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

项目摘要/摘要 高质量的(前期)临床研究依赖于高质量生物样品的使用；如果没有后者，它就是 不可能有前者。不幸的是，来自所有主要生物分子的与癌症相关的临床分析 班级暂时暴露于血浆或血清(P/S)时表现出不稳定性 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一般较低(如2-20%)，真正解冻的数量 等待发现的生物标记物的数量要低几个数量级--这意味着在被虐待的样本中 虚假线索的数量(即潜在的虚假发现)比博纳的数量大几个数量级 真正的生物标志物等着被发现。因此，每年，分析前样本处理不善和 存储在生物医学研究中产生了大量代价高昂的虚假线索，这是不可接受的。不出所料， 该领域的专家一致认为，必须立即将这一问题降至最低。 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，需要 10微米P/S的L称为ΔS-半胱氨酸-白蛋白，它提供了对血浆存档时间的估计 或者血清样本在相当于室温的条件下保存。这个测试效果很好，但它需要 昂贵的液色谱-质谱仪(LC-MS)仪器和运行它的用户专业知识。这个 对LC-MS的需求限制了大多数生物医学研究实验室的可获得性，这进一步阻碍了它的使用 除了大多数调查人员已经有了进行质量控制(QC)分析的有限激励之外 他们的P/S样本。幸运的是，我们发现控制ΔS-半胱氨酸的体外生物化学- 白蛋白标记物可以通过平板阅读器的吸光度或荧光测量来访问。 将ΔS半胱氨酸白蛋白分析转换为平板阅读器格式，将使P/S QC对 几乎所有的生物医学研究科学家。因此，该项目的目标是开发和分析验证 基于平板阅读器的吸光度和荧光法有效替代ΔS-半胱氨酸-白蛋白 化验。这将通过两个具体目标来实现： 具体目标1：开发基于平板阅读器的吸光度和荧光分析，通过量化Low 在分子量硫醇和二硫化物分子的P/S中，将能够检测到暴露的血浆和 将血清标本恢复到解冻条件(即温度&30°C)。 具体目标2：a)对分析方法进行分析，并对其进行初步分析验证。b) 胃肠道癌新鲜P/S样本及其时程等值的小人群调查 患者和非癌症年龄/性别匹配的对照组开始将这些检测与P/S接触时间联系起来 -20°C、4°C和23°C的解冻条件。

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.