A novel keratanase‐generated keratan sulphate antibody and its applications

Abstract

Introduction The sequencing of the genome has provided us with important information regarding the primary structure of many matrix proteins. This in turn has lead to advances in studies of the functions of post‐translational modifications on connective tissue proteoglycans (PGs). Changes in GAG structure with ageing and disease have been well documented (Thonar et al. 1986; Brown et al. 1998). However, little is known about the exact sites of and differential substitution of GAGs on the aggrecan core protein and how these substitutions facilitate normal function or the changes seen with disease. The CS : KS ratio of substitution change significantly, with KS levels increasing with age and decreasing with the onset of disease. Objective was to produce monoclonal antibody (MAb) reagents to keratanase (k'ase) generated stub epitopes, that could be used to help identify, characterize and quantify sites of KS substitution on PGs, providing the potential to determine how the arrangement of such substitutions change with development, ageing and pathology.Methods Bovine Nasal Cartilage aggrecan (BNC A1D1) was trypsin digested, generating a range of glycosaminoglycan (GAG) fragments. The sample was then subjected to anion‐exchange and size exclusion chromatography to separate KS from CS fragments. Fractions collected were analysed by SDS‐PAGE and Western blotting. Fractions positive for KS were pooled and kinase digested to expose the KS stub antigens. Immunization and fusions were carried out as previously described (Nieduszynski et al. 1990). Initial screenings were carried out using ELISA. Briefly, 96‐well microtitre plates were coated with the immunizing antigen overnight at 37 °C. The plates were then blocked prior to the addition of hybridoma media for 1–2 h at 37 °C. Binding was detected using an alkaline phosphatase‐conjugated secondary antibody for 1 h at 37 °C prior to the addition of the substrate. Positive wells were further screened by ELISA and SDS‐PAGE using the immunizing antigen, chondroitinase‐digested BNC and an A1D1 BNC preparation to establish the kinase stub specificity of the hybridomas. Further screenings by Western blotting was carried out on positive hybridomas selected. Antigens used included keratanase‐digested bovine corneal KS‐PGs, keratanase‐II‐digested KS‐PGs and a nonkeratanase‐digested corneal KS‐PG sample.Results Screening: Screening identified two positive hybridomas, B‐KS‐I and B‐KS‐II, which were specific for kinase‐generated KS stub. On screening, these antigens showed reactivity specifically for kinase‐digested BNC abc core, with no reactivity to the nonkinased linear KS GAG epitopes. Reactivity to kinase‐digested corneal KS‐PGs indicated that the MAbs generated were indeed to a stub structure in the KS chain and not to some linkage region epitope, amino acid sequence or oligosaccharide present on the core protein.Application: Immunohistochemistry utilizing B‐KS‐I was used to localize KS in a range of tissues along side anti‐KS 5D4. In human articular cartilage engineered grafts, labelling showed B‐KS‐I and 5D4 to have broadly overlapping labelling patterns for KS; however, label for B‐KS‐I had a much more restricted and subtle tissue distribution than that of antibody 5D4.Discussion These new KS stub MAbs have potential to be used in many different areas of research. They may be used in analysis of trypsin‐digested purified aggrecan from cattle joints of different ages to determine sites of KS substitution, which remain common or change with development and ageing. They may also be used in analysis of cartilage explant culture metabolites to assess KS substitution on the aggrecan fragments generated after stimulation of these cultures with cytokines such as IL‐1 or TNF‐α. Collectively it will provide important new information on the changing pattern of KS substitution in connective tissue PGs with development, ageing and the onset of pathology.