Bevan Ruth J, Durand Mireille F, Hickenbotham Peter T, Kitas George D, Patel Parul R, Podmore Ian D, Griffiths Helen R, Waller Helen L, Lunec Joseph
Department of Clinical Biochemistry, University of Leicester, Leicester, UK
Free Radic Biol Med. 2003 Sep 1;35(5):517-27. doi: 10.1016/s0891-5849(03)00359-9.
The involvement of oxidatively modified low density lipoprotein (LDL) in the development of CHD is widely described. We have produced two antibodies, recognizing the lipid oxidation product malondialdehyde (MDA) on whole LDL or ApoB-100. The antibodies were utilized in the development of an ELISA for quantitation of MDA-LDL in human plasma. Intra- and inter-assay coefficients of variation (% CV) were measured as 4.8 and 7.7%, respectively, and sensitivity of the assay as 0.04 micro g/ml MDA-LDL. Recovery of standard MDA-LDL from native LDL was 102%, indicating the ELISA to be specific with no interference from other biomolecules. Further validation of the ELISA was carried out against two established methods for measurement of lipid peroxidation products, MDA by HPLC and F(2)-isoprostanes by GC-MS. Results indicated that MDA-LDL is formed at a later stage of oxidation than either MDA or F(2)-isoprostanes. In vivo analysis demonstrated that the ELISA was able to determine steady-state concentrations of plasma MDA-LDL (an end marker of lipid peroxidation). A reference range of 34.3 +/- 8.8 micro g/ml MDA-LDL was established for healthy individuals. Further, the ELISA was used to show significantly increased plasma MDA-LDL levels in subjects with confirmed ischemic heart disease, and could therefore possibly be of benefit as a diagnostic tool for assessing CHD risk.
氧化修饰的低密度脂蛋白（LDL）参与冠心病的发展已被广泛描述。我们制备了两种抗体，可识别完整LDL或载脂蛋白B-100（ApoB-100）上的脂质氧化产物丙二醛（MDA）。这些抗体被用于开发一种酶联免疫吸附测定法（ELISA），用于定量人血浆中的MDA-LDL。测定的批内和批间变异系数（%CV）分别为4.8%和7.7%，该测定法的灵敏度为0.04μg/ml MDA-LDL。从天然LDL中回收标准MDA-LDL的回收率为102%，表明该ELISA具有特异性，不受其他生物分子的干扰。针对两种已确立的脂质过氧化产物测定方法对该ELISA进行了进一步验证，即通过高效液相色谱法（HPLC）测定MDA，通过气相色谱-质谱联用仪（GC-MS）测定F2-异前列腺素。结果表明，MDA-LDL在氧化后期形成，比MDA或F2-异前列腺素都晚。体内分析表明，该ELISA能够测定血浆MDA-LDL的稳态浓度（脂质过氧化的终末标志物）。为健康个体建立了34.3±8.8μg/ml MDA-LDL的参考范围。此外，该ELISA用于显示确诊为缺血性心脏病的受试者血浆MDA-LDL水平显著升高，因此可能作为评估冠心病风险的诊断工具具有益处。
