CS846 ELISA Aggregan Cartilage Synthesis Assay

Intended use:

The CS846, also known as aggrecan chondroitin sulfate 846 epitope, measures newly formed aggrecan. This ELISA assay with specific IgM monoclonal antibody recognizes an epitope present in fetal aggrecan and large aggrecan produced to repair cartilage damage when tissue is affected by RA or OA.

Click here – to download the CS846 Specification Sheet

Method:                      ELISA competitive, 96 wells

Sample type:              Optimized for serum (may also be used for plasma, synovial fluid, cell culture media)

Sample volume:        10 µL per replicate

Calibration range:     20 – 1000 ng/mL

Incubation time:        3 hours

Species:                     Optimized for human (may also be used for various animal species)

1.1 PRINCIPLE OF PROCEDURE This assay uses a mouse IgM monoclonal antibody to measure an epitope on chondroitin sulfate chains of the cartilage proteoglycan aggrecan which is increased in content in arthritis. In arthritis, articular cartilage is progressively destroyed. This is characterized by degradation of key molecules such as type II collagen and the proteoglycan aggrecan. It is accompanied by attempts at repair with an increase in synthesis of these molecules. This assay is for in vitro research use only and has been optimized for analyzing human serum. It has also been used to analyze various types of samples and species. It has been designed to be used for comparative analysis only, and is not to be used for absolute diagnostic purposes. All samples must be treated the same way and in the recommended manner.

1.2 PRINCIPLE OF THE ASSAY This assay is a competitive enzyme immunoassay in a 96-well format. Unlabelled CS846 standards and unknown diluted serum samples are added to the appropriate wells of the goat anti-mouse antibody (GAM) pre-coated plate. Biotin-labelled CS846 is added to all the wells followed by monoclonal mouse CS846 antibody to start the competition. This mixture is incubated to allow binding between biotin-labeled aggrecan (CS846-Biotin) and unlabeled aggrecan (Standards and samples) to the CS846 antibody as well as binding of the CS846 antibody to the goat anti-mouse IgM coated on the plate. After washing the plate, Streptavidin conjugated to horseradish peroxidase (SA-HRP) is added to bind to any bound biotinylated CS846. After washing the plate again, Tetramethylbenzidine substrate (TMB) is added to each well which react with HRP to form a blue product. The reaction is stopped and the signal 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 present in the sample.