CHROMATOGRAPHIC AUTOMATION OF IMMUNOASSAYS

在临床样品中，药物的游离或非蛋白质结合部分，或 激素是非常感兴趣的，因为它代表了化合物的 生物活性形式。 这使得测量这一部分 是病人诊断和治疗的理想选择。 但目前的分析方法 对于这些测量（通常基于免疫测定）是缓慢的，费力的， 这是一个非常复杂的过程，不能通过现有的自动化系统来完成。 这是 由于必须测定的低浓度和需要 在分析之前分离分析物的结合部分和游离部分。 在 此外，在分离过程中结合的分析物解离的影响 这个过程并没有很好地理解。 本研究的目的是获得一个 更好地理解这种分离，并开发新的，自动化的 免费药物/激素测量系统。 解离效应将是 通过检测两种模型化合物甲状腺素 和皮质醇 用于自动化这些的游离药物/激素测定的系统 将基于高性能免疫亲和性开发化合物 色谱和荧光检测（HPIAC/CL）。 在该方法中， 将使用固定化抗体柱快速提取游离分析物 随后使用荧光法进行分析物检测， 吖啶酯标记。 本工作的第一部分将研究的相互作用动力学 甲状腺素和皮质醇及其结合蛋白。 会来做这项工作 研究其结合蛋白的保留和带宽， 它们通过含有固定的甲状腺素或皮质醇的柱。 目的是为了更好地理解解离效应， 免费药物/激素检测，并提供数据，以尽量减少这些影响 在HPIAC/CL系统中。 第二部分将研究所涉及的过程 在分析物吸附到固定化抗体柱中。 目标是 以确定所需的最佳色谱条件， 使用HPIAC/CL自动化游离药物/激素免疫测定。 这将是 研究使用计算机模拟和工作与模型系统， 伴刀豆球蛋白A与荧光和非荧光糖的结合。 的 最后一节将使用以前研究的数据来设计系统 基于HPIAC/CL进行游离甲状腺素和皮质醇测量。 一 将此方法与当前方法进行比较，以确定 潜在的优势和局限性。 该系统在 然后还将考虑测量其他药物/激素。

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.