Murine monoclonal antibodies recognizing rabbit proteoglycans

Alterations in the structure and composition of sulfated proteoglycans are found in aging and osteoarthritic rabbits. Monoclonal antibodies (mAB) recognizing specific epitopes of rabbit cartilage proteoglycans would be useful in documenting proteoglycan changes during pathophysiological responses resulting in osteoarthritic pathology in rabbit synovial joints after partial medial meniscectomy. To this point, Balb/c mice were immunized with rabbit proteoglycan (fraction A1D1D1) extracted from xiphoid process. Murine spleen cells were used to prepare hybridomas by fusion with the tumor cell line SP 2/0-Ag 14. Nine mAbs were found to bind to A1D1D1 in a solid phase radioimmunoassay. Binding curves, utilizing A1D1D1 as ligand, resulted in the assignment of mAbs to 3 classes - high, moderate and poor binding mAbs. Binding avidity was independent of immunoglobulin subclass. A1D1D1 was digested with trypsin, chromatographed on DEAE-cellulose and tryptic peptides further resolved by dissociative CsCl density gradient centrifugation. The mAbs were studied in detail utilizing competitive inhibition assays of the resolved peptide fragments. Three types of antigenic fine specificity were observed; a mAb (2G2) which recognized a recurrent epitope on the native A1D1D1, a mAb (2E9) which recognized a single protein epitope, in that it bound to a tryptic peptide that contained a high gluNH2:galNH2 and a mAb (6C9) which preferentially recognized a recurring epitope on heat-treated (50 degrees C minutes) A1D1D1. In this analysis, the epitopes of these mAbs appear to be associated with the core protein since only one mAb (2C7) was competitively inhibited from binding to native A1D1D1 by glycosaminoglycans, hyaluronic acid and oligosaccharides of hyaluronic acid. Direct immunofluorescence staining of rabbit hip, shoulder and knee cartilage showed a differential staining pattern of extracellular matrix with the various mAbs. FITC-2G2 stained the interterritorial matrix intensely; and also the perilacunae zones, whereas FITC-2E9 and FITC-6C9 appeared restricted to the perilacunae regions.