A microplate assay specific for the enzyme aggrecanase.
We have identified a 41-residue peptide, bracketing the aggrecanase cleavage site of aggrecan, that serves as a specific substrate for this enzyme family. Biotinylation of the peptide allowed its immobilization onto streptavidin-coated plates. Aggrecanase-mediated hydrolysis resulted in an immobilized product that reveals an N-terminal neoepitope, recognized by the specific antibody BC-3. This assay is highly specific for aggrecanases; MMPs were inactive in this assay. Reduction of the peptide size below 30 amino acids resulted in a significant diminution of activity. Using the immobilized 41-residue peptide as a substrate, we have developed a 96-well microplate-based assay that can be conveniently used for high-throughput screening of samples for aggrecanase activity and for discovery of inhibitors of aggrecanase activity.[1]References
- A microplate assay specific for the enzyme aggrecanase. Miller, J.A., Liu, R.Q., Davis, G.L., Pratta, M.A., Trzaskos, J.M., Copeland, R.A. Anal. Biochem. (2003) [Pubmed]
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