Itabe H, Yamamoto H, Imanaka T, Shimamura K, Uchiyama H, Kimura J, Sanaka T, Hata Y, Takano T
Department of Microbiology and Molecular Pathology, Teikyo University, Kanagawa, Japan.
J Lipid Res. 1996 Jan;37(1):45-53.
We have established a new method capable of measuring the very low concentrations of oxidized low density lipoprotein (OxLDL). In our previous study, we obtained a novel murine monoclonal antibody against oxidized lipoproteins (Itabe, H. et al. 1994. J. Biol. Chem. 269: 15274-15279). The epitope of this antibody resides in oxidized products of phosphatidylcholine that can form complexes with polypeptides, including apolipoprotein B. When the monoclonal antibody was precoated onto microtiter wells prior to carrying out a sandwich ELISA using an anti-human apolipoprotein B antibody, it was possible to detect 0.5 ng protein of copper-induced OxLDL. The detection of OxLDL was dependent on the presence of monoclonal antibody and was blocked by oxidized phosphatidylcholine (OxPC). Under the same sandwich ELISA condition, native LDL showed a dose-dependent increase of absorbance that was inhibited by complex of OxPC with BSA. These results suggest the possible occurrence of oxidative modification of human plasma LDL, which is recognized by the antibody against OxPC. The level of LDL oxidation of normal human subjects was found to be 0.52 +/- 0.35 units per 5 mu g protein of LDL, where one unit was defined as the reactivity corresponding to 1 ng of copper-induced OxLDL by this assay. Furthermore, we found that the LDL oxidation level in patients who had been receiving hemodialysis treatment was increased more than eightfold over that of normal subjects. We suggest that LDL in human plasma is oxidatively modified under certain conditions and this method for measurement of OxLDL could be used to study the relationship between in vivo oxidation reaction and various pathological conditions.
我们建立了一种能够测量极低浓度氧化型低密度脂蛋白（OxLDL）的新方法。在我们之前的研究中，我们获得了一种针对氧化脂蛋白的新型鼠单克隆抗体（伊塔贝，H.等人，1994年。《生物化学杂志》269: 15274 - 15279）。该抗体的表位存在于磷脂酰胆碱的氧化产物中，这些氧化产物可与包括载脂蛋白B在内的多肽形成复合物。在使用抗人载脂蛋白B抗体进行夹心ELISA之前，将单克隆抗体预包被在微量滴定孔上时，能够检测到0.5 ng蛋白质的铜诱导OxLDL。OxLDL的检测依赖于单克隆抗体的存在，并被氧化磷脂酰胆碱（OxPC）阻断。在相同的夹心ELISA条件下，天然LDL显示出吸光度的剂量依赖性增加，这被OxPC与牛血清白蛋白的复合物所抑制。这些结果表明人血浆LDL可能发生氧化修饰，这种修饰可被抗OxPC抗体识别。发现正常人类受试者的LDL氧化水平为每5μg LDL蛋白质0.52±0.35单位，其中一个单位被定义为通过该测定法与1 ng铜诱导的OxLDL相对应的反应性。此外，我们发现接受血液透析治疗的患者的LDL氧化水平比正常受试者增加了八倍以上。我们认为人血浆中的LDL在某些条件下会发生氧化修饰，这种测量OxLDL的方法可用于研究体内氧化反应与各种病理状况之间的关系。
