Molecular Dynamics Section, National Institute on Aging, Baltimore, MD 21224, USA.
Anal Biochem. 2010 May 15;400(2):289-94. doi: 10.1016/j.ab.2010.01.032. Epub 2010 Feb 1.
Oxidative stress, originating from reactive oxygen species (ROS), has been implicated in aging and various human diseases. The ROS generated can oxidize proteins producing protein carbonyl derivatives. The level of protein carbonyls in blood plasma has been used as a measure of overall oxidative stress in the body. Classically, protein carbonyls have been quantitated spectrophotometrically by directly reacting them with 2,4-dinitrophenylhydrazine (DNPH). However, the applicability of this method to biological samples is limited by its low inherent sensitivity. This limitation has been overcome by the development of sensitive enzyme-linked immunosorbent assay (ELISA) methods to measure protein carbonyls. As part of the Healthy Aging in Neighborhoods of Diversity across the Lifespan (HANDL) study, oxidative stress in humans was quantified by measuring blood plasma protein carbonyls using the two commercially available ELISA kits and the spectrophotometric DNPH assay. Surprisingly, two ELISA methods gave very different values for protein carbonyls, both of which were different from the value of the spectrophotometric method. We have developed a fluorescent semi-microplate format assay of protein carbonyls involving direct reaction of protein carbonyls with fluorescein thiosemicarbazide that correlates (R=0.992) with the direct spectrophotometric method. It has a coefficient of variation of 4.99% and is at least 100 times more sensitive than the spectrophotometric method.
氧化应激源于活性氧（ROS），与衰老和各种人类疾病有关。生成的 ROS 可以氧化蛋白质，产生蛋白质羰基衍生物。血浆中蛋白质羰基的水平已被用作衡量体内整体氧化应激的指标。经典方法是通过直接与 2,4-二硝基苯肼（DNPH）反应来分光光度法定量蛋白质羰基。然而，由于其固有灵敏度低，该方法在生物样本中的适用性受到限制。通过开发敏感的酶联免疫吸附测定（ELISA）方法来测量蛋白质羰基，克服了这一限制。作为多样性在整个生命周期中的邻里健康老龄化（HANDL）研究的一部分，通过使用两种市售的 ELISA 试剂盒和分光光度法 DNPH 测定法来测量血浆蛋白羰基，定量了人类的氧化应激。令人惊讶的是，两种 ELISA 方法对蛋白质羰基的测量值差异很大，两者都与分光光度法的值不同。我们开发了一种涉及蛋白质羰基与荧光素硫代卡巴肼直接反应的荧光半微量板格式蛋白质羰基测定法，与直接分光光度法相关（R=0.992）。它的变异系数为 4.99%，比分光光度法至少灵敏 100 倍。
