Measurement of aggrecanase-generated interglobular domain catabolites in the medium and extracts of cartilage explants using Western blot analysis

Cartilage explant cultures and chondrocyte cultures have been routinely used to study the degradative mechanisms involved in joint destruction that lead to disease states such as osteoarthritis and rheumatoid arthritis. Stimulation of these culture systems with catabolic cytokines or chemical agents such as retinoic acid leads to an increase in the release of catabolites and enzymes into the culture medium. Detection of these catabolites using neoepitope antibodies can be used as indicators for the activity of degradative enzymes responsible for the breakdown of the tissue. Catabolites released include aggrecan interglobular domain (IGD) fragments, whereas enzymes include the aggrecanases (ADAMTS-4, TS-5, and-TS-1) as well as a number of matrix metalloproteinases. Monoclonal antibodies have been developed that specifically recognize the new N- and C-terminal amino acid sequences generated by aggrecanase cleavage of the IGD in aggrecan between 373E-374A (human sequence enumeration). These mouse monoclonal antibodies have been named BC-3 and BC-13 (1,2). BC-3 recognizes the new N-terminal sequence ARGSVIL... and BC-13 recognizes the new C-terminal...NITEGE in the IGD of aggrecan. These same amino acid sequences are not recognized in the intact molecule. Furthermore, the specific N- or C-terminal amino acid is essential for epitope recognition by these antibodies. Catabolites are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane, which are then immuno-blotted with either the N- or C-terminal neoepitope antibodies