CHROMATOGRAPHIC AUTOMATION OF IMMUNOASSAYS

免疫分析的色谱自动化

• 批准号: 2182873
• 负责人: DAVID S HAGE
• 金额: $ 9.16万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-13 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182873
• 关键词: adsorption; affinity chromatography; biomedical automation; blood chemistry; chemical association; chemical kinetics; computer simulation; concanavalin A; corticosteroid binding protein; cortisol; drug interactions; immunologic assay /test; luminescence; thyroid hormone binding protein; thyroxine

In clinical samples the free, or non-protein bound, fraction of a drug or hormone is of great interest since it represents the compound's biologically-active form. This makes the measurement of this fraction ideal for patient diagnosis and treatment. But current analytical methods for these measurements (typically based on immunoassays) are slow, labor- intensive, and cannot be performed by existing automated systems. This is due to the low concentrations which must be determined and the need to separate the bound and free fractions of the analyte prior to analysis. In addition, the effects of bound analyte dissociation during the separation process are not well understood. The aim of this study is to obtain a better understanding of this dissociation and to develop new, automated systems for free drug/hormone measurements. Dissociation effects will be studied by examining the protein binding of two model compounds, thyroxine and cortisol. Systems for automating free drug/hormone assays for these compounds will be developed based on high-performance immunoaffinity chromatography and chemiluminescent detection (HPIAC/CL). In this method, an immobilized antibody column will be used to rapidly extract free analyte from a sample, followed by analyte detection using chemiluminescent acridinium ester labels. The first section of this work will examine the interaction kinetics of thyroxine and cortisol with their binding proteins. This will be done by studying the retention and band-broadening of their binding proteins as they pass through columns containing immobilized thyroxine or cortisol. The goal is to obtain a better understanding of dissociation effects in free drug/hormone assays and to provide data for minimizing these effects in HPIAC/CL systems. The second section will study the processes involved in the adsorption of analytes to immobilized antibody columns. The goal is to determine what optimum chromatographic conditions are needed for automating free drug/hormone immunoassays using HPIAC/CL. This will be studied using computer simulations and work with a model system, the binding of concanavalin A to fluorescent and nonfluorescent sugars. The last section will use data from the previous studies to design systems based on HPIAC/CL for free thyroxine and cortisol measurements. A comparison of this approach to current methods will be made to determine its potential advantages and limitations. The use of this system in measuring other drugs/hormones will then also be considered.

在临床样品中，药物的免费或非蛋白质结合的部分或 激素引起了极大的兴趣，因为它代表了该化合物的 生物活性形式。 这使得该部分的测量 是患者诊断和治疗的理想选择。 但是当前的分析方法 对于这些测量（通常是基于免疫测定），是缓慢的，劳动的 密集，无法由现有自动化系统执行。 这是 由于必须确定的低浓度，并且需要 在分析之前，分析物的结合和游离分数。 在 此外，分离期间结合分析物解离的影响 过程不太了解。 这项研究的目的是获得 更好地理解这种解离并发展新的自动化 免费药物/激素测量的系统。 解离效应将是 通过检查两种模型化合物甲状腺素的蛋白质结合来研究 和皮质醇。 自动化免费药物/激素测定的系统 化合物将根据高性能免疫亲和力开发 色谱和化学发光检测（HPIAC/CL）。 在这种方法中， 固定抗体柱将用于快速提取免费分析物 从样品中，然后是使用化学发光的分析物检测 丙啶酯标签。 这项工作的第一部分将检查 甲状腺素和皮质醇及其结合蛋白。 这将由 研究其结合蛋白的保留率和带囊 它们穿过含有固定的甲状腺素或皮质醇的柱。 目的是更好地了解分离效应 免费药物/激素分析，并提供数据以最大程度地减少这些影响 在HPIAC/CL系统中。 第二部分将研究涉及的过程 在吸附分析物对固定抗体柱的吸附。 目标是 确定需要哪些最佳色谱条件 使用HPIAC/CL自动化免费药物/激素免疫测定。 这将是 使用计算机模拟研究并与模型系统一起工作 乳褐化膜A与荧光和非荧光糖的结合。 这 最后一部分将使用以前研究的数据来设计系统 基于HPIAC/CL的游离甲状腺素和皮质醇测量。 一个 将对这种方法与当前方法进行比较以确定 它的潜在优势和局限性。 该系统在 然后，还将考虑测量其他药物/激素。