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Aggrecanase Activity, Sensitive ELISA

Sensitive Aggrecanase ELISA
Catalog Number: 
M046009
Qty/Size: 
96 wells

Sensitive Aggrecanase ELISA from MD Bioproducts

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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

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

Sample Preparation: Proteolysis

Standard Curve Range:  0.022 - 1.4 nM

Sensitivity:  2 pM

Assay Length:   3.75 hours

Literature/Support

Literature/Support: 

Product Insert:

Aggrecanase Activity Assay Insert (PDF)

 

Articles/Troubleshooting:

ELISA Troubleshooting Guide

ELISA Data Reduction Guide

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.

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.


How It Works

How It Works: 

The Sensitive Aggrecanase Activity Assay measures activities of aggrecanases in the pM concentration range. This high sensitivity is acheived with an engineered aggreganase substrate derived from aggrecan interglobulin domain. It is used to measure aggrecanase activity in serum-free cell culture supernates. This assay consists of two modules, the Aggrecanase Module and the ELISA Module. The modified aggrecan in- terglobular domain (aggrecan-IGD-s) is first digested with aggrecanase. Proteolytic cleavage of the substrate releases an aggrecan peptide with the N-terminal sequence ARGSVIL (ARGSVIL-peptide-s). 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-s standard, proteolytic digestions of aggrecan-IGD-s 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-s 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.