Rabbit antibodies against the human milk sialyloligosaccharide alditol of LS-tetrasaccharide a (NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc OH)

Abstract

The sialyloligosaccharide, NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LS-tetrasaccharide a), a minor component of human milk, is obtained in relatively large quantities from autohydrolysates of the major milk disialyloligosaccharide, NeuAcα2-3Galβ1-3[NeuAcα2-6]GlcNAcβ1-3Galβ1-4Glc (disialyllacto- N-tetraose). Rabbits immunized with an oligosaccharide-protein conjugate prepared from keyhole limpet hemocyanin and LS-tetrasaccharide a produce antibodies directed against the corresponding oligosaccharide alditol. The anti-LS-tetrasaccharide a sera bind 3H-labeled LS-tetrasaccharide a in a direct-binding radioimmunoassay on nitrocellulose filters. The specificities of these antibodies are determined by comparing inhibitory activities of structurally related oligosaccharides. Strong hapten-antibody binding ( K a > 10 6 m −1) requires sialic acid linked α2–3 to the nonreducing terminal galactose residue of reduced lacto- N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc OH). Specificities of antibodies prepared against keyhole limpet hemocyanin conjugates of LS-tetrasaccharide b (Galβ1-3[NeuAcα2-6]GlcNAcβ1-3Galβ1-4Glc) and LS-tetrasaccharide c (NeuAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-4Glc) differ only slightly from rabbit antibodies prepared against the corresponding bovine serum albumin conjugates described previously [ D. F. Smith and V. Ginsburg (1980) J. Biol. Chem. 255, 55–59 ].