Aggrecanase Activity, Sensitive ELISA
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The Sensitive Aggrecanase Activity ELISA Kit measures activities of aggrecanase in the pM concentration range. The high sensitivity is acheived with an engineered aggreganase substrate derived from aggrecan interglobular domain.
The 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.
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)
Measuring aggrecanase activity to identify potential therapeutics for osteoarthritis. (Blog Post)
| References/Citations: | How the sensitive aggrecanase activity ELISA kit was used: |
| In vitro inhibition of aggrecanase activity by tetracyclines and proteoglycan loss from osteoarthritic human articular cartilage. Steinmeyer J, et al. J Orthop Res. 2010 Jun; 28(6):828-33. | Measure aggrecanase activity in in situ human articular cartilage explants from OA patients using serum-free culture conditions. |
| Aggrecanase- and matrix metalloproteinase-mediated aggrecan degradation is associated with different molecular characteristics of aggrecan and separated in time ex vivo. Madsen SH, et al. Biomarkers. 2010 May; 15(3):266-76. | |
| 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. | Measure aggrecanase activity in primary human knee articular chondrocyte cultures. Supernatents were collected from cultured cells and stored at -20C until analysis. |
| Syndecan-4 regulates ADAMTS-5 activation and cartilage breakdown in osteoarthritis. Echtermeyer F, et al. Nat Med. 2009 Sep; 15(9): 1072-6. | Measure aggrecanase activity in the supernatants of cartilage explant cultures after treatment with IL-1. Cartilage explant cultures are from wild-type and Sdc4 knock-out mice. |
| The carbon monoxide-releasing molecule tricarbonyldichlororuthenium(II) dimer protects human osteoarthritis chondrocytes and cartilage from the catabolic actions of interleukin-1β. Megias J, et al. J Pharmacol Exp Ther. 2008 Apr; 325(1):56-61. | <!--StartFragment-->Measure aggrecanase activity from the supernatents of chondrocytes in primary culture. Chondrocytes were isolated by sequential enzymatic digestion of cartilage specimens obtained from osteoarthritis patients. |
| Methods in studying ECM degradation. Everts V, et al. Methods in Extracellular Matrix Research. 2008 May;45(1):86-92 | Review article that acknowledges our aggrecanase activity ELISA. |