【摘要】 目的建立一种单克隆抗体介导的、经生物素—亲和素系统放大的H5亚型禽流感病毒捕获ELISA检测方法,为进一步研究检测试剂盒提供基础。方法用亲和层析法纯化抗禽流感病毒H5亚型血凝素单克隆抗体,包被微量反应板,用于捕获病毒抗原,再用生物素标记的单抗和酶标亲和素来检测病毒血凝素抗原,经方阵试验优化ELISA反应体系。用该方法检测H1—H15亚型AIV标准毒株和H5、H7、H9亚型AIV分离株,并与血凝和血凝抑制试验比较,评价其敏感性和特异性。结果纯化后的单抗具有良好的反应活性,生物素标记单抗工作浓度为1∶5000;ELISA对H5亚型AIV的检出限为025个血凝单位。该ELISA反应体系能检出H5N3标准株和所有20株国内H5亚型AIV分离株,而与其他14个血凝素亚型的AIV标准株、15个H9亚型AIV分离株和2个H7亚型AIV分离株均无交叉反应。结论初步建立了检测H5亚型禽流感病毒的单抗—生物素捕获ELISA方法,为研制试剂盒和进一步应用试验提供了基础。更多还原

【Abstract】 Objective To establish H5 avian influenza virus (AIV) capture ELISA technique employing biotin labeled monoclonal antibody (Mab) as the basic work to develop ELISA Kit for detecting of H5 avian influenza. Method The anti_H5 AIV Mab was purified with SPG column Chromatogram, and coated in ELISA plates as capturing antibody. The captured AIV was detected by biotin_Mab and streptavidin_HRP. The optimal reaction system was selected by matrix assay. AIV reference strains H1_H15, and H5, H7, H9 isolates were detected to estimate the ELISA system. The result was compare to HA and HI tests. Results After purification, Mab remains good activation. The working dilution ratio of Biotin_Mab was 1∶5000 The sensitivity of the capture ELISA system was 0 25HA unit. And the ELISA could detect H5N3 reference strain and all the 20 H5 AIV isolates, but not react with all the other 15 H9 and 2 H7 isolates. Conclusion The H5 Avian Influenza Virus Capture ELISA was basically founded to enable the ELISA Kit come into being.更多还原