Neoglycolipid micro-immunoassays applied to the oligosaccharides of human milk galactosyltransferase detect blood-group related antigens on both O- and N-linked chains.

Reduced O-linked chains and reducing N-linked chains were obtained from human milk galactosyltransferase by degradation with alkaline borohydride and hydrazinolysis, and then purified by ion-exchange chromatography. The reactivities of the conjugates of the oligosaccharides with L-alpha-phosphatidyl ethanolamine dipalmitoyl (PPEADP) towards monoclonal anti-Lea and anti-SSEA-1 were then determined, either by antibody-binding assays after absorbing the neoglycolipids onto plastic wells, or by inhibition assays after incorporating the neoglycolipids into liposomes and testing them as inhibitors of antibody binding. The oligosaccharides were also immunostained with monoclonal anti-Lea after h.p.t.l.c. and coupling to PPEADP. Antigenic activities were detected in the O-linked chains by all three assay systems, whereas, for the less abundant N-linked chains, reactivities were detected by the inhibition assays only. The results provide evidence for the expression of Lea and SSEA-1 antigen activities on both the O- and N-linked chains of this enzyme glycoprotein.