免疫磁分离技术结合阻抗测量法快速检测禽流感病毒

Immunomagnetic Separation and Impedance Measurement for Rapid Detection of Avian Influenza Virus

采用免疫磁分离和阻抗测量技术联用的方法,实现了对禽流感H5亚型病毒的特异性快速检测。将偶联生物素的H5抗体固定于链霉亲和素修饰的纳米磁珠表面,制备成免疫磁珠,用于样品中禽流感H5N1病毒的分离和浓缩。使用金叉指阵列微电极测量样品阻抗,在特征频率(100 kHz)下,阻抗模值随样品中H5N1病毒浓度增加而增大。研究了抗体浓度、免疫反应时间和磁分离时间对阻抗信号的影响,结果表明,在优化的实验条件(抗体浓度0.25 g/L、免疫反应时间60 min、磁分离时间3 min)下,纯病毒样品和拭子样品中H5N1病毒浓度分别在2!1～24HA unit/50μL和20～24HA unit/50μL范围时,病毒浓度对数值与阻抗信号值呈线性相关关系,检出限分别为0.5 HA unit/50μL和2 HA unit/50μL。

A method for specific and rapid detection of H5 subtype avian influenza virus was developed based on immunomagnetic separation coupled with impedance measurement.The biotin-labeled anti-H5 monoclonal antibodies were immobilized onto the streptavidin-coated magnetic nanobeads to separate and concentrate avian influenza virus H5N1.Impedance measurement was performed by the gold interdigitated array microelectrodes.Impedance magnitude increased with the increase of the concentration of H5N1 virus in the sample solution at the frequency of 100 kHz.The effects of antibody concentration,immunoreaction time and magnetic separation time were investigated and the optimized conditions were 0.25 g/L antibody,60 min immunoreaction time and 3 min magnetic separation time.A linear relationship between the logarithmic value of concentration of H5N1 virus and the value of impedance signal was found in pure virus sample with concentration range of 21-24 HA unit per 50 μL and in swab sample with concentration of 20-24 HA unit per 50 μL.The detection limits in pure virus sample and swab sample were 0.5 HA unit per 50 μL and 2 HA unit per 50 μL,respectively.