压电免疫芯片检测禽流感病毒H9亚型抗原的方法研究

Study on the Detection Methods of H9 Subtype Avian Influenza Viruses Antigens by a Piezoelectric Immunosensor

目的为用压电免疫芯片检测禽流感病毒抗原寻找一种可行的抗体固定方法。方法以AT切型、基频10mHz的石英晶体为材料，分别采用聚乙烯亚胺～戊二醛交联法、葡萄球菌A蛋白法将禽流感病毒H9亚型单克隆抗体固定在石英晶体的金膜电极表面构建禽流感压电免疫芯片，用于检测禽流感病毒H9抗原。结果①聚乙烯亚胺-戊二醛交联法构建的压电免疫芯片检测禽流感病毒H9抗原的线性范围为稀释度1：10～1：160，Y=-8．868X＋73．514，r^2=0．9666，与H5抗原不存在交叉反应，与鸡胚分离法的符合率达到91．7％。②葡萄球菌A蛋白法若先固定抗体，则无法检测到抗原；如先进行抗原抗体反应再与葡萄球菌A蛋白结合，可以达到定性检测的效果。结论聚乙烯亚胺-戊二醛交联法适合于构建禽流感病毒H9亚型抗原压电免疫芯片，其检测特异度、重复性和线性均满足检测要求；葡萄球菌A蛋白法在检测抗原时存在局限性，尚不能直接检测抗原。

Objective To find a feasible immobilization method of H9 subtype avian influenza viruses （AIV） antibody for the detection of H9 antigen by a piezoelectric immunosensor. Methods H9 subtype AIV monoclonal antibody was deposited on the gold electrode＇s surface of 10 mHz quartz AT-cut crystal by polyethyleneimine adhesion and glutaraldehyde cross-linking method and SPA method to make the immunosensor. Results （1）PEI method ：the linear range of H9 antigen dilution was between 1 ： 10-1 ： 160,Y=-8. 868X＋73. 514,r^2=0. 966 6,no cross-reaction to H5 antigen,the coincidence was 91.7 % compared with chick embryo separation. （2）SPA method ：It wouldn＇t detect the corresponding antigen yet if the antibody had been immobilized to the gold electrode;the qualitative detection could be gotten by combining SPA quartz crystal with antigen-antibody compounds. Conclusion The PEI method is applicable to establish the H9 subtype AIV immunosensors,and it has better response to it＇s antigen,while the SPA method couldn＇t detect the antigen directly yet.