Aggrecanase Activity, Sensitive ELISA
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The Aggrecanase Activity Assay measures activity of aggrecanases and is used to screen and characterize aggrecanase inhibitors. This assay consists of two modules, the Aggrecanase Module and the ELISA Module. In the Aggrecanase Module, a recombinant fragment of human aggrecan interglobular domain (aggrecan-IGD) is first digested with aggrecanase. Proteolytic cleavage of the substrate releases an aggrecan peptide with the N-terminal sequence ARGSVIL (ARGSVIL-peptide). The ARGSVIL-peptide is then quantified in the ELISA Module using two monoclonal anti-peptide sequence antibodies.
The Sensitive Aggrecanase Activity assay measures activities of aggrecanase in the pM concentration range. The high sensitivity is acheived with an engineered aggreganase substrate derived from aggrecan interglobular domain.
Overview
Aggrecanases ADAMTS1, ADAMTS4 and ADAMTS5 are members of the ADAMTS-family of proteases (ADAMTS = A disintegrin and metalloprotease with thrombospondin motif). They are synthesized as multidomain proteins consisting of prodomains, protease, disintegrin, thrombospondin and cys-rich domains followed by spacer regions and additional thrombospondin motifs. The prodomains are cleaved-off intracellularly and the enzymes are processed further from their C-terminal ends after secretion.
Aggrecanases cleave specific peptide bonds in aggrecan, the main proteoglycan of articular cartilage. They hydrolyze also other lecticanes as versican and brevican. Convincing evidence indicates that aggrecanases are mainly responsible for excessive aggrecan degradation in osteoarthritic joints.
Data/Specifications
Species: human
Sample Type: cell culture supernates
Sample Size: 100 uL
Sample Preparation: Proteolysis
Standard Curve Range: 0.022 - 1.4 nM
Sensitivity: 2 pM
Assay Length: 3.75 hours
Literature/Support
Aggrecanase Activity Assay Insert (PDF)
References/Citations
Adaptor Proteins and Ras Synergistically Regulate IL-1-Induced ADAMTS-4 Expression in Human Chondrocytes
Rasheed Ahmad et al., J. Immunol., Apr 2009; 182: 5081 - 5087.