Identification of blood group A and B antigens in human glycophorin

Glycophorin A (GPA), the major sialoglycoprotein of human erythrocyte membranes, was isolated separately from blood group A and B erythrocytes using phenol-water extraction. After purification, performed as gel filtration in the presence of SDS, two glycophorin samples GPA-A and GPA-B were run, in duplicate, in SDS-PAGE and electroblotted onto Immobilon P. After staining with 1) anti-glycophorin antibody and 2) with relevant anti-blood group (A or B) antibody it was shown that the band pattern of the samples in each duplicate was the same. GPA-A and GPA-B samples were also degraded using Carlson degradation (beta-elimination in mild alkaline/strong reducing conditions) and from reaction products the fractions of O-glycans and N-glycans were isolated; they were used in hemagglutination inhibition test. It was shown that both sugar fractions derived from GPA-A did inhibit agglutination of blood group A erythrocytes by anti-A antibody, whereas oligosaccharide fractions derived from GPA-B inhibited agglutination of blood group B erythrocytes by anti-B antibody. These results, obtained using immunochemical methods, confirm the presence of blood group A and B determinants in the carbohydrate moiety of human glycophorin, derived from the blood group A or B erythrocytes, respectively.