Selective targeting of human alkaline phosphatase isozymes

项目摘要 该项目的目标是开发能够区分四种人类的核酸适配子。 用于加强血液检测的碱性磷酸酶同工酶。碱性 磷酸酶催化从蛋白质、核酸、 碳水化合物和无机焦磷酸盐。人类表达四种不同的碱性磷酸酶 同工酶；生殖细胞碱性磷酸酶、肠道碱性磷酸酶、胎盘碱性 磷酸酶和组织非特异性碱性磷酸酶。由于结构上的高度相似性 在这些同工酶中，还不可能产生具有 严格的同工酶专一性。目前可用的碱性磷酸酶抗体都显示 不同程度的同工酶交叉反应，这降低了它们在诊断中的有效性 申请。为了解决这个问题，我们将产生具有严格的 利用指数富集法系统进化配基的同工酶专一性(SELEX)。这个 使用表面等离子体共振技术测量适配子的结合动力学，并对其进行纯化 碱性磷酸酶同工酶。适配子的检测下限将使用 酶联适配子试验。

Project Summary The goal of this project is to develop nucleic acid aptamers that can discriminate the four human alkaline phosphatase isozymes for use in the development of enhanced blood tests. Alkaline phosphatases catalyze the removal of a phosphate group from proteins, nucleic acids, carbohydrates, and inorganic pyrophosphate. Humans express four distinct alkaline phosphatase isozymes; germ cell alkaline phosphatase, intestinal alkaline phosphatase, placental alkaline phosphatase, and tissue non-specific alkaline phosphatase. Due to the high structural similarity of these isozymes, it has not been possible to generate monoclonal antibodies that have a stringent isozyme specificity. Currently available alkaline phosphatase antibodies all display varying degrees of isozyme cross reactivity, which reduces their usefulness in diagnostic applications. To address this issue, we will generate nucleic acid aptamers that possess stringent isozyme specificity using systematic evolution of ligands by exponential enrichment (SELEX). The binding kinetics of the aptamers will be measured using surface plasmon resonance and purified alkaline phosphatase isozymes. The aptamer’s detection limit will be determined using an enzyme-linked aptamer assay.