喷射式流动注射电化学发光免疫检测禽流感H9亚型

The detection of avian influenza virus H9 with jet flow injection electrochemiluminescence

利用磁分离和生物素亲和素技术,形成亲和素化磁微球-生物素化抗体-抗原-钌标抗体的免疫夹心复合物,初步建立了体外免疫诊断试剂的制备方法,并利用喷射式流动注射电化学发光体系对制备出的禽流感病毒H9免疫复合物进行检测。实验选择最适的包被抗体,检测抗体和封闭剂,优化Ru标抗体的最佳稀释度。在生物素化的兔抗H9多抗作为亲和素化的磁微球的结合抗体,鼠抗H9单抗作为Ru(bpy)32+标记抗体,2%BSA作为封闭剂,1:50倍稀释的Ru-鼠抗H9单抗条件下,非特异性吸附最低。测定不同浓度的H9抗原,发现抗原浓度在3.125～100μg/mL范围内与电化学发光强度呈较好的线性关系。实验还测定了不同亚型的禽流感病毒、不同来源的毒株和鸡的棉拭子样品。

Based on magnetic separation technology and biotin-avidin technology,the article prepared the streptavidin-coated magneticbeads-biotinylated antibody-antigen-Ru（bpy）32＋ labeled antibody was prepared,and a method for the detection of the prepared avian influenza virus H9 sandwich complex with jet flow injection electrochemiluminescence system was proposed.The choices of antibody and blocking reagent have been studied,and the results showed that choices of biotinylated rabbit polyclonal H9 antibody,Ru（bpy）32＋ labeled mouse monoclonal H9 antibody,2% BSA as blocking reagents could reduce the nonspecific adsorption.Different concentration of H9 have been detected,and the results showed that the ECL intensity was linear with the concentration of H9 in the range of 3.125～100 μg/mL.The high specificity and veracity of this method had been confirmed by detecting the avian influenza virus of different subtypes,avian influenza H9 virus of different sources and avian influenza samples.