新型鸭呼肠孤病毒σB蛋白原核表达及间接ELISA抗体检测方法的建立

Prokaryotic Expression of Novel Duck Reovirus σB Protein and Establishment of an Indirect ELISA Antibody Detection Method

关键词:  新型鸭呼肠孤病毒  σB蛋白  原核表达  间接ELISA

Key words:  Novel duck reovirus  σB protein  Prokaryotic expression  Indirect ELISA

摘要:

为建立一种高效、便捷的新型鸭呼肠孤病毒(Novel duck reovirus，NDRV)抗体检测方法，以大肠杆菌表达系统表达的重组σB蛋白作为包被抗原，建立了NDRV抗体的间接ELISA检测方法。对该检测方法的反应条件进行优化，并进行特异性、敏感性和重复性试验以及临床样本检测。结果显示建立的间接ELISA检测方法的最佳反应条件为：0.5 μg/mL抗原4 ℃包被过夜，待检血清1:400稀释37 ℃孵育60 min，显色时间为15 min，Cut off值为0.432。该方法对新城疫病毒(NDV)、鸭圆环病毒(DuCV)、新型鹅细小病毒(NGPV)和鸭坦布苏病毒(DTMUV)阳性血清均不存在交叉反应，且检测阳性血清的敏感度为1:6400，批间和批内重复试验的变异系数均小于8％，该检测方法与市售NDRV抗体ELISA检测试剂盒的总符合率为96.88%。本研究成功建立了间接ELISA检测方法，为NDRV抗体检测提供了有效便捷的方法。

Abstract:

In order to establish an efficient and convenient method for the detection of novel duck reovirus (NDRV) antibodies, an indirect ELISA method for the detection of NDRV antibodies was developed using recombinant σB protein expressed by the E. coli expression system as the encapsulated antigen. The reaction conditions of the assay were optimized, and specificity, sensitivity and reproducibility tests as well as clinical samples were performed. The results showed that the optimal reaction conditions of the established indirect ELISA method were as follows: 0.5 μg/mL antigen was encapsulated at 4 ℃ overnight, the serum to be examined was diluted 1:400 and incubated at 37 ℃ for 60 min, the color development time was 15 min, and the value of Cut off was 0.432.The method did not cross-react to the positive sera of Newcastle Disease Virus (NDV), Duck Circovirus (DuCV), Novel Goose parvovirus (NGPV) and Duck Tambusu Virus (DTMUV), and the sensitivity of detecting the positive sera was 1:6400, with coefficients of variation of the inter- and intra-batch replicate tests of less than 8%, and the overall compliance rate of this assay with the commercially available ELISA kit for the antibody against NDRV was 96.88%.In this study, an indirect ELISA assay was successfully established, providing an effective and convenient method for NDRV antibody detection.