【摘要】 目的:建立一种准确、快速的双抗体夹心酶免疫吸附的方法,以定量检测组织中过氧亚硝基阴离子的水平。方法:分别以小鼠源性抗3-硝基酪氨酸单克隆IgG抗体和兔源性抗3-硝基酪氨酸IgG抗体为包被抗体和检测抗体,采用正交设计方法摸索以上各抗体的浓度,建立定量检测3-硝基酪氨酸(3-NT)的双抗体夹心ELISA法。同时,测定心肌缺血再灌注大鼠心肌组织3-NT的含量。结果:本研究建立的双抗体夹心ELISA法检测3-NT的最低检测下限为0.10 ng.ml-1,具有良好线性关系的检测范围是(0.15～7.50)ng.ml-1(r2=0.995);心肌缺血再灌注组大鼠的心肌组织中的3-NT水平为(1022.42±97.35)ng.mg pro-1,明显高于假手术组(246.58±56.52 ng.mg pro-1,P<0.01)。结论:本研究建立的定量检测3-NT的双抗体夹心ELISA法能够方便、准确、快速地检测组织中3-NT的含量,为定量检测组织中ONOO-的水平提供了新的方法。更多还原

【Abstract】 Aim: 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.Methods: 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.Results: 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·ml-1 and the linear range of standard work curve was 0.15～7.50 ng·ml-1(r2=0.995).The 3-NT concentration in cardiac tissue from rats subjected to MI/R(1022.42±97.35 ng·mg pro-1) was significantly higher than that in the sham group(246.58±56.52 ng·mg pro-1,P<0.01).Conclusion: 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.更多还原