Abstract

This study measured the possible cross-reactivity of hapten-specific IgG antibodies purified from the sera of rabbits sensitized to an albumin-acetaldehyde conjugate [N-ethyl-rabbit serum albumin (N-ethyl-RSA)] with acetaldehyde-phosphatidylethanolamine adducts. The N-ethyl-RSA was coupled to an Affigel-10 column to affinity purify the IgG (anti-N-ethyl-RSA IgG). Dioleoyl-phosphatidylethanolamine (DOPE) was reacted with acetaldehyde to form a Schiff base, which was reduced to N-ethyl-DOPE, purified by high pressure liquid chromatography, and analyzed with direct chemical ionization mass spectrometry. Lamellar liposomes containing either 5% by weight N-ethyl-DOPE and 95% egg phosphatidylcholine or a mixture of 5% N-ethyl-DOPE, 71% DOPE, and 24% dioleoylphosphatidylcholine, as well as hexagonal phase micelles containing 5% N-ethyl-DOPE and 95% DOPE, were prepared by sonication. Anti-N-ethyl-RSA IgG was then incubated with each of these lipid mixtures for 30 min, a fluorescein-conjugated goat anti-rabbit IgG was added for an additional 30 min, and then binding of anti-N-ethyl-RSA IgG to N-ethyl-DOPE in the liposomes or micelles was measured by flow cytometry. Anti-N-ethyl-RSA IgG bound to N-ethyl-DOPE in both vesicles and hexagonal phase micelles, but the affinity was 16 times greater for the hapten in the hexagonal phase. This result demonstrates that physical presentation of the hapten can affect antibody recognition and that antibodies raised against N-ethyl-RSA can cross-react with acetaldehyde-phospholipid adducts.