Aggrecanase Sensitive Activity Assay


The Aggrecanase Sensitive Activity Assay Kit measures activities of aggrecanases in the pM-concentration range. This high sensitivity is achieved with an engineered aggrecanase substrate derived from aggrecan interglobular domain. The Sensitive Aggrecanase Activity Assay allows measurement of aggrecanase activity in serum-free cell culture supernatants.

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Aggrecanase Sensitive Activity Assay

The Aggrecanase Sensitive Activity Assay is For Research Use Only

Size: 1×96 wells
Sensitivity: 12pM
Dynamic Range: 0.56 – 100 pM
Incubation Time: 4 hours
Sample Type: cell culture
Sample Size: 5 µL
Controls Included

Assay Principle

ARGSVIL-peptide-s standard, proteolytic digested aggrecan-IGD-s with standard aggrecanase and test samples are incubated in microtiter wells pre-coated with anti-ARGSVIL-neoepitope antibody. ARGSVIL-peptide-s is bound to the coated antibody, while other components are removed with washing and aspiration. The bound ARGSVIL-peptide-s is detected with a second peroxidase-labeled antibody. Any excess of the conjugate is removed with washing and aspiration. The amounts of peroxidase bound to different wells are determined in reactions with peroxidase substrate TMB. The reactions are stopped by addition of sulfuric acid solution and absorbance is read at 450 nm in a microtiter plate spectrophotometer.
The assay kit consists of two modules, the Aggrecanase module and the ELISA Module. The modified aggrecan interglobular domain (aggrecan-IGD-s) is first digested with Aggrecanase (ADAMTS4). Proteolytic cleavage of the substrate releases an aggrecan peptide with the Nterminal sequence ARGSVIL (ARGSVIL-peptide-s). The ARGSVIL-peptide-s is then quantified with two monoclonal anti-peptide antibodies.

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Additional Information

Assay Background

Aggrecan is a large aggregating proteoglycan of articular cartilage [1]. It is found also in aorta tissue, discs, tendons [1] and in the perineuronal net [2]. The aggrecan core protein consists of 2317 amino acids [3]. Up to 130 glucosaminoglycan chains are attached to the core protein and the total molecular mass can reach 2.2 – 3.0 x 106 Daltons [4].
Within the aggrecan molecule 3 globular domains G1, G2 and G3 can be distinguished. Domains G1 and G2 are connected by a rod-shaped polypeptide called interglobular domain (IGD), while the sequence between domains G2 and G3 contains attachment regions for keratan sulfate and chondroitin sulfate chains. Aggrecan interacts via the G1 domain with hyaluronan and link protein to form large aggregates. Such aggregates can contain up to 50 -100 aggrecan monomers noncovalently bound to a single hyaluronan chain through 2 link proteins [1, 4]. Aggrecan degradation is catalyzed by proteinases of the matrix metalloproteinase and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) families. Aggrecan-cleaving ADAMTS4 and ADAMTS5, named also aggrecanase 1 and aggrecanase 2, hydrolyze aggrecan at five different sites in vitro and in vivo [5, 6, 7, 8]. Four cleavage sites are located in the chondroitin sulfate-rich region between aggrecan globular domains G2 and G3 (sites E1667 – G1668, E1480 – G1481, E1771 – A1772, E1871 – L1872), while one site is placed in the rod-like polypeptide between globular domains G1 and G2 (E373 – A374). In experiments with isolated proteins additional cleavage sites in aggrecan were identified both for ADAMTS4 [9] and ADAMTS5 [6]. A third proteinase of the ADAMTS family, ADAMTS1, also hydrolyses aggrecan at multiple sites including the unique site in the aggrecan interglobular domain [10].
The enzymatic activity of aggrecanases has been analyzed with isolated aggrecan preparations [11], recombinant aggrecan fragments [12] and a 41-residue peptide immobilized onto strepavidin-coated microplates [13]. The Sensitive Aggrecanase Activity Assay provides an improved and ready-to-use method for sensitive determination of aggrecanase activity.

Typical Standard Curve


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