CPII Cartilage Synthesis Competition Assay
(formerly known as "ProCollagen II C-Propeptide Assay")
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This assay, optimized for serum, monitors changes in the rate of synthesis of cartilage components (type II collagen) indicative of Osteoarthritis (OA) and Rheumatoid Arthritis (RA).
The precursor of newly formed collagen is procollagen. Collagenases cleave the amino and carboxy propeptides to transform procollagen to collagen. The CPII assay measures the released carboxy propeptide in the formation of collagen.
How the Assay Works:
The assay is a two-step competitive immunoassay in a 96-well format. In a polypropylene mixing plate, bovine CPII standards and diluted unknown serum samples (1/2) are added followed by a rabbit polyclonal antibody specific for CPII. This mixture is pre-incubated on a plate shaker to allow the binding of rabbit polyclonal antibody to the free CP II. Then the pre-incubated samples are transferred onto the ELISA plate and incubated for 2 hours with shaking to allow the CPII antibody to bind either to the CP II coated onto the bottom of the ELISA plate or to the CP II in solution. After washing, goat anti-rabbit horseradish peroxidase (HRP) conjugate is then added which binds to any rabbit 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 CPII epitope.