OxPLs-Masking/Degradation Immune Assay: An All-Included Analysis of Mechanisms Detoxifying Oxidized Phospholipids

The manuscript discusses the use of circulating oxidized phospholipids (OxPLs) as potential disease biomarkers. Blood levels of OxPLs have been previously demonstrated to be strongly associated with cardiovascular disease. These data were obtained using an immune assay based on monoclonal antibody E06 which detects oxidized phosphatidylcholine. The impact of other classes of OxPLs on disease progression is not known. It is postulated that 1) other classes of OxPLs may contribute to cardiovascular disease and 2) both the absolute concentrations of OxPLs and the activity of detoxifying mechanisms are essential factors linking OxPLs with disease progression. Among such mechanisms is opsonization of OxPLs by endogenous antibodies and other plasma proteins. Furthermore, plasma contains enzymes degrading OxPLs. Experimental data from this study show that highly diluted blood plasma significantly reduces (masks) binding of E06, as well as another anti-OxPL monoclonal antibody that recognizes oxidized phosphatidylethanolamine, to OxLDL. Potentially, sequestration or degradation of OxPLs by plasma proteins, antibodies or enzymes can ameliorate pathogenic effects of OxPLs, thus preventing disease onset. Based on these considerations, it is hypothesized that analysis of blood levels of masking/degrading proteins may provide important additional information complementary to the E06-based assay and therefore improve its diagnostic value. Practical Applications: The manuscript discusses application of antibodies against OxPLs for their analysis as potential disease biomarkers. Plates are coated with OxLDL, blocked and incubated with either control buffer or plasma, which contains proteins and enzymes binding or degrading OxPLs present on OxLDL. Then the amount of remaining OxPLs is determined using monoclonal antibodies detecting OxPCs or OxPEs. The difference in signal intensities between the buffer- and plasma-treated wells represents the activity of OxPLs-masking/degradation.