Cholesterol-dependent complement activation has been proposed as a factor that might influence the pathogenesis of atherosclerosis. Although antibodies to cholesterol conjugates have been reported, cholesterol is widely regarded as a poorly immunogenic substance. Monoclonal IgM complement-fixing antibodies to cholesterol were obtained in the present study after immunizing mice with liposomes containing high amounts of cholesterol (71 mol % relative to phosphatidylcholine) and lipid A as an adjuvant.Clones were selected for the ability of secreted antibodies to react with liposomes containing 71% cholesterol but not with liposomes containing 43% cholesterol. The antibodies also reacted with crystalline cholesterol in a solid-phase enzymelinked immunosorbent assay. Binding of monoclonal antibodies to the surface of crystalline cholesterol was demonstrated by electron microscopy by utilizing a second antibody (antilgM) labeled with colloidal gold. The immunization period required to induce monoclonal antibodies was very short (3 days) and a high fraction of the hybrid cells (at least 70%) were secreting detectable antibodies to cholesterol. The results demonstrate that cholesterol can be a highly immunogenic molecule and that complement-flxing antibodies to cholesterol can be readily obtained.Cholesterol has been widely studied as a fundamental and ubiquitous constituent of cell membranes and lipoproteins (1,2). Despite occasional reports that conjugates of cholesterol can be immunogenic (3)(4)(5), because of its widespread distribution and important biological roles cholesterol has generally been assumed to be a nonimmunogenic or poorly immunogenic molecule. However, since 1977 several laboratories have reported activation of the classical and alternative pathways of complement by cholesterol (6-11). The exact mechanism (or mechanisms) of complement activation by cholesterol has not been determined. In one case human IgG antibodies to cholesterol were implicated in the activation of the classical pathway (9). On the other hand, it has been proposed that nonimmune activation of the alternative pathway of rabbit complement (7) or human complement (8, 9) also occurs. The issue of the immunogenicity of cholesterol is an important one, since antibodies to cholesterol have been reported to protect against induced atherosclerosis in rabbits (4). On the other hand, activation of the classical pathway of complement by cholesterol has been proposed as a possible mechanism in the pathogenesis of atherosclerosis (6,9,11).In previous studies we induced antibodies to the phospholipid constituents of lipid model membranes (liposomes) (reviewed in ref. 12). Production of antibodies to liposomes was achieved by utilizing lipid A as a powerful adjuvant in the liposomal lipid bilayer. In the present work we used lipid A as an adjuvant to induce antibodies to liposomes containing high concentrations of cholesterol. We found that cholesterol is an excellent immunogen and that murine monoclonal antibodies to cholesterol are eas...