GENERATION, CHARACTERIZATION, AND HISTOCHEMICAL APPLICATION OF MONOCLONAL-ANTIBODIES SELECTIVELY RECOGNIZING OXIDATIVELY MODIFIED APO-B-CONTAINING SERUM-LIPOPROTEINS

To investigate either the role oxidized LDL plays in atherosclerosis o r structural changes on the surface of oxidized LDL, monoclonal antibo dies (mAbs) are an important tool. After immunizing mice with Cu2+-oxi dized LDL (oxLDL) and fusion of splenocytes, hybridoma supernatants we re screened and cloned. Two mAbs, OB/04 and OB/09 (IgG and IgM), were further characterized. In solid-phase fluorescence immunoassays and We stern blot analysis both mAbs reacted with oxLDL, LDL oxidized by a fr ee radical-generating azo compound, or oxVLDL but not with native LDL, acetylated LDL, oxHDL(3), azo-oxidized HDL(3), or HDL(3) modified wit h 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 follow ed by 4-hydroxynonenal but not with 4-hydroxyoctenal or hepta-2,4-dien al. mAb OB/04 had a weak affinity for LDL after modification with thes e aldehydes except for MDA-LDL. LDL modified with arachidonic acid oxi dation products (AAOPs) was also recognized by this mAb. However, albu min modified either by the aldehydes applied or by AAOPs did not react with either mAb. Thus, the data indicate that each of the mAbs recogn izes a different epitope that is expressed only on apoB-containing lip oproteins upon oxidative modification. An immunostaining with mAb OB/0 4 was obtained in areas rich in macrophages and in connective tissue o f a human atherosclerotic lesion.