Synthesis of a dimeric Lewis antigen and the evaluation of the epitope specificity of antibodies elicited in mice

The Lewis^y-Lewis^x heptasaccharide, modified by an artificial aminopropyl spacer, was synthesized by an approach that employed two orthogonally protected lactosamine building blocks. A p-(benzoyl)-benzyl glycoside was used as a novel anomeric protecting group, which could be selectively removed at a late stage in the synthesis, thus offering the benefit of enhanced flexibility. The artificial aminopropyl moiety was modified by a thioacetyl group, which allowed an efficient conjugation to keyhole limpet hemocyanin (KLH) that had been activated with electrophilic 3-(bromoacetamido)- propionyl groups. Mice were immunized with the Le^yLe ^x-BrAc-KLH antigen. Analysis of the sera by ELISA established that a strong helper T-cell immune response was raised against the Le ^yLe^x saccharide. Further ELISA analysis showed that the titer for monomeric Le^y tetrasaccharide was tenfold lower whereas recognition of the Le^x trisaccharide was negligible.