Plate reader assays to forensically assess exposure of plasma and serum to thawed conditions

项目总结/摘要 高质量的临床（前）研究依赖于使用高质量的生物标本;没有后者， 不可能有前者。不幸的是，来自所有主要生物分子的癌症相关临床分析物 当它们所在的血浆或血清（P/S）暂时暴露于 解冻条件（即，温度>-30 °C）。尽管表现出不稳定性的生物分子的百分比 在通常遇到的解冻条件下通常是低的（例如2 - 20%）， 等待被发现的生物标志物的数量级更低，这意味着在错误处理的样本中， 错误引导的数量（即，潜在的错误发现）的数量比博纳的数量大几个数量级。 真正的生物标志物等待被发现。因此，每年，分析前样品处理不当， 存储在生物医学研究中产生了大量代价高昂错误线索，这是不可接受的。不出意料的是， 该领域的专家一致认为，必须立即尽量减少这一问题。 在NCI（IMAT）的支持下（R33CA217702），我们最近开发了一种简单、廉价、快速的检测方法， 10 µ L P/S（称为Δ S-Cys-白蛋白），提供血浆存档时间的估计值 或血清样本在室温下保存。该分析工作得很好，但它需要 昂贵的液相色谱-质谱仪（LC-MS）仪器和用户专业知识来运行它。 对LC-MS的需求限制了其对大多数生物医学研究实验室的可用性，这进一步抑制了其使用 除了大多数研究人员已经拥有的有限激励之外， 他们的P/S样本幸运的是，我们已经发现，控制Δ S-Cys- 白蛋白标记物可通过基于酶标仪的吸光度或荧光测量来获得。 将Δ S-Cys-白蛋白测定转换为基于酶标仪的格式将使P/S QC变得触手可及， 几乎所有的生物医学研究科学家。因此，本项目的目标是开发和分析验证 有效替代Δ S-Cys-白蛋白的基于酶标仪的吸光度和荧光测定 比色法这将通过两个具体目标来实现： 具体目标1：开发基于酶标仪的吸光度和荧光测定法，通过定量低 分子量硫醇和二硫化物轴承分子在P/S，将能够检测暴露的血浆和 将血清样品解冻（即，温度>-30 °C）。 具体目标2：A）分析表征检测试剂盒，并对其进行初步分析验证。（B） 对来自GI癌症的新鲜P/S样品及其时间过程等分试样进行小群体调查 患者和无癌症年龄/性别匹配的对照开始将这些测定与P/S暴露时间联系起来， 在-20 °C、4 ° C和23 ° C下解冻。

Project Summary/Abstract High quality (pre)clinical research relies on the use of high quality biospecimens; without the latter it is impossible to have the former. Unfortunately, cancer-relevant clinical analytes from all major biomolecular classes exhibit instability when the plasma or serum (P/S) in which they reside is temporarily exposed to thawed conditions (i.e., temperatures > -30 °C). Though the percentage of biomolecules that exhibit instability under commonly encountered thawed conditions is generally low (e.g. 2-20%), the number of genuine biomarkers waiting to be discovered is orders of magnitude lower—meaning that in mistreated samples the number of false leads (i.e., potential false discoveries) is orders of magnitude greater than the number of bona fide biomarkers waiting to be found. For this reason, every year, poor pre-analytical sample handling and storage generate unacceptably large numbers of costly false leads in biomedical research. Unsurprisingly, experts in the field agree that this problem must be minimized immediately. Under NCI (IMAT) support (R33CA217702) we recently developed a simple, inexpensive, rapid assay requiring 10 µL of P/S known as ΔS-Cys-Albumin that provides an estimate of the amount of time that archived plasma or serum samples have spent at the equivalent of room temperature. This assay works very well but it requires an expensive liquid chromatograph-mass spectrometer (LC-MS) instrument and user expertise to run it. The need for LC-MS limits its availability to most biomedical research labs which further dis-incentivizes its use beyond the limited incentives that most investigators already have to conduct quality control (QC) analysis on their P/S samples. Fortuitously, we have discovered that the ex vivo biochemistry that governs the ΔS-Cys- Albumin marker can be accessed via plate reader-based absorbance or fluorescence measurements. Converting the ΔS-Cys-Albumin assay to a plate reader-based format would put P/S QC at the fingertips of nearly all biomedical research scientists. As such, the goal of this project is to develop and analytically validate plate reader-based absorbance and fluorescence assays that effectively substitute for the ΔS-Cys-Albumin assay. This will be done via two Specific Aims: Specific Aim 1: Develop plate reader-based absorbance and fluorescence assays that, by quantifying low molecular weight thiol and disulfide-bearing molecules in P/S, will be able to detect exposure of plasma and serum specimens to thawed conditions (i.e., temperatures > -30 °C). Specific Aim 2: A) Analytically characterize the assays and perform an initial analytical validation of them. B) Conduct a small population survey of fresh P/S samples and time course aliquots thereof from GI cancer patients and cancer-free age/gender matched controls to begin to link these assays to P/S exposure time to thawed conditions at -20 °C, 4 °C, and 23 °C.