【摘要】 为建立一种快速检测鸭甲肝病毒(DHAV)1型和3型(DHAV-1、DHAV-3)血清抗体的方法,本研究诱导表达了DHAV-1、DHAV-3重组蛋白VP0、VP1、VP3,并采用ELISA方法比较了3者的反应原性,结果显示DHAV-1 VP0更适合作为检测DHAV-1、DHAV-3血清抗体的通用型诊断抗原。经各反应条件优化,建立了一种基于DHAV-1 VP0重组蛋白的可同时检测DHAV-1、DHAV-3血清抗体的间接ELISA方法。优化的主要反应条件为:抗原包被浓度为0.25μg/m L,待检血清稀释度为1100,山羊抗鸭HRP-IgG 1400稀释。该方法特异性较强,只与DHAV-1、DHAV-3阳性血清反应,与鸭源小鹅瘟病毒(GPV)、鸭病毒性肠炎病毒(DEV)、H9N2亚型禽流感病毒(H9N2 AIV)、鸭坦布苏病毒(DTMUV)等阳性血清均无交叉反应;敏感性为1320;批内变异系数小于4%,批间变异系数小于6%;与商品化的ELISA抗体检测试剂盒比较,二者阳性符合率为93.3%,阴性符合率为88.9%,总符合率为95.7%,且该方法可用于临床血清样本的检测。本研究为检/监测DHAV血清抗体水平提供了技术手段。更多还原

【Abstract】 In order to establish a rapid serological method for detecting the antibodies against Duck hepatitis A virus(DHAV)type 1 and type 3(DHAV-1, DHAV-3), the reactionogenicities of recombinant proteins VP0, VP1 and VP3 with DHAV-1 and DHAV-3 were compared by using ELISA assays. The result showed that the DHAV-1 VP0 could react with serum antibodies against both DHAV-1 and DHAV-3 serving as a universal antigen. After the optimization of reaction conditions, an indirect ELISA was established based on DHAV-1 VP0 recombinant protein for the antibodies detection of DHAV-1, DHAV-3. The mainly optimized reaction conditions were as following: the concentration of coated antigen was 0.25 μg/m L, and the dilutions of serum and goat against duck HRP-Ig G were 1.100 and 1.400 respectively. The specificity assay showed that the ELISA only detected antibodies against DHAV-1 and DHAV-3 but had no cross-reaction with other serum antibodies against goose parvovirus(GPV), duck enteritis virus(DEV), H9 N2 subtype avian influenza virus(H9 N2 AIV) and duck tambusu virus(DTMUV). The sensitivity was 1.320 of DHAV-3 positive serum, and the coefficient variability of intra-batch was less than 4% and inter-batch was less than 6%. Compared with commercial DHAV ELISA antibody detect kit, the coincidences of positive and negative rate were 93.3% and 88.9%,respectively, and the total coincidence rate was 95.7%. The results suggested that the indirect ELISA could detect the clinical DHAV serum samples. This study provid a technological method to detect and/or monitor DHAV serum antibody level for drafting effective preventative measure.更多还原