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Cartilage Degradation Assays
Clinical Applications:
This serum assay measures the amount of real-time collagen (types I and 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 two short peptide epitopes (peptide 287 and 378) that are recognized by rabbit polyclonal antibodies. In a polypropylene mixing plate, C1-2C standards and diluted unknown serum samples (1/2) are addedfollowed by rabbit polyclonal antibody. The pre-incubated mixture is transferred onto the ELISA plate and incubated to allow the antibody to bind either to the BSA-287 conjugate coated on the ELISA plate, to the free 378 peptide (Standards) or to endogenous neoepitopes (samples). After washing, goat anti-rabbit horseradish peroxidase (GAR-HRP) conjugate is added which binds to any rabbit antibody on the plate. Tetra-methylbenzidine (TMB) substrate is added and then HRP degrades H202 and oxidizes TMB to form a blue product. The reaction is stopped and 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 epitope.
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