一种更灵敏的特异性检测H9亚型禽流感抗体的间接ELISA方法的建立

摘要

H9 subtype avian influenza virus causes worldwide epidemic,resulting in enormous economic losses of poultry production.In the present study,an indirect ELISA method was established for more accurate and specific detection.The recombinant protein of the globular head domain of HA of H9 subtype avian influenza virus was used as antigen.Specific blocking buffers and dilution buffers were determined to increase the sensitivity and specificity.The sensitivity of ELISA was higher than that of hemagglutination inhibition (HI) test.The coating antigen is very specific and no cross-reactivity with positive serum against H3N2,H5N2 and H7N9 subtype influenza viruses,Newcastle disease virus,avian infectious bronchitis virus,avian infectious disease virus,and egg drop syndrome virus.Two hundred of clinical sera samples were examined.The results indicate the coincidence rate between ELISA and HI test reached 97％.In addition,there was a positive correlation between OD450 values and the logarithm of HI titer to the base 2 of an individual serum sample (R2=0.981 1).%H9亚型禽流感在全球范围内广泛流行,控制其传播需监测H9亚型禽流感病毒的感染情况及疫苗的免疫效果.为了建立便于检测且灵敏特异的H9亚型禽流感抗体间接ELISA方法,本实验利用不同亚型之间变异较大的H9亚型禽流感病毒HA蛋白的头部球状区作为包被抗原,确定了最佳复合封闭液和抗体稀释液,提高了其特异性.结果显示建立的ELISA方法灵敏度高于血凝抑制试验(HI),且与H3N2、H5N2、H7N9亚型流感病毒及新城疫病毒(NDV)、鸡传染性支气管炎病毒(IBV)、鸡传染性法氏囊病毒(IBDV)和产蛋下降综合征病毒(EDSV)的阳性血清均无交叉反应.另外,利用该方法及HI试验对200份临床鸡血清样本进行检测,两种检测方法的符合率达97％,且存在较高的相关性(R2=0.9811).