Generation of anti-oligosaccharide antibodies that recognize mammalian glycoproteins by immunization with a novel artificial glycosphingolipid

Here we report a new method for the efficient generation of antibodies that recognize the fine structures of oligosaccharides on glycoproteins. We found a newly designed artificial glycosphingolipid carrying a very long chain fatty acid to be a strong immunogen in mice, with the serum of immunized mice containing antibodies recognizing the oligosaccharide structure of the immunogen. First, we found that conjugation of a simple ceramide analogue to target oligosaccharides could enhance the immunogenicity of these oligosaccharides in these immunized mice. This effect was confirmed in mice immunized with the artificial glycosphingolipids carrying 6′-Sialyl-LacNAc, 3′-Sialyl-LacNAc and LacNAc. Next, we tried to improve the immunogenic enhancing effect of the ceramide analogue. In a model experiment using 6′-Sialyl-LacNAc oligosaccharide, we manipulated the alkyl chains to several lengths, and found that a longer alkyl chain length of the fatty acid correlated with high immunogenicity. Among these we examined, artificial glycosphingolipids conjugated with a ceramide analogue carrying a very long chain fatty acid (lignoceric acid) showed the strongest immunogenicity. By using the artificial glycosphingolipid containing 6′-Sialy-LacNAc and lignoceric acid, we succeeded in the generation of a new anti-6′-Sialyl-LacNAc antibody that recognizes 6′-Sialyl-LacNAc carrying glycoproteins but does not bind to 6′-Sialyllactose, asialo-glycoporoteins and glycoproteins carrying 3′-Sialyl-LacNAc. These results indicate that the established technology is valuable for the targeted generation of monoclonal antibodies against glycoproteins containing specific oligosaccharide structures.