Analytical validation of monoclonal antibody-based ELISA methods for OxPL-apoB and OxPL-apo(a)

Oxidized phospholipids (OxPL) are bioactive lipid species that circulate bound to apolipoprotein B-100 [apoB] and apolipoprotein(a) [apo(a)] and have been widely studied as biomarkers of oxidative lipid burden. When bound to apolipoprotein B-100 [OxPL-apoB] and apolipoprotein(a) [OxPL-apo(a)], they serve as informative biomarkers for CVD risk prediction, risk reclassification, and therapeutic monitoring, particularly in studies involving RNA-targeted therapies against lipoprotein(a). To date, measurement of OxPL-apoB and OxPL-apo(a) has been limited to research-use assays performed in an academic laboratory without formal clinical laboratory validation. Here we report the first full CLIA-compliant analytical validation of chemiluminescent ELISA methods for OxPL-apoB and OxPL-apo(a), enabling their implementation in a regulated clinical reference laboratory setting. The OxPL-apoB ELISA employs murine monoclonal IgG antibody MB47 to capture apoB-100-containing lipoproteins, while the OxPL-apo(a) employs murine monoclonal IgG antibody LPA4 to capture apo(a)-containing particles. In both assays, OxPL is detected by murine monoclonal IgM antibody biotin-E06. The concentration of OxPL is determined against a standard curve of phosphocholine (PC) equivalents using PC-modified bovine serum albumin. The analytical measuring range of both assays is 1.48-148.48 nmol/L PC-OxPL. Serum and plasma matrices showed minimal bias and were analytically equivalent. In healthy donors, OxPL-apoB levels ranged from <1.48 to 25.23 nmol/L PC-OxPL (mean 4.18, median 1.79 nmol/L), while OxPL-apo(a) levels ranged from <1.48 to 126.94 nmol/L PC-OxPL (mean 31.04, median 6.90 nmol/L), with strong correlation to Lp(a) concentrations (R² = 0.82). These assays provide robust tools for quantifying proatherogenic and pro-inflammatory OxPL-lipoprotein complexes in clinical, translational, and pharmacological research settings.