4种禽病毒抗体可视化蛋白芯片的制备及应用

Production and Preliminary Application of a Visual Protein Chip to Differentiate Antibodies of Four Poultry Diseases

【目的】研制可同时鉴别检测AI、ND、IB、IBD等4种禽病血清抗体的可视化蛋白芯片。【方法】用超速离心法和蔗糖密度梯度法分别纯化了4种病毒蛋白抗原,调整到合适浓度后点于醛基修饰的片基上,制备蛋白芯片;分别用4种禽病阳性血清杂交,再与金标二抗杂交,银染显色后读取结果,建立4种禽病的可视化蛋白芯片工作平台;进一步对此芯片的特异性以及灰度值与抗体浓度的相关性进行了研究;最后对18个现地血清样品进行了初步应用检测。【结果】结果显示经过条件优化后,芯片检测探针可特异性的相应的抗体进行杂交,呈现较强的杂交信号,且无交叉杂交。信号灰度值在抗体浓度为50～300μg·ml-1范围内成线性关系。用芯片和其它传统抗体检测方法同时检测18份现地样品,结果发现可视化蛋白芯片具有很好的特异性,并且灵敏性明显高于传统检测手段。【结论】研究表明该方法可用于同步鉴别4种禽病抗体,是一种有效的抗体检测新方法。

【Objective】 To produce a visual protein chip that can detect antibodies against avian influenza virus, newcastle disease virus, infectious bronchitis virus and infectious bursal disease virus simultaneously. 【Method】 To prepare protein chips, the four kinds of virus protein were purified, respectively, by ultracentrifugation and sucrose density gradient centrifugalization method , and then they were spotted on a aldehyde group-modified glass slides with a proper concentration. The four kinds of virus protein on the protein chip were reacted with the corresponding positive serum against these viruses, and then were hybridized with nano-gold labeled second antibody. The result was obtained after silver staining. The platform of a visual protein chip to differentiate antibodies of four poultry diseases was constructed. Furthermore, specificity and the correlation of the gray value scale with the antibody concentration of this visual protein chip were studied. Finally, the visual protein chip was used to test 18 field serum specimens. 【Result】 The results showed that after the conditions were optimized, the probe of this protein chip could be hybridized with corresponding antibodies specifically and strong signal was presented without cross-hybridization. The gray value scale showed linear in the concentration range of antibodies at 50-300 μg·ml^-1. This protein chip showed good sensibility and sensitivity compared with traditional method when they were used to detect 18 serum samples simultaneouly. 【Conclusion】 The protein chip reported here can be used to differentiate antibodies of four poultry diseases.