检测禽流感病毒的压电免疫芯片两种抗体固定方法的研究

Study on two immobilization methods of antibodies to avian influenza viruses for a piezoelectric immunosensor

目的探讨构建检测禽流感病毒压电免疫芯片的2种抗体固定方法。方法以AT切型、基频10 MHz的石英晶体为材料,分别采用聚乙烯亚胺-戊二醛交联法（简称戊二醛法）、葡萄球菌A蛋白（SPA）法将禽流感病毒H5亚型单克隆抗体固定在石英晶体的金膜电极表面,构建检测禽流感病毒压电免疫芯片。结果戊二醛法构建的压电免疫芯片检测H5抗原的线性范围为稀释度1∶2~1∶40,Y=75.814-13.748X,r2=0.978 9,与H9抗原不存在交叉反应,与鸡胚分离法的符合率达到91.7%。SPA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SPA结合,可以达到定性检测的效果。结论戊二醛法适合于构建禽流感病毒压电免疫芯片,其检测线性满足检测要求;SPA法在检测抗原时存在局限性,尚不能直接检测病毒抗原。

Objective To study two immobilization methods of antibodies to avian influenza viruses for a piezoelectric immunity protein chips.Methods Monoclonal antibody to avian influenza virus H5 subtype was deposited on the gold electrode′s surface of AT-cut 10 MHz quartz crystal by polyethyleneimine adhesion and glutaraldehyde cross-linking method（glutaraldehyde method） and staphylococcal protein A（SPA） method.Results By glutaraldehyde method,the linear range of H5 antigen dilution was between 1∶2-1∶40,Y=75.814-13.748X,r2=0.978 9.There was no cross-reaction to H9 antigen,and the coincidence was 91.7% compared with chick embryo separation.By SPA method,it wouldn′t detect the corresponding antigen if the antibody had been immobilized to the gold electrode first.However,the qualitative detection could be gotten by combining SPA quartz crystal with antigen-antibody compounds.Conclusions The glutaraldehyde method is applicable to establish the avian infuenza viruses piezoelectric immunity protein chip.It has better linearity.The SPA method has limitation and can′t detect the antigen directly.