Chromatographic Automation Of Immunoassays

申请人描述：许多药物和药品的免费或非绑定形式 血液或血清中的激素引起了极大的兴趣，因为这被认为是 代表这些化合物的活性形式，使其成为理想的分析工具 用于病人诊断或治疗的工具。但目前还没有将军， 测量体内游离溶质水平的快速方法。近期工作 用L-甲状腺素和R/S-华法林证明了自由溶质 通过使用毫秒级的亲和力提取和 在线层析免疫分析。这项建议旨在进一步发展 并改进这一方法，以便与其他分析物一起使用。第一部分 该项目将考虑这些分析的几个新应用(例如， 治疗监测和药理学研究)。 还将研究替代的分析格式，例如1)使用LC/MS或 近红外荧光标记用于检测，2)使用整体式 亲和萃取柱中的载体，3)适配子作为配基的使用 对于这些柱，以及4)高效液相置换或免疫测定法的发展 提取的药物和激素的定量分析。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：研究方法。具体审查项目包括：1) 亲和分析游离药物/激素所需的提取 提取，2)当多个结合剂用于一种 溶质在样品中，以及3)亲和力和解离动力学 在这类分析中使用的配体是必需的。最后一节将研究 新分析物与其在血清或血浆中的结合剂相互作用 协助开发自由溶质分析方法。这将涉及到使用 基于高效液相、毛细管电泳法或液质联用法的几种亲和力方法：1)鉴定药物 在血清或血浆中与每个新的游离组分候选相互作用 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 分析物从其结合剂中释放)。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.