Monoclonal antibodies against the protein core and glycosaminoglycan side chain of glomerular basement membrane heparan sulfate proteoglycan: characterization and immunohistological application in human tissues

Abstract

We raised monoclonal antibodies (MAb) against the core protein and the heparan sulfate (HS) side chain of heparan sulfate proteoglycan (HSPG) from glomerular basement membranes (GBM). Anti-HSPG-core MAb were obtained after immunization of mice with HSPG purified from human GBM and the anti-HS MAb after immunization of mice with HSPG from rat glomeruli, which crossreacted with human HS and GBM HSPG. The specificity of the MAb was demonstrated by ELISA studies, Western blotting, inhibition experiments, and indirect immunofluorescence (IF) on kidney cryostat sections pre-treated with glycosaminoglycan (GAG)-degrading enzymes. Indirect IF on normal human kidney tissue showed prominent GBM staining for both MAb, with variable staining of the other renal basement membranes (BMs). By indirect immunoelectron microscopy (IEM), most intense staining was observed at the endothelial side of the GBM for both MAb, although the staining patterns were not identical. Both MAb were used to localize HSPG in human tissues by indirect IF. They bound to antigens present in the BMs of most tissues examined, including those of epithelia and endothelia. Differences between both MAb were observed for BMs of muscle cells, since the anti-HSPG core protein MAb (JM-72) staining was negative, whereas the anti-HS MAb (JM-403) clearly stained these structures. Comparison of our staining patterns in human tissues with the distribution of other anti-BM HSPG antibodies suggests that there are at least two types of BM HSPG, which have common epitopes on the HS side chains recognized by JM-403.