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Aggrecanase Activity ELISA

Aggrecanase Activity Assay - ELISA from MD Bioproducts
Catalog Number: 
M046008
Qty/Size: 
96 wells

Aggrecanase Activity ELISA from MD Bioproducts

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The Aggrecanase Activity ELISA Kit 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

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

Data/Specifications: 

Species: human

Sample Type:  cell culture supernates

Sample Size: 100 uL

Standard Curve Range:  0.062 - 4 nM

Sensitivity:  0.025 nM

Assay Length:   3.75 hours

How It Works

How It Works: 

The Aggrecanase Activity Assay measures activity of aggrecanases. It is used to screen and characterize aggrecanase inhibitors. This assay consists of two modules, the Aggrecanase Module and the ELISA Module. A recombinant fragment of human aggrecan interglobular domain (aggrecan-IGD) is first digested with ag- grecanase. Proteolytic cleavage of the substrate releases an aggrecan peptide with the N-terminal sequence ARGSVIL (ARGSVIL-peptide). The ARGSVIL-peptide is then quantified with two monoclonal anti-peptide antibodies.

 

Aggrecanase module: Proteolysis of aggrecan-IGD by aggrecanase

Aggrecan-IGD is incubated with standard aggrecanase and samples of unknown aggrecanase activity. In addition to aggrecanase, different concentrations of aggrecanase inhibitors can be added. The reaction is then stopped by dilution with EDTA-containing buffer.

 

ELISA module: Aggrecan peptide ELISA

ARGSVIL-peptide standard, proteolytic digestions of aggrecan-IGD with standard aggrecanase and samples are incubated in microtiter wells precoated with anti-ARGSVIL-neoepitope antibody. ARGSVIL-peptide is bound to the coated antibody, while other components are removed by washing and aspiration. The bound ARGSVIL-peptide is detected with a second peroxidase-labeled antibody. Any excess of the conjugate is removed by 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.