Antiphospholipid antibodies are directed against epitopes of oxidized phospholipids. Recognition of cardiolipin by monoclonal antibodies to epitopes of oxidized low density lipoprotein.

Abstract

The optimal clinical management of patients with antiphospholipid antibody syndrome (APS) is uncertain because of a lack of an underlying hypothesis to explain why antiphospholipid autoantibodies (aPL) form to such ubiquitous compounds as phospholipids (PL). In this paper, we demonstrate that many, if not most, aPL are actually directed at neoepitopes of oxidized PL, or neoepitopes generated by adduct formation between breakdown products of oxidized PL and associated proteins. Each cardiolipin (CL) molecule contains four unsaturated fatty acids and is highly susceptible to oxidation, particularly upon exposure to air. Yet, standard anticardiolipin antibodies (aCL) immunoassays routinely bind CL to microtiter wells by evaporation of the ethanol solvent overnight at 4 Њ C. Using a variety of techniques, we demonstrated that rapid oxidation occurs when CL is plated and exposed to air. Sera from apo E-deficient mice, which have high autoantibody titers to oxidized low density lipoprotein, showed a striking time-dependent increase in binding to CL that was exposed to air for increasing periods of time. Monoclonal antibodies to oxidized LDL, cloned from the apo E-deficient mice, also bound to oxidized CL. Both sera and affinity-purified aCL-IgG from APS patients bound to CL progressively as it was oxidized. However, the monoclonal antibodies from apo E-deficient mice, or sera or aCL-IgG from APS patients did not bind to a reduced CL analog that was unable to undergo peroxidation. These data demonstrate that many aPL are directed at neoepitopes of oxidized phospholipids, and suggest that oxidative events may be important in the pathophysiology of APS. In turn, this suggests new therapeutic strategies, possibly including intensive antioxidant therapy.

C-reactive protein binds to both oxidized LDL and apoptotic cells through recognition of a common ligand: Phosphorylcholine of oxidized phospholipids

“…CRP binding to antigens was determined by using a chemiluminescent immunoassay (20). In brief, antigens in PBS containing 0.27 mM EDTA were added to each well of a 96-well, white, roundbottomed microtitration plate (Dynex Technologies, Chantilly, VA) and incubated overnight at 4°C.…”

Immunization of LDL Receptor–Deficient Mice With Homologous Malondialdehyde-Modified and Native LDL Reduces Progression of Atherosclerosis by Mechanisms Other Than Induction of High Titers of Antibodies to Oxidative Neoepitopes

“…16,38 In this assay, 5 g/mL of the antigen in 50 mmol/L Tris-buffered saline (TBS), pH 7.5, containing 0.27 mmol/L EDTA, 0.02% NaN 3 , and 20 mol/L BHT (dilution buffer) was added to each well of a 96-well, white, round-bottomed MicroFluor microtitration plate (Dynex Technologies) and incubated overnight at 4°C. Plates were washed 4 times with washing buffer (TBS containing 0.27 mmol/L EDTA, 20 mol/L BHT, 0.02% NaN 3 , and 0.001% aprotinin) in an automated plate washer.…”

“…We recently demonstrated that the standard assays used to measure "anticardiolipin" antibodies actually measure antibodies to oxidized cardiolipin (OxCL). 38 In standard assays, cardiolipin is added to microtiter wells in ethanol and is plated by overnight evaporation of the solvent. Plated cardiolipin exposed to air is rapidly oxidized within a few minutes.…”

“…Plated cardiolipin exposed to air is rapidly oxidized within a few minutes. 38,39 To standardize the extent of oxidation, bovine heart cardiolipin (Avanti Polar Lipids) was diluted to 20 g/mL in 100% ethanol, and 25 L was added to each well. Plates were dried under air at room temperature (Ϸ30 minutes) and air exposure was then continued for another hour.…”

Determinants of Bioactivity of Oxidized Phospholipids

“…7,8 The levels of these lipids were increased in atherosclerotic lesions in rabbit aortas, and natural antibodies to these oxidized phospholipids were present in the sera of apo E-null mice. 8,9 We have observed several differences in action between POVPC and PGPC. POVPC induced monocyte binding and connecting segment-1 fibronectin expression, increased the levels of cAMP, and inhibited lipopolysaccharide (LPS)-induced E-selectin expression and neutrophil binding.…”