Chromatographic Automation of Immunoassays

描述(由申请人提供)：血液或血清中许多药物和激素的自由或非结合部分引起临床和药物化学家的极大兴趣，因为这一部分被认为代表许多此类药物的活性形式。因此，理想情况下，应该测量这些药物和激素的游离部分，作为患者诊断和治疗的工具。然而，目前还没有一种通用的、快速的方法来测量临床样品中药物和激素的游离部分。这项建议的总体目标是1)更好地了解药物和激素与其在血液或血清中的结合剂之间的相互作用，以及2)利用这些信息开发快速、可靠的免费药物和激素检测方法。该项目的基本假设是，可以基于超快亲和提取和层析免疫分析技术创建一类新的改进的游离药物和激素分析，就像最近对华法林、甲状腺素、苯妥英和卡马西平等化合物所证明的那样。未来的研究将在这些先前努力的基础上，考虑可用于此类工作的新的分析物和方法。将特别强调可适用于检测各种分析物的高通量方法和技术。在结合研究领域有待探索的进展将包括开发快速亲和方法来检查药物或激素与血清试剂相互作用的程度和速度，以及将亲和微柱用作个性化医学中研究药物与血清蛋白质变体相互作用的变化的工具。用于免费药物和激素检测的改进工具将包括用于快速提取药物或激素的超高容量亲和力载体、带柱上检测的层析免疫分析、用于免费药物/激素检测的多维系统、使用血清蛋白质柱进行免费药物/激素检测，以及改进分析平台，以提高使用带有近红外荧光标记的层析免疫分析的灵活性和样品吞吐量。这项工作的直接影响将是更好地理解药物和激素如何与血清结合剂相互作用，以及如何在循环系统中运输。此外，将为免费药物/激素测量创造的新技术将极大地提高临床医生和药物化学家研究和检查药物和激素在体内的生物效应的能力。这一能力将导致更好的临床测试方案和个性化药物，用于治疗和诊断患有各种疾病的患者。在这个项目中开发的各种基于亲和力的方法，用于检查溶质-配体结合，从生物样品中快速提取分析物，并测量这种分析物的痕量水平，在许多生化研究领域也应该是有价值的，包括蛋白质组学、代谢组学和候选药物的表征或筛选。

DESCRIPTION (provided by applicant): The free, or non-bound, fraction of many drugs and hormones in blood or serum is of great interest to clinical and pharmaceutical chemists because this fraction is believed to represent the active form of many such agents. As a result, it is the free fraction of these drugs and hormones that should ideally be measured as a tool for patient diagnosis and treatment. However, there is currently no general and fast approach for measuring the free fractions of drugs and hormones in clinical samples. The overall goals of this proposal are 1) to obtain a better understanding of drug and hormone interactions with their binding agents in blood or serum and 2) to use this information to develop fast, reliable free drug and hormone assays. The underlying hypothesis of this project is that a new class of improved free drug and hormone assays can be created based on the techniques of ultrafast affinity extraction and chromatographic immunoassays, as recently demonstrated for compounds such as warfarin, thyroxine, phenytoin and carbamazepine. Future studies will build on these previous efforts by considering new analytes and approaches that can be employed in such work. Particular emphasis will be given to high-throughput methods and techniques that can be adapted for the detection of a broad range of analytes. Advances to be explored in the field of binding studies will include the development of rapid affinity methods for examining both the extent and rate of drug or hormone interactions with serum agents, and the use of affinity microcolumns as tools in personalized medicine to study changes in drug interactions with variants of serum proteins. Improved tools to be created for free drug and hormone measurements will include ultra-high capacity affinity supports for use in fast drug or hormone extractions, chromatographic immunoassays with on-column detection, multi-dimensional systems for free drug/hormone assays, the use of serum protein columns to carry out free drug/hormone assays, and improved analytical platforms for improving the flexibility and sample throughput when using chromatographic immunoassays with near infrared fluorescent labels. An immediate impact of this work will be a better understanding of how drugs and hormones interact with serum binding agents and are transported in the circulatory system. In addition, the new techniques that will be created for free drug/hormone measurements should greatly improve the ability of clinicians and pharmaceutical chemists to study and examine the biological effects of drugs and hormones in the body. This capability should lead to better protocols in clinical testing and in personalized medicine for the treatment and diagnosis of patients suffering from a variety of disorders. The various affinity-based approaches developed in this project for examining solute-ligand binding, rapidly extracting analytes from biological samples, and measuring trace levels of such analytes should also be valuable in many areas of biochemical research, including proteomics, metabolomics and the characterization or screening of drug candidates.