Sensitive detection of hydrophobic antigens using a novel lipid-aggregate based ELISA

Antibodies against hydrophobic antigens are common in several autoimmune diseases. However, detection of such antibodies by standard immune-assays, such as ELISA, is problematic, in part because of the problems with coating hydrophobic molecules onto polystyrene multi-well plates. We describe a novel method of stably associating hydrophobic antigens to ELISA plates. By mixing the antigen with a hydrophobic molecule containing a hydrophilic anchor, we generate mixed lipid aggregates that can attach to ELISA plates, and are resistant to detergent wash. Using the ganglioside GM-1 and phosphatidylethanolamine conjugated to the hapten DNP (dinitrophenyl) as model antigens, we show that hydrophobic antigens incorporated into mixed lipid aggregates expose their antigenic determinants in a correct configuration. The detection limit of both GM-1 and DNP-PE was considerably improved compared to when these antigens were coated on ELISA plates using organic solvents. Furthermore GM-1 incorporated into mixed lipid aggregates can be detected by specific antibodies in patient serum. The method of incorporating hydrophobic antigens into mixed lipid aggregates for stable association to ELISA plates can presumably be applied to a vast array of hydrophobic antigens, and may well be developed into a large scale screening system for serum reactivity towards different hydrophobic antigens.