Antibody labeling of cholesterol/ceramide ordered domains in cell membranes

A monoclonal antibody raised against mixed monolayers of 60:40 mol % cholesterol/C16-ceramide of known structure was used to label cholesterol/ceramide-rich domains in cell membranes. The antibody, Cer-Chol 405F specifically recognizes the mixed crystalline and homogeneous phase in monolayers, but it does not interact with either of the components separately. It interacts differently with mixed monolayers that contain ceramides of different acyl chain length. When used on cells, the antibody labeling is sensitive to changes in cholesterol and ceramide levels, as well as to over-expression of specific ceramides; this is in agreement with the results that were obtained on lipid monolayers. This represents a proof of concept of the applicability of a new approach to the structural characterization of lipid microdomains in cell membranes. The approach consists of raising antibodies that recognize specific structural organizations of lipids in artificial mixtures, characterizing the antibody/ordered domain complexes in vitro, and subsequently using them to detect the presence of the same (or similar) domains in cell membranes.