Kotani K, Kondo A, Manabe M, Maekawa M, Kanno T
Department of Laboratory Medicine, Hamamatsu University School of Medicine.
Rinsho Byori. 1997 Jan;45(1):47-54.
Numerous studies have indicated that oxidative modification of low-density lipoprotein(LDL) plays a critical role in the pathogenesis of atherosclerosis. Malondialdehyde-modified LDL(MDA-LDL) is one of the candidates which is the oxidative product of LDL. However, the existence of MDA-LDL in the circulation has been in dispute. Therefore, for the assessment of oxidized-LDL in human serum, we developed a sensitive enzyme-linked-immunosorbent assay(ELISA) for the detection of MDA-LDL. In our method, monoclonal antibody against MDA-LDL, ML25 was used. ML25 recognized MDA-LDL as well as MDA-modified proteins by a solid-phase competitive enzyme immunoassay. Therefore, to establish an ELISA method which is specific for detection of MDA-LDL, ML25 was combined with apoB-specific antibody (AB16) as the second antibody. Using this method, MDA-LDL was detectable in the sera of 314 healthy individuals. The concentration of MDA-LDL preferably ranged from 20 to 80 units/l when the absorbance with artificially prepared MDA-LDL at the concentration of 1 mg/l was defined as 1 unit/l. Furthermore, assays for lipoprotein subfractions separated by density-gradient ultracentrifugation revealed that MDA-LDL was mainly distributed in the LDL fraction as expected, and MDA-LDL/apoB ratio showed a peak at small, dense LDL fractions. This finding seems to be quite interesting since an elevated level of small, dense LDL has been reported to be associated with an increased risk of atherosclerosis. We concluded that our method is useful for specific detection of MDA-LDL in human serum and might be an elevation method for atherogenicity.
众多研究表明，低密度脂蛋白（LDL）的氧化修饰在动脉粥样硬化的发病机制中起关键作用。丙二醛修饰的LDL（MDA-LDL）是LDL的氧化产物之一。然而，循环中MDA-LDL的存在一直存在争议。因此，为了评估人血清中的氧化型LDL，我们开发了一种灵敏的酶联免疫吸附测定法（ELISA）来检测MDA-LDL。在我们的方法中，使用了抗MDA-LDL的单克隆抗体ML25。ML25通过固相竞争酶免疫测定法识别MDA-LDL以及MDA修饰的蛋白质。因此，为了建立一种特异性检测MDA-LDL的ELISA方法，将ML25与载脂蛋白B特异性抗体（AB16）作为二抗结合使用。使用这种方法，在314名健康个体的血清中可检测到MDA-LDL。当将浓度为1 mg/l的人工制备的MDA-LDL的吸光度定义为1单位/l时，MDA-LDL的浓度优选在20至80单位/l范围内。此外，对通过密度梯度超速离心分离的脂蛋白亚组分的分析表明，MDA-LDL如预期的那样主要分布在LDL组分中，并且MDA-LDL/载脂蛋白B比值在小而密的LDL组分处显示出峰值。这一发现似乎相当有趣，因为据报道小而密的LDL水平升高与动脉粥样硬化风险增加有关。我们得出结论，我们的方法可用于特异性检测人血清中的MDA-LDL，并且可能是一种评估动脉粥样硬化性的方法。
