Chromatographic Automation Of Immunoassays

申请人的描述：许多药物的游离或非结合形式， 血液或血清中的激素是非常感兴趣的，因为这被认为 代表这些化合物的活性形式，使其成为理想的分析方法。 用于患者诊断或治疗的工具。但目前还没有一个将军， 快速测量体内游离溶质水平的方法。最近的工作 与L-甲状腺素和R/S-华法林的联合应用证明， 通过使用毫秒级的亲和提取可以进行测量， 在线层析免疫测定。该提案旨在进一步发展 并改进该方法以用于其他分析物。的第一部分 该项目将考虑这些测定的几种新应用（例如， 治疗监测和药理学研究）。 还将检查替代测定形式，例如1）使用LC/MS或 近红外荧光标记用于检测，2）使用单片 在亲和提取柱中的支持物，3）适体作为配体I的用途 对于这些列，和4）HPLC置换或免疫测定的发展 用于定量提取的药物和激素的测定。的第二部分 该项目将使用计算机模拟和模型系统来优化这些 方法.具体检查项目包括：1） 通过亲和色谱法分析游离药物/激素所需的提取 提取，2）当用于提取的多种结合剂时对这些测定的影响 溶质在样品中，和3）亲和力和解离动力学， 是用于这种测定的配体所需要的。最后一节将研究 血清或血浆中新分析物与其结合剂的相互作用， 有助于游离溶质测定的发展。这将涉及使用 基于HPLC、CE或LC/MS的几种亲和方法，用于1）鉴定试剂 在血清或血浆中与每种新游离组分候选物相互作用 测量，2）确定这些相互作用的平衡常数， 以及3）估计这些过程的稀有常数（例如，罕见的 分析物从其结合剂释放）。这项工作的结果应该是 创造新的、更快速的工具来确定游离药物或激素水平， 为研究这些化合物的行为和相互作用提供了更好的手段 在我们的身体里。

APPLICANT'S DESCRIPTION: The free, or non-bound, form of many drugs and hormones in blood or serum is of great interest since this is believed to represent the active form of these compounds, making it the ideal analytical tool for patient diagnosis or treatment. But there is currently no general, fast approach for measuring free solute levels within the body. Recent work with L-thyroxine and R/S-warfarin has demonstrated that free solute measurements are possible by using millisecond-scale affinity extractions and on-line chromatographic immunoassays. This proposal seeks to further develop and refine this approach for its use with other analytes. The first part of this project will consider several new applications for these assays (e.g., therapeutic monitoring and pharmacological studies). Alternative assay formats will also be examined, such as 1) the use of LC/MS or near-infrared fluorescent labels for detection, 2) the use of monolithic supports in affinity extraction columns, 3) the use of aptamers as ligands I for these columns, and 4) the development of HPLC displacement or immunometric assays for quantitating the extracted drugs and hormones. The second part of this project will use computer simulations and model systems to optimize these methods. Specific items to be examined will include: 1) the degree of extraction that is needed for free drug/hormone analysis by affinity extraction, 2) the effect on these assays when multiple binding agents for a solute are in a sample, and 3) the affinities and dissociation kinetics that are needed for ligands used in such assays. The last section will examine the interactions of new analytes with their binding agents in serum or plasma to aid in the development of the free solute assays. This will involve the use of several affinity methods based on HPLC, CE or LC/MS to 1) identify' the agents in serum or plasma that interact with each new candidate for free fraction measurements, 2) determine the equilibrium constants for these interactions, and 3) estimate the rare constants for these processes (e.g., the rare of analyte release from its binding agents). The result of this work should be the creation of new, more rapid tools for determining free drug or hormone levels, providing a better means for studying how such compounds behave and interact within our bodies.