禽流感、新城疫可目视化诊断蛋白芯片的制备及初步应用

Preparation and preliminary application of a visual diagnostic protein chip for avian influenza and Newcastle disease

本研究制备了可以同时鉴别诊断禽流感(AI)、新城疫(ND)2种禽病血清抗体的可视化蛋白芯片。以超速离心法和蔗糖密度梯度法分别纯化了2种病毒蛋白抗原,用点样缓冲液稀释后点于醛基修饰的片基上,制备蛋白芯片;将芯片与待检血清杂交后,再与胶体金标记的二抗杂交,银染显色后根据灰度值判定结果,从而建立了2种禽病的可视化蛋白芯片鉴别诊断方法。采用该方法对18份现地血清样品进行初步检测,结果显示该芯片可特异性的与相应的抗体杂交,无交叉反应,而且呈现较强的杂交信号,其信号灰度值在阳性血清浓度为50μg/mL～300μg/mL范围内成线性关系(Y=0.426X+4.225)。用该芯片方法和琼扩(AGP)抗体检测方法对18份现地样品进行符合率检测,结果表明该可视化蛋白芯片具有很好的特异性,并且检测灵敏度为是AGP方法的400倍以上。研究表明该方法可用于同步鉴别2种禽病,是一种有效的抗体检测新方法。

To prepare a visual protein chip capable of detecting antibodies against avian influenza and Newcastle disease simultaneously, purified proteins from both viruses were spotted on a aldehyde group modified glass slides and hybridized with positive serum against each virus. The bound antibodies were detected with nano-gold labelled second antibody and developed by silver staining. This method was compared with HI assays by testing on 18 specimens. The results showed that antigens of both virus could be hybridized specifically with the probe of this protein chip under optimized conditions. The signal intensity showed a linear range in the concentration of 50 μg/mL to 300 μg/mL of antibodies. The results of protein chip and HI assay are highly consistent.