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C2C Cartilage Degradation Competition Assay

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Clinical Applications:

This serum assay measures real-time collagen TYPE ii degradation, which may allow physicians/researchers to monitor the progression of Osteoarthritis (OA) and Rheumatoid Arthritis (RA).

Physiology

Enzymes known as Matrix Mettalloproteinases or MMPs (1,8,13) cleave Type l and ll collagen (bone and cartilage respectively) to form degradation fragments.

These fragments form two NEOEPITOPES which can be detected by IBEX immunoassays, C1–2C and C2C in serum. C1-2C and C2C assays detect different molecular structures of one of these neoepitopes.

How the Assay Works:

The assay is a two-step competitive immunoassay in a 96-well plate format using a short peptide epitope (peptide 287) that is recognized by a mouse monoclonal antibody. In a polypropylene mixing plate, C2C standards and diluted unknown serum samples (1/2) are added followed by monoclonal mouse IgG antibody. This pre-incubated mixture is transferred onto the ELISA plate and incubated to allow the free antibody to either bind to the BSA-287 conjugate coated on the plate, to the free 287 peptide (in the standards) or to the endogenous neoepitopes (samples). After washing, goat anti-mouse horseradish peroxidase (GAM-HRP) conjugate is added, which binds to any mouse antibody on the plate. Tetramethylbenzidine (TMB) substrate is added, then HRP degrades H202 and oxidizes TMB to form a blue product. The reaction is stopped and signal is amplified with an acid, which converts the product from a blue to a yellow colour that can be quantified at 450 nm.The optical density (OD) at 450 nm is inversely proportional to the amount of C2C epitopes.