双抗体夹心ELISA检测羊肠道病毒抗原方法的建立及病原流行病学调查

Development of a sandwich ELISA for detection of CEV antigen and epidemiological investigation

应用本实验室分离的国内首株羊肠道病毒(CEV)-JL14VP1重组蛋白制备的兔源多克隆抗体和抗CEV-JL14病毒的单克隆抗体,建立了检测CEV抗原的双抗体夹心ELISA方法,并对吉林省和内蒙古地区羊群感染CEV进行了病原流行病学调查。方阵试验确定了CEV VP1鼠源IgG捕获抗体的最佳包被量为0.2μg/孔,酶标抗体的最佳稀释倍数为1∶1 000。对大量CEV阴性粪便样品进行检测与统计学处理,确定了双抗体夹心ELISA检测CEV的判定标准为样品D490值≥0.216判定为阳性。特异性、敏感性和重复性试验结果表明,所建立的检测CEV抗原方法具有特异、敏感、快速和重复性好等特点。以建立的双抗体夹心ELISA方法,对吉林省和内蒙古不同地区的羊群粪便样品进行了检测,发现不同地区的羊群都存在着严重的CEV感染,感染率高达12%~100%。本研究为今后CEV感染的诊断、检疫及防制提供了有效的手段和流行病学理论依据。

Enterovirus infection in sheep/goat is an emerging infectious disease worldwide, which poses a severe threat to the development of sheep/goat industry. In this study,we reported the de- velopment of a double antibody sandwich ELISA for detecting caprine/ovine enterovirus antigens using the antibodies generated against the recombinant proteins VP1 of caprine enterovirus JL14 （CEV-JL14） ,and investigated the CEV infections in sheep/goat herds in Jilin province and Inner Mongolia Automonous region. The concentrations of coating-antibody and HRP-conjugated anti- VP1 were optimized using square matrix titrimetry. The criterion for sandwich ELISA was deter- mined and sample was assessed as positive when its D490≥0. 216. The established ELISA had high sensitivity,specificity and simplicity. Epidemiological survey demonstrated that CEV infection in sheep/goat herds in Jilin province and Inner Mongolia Automonous region was up to 12%-100%. The findings in this study will provide a sensitive, specific, simple and rapid antigen detection method for diagnosis and epidemiologic survey of CEV infection,and an invaluable data for preven tion and control of this emerging infectious diseases.