基于新城疫病毒NP蛋白的间接ELISA抗体检测方法的建立

Development of an indirect ELISA for NDV antibody detection based on NP protein

采用原核表达纯化方法,获得建立新城疫病毒(NDV) LaSota株的核衣壳蛋白(NP)。将其作为包被抗原,通过优化反应条件,建立NDV间接ELISA抗体检测方法,并对其敏感性、重复性和特异性进行测定分析。该方法的检测灵敏度是血凝抑制试验(HI)的50倍,与H9亚型禽流感病毒等其他禽源病毒或细菌的阳性血清无交叉反应,组间重复性试验的变异系数小于6%。200份养殖场采集的鸡血清检测结果表明,ELISA方法与HI试验的检测符合率为97%。表明该方法可用于临床鸡血清的新城疫抗体检测。

An indirect ELISA assay for the detection of antibodies against Newcastle disease virus(NDV) was developed and applied to clinical serum samples.The nucleocapsid protein(NP) of NDV LaSota strain was obtained by prokaryotic expression and purification.The purified NP protein was used as coating antigen, an indirect ELISA for NDV antibody detection was established by optimizing the reaction conditions.The sensitivity of the ELISA was 50-fold higher than that of hemagglutination inhibition test(HI).There was no cross-reactivity with the positive serums against other avian pathogens, such as H9 subtype avian influenza virus.The co-efficient of variation of the ELISA was lower than 6%.A total of 200 chicken serum samples were collected from chicken farms and detected for NDV antibody using this assay.Results showed that the agreement between ELISA and HI test was 97%.Therefore, the NP-EILSA assay is sensitive and specific, it can be used for the detection of serum antibodies against NDV.