Production and Characterization of Monoclonal Antibodies against Keratan Sulfate-containing Glycoconjugates from Mineralized Tissue

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Keratan sulfate-containing glycoconjugates (KSGs) were partially purified from the mineral phase of rabbit alveolar bone. The KSGs were injected intravenously into BALB/c mice, and spleen cells were collected from the hyperimmune mice. The spleen cells were fused with murine myeloma cells (P3X63-Ag8-U1), and hybridomas secreting monoclonal antibodies (MAbs) against the KSGs were obtained. We established two cell lines which strongly secreted at IgM type immunoglobulin out of six hybridomas selectedl the other four produced an IgG^3 type. These MAbs recognized KSGs from the mineral and matrix phases of rabbit and human alverlar bones. To determine the location of the binding sites of the MAbs on the KSG molecules, they were treated with several enzymes to examine the kind of glycosaminoglycan chains and glycoside residues on the core proteins. SDS-PAGE and Western blotting analyses revealed that one of the MAbs, NK1, reacted most strongly with an epitope associated with the keratan sulfate (KS) chains. Comparative studies with NK1 showed that its antigenic binding site and immunoglobulin class were different from those of a previously used monoclonal antibody, 5D4. NK1 was an IgM type immunoglobulin while 5D4 was an IgG^1 type. Furthermore, the titer of NK1 was approximately 20-fold higher than that of 5D4. NK1 antibody could have a potentially useful role in biochemical and immunohistochemical studies to detect low levels of KSGs in mineralized tissues previously not detectable with 5D4.
硬組織再生生物学会の論文

Production and Characterization of Monoclonal Antibodies against Keratan Sulfate-containing Glycoconjugates from Mineralized Tissue

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