ML 2-5

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.

We developed a sensitive enzyme-linked immunosorbent assay (ELISA) for detection of malondialdehyde-modified low-density lipoprotein (MDA-LDL) in human serum. A monoclonal antibody against MDA-LDL (ML25) used in our method recognized not only MDA-LDL but also other MDA-modified proteins. However, MDA-LDL was able to be detected specifically by using a combination of ML25 and an antibody specific for apolipoprotein B (apo B) (AB16), which was conjugated with beta-galactosidase. Using this method, measurable amounts of MDA-LDL were detected in the sera of 40 healthy individuals. MDA-LDL was observed to be mainly distributed in the human LDL fraction separated by density gradient ultracentrifugation, while in each lipoprotein subfraction the largest amount of MDA-LDL per protein was found at a subfraction between LDL and HDL. The particle size of LDL in this fraction was smaller than that of LDL in the main LDL fraction, as assessed by electrophoresis. In addition, LDL oxidized by Cu2+ was also detectable with this method. We conclude that our method is sensitive and specific for MDA-LDL and might be useful for investigating MDA-LDL in the human circulation.