免疫旋转生物传感器高灵敏检测HIV-1 P24抗原方法的研究

摘要

目的 通过构建免疫旋转生物传感器,建立一种高灵敏度的HIV-1 P24定量检测方法.方法 将生物素标记的HIV-1 P24单克隆抗体通过亲和素-生物素-β亚基抗体(Streptavidin-biotin-β antibody)的方式连接到光合作用复合体(Chromatophores)的β亚基上,同时用pH敏感的荧光索F1300标记到Chromatophores上,构建了用于检测P24的免疫旋转生物传感器,并用原核表达的P24抗原对该传感器进行了灵敏度、特异性实验.结果 构建的免疫旋转生物传感器可以将P24抗原结合ATPase造成的旋转速率变化转变为光强度的变化,可以高灵敏的检测HIV-1P24抗原,最低检出度可达0.1 fg/ml.结论 成功建立了基于免疫生物传感器(Immuno-rotary Biosensor,IRB)系统的HIV-1 P24检测方法,该方法操作简单,灵敏度高,为建立基于分子马达HIV-1 P24定量检测方法打下基础.%Objective To build a high sensitive method to detect HIV-1 P24 antigen quantitatively by Immuno-rotary Biosensor (IRB) based on F0F1-ATPase. Methods The immuno-rotary biosensor based on F0F1-ATPase in chromatophores for detecting HIV-1 P24 was assembled by label F0F1-ATPase chromatophores with the biotin-labled HIV-1 P24 antibody through the streptavidin-biotin-β antibody and the pH sensitivity of fluorescence F1300. Then it's sensitivity and specificity was tested by the prokaryotic expressed HIV-1 P24 antigen. Results Immuno-rotary biosensor based on F0F1-ATPase in chromatophores can detect P24 antigen sensitivily, the lowest level that could be detected was 0.1 fg/ml. Conclusion IRBbased system was successfully assembled and used for the detection of P24 antigen, the rapid and sensitive technique will be useful for detecting HIV-1 P24 antigen quantitatively.