Generation and characterization of a series of monoclonal antibodies that specifically recognize [HexA(±2S)-GlcNAc]n epitopes in heparan sulfate

Abstract

Five monoclonal antibodies AS17, 22, 25, 38 and 48, a single monoclonal antibody ACH55, and three monoclonal antibodies NAH33, 43, 46, that recognize acharan sulfate (IdoA2S-GlcNAc)n, acharan (IdoA-GlcNAc)n and N-acetyl-heparosan (GlcA-GlcNAc)n, respectively, were generated by immunization of mice with keyhole limpet hemocyanin-conjugated polysaccharides. Specificity tests were performed using a panel of biotinylated GAGs that included chemically modified heparins. Each antibody bound avidly to the immunized polysaccharide, but did not bind to chondroitin sulfates, keratan sulfate, chondroitin nor hyaluronic acid. AS antibodies did not bind to heparan sulfate or heparin, but bound to 6-O-desulfated, N-desulfated and re-N-acetylated heparin to varying degrees. ACH55 bound to tri-desulfated and re-N-acetylated heparin but hardly bound to other modified heparins. NAH antibodies did not bind to heparin and modified heparins but bound to heparan sulfate to varying degrees. NAH43 and NAH46 also bound to partially N-de-acetylated N-acetyl-heparosan. Immunohistochemical analysis in rat cerebella was performed with the antibodies. While NAH46 stained endothelia, where heparan sulfate is typically present, neither ACH55 nor AS25 stained endothelia. On the contrary ACH55 and AS25 stained the molecular layer of the rat cerebella. Furthermore, ACH55 specifically stained Purkinje cells. These results suggest that there is unordinary expression of IdoA2S-GlcNAc and IdoA-GlcNAc in specific parts of the nervous system.