Discrimination between minimally modified LDL and fully oxidized LDL using monoclonal antibodies.

Abstract Low density lipoprotein (LDL) can be oxidized in a stepwise process that leads to the production of minimally modified low density lipoprotein (mm-LDL), in which only the lipid component is oxidized, and then of fully oxidized LDL (oxLDL), in which both the lipids and the protein are oxidized. The thiobarbituric reactive substances (TBARS) assay is a recognized method for determination of oxidized LDL, however this method is unable to distinguish between mm-LDL and oxLDL. In this study, seven specific monoclonal antibodies (mAbs) against human LDL were generated and selectively bound to the apolipoprotein B-100 (apoB-100) component of LDL. Oxidized LDL was produced by incubation of human LDL with 10 μM CuSO4 for various times. The TBARS assay revealed that the optimal incubation time to achieve maximal lipid oxidation was 9 h. Indirect ELISA using the newly generated mAbs was implemented to differentiate between mm-LDL and oxLDL and it was found that binding of the mAbs to oxLDL was significantly decreased after 48 h of incubation, reflecting the oxidative modification of apoB-100. Our results suggest that the optimal times for incubation of LDL with CuSO4 for generation of mm-LDL and oxLDL were 9 h and 48 h, respectively.