基于核酸分子杂交的免疫芯片抗体固定新方法  

A New Method for Immobilizing Antibody Probes in Immunochips Based on DNA Hybridization

作　　者：沙莎[1] 殷赟[1] 高晓莲[2] 黄庄荣[3] 余挺[1] 郑晓冬[1] 

机构地区：[1]浙江大学生物系统工程与食品科学学院,杭州310058 [2]美国休斯顿大学化学系,德克萨斯州77004 [3]浙江大学农业与生物技术学院,杭州310058

出　　处：《分析化学》2013年第2期199-204,共6页Chinese Journal of Analytical Chemistry

基　　金：国家863计划目标导向性课题(No.2007AA10Z436);国家自然科学基金(No.30972051);国家转基因生物新品种培育重大专项(No.2009ZX08012-010B)资助

摘　　要：基于核酸分子杂交原理构建了一种新型抗体固定方法。先将抗体与寡核苷酸单链交联,再将两者的复合物与固相载体表面上的互补寡核苷酸链结合,从而将抗体固定到载体的表面。在磁珠表面对该固定方法进行实验,证明了方法的可行性。以本方法构建了针对转基因Bt Cry1Ac蛋白的免疫芯片,用Cy3标记二抗对其探针固定效果进行分析,并且在芯片上对Bt Cry1Ac蛋白进行梯度浓度检测试验。结果表明,以本方法构建的抗体芯片,探针分布具有良好的特异性;探针层分布均匀,非特异吸附小;检测灵敏度达到0.01～0.05!g/L;此外,通过杂交核酸双链的解离成功实现了芯片的再生,有助于解决传统抗体固定方法中芯片不可再生的问题。Based on complementary DNA strands hybridization, a method for immobilizing antibodies on immunochips was established. In this method, each kind of antibodies was conjugated to a specific sequence of oligonucleotide, and then was immobilized onto a complementary-oligonucleotide coated solid surface via base pairing. The method was first testified on magnetic beads surface. An immunochip against transgenic Bt CrylAc protein was further developed based on this in mobilization method. The immobilization outcome was analyzed using Cy3 labeled secondary antibody, and gradient concentrations of Bt Cryl Ac protein were tested on the developed chip. The result showed the chip using this immobilization method achieved good specificity, even distribution of antibody probes, little non-specific adsorption, and a detection limit of 0.01-0.05μg/L. Finally, the immunochip could be regenerated through dissociating the two DNA strands.

关 键 词：免疫芯片 抗体固定 抗体-寡核苷酸复合物 核酸杂交 

分 类 号：Q503[生物学—生物化学]