Selective targeting of human alkaline phosphatase isozymes

选择性靶向人碱性磷酸酶同工酶

• 批准号: 10117265
• 负责人: Chu-Young Kim
• 金额: $ 15.1万
• 依托单位: UNIVERSITY OF TEXAS EL PASO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-02 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10117265
• 关键词: Address; Affect; Affinity; Alcoholism; Alkaline Phosphatase; Antibodies; Aptamer Technology; Binding; Biological; Biological Assay; Blood; Blood Tests; Blood specimen; Carbohydrates; Chronic; Chronic Kidney Failure; Cirrhosis; Complex; DNA; Detection; Development; Diabetes Mellitus; Diagnosis; Diagnostic; Diphosphates; Disease; Enzymes; Evolution; Excision; Future; Germ Cells; Goals; Hepatitis; Human; Individual; Intestines; Isoenzymes; Kinetics; Laboratories; Lead; Ligands; Link; Liver Failure; Malignant neoplasm of liver; Malignant neoplasm of prostate; Measurement; Measures; Methods; Monoclonal Antibodies; Nucleic Acids; Organ; Patients; Pattern; Physicians; Protein Isoforms; Proteins; Reading; Reporting; Resolution; Sampling; Serum; Specificity; Spectrum Analysis; Structure; Surface Plasmon Resonance; Techniques; Testing; Tissues; United States National Institutes of Health; Work; aptamer; carbonate dehydratase; clinically relevant; cross reactivity; detection limit; disease diagnosis; gel electrophoresis; inorganic phosphate; novel; osteosarcoma; tool

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.

项目概要 该项目的目标是开发能够区分四种人类的核酸适体 碱性磷酸酶同工酶用于开发增强的碱性血液测试。 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高度相似。 在这些同工酶中，不可能产生具有 目前可用的碱性磷酸酶抗体均显示严格的同工酶特异性。 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 为了解决这个问题，我们将产生具有严格性的核酸适体。 使用指数富集配体系统进化 (SELEX) 来确定同工酶特异性。 适体的结合动力学将使用表面等离振子共振进行测量并纯化 适配体的检测限将使用 酶联适体测定。