Generation of a polyclonal antibody against lipid peroxide-modified proteins.
Generation of a polyclonal antibody against lipid peroxide-modified proteins.
复制标题
生成抗脂质过氧化物修饰蛋白的多克隆抗体。
DOI：
10.1016/s0891-5849(96)00615-6
复制
发表时间：
1997
影响因子：
7.4
通讯作者：
Parthasarathy,S
中科院分区：
文献类型：
--
作者：
Kim,JG;Sabbagh,F;Santanam,N;Wilcox,JN;Medford,RM;Parthasarathy,S
在兔中产生针对脂质过氧化物（LOOH）修饰的兔血清白蛋白（RSA）的特异性多克隆抗体。该抗体以浓度依赖性方式选择性地识别修饰的蛋白质，并且不与环糊精修饰的蛋白质或用自由基产生剂2，2 ′-偶氮二（2-脒基丙烷）盐酸盐（AAPH）直接氧化的蛋白质交叉反应。抗体也以浓度依赖性方式识别氧化低密度脂蛋白（Ox-LDL），但不识别天然LDL。该抗体还与几种其他由LOOH修饰的蛋白质发生交叉反应，表明该抗体针对共同的表位，而不是针对蛋白质序列。正常人血浆的蛋白质印迹分析表明，至少有三种不同的蛋白质被抗体识别。RAW细胞，与LOOH预孵育，免疫染色的抗体和抗原表位存在于细胞内，而控制缺乏的一级抗体未能显示免疫反应性。来自胆固醇喂养的猴子和人类动脉粥样硬化病变的动脉粥样硬化动脉也被抗体免疫染色。免疫反应性共定位于富含泡沫细胞巨噬细胞的区域。这些结果表明，LOOH修饰的蛋白质呈现独特的抗原表位，其可能代表LOOH与蛋白质相互作用的主要产物。
A specific polyclonal antibody against the lipid peroxide (LOOH)-modified rabbit serum albumin (RSA) was generated in rabbits. The antibody selectively recognized the modified protein in a concentration-dependent manner and did not cross react with aldehyde-modified proteins or proteins directly oxidized with the free radical generator 2,2′-azobis (2-amidinopropane) hydrochloride (AAPH). Oxidized low-density lipoprotein (Ox-LDL), but not native LDL, was also recognized by the antibody in a concentration-dependent manner. The antibody also cross reacted with several other proteins modified by LOOH suggesting that the antibody is directed towards a common epitope and not towards the protein sequence. Western blot analysis of normal human plasma showed that at least three different proteins are recognized by the antibody. RAW cells, preincubated with LOOH, were immunostained with the antibody and the antigenic epitopes were present intracellularly, while controls lacking in the primary antibodies failed to show immunoreactivity. Atherosclerotic arteries from cholesterol-fed monkeys and human atherosclerotic lesions were also immunostained by the antibody. The immunoreactivity was co-localized in areas rich in foam cell macrophages. These results suggest that LOOH-modified proteins present an unique antigenic epitope that may represent a primary product of interaction of LOOH with proteins.