A Method for In Vitro Measurement of Oxidized Low-Density Lipoprotein in Blood,Using Its Antibody,Fluorescence-Labeled Heptapeptide and Polyethylene Glycol

Two oxidized forms of low-density lipoprotein (LDL), oxidized (Ox-LDL) and minimally modified (MM-LDL), and the immune complexes (LDL-ICs) that they form with their corresponding antibodies, play a major role in the pathogenesis of atherosclerosis. Recently, we reported that the heptapeptide KP6 (Lys-Trp-Tyr-Lys-Asp-Gly-Asp) coupled through its ε-amino group present on the N-terminal Lys to fluorescein isothiocyanate (FITC)— (FITC)KP6— binds specifically to Ox-LDL and MM-LDL, but not to native LDL. Here, to develop a novel method for measuring the levels of oxidatively modified LDL in blood, using (FITC)KP6, we analyzed the latter’s binding with MM-LDL-IC and Ox-LDL-IC. Polyacrylamide gel electrophoresis analysis revealed that (FITC)KP6 could efficiently and specifically bind to polyethylene glycol (PEG)-precipitated MM-LDL-IC and Ox-LDL-IC in a dose-dependent manner with high sensitivity in plasma and serum. Our results indicate that the above method for measuring the levels of PEG-precipitated, oxidatively modified LDL-ICs, formed by the addition of anti-Ox-LDL antibody to blood, using (FITC)KP6, can aid the diagnosis of atherosclerosis.