Selective targeting of human alkaline phosphatase isozymes

项目摘要 本项目的目标是开发能够区分四种人类基因组的核酸适体。 碱性磷酸酶同工酶用于开发增强的血液测试。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 生殖细胞碱性磷酸酶，肠碱性磷酸酶，胎盘碱性磷酸酶 磷酸酶和组织非特异性碱性磷酸酶。由于结构高度相似 在这些同工酶中，还不可能产生具有特异性的单克隆抗体。 严格同工酶特异性。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性，这降低了它们在诊断中的有用性。 应用.为了解决这一问题，我们将产生具有严格限制性的核酸适体。 同工酶特异性使用指数富集配体系统进化（SELEX）。的 将使用表面等离子体共振测量适体的结合动力学 碱性磷酸酶同工酶适体的检测限将使用 酶联适体测定。

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.