Specificity of human anti-NOR antibodies, a distinct species of "natural" anti-α-galactosyl antibodies

Natural anti-NOR antibodies are common in human sera and agglutinate human erythrocytes of a rare NOR phenotype. The NOR phenotype-related antigens are unique neutral glycosphingolipids recognized by these antibodies and Griffonia simplicifolia IB4 isolectin (GSL-IB4). The oligosaccharide chains of NOR glycolipids are terminated by Galalpha1-4GalNAcbeta1-3Galalpha units. To characterize the specificity of anti-NOR antibodies and compare it with specificities of GSL-IB4 and known anti-Galalpha1,3Gal antibodies, alpha-galactosylated saccharides and saccharide-polyacrylamide conjugates were used. New synthetic oligosaccharides, corresponding to the terminal di- and trisaccharide sequence of NOR glycolipids and the conjugate of the NOR-tri with HSA were included. These compounds were tested by microtiter plate ELISA and hemagglutination inhibition. Anti-NOR antibodies reacted most strongly with Galalpha1-4GalNAcalpha1-3Gal (NOR-tri), and over 100 times less strongly with Galalpha1-4GalNAc (NOR-di). The antibodies reacted also with Galalpha1-4Gal and Galalpha1-4Galbeta1-4GlcNAc, similarly as with NOR-di but not with other tested compounds. In turn, anti-Galalpha1,3Gal antibodies reacted most strongly with Galalpha1-3Gal and were very weakly inhibited by the NOR-related oligosaccharides (weaker than by galactose), and NOR-tri was less active than NOR-di. GSL-IB4 reacted with all tested alpha-galactosylated saccharides and conjugates, including the similarly active NOR-tri and NOR-di. These results showed that anti-NOR represent a new species of anti-alpha-galactosyl antibodies with high affinity for the Galalpha1-4GalNAcbeta1-3Gal sequence present in rare NOR erythrocytes.