Borovic Suzana, Rabuzin Filip, Waeg Georg, Zarkovic Neven
Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia.
Free Radic Res. 2006 Aug;40(8):809-20. doi: 10.1080/10715760600693422.
Highly reactive aldehyde 4-hydroxynonenal (HNE) is the final product of lipid peroxidation, known as a second messenger of free radicals and a signaling molecule. It forms protein conjugates involved in pathology of various diseases. To determine cellular HNE-protein conjugates we developed indirect ELISA based on well-known, monoclonal antibody against HNE-histidine (HNE-His) adducts. The method was calibrated using HNE-albumin conjugates as standards (R(2) = 0.999) and validated on human osteosarcoma cell cultures (HOS). The ELISA showed good sensitivity (8.1 pmol HNE-His/mg of protein), precision ( +/- 8% intra-assay and +/- 12% inter-assay) and spiking recovery ( +/- 9%). The assay revealed 60-fold increase of cellular HNE-His adducts upon copper-induced lipid peroxidation of HOS. The ELISA matched HNE-immunocytochemistry of HNE-treated HOS cells and quantified the increase of cellular HNE-His conjugates in parallel to the decrease of free HNE in culture medium. The ELISA was developed as ELISA Stress for severe lipid peroxidation and ELISA Fine for studies on HNE physiology.
高反应性醛4-羟基壬烯醛（HNE）是脂质过氧化的终产物，被称为自由基的第二信使和信号分子。它会形成参与各种疾病病理过程的蛋白质缀合物。为了测定细胞中的HNE-蛋白质缀合物，我们基于针对HNE-组氨酸（HNE-His）加合物的知名单克隆抗体开发了间接ELISA。该方法以HNE-白蛋白缀合物作为标准品进行校准（R² = 0.999），并在人骨肉瘤细胞培养物（HOS）上进行了验证。ELISA显示出良好的灵敏度（8.1 pmol HNE-His/毫克蛋白质）、精密度（批内±8%，批间±12%）和加标回收率（±9%）。该检测方法显示，在铜诱导的HOS脂质过氧化过程中，细胞中的HNE-His加合物增加了60倍。ELISA与HNE处理的HOS细胞的HNE免疫细胞化学结果相符，并定量了细胞中HNE-His缀合物的增加，同时培养基中游离HNE减少。该ELISA被开发为用于严重脂质过氧化的ELISA Stress和用于HNE生理学研究的ELISA Fine。
