDRV,DHV,MPV,or MD-GPV.The coincidence rate between the indirect ELISA coated with the recombinantσB andσC proteins and that coated with NDRVwas 92.5%.It appeared that the newly developed ELISA exhibited satisfactory sensitivity and specificity on measurements,and could be an alternative means for detecting anti-NDRV antibodies.