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。
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 （ r^2= 0. 995）. The 3-NT concentration in cardiac tissue from rats subjected to MI/R （ 1022.42 ±97.35 ng·ml^-1 ） was significantly higher than that in the sham group（246.58 ±56.52 ng·ml^-1, P 〈 0.01）. Conclusion： A two-antibody-sandwich ELISA was established for determiming 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.
出处
《中国应用生理学杂志》
CAS
CSCD
北大核心
2009年第4期569-572,共4页
Chinese Journal of Applied Physiology
基金
国家自然科学基金青年科学基金项目(30700276)
山西省青年科技研究基金项目(2007021048)
