Muller Charles H, Lee Tiffany K Y, Montaño Michalina A
Male Fertility Lab, Department of Urology, University of Washington School of Medicine, Seattle, WA, USA.
Methods Mol Biol. 2013;927:363-76. doi: 10.1007/978-1-62703-038-0_31.
An improved enhanced chemiluminescence antioxidant assay utilizes horseradish peroxidase conjugate and luminol to produce a cell-free oxygen radical generating system. We introduce the use of a peroxidase enzyme stabilizer to prolong the production of oxygen radicals at a steady rate. Addition of antioxidants temporarily interrupts oxygen radical generation, resulting in an inhibition curve. A linear relationship exists between the area of the inhibition curve and the molar quantity of added antioxidant used to quantify total nonenzymatic antioxidant capacity (TAC) in biological fluids including seminal plasma. We streamline the existing enhanced chemiluminescence technique by using a microtiter plate luminometer. A plate luminometer is as accurate as a tube luminometer in measuring TAC, using identical reaction volumes. As little as 1-50 μL of sample may be analyzed. A plate luminometer can detect molar Trolox equivalents as low as 12.5 μM, compared to 25 μM in tube luminometer, using identical volumes. The plate luminometer assay is made even more rapid with use of an injector.
一种改进的增强化学发光抗氧化剂测定法利用辣根过氧化物酶偶联物和鲁米诺来产生无细胞的氧自由基生成系统。我们引入了一种过氧化物酶稳定剂的使用，以稳定速率延长氧自由基的产生。添加抗氧化剂会暂时中断氧自由基的生成，从而产生一条抑制曲线。抑制曲线的面积与用于量化包括精浆在内的生物流体中总非酶抗氧化能力（TAC）的添加抗氧化剂的摩尔量之间存在线性关系。我们通过使用微孔板发光计简化了现有的增强化学发光技术。在使用相同反应体积的情况下，平板发光计在测量TAC方面与管式发光计一样准确。样品量低至1 - 50μL即可进行分析。与管式发光计使用相同体积时检测到的25μM相比，平板发光计可以检测低至12.5μM的摩尔Trolox当量。使用注射器可使平板发光计测定法更加快速。
