Immunogenicity of malondialdehyde-modified low density lipoproteins: Studies with monoclonal antibodies

Abstract

Malondialdehyde (MDA)-modified low density lipoprotein (LDL) can stimulate the accumulation of cholesteryl esters in cultured macrophages through its interaction with specific scavenger receptors. It has been speculated that such interaction occurs in vivo thus contributing to the formation of foam cells within atherosclerotic lesions. This report describes the development of new tools in the form of a specific assay for MDA-LDL to investigate this hypothesis. We have immunized BALB/c mice with malondialdehyde mouse low density lipoproteins and antibodies against malondialdehyde human low density lipoproteins were generated. Monoclonal antibodies were produced using hybridoma techniques and one particular clone (EB 7-3) was expanded for further studies. The immunoreactivity of several antigens was tested using antibody EB 7-3 in an enzyme-linked immunosorbent assay (ELISA). In a typical assay malondialdehyde human LDL (with at least 40% of lysines modified) was coated (2 μg/ml, 100 lt,l) in 96-well microtiter plates. Antibody plus one of several antigens were then added and the interaction between the antibody and coated antigen was measured using alkaline phosphatase-conjugated affinity purified goat anti-mouse immunoglobulin. The binding of antibody EB 7-3 to wells coated with malondialdehyde-LDL was competitively inhibited by malondialdehyde-LDL added in solution, with half maximal inhibition occurring at 150 ± 80 ng/ml. In addition, the ability of malondialdehyde-LDL to inhibit this interaction was proportional to the degree of modification: the more lysines were modified the more did malondialdehyde-LDL inhibit the binding of antibody EB 7-3 to coated malondialdehyde-LDL. On the other hand, other modified LDL preparations - glucosylated, carbamylated, acetylated, acetoacetylated, reductively methylated, or cyclohexanedione modified as well as native LDL - failed to inhibit the binding of antibody EB 7-3 to malondialdehyde-LDL coated plates. Malondialdehyde-high density lipoprotein (HDL) as well as polyinosinic acid (but not polyvinyl sulfate) inhibited competitively the interaction between malondialdehyde-LDL and antibody EB 7-3. Aortic intimal or medial extracts obtained from 24 autopsy specimens failed to show any MDA-modified LDL. We conclude that (a) malondialdehyde-LDL is immunogenic in mice; (b) specific antibodies, most likely directed against the malondialdehyde-lysine-containing region have been generated and were used in a sensitive assay to measure MDA-LDL concentrations; and (c) MDA-LDL does not seem to be present in human aortic intima, thus casting significant doubts on the role of MDA-LDL in formation of foam cells.