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游离或非结合部分引起临床和药物化学家的极大兴趣，因为认为该部分代表许多此类试剂的活性形式。因此，这些药物和激素的游离部分应该理想地作为患者诊断和治疗的工具进行测量。然而，目前还没有通用的和快速的方法来测量临床样品中的药物和激素的游离分数。本提案的总体目标是：1）更好地了解药物和激素与其在血液或血清中的结合剂的相互作用; 2）利用这些信息开发快速、可靠的游离药物和激素测定法。该项目的基本假设是，一个新的类的改进的游离药物和激素测定可以创建基于超快亲和提取和色谱免疫测定技术，如最近证明的化合物，如华法林，甲状腺素，苯妥英钠和卡马西平。未来的研究将建立在这些以前的努力，考虑新的分析物和方法，可以在这样的工作。特别强调的是高通量的方法和技术，可以适用于检测范围广泛的分析物。结合研究领域的进展将包括快速亲和方法的发展，用于检查药物或激素与血清试剂相互作用的程度和速率，以及使用亲和微柱作为个性化医疗工具来研究药物与血清蛋白变体相互作用的变化。用于游离药物和激素测量的改进工具将包括用于快速药物或激素提取的超高容量亲和支持物、具有柱上检测的色谱免疫测定、用于游离药物/激素测定的多维系统、使用血清蛋白柱进行游离药物/激素测定、以及当使用具有近红外荧光标记的色谱免疫测定时用于提高灵活性和样品通量的改进的分析平台。这项工作的直接影响将是更好地了解药物和激素如何与血清结合剂相互作用并在循环系统中运输。此外，为免费药物/激素测量而创建的新技术应大大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。这一能力应导致临床测试和个性化医疗方面更好的协议，用于治疗和诊断患有各种疾病的患者。本项目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，并测量痕量水平的此类分析物，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.