细胞酶联反应法 (cell-ELA) 测定特异结合U87-EGFRvⅢ细胞的适配子的亲和力

Cell-ELA-based determination of binding affinity of DNA aptamer against U87-EGFRvIII cell

摘要:

通过细胞-指数级富集的配基系统进化 (Cell based systematic evolution of ligands by exponential enrichment，cell-SELEX) 方法从随机文库中筛选得到与稳定过表达人表皮生长因子受体Ⅲ型突变体 (Epidermal growth factor receptor variant Ⅲ，EGFRvⅢ) 的胶质瘤细胞U87细胞株 (U87-EGFRvⅢ) 特异结合的DNA适配子。以U87-EGFRvⅢ细胞作为检测对象，筛选到的适配子A15作为检测分子，建立一种细胞酶联反应 (Cell enzyme-linked assay，cell-ELA) 方法测定适配子的亲和力，并用EGFR抗体作为对照来比较此种DNA适配子的亲和力。结果显示，所测定的DNA适配子A15的解离平衡常数 (Equilibrium dissociation constants，Kd) 小于100 nmol/L，对U87-EGFRvⅢ细胞的结合能力与抗体相似。Cell-ELA法可较方便地用于cell-SELEX中适配子亲和力的测定。

关键词:细胞酶联反应法，细胞-指数级富集的配基系统进化，DNA适配子，解离平衡常数

Abstract:

A15, a DNA aptamer with binding specificity for U87 glioma cells stably overexpressing the epidermal growth factor receptor variant III (U87-EGFRvIII), was generated by cell systematic evolution of ligands by exponential enrichment (cell-SELEX) using a random nucleotide library. Subsequently, we established a cell enzyme-linked assay (cell-ELA) to detect the affinity of A15 compared to an EGFR antibody. We used A15 as a detection probe and cultured U87-EGFRvIII cells as targets. Our data indicate that the equilibrium dissociation constants (Kd) for A15 were below 100?nmol/L and had similar affinity compared to an EGFR antibody for U87-EGFRvIII. We demonstrated that the cell-ELA was a useful method to determine the equilibrium dissociation constants (Kd) of aptamers generated by cell-SELEX.

Key words:cell enzyme-linked assay (cell-ELA), cell based systematic evolution of ligands by exponential enrichment (cell-SELEX), DNA aptamer, Kd