Production and characterization of a monoclonal antibody against human glomerular heparan sulfate.
After immunization with heparan sulfate proteoglycan (HSPG) isolated from human glomeruli, two mouse monoclonal antibodies (mAbs) against heparan sulfate (HS) were obtained. Both mAbs were of the IgM isotype and showed identical specificity. One of these, mAb JM-13 is described in detail. In enzyme-linked immunosorbent assay and Western blotting, reactivity was found with human glomerular basement membrane HSPG and HS. No binding occurred to the core protein of HSPG obtained after removal of HS with trifluoromethanesulfonic acid. In enzyme-linked immunosorbent assay, mAb JM-13 did neither bind to other proteoglycans, nor to other basement membrane components like collagen type IV, laminin, or fibronectin. In indirect immunofluorescence on cryostat sections of human kidneys, a restricted staining of tubular basement membranes was observed along with staining of the vascular basement membranes. In the glomerulus, a weak, fine granular staining was seen along the capillary wall and in the mesangium. MAb JM-13 bound also to the basolateral cell membranes of proximal tubular cells, to the cell membranes of cultured human and rat glomerular visceral epithelial cells, rat mesangial cells, human hepatocytes in culture, and in liver cryostat sections, indicating also a recognition of cell surface-associated HS. Pretreatment of the sections with heparitinase abolished binding of JM-13, whereas treatment with chondroitinase ABC had no effect. Inhibition studies in enzyme-linked immunosorbent assay as well as in indirect immunofluorescence corroborated the HS specificity of mAb JM-13. In conclusion, mAb JM-13 binds to an epitope on the HS chains of glomerular, tubular, and cell surface-associated HSPG.
van den Born
J
J
Department of Medicine, University of Nijmegen, The Netherlands.
van den Heuvel
L P
LP
Bakker
M A
MA
Veerkamp
J H
JH
Assmann
K J
KJ
Berden
J H
JH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D001485:Basement Membrane / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006497:Heparan Sulfate / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007158:Immunologic Techniques; D007678:Kidney Glomerulus / Q000378:metabolism*; D051379:Mice
D000911:Antibodies, Monoclonal; D006497:Heparan Sulfate