Improved method for the immunological detection of malondialdehyde-modified low-density lipoproteins in human serum

Oxidized low-density lipoproteins (ox-LDL) such as malondialdehyde-modified LDL (MDA-LDL) play an important role in the pathogenesis of atherosclerosis. This study is aimed to establish a standard enzyme-linked immunosorbent assay (ELISA) for measuring serum MDA-LDL, and evaluate its usefulness by analyzing serum samples of diabetes mellitus (DM) patients. Serum sample stability, analytical sensitivity, intra- and inter-assay precision, dilution linearity, supplementation and recovery, and interfering substances were examined. Normal reference levels in 86 healthy subjects (33 males and 53 females; average age 34.4(plus-minus)7.8 years) with normal lipid profiles were also determined. MDA-LDL levels in blood and serum were unstable and gradually increased during storage. However, it could be stabilized by the addition of a reagent, which included sucrose. The detection limit of the assay was 6.3 U/l. Intra- and inter-assay imprecisions were <5.6 and <9.4%, respectively. Excellent dilution linearity was observed, and the average recovery was 105%. None of the test substances, for example, hemoglobin, lipid and bilirubin interfered with the assay. The normal reference levels of MDA-LDL and MDA-LDL/LDL-C in the 86 subjects with normal lipid levels were calculated as 58.8(plus-minus)17.9 U/l and 60.4(plus-minus)11.9 mU/mg, respectively. The both serum levels of MDA-LDL (122(plus-minus)62.8 U/l) and MDA-LDL/LDL-C (96.8(plus-minus)48.5 mU/mg) in the DM patients were significantly higher than those of controls (P<0.0001). Moreover, DM patients with atherosclerosis complications showed significantly higher MDA-LDL and MDA-LDL/LDL-C levels than those without complications, 174(plus-minus)81.3 versus 101(plus-minus)39.2 U/l, (P<0.01) and 138(plus-minus)59.0 versus 80.3(plus-minus)32.1 mU/mg, (P<0.005), respectively. These results suggest that the ELISA method for MDA-LDL described in this study fulfilled the sensitivity, reproducibility, and accuracy requirements for routine clinical assays and might therefore be a useful tool for evaluating atherogenicity.