GENERATION AND CHARACTERIZATION OF ANTI-SULFOGLUCURONOSYL PARAGLOBOSIDE MONOCLONAL-ANTIBODY NGR50 AND ITS IMMUNOREACTIVITY WITH PERIPHERAL-NERVE

Sulfoglucuronosyl paragloboside (SGPG) is a member of the sulfated glu curonic acid-containing glycolipid (SGGL) family found primarily in pe ripheral nerves, These glycolipids contain the HNK-1 carbohydrate epit ope and are recognized by monoclonal IgM from patients with chronic de myelinating neuropathy and paraproteinemia. Recent studies indicate th at SGGLs may serve as ligands for selectins, amphoterin, and laminin, suggesting that these glycolipids may play an important role in cellul ar adhesion, To elucidate the biological function of these glycolipids , we produced a murine monoclonal antibody (mAb) and studied its antig enic specificity, Using an enzyme-linked immunosorbent assay (ELISA), we found that the mAb designated as NGR50 belonged to the IgG(2a) subc lass, and that the minimal titer (2 SD above the mean optical density value of control) of this mAb was 1:640, with 20 ng of purified SGPG a s the antigen, Thin-layer chromatography (TLC) immunoblotting revealed that this mAb reacted specifically with SGPG and sulfoglucuronosyl la ctosaminyl paragloboside (SGLPG), which is a structural analogue of th e former, but not with other glycolipids, Desulfated derivates of SGPG and SGLPG did not react with mAb NGR50, Western blot analysis showed crossreactivity with human myelin-associated glycoprotein (MAG), but n ot with rat MAG or rat glycoprotein P0. Unlike anti-HNK-1 monoclonal a ntibody, however, NGR50 reacted only weakly with several proteins in t he 20-30-kD regions, including human P0, suggesting that mAb50 has a d ifferent fine specificity as an anti-HNK-1 antibody, Immunocytochemica l study of rat sciatic nerve using mAb NGR50 revealed positive stainin g at the outer surface of the myelin sheath and Schwann cells, as well as in the intervening connective tissues, Faint staining was also vis ible at the axolemmal-myelin interface; however, compact myelin was no t stained. (C) 1996 Wiley-Liss, Inc.