Kresina T F, Malemud C J
Coll Relat Res. 1986 Mar;6(1):15-39. doi: 10.1016/s0174-173x(86)80030-9.
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.
在衰老和患骨关节炎的兔子中发现了硫酸化蛋白聚糖的结构和组成变化。识别兔软骨蛋白聚糖特定表位的单克隆抗体（mAB），对于记录部分内侧半月板切除术后兔滑膜关节发生骨关节炎病理过程中蛋白聚糖的变化将很有用。至此，用从剑突提取的兔蛋白聚糖（A1D1D1组分）免疫Balb/c小鼠。用鼠脾细胞与肿瘤细胞系SP 2/0-Ag 14融合制备杂交瘤。在固相放射免疫分析中发现9种单克隆抗体与A1D1D1结合。以A1D1D1作为配体的结合曲线，将单克隆抗体分为3类——高结合、中等结合和低结合单克隆抗体。结合亲和力与免疫球蛋白亚类无关。用胰蛋白酶消化A1D1D1，在DEAE-纤维素上进行层析，并用解离性CsCl密度梯度离心进一步分离胰蛋白酶肽段。利用解析肽段的竞争性抑制试验对单克隆抗体进行了详细研究。观察到三种类型的抗原精细特异性；一种单克隆抗体（2G2）识别天然A1D1D1上的一个重复表位，一种单克隆抗体（2E9）识别一个单一蛋白质表位，因为它与一个含有高比例的谷氨酰胺:半乳糖胺的胰蛋白酶肽段结合，还有一种单克隆抗体（6C9）优先识别经热处理（50℃ 分钟）的A1D1D1上的一个重复表位。在该分析中，这些单克隆抗体的表位似乎与核心蛋白相关，因为只有一种单克隆抗体（2C7）被糖胺聚糖、透明质酸和透明质酸寡糖竞争性抑制与天然A1D1D1的结合。兔髋、肩和膝关节软骨的直接免疫荧光染色显示，不同的单克隆抗体对细胞外基质有不同的染色模式。FITC-2G2强烈染色细胞间基质；以及陷窝周围区域，而FITC-2E9和FITC-6C9似乎局限于陷窝周围区域。
