Generation, Characterization, and Histochemical Application of Monoclonal Antibodies Selectively Recognizing Oxidatively Modified ApoB-Containing Serum Lipoproteins

Abstract

To investigate either the role oxidized LDL plays in atherosclerosis or structural changes on the surface of oxidized LDL, monoclonal antibodies (mAbs) are an important tool. After immunizing mice with Cu(2+)-oxidized LDL (oxLDL) and fusion of splenocytes, hybridoma supernatants were screened and cloned. Two mAbs, OB/04 and OB/09 (IgG and IgM), were further characterized. In solid-phase fluorescence immunoassays and Western blot analysis both mAbs reacted with oxLDL, LDL oxidized by a free radical-generating azo compound, or oxVLDL but not with native LDL, acetylated LDL, oxHDL3, azo-oxidized HDL3, or HDL3 modified with malondialdehyde (MDA). In competitive immunoassays with LDL modified by oxidized fatty acid-derived aldehydes, mAb OB/09 strongly reacted with MDA-LDL or MDA-VLDL and LDL modified with 4-hydroxyhexenal followed by 4-hydroxynonenal but not with 4-hydroxyoctenal or hepta-2,4-dienal. mAb OB/04 had a weak affinity for LDL after modification with these aldehydes except for MDA-LDL. LDL modified with arachidonic acid oxidation products (AAOPs) was also recognized by this mAb. However, albumin modified either by the aldehydes applied or by AAOPs did not react with either mAb. Thus, the data indicate that each of the mAbs recognizes a different epitope that is expressed only on apoB-containing lipoproteins upon oxidative modification. An immunostaining with mAb OB/04 was obtained in areas rich in macrophages and in connective tissue of a human atherosclerotic lesion.

Immunohistochemical Demonstration of Enzymatically Modified Human LDL and Its Colocalization With the Terminal Complement Complex in the Early Atherosclerotic Lesion

“…27,55,56 Hammer et al 57 similarly showed cellular staining for ox-LDL in macrophage foam cells in one cryosection of human atherosclerotic tissue from the femoral artery in a case of advanced arterial occlusive disease. In rabbit studies by Haberland et al 13 and Boyd et al, 12 monoclonal antibodies against MDA-LDL were used to demonstrate extracellular deposition of MDA-LDL adducts in early lesions.…”

“…Immunohistochemical analyses of atherosclerotic lesions have been performed on rabbit and human tissues. 12,13,27,[55][56][57] Additionally, there is one study on focal glomerulosclerotic lesions in the rat. 58 In considering the available data, it is important to heed two questions.…”

Large Scale Gene Expression Analysis of Cholesterol-loaded Macrophages

“…For Ox-LDL loading experiments, cells were seeded at a density of 1 ϫ 10 6 /ml in medium containing PMA (Research Biochemical International, Natick, MA) at 1 ϫ 10 Ϫ7 M for 24 h. The medium was then replaced by culture medium with or without 100 g/ml CuSO 4 "fully" oxidized LDL (Intracel, Rockville, MD) according to the method of Hammer et al (16). Medium was replaced every 2 days during the time of culture.…”

Antibodies to Oxidized Low-Density Lipoproteins and Angiographically Assessed Coronary Artery Disease in White Patients

“…7 Oxidative modification of LDL induces immunogenic epitopes in the LDL molecule, 8 thus leading to formation of antibodies against oxLDL (oxLDLAbs) that are detectable in sera in the majority of patients with advanced atherosclerotic lesions 9 and can be measured as a tool for investigating the mechanisms of atherogenesis. 10 Several studies demonstrated an increased titer of oxLDLAbs in patients with atherosclerotic coronary artery disease (CAD), 9,[11][12][13] acute myocardial infarction (MI), 14 and cerebral or peripheral artery disease. 15,16 In contrast, other studies found no such positive relationship between oxLDLAb titers and cardiovascular (CV) phenotypes.…”