Yan Li, Xu Yan-Wu, Wang Xiao-Liang, Wu Ye, Tian Jue, Yang Guang-Zhao, Ma Xiu-Rui, Liu Hui-Rong
Department of Physiology, Shanxi Medical University, Taiyuan 030001, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2009 Nov;25(4):569-72.
To prepare the working standards of 3-nitrotyrosine (3-NT) and establish a two-antibody-sandwich ELISA for determining the concentration of peroxynitrite in the tissue.
Nitrated bovine serum albumin was prepared by additions of an alkaline stock solution of peroxynitrite which was synthesized by a quenched-flow reactor. The monoclone anti-3-NT antibody from mouse was used as coating antibody and the polyclone anti-3-NT antibody from as labeling antibody to prepare the standard work curve by orthogonal design. The concentrations of 3-NT in cardiac tissue from rats subjected to myocardial ischemia and reperfusion (MI/R) were analyzed.
A two-antibody-sandwich ELISA method for measuring 3-NT content in biological fluids and homogenates was successfully established. The detecting limit was 0.1 ng x ml(-1) and the linear range of standard work curve was 0.15 - 7.50 ng x ml(-1) (r2 = 0.995). The 3-NT concentration in cardiac tissue from rats subjected to MI/R (1022.42 +/- 97.35 ng x mg pro(-1)) was significantly higher than that in the sham group (246.58 +/- 56.52 ng x mg pro(-1), P < 0.01).
A two-antibody-sandwich ELISA was established for determining the 3-NT concentration in the tissue and conveniently, quickly, accurately quantitative analysis of the content of 3-NT. The assay provides a new method for quantitative analysis of the peroxyinitrite in the future.
制备3-硝基酪氨酸（3-NT）工作标准品，建立双抗体夹心酶联免疫吸附测定法（ELISA）检测组织中过氧亚硝酸盐浓度。
通过加入由猝灭流动反应器合成的过氧亚硝酸盐碱性储备液制备硝化牛血清白蛋白。以小鼠抗3-NT单克隆抗体为包被抗体，抗3-NT多克隆抗体为标记抗体，采用正交设计制备标准工作曲线。分析心肌缺血再灌注（MI/R）大鼠心脏组织中3-NT的浓度。
成功建立了用于检测生物体液和匀浆中3-NT含量的双抗体夹心ELISA法。检测限为0.1 ng x ml(-1)，标准工作曲线的线性范围为0.15 - 7.50 ng x ml(-1)（r2 = 0.995）。MI/R大鼠心脏组织中3-NT浓度（1022.42 +/- 97.35 ng x mg pro(-1)）显著高于假手术组（246.58 +/- 56.52 ng x mg pro(-1)，P < 0.01）。
建立了双抗体夹心ELISA法测定组织中3-NT浓度，可方便、快速、准确地对3-NT含量进行定量分析。该测定法为今后过氧亚硝酸盐的定量分析提供了一种新方法。
