Toyokuni S, Miyake N, Hiai H, Hagiwara M, Kawakishi S, Osawa T, Uchida K
Department of Pathology, Faculty of Medicine, Kyoto University, Japan.
FEBS Lett. 1995 Feb 13;359(2-3):189-91. doi: 10.1016/0014-5793(95)00033-6.
Monoclonal antibodies directed against proteins modified with the major membrane lipid peroxidation product, 4-hydroxy-2-nonenal, have been established and characterized. The monoclonal antibodies specific for HNE-modified proteins were raised by immunizing mice with a HNE-keyhole limpet hemocyanin conjugate. The resulting five monoclonal antibodies (mAbs HNEJ-1-5) recognized HNE-modified bovine serum albumin (BSA), but not native BSA in Western blot studies. Of the five mAbs, HNEJ-2 exhibited the highest affinity for HNE-modified proteins and a much higher affinity for the HNE-histidine adduct than the HNE-lysine or HNE-cysteine adducts. mAb HNEJ-2 did not cross-react with proteins that had been treated with other aldehydes, such as 1-hexenal, 2-hexenal, 4-hydroxy-2-hexenal, 2-nonenal, formaldehyde, or glutaraldehyde. These results suggest that the major epitope recognized by mAb HNEJ-2 is the Michael addition-type HNE-histidine adduct.
针对由主要膜脂质过氧化产物4-羟基-2-壬烯醛修饰的蛋白质的单克隆抗体已被制备并进行了表征。通过用4-羟基-2-壬烯醛-钥孔血蓝蛋白缀合物免疫小鼠，产生了对4-羟基-2-壬烯醛修饰蛋白具有特异性的单克隆抗体。在蛋白质印迹研究中，所产生的五种单克隆抗体（单克隆抗体HNEJ-1-5）识别4-羟基-2-壬烯醛修饰的牛血清白蛋白（BSA），但不识别天然BSA。在这五种单克隆抗体中，单克隆抗体HNEJ-2对4-羟基-2-壬烯醛修饰的蛋白表现出最高亲和力，并且对4-羟基-2-壬烯醛-组氨酸加合物的亲和力比对4-羟基-2-壬烯醛-赖氨酸或4-羟基-2-壬烯醛-半胱氨酸加合物的亲和力高得多。单克隆抗体HNEJ-2与用其他醛类处理过的蛋白不发生交叉反应，这些醛类包括1-己烯醛、2-己烯醛、4-羟基-2-己烯醛、2-壬烯醛、甲醛或戊二醛。这些结果表明，单克隆抗体HNEJ-2识别的主要表位是迈克尔加成型的4-羟基-2-壬烯醛-组氨酸加合物。
