鹅细小病毒NS2蛋白间接ELISA方法的建立

Establishment of an indirect ELISA for detection of antibodies against NS2 protein of Gosling plague

为建立检测鹅细小病毒(GPV)的间接ELISA方法,本研究表达了GPV重组NS2蛋白,并以Ni-NTA亲和层析柱纯化的蛋白作为检测抗原,建立间接ELISA检测方法,并进行抗体消长规律的检测。用该方法检测阴性血清样本30份,确定检测临界值为0.281。与琼脂扩散试验结果的阳性符合率为100%,阴性符合率为86.67%。与抗鹅H5、H7、H9亚型禽流感病毒阳性血清和抗鹅副粘病毒阳性血清无交叉反应。在此基础上组装检测试剂盒,试剂盒批内变异系数为4.2%,批间变异系数为8.3%,包被抗原的酶标板在4℃可保存6个月。该方法对人工感染GPV血清检测结果表明,接种病毒后第8周,NS2蛋白的抗体水平达到峰值。本研究为GPV流行病学调查提供了一种快速、方便、敏感的抗体检测方法。

In this study, an indirect ELISA method for detection of Goose parvovirus （GPV） was developed by using purified recombinant NS2 protein of the virus as coating antigen. The assay had a sensitivity of 100 % for positive samples and 86.67 % for negative samples. It had no cross reaction with goose positive serum of H5, H7, H9 subtypes of avian influenza virus, and goose paramyxovirus. The coefficient variation of intro-batch was 4.2 % and the coefficient variation of inter-batches was 8.3 %. The ELISA plate could be validity for storage at 4 ℃ up to 6 months. This method was a rapidly and sensitive for the detection of GPV antibody and epidemiological survey.